characterization and application of monoclonal antibody

Louisiana State UniversityLSU Digital Commons

LSU Historical Dissertations and Theses Graduate School

1988

Characterization and Application of MonoclonalAntibody and Bovine Neutrophil Reactivity toPasteurella Haemolytica Antigens.Frank William AustinLouisiana State University and Agricultural & Mechanical College

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Recommended CitationAustin, Frank William, "Characterization and Application of Monoclonal Antibody and Bovine Neutrophil Reactivity to PasteurellaHaemolytica Antigens." (1988). LSU Historical Dissertations and Theses. 4556.https://digitalcommons.lsu.edu/gradschool_disstheses/4556

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O rder N um ber 8904525

C haracterization and application o f m onoclonal antibody and bovine neutrophil reactiv ity to Pasteurella haemolytica antigens

Austin, Frank William, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1988

U M I300 N. Zeeb Rd.Ann Arbor, MI 48106

CHARACTERIZATION AND APPLICATION OF MONOCLONAL ANTIBODY AND BOVINE NEUTROPHIL REACTIVITY TO

PASTEURELLA HAEMOLYTICA ANTIGENS

A D i s s e r t a t i o n

Submi t t ed t o t h e Gradua te F a c u l t y o f t h e Lo u i s i a n a S t a t e U n i v e r s i t y and

A g r i c u l t u r a l and Mechanical Co l l ege in p a r t i a l f u l f i l l m e n t o f t h e

r e q u i r e m e n t s f o r t h e de g r e e o f Doctor o f Ph i l o s ophy

in t h e

V e t e r i n a r y Mi c r ob i o l ogy and P a r a s i t o l o g y Opt ion o f

The I n t e r d e p a r t m e n t a l Program in

V e t e r i n a r y Medical Sc i e nces

byFrank Wi l l i am A u s t i n

B . S . , Oklahoma S t a t e U n i v e r s i t y , 1979D.V.M. , Oklahoma S t a t e U n i v e r s i t y , 1983

August 1988

ACKNOWLEDGEMENTS

I am s i n c e r e l y g r a t e f u l t o t h e peop l e who have p a r t i c i p a t e d in my

e d u c a t i o n as a v e t e r i n a r y m i c r o b i o l o g i s t a t Lo u i s i an a S t a t e U n i v e r s i t y .

They no t on l y p r ov i d e d t h e o p p o r t u n i t i e s f o r academic g rowt h , b u t a l s o

c o n t r i b u t e d t o my growth as a human b e i ng . Dr. Richard E. C o r s t v e t , my

major p r o f e s s o r , s u p p l i e d s u p p o r t , g u i d a n c e , and f reedom which

s t i m u l a t e d p r o f e s s i o n a l and p e r s o n a l deve l opment . I d e e p l y a p p r e c i a t e

h i s t h o u g h t f u l s u g g e s t i o n s and a c t i o n s . I am a l s o t h a n k f u l t o t h e

f a c u l t y members who have s e r v e d on my g r a d u a t e a d v i s o r y commi t t ee ,

D r . s J . Turk , F. Lozano A . , F.M. E n r i g h t , G.F. Amborski , K.L. S ch no r r ,

M. Newman, and H.U. Cox.

I t hank t h e U n i v e r s i t y , t h e School o f V e t e r i n a r y Med i c i ne ,

Depar tment o f V e t e r i n a r y Mi c r ob i o l ogy and P a r a s i t o l o g y , and Depar tment

o f V e t e r i n a r y P a t ho l ogy f o r t h e p r i v i l e g e o f a l l ow i n g me t o e a r n t h i s

d eg r e e . I wi sh t o thank t h e r e s e a r c h a s s o c i a t e s whose t e c h n i c a l

a s s i s t a n c e and p r a c t i c a l working knowledge he l ped t u r n i d e a s i n t o

r e s u l t s . S pe c i a l t ha nks go t o D. Nobles , A. Smi th , and L. S h a f f e r f o r

t h e i r e n d e v o r s . M. Br ous sa r d and H. Cowgil l d e s e r ve r e c o g n i t i o n f o r

t h e i r e x p e r t i s e i n g r a p h i c a r t and medical pho t og r aphy . I am i n de b t e d

t o S. Whi t l ey f o r t y p i n g t h i s m a n u s c r i p t . Many thanks t o t h e g r a d u a t e

s t u d e n t s who gave s u p p o r t , a d v i c e and f r i e n d s h i p on an u n c o n d i t i o n a l

b a s i s . I hope t o r e f l e c t t h e knowledge, f r i e n d s h i p and love

e n c o u n t e r e d d u r i n g t h i s t r a i n i n g .

TABLE OF CONTENTSPage

TITLE PAGE............................................................................................................................ i

ACKNOWLEDGEMENTS ............................................................................................................. i i

TABLE OF CONTENTS.................................................................................................................. i i i

LIST OF TABLES...................................................................................................................... v

LIST OF FIGURES.................................................................................................................. vi

ABSTRACT..................................................................................................................................... v i i i

CHAPTER I . Development and C h a r a c t e r i z a t i o n o f MonoclonalA n t i b o d i e s A g a i n s t P a s t e u r e l l a ha e mol y t i ca S e r o t ype 1 . . . 1

A. Summary............................................................................................................... 1B. I n t r o d u c t i o n .............. ...................................................................................... 1C. M a t e r i a l s and Methods .............................................................................. 3D. R e s u l t s ............................................................................................................... 8E. D i s c u s s i o n ............................................................................................................15F. B i b l i o g r a p h y .......................................................................................................19

CHAPTER I I . Bovine N e u t r op h i l Response t o P a s t e u r e l l aha e mol y t i ca A n t i g e n s : Measurement o f Cy t o t ox i n A c t i v i t yin a C o l o r i m e t r i c A s s a y .......................................................................................23

A. Summary.....................................................................................................................23B. I n t r o d u c t i o n .......................................................................................................24C. M a t e r i a l s and Methods ................................................................................... 26D. R e s u l t s .....................................................................................................................31E. D i s c u s s i o n ........................................................................................................... 46F. B i b l i o g r a p h y .......................................................................................................50

CHAPTER I I I . C h a r a c t e r i z a t i o n o f a N e u t r a l i z i n g Monoclonal Ant ibody P r e pa r e d A g a i n s t P a s t e u r e l l a h a e mol y t i ca S e r o t y p e 1 C y t o t o x i n .................................................................................................53

A. Summary.....................................................................................................................53B. I n t r o d u c t i o n .......................................................................................................54C. M a t e r i a l s and Methods ...................................................................................56D. R e s u l t s .....................................................................................................................59E. D i s c u s s i o n ........................................................................................................... 65F. B i b l i o g r a p h y ...................................................................................................... 68

CHAPTER IV. S t r u c t u r a l Changes in Bovine N e u t r o p h i l s Exposed t o P a s t e u r e l l a ha e mol y t i ca S e r o t y p e 1 C y t o t o x i n ........................................................................................................................ 70

A. Summary.....................................................................................................................70B. I n t r o d u c t i o n .......................................................................................................70C. M a t e r i a l s and Methods ...................................................................................72

i i i

PAGE

D. R e s u l t s ......................... 74E. D i s c u s s i o n ...................................................................................................... 88F. B i b l i o g r a p h y ................................................................................................. 91

CHAPTER V. P u r i f i c a t i o n o f t h e S e r o t y p e 1 S p e c i f i c Ep i t ope From P a s t e u r e l l a h a e m o l y t i c a u s i n g Monoclonal Ant ibody I mm u n o a f f i n i t y ......................................................................................................... 96

A. Summary............................................................................................................... 96B. I n t r o d u c t i o n ................................................................................................. 97C. M a t e r i a l s and Methods ............................................................................. 99D. R e s u l t s .....................................................................................................................101E. D i s c u s s i o n ........................................................................................................... I l lF. B i b l i o g r a p h y .......................................................................................................116

CHAPTER VI. Conc l us i ons and Fu t u r e P e r s p e c t i v e s ................................... 120

VITAE................................................................................................................................................ 123

iv

L IS T OF TABLES

TABLE PAGE

1.1 D e t e r m i n a t i on o f Monoclonal Ant ibody I s o t y p e .......................... 9

1 . 2 R e a c t i v i t y o f Monoclonal A n t i b o d i e s w i t h£ . Haemolyt i ca Whole Cel l S e r o t y p e s ......................................................10

1 . 3 R e a c t i v i t y o f Monoclonal A n t i b o d i e s w i t hP. mu l t o c i d a Whole Cel l S e r o t y p e s ...........................................................11

1 . 4 R e a c t i v i t y o f Monoclonal A n t i b o d i e s w i t hFormal in o r Pa r a f o r ma l dehyde T r e a t e dP. h a e m o l y t i ca Whole C e l l s o r Ca p s u l a rM a t e r i a l ....................................................................................................................12

1 . 5 C h a r a c t e r i z a t i o n o f Monoclonal A n t i b o d i e sP r e pa r e d A g a i n s t P. h a e mo l y t i ca S e r o t y p e 1 ............................. 14

I I . 1 E v a l u a t i o n o f MTT Formazan S o l v e n t s .............................................32

1 1 . 2 C y t o t o x c i t y o f t h e Cy t o t ox i n P r e p a r a t i o n s ......................................... 38

1 1 . 3 Chemical and Immunologic C h a r a c t e r i z a t i o n o ft h e Cy t o t ox i n P r e p a r a t i o n s ................................................................... 45

I I I . l Ant ibody S o l u t i o n s Capable o f Cy t o t ox i nN e u t r a l i z a t i o n in t h e MTT C o l o r i m e t r i c Assay ........................ 61

V . l R e t e n t i o n o f Ant ibody A c t i v i t y and P r o t e i nC o n c e n t r a t i o n o f P u r i f i e d MonoclonalA n t i b o d y ................................................................................................................. 102

V.2 C h a r a c t e r i z a t i o n o f S a l i n e E x t r a c t a b l e Ca p s u l a rM a t e r i a l from P. h a e m o l y t i ca S e r o t y pe 1 ...................................... 103

V.3 S e l e c t i o n o f an Opt imal E l u a n t f o r I mmunoa f f i n i t yBased on D i s s o c i a t i o n o f B a c t e r i a l A g g l u t i n a t i o n . . . . 104

V.4 Monoclonal Ant ibody Coupl ing E f f i c i e n c y t o CNBrA c t i v a t e d Sepharose 4B ............................................................................ 105

V.5 Chemical D e t e r m i n a t i o n o f I mmunoaf f i n i t yAnt igen S e p a r a t i o n ...................................................................................... 109

V.6 A n a l y s i s o f t h e I mmunoa f f i n i t y Pr oduc t ....................................... 110

v

L IS T OF FIGURES

FIGURE PAGE

1 1 . 1 I n t r a c e l l u l a r a g g r e g a t e s and e x t r a c e l l u l a r c r y s t a l so f MTT formazan produced by bovine n e u t r o p h i l s ..............................33

1 1 . 2 N o n l i n e a r i t y o f abs o r ba nc e v e r s e s n e u t r o p h i l number a t v a r i o u s t ime i n t e r v a l s in t h e MTTc o l o r i m e t r i c a s s a y ................................................................................................ 34

1 1 . 3 L i n e a r r e l a t i o n s h i p between a b s o r ba n c e and MTT i n c u b a t i o n t ime a t d i f f e r e n t n e u t r o p h i l numbersp e r wel 1 ........................................................................................................................35

1 1 . 4 E f f e c t o f methanol f i x e d n e u t r o p h i l s mixed wi t hnormal n e u t r o p h i l s in t h e MTT c o l o r i m e t r i c a s s a y .........................36

1 1 . 5 Dose r e s po n s e o f bov ine n e u t r o p h i l s exposed t o t h ep r e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n in t h e MTT c o l o r i m e t r i c a s s a y ................................................................................................ 37

1 1 . 6 Sigmoidal dose r e s po n s e o f bov ine n e u t r o p h i l s exposed t o t h e 15:1 c o n c e n t r a t e d c y t o t o x i np r e p a r a t i o n in t h e MTT c o l o r i m e t r i c a s s a y .................................. 39

1 1 . 7 Dose r e s po n s e o f bov ine n e u t r o p h i l s t o c y t o t o x i nu l t r a f i l t r a t e in t h e MTT c o l o r i m e t r i c a s s a y ............................. 40

1 1 . 8 Dose r e s po n s e o f n e u t r o p h i l s exposed t o 15:1c o n c e n t r a t e d c y t o t o x i n i n a c t i v a t e d wi t h mi ld h e a t . . . . 42

1 1 . 9 Dose r e s po n s e o f n e u t r o p h i l s t o b o i l e d andc l a r i f i e d 15:1 c o n c e n t r a t e d c y t o t o x i n ........................................... 43

111.1 Cy t o t ox i n a c t i v i t y p r e s e n t in t h e 15:1 c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n u s i n g t heMTT c o l o r i m e t r i c a s s a y .......................................................................................59

1 1 1 . 2 Cy to t o x i n n e u t r a l i z a t i o n by monoclonal a n t i b o d i e sin t h e MTT c o l o r i m e t r i c a s s a y ....................................................................63

1 1 1 . 3 E x t e n t o f c y t o t o x i n n e u t r a l i z a t i o n by t h e nMcAbin a s c i t i c f l u i d compared t o o t h e r n o n - n e u t r a l i z i n g a s c i t i c f l u i d s in t h e MTT c o l o r i m e t r i c a s s a y ....................... . 64

I V . 1 T r an s m i s s i o n e l e c t r o n mi c rograph o f a normal bovinen e u t r o p h i l ................................................................................................................... 76

I V . 2 E l e c t r o n mi c r ogr aph o f a bov ine n e u t r o p h i l exposedt o c y t o t o x i n ( 1 : 8 d i l u t i o n ) f o r 5 m i n u t e s ....................................... 77

vi

FIGURE PAGE

I V . 3 A l t e r a t i o n o f bov ine n e u t r o p h i l g r a n u l e i n t e g r i t yo c c u r i n g a t 5 mi nu t e s c y t o t o x i n ( 1 : 8 d i l u t i o n ) e x p o s u r e ........................................................................................................................78

IV. 4 Bovine n e u t r o p h i l w i t h 2 l a r g e uropods t y p i c a l o fc e l l s f o l l o wi n g a 10 mi nute c y t o t o x i n ( 1 : 8 d i l u t i o n ) e x p o s u r e ................................................................................ 79

I V . 5 T r a n s mi s s i on e l e c t r o n mi c r og r aphs showing g r a n u l e sin n e u t r o p h i l s exposed t o c y t o t o x i n ( 1 : 8 d i l u t i o n ) f o r 10 m i n u t e s ......................................................................................................... 80

IV. 6 Ne u t r o p h i l f o l l o w i n g a 15 mi nute c y t o t o x i n( 1 : 8 d i l u t i o n ) e xpos u r e ............................................................................ 81

I V . 7 C o n c e n t r i c a l l y l a mi n a t ed myel in f i g u r e formed fromt h e l a r g e p e r s i s t e n t g r a n u l e s .............................................................. 82

IV. 8 Ne u t r op h i l a t 20 mi nu t e s p o s t c y t o t o x i n ( 1 : 8 d i l u t i o n )e x p o s u r e ....................... 83

IV . 9 T r a n s mi s s i on e l e c t r o n mi crograph o f a bov inen e u t r o p h i l exposed t o a 1:512 c y t o t o x i n d i l u t i o nf o r 20 m i n u t e s ......................................................................................................... 84

I V . 10 Ne u t r op h i l exposed t o a 1:256 c y t o t o x i n d i l u t i o nf o r 20 m i n u t e s ......................................................................................................... 85

I V . 11 N e u t r op h i l exposed t o a 1:64 c y t o t o x i n d i l u t i o nf o r 20 m i n u t e s ......................................................................................................... 86

I V . 12 N e u t r op h i l exposed t o a 1:32 c y t o t o x i n d i l u t i o nf o r 20 m i n u t e s ..........................................................................................................87

V. l Phase c o n t r a s t mi c rograph o f e n c a p s u l a t e d P. h a e m o l y t i ca s e r o t y p e 1 immobi l i zed onimmunobead s u r f a c e .............................................................................................. 106

V.2 Phase c o n t r a s t mi c rograph o f e n c a p s u l a t e d P.h a e m o l y t i ca s e r o t y p e 2 and immunobeads d e m o n s t r a t i n g s p e c i f i c i t y by a l a ck o fi m m o b i l i z a t i o n ........................................................................................................108

v i i

ABSTRACT

Monoclonal a n t i b o d i e s (McAbs) were p r ep a r e d a g a i n s t P a s t e u r e l l a

ha e mol y t i ca s e r o t y p e 1 (Ph-1) c a p s u l a r m a t e r i a l t o i d e n t i f y , q u a n t i t a t e

and p u r i f y a n t i g e n s . McAbs were s e l e c t e d by ELISA and c h a r a c t e r i z e d

ac c o r d i n g t o t h e i r i s o t y p e , c r o s s - r e a c t i v i t y wi t h P. h a emol y t i ca and P.

mu 1 t o e i d a s e r o t y p e s in ELISA, a n t i g e n f o r ma l i n s e n s i t i v i t y and a b i l i t y

t o cause b a c t e r i a l a g g l u t i n a t i o n . Four groups o f McAbs were

e s t a b l i s h e d each r e c o g n i z i n g a d i f f e r e n t e p i t o p e . Var ious P.

h a emol y t i ca and P. mu 1 t o e i d a s e r o t y p e s were found t o s h a r e s e v e r a l

e p i t o p e s d e m o n s t r a t i n g t h e i r a n t i g e n i c r e l a t e d n e s s .

A c o l o r m e t r i c a s s a y , based on t e t r a z o l i u m dye r e d u c t i o n by bov ine

n e u t r o p h i l s , was adap t ed f o r t h e measurement and c h a r a c t e r i z a t i o n o f

Ph-1 c y t o t o x i n a c t i v i t y . Using d i f f e r e n t c y t o t o x i n p r e p a r a t i o n s which

v a r i e d in c y t o l y t i c a c t i v i t y , c y t o t o x i n a c t i v i t y was i d e n t i f i e d as t h e

i n h i b i t i o n o f dye r e d u c t i o n . S t i m u l a t i o n o f dye r e d u c t i o n by

n e u t r o p h i l s in r e s p o n s e t o t h e c y t o t o x i n p r e p a r a t i o n s was a l s o d e t e c t e d

in t h e a s s a y s u g g e s t i n g n e u t r o p h i l a c t i v a t i o n i n v o l v i n g t h e r e s p i r a t o r y

b u r s t . McAbs and chemica l methods were used t o i d e n t i f y and q u a n t i t a t e

c y t o t o x i n p r e p a r a t i o n a n t i g e n s .

The c o l o r i m e t r i c a s s a y was f u r t h e r a p p l i e d t o s e l e c t and

c h a r a c t e r i z e a c y t o t o x i n - n e u t r a l i z i n g monoclonal a n t i b o d y (nMcAb).

The nMcAb was no t r e a c t i v e i n ELISA, bu t produced s u b s t a n t i a l

n e u t r a l i z a t i o n in t h e c o l o r i m e t r i c a s s a y . The nMcAb was i d e n t i f i e d as

an IgM. McAbs s e l e c t e d ELISA a g a i n s t c a p s u l a r m a t e r i a l d i d no t a f f e c t

n e u t r o p h i l dye r e d u c t i o n in r e s po n s e t o c y t o t o x i n .

U l t r a s t r u c t u r a l changes in bov ine n e u t r o p h i l s exposed t o c y t o t o x i n

were examined o ve r d i f f e r e n t t i me i n t e r v a l s and c o n c e n t r a t i o n s t o

c h a r a c t e r i z e i t s e f f e c t s a t t h e c e l l u l a r l e v e l . I d e n t i c a l t ime and

c o n c e n t r a t i o n dependen t changes i n v o l v i n g c e l l p o l a r i z a t i o n wi t h uropod

f o r m a t i o n , plasma membrane d e f e c t s , and d i f f e r e n t i a l i n t r a c e l l u l a r

d e g r a n u l a t i o n p r o g r e s s i n g t o comple t e l y s i s were o b s e r ve d .

P o l a r i z a t i o n wi t h uropod f o r m a t i on s u gg es t e d t h e p r e s en c e o f a

c h e m o t a c t i c f a c t o r and c o r r e s po n d e d wi t h a c t i v a t i o n obs e rved in t h e

c o l o r i m e t r i c a s s a y .

A McAb was a p p l i e d in i mmu n o a f f i n i t y t o p u r i f y t he Ph-1 s p e c i f i c

e p i t o p e from c a p s u l a r m a t e r i a l . The i mmunoaf f i n i t y p r od u c t c o n t a i n e d

no d e t e c t a b l e p r o t e i n and a t l e a s t one h a l f t h e o r i g i n a l hexosamine

c o n t e n t . McAbs were used in ELISA t o i d e n t i f y and q u a n t i t a t e t h e

p r o d u c t a n t i g e n s . L i p o p o l y s a c c h a r i d e and p a n - p a s t e u r e l l a c a r b o h y d r a t e

were s e l e c t i v e l y r e t a i n e d in a d d i t i o n t o Ph-1 s p e c i f i c c a p s u l a r

p o l y s a c c h a r i d e s u g g e s t i n g e p i t o p e s h a r i n g among p o l y s a c c h a r i d e

a n t i g e n s .

i x

CHAPTER 1

DEVELOPMENT AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES

AGAINST PASTEURELLA HAEMOLYTICA SEROTYPE 1

SUMMARY

P a s t e u r e l l a h a e mo l y t i ca s e r o t y p e 1 (Ph-1) c a p s u l a r m a t e r i a l was

e x t r a c t e d f rom l o g a r i t h m i c growth phase c e l l s and used t o immunize

BALB/c mice f o r monoclonal a n t i b o d y p r o d u c t i o n . Hybridomas p roduc i ng

a n t i b o d y t o Ph-1 c a p s u l a r m a t e r i a l were s e l e c t e d u s i n g an ELISA

p r oc e d u r e and r e p e a t e d l y c l oned by l i m i t i n g d i l u t i o n . Monoclonal

a n t i b o d i e s o b t a i n e d were c h a r a c t e r i z e d and grouped a c c o r d i n g t o t h e i r

i s o t y p e , c r o s s - r e a c t i v i t y w i t h 6 and 24 hour P. h a e m o l y t i c a (Ph) and P.

mu1 t o e i d a (Pm) s e r o t y p e s in ELISA, a n t i g e n f o r m a l i n s e n s i t i v i t y and

a b i l i t y t o cause r a p i d p l a t e a g g l u t i n a t i o n . Four g roups o f a n t i b o d i e s

were e s t a b l i s h e d which were (1) IgM Ph-1 s p e c i f i c a g g l u t i n a t i n g , (2)

IgG^ c r o s s - r e a c t i v e w i t h v a r i o u s Ph s e r o t y p e s , (3) IgM c r o s s - r e a c t i v e

wi t h many Ph and Pm s e r o t y p e s , and (4) IgG^A c r o s s - r e a c t i v e wi t h a l l Ph

and Pm s e r o t y p e s . The Ph-1 s p e c i f i c a n t i g e n and t h e a n t i g e n common t o

a l l Ph and Pm s e r o t y p e s were f o r ma l i n s e n s i t i v e . These s t u d i e s

d e m o n s t r a t e t h e use o f d i f f e r e n t i a l a n t i g e n e x p r e s s i o n by b a c t e r i a l

s e r o t y p e s t o c h a r a c t e r i z e monoclonal a n t i b o d i e s which were produced

a g a i n s t a complex a n t i g e n s o u r ce and i n d i c a t e s t h a t t h e v a r i o u s Ph and

Pm s e r o t y p e s s h a r e s e v e r a l common e p i t o p e s .

INTRODUCTION

P a s t e u r e l l a h a e m o l y t i c a b i o t y p e A, s e r o t y p e 1 (Ph-1) i s t h e

p a t h o g e n i c a g en t most f r e q u e n t l y a s s o c i a t e d wi t h s e v e r e f i b r i n o u s

1

2

1 7pnemonia in pnemonic p a s t e u r e l l o s i s ( s h i p p i n g f e v e r ) o f c a t t l e . *

S u c c e s s f u l d i v i s i o n o f P. h a e m o l y t i ca i n t o d i s c r e t e s e r o t y p e s was

accompl i shed u s i n g s p e c i f i c r a b b i t a n t i s e r a in i n d i r e c t

h e m a g g l u t i n a t i o n and r a p i d p l a t e a g g l u t i n a t i o n r e a c t i o n s . 3 , 4 S o l u b l e

c a p s u l a r p o l y s a c c h a r i d e a n t i g e n s a r e a c c e p t e d t o c o n f e r s e r o t y p e

s p e c i f i c i t y r e c o gn i z e d by a n t i s e r a in t h e s e r e a c t i o n s . 4 , 6 G r e a t e r t ha n

12 s e r o t y p e s have been i d e n t i f i e d which can be d i v i d e d i n t o 2 b i o t y p e s ,

A and T, r e s p e c t i v e o f t h e i r a b i l i t y t o f e r me n t a r a b i n o s e and

7 8t r e h a l o s e . 5 S e r o t y p e s 3 , 4 and 10 be l ong t o b i o t y p e T, w h i l e t h e

rema i n i ng s e r o t y p e s do no t f e r me n t t r e h a l o s e and be l ong t o b i o t y p e A.

S e r o l o g i c a l c r o s s - r e a c t i o n s among t h e v a r i o u s s e r o t y p e s , mo s t l y w i t h i n

b i o t y p e s , have been r e p o r t e d u s i n g s e v e r a l a n a l y t i c a l

3 4 9-12t e c h n i q u e s . * ’ These r e a c t i o n s and c r o s s r e a c t i o n s have d e f i n e d a

complex, y e t d i s t i n g u i s h a b l e a n t i g e n i c p r o f i l e among t h e P a s t e u r e l l a e .

With t h e development o f monoclonal a n t i b o d y t e c h n i q u e s , more t ha n

13a decade ago by Kohler and M i l s t e i n , i t has become p o s s i b l e t o s t u d y

complex a n t i g e n i c sys t ems u s i n g s e r o l o g i c methods . Ce l l s u r f a c e

a n t i g e n s o f l ymphocytes and b a c t e r i a r e p r e s e n t a complex sys t em t o

which monoclonal a n t i b o d i e s have been e x t e n s i v e l y a p p l i e d . Whole c e l l s

o r e x t r a c t s a r e commonly i n j e c t e d i n t o mice and monoclonal a n t i b o d i e s

can be o b t a i n e d which d e f i n e s p e c i f i c s u r f a c e a n t i g e n s in a complex

14-20s u b p o p u l a t i o n o f c e l l s . Because t h e s e r o t y p e s and b i o t y p e s o f

P a s t e u r e l l a h a e m o l y t i ca and o t h e r P a s t e u r e l l a s p e c i e s r e p r e s e n t

a n t i g e n i c a l l y complex s u b p o p u l a t i o n s o f c e l l s , i t s hou l d be p o s s i b l e t o

p roduce monoclonal a n t i b o d i e s which can d i f f e r e n t i a t e s o l u b l e o r

s u r f a c e e x p r e s s e d a n t i g e n s among t h e s e p o p u l a t i o n s . R e a c t i v i t y o f

monoclonal a n t i b o d i e s w i t h c e l l s u r f a c e a n t i g e n s i s commonly measured

3

u s i n g e n zyme- l i nked immunosorbent a s s a y s (ELISA) f o r which many

14-22v a r i a t i o n s e x i s t . U s u a l l y c e l l s a r e a t t a c h e d t o a s o l i d phase (96

wel l p l a t e s ) u s i n g a c r o s s - l i n k i n g a g en t such as g l u t a r a l d e h y d e ,o n p i

p a r a f o r ma l d e h yd e , o r o t h e r f i x a t i v e s . ’ These p ro c e d u r es

o f t e n a l t e r a n t i g e n i c i t y o f c e l l s u r f a c e c o n s t i t u e n t s and t h e r e f o r e t h e

a s s a y r e s u l t s o b t a i n e d . " Ant igen r e a c t i v i t y f o l l o w i n g d i f f e r e n t

t r e a t m e n t s can t h e r e f o r e be used t o d i s t i n g u i s h v a r i o u s a n t i g e n s and

t o d i f f e r e n t i a t e monoclonal a n t i b o d y s p e c i f i c i t i e s . I t i s e a s i l y

e n v i s i o n e d t h a t t h i s cou l d be an i m p o r t a n t phenomena wi t h r e s p e c t t o

t h e immunogenic i ty and e f f i c a c y o f b a c t e r i n s and v a c c i n e s t r e a t e d wi t h

d i f f e r e n t f i x a t i v e a g e n t s .

F u r t h e r a n t i b o d y c h a r a c t e r i z a t i o n s f r e q u e n t l y i n vo l ve

d e t e r m i n a t i o n o f i s o t y p e , s u b i s o t y p e , t h e c a p a c i t y t o n e u t r a l i z e

b i o l o g i c a l a c t i v i t i e s , b a c t e r i o c i d a l o r b a c t e r i o s t a t i c e f f e c t s in t h e

p r e s e n c e o f complement , and i n s o l u b i l i z a t i o n o f a n t i g e n s t h r ough

a g g l u t i n a t i o n o r p r e c i p i t a t i o n . Monoclonal a n t i b o d y t e ch n o l o g y has no t

been e x t e n s i v e l y a p p l i e d t o F\ h a emol y t i ca o r bovine pnemonic

p a s t e u r e l l o s i s . The pu r pos e o f t h i s r e p o r t i s t o d e s c r i b e t he

deve l opment and c h a r a c t e r i z a t i o n o f monoclonal a n t i b o d i e s a g a i n s t F\

h a e mo l y t i ca s e r o t y p e 1 c a p s u l a r m a t e r i a l .

MATERIALS AND METHODS

P. h a e mo l y t i ca a n t i g e n p r e p a r a t i o n :

P a s t e u r e l l a h a e m o l y t i ca b i o t y p e A, s e r o t y p e 1, was m a i n t a i n e d by

l y o p h i l i z a t i o n in skim mi lk and p e r i o d i c animal pas sage as p r e v i o u s l y

31d e s c r i b e d . Agar p l a t e s c o n t a i n i n g 10% ( v / v ) bovine b l ood , 1% ( v / v )

h o r se serum, and 1% ( v / v ) y e a s t h y d r o l y s a t e were used t o p r op a g a t e t h e

b a c t e r i u m in c a n d l e j a r s a t 37C. C a p s u l a r m a t e r i a l was e x t r a c t e d from

326 hour c o n f l u e n t c u l t u r e s a c c o r d i n g t o t h e method o f Gen t ry . The

b a c t e r i a were removed from t h e s o l u b i l i z e d c a p s u l a r m a t e r i a l by

c e n t r i f u g a t i o n a t 12,000 xg f o r 30 min a t 4C. The s u p e r n a t e c o n t a i n i n g

c a p s u l a r a n t i g e n s was f i l t e r s t e r i l i z e d (0.22ym) and d i a l y z e d (8 -10 k

MWC0, F i s h e r S c i . Co. ) e x t e n s i v e l y a g a i n s t d i s t i l l e d w a t e r a t 4C ove r

48 h r s . The d i a l y z e d c a p s u l a r a n t i g e n s were f r o z e n a t -70C and

l y o p h i 1i z ed p r i o r to use .

Mice and immuniza t ions :

The BALB/c mice which were used f o r t h e p r o d u c t i o n o f monoclonal

a n t i b o d i e s were br ed in o u r f a c i l i t i e s . One h a l f mg c a p s u l a r m a t e r i a l

in F r e u n d ' s comple t e a d j u v a n t ( 0 . 5 ml) was used t o immunize 6-8 week

o l d f emale mice by i n t r a p e r i t o n e a l i n j e c t i o n . One month l a t e r , mice

were immunized ag a i n wi t h 0 . 5 mg c a p s u l a r m a t e r i a l in 0 . 5 ml phospha te

b u f f e r e d s a l i n e (PBS, pH 7 . 2 ) by t h e same r o u t e . Blood samples were

wi thdrawn from t h e r e t r o - o r b i t a l p l exus p r i o r t o immunizat ion and 3

days f o l l o w i n g t h e b o o s t e r immuniza t ion f o r measurement o f serum

a n t i b o d y . Su cc e s s f u l immuniza t ion and s e l e c t i o n o f mice f o r monoclonal

a n t i b o d y p r o d u c t i o n was e s t a b l i s h e d u s i n g ELISA ( se e Assay f o r a n t i bo dy

p r o d u c t i o n and hybridoma c l o n i n g ) . Mice havi ng t he h i g h e s t a n t i b o d y

t i t e r t o c a p s u l a r m a t e r i a l were choosen f o r c e l l f u s i o n .

5

Cel l f u s i o n and h y b r id s e l e c t i o n :

Four days f o l l o w i n g t h e b o o s t e r immuni za t i on , s p l e e n c e l l s from

one mouse were f u sed wi t h SP-2 /o mur ine myeloma c e l l s a t a 4 :1 r a t i o in

t h e p r e s e n c e o f 50% ( v / v ) 1450 MW p o l y e t h y l e n e g l y c o l . A f t e r t he

f u s i o n , c e l l s were g e n t l y washed wi t h and d i l u t e d in RPMI-1640

c o n t a i n i n g 15% ( v / v ) f e t a l c a l f serum, O.OlmM h yp ox a n t h i n e , 1 . 6x l0~ mM

t h y m i d i n e , 10 mM sodium b i c a r b o n a t e , 1% ( v / v ) n o n - e s s e n t i a l amino

a c i d s , and lOmM L- g l u t a mi ne (HT-medium) t o a c o n c e n t r a t i o n o f 7 . 5x10^

s p l e n o c y t e s / m l . A l i q u o t s ( 0 . 1ml ) were d e l i v e r e d t o t h e w e l l s o f f o u r

96 we l l p l a t e s which c o n t a i n e d 5x10^ BALB/c t h y mo c y t e s / we l l as f e e d e r

c e l l s . The mixed c e l l p o p u l a t i o n was c u l t u r e d f o r 24 h r a t 37C in t h e

p r e s e n c e o f 5% CO2 . Hybr id c e l l s were s e l e c t e d by t h e d a i l y f e e d i n g of

0 . 1 ml volumes o f HT-medium c o n t a i n i n g 4X10"^ mM a m i n o p t e r i n

(HAT-medium) f o r f o u r c o n s e c u t i v e d a y s . Fo l lowing h y b r id s e l e c t i o n t h e

c e l l s were m a i n t a i n e d and expanded in HT-medium. P l a t e s were v i s u a l l y

a s s e s s e d f o r c l o n a l growth on a d a i l y b a s i s d u r i n g hy b r id c e l l

s e l e c t i o n u s i n g an i n v e r t e d mi c r o s c o p e .

Assay f o r a n t i b o d y p r o d u c t i o n and hybridoma c l o n i n g :

S u pe r n a t e s from hybr idoma c e l l c u l t u r e s o r mouse s e r a were

e v a l u a t e d f o r s p e c i f i c a n t i b o d y c o n t e n t by ELISA u s i n g c a p s u l a r

m a t e r i a l as t h e a n t i g e n . B r i e f l y , 50 pi volumes c o n t a i n i n g 5pg

c a p s u l a r m a t e r i a l i n TEN b u f f e r 0.05M T r i s , 0.001M Na^EDTA, 0.15M NaCl ,

pH 7 . 4 were a i r d r i e d in 96 wel l p o l y s t y r e n e p l a t e s o v e r n i g h t . The

d e s s i c a t e d a n t i g e n was f i x e d wi t h 50 pi pa r a f o r ma l dehyde pe r wel l f o r 5

min and washed 3 t i mes wi t h TEN b u f f e r . Wel ls were b locked wi t h

200 yl a l i q u o t s o f 2% (w/v) BSA in phos pha t e b u f f e r e d s a l i n e (PBS,

0.15M, pH 7 . 2 ) f o r 2 h r and washed. Hybridoma c u l t u r a l s u p e r n a t e s o r

mouse s e r a (50 y l ) were added and a l l owed t o b ind f o r 45 min a t room

t e m p e r a t u r e . The w e l l s were washed 3 t i me s and 50 yl volumes o f g o a t

a n t i - m o us e IgG, IgM h o r s e - r a d i s h p e r o x i d a s e c o n j u g a t e d s e c onda r y

a n t i b o d y (1 : 1500 d i l u t i o n i n TEN b u f f e r ) was added t o t h e w e l l s and

i n c u b a t e d f o r 45 min a t room t e m p e r a t u r e . Unbound c o n j u g a t e d a n t i b o d y

was washed from t h e w e l l s 3 t i mes and hybr idomas s e c r e t i n g a n t i b o d y

s p e c i f i c f o r c a p s u l a r m a t e r i a l were i d e n t i f i e d u s i n g

o r t h op h e n y l e n e d i a mi n e as a s u b s t r a t e . P l a t e s were v i s u a l l y i n s p e c t e d

f o r p o s i t i v e r e a c t i o n s and 10 w e l l s were s e l e c t e d f o r c l o n i n g .

Fo l lowing s e l e c t i o n o f t h o s e hybr idomas s e c r e t i n g a n t i b o d y

s p e c i f i c f o r c a p s u l a r a n t i g e n s , c e l l s were c l oned by l i m i t i n g d i l u t i o n

in 96 wel l p l a t e s . BALB/c thymocytes ( 5x10^ / we l 1) were used as f e e d e r2

c e l l s . Cloned hybr idomas were expanded in 25 cm f l a s k s in HT-medium

and c r y o p r e s e r v e d in t h e same medium c o n t a i n i n g 20% ( v / v ) FCS and 10%

( v / v ) DMS0.

P e r i t o n e a l a s c i t i c f l u i d , f o r each o f t h e c l one d hybr idomas , was

produced in a d u l t BALB/c mice c o n d i t i o n e d one week in advance wi t h 0 . 5

ml p r i s t a n e . P r e c o n d i t i o n e d mice were i n o c u l a t e d w i t h 106 v i a b l e

h yb r i d c e l l s . P e r i t o n e a l a s c i t i c f l u i d was h a r v e s t e d by p e r i o d i c

ab d o mi n o ce n t e s i s b e g i nn i n g 7 days a f t e r i n o c u l a t i o n , c l a r i f i e d by

c e n t r i f u g a t i o n , and s t o r e d a t -70C.

D e t e r m i n a t i o n o f monoclonal a n t i b o d y i s o t y p e :

I s o t y p e s o f t h e 10 s e l e c t e d monoclonal a n t i b o d i e s were d e t e r m i n e d ,

u s i n g hybr idoma c u l t u r a l s u p e r n a t e s , w i t h two d i f f e r e n t co m me r c i a l l y

7

a v a i l a b l e i s o t y p i n g k i t s based on immunodi f fus ion and a n t i b o d y c a p t u r e .

Assay o f monoclonal a n t i b o d y s p e c i f i c i t y :

Monoclonal a n t i b o d i e s , were a s s a y e d f o r t h e i r s p e c i f i c i t y t o o t h e r

P. h a e m o l y t i c a and P. m u l t o c i d a s e r o t y p e s u s i n g t h e ELISA p r oc e d u r e

wi t h s l i g h t m o d i f i c a t i o n s . P. ha e mol y t i ca s e r o t y p e s and b i o t y p e s

4 7 331-12 ' and P. m u l t o c i d a s e r o t y p e s 1-16 were d e f i n e d and p r o p a g a t e d

as d e s c r i b e d f o r Ph-1. Both 6 and 24 h r c u l t u r e s o f t h e b a c t e r i a were

p r e p a r e d and suspended in PBS. B a c t e r i a l s u s p e n s i o n s were a d j u s t e d t o9

1x10 CFU/ml u s i n g a s p e c t r o p h o t o m e t e r and 50 yl a l i q u o t s o f a 1:26

d i l u t i o n , d e t e r mi n e d opt imum by c h ec k e r bo a r d t i t r a t i o n , were used as

a n t i g e n s i n t h e ELISA.

D e t e r m i n a t i o n o f a n t i g e n f o r m a l i n s e n s i t i v i t y :

Ph-1 whole c e l l s o r homologous c a p s u l a r m a t e r i a l were p r e p a r e d as

d e s c r i b e d and d e s s i c a t e d i n t o 96 wel l p l a t e s . The d e s s i c a t e d a n t i g e n

were e i t h e r u n t r e a t e d o r t r e a t e d wi t h 50 yl 10% ( v / v ) b u f f e r e d f o r m a l i n

a n d / o r p a r a f o r ma l d e h y d e f o r 5 min. The w e l l s were washed t h r e e t i mes

and t h e ELISA comple t ed as d e s c r i b e d .

D e t e r m i n a t i o n o f r a p i d s l i d e a g g l u t i n a t i o n o f Ph-1 by a s c i t i c f l u i d s :

P e r i t o n e a l a s c i t i c f l u i d s were c l a r i f i e d by c e n t r i f u g a t i o n a t 2500

xg f o r 5 min, r e s i d u a l p r i s t a n e removed wi t h a p i p e t t e and t h e a s c i t i c

f l u i d pas sed t h r ou g h a 0 . 2 2 urn f i l t e r . F i f t y yl a l i q u o t s o f t h e

c l a r i f i e d a s c i t i c f l u i d s f rom each c l one d hybridoma were mixed wi t h 50g

yl volumes o f 1x10 CFU/ml o f t h e P. h a e m o l y t i c a s e r o t y p e s on a c l e a n e d

8

s l i d e . The s l i d e s were s l o w l y rocked a t room t e m p e r a t u r e and obse r ved

f o r r a p i d a g g l u t i n a t i o n .

RESULTS

Fol lowing h y b r i d c e l l s e l e c t i o n and e xpa ns i on in 96 wel l p l a t e s ,

n e a r l y e v e r y wel l c o n t a i n e d r a p i d l y growing hybr idomas 10 days a f t e r

t h e f u s i o n . S p e c i f i c a n t i b o d y p r o d u c t i o n a g a i n s t P. h a emol y t i ca

s e r o t y p e 1 c a p s u l a r a n t i g e n s was i d e n t i f i e d in 250 c u l t u r e s (74% o f

t o t a l ) . These p o s i t i v e w e l l s were v a r i a b l e in t h e i r de g r e e o f hy b r id

c e l l growth and a n t i b o d y r e a c t i v i t y based on ELISA s u b s t r a t e

a b s o r b a n c e . Ten w e l l s t h a t v a r i e d in a b s o r b a n c e , a l l which c o n t a i n e d

v i g o r o u s l y growing hybr i domas , were chosen f o r c l o n i n g by l i m i t i n g

d i l u t i o n and f u r t h e r s t u d y . These hybr idomas were numbered 1 t h rough

10 and c l oned g r e a t e r t ha n 5 t i mes and s e l e c t e d by ELISA o ve r s e v e r a l

months . Dur ing c l o n i n g 2 hybr idomas proved t o be u n s t a b l e and f a i l e d

t o produce a n t i b o d y as d e t e c t e d by ELISA. A t h i r d hybridoma became

c o n t ami n a t e d and was d i s c a r d e d .

I s o t y p e s o f t h e 10 o r i g i n a l l y s e l e c t e d hybr idomas were de t e r mi n e d

us i n g c u l t u r a l s u p e r n a t e in two d i f f e r e n t methods (Tab l e 1 . 1 ) . The

r e s u l t s o f immunodi f fus ion and a n t i b o d y c a p t u r e were in e x a c t

a gg r ee men t , however , i mmunodi f fus ion was a p p a r e n t l y no t as s e n s i t i v e as

a n t i b o d y c a p t u r e and f a i l e d t o i d e n t i f y many a n t i b o d i e s . A n t i b o d i e s

were i d e n t i f i e d i n e i t h e r t h e IgM o r IgG c l a s s and many hybr idomas were

found t o produce t h e same i s o t y p e and s u b - i s o t y p e o f a n t i b o d y .

9

Table 1.1 - D e t e r m i n a t i o n o f Monoclonal Ant ibody I s o t y p e

Monoclonal I s o t y p e

Ant ibody Immunodi f fus ion Ant ibody Capt ure

1 I gG3 I gG3

2 ND* IgG2A

3 IgM IgM

4 ND IgG2A

5 ND IgG3

6 ND I gG3

7 ND ND

8 IgGl IgGl

9 IgM IgM

10 IgM IgM

* ND = Not de t e rmi ne d

R e a c t i v i t y o f t he seven r ema i n i ng monoclonal a n t i b o d i e s wi th P.

ha e m o l y t i ca s e r o t y p e s ( Tab l e 1 . 2 ) and P. mu l to c i da s e r o t y p e s (Table

1 . 3 ) was d e t e r mi ne d u s i n g whole b a c t e r i a l c e l l s as t h e a n t i g e n s in

ELISA. Monoclonal a n t i b o d i e s were grouped a c c o r d i n g t o t h e i r

r e a c t i v i t y among t h e s e s e r o t y p e s . Four p a t t e r n s o f r e a c t i v i t y were

i d e n t i f i e d : (1) P. ha e mol y t i ca s e r o t y p e 1 s p e c i f i c , (2) c r o s s r e a c t i v e

wi t h P. h a e m o l y t i ca s e r o t y p e s 1, 5 - 8 , and 12 no t c r o s s r e a c t i v e wi th

any P. mu l to c i d a s e r o t y p e s , (3) c r o s s r e a c t i v e wi t h P. haemol y t i ca

s e r o t y p e s 1, 2 , 5 - 8 , and 12 wi t h 6 and 24 h r c u l t u r e v a r i a b i l i t y a l s o

10

c r o s s r e a c t i v e wi t h P. m u l t o c i d a s e r o t y p e s 1 - 7 , 9 , 12, 15, 16, and

(4) c r o s s r e a c t i v e wi t h a l l P. h a e m o l y t i ca and P. m u l t oc i d a s e r o t y p e s .

Monoclonal a n t i b o d i e s grouped on t h e b a s i s o f s e r o t y p e r e a c t i v i t y were

o f t h e same i s o t y p e and s u b - i s o t y p e .

Tab l e 1 .2 - R e a c t i v i t y o f Monoclonal A n t i b o d i e s w i t h P. h a e m o l y t i ca

Whole Cel l S e r o t y p e s

P. h a e mo l y t i ca Monoclonal A n t i b od i e s

s e r o t y p e 3 1 , 5 , 6 9 , 10 2

1 + + + +

2 - - - ( 6 ) + ( 2 4 ) * +

3 - - - +

4 - - - +

5 - + + +

6 - + + +

7 - + + +

8 - + + +

9 - - + ( 6 ) - ( 2 4 ) * +

10 - - - +

11 - - - +

12 - + + +

Denotes d i f f e r e n c e s in r e a c t i v i t y between 6 and 24 h r c u l t u r e s wi t h

b o t h a n t i b o d i e s .

11

Tabl e 1 . 3 - R e a c t i v i t y o f Monoclonal A n t i b o d i e s w i t h P. m u l t o c i d a Whole

Cel l Se r o t y p e s

P. mu l t oc i da Monoclonal A n t i b o d i e s

s e r o t y p e t 3 1 , 5 , 6 9 , 10 2

1 - + +

2 - + +

3 - + +

4 - + +

5 - + +

6 - + +

7 _ + +

8 - - +

9 - + +

12 - + +

14 - - +

15 - + +

16 - ( + . 6 ) * + +

+No d i f f e r e n c e in r e a c t i v i t y between 6 and 24 hr c u l t u r e s .★

Number 6

monoclonal a n t i b o d y p o s i t i v e f o r s e r o t y p e 16 o n l y .

Formal in t r e a t m e n t o f P. ha e mol y t i ca s e r o t y p e 1 whole c e l l s o r

c a p s u l a r m a t e r i a l was found t o c o m p l e t e l y a l t e r t h e a n t i g e n i c i t y ,

r e d u c i n g t h e r e a c t i v i t y o f some monoclonal a n t i b o d i e s in ELISA (Table

1 . 4 ) . The P. h a e m o l y t i ca s e r o t y p e 1 s p e c i f i c a n t i g e n , r e c o g n i z e d by

12

monoclonal a n t i b o d y number 3 , and t h e a n t i g e n common t o a l l P.

ha e mol y t i ca and P. m u l t o c i d a s e r o t y p e s , r ec o g n i ze d by monoclonal

a n t i b o d y number 2 , was a p p a r e n t l y d e s t r o y e d by f o r ma l i n t r e a t m e n t .

Pa ra fo rma l dehyde t r e a t m e n t d i d no t a d v e r s e l y a f f e c t a n t i g e n i c i t y i n

t h i s sys t em. N e i t h e r f o r m a l i n nor pa r a f o r ma l dehyde was n e c e s s a r y f o r

c a p s u l a r m a t e r i a l a n t i g e n i c i t y in ELISA.

Table 1.4 - R e a c t i v i t y o f Monoclonal A n t i b o d i e s wi t h Formal in o r

Pa ra fo rma l dehyde T r e a t e d P. ha e mol y t i ca s e r o t y p e 1 Whole

C e l l s o r Ca p s u l a r M a t e r i a l in ELISA

Ant igenie

Trea tment

3

Monoclonal

1 , 5 , 6

A n t i b od i e s

9 , 10 2

Formal i n / p a r a f o r m a l d e h y d e - + + -

For ma l i n / no p a r a f o r ma l de hyde - + + -

No formal i n / p a r a f o r m a l d e h y d e + + + +

No f o r m a l i n / n o pa r a f o r ma l d e h y d e + + + +

*Whole c e l l s o r c a p s u l a r m a t e r i a l were t r e a t e d f o l l o w i n g a d s o r p t i o n t o

96 wel l p l a t e s .

Rapid s l i d e a g g l u t i n a t i o n o f P. h a e m o l y t i ca s e r o t y p e 1 by

c l a r i f i e d p e r i t o n e a l a s c i t i c f l u i d s was produced on l y by monoclonal

a n t i b o d y number 3. Ot he r monoclonal a n t i b o d i e s were i n c a p a b l e o f

a g g l u t i n a t i n g P. h a e m o l y t i c a s e r o t y p e 1 o r t h e r ema i n i ng s e r o t y p e s .

13

The a b i l i t y o f monoclonal a n t i b o d y number 3 t o a g g l u t i n a t e s e r o t y p e 1

c o r r e s po n d e d w i t h i t s ELISA r e a c t i v i t y (Tab l e s 1.2 and 1 . 3 ) .

A summary c h a r a c t e r i z i n g t h e monoclonal a n t i b o d i e s p r e p a r e d

a g a i n s t F\ h a e m o l y t i c a s e r o t y p e 1 c a p s u l a r m a t e r i a l i s p r e s e n t e d in

Tab l e 1 . 5 . Monoclonal a n t i b o d i e s were p l a c e d in f o u r groups based on

t h e i r c h a r a c t e r i s t i c i s o t y p e s , s e r o t y p e r e a c t i v i t y , a n t i g e n f o r m a l i n

s e n s i t i v i t y , and a g g l u t i n a t i o n . The r e s u l t s o f t h e s e d e t e r m i n a t i o n s

i n d i c a t e d s e v e r a l o f t h e a n t i b o d i e s r e c o g n i z e d t h e same e p i t o p e .

Table 1.5 - C h a r a c t e r i z a t i o n o f Monoclonal A n t i b o d i e s P r epa r ed A g a i n s t

P a s t e u r e l l a haemol y t i ca s e r o t y p e 1

Group

Monoclonal

Ant ibody

Number(s)

I s o t y p e Se r o t ype R e a c t i v i t y

P. haemol y t i ca P. mu l t oc i da

Ant igen

Formal in

S e n s i t i v i t y

A g g l u t i n a t i o n

1 3 IgM 1 None + +

2 1 , 5 , 6 IgG3 1 , 5 - 8 , 1 2*

None - -

3 9 , 10 IgM 1 , 3 , 5 - 8 , 1 2 1 - 7 , 9 , 1 2 , 1 5 , 1 6 - -

4 2 >9G2A a l l a l l + -

Monoclonal a n t i b od y number 6 r e a c t i v e wi t h P. mu l t o c i da s e r o t y p e 16 on l y .

15

DISCUSSION

Monoclonal a n t i b o d i e s were deve l oped a g a i n s t P haemol y t i ca

s e r o t y p e 1 c a p s u l a r m a t e r i a l . The hydr idomas p r oduc ing t h e s e

monoclonal a n t i b o d i e s were s e l e c t e d from a t o t a l o f 250 a n t i bo dy

produc i ng w e l l s g e n e r a t e d by f u s i n g t h e immune s p l e e n c e l l s w i t h SP-2/o

myeloma c e l l s . Ten d i f f e r e n t w e l l s c o n t a i n i n g v i g o r o u s l y growing

hybr idomas were chosen on t h e b a s i s o f a n t i b o d y b i nd i ng wi t h s e r o t y p e 1

c a p s u l a r m a t e r i a l . Monoclonal a n t i b o d y number 3 had t h e h i g h e s t ELISA

r e a c t i v i t y , wh i l e a n t i b o d i e s 1, 5 , 6, and 2 produced modera t e ELISA

a b s o r b a n c e s , and a n t i b o d i e s 9 and 10 d i s p l a y e d low ELISA r e a c t i v i t y

( da t a no t shown). These a n t i b o d i e s were c h a r a c t e r i z e d a c c o r d i n g t o

i s o t y p e , b a c t e r i a l s e r o t y p e s p e c i f i c i t y , a n t i g e n s e n s i t i v i t y t o

f o r ma l i n and a g g l u t i n a t i o n p r o p e r t i e s . The s e r o t y p e d i s t r i b u t i o n of

a n t i g e n s r e c o g n i ze d by t h e monoclonal a n t i b o d i e s were de t e rmi ne d u s i n g

p r i m a r i l y ELISA wi t h d e f i n e d b a c t e r i a l c e l l s . These d e t e r m i n a t i o n s

a l l owed e s t a b l i s h m e n t o f f o u r d i f f e r e n t groups o f a n t i b o d i e s , each

r e c o g n i z i n g an a p p a r e n t l y d i f f e r e n t e p i t o p e . The f i r s t e p i t o p e ,

r e c o g n i ze d by monoclonal a n t i b o d y number 3 , i s P. haemol y t i ca s e r o t y p e

1 s p e c i f i c and i nvo l ved wi t h b a c t e r i a l a g g l u t i n a t i o n . The second

e p i t o p e , d e f i n e d by a n t i b o d i e s 1, 5 , and 6 , i s p r e s e n t on many P.

h a e m o l y t i ca s e r o t y p e s , bu t no t P. mu l to c i d a s e r o t y p e s . The t h i r d

e p i t o p e , bound by a n t i b o d i e s 9 and 10, i s p r e s e n t on many P.

ha e mol y t i ca and P. m u l t oc i d a s e r o t y p e s . F i n a l l y , t h e f o u r t h e p i t o p e ,

p r e s e n t on a l l P. ha e mol y t i ca and P. m u l t oc i d a s e r o t y p e s , i s r e c o g n i ze d

by monoclonal a n t i b o d y number 2. In t h i s s t u d y , on l y P. haemol y t i ca

and P. mu l to c i d a s e r o t y p e s were examined , however , t h i s sys t em may be

ex t ended t o and p o s s i b l y i n c l u d e o t h e r s p e c i e s o f P a s t e u r e l l a , o r o t h e r

16

genera w i t h i n t h e B r u c e l l a c e a e f a m i l y , o r even o t h e r Gram-nega t ive

b a c t e r i a . Thi s sys t em e x e m p l i f i e s t h e use o f a n t i g e n d i s t r i b u t i o n

among b a c t e r i a l s e r o t y p e s t o d i s t i n g u i s h d i f f e r e n t monoclonal

a n t i b o d i e s .

The P. h a emol y t i ca s e r o t y p e 1 s p e c i f i c a n t i g e n i s d e f i n e d

r o u t i n e l y by i n d i r e c t h e m a g g l u t i n a t i o n and r a p i d p l a t e

3 4a g g l u t i n a t i o n . ’ S e r o t y p e s p e c i f i c i t y i s b e l i e v e d t o r e s i d e in

c a p s u l a r p o l y s a c c h a r i d e a n t i g e n s o f P. h a e m o l y t i c a . 4 , 6 S e r o t ype 1

s p e c i f i c a g g l u t i n a t i n g a n t i b o d y has been s u g g e s t e d as n e c e s s a r y f o r

34p r o t e c t i o n from e x p e r i me n t a l d i s e a s e . Immunizat ion o f c a t t l e wi th

c r ude c a p s u l a r e x t r a c t s has a f f o r d e d some de g r e e o f r e s i s t a n c e t oo c oo

e x p e r i me n t a l c h a l l e n g e , ’ however , f o r m a l i n i z e d b a c t e r i n s have been

39 41shown t o have a n e u t r a l o r d e l e t e r i o u s e f f e c t . * The r e s u l t s o f

t h i s s t u d y i n d i c a t e s monoclonal a n t i b o d y number 3 r e c o gn i z e s t he

s e r o t y p e 1 s p e c i f i c c a p s u l a r p o l y s a c c h a r i d e o f P. h a e mol y t i ca and

c a u s e s b a c t e r i a l a g g l u t i n a t i o n . F u r th e r mo r e , i t i s s u gg es t e d t h a t t h e

s e r o t y p e 1 s p e c i f i c a n t i g e n i s f o r ma l i n s e n s i t i v e , which may a c c o u n t ,

a t l e a s t in p a r t , f o r t h e i n e f f e c t i v e n e s s o f f o r m a l i n i z e d b a c t e r i n s t o

i mpa r t d i s e a s e r e s i s t a n c e .

S e r o l o g i c c r o s s - r e a c t i v i t y among t h e e s t a b l i s h e d s e r o t y p e s , w i t h i n

each b i o t y p e , o f P. h a e m o l y t i ca has been d i s c o v e r e d by i n d i r e c t

3 11 4 10h e m a g g l u t i n a t i o n , ’ r a p i d p l a t e a g g l u t i n a t i o n , ELISA, and c r o s s e d

12I m m u n o e l e c t r o p h o r e s i s . Crossed i m mu n o e l ec t r op h o r es i s f u r t h e r

12d em o n s t r a t e d c r o s s - r e a c t i o n s between b i o t y p e s o f P. h a e m o l y t i c a .

However, t h e a n t i g e n s r e s p o n s i b l e f o r t h i s c r o s s - r e a c t i v i t y have

remained u n c h a r a c t e r i z e d . Monoclonal a n t i b o d i e s 1, 5, and 6

c h a r a c t e r i z e d in t h i s s t u d y were c o n s i d e r a b l y c r o s s - r e a c t i v e wi t h many

17

P. h a e m o l y t i ca s e r o t y p e s , bu t n o t between t h e b i o t y p e s . In a n o t h e r

s t u d y , t h e s e monoclonal a n t i b o d i e s were shown t o be s p e c i f i c f o r

l i p o p o l y s a c c h a r i d e s by immunoblo t t i ng and t h e a n t i g e n was l o c a l i z e d

w i t h i n t h e b a c t e r i a l c a p s u l e u s i n g p r o t e i n A-gold l a b e l e d c o n j u g a t e in

42e l e c t r o n mi c r os c o p y . The 1i p o p o l y s a c c h a r i d e e p i t o p e r e c o gn i z e d by

t h e s e a n t i b o d i e s was no t s e n s i t i v e t o f o r m a l i n . These r e s u l t s supp l y

ev i d e n ce t h a t a 1i p o p o l y s a c c h a r i d e e p i t o p e i s common among many

s e r o t y p e s , bu t no t between b i o t y p e s o f P. h a e m o l y t i c a . Thus , some

deg r ee o f c r o s s - r e a c t i v i t y among s e r o t y p e s bu t no t between b i o t y p e s , o f

P. h a e m o l y t i c a ma.y be a t t r i b u t a b l e t o a common 1 i p o p o l y s a c c h a r i d e

e p i t o p e .

The s i m i l a r i t y o f a n t i b o d i e s 1, 5 , and 6 and a n t i b o d i e s 9 and 10

r e a c t i n g w i t h P. h a e m o l y t i ca s e r o t y p e s was f u r t h e r d i s t i n g u i s h e d u s i n g

P. m u l t oc i d a s e r o t y p e s r e c o gn i z e d on l y by t h e l a t t e r g roup . S e r o l o g i c

c r o s s - r e a c t i v i t y among P. h a e m o l y t i ca and P. mu l to c i da s e r o t y p e s has

no t been r e p o r t e d i n t h e l i t e r a t u r e . Monoclonal a n t i b o d i e s 9 and 10

were c r o s s - r e a c t i v e among many s e r o t y p e s o f t h e s e s p e c i e s , bu t not

between b i o t y p e s o f P. h a e m o l y t i c a . Immunoblot a n a l y s i s o f t h e a n t i g e n

r e c o g n i z e d by t h e s e a n t i b o d i e s r e v e a l e d a 29KD p r o t e i n , most l i k e l y o f

o u t e r membrane o r g i n , however , i t s e x a c t l o c a l e c ou l d no t be d e t e r mi ne d

42u s i n g p r o t e i n A-gold p robes due t o t h e a n t i b o d y i s o t y p e .

A n t i g e n i c i t y o f t h e 29KD p r o t e i n was no t a f f e c t e d by f o r ma l i n

t r e a t m e n t . The 29KD p r o t e i n r e p r e s e n t s a p o t e n t i a l sou r ce o f

c r o s s - r e a c t i v i t y among P. h a e m o l y t i ca and P. mu l to c i da s e r o t y p e s .

Common a n t i g e n s o f Gram-nega t i ve b a c t e r i a have been r e p o r t e d in

12 43 45h a e m o l y t i c a and o t h e r b a c t e r i a . ’ Monoclonal a n t i b o d y number 2

18

was c r o s s - r e a c t i v e w i t h a l l P. h a e m o l y t i c a and P. mu l t o c i d a s e r o t y p e s

examined. I t i s unknown wh e t h e r a n t i b o d y number 2 r ec o g n i z e s o t h e r

s p e c i e s o f P a s t e u r e l l a , o r o t h e r members o f t h e f am i ly B r u c e l l a c e a e , o r

o t h e r Gram-nega t i ve b a c t e r i a ; however , t h i s a n t i b o d y d i d no t r e a c t wi t h

E s c h e r i c h i a c o l i , used as b a c t e r i a l a n t i g e n c o n t r o l in t h e ELISA ( d a t a

no t shown) . The a n t i g e n c ou l d no t be d e t e c t e d by i mmunoblo t t i ng and

i t s n a t u r e remains u nd e t e r mi n e d . I t was f o r m a l i n s e n s i t i v e and t h i s

p r o p e r t y may a l s o a c c o u n t , a t l e a s t i n p a r t , f o r t h e f a i l u r e o f

f o r m a l i n i z e d b a c t e r i n s t o i m p a r t p r o t e c t i o n from d i s e a s e .

N e v e r t h e l e s s , t h i s a n t i g e n r e p r e s e n t s a s o u r ce o f c o n s i d e r a b l e

c r o s s - r e a c t i v i t y among P. h a e m o l y t i ca s e r o t y p e s , b i o t y p e s , and P.

m u l t o e i d a .

In summary, deve l opment and c h a r a c t e r i z a t i o n o f t h e r e p o r t e d

monoclonal a n t i b o d i e s has l e a d t o t h e a c q u i s i t i o n o f powerful t o o l s t o

s t u d y t h e a n t i g e n s o f P. h a e m o l y t i c a . Four groups o f a n t i b o d i e s were

e s t a b l i s h e d based on t h e i r c h a r a c t e r i s t i c s each c o r r e s p o n d i n g t o a

d i f f e r e n t a n t i g e n . Th i s s t u d y e x e m p l i f i e d t h e use o f d i f f e r e n t i a l

a n t i g e n e x p r e s s i o n among P a s t e u r e l l a s e r o t y p e s t o d i s t i n g u i s h

monoclonal a n t i b o d i e s p r e p a r e d and s e l e c t e d from a complex a n t i g e n

s o u r c e . F u r t h e r immunologic , b i o c h e m i c a l , and b i o l o g i c a l e x pe r i me n t s

a r e needed t o more f u l l y c h a r a c t e r i z e t h e a n t i b o d y g r ou ps , t h e s p e c i f i c

a n t i g e n s r e c o g n i z e d , and t h e i r r e l a t i o n s h i p t o d i s e a s e .

BIBLIOGRAPHY

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23. Levy R, D i l l e y J . The in v i t r o a n t i b o d y r e s p o n s e t o c e l l s u r f a c e a n t i g e n s . I I . Monoclonal a n t i b o d i e s t o human leukemia c e l l s . J Immunol 1977; 119 :394-400 .

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28. Dunn SD, Tozer RG, Ant c z a k , DF, Heppel LA. Monoclonal a n t i b o d i e s t o E s c h e r i c h i a c o l i Fj -ATPase. J Biol Chem 1985; 260:10418-10425.

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32. Gent ry MJ, C o r s t v e t RE, P a n c i e r a RJ. E x t r a c t i o n o f c a p s u l a rm a t e r i a l f rom P a s t e u r e l l a h a e m o l y t i c a . Am J Vet Res 1982; 43 :2070-2073 .

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35. Confer AW, L e s s l e y , BA, P a n c i e r a RJ, Fu l t on RW, Kreps JA. Serum a n t i b o d i e s t o a n t i g e n s d e r i v e d from a s a l i n e e x t r a c t o f P a s t e u r e l l a h a e m o l y t i c a : c o r r e l a t i o n wi t h r e s i s t a n c e t o e x p e r i me n t a l bov ine pneumonic p a s t e u r e l l o s i s . Vet Immunol Immunopathol 1985; 10 :265-278 .

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37. Matsumota M, Schmi tz JA, Syuto B, Watrons BJ, Mat t son DE. Immunogenici ty o f a s o l u b l e a n t i g e n a g a i n s t P a s t e u r e l l a h a e m o l y t i c a - a s s o c i a t e d pneumonia in c a l v e s . Vet Res Commun 1984; 8 : 117 - 130 .

38. Yates WDG, S t o c k d a l e PHG, Babiuk LA, Smith RJ. P r e v en t i on o f e x pe r i me n t a l bov ine pneumonic p a s t e u r e l l o s i s wi t h an e x t r a c t o f P a s t e u r e l l a h a e m o l y t i c a . Can J Comp Med 1983; 47 :250-256 .

39. F r i end SCE, Wi lk i e BN, Thomson RG, Barnuum DA. Bovine pneumonic p a s t e u r e l l o s i s : e x p e r i me n t a l i n d u c t i o n in v a c c i n a t e d and n o n v ac c i n a t e d c a l v e s . Can J Comp Med 1977; 4 1 : 7 7 - 83 .

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40. Mar t i n SW, Meek AH, Davis DG, Johnson JA, C u r t i s RA. F ac t o r s a s s o c i a t e d wi t h m o r b i d i t y and m o r t a l i t y in f e e d l o t c a l v e s : t h e Bruce County b e e f p r o j e c t , y e a r two. Can J Comp Med 1981; 45 : 103 - 112 .

41. Confer AW, P a n c i e r a RJ, Fu l t on RW, e t a l . E f f e c t o f v a c c i n a t i o n w i t h l i v e o r k i l l e d P a s t e u r e l l a h a e m o l y t i ca on r e s i s t a n c e t o e x pe r i me n t a l bov ine pneumonic p a s t e u r e l l o s i s . Am J Vet Res 1985; 4 6 : 342 - 347 .

42. Schno r r KL, Todd WJ, C o r s t v e t , RE, A u s t i n FW, Nobles D. I d e n t i f i c a t i o n and l o c a l i z a t i o n o f P a s t e u r e l l a ha e mol y t i ca a n t i g e n s by monoclonal a n t i b o d i e s . The F i r s t I n t e r n a t i o n a l V e t e r i n a r y Immunology Symposium, pg 133.

43. P e t e r s H, J u r s M, J ann B e t a l . Monoclonal a n t i b o d i e s t o e n t e r o b a c t e r i a l common a n t i g e n and t o E s c h e r i c h i a c o l i1i p o p o l y s a c c h a r i d e o u t e r c o r e : Demons t r a t i on o f an a n t i g e n i c d e t e r m i n a n t s h a r e d by e n t e r o b a c t e r i a l common a n t i g e n and E. c o l i K5 c a p s u l a r p o l y s a c c h a r i d e . I n f e c t Immun 1985; 50 :459-466 .

44. Makela PH, Mayer H. E n t e r o b a c t e r i a l common a n t i g e n . B a c t e r i o l Rev 1976; 40 : 591 - 632 .

45. Mayer H, Schmidt G. Chemis t ry and b i o l o g y o f t h e e n t e r o b a c t e r i a l common a n t i g e n (ECA). Cur r Top Mi c rob i o l Immunol 1979; 8 5 : 99 - 153 .

46. Lugowski C, Romanowska E, Keene L, Li ndberg B. I d e n t i f i c a t i o n o f a t r i s a c c h a r i d e r e p e a t i n g u n i t in t h e e n t e r o b a c t e r i a l common a n t i g e n . C a r bohydr a t e Res 1983; 118:173-181 .

CHAPTER 2

BOVINE NEUTROPHIL RESPONSE TO PASTEURELLA HAEMOLYTICA ANTIGENS:

MEASUREMENT OF CYTOTOXIN ACTIVITY IN A COLORIMETRIC ASSAY

SUMMARY

A c o l o r i m e t r i c a s s a y , based on m i t o c h o n d r i a l r e d u c t i o n o f a

t e t r a z o l i u m dye ( 3 - ( 4 , 5 - d i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m

bromide) (MTT) was a d ap t e d f o r t h e measurement o f P a s t e u r e l l a

h ae m o l y t i c a s e r o t y p e 1 c y t o t o x i n u s i n g bovine n e u t r o p h i l s . Cy t o t ox i n

was produced in a d e f i n e d medium and c o n c e n t r a t e d by u l t r a f i l t r a t i o n .

Five c y t o t o x i n p r e p a r a t i o n s , a p r e c o n c e n t r a t e , a 15:1 c o n c e n t r a t e , an

u l t r a f i I t r a t e and 15:1 c o n c e n t r a t e s i n a c t i v a t e d a t e i t h e r 56C o r by

b o i l i n g f o r 1 h r f o l l owe d by c l a r i f i c a t i o n , were used t o measure

c y t o t o x i n a c t i v i t y in t h e a s s a y . Chemical and immunologic methods

were used t o i d e n t i f y and q u a n t i t a t e a n t i g e n s in t h e c y t o t o x i n

p r e p a r a t i o n s . N e u t r o p h i l c y t o t o x i c i t y was i d e n t i f i e d as i n h i b i t i o n o f

t e t r a z o l i u m dye r e d u c t i o n which i n d i c a t e d t h a t n e u t r o p h i l s were

k i l l e d . At d i f f e r e n t c o n c e n t r a t i o n s each o f t h e c y t o t o x i n

p r e p a r a t i o n s , e x c e p t t h e u l t r a f i l t r a t e , caused n e u t r o p h i l s t i m u l a t i o n

o f t e t r a z o l i u m dye r e d u c t i o n r e l a t i v e t o unexposed c e l l s whe t he r

c y t o t o x i c i t y was p r e s e n t o r n o t . R e s u l t s o f t h i s s t u d y i n d i c a t e t h e

MTT c o l o r i m e t r i c a s s a y can measure bov ine n e u t r o p h i l c y t o t o x i c i t y o r

s t i m u l a t i o n . N e u t r op h i l s t i m u l a t i o n o f t e t r a z o l i u m dye r e d u c t i o n may

r e f l e c t a c t i v a t i o n v i a t h e r e s p i r a t o r y b u r s t in r e s po n s e t o c y t o t o x i n

p r e p a r a t i o n a n t i g e n s .

23

24

INTRODUCTION

The p a t h o g e n e s i s o f bov ine pneumonic p a s t e u r e l l o s i s ( s h i p p i n g

f e v e r ) i s c u r r e n t l y r ec o g n i z e d t o i n v o l v e t h e i n t e r a c t i o n o f many

1 2h o s t , pa t hogen and env i ro n me n t a l f a c t o r s . 5 The s e v e r e f i b r i n o u s

pneumonia c h a r a c t e r i z i n g t h i s d i s e a s e i s most commonly a s s o c i a t e d wi t h

lower r e s p i r a t o r y t r a c t i n f e c t i o n by P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e

1-31. In t h e a c u t e s t a g e s o f pneumonic p a s t e u r e l l o s i s , a s i g n i f i c a n t

s t e p in t h e p a t h o g e n e s i s i s t h e r a p i d acc u mu l a t i o n o f p r i m a r i l y

4-7n e u t r o p h i l s in a l v e o l a r s p a c e s .

During l o g a r i t h m i c - p h a s e g rowt h , P. ha e mol y t i ca has been shown t o

l i b e r a t e a s o l u b l e c y t o t o x i n ( l e u k o t o x i n ) which i s s p e c i f i c a l l y

c y t o l y t i c f o r rumi nan t pulmonary macrophages , p e r i p h e r a l blood

8-12mononuclear c e l l s , and n e u t r o p h i l s . ~ Cy t o t ox i n i s b e l i e v e d t o

c o n t r i b u t e t o t h e p a t h o g e n e s i s o f t h e pulmonary l e s i o n by d i r e c t l y

i mp a i r i n g phagocy t e f u n c t i o n and hence b a c t e r i a l c l e a r a n c e from t he

l ung . Cy t o t ox i n may f u r t h e r c o n t r i b u t e t o t h e p a t h o g e n e s i s by c a u s in g

t h e r e l e a s e a n d / o r g e n e r a t i o n o f i n f l ammat ory m e d i a t o r s from

n e u t r o p h i l s . P. h a e m o l y t i c a c y t o t o x i n has been c h a r a c t e r i z e d as a

h e a t l a b i l e , t r y p s i n s e n s i t i v e p r o t e i n wi t h an a p p r ox i ma t e m o l e c u l a r

we i gh t o f 105 ,000. The c y t o l y t i c a c t i v i t y can be n e u t r a l i z e d by1 1 fi

a n t i b o d y from a v a r i e t y o f s o u r c e s . " R e s i s t a n c e t o e x p e r i m e n t a l l y

induced and n a t u r a l l y o c c u r i n g pneumonic p a s t e u r e l l o s i s has been

c o r r e l a t e d wi t h h igh t i t e r s o f c y t o t o x i n n e u t r a l i z i n g serum

17-19a n t i b o d i e s . However, c y t o t o x i n p r e p a r a t i o n s c o n t a i n s e v e r a l

s o l u b l e a n t i g e n s o f P. h a e m o l y t i c a i n c l u d i n g c a p s u l a r p o l y s a c c h a r i d e s ,

20-221i p o p o l y s a c c h a r i d e s and p o s s i b l y o t h e r u n i d e n t i f i e d components .

The d i v e r s i t y o f serum a n t i b o d i e s and t h e i m p u r i t i e s o f c y t o t o x i n

25

p r e p a r a t i o n s a n t i g e n s have hampered i d e n t i f i c a t i o n o f many, p o s s i b l y

i m p o r t a n t i n t e r a c t i o n s between n e u t r o p h i l s and t h e s e components .

Most a s s a y s d e s i gne d t o q u a n t i t a t e c y t o t o x i n have r e l i e d on

plasma membrane damage as an i n d i c a t o r o f a c t i v i t y . These a s s a y s

8 9 11 12 14i n c l u d e measurement o f t r y p a n b l u e dye e x c l u s i o n , *

^ c h r o mi u m r e l e a s e , 8 > 1 0 , 1 1 , 1 4 , 1 6 mi c r o t j - t e r n e u t r a l r ed

19 23 24d y e - e l u t i o n , ’ and monol ayer i n t e g r i t y . A d d i t i o n a l l y , a luminol

depende n t ch e mi l u mi n e s c e n t ( LDCL) - i nhb i t i on a s s a y has been d e s c r i b e d ,or n c

u s i n g bovine n e u t r o p h i l s , f o r d e t e c t i n g c y t o t o x i n a c t i v i t y . *

These a s s a y s have a l s o been used t o measure c y t o t o x i n n e u t r a l i z a t i o n

by a n t i b o d y in serum and n a s a l o r b r o n c h o a l v e o l a r wa s h i ngs . The

LDCL- inhbi t i on a s s a y was found t o be t h e most s e n s i t i v e in d e t e c t i n g

c y t o t o x i n a c t i v i t y when compared t o a s s a y s depende n t on c e l l l y s i s and

25membrane i n t e g r i t y as i n d i c a t o r s o f c y t o t o x i c i t y . I t was

h y p o t h e s i z e d t h a t t h e LDCL - i n h b i t i o n a s s a y was more s e n s i t i v e

beca use i t measured m e t a b o l i c impai rment o f t h e n e u t r o p h i l .

Most r e c e n t l y a m i c r o t i t e r c o l o r i m e t r i c a s s a y based on MTT

( 3 - ( 4 , 5 - d i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m bromide) dye

r e d u c t i o n by bov ine n e u t r o p h i l s was r e p o r t e d t o q u a n t i t a t e c y t o t o x i n

27and i t s n e u t r a l i z a t i o n . The MTT c o l o r m e t r i c a s s a y was a da p t ed from

28t h e methods o r i g i n a l l y d e s c r i b e d by Mossmann f o r t h e q u a n t i t a t i o n o f

c e l l u l a r p r o l i f e r a t i o n and c y t o t o x i c i t y . The MTT c o l o r i m e t r i c a s s a y

has s i n c e been r e p o r t e d to measure c e l l u l a r a c t i v a t i o n i nde pe nde n t o f

29p r o l i f e r a t i o n . The p r i n c i p l e o f t h i s a s s a y i s t h e r e d u c t i o n o f

y e l l o w MTT dye t o a da rk b l u e i n s o l u b l e formazan by m e t a b o l i c a l l y

a c t i v e m i t o c h o n d r i a and i t s s ub se q u e n t p h o t o m e t r i c q u a n t i t a t i o n .

MTT c o l o r i m e t r i c a s s a y s o f f e r a un i que means o f measu r i ng n e u t r o p h i l

26

r e s po n s e t o t o x i n s and a n t i g e n s because n e u t r o p h i l s undergo a

3 0 -3 2r e s p i r a t o r y b u r s t i n v o l v i n g i n c r e a s e d m i t o c h o n d r i a l a c t i v i t y .

Assays o f n e u t r o p h i l me tabo l i sm i n r e s p o n s e t o P. ha e mol y t i ca

a n t i g e n s i n c y t o t o x i n p r e p a r a t i o n s a r e needed t o ga i n a b e t t e r

u n d e r s t a n d i n g o f t h e p a t h o g e n e s i s in pneumonic p a s t e u r e l l o s i s . The

p u r pos e s o f t h i s s t u d y were t o a d a p t , o p t i m i z e , and v a l i d a t e t h e MTT

c o l o r i m e t r i c a s s a y u s i n g bovine n e u t r o p h i l s f o r t h e measurement o f

c y t o t o x i n a c t i v i t y and t o i d e n t i f y component a n t i g e n s in c y t o t o x i n

p r e p a r a t i o n s u s i n g monoclonal a n t i b o d i e s and chemica l methods . The

MTT c o l o r i m e t r i c a s s a y was a l s o adap t ed f o r t h e s e l e c t i o n and

a s s e s s m e n t o f a c y t o t o x i n n e u t r a l i z i n g monoclonal a n t i b o d y and t o

d e t e r m i n e t h e e f f e c t s o f o t h e r monoclonal a n t i b o d i e s a g a i n s t a n t i g e n s

in c y t o t o x i n p r e p a r a t i o n s on n e u t r o p h i l r e s p o n s e s .


B a c t e r i a l c u l t u r e s and c y t o t o x i n p r o d u c t i o n :

P a s t e u r e l l a h a e m o l y t i ca b i o t y p e A, s e r o t y p e 1, o r i g i n a l l y

i s o l a t e d from t h e t r a c h e a o f a f e e d l o t c a l f , was m a i n t a i n e d by

l y o p h i l i z a t i o n in skim mi lk and p e r i o d i c animal pas s age as p r e v i o u s l y

33d e s c r i b e d . L y o p h i l i z e d c u l t u r e s were r e c o n s t i t u t e d and s t r e a k e d f o r

i s o l a t i o n on a g a r p l a t e s c o n t a i n i n g 10% ( v / v ) c i t r a t e d bov ine b l ood ,

1% ( v / v ) h o r s e serum, and 1% ( v / v ) y e a s t h y d r o l y s a t e . I s o l a t e d

c o l o n i e s were s e l e c t e d and suspended in D u l b e c c o ' s phospha t e b u f f e r e d

s a l i n e (PBS, pH7.4) f o r c o n f l u e n t i n c o l u a t i o n o f f r e s h media . Agar

p l a t e s were i n c u b a t e d in c a n d l e j a r s a t 37C f o r 18 t o 24 h r .

Co n f l u e n t c u l t u r e s were h a r v e s t e d , suspended in D u l b e c c o ' s PBS, and

a d j u s t e d t o an o p t i c a l d e n s i t y o f 0 . 25 a t 650 nm u s i n g a 1:10

27

gd i l u t i o n . Thi s s u s p e n s i o n c o n t a i n e d 1X10 CFU/ml as d e t e r mi ne d by

p l a t e c o u n t .

P. h a emol y t i ca c y t o t o x i n was produced in a d e f i n e d medium u s i n g

m o d i f i c a t i o n s o f p r e v i o u s l y d e s c r i b e d m e t h o d s . ^ A 10 .0 ml sample o f

t h e b a c t e r i a l s u s p e n s i o n was used t o i n o c l u l a t e 500 ml RPMI-1640,

c o n t a i n i n g lOmM L- g l u t a mi ne and 10 mM sodium b i c a r b o n a t e , g i v i n g a

f i n a l i n o c u l a t i o n d e n s i t y o f 2X10^ CFU/ml. Fo l lowing i n c u b a t i o n f o r

5 . 5 h r a t 37C in t h e p r e s e n c e o f 5% CO2 , t h e b a c t e r i a were p e l l e t e d by

c e n t r i f u g a t i o n a t 12 ,000 xg f o r 30 min a t 4C. The s u p e r n a t e was

c o l l e c t e d , pa s sed t h r ough a 0 . 22 um f i l t e r , and c o n c e n t r a t e d 15:1

u s i n g a PM-30 membrane f i t t e d in an u l t r a f i l t r a t i o n c e l l a t 4C.

Samples o f p r e c o n c e n t r a t e d c y t o t o x i n and c y t o t o x i n u l t r a f i l t r a t e were

r e t a i n e d f o r s u b se q u e n t a n a l y s i s . A l i q u o t s o f c o n c e n t r a t e d c y t o t o x i n

were h e a t i n a c t i v a t e d a t e i t h e r 56C f o r 1 hr o r by b o i l i n g f o r 1 hr

f o l l owe d by a h igh speed c e n t r i f u g a t i o n s t e p and 0 . 22 urn f i l t r a t i o n .

The b a c t e r i a l p e l l e t s and c y t o t o x i n p r e p a r a t i o n s were c u l t u r e d on t h e

a p p r o p r i a t e media t o e n s u r e p u r i t y and s t e r i l i t y r e s p e c t i v e l y .

Cy t o t o x in p r e p a r a t i o n s were s t o r e d a t -70C.

A n a l y s i s o f t h e c y t o t o x i n p r e p a r a t i o n s :

P r o t e i n c o n c e n t r a t i o n s o f t h e c y t o t o x i n p r e p a r a t i o n s were

34d e t e r mi n e d u s i n g t h e method o f B r ad f o r d . Q u a n t i t a t i o n o f

35hexosamines was per fo rmed a c c o r d i n g t o p r e v i o u s l y p u b l i s h e d methods .

An t i ge ns p r e s e n t in t h e c y t o t o x i n p r e p a r a t i o n s were q u a n t i t a t e d in

ELISA u s i n g p r e v i o u s l y d e f i n e d monoclonal a n t i b o d i e s ( Chap t e r 1) .

F i f t y pi d i l u t i o n s o f t h e c y t o t o x i n p r e p a r a t i o n s i n D u l b e c c o ' s PBS

were d e s s i c a t e d in t h e w e l l s o f 96 wel l p l a t e s o v e r n i g h t . The

ads o r bed a n t i g e n s were n o t t r e a t e d wi t h a f i x a t i v e and monoclonal

a n t i b o d i e s i n a s c i t i c f l u i d ( 1 : 2 0 d i l u t i o n ) were used t o q u a n t i t a t e

t h e a n t i g e n s . The r ema i n i ng ELISA s t e p s were comple ted as d e s c r i b e d

( Ch a p t e r 1) . The a n t i g e n t i t e r was e x p re s s e d as t h e i n v e r s e o f t h e

h i g h e s t d i l u t i o n p r oduc i ng an abs o r ba nc e va l ue 1.5 t i mes g r e a t e r t ha n

normal mouse serum used as a c o n t r o l . C y t o t ox i c a c t i v i t y p r e s e n t in

t h e 5 c y t o t o x i n p r e p a r a t i o n s was de t e r mi ne d u s i n g bovine n e u t r o p h i l s

8 9 11and t r y p a n b l u e dye e x c l u s i o n . ’ * E q u i v a l e n t p r o p o r t i o n s o f

n e u t r o p h i l s and c y t o t o x i n p r e p a r a t i o n s were used i n t h e t r y p a n b lu e

dye e x c l u s i o n and MTT c o l o r i m e t r i c a s s a y s f o r c y t o t o x i n a c t i v i t y .

P r e p a r a t i o n o f bov ine n e u t r o p h i l s :

Bovine n e u t r o p h i l s were p r e p a r e d u s i n g m o d i f i c a t i o n s o f t h e

36method d e s c r i b e d by Boyum. B r i e f l y , h e p a r i n i z e d b lood (10 U/ml) was

c e n t r i f u g e d a t 500xg f o r 30 min a t 22C and t h e b u f f y c o a t s and upper

t h i r d o f t h e packed c e l l volume were c o l l e c t e d . The c o n t a mi n a t i n g

e r y t h r o c y t e s were l y s ed w i t h an equal volume o f i c e co l d 0.87% NH^Cl

(pH 7 . 2 ) f o r 15 min. The r e s u l t i n g l e u k o cy t e s were p e l l e t e d a t 200 xg

f o r 5 min and washed t w i c e in cal c ium-magnes ium f r e e Hank' s b a l an c e d

s a l t s o l u t i o n ( pH7 . 4 ) . The l e u k o c y t e s u s p e n s i o n was c a r e f u l l y l a y e r e d

on d i s c o n t i n u o u s d e n s i t y g r a d i e n t s c o n t a i n i n g F i c o l 1 - sod i um

d i a t r i z o a t e 1 .077 g/ml on t o p o f 1 .119 g / m l . N e u t r o p h i l s were

i s o l a t e d a t t h e 1 . 0 7 7 / 1 . 1 1 9 g/ml i n t e r p h a s e by c e n t r i f u g a t i o n a t 400xg

f o r 15 min a t 22C. The mononuc lea r c e l l l a y e r a t t h e upper g r a d i e n t

i n t e r p h a s e was d i s c a r d e d . N e u t r o p h i l s were washed t h r e e t i me s p r i o r

t o s u s p e n s i o n in D u l b e c c o ' s PBS f o r enumera t i on and u s e . Thi s

p r o ce d u r e r o u t i n e l y produced c e l l s u s p e n s i o n s c o n s i s t i n g o f a t l e a s t

29

90% n e u t r o p h i l s w i t h an o v e r a l l v i a b i l i t y o f g r e a t e r t ha n 95% when

e v a l u a t e d by d i f f e r e n t i a l s t a i n s and t r y pa n b l ue dye e x c l u s i o n . The

n e u t r o p h i l s u sp e n s i o n was a d j u s t e d t o a f i n a l working c o n c e n t r a t i o n o f

5 X 106 c e l l s / m l .

E v a l u a t i o n o f MTT formazan s o l v e n t s :

One hundred ul a l i q u o t s o f t h e n e u t r o p h i l s u s p e n s i o n (5 X 10^

c e l l s / m l ) were d e l i v e r e d t o t h e w e l l s o f a 96 wel l f l a t bot tom t i s s u e

c u l t u r e p l a t e u s i n g a m u l t i c h a n n e l p i p e t t o r . The p l a t e was

c e n t r i f u g e d f o r 5 min a t 500xg t o f a c i l i t a t e n e u t r o p h i l a d h e r en c e .

A l i q u o t s (25 u l ) o f MTT dye a t 5 mg/ml Du l b e c c o ' s PBS were added t o

t h e w e l l s and t h e n e u t r o p h i l s were a l l owed t o r educe t h e dye f o r 4

h r a t 37C. D i f f e r e n t o r g a n i c s o l v e n t s , d e t e r g e n t s , and combi na t i ons

( lOOp1) were added t o t h e w e l l s and t h e p l a t e was a g i t a t e d on a

micro-ELISA s h a k e r . S o l v e n t s were v i s u a l l y e v a l u a t e d f o r t h e i r

a b i l i t y t o r a p i d l y and c o m p l e t e l y s o l u b i l i z e MTT formazan formed by

n e u t r o p h i l monol aye r s in 96 wel l p l a t e s .

V a l i d a t i o n and o p t i m i z a t i o n o f t h e MTT c o l o r i m e t r i c a s s a y :

Two f o l d t e s t t u b e d i l u t i o n s o f t h e n e u t r o p h i l s u s p e n s i o n were6 4

per formed between 5X10 c e l l s / m l and 7.81X10 c e l l s / m l in D u l b e c c o ' s

PBS. A l i q u o t s (100 u l ) o f t h e s e s u sp e n s i o n s were d i s p e n s e d in

r e p l i c a t e s o f 5 i n t o 96 we l l p l a t e s which were t hen c e n t r i f u g e d t o

encour a ge c e l l a d he r e n c e . Twenty f i v e pi a l i q u o t s o f MTT dye (5

mg/ml) were added t o each w e l l . P l a t e s were i n c u b a t e d a t 1 hour

i n t e r v a l s between 1 . 5 and 4 . 5 h r a t 37C. Fol lowing MTT i n c u b a t i o n

wi t h n e u t r o p h i l s , MTT formazan was s o l u b i l i z e d wi t h hexamethyl

30

p h os ph o r i c t r i a m i d e (HMPT) (100 u l ) by v o r t e x i n g f o r 7 min.

S o l u b i l i z e d formazan was q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y w i t h an

ELISA p l a t e r e a d e r s e t a t a t e s t wave l e ng t h o f 570 nm, a r e f e r e n c e

wave l e ng t h o f 630 nm, and a c a l i b r a t i o n s e t t i n g o f 1 . 00 . Cont ro l

b l a n k s , c o n s i s t i n g o f r e p l i c a t e w e l l s i n which MTT was added t o t h e

maximum c e l l # /we l l i mmed i a t e l y p r i o r t o s o l u b i l i z a t i o n , were

s u b t r a c t e d from a b s o r ba n c e v a l u e s and t h e n e t d e t e r m i n e d . The mean

and s t a n d a r d d e v i a t i o n o f t h e r e p l i c a t e t e s t s were de t e r mi n e d and

p l o t t e d .

In a s e p a r a t e s i m i l a r e x p e r i m e n t , methanol o r f o r m a l i n f i x e d and

washed n e u t r o p h i l s were mixed wi t h v i a b l e n e u t r o p h i l s a t v a r i o u s

p r o p o r t i o n s w h i l e keep i ng t h e t o t a l number c o n s t a n t a t e i t h e r 5X 10^

c e l l s / m l o r 2 . 5 X 10^ c e l l s / m l . Absorbance was measured a t 1 hour

i n t e r v a l s between 2 . 5 and 4 . 5 h r a t 37C. The mean and s t a n d a r d

d e v i a t i o n o f 4 r e p l i c a t e w e l l s was d e t e r mi n e d and p l o t t e d .

MTT c o l o r i m e t r i c a s s a y f o r d e t e r m i n a t i o n o f c y t o t o x i n a c t i v i t y :

S e r i a l two f o l d (50 y l ) d i l u t i o n s o f t h e c y t o t o x i n p r e p a r a t i o n s

i n D u l b e c c o ' s PBS were pe r fo rmed i n s e p e r a t e 96 wel l p l a t e s . F i f t y yl

SP-2 /o mur ine myeloma c e l l c o n d i t i o n e d medium (RPMI-1640 wi t h 15%

( v / v ) FCS, 0 . 01 mM h y p o x a n t h i n e , 0 . 0 1 mM t h y m i d i n e , 10 mM sodium

b i c a r b o n a t e , 1% ( v / v ) n o n - e s s e n t i a l amino a c i d s and L - g l u t a m i n e ) was

added t o each w e l l . A l i q u o t s (100 u l ) o f t h e n e u t r o p h i l s u s p e n s i o n (5

X 10^ c e l l s / m l ) were added t o t h e w e l l s and t h e p l a t e s were i n c u b a t e d

f o r 20 min a t 37C. Fo l lowing i n c u b a t i o n , t h e p l a t e s were c e n t r i f u g e d

and g e n t l y washed wi t h t h r e e 150 yl volumes o f D u l b e c c o ' s PBS and

a s p i r a t e d t o remove i n t r a c e l l u l a r p r o d u c t s r e l e a s e d from t h e

31

n e u t r o p h i l s . N e u t r op h i l c y t o l y s i s was a l s o examined in w e l l s u s i n g an

i n v e r t e d l i g h t m i c r os co p e . T w e n t y - f i ve ul a l i q u o t s o f MTT s o l u t i o n

were added t o t h e w e l l s and t h e n e u t r o p h i l s were a l l owed t o r educe t h e

dye f o r 4 h r a t 37C. The r e s u l t i n g formazan was s o l u b i l i z e d and

q u a n t i t a t e d as d e s c r i b e d . C o n t r o l s c o n s i s t e d o f r e p l i c a t e w e l l s

t r e a t e d i d e n t i c a l l y bu t u s i n g D u l b e c c o ' s PBS in p l a c e o f t h e c y t o t o x i n

p r e p a r a t i o n s and w e l l s in which MTT was added i mmedi a t e l y p r i o r t o

t h e s o l u b i l i z a t i o n s t e p . The mean and s t a n d a r d d e v i a t i o n o f g r o s s

d a t a from 4 r e p l i c a t e s f o r each c y t o t o x i n p r e p a r a t i o n d i l u t i o n was

d e t e r mi n e d and p l o t t e d .

RESULTS

I n i t i a l e x p e r i m e n t s d em o n s t r a t e d t h a t bov ine n e u t r o p h i l s r educed

MTT t o a deep b l u e i n s o l u b l e formazan as has been p u b l i s h e d f o r

l ymphocytes ( F i g u r e I I . 1 ) . However, a c i d i s o p r op a n o l o n l y p a r t i a l l y

d i s s o l v e d MTT formazan u s i n g t h e d e s c r i b e d e x pe r i m e n t a l c o n d i t i o n s .

D i f f e r e n t o r g a n i c s o l v e n t s and d e t e r g e n t s were t h e r e f o r e e v a l u a t e d f o r

t h e i r a b i l i t y t o d i s s o l v e MTT formazan produced by a d h e r e n t

n e u t r o p h i l s . Rapid and compl e t e s o l u b i l i z a t i o n o f MTT formazan was

a c h i e v e d u s i n g HMPT (50% f i n a l c o n c e n t r a t i o n ) and s ha k i ng f o r 7 min.

However, p r o lo n g e d i n c u b a t i o n (>1 hour ) o f HMPT wi t h MTT s o l u t i o n

r e s u l t e d i n s po n t a n e o u s r e d u c t i o n ( da t a no t shown) . O t he r s o l v e n t s

were found t o be u n s a t i s f a c t o r y f o r a v a r i e t y o f r ea s o n s (Tab l e I I . 1 ) .

32

Tabl e I I . 1 - E v a l u a t i o n o f MTT Formazan S o l v e n t s .

★S o l v e n t S o l u b i l i z a t i o n

methanol none

e t h a n o l none

propanol incompl e t e

i s o pr op a n o l (100%,70%,50%) incompl e t e

0 . 0 4 N HC1 i n i s o p r op a n o l incompl e t e

i s o pr op a n o l w i t h 1 t o 10% Tr i t onX-100 i nc ompl e t e

i s o p r op a n o l w i t h 1 t o 10% SDS+ g e l a t i o n o f w e l l s

i s o p r op a n o l i n DMSO ( 7 5 : 2 5 , 50 :50) i nc ompl e t e

DMSO incompl e t e

DMF none

Acetone none

P y r i d i n e o f p l a t e

HMPT r a p i d and comple t e

*used a t 100% u n l e s s i n d i c a t e d

A b b r e v i a t i o n s : SDS = sodium dodecyl s u l f a t e ; DMSO = d imethyl

s u l f o x i d e ; DMF = d imethy l formamide; HMPT = hexamethyl p h os pho r i c

t r i a m i d e .

33

#

#

Fi g u r e I I . 1 - I n t r a c e l l u l a r a g g r e g a t e s and e x t r a c e l l u l a r c r y s t a l s o f

MTT formazan produced by bov i ne n e u t r o p h i l s ; l i g h t mi c r os c o p y , X

1, 000.

570n

m

34

A p l o t o f n e u t r o p h i l number p e r wel l a g a i n s t a b s o r ba nc e a t 570 nm

r e v e a l e d a n o n - l i n e a r r e l a t i o n s h i p a t each c o n c e n t r a t i o n o ve r t ime

( F i g u r e I I . 2 ) .

0 .2 7 0 4 .5 hours

3 .5 hours

0.2022 .5 hours

1.5 hours0 .1 3 5

0 .0 6 7

0 .0 0 00 .0 7 8 0 .1 5 6 0 .3 1 2 0 .6 2 5 1.25 2 .5 0 5 .0 0

Neutrophil Numbers (X 106 )/well

F i g u r e I I . 2: N o n l i n e a r i t y o f a bs o r ba nc e v e r s e s n e u t r o p h i l number a t

v a r i o u s t i me i n t e r v a l s in t h e MTT c o l o r i m e t r i c a s s a y .

uiuozs

Rear rangement o f t h i s d a t a showed t h e r e was a l i n e a r r e l a t i o n s h i p

between abs o r ba nc e and t i me a t a c o n s t a n t n e u t r o p h i l number ( F i g u r e

I I . 3 ) .

0 .2 7 0 5.0X1 OVwell

0 .2 0 2

0 .1 3 52.5X1 OVwell

0 .0 6 7■fl.25 X 1 0 /well* 6 .2 5 X 1 o V w e l l * 3 .1 2 X 1 0 /well0 .0 0 0

1.5 2 .5 3 .5 4.5Incubation Time - Hours

F i g u r e I I . 3 - L i n e a r r e l a t i o n s h i p between abs o r ba nc e and MTT

i n c u b a t i o n t i me a t d i f f e r e n t n e u t r o p h i l numbers p e r w e l l .

“ - - - ~ - -s sys t em absorbance resu ltc

a d v e r s e l y a f f e c t e d ( F i g u r e I , . 4 ) . " 0 t

0 .2 2 5

0 .1 6 8

c n v l 0 5/w e l l live

g 0.112ID

<

0 .0 5 6

0 . 0 0 02.5

3 .7 5 X 1 0 s/well live 1.25X lo V w e l l fixed 1

2 .5 X 1 0 s/w e l l live: 2 .5 X 1 0 s/w e i l f ix e d ,2 .5 X1 OVwell live- 1 .8 7 X 1 0 s/well l i v e .

6 .25X 10V w el l fixed1 .25 X 1 0s/well live

l".25X10*/well fixed

3.5Incubation Time - Hours 4.5

neutropfiiis ^

produced iden t ica i r e su i ts (not sdoun). " " ^ neUtr°phils

The optimum conditions for the MTT •

* 5 X 10* neutrophils per weH w i t h a m i 7 ^ ^ ^ *

u s i n g HMPT as a formazan s o l v e n t . ^ a t l ° " ^ ° f 4 ^ ^

37

D i f f e r e n t dose r e s p o n s e cu r ves were produced u s i n g each o f t h e 5

c y t o t o x i n p r e p a r a t i o n s . P r e c o n c e n t r a t e d c y t o t o x i n r e s u l t e d in an

immediate l i n e a r c o n c e n t r a t i o n dependent i n c r e a s e in abso r bance

f o l l owe d by i n c r e a s e d dye r e d u c t i o n r e l a t i v e t o unexposed n e u t r o p h i l s

( F i g u r e I I . 5 ) .

0 . 1 6 0

0 .1 4 0

0 .1 2 0

EeoN

< 0.100 PreconcentratedCytotoxin

0 . 0 8 0

0 . 0 6 0 ■

0 1 2 3 4 5 6 7 8 9 10 11Log2 -Cytotoxin Dilution

F i g u r e 1 1 . 5. Dose r e s p o n s e o f bov ine n e u t r o p h i l s exposed t o t h e

p r e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n in t h e MTT c o l o r i m e t r i c a s s a y ,

♦ i n d i c a t e s a b s o r b a n c e t e s t va l ue o b t a i n e d u s i n g unexposed n e u t r o p h i l s .

38

N e u t r o p h i l s r esponded w i t h more v a r i a n c e t o t h e h i g h e r d i l u t i o n s o f

p r e c o n c e n t r a t e d c y t o t o x i n . Ce l l l y s i s was no t e v i d e n t in p l a t e w e l l s

u s i n g an i n v e r t e d m i c r o s c o p e , even a t low d i l u t i o n s o f p r e c o n c e n t r a t e d

c y t o t o x i n . Trypan b l u e dye e x c l u s i o n a s s a y s r e v e a l e d minimal

c y t o t o x i c i t y and mor pho l og i c a l change upon exposu r e t o t he

p r e c o n c e n t r a t e d p r e p a r a t i o n (Tab l e I I . 2 ) .

Tab l e 1 1 . 2. C y t o t o x i c i t y o f t h e Cy t o t ox i n P r e p a r a t i o n s Determined

Using Trypan Blue Dye Ex c l u s io n .

Cy t o t ox i n P r e p a r a t i o n *•k

% C y t o t o x i c i t y

P r e c o n c e n t r a t e 20

15x C o n c e n t r a t e 90-95

U l t r a f i l t r a t e 5

56C/ l ho u r - 15x C o n c e n t r a t e 10

B o i l e d / l h o u r - 15x C o n c e n t r a t e 5

*Trypan b l u e dye e x c l u s i o n a s s a y per fo rmed u s i n g same p r o p o r t i o n o f

n e u t r o p h i l s and c y t o t o x i n p r e p a r a t i o n s as in c o l o r i m e t r i c a s s a y .

tUsed a t 100%, no d i l u t i o n .

A s igmoi da l r e l a t i o n s h i p between a b s o r ba n c e and c o n c e n t r a t e d

c y t o t o x i n d i l u t i o n was e s t a b l i s h e d u s i n g t h e MTT c o l o r i m e t r i c a s s a y .

I n c r e a s i n g t h e c y t o t o x i n c o n c e n t r a t i o n lowered t h e i n i t i a l s t a r t i n g

a b s o r ba n c e o f t h e p r e c o n c e n t r a t e d p r e p a r a t i o n and deve l oped a b a s e l i n e

up t o a 1 :8 d i l u t i o n ( F i g u r e I I . 6 ) .

39

0 .1 4 515x Concentrated

Cytotoxin

0 .1 2 5

0 .1 0 5

Eeor*

< 0 .0 8 5

0 .0 6 5

0 .0 4 5


Fi g u r e I I . 6 - Sigmoidal dose r e s p o n s e o f bovine n e u t r o p h i l s exposed t o

t h e 15:1 c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n in t h e MTT c o l o r i m e t r i c

a s s a y . * i n d i c a t e s a b s o r ba n c e produced by normal n e u t r o p h i l s .

Thi s b a s e l i n e was f o l l owe d by a l i n e a r c o n c e n t r a t i o n dependen t

i n c r e a s e in ab s o r ba n c e wi t h i n c r e a s i n g c y t o t o x i n d i l u t i o n s i m i l a r t o

t h e p r e c o n c e n t r a t e d p r e p a r a t i o n . The c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n a l s o caused enhanced dye r e d u c t i o n a t h igh d i l u t i o n s

r e l a t i v e t o unexposed c e l l s . The b a s e l i n e o f t h i s cu r ve c o n s i s t e d o f

40

t o t a l l y l y s ed c e l l s and MTT r e d u c t i o n t o formazan was no t e v i d e n t when

viewed wi t h an i n v e r t e d m i c r o s c o pe . The c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n produced 90-95% c y t o t o x i c i t y as a s s e s s e d by t r y p a n b l ue

dye e x c l u s i o n (Tab l e I I . 2 ) .

The u l t r a f i l t r a t e f rom c y t o t o x i n c o n c e n t r a t i o n , c o n t a i n i n g

a n t i g e n s l e s s t ha n 30 KD, produced a l i n e a r , c o n c e n t r a t i o n i nde penden t

r e l a t i o n s h i p when ab s o r ba n c e was p l o t t e d a g a i n s t d i l u t i o n ( F i g u r e

I I . 7 ) .

0 . 1 5 0Cytotoxin

Ultrafiltrate

0 .1 4 0

0 . 1 3 0

0 . 1 2 0

0.110

0.100

0 . 0 9 0 0 1 2 3 4 5 6 7 8 9 10 11Log2 -Cytotoxin Dilution

F i g u r e I I . 7. Dose r e s p o ns e o f bovine n e u t r o p h i l s t o c y t o t o x i n

u l t r a f i l t r a t e in t h e MTT c o l o r i m e t r i c a s s a y . *abso r bance v a l ue o f

n e u t r o p h i l s no t exposed t o c y t o t o x i n .

41

Dye r e d u c t i o n by n e u t r o p h i l s appe a r e d t o be i nd e pe n d e n t o f

u l t r a f i l t r a t e d i l u t i o n and ex t r eme v a r i a t i o n was o b t a i n e d a t each

a s s a y p o i n t . No d i f f e r e n c e in n e u t r o p h i l morphology was obs e r ve d

among t h e w e l l s u s i n g an i n v e r t e d mi c r os c o p e . N e u t r o p h i l s exposed t o

c y t o t o x i n u l t r a f i l t r a t e were no t undergo i ng c y t o l y s i s as e v i de n c e d by

t r y p a n b l u e dye e x c l u s i o n ( Tab l e I I . 2 ) .

I n a c t i v a t i o n o f t h e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n , by

h e a t i n g a t 56C f o r 1 h r produced a b e l l shaped cu r ve in t he

c o l o r i m e t r i c a s s a y ( F i g u r e I I . 8 ) .

42

0 . 1 5 5

0 . 1 4 5

0 . 1 3 5EcoNU»

< 0 . 1 2 5

0 . 1 1 5 15x Concentrated Cytotoxin

56°C 1 hour0 .1 0 5


Fi g u r e I I . 8. Dose r e s p o n s e o f bov i ne n e u t r o p h i l s exposed t o 15:1

c o n c e n t r a t e d c y t o t o x i n which had been i n a c t i v a t e d wi t h mi l d h e a t (56°C

f o r 1 h r ) . *de no t e s a b s o r b a n c e v a l u e produced by unexposed c e l l s .

The b a s e l i n e and lowered i n i t i a l s t a r t i n g a b s o r ba n c e o f t h e f u l l y

a c t i v e c o n c e n t r a t e d p r e p a r a t i o n was a b s e n t . However, MTT r e d u c t i o n by

n e u t r o p h i l s i n c r e a s e d w i t h r e s p e c t t o d i l u t i o n , t he n d e c r e a s e d t o an

ab s o r ba n c e v a l u e c h a r a c t e r i s t i c o f unexposed c e l l s . Ne u t r op h i l l y s i s

was n o t a p p a r e n t i n t h e p l a t e w e l l s and c y t o t o x i c i t y was no t e v i d e n t

v i s u a l l y u s i n g t r y p a n b l u e dye .

43

B o i l i n g t h e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n f o r 1 h r , f o l l owed

by c l a r i f i c a t i o n , r e s u l t e d i n a d i f f e r e n t dose r e s po ns e by t h e

n e u t r o p h i l s ( F i g u r e I I . 9 ) .

0 .1 5 5

0 .1 4 5

0 .1 3 5

EeON10

< 0 .1 2 5

15x Concentrated Cytotoxin

Boiled 1 hour * = 0 . 0 9 5 +/-0.007

0 .10 5

Log 2 "Cytotoxin Dilution

Fi g u re I I . 9. Dose r e s p o n s e o f n e u t r o p h i l s t o b o i l e d and c l a r i f i e d

c o n c e n t r a t e d ( 1 5 : 1 ) c y t o t o x i n . * i n d i c a t e s normal n e u t r o p h i l s .

MTT r e d u c t i o n was g r e a t e s t when t h e b o i l e d p r e p a r a t i o n was used a t t h e

h i g h e s t t e s t c o n c e n t r a t i o n and t he n d e c r e a s e d w i t h d i l u t i o n o f t h e

c y t o t o x i n p r e p a r a t i o n . The c y t o t o x i n u l t r a f i l t r a t e and h e a t

i n a c t i v a t e d p r e p a r a t i o n s caused n e u t r o p h i l s t o r es pond w i t h more

v a r i a n c e as i n d i c a t e d by t h e l a r g e s t a n d a r d d e v i a t i o n s c h a r a c t e r i z i n g

44

each d i l u t i o n . Cel l l y s i s was no t e v i d e n t in p l a t e w e l l s and

c y t o t o x i c i t y was n o t e v i d e n t u s i n g t r y p a n b l ue dye wi t h t h e s e

p r e p a r a t i o n s .

Chemical and immunologic a n a l y s i s o f t h e c y t o t o x i n p r e p a r a t i o n s

d e m o n s t r a t e d d i f f e r e n c e s i n t h e p r o t e i n and a n t i g e n c o n t e n t wh i l e

hexosamine c o n c e n t r a t i o n remained r e l a t i v e l y c o n s t a n t (Table I I . 3 ) .

The u l t r a f i l t r a t e , f rom c y t o t o x i n c o n c e n t r a t i o n , c o n t a i n e d a n e a r l y

e q u i v a l e n t hexosamine c o n c e n t r a t i o n r e l a t i v e t o t h e c o n c e n t r a t e d

p r e p a r a t i o n s , bu t o n l y t h e p a n - p a s t e u r e l l a c a r b o h y d r a t e a n t i g e n was

d e t e c t e d a t s i g n i f i c a n t l e v e l s as d e t e c t e d by monoclonal a n t i b o d i e s in

ELISA. Hea t i ng t h e c o n c e n t r a t e d p r e p a r a t i o n a t 56C f o r 1 h r d id not

a l t e r t h e ELISA r e a c t i v i t y o f t h e a n t i g e n s . B o i l i n g t h e c o n c e n t r a t e d

p r e p a r a t i o n , f o l l owe d by c l a r i f i c a t i o n , s i g n i f i c a n t l y reduced t h e

amount o f 29 k i l o d a l t o n p r o t e i n and i t was u n d e t e c t a b l e u s i n g ELISA.

The r ema i n i ng a n t i g e n s were no t a f f e c t e d by b o i l i n g .

45

Table I I . 3 - Chemical and Immunologic C h a r a c t e r i z a t i o n o f t h e

Cy t o t ox i n P r e p a r a t i o n s .

Assay

C y t o t ox i n P r e p a r a t i o n s

Pre-Con

c e n t r a t e

15:1 Con

c e n t r a t e

U l t r a

f i l t r a t e 5 6 C / l h r

Bo i l ed

/ I h r

P r o t e i n (ug/ml ) <5. 0 70 . 0 0 . 0 6 6 . 0 <10.0

Hexosamine (yM/ml) 0 . 038 0 . 050 0 . 045 0 . 050 0 . 050

McAb ELISA:*

L i p o p o l y s a c c h a r i d e 64 >256 0 >256 >256

P a n - p a s t e u r e l l a CH^O >256 >256 256 >256 >256

Ser o t ype 1 s p e c i f i c Ag 32 >256 0 >256 >256

29KD p r o t e i n 64 >256 4 >256 0

★Monocolonal a n t i b o d i e s were used a t c o n s t a n t c o n c e n t r a t i o n s (1 : 20

a s c i t i c f l u i d d i l u t i o n ) in ELISA. Ant i ge n t i t e r was e x p r e s s e d as t he

i n v e r s e o f t h e h i g h e s t d i l u t i o n p r o d u c i n g an a ds o r ba nc e va l ue 1.5 t imes

g r e a t e r t han t h e a bs o r ba nc e o b t a i n e d wi t h normal mouse serum.

46

DISCUSSION

In t h e p r e s e n t s t u d y , bov ine n e u t r o p h i l s were obs e r ved t o reduce

MTT dye t o a deep b l u e formazan under d e f i n e d c o n d i t i o n s in D u l b e c c o ' s

PBS. The MTT formazan o c c u r r e d as i n t r a c e l l u l a r a g g r e g a t e s and

e x t r a c e l l u l a r c r y s t a l s . E x t r a c e l l u l a r c r y s t a l f o rm a t i on i n d i c a t e d

p a r t i a l s o l u b i l i t y o f MTT formazan in D u l b e c c o ' s PBS. T h e r e f o r e , a

s o l v e n t was sough t t o d i s s o l v e MTT formazan w i t h o u t removing t h e wel l

c o n t e n t s t o e n s u r e measurement o f a l l r educed MTT. Acid i s o p r o p a n o l ,

28 29used s u c c e s s f u l l y by o t h e r i n v e s t i g a t o r s , 5 was no t e f f e c t i v e under

our e x p e r i me n t a l c o n d i t i o n s because i t would not c o mp l e t e l y s o l u b i l i z e

MTT formazan when d i l u t e d by t he wel l c o n t e n t s . HMPT was found t o be a

r a p i d and comple t e formazan s o l v e n t t h a t was e f f e c t i v e in t h e p r e s en c e

o f t h e wel l c o n t e n t s . HMPT d i d not i n t e r f e r e wi t h a b s o r ba n c e a t a t e s t

wave l eng t h o f 570 nm and a r e f e r e n c e wave leng th o f 630 nm and

e f f e c t i v e l y s o l u b i l i z e d n e u t r o p h i l s and t h e formazan t h e y p roduced .

A n o n - l i n e a r r e l a t i o n s h i p between a b s o r ba n c e and n e u t r o p h i l number

p e r wel l was e s t a b l i s h e d u s i n g t h e d e s c r i b e d p r o c e d u r e s . Thi s i s in

d i r e c t c o n t r a d i c t i o n t o o t h e r r e p o r t s which d e s c r i b e a l i n e a r

28 29r e l a t i o n s h i p between t h e s e p a r a m e t e r s . ’ Al though many c o n d i t i o n s

i n t h i s s t u d y were v a r i e d from t h o s e o r i g i n a l l y d e s c r i b e d , t h e r ea s on

f o r t h i s n o n - l i n e a r i t y remains l a r g e l y un d e t e r mi n e d . However, a

c o o p e r a t i v e e f f e c t by t h e n e u t r o p h i l s i n MTT r e d u c t i o n i s a p l a u s a b l e

e x p l a n a t i o n . Our a s s a y p r oc e d u r e p roduces a l i n e a r r e l a t i o n s h i p

between abs o r ba nc e and MTT i n c u b a t i o n t ime a t c o n s t a n t c e l l numbers .

The s l o p e o f t h e s e l i n e a r r e l a t i o n s h i p s a t d i f f e r e n t c e l l numbers

i n c r e a s e d wi t h r e s p e c t t o i n c r e a s i n g c e l l c o n c e n t r a t i o n which s u p p o r t s

a c o o p e r a t i v e e f f e c t o f t h e n e u t r o p h i l s in dye r e d u c t i o n . When

47

ab s o r ba n c e was p l o t t e d a g a i n s t MTT i n c u b a t i o n t i me by o t h e r

28 29i n v e s t i g a t o r s , n o n - l i n e a r r e l a t i o n s h i p s wre o b t a i n e d . ’ I t was

h y p o t h e s i z e d t h a t p r o g r e s s i v e c e l l de a t h d u r i n g MTT i n c u b a t i o n was

r e s p o n s i b l e f o r t h e n o n - l i n e a r i t y . L a t e r t h i s was proven and c o r r e c t e d

29v a l u e s r e v e a l e d a l i n e a r r e l a t i o n s h i p ove r t i me . The l i n e a r

r e l a t i o n s h i p between a d s o r ba n c e and t ime in t h i s s t u d y i n d i c a t e d a

c o n s t a n t r a t e o f MTT r e d u c t i o n . The r a t e o f MTT r e d u c t i o n was no t

a f f e c t e d by mixing e i t h e r methanol o r f o r ma l i n f i x e d and washed

n e u t r o p h i l s w i t h normal c e l l s in t h e a s s a y .

O p t i m i z a t i o n and v a l i d a t i o n s t u d i e s o f t h e MTT c o l o r i m e t r i c a s s a y

u s i n g bovine n e u t r o p h i l s r e s u l t e d in t h e d e c i s i o n t o use HMPT as a MTT5

formazan s o l v e n t , 5X10 n e u t r o p h i l s p e r w e l l , and a MTT i n c u b a t i o n t ime

o f 4 h r f o r t h e measurement o f c y t o t o x i n a c t i v i t y . The l a t t e r two

p a r am e t e r s were chosen because t h e y produced a n e a t l y c o n f l u e n t

monolayer and me a s u re a b le a b s o r ba n c e v a l u e s r e s p e c t i v e l y .

C y t o t ox i n a c t i v i t y i n t h e MTT c o l o r i m e t r i c a s s a y was measured

u s i n g d i f f e r e n t c y t o t o x i n p r e p a r a t i o n s which v a r i e d in t h e i r a b i l i t y t o

cause c y t o t o x i c i t y as a s s e s s e d by t r y p a n b l u e dye e x c l u s i o n a s s a y s .

The p r e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n i n h i b i t e d MTT r e d u c t i o n a t

low d i l u t i o n s r e l a t i v e t o unexposed n e u t r o p h i l s . I n c r e a s i n g t h e

c y t o t o x i n c o n c e n t r a t i o n caused f u r t h e r i n h i b i t i o n o f MTT r e d u c t i o n and

t o t a l l y l y s ed c e l l s . I n a c t i v a t e d c y t o t o x i n p r e p a r a t i o n s d i d n o t cause

i n h i b i t i o n o f MTT r e d u c t i o n . The i n h i b i t i o n o f MTT r e d u c t i o n was

i n t e r p r e t e d as c y t o t o x i n a c t i v i t y i n t h e MTT c o l o r i m e t r i c a s s a y .

Enhanced MTT r e d u c t i o n by t h e n e u t r o p h i l s o cc u r r e d in r e s po n s e t o

a l l c y t o t o x i n p r e p a r a t i o n s e x c e p t t h e u l t r a f i l t r a t e . N e u t r o p h i l s were

s t i m u l a t e d t o r educe more MTT a t d i f f e r e n t c o n c e n t r a t i o n s among t h e

48

c y t o t o x i n p r e p a r a t i o n s . A p p a r e n t l y enhanced m i t o c h o n d r i a l a c t i v i t y

o c c u r r e d a t d i f f e r e n t c o n c e n t r a t i o n s o f t h e p r e p a r a t i o n s whe t he r

c y t o t o x i c a c t i v i t y was p r e s e n t o r n o t . These f i n d i n g s i n d i c a t e t h e MTT

c o l o r i m e t r i c a s s a y i s c a p a b l e o f measu r i ng more t han n e u t r o p h i l

c y t o l y s i s . Enhanced MTT dye r e d u c t i o n most l i k e l y r e f l e c t s n e u t r o p h i l

a c t i v a t i o n v i a t h e r e s p i r a t o r y b u r s t . Ne u t r op h i l a c t i v a t i o n

may a c c o u n t f o r t h e i r r a p i d and i n t e n s e i n f l u x i n t o pulmonary t i s s u e

4-7f o l l o w i n g e x p e r i m e n t a l c h a l l e n g e .

From t h e r e s u l t s o b t a i n e d , i t i s u n c l e a r whe t he r c y t o t o x i n a t low

c o n c e n t r a t i o n s , i n a c t i v a t e d c y t o t o x i n , o t h e r component a n t i g e n s , o r

comb i n a t i on s o f them a r e r e s p o n s i b l e f o r n e u t r o p h i l s t i m u l a t i o n o f MTT

r e d u c t i o n . S i nce most p r o t e i n was removed from t h e b o i l e d p r e p a r a t i o n ,

which caused n e u t r o p h i l s t i m u l a t i o n o f MTT r e d u c t i o n , i t i s u n l i k e l y

t h a t p r o t e i n in i t s e l f was r e s p o n s i b l e f o r n e u t r o p h i l a c t i v a t i o n .

S i m i l a r l y , beca use t he o n l y d e t e c t a b l e a n t i g e n in t h e u l t r a f i l t r a t e was

p a n - p a s t e u r e l l a c a r b o h y d r a t e , which d i d no t cause n e u t r o p h i l

a c t i v a t i o n , i t i s u n l i k e l y t h i s a n t i g e n was s o l e l y r e s p o n s i b l e f o r

enhanced MTT r e d u c t i o n . L i p o p o l y s a c c h a r i d e and c a p s u l a r p o l y s a c c h a r i d e

were t h e on l y two a n t i g e n s , d e t e c t a b l e u s i n g t h e d e s c r i b e d methods ,

which were common t o a l l p r e p a r a t i o n s r e s u l t i n g in n e u t r o p h i l

s t i m u l a t i o n . C l e a r l y , more e x t e n s i v e f r a c t i o n a t i o n o r p u r i f i c a t i o n o f

a n t i g e n s in t h e c y t o t o x i n p r e p a r a t i o n s a r e needed t o i d e n t i f y

components r e s p o n s i b l e f o r n e u t r o p h i l a c t i v a t i o n .

Because c y t o t o x i n a c t i v i t y was c h a r a c t e r i z e d in t h e p r e s en ce o f

SP-2 / o mur ine myeloma c e l l c o n d i t i o n e d medium, t h e MTT c o l o r i m e t r i c

a s s a y shou l d be r e a d i l y a d a p t a b l e f o r t h e s e l e c t i o n o f a c y t o t o x i n

n e u t r a l i z i n g monoclonal a n t i b o d y . Measurement o f c y t o t o x i n a c t i v i t y in

49

t h e p r e s e n c e o f o t h e r body f l u i d s , such as immune and non-immune

s e rums , na s a l o r b r o n c h o a l v e o l a r wa s h i n g s , may e x t e n d t h e use o f t h i s

a s s a y f o r s t u d y i n g o t h e r i n t e r a c t i o n s . F u r th e r mo r e , t h e i n c o r p o r a t i o n

o f monoclonal a n t i b o d i e s t o component a n t i g e n s o f c y t o t o x i n

p r e p a r a t i o n s may a i d i d e n t i f i c a t i o n o f n e u t r o p h i l m e t a b o l i c r e s p o n s e s

t o p a r t i c u l a r a n t i g e n s o r t h e i r immune complexes . The MTT c o l o r i m e t r i c

a s s a y has p o t e n t i a l a p p l i c a t i o n f o r i n v e s t i g a t i n g t h e m e t a b o l i c

r e s po ns e o f n e u t r o p h i l s f rom o t h e r s p e c i e s t o a wide v a r i e t y o f

a n t i g e n s , t o x i n s and a n t i b o d i e s .

BIBLIOGRAPHY

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CHAPTER 3

CHARACTERIZATION OF A NEUTRALIZING MONOCLONAL ANTIBODY PREPARED

AGAINST PASTEURELLA HAEMOLYTICA SEROTYPE 1 CYTOTOXIN

SUMMARY

P a s t e u r e l l a h a e m o l y t i c a s e r o t y p e 1 (Ph-1) c y t o t o x i n was produced

in a d e f i n e d medium, c o n c e n t r a t e d by u l t r a f i l t r a t i o n and used t o

immunize BALB/c mice f o r monoclonal a n t i b o d y p r o d u c t i o n . A hybridoma

produc i ng a n t i b o d y c a p a b l e o f c y t o t o x i n n e u t r a l i z a t i o n was s e l e c t e d

u s i n g a t e t r a z o l i u m dye (MTT) c o l o r i m e t r i c a s s a y wi t h i s o l a t e d bov ine

p e r i p h e r a l b lood n e u t r o p h i l s as t a r g e t c e l l s . Ne u t r o ph i l r e s po n s e t o

o t h e r d e f i n e d monoclonal a n t i b o d i e s , s e l e c t e d by ELISA, a g a i n s t

component a n t i g e n s in t h e c y t o t o x i n p r e p a r a t i o n was a l s o e v a l u a t e d i n

t h e MTT c o l o r i m e t r i c a s s a y . R e a c t i v i t y o f t h e c y t o t o x i n n e u t r a l i z i n g

a n t i b o d y t o 6 and 24 h r c u l t u r e s o f known P. h a e m o l y t i ca (Ph)

s e r o t y p e s as wel l as Ph-1 whole c e l l s , c a p s u l a r m a t e r i a l , and

c y t o t o x i n p r e p a r a t i o n s t r e a t e d in v a r i o u s ways was d e t e r mi n e d u s i n g an

ELISA p r o c e d u r e . A s c i t i c f l u i d c o n t a i n i n g t h e n e u t r a l i z i n g a n t i b o d y

was e v a l u a t e d f o r t h e e x t e n t o f c y t o t o x i n n e u t r a l i z a t i o n u s i n g t h e MTT

c o l o r i m e t r i c a s s a y . Using s t a n d a r d s e r o l o g i c methods t h e i s o t y p e o f

t h e n e u t r a l i z i n g a n t i b o d y was i d e n t i f i e d as IgM. The n e u t r a l i z i n g

a n t i b o d y was no t r e a c t i v e in ELISA wi t h Ph whole c e l l s , c a p s u l a r

m a t e r i a l , o r c y t o t o x i n p r e p a r a t i o n s r e g a r d l e s s o f s e r o t y p e , c u l t u r e

a g e , o r t r e a t m e n t . N e u t r a l i z i n g a c t i v i t y was d e t e c t a b l e a t a

1 :200 , 000 d i l u t i o n o f a s c i t i c f l u i d i n t h e MTT c o l o r i m e t r i c a s s a y .

Monoclonal a n t i b o d i e s s e l e c t e d by ELISA d id no t a f f e c t n e u t r o p h i l

r e s po ns e t o t h e c y t o t o x i n p r e p a r a t i o n in t h e MTT c o l o r i m e t r i c a s s a y .

53

54

INTRODUCTION

Bovine pneumonic p a s t e u r e l l o s i s , commonly known as s h i p p i n g f e v e r ,

i s a s e v e r e f i b r i n o u s bronchopneumonia c a u s i n g ma jo r economic l o s s e s t o

1 2t h e Nor th American f e e d l o t c a t t l e i n d u s t r y . P a s t e u r e l l a h a e m o l y t i c a

s e r o t y p e 1 (Ph-1) i s t h e p a t h o g e n i c a g e n t most f r e q u e n t l y i s o l a t e d in

1-3a s s o c i a t i o n wi t h t h i s d i s e a s e . P. ha e mol y t i ca has been shown t o

s e c r e t e , i n t o c u l t u r a l s u p e r n a t e , a h e a t l a b i l e , immunogenic p r o t e i n

c y t o t o x i n ( l e u k o t o x i n ) which i s s p e c i f i c a l l y c y t o l y t i c f o r r umi nan t

a l v e o l a r macrophages and p e r i p h e r a l b lood l e u k o c y t e s . ^ " ® Cy to t o x i n i s

c u r r e n t l y viewed as a p o t e n t i a l v i r u l e n c e f a c t o r o f P. h a e m o l y t i c a ,

b e l i e v e d t o c o n t r i b u t e t o t h e p a t h o g e n e s i s o f pneumonic p a s t e u r e l l o s i s

t h r ough i t ' s l y t i c e f f e c t on pulmonary d e f e n s e c e l l s . The i n t e r a c t i o n

o f c y t o t o x i n w i t h n e u t r o p h i l s i s t h o u g h t t o be i m p o r t a n t beca use o f t h e

n e u t r o p h i l ' s e a r l y and p r edomi nan t i n f l u x i n t o lung t i s s u e f o l l o w i n g

7 8e x p e r i m e n t a l c h a l l e n g e , 5 a b i l i t y t o c l e a r b a c t e r i a t h r ough

9 10p h a g o c y t o s i s , and p o t e n t i a l t o c a us e t i s s u e damage. * The

a s s o c i a t i o n o f h igh c y t o t o x i n n e u t r a l i z i n g serum a n t i b o d y t i t e r s w i t h

d i s e a s e r e s i s t a n c e may i n d i c a t e t h e i mpor t ance o f c y t o t o x i n in t he

11-13p a t h o g e n e s i s o f pneumonic p a s t e u r e l l o s i s . L i k e wi s e , t h e l ack of

p a t h o p h y s i o l o g i c changes in n e u t r o p e n i c c a t t l e e x p e r i m e n t a l l y

c h a l l e n g e d wi t h P. h a e m o l y t i c a a r e t h o ug h t t o r e f l e c t t h e impor t anceg

o f n e u t r o p h i l s i n t h e d i s e a s e .

However, Ph-1 l i b e r a t e s s e v e r a l s o l u b l e a n t i g e n s i n t o c u l t u r a l

s u p e r n a t e in a d d i t i o n t o c y t o t o x i n which a r e known t o a f f e c t t h e

n e u t r o p h i l . O t he r component a n t i g e n s o f c y t o t o x i n p r e p a r a t i o n s

i n c l u d e 1i p o p o l y s a c c h a r i d e s , c a p s u l a r p o l y s a c c h a r i d e s , o t h e r

c a r b o h y d r a t e s and p r o t e i n s ( Ch a p t e r 2 ) . At t empt s t o p u r i f y c y t o t o x i n

55

from o t h e r s o l u b l e a n t i g e n s o f P. ha e mol y t i ca have r e s u l t e d i n a

14 15s u b s t a n t i a l l o s s o f c y t o t o x i c a c t i v i t y . * Co n s e q u e n t l y , d i r e c t

a s s a y s f o r c y t o t o x i n , i n d e p e n d e n t o f i t s c e l l u l a r e f f e c t s , have no t

been d ev e l op e d .

Because c y t o t o x i n i s immunogenic and i t s e f f e c t s a r e n e u t r a l i z a b l e

by a n t i b o d y we d ec i de d t o use t h e s e p r o p e r t i e s t o deve l op a monoclonal

a n t i b o d y a g a i n s t c y t o t o x i n . S p e c i f i c monoclonal a n t i b o d y a g a i n s t

c y t o t o x i n c ou l d a i d i n i t s i d e n t i f i c a t i o n , q u a n t i t a t i o n , and l a r g e

s c a l e p u r i f i c a t i o n u s i n g con t empor a r y immunologic t e c h n i q u e s .

Fu r th e r m o r e , s p e c i f i c n e u t r a l i z a t i o n o f c y t o t o x i n may a l l ow

i d e n t i f i c a t i o n o f novel i n t e r a c t i o n s o f n e u t r o p h i l s wi t h o t h e r

component a n t i g e n s in c y t o t o x i n p r e p a r a t i o n s . A d d i t i o n a l l y , t h e e f f e c t

o f s p e c i f i c a n t i b o d i e s a g a i n s t component a n t i g e n s o f c y t o t o x i n

p r e p a r a t i o n s i s unknown and c ou l d a l s o i d e n t i f y more s u b t l e

i n t e r a c t i o n s wi t h n e u t r o p h i l s . Assays and r e a g e n t s c o n c e r n i n g t he

i n t e r a c t i o n o f s p e c i f i c a n t i b o d i e s and a n t i g e n s w i t h n e u t r o p h i l s a r e

needed t o ga i n a b e t t e r u n d e r s t a n d i n g o f t h e p a t h o g e n e s i s in pneumonic

p a s t e u r e l l o s i s .

The r e s p o n s e o f n e u t r o p h i l s t o a c t i v e and h e a t i n a c t i v a t e d

c y t o t o x i n p r e p a r a t i o n s in t h e MTT c o l o r i m e t r i c a s s a y has been

c h a r a c t e r i z e d ( Ch a p t e r 2 ) . At d i f f e r e n t c o n c e n t r a t i o n s , both a c t i v e

and i n a c t i v e p r e p a r a t i o n s were found t o s t i m u l a t e n e u t r o p h i l r e d u c t i o n

o f t e t r a z o l i u m dye. Thi s a s s a y was c a r r i e d ou t in t h e p r e s en c e o f

SP-2 /o mur ine myeloma c e l l c o n d i t i o n e d medium f o r e q u i v a l e n c e wi th

n e u t r a l i z a t i o n by monoclonal a n t i b o d y . The p r imary purpose o f t h i s

s t u d y was t o s e l e c t and c h a r a c t e r i z e a n e u t r a l i z i n g monoclonal

a n t i b o d y a g a i n s t Ph-1 c y t o t o x i n u s i n g t h e MTT c o l o r i m e t r i c a s s a y .

56

A d d i t i o n a l o b j e c t i v e s o f t h i s i n v e s t i g a t i o n were t o d e t e r mi n e t h e

e f f e c t o f o t h e r component a n t i g e n s in c y t o t o x i n p r e p a r a t i o n s (Ch a p t e r

2) on bov ine n e u t r o p h i l r e s p o n se s u s i n g d e f i n e d monoclonal a n t i b o d i e s

i n t h e MTT c o l o r i m e t r i c a s s a y .


P. h a e mo l y t i ca s e r o t y p e 1 c y t o t o x i n :

Ph-1 c y t o t o x i n was produced in RPMI-1640 c o n t a i n i n g lOmM sodium

b i c a r b o n a t e and lOmM L - g l u t a m i n e , c o n c e n t r a t e d 15:1 by

u l t r a f i l t r a t i o n , and s t o r e d a t -70C as p r e v i o u s l y d e s c r i b e d (Chap t e r

2 ).

MTT c o l o r i m e t r i c a s s a y o f c y t o t o x i n a c t i v i t y :

C y t o t o x i c a c t i v i t y in t h e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n was

measured and s t a n d a r d i z e d u s i n g t h e MTT c o l o r i m e t r i c a s s a y d e s c r i b e d in

Ch ap t e r 2. The a s s a y was per formed in t h e p r e s en c e o f SP-2/o mur ine

myeloma c e l l c o n d i t i o n e d medium f o r e q u i v a l e n c e wi t h c y t o t o x i n

n e u t r a l i z a t i o n a s s a y s .

MMT c o l o r i m e t r i c a s s a y f o r c y t o t o x i n n e u t r a l i z a t i o n :

A 1 :8 d i l u t i o n o f t h e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n was used

in t h e MTT c o l o r i m e t r i c a s s a y f o r c y t o t o x i n n e u t r a l i z a t i o n . C y t o t ox i n

n e u t r a l i z a t i o n was de t e r mi n e d by t h e i n t r o d u c t i o n and compar i son o f

e i t h e r n e u t r a l i z i n g o r n o n - n e u t r a l i z i n g se rums , hybridoma c u l t u r a l

s u p e r n a t e s , o r p e r i t o n a l a s c i t i c f l u i d s (50^1) i n p l a c e o f t h e SP-2 / o

mur ine myeloma c e l l c o n d i t i o n e d medium in t h e a s s a y o f c y t o t o x i n

57

a c t i v i t y . N e u t r a l i z a t i o n was a l l owed t o o c c u r f o r 15 min p r i o r t o

a dd i ng t h e n e u t r o p h i l s .

The e x t e n t o f c y t o t o x i n n e u t r a l i z a t i o n :

N e u t r a l i z a t i o n by p e r i t o n e a l a s c i t i c f l u i d s was d e t e r mi n e d u s i n g a

10 f o l d d i l u t i o n s e r i e s i n t h e c o l o r i m e t r i c a s s a y . Def ined monoclonal

a n t i b o d i e s g e n e r a t e d a g a i n s t Ph-1 c a p s u l a r m a t e r i a l (Ch a p t e r 1) were

a l s o e v a l u a t e d in t h e a s s a y . Checkerboard combi na t i ons o f t h e s e

a n t i b o d i e s , in equal p r o p o r t i o n s o f hybridoma c u l t u r a l s u p e r n a t e , were

f u r t h e r examined f o r c y t o t o x i n n e u t r a l i z a t i o n . Wel ls i n which

n e u t r o p h i l s were exposed t o RPMI-1640 in p l a c e o f c y t o t o x i n were used

as p o s i t i v e n e u t r a l i z a t i o n c o n t r o l s t o r e p r e s e n t l i v e c e l l s . Nega t i ve

c o n t r o l s c o n s i s t e d o f n e u t r o p h i l s exposed t o a 1 :8 d i l u t i o n o f

c o n c e n t r a t e d c y t o t o x i n . R e s u l t s were e x p r e s s e d as t h e mean o f

q u a d r u p l i c a t e s p l u s o r minus one s t a n d a r d d e v i a t i o n .

P ro d u c t i o n o f monoclonal a n t i b o d y :

Six t o e i g h t week o l d f ema le BALB/c mice were immunized by

i n t r a p e r i t o n e a l i n j e c t i o n w i t h 0 . 5 ml c o n c e n t r a t e d c y t o t o x i n . Four

weeks l a t e r , mice were immunized a s econd t i me u s i n g t h e same r o u t e

and inoculum. Two weeks f o l l o w i n g t h e second i mmuni z a t i on , mice were

immunized a g a i n by i n t r a v e n o u s i n o c u l a t i o n wi t h 0 . 1 ml c o n c e n t r a t e d

c y t o t o x i n . Three days a f t e r t h e l a s t i mmuni za t i on , s e r a was c o l l e c t e d

f rom t h e r e t r o - o r i b i t a l p l e x u s and e v a l u t e d f o r c y t o t o x i n

n e u t r a l i z a t i o n in t h e MTT c o l o r i m e t r i c a s s a y . A s i n g l e mouse,

p r od u c i n g t h e h i g h e s t a b s o r ba n c e v a l u e in t h e c o l o r i m e t r i c a s s a y f o r

c y t o t o x i n n e u t r a l i z a t i o n , was s e l e c t e d f o r hybridoma p r o d u c t i o n .

58

S p l e n o c y t e s , from t h e mouse n e u t r a l i z i n g t h e most c y t o t o x i n , were

fu s e d wi t h SP-2/o mur ine myeloma c e l l s on t h e f o u r t h day f o l l o wi n g t h e

l a s t immuniza t ion and hybr idomas were s e l e c t e d wi t h HAT medium as

p r e v i o u s l y d e s c r i b e d ( C h a p t e r 1 ) . Hybridoma c u l t u r e s u p e r n a t e s were

e v a l u a t e d f o r c y t o t o x i n n e u t r a l i z a t i o n u s i n g t h e MTT c o l o r i m e t r i c

a s s a y . Hybridomas s e c r e t i n g a n t i b o d y c a p a b l e o f c y t o t o x i n

n e u t r a l i z a t i o n were c l oned by l i m i t i n g d i l u t i o n . A s c i t i c f l u i d s were

produced by i n j e c t i n g c l on e d hybr idomas i n t o t h e p e r i t o n e a l c a v i t y o f

BALB/c mice p r e c o n d i t i o n e d one week in advance wi t h 0 . 5 ml p r i s t a n e .

ELISA r e a c t i v i t y o f t h e c y t o t o x i n n e u t r a l i z i n g monoclonal a n t i b o d y :

R e a c t i v i t y o f t h e c y t o t o x i n n e u t r a l i z i n g monoclonal a n t i b od y wi th

Ph-1 whole c e l l s , c a p s u l a r m a t e r i a l , and c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n s which had been e i t h e r u n t r e a t e d , f i x e d wi t h f o r m a l i n ,

p a r a f o r ma l d e h yd e , o r both were de t e r mi n e d in an ELISA p rocedure

( Chap t e r s 1 and 2 ) . In a s e p a r a t e ELISA e x p e r i me n t , P. haemol y t i ca

s e r o t y p e s 1 t h r ough 12 whole c e l l s from 6 and 24 h r c u l t u r e s were used

as a n t i g e n s . Monoclonal a n t i b o d i e s deve l oped a g a i n s t s e r o t y p e 1

c a p s u l a r m a t e r i a l were used as p o s i t i v e c o n t r o l s . R e a c t i v i t y in ELISA

was de t e r mi n e d u s i n g hybridoma c u l t u r a l s u p e r n a t e s as t h e pr imary

a n t i b o d y s o l u t i o n .

I s o t y p e d e t e r m i n a t i o n :

The i s o t y p e o f t h e c y t o t o x i n n e u t r a l i z i n g monoclonal a n t i b o d y was

d e t e r mi n e d u s i n g c o m m e r i c i a l l y a v a i l a b l e k i t s based on a n t i b o d y c a p t u r e

ELISA and immunod i f fus i on . Hybridoma c u l t u r a l s u p e r n a t e was used in

i s o t y p e d e t e r m i n a t i o n s .

RESULTS

F i g u r e I I I . l i l l u s t r a t e s t h e MTT c o l o r i m e t r i c a s s a y r e s u l t s o f

c y t o t o x i c a c t i v i t y p r e s e n t i n t h e 15:1 c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n .

0 .1 4 515x Concentrated

Cytotoxin

0 .1 2 5

0 .1 0 5

0 .0 8 5

0 .0 6 5

0 .0 4 5

2 3 4 5 6 7 8 9 10 110 1Log2 -Cytotoxin Dilution

F ig u r e I I I . l - Cy t o t o x i c a c t i v i t y p r e s e n t in t h e 15:1 c o n c e n t r a t e d

c y t o t o x i n p r e p a r a t i o n u s i n g t h e MTT c o l o r i m e t r i c a s s a y . A 1:8

d i l u t i o n o f t h e c o n c e n t r a t e d c y t o t o x i n p r e p a r a t i o n was used in t h e

a s s a y f o r c y t o t o x i n n e u t r a l i z a t i o n , i n d i c a t e s a bs o r ba nc e v a l ue

p roduced by normal n e u t r o p h i l s .

60

C y t o t o x i c a c t i v i t y was i n e x c e s s o f n e u t r o p h i l number up t o a 1:8

c y t o t o x i n d i l u t i o n , p r o d u c i n g a b a s e l i n e o f t o t a l l y l y s e d c e l l s . At a

1 : 8 and h i g h e r c y t o t o x i n d i l u t i o n s , ab s o r ba nc e i n c r e a s e d wi t h r e s p e c t

t o d i l u t i o n . T h e r e f o r e , a 1 :8 d i l u t i o n o f t h e c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n was used in t h e MTT c o l o r i m e t r i c a s s a y f o r c y t o t o x i n

n e u t r a l i z a t i o n .

Normal mouse s e r a was i n c a p a b l e o f c y t o t o x i n n e u t r a l i z a t i o n in t h e

MTT c o l o r i m e t r i c a s s a y ( Tab l e I I I . l ) .

61

Tab l e I I I . l - Ant ibody S o l u t i o n s Capable o f Cy t o t ox i n N e u t r a l i z a t i o n

in t h e MTT C o l o r i m e t r i c Assay.

An t ibody S o l u t i o n Cy t o t ox i n N e u t r a l i z a t i o n

Normal mouse serum -

C a p s u l a r m a t e r i a l immune

mouse serum

+

Cy t o t ox i n immune mouse

serum

C a p s u l a r m a t e r i a l monoclonal

a n t i b o d i e s : a n t i - * +

+

1i p o p o l y s a c c h a r i d e -

p a n - p a s t e u r e l l a c a r b o h y d r a t e -

s e r o t y p e 1 c a p s u l a r p o l y s a c c h a r i d e -

29 KD p r o t e i n -

♦Monoclonal a n t i b o d i e s p r e s e n t in e i t h e r hybridoma c u l t u r a l s u p e r n a t e

o r p e r i t o n e a l a s c i t i c f l u i d .

t Ch e cke r boa r d c o m b i n a t i on s o f monoclonal a n t i b o d i e s a g a i n s t c a p s u l a r

m a t e r i a l a n t i g e n s a l s o e v a l u a t e d .

62

Sera from mice immunized w i t h e i t h e r c a p s u l a r m a t e r i a l (Ch a p t e r 1) or

c o n c e n t r a t e d c y t o t o x i n e f f e c t i v e l y n e u t r a l i z e d c y t o t o x i n a c t i v i t y .

T h e r e f o r e , d e f i n e d monoclonal a n t i b o d i e s g e n e r a t e d a g a i n s t P.

h a e m o l y t i c a s e r o t y p e 1 c a p s u l a r m a t e r i a l a n t i g e n s (Chap t e r 1) were

e v a l u a t e d f o r c y t o t o x i n n e u t r a l i z a t i o n . These a n t i b o d i e s d i d not

n e u t r a l i z e c y t o t o x i c a c t i v i t y . Checkerboard c ombi na t i ons o f t h e s e

a n t i b o d i e s were a l s o e v a l u a t e d f o r n e u t r a l i z a t i o n w i t h o u t s uc c e s s

( da t a not shown).

Fo l lowing t h e f u s i o n o f s p l e n o c y t e s , from c y t o t o x i n immune mice ,

wi t h SP-2 /o mur ine myeloma c e l l s , s e l e c t i o n o f a p p r o x i m a t e l y 1000

h y b r i d c e l l s was a cc ompl i she d u s i ng HAT medium. One l i n e from t h e s e

h y b r i d s , t ermed n e u t r a l i z i n g monoclonal a n t i b o d y (nMcAb), was s e l e c t e d

u s i n g c u l t u r e s u p e r n a t e s in t h e MTT c o l o r i m e t r i c a s s a y f o r c y t o t o x i n

n e u t r a l i z a t i o n ( F i g u r e I I I . 2 ) . The hybridoma p roduc i ng t h e nMcAb was

c l oned by l i m i t i n g d i l u t i o n and s e l e c t e d u s i n g t h e MTT c o l o r i m e t r i c

a s s a y f o r c y t o t o x i n n e u t r a l i z a t i o n .

63

0 .295-

0 . 2 2 1 -

£ 0.147- <

0.073- T

0 . 0 0 0 1 i '— — — i— — — i— — — i— i inMcAb *1 * 2 * 3 # 1 0 control 1 control2

Hybridoma Cultural Supernates

Fi g u r e I I I . 2 - Cy t o t ox i n n e u t r a l i z a t i o n by monoclonal a n t i b o d i e s in

t h e MTT c o l o r i m e t r i c a s s a y . nMcAb = n e u t r a l i z i n g monoclonal a n t i b o d y ;

#1 = a n t i - 1 i p o p o l y s a c c h o r i d e ; #2 = a n t i - p a n - p a s t e u r e l l a c a r b o h y d r a t e ;

#3 a n t i - s e r o t y p e 1 c a p s u l a r p o l y s a c c h a r i d e ; #10 = a n t i - 2 9 KD p r o t e i n .

Hybridoma c u l t u r a l s u p e r n a t e s were no t d i l u t e d . Cont ro l A r e p r e s e n t s

l i v e c e l l s no t exposed t o c y t o t o x i n . Cont ro l B r e p r e s e n t s dead c e l l s

exposed t o a 1:8 d i l u t i o n o f c o n c e n t r a t e d c y t o t o x i n .

The e x t e n t o f c y t o t o x i n n e u t r a l i z a t i o n by t h e nMcAb in p e r i t o n e a l

a s c i t i c f l u i d i s d i s p l a y e d in F i g u r e I I I . 3.

Colorimetric Assay for Cytotoxin NeutralizationNeutrophils 5x10s/well Cytotoxin dilution 1:8

_L

64

Colorimetric Assay for Cytotoxin Neutralization0.2951

Neutrophils 5x10a/weli Cytotoxin Dilution 1:8

0 . 2 2 1 -

Eeo 0.147-

0.073 ■

0 .0 0 01:200 1:2000 1:20000 1 :2 00000

Ascitic Fluid Dilution1:201:2

Fi g u r e I I I . 3 - E x t e n t o f c y t o t o x i n n e u t r a l i z a t i o n by t h e nMcAb (--------- )

in a s c i t i c f l u i d compared t o o t h e r n o n - n e u t r a l i z i n g a s c i t i c f l u i d s

( -------) in t h e MTT c o l o r i m e t r i c a s s a y .

Cy t o t ox i n n e u t r a l i z a t i o n was no t d e t e c t a b l e u s i n g a s c i t i c f l u i d s

c o n t a i n i n g monoclonal a n t i b o d i e s a g a i n s t s e r o t y p e 1 c a p s u l a r m a t e r i a l

a n t i g e n s ( Ch a p t e r 1 ) . The n o n - n e u t r a l i z i n g a s c i t i c f l u i d s , c o n t a i n i n g

a n t i b o d i e s a g a i n s t e i t h e r 1i p o p o l y s a c c h a r i d e , p a n - p a s t e u r e l l a

c a r b o h y d r a t e , s e r o t y p e I c a p s u l a r p o l y s a c c h a r i d e o r t h e 29KD p r o t e i n o f

P. h a e m o l y t i c a , produced i d e n t i c a l r e s u l t s in t h e MTT c o l o r i m e t r i c

a s s a y f o r n e u t r a l i z a t i o n . C y t o t ox i n n e u t r a l i z a t i o n by t h e nMcAb was

d e t e c t a b l e o u t t o a 1 : 2 0 0 , 00 0 a s c i t i c f l u i d d i l u t i o n where one

s t a n d a r d d e v i a t i o n o f t h e n e u t r a l i z i n g and n o n - n e u t r a l i z i n g a s c i t i c

f l u i d s conve r ge d .

65

When t h e nMcAb in hybr idoma c u l t u r a l s u p e r n a t e was e v a l u a t e d f o r

r e a c t i v i t y w i t h P. h a e m o l y t i ca a n t i g e n s i n ELISA c o n s i s t a n t l y n e g a t i v e

r e s u l t s were o b t a i n e d . The nMcAb was no t r e a c t i v e wi t h P. ha e mol y t i ca

s e r o t y p e 1 whole c e l l s , c a p s u l a r m a t e r i a l , o r c o n c e n t r a t e d c y t o t o x i n

p r e p a r a t i o n s . The nMcAb was no t r e a c t i v e wi t h P. h a e m o l y t i ca s e r o t y p e s

1 t h r ough 12 whole c e l l s . N e i t h e r a n t i g e n f o r m a l i z a t i o n nor f i x a t i o n

w i t h p a r a f o r ma l de hyde i mpa r t e d ELISA r e a c t i v i t y . Monoclonal a n t i b o d i e s

g e n e r a t e d a g a i n s t Ph-1 c a p s u l a r m a t e r i a l (Ch a p t e r 1) were used as

p o s i t i v e c o n t r o l s , s u b s t a n t i a t i n g t h e n e g a t i v e ELISA r e a c t i v i t y o f t h e

nMcAb.

Both a n t i b o d y c a p t u r e ELISA and immunodi f fus ion conf i rmed t h e

p r e s en ce o f mur ine a n t i b o d y in hybridoma c u l t u r a l s u p e r n a t e s w i t h

n e u t r a l i z i n g a c t i v i t y and i d e n t i f i e d t h e i s o t y p e as an IgM.

DISCUSSION

The e x pe r i me n t a l r e s u l t s d e m o n s t r a t e d t h a t P. h a emol y t i ca

c y t o t o x i n a c t i v i t y can be n e u t r a l i z e d by monoclonal a n t i b o d y .

Cy t o t ox i n n e u t r a l i z i n g a c t i v i t y was p r e s e n t in c u l t u r a l s u p e r n a t e and

p e r i t o n e a l a s c i t i c f l u i d d e r i v e d o n l y f rom t h e hybr idoma s e l e c t e d u s i ng

t h e MTT c o l o r i m e t r i c a s s a y . The a n t i b o d y n a t u r e o f c y t o t o x i n

n e u t r a l i z i n g a c t i v i t y i s s u p p o r t e d by t h e p r e s e n c e o f mur ine IgM in

hybridoma c u l t u r a l s u p e r n a t e which n e u t r a l i z e d c y t o t o x i c a c t i v i t y w h i l e

o t h e r monoclonal a n t i b o d i e s and SP-2 /o mur ine myeloma c e l l c o n d i t i o n e d

medium cou l d n o t . High c y t o t o x i n n e u t r a l i z i n g t i t e r s were o b t a i n e d

w i t h p e r i t o n e a l a s c i t i c f l u i d from hybr idomas p r o d u c i n g t h e nMcAb

compared t o o t h e r n o n - n e u t r a l i z i n g monoclonal a n t i b o d i e s u s i n g t h e MTT

c o l o r i m e t r i c a s s a y .

66

Although nMcAb b i n d i n g t o P. h a e m o l y t i c a s e r o t y p e 1 a n t i g e n

p r e p a r a t i o n s in ELISA c ou l d n o t be d e m o n s t r a t e d , r e g a r d l e s s o f t h e

c o n d i t i o n s e v a l u a t e d , a c o o p e r a t i v e r e s e a r c h team has shown nMcAb

b i n d i n g t o a 105 k i l o d a l t o n p r o t e i n s e p a r a t e d by e l e c t r o p h o r e s i s and

b l o t t e d t o n i t r o c e l l u l o s e . 9 Th i s 105 k i l o d a l t o n p r o t e i n has r e c e n t l y

been shown t o c o n s t i t u t e a ma j o r component o f c y t o t o x i n . ^ I t i s

unknown why nMcAb b i n d i n g c ou l d no t be d e m o ns t r a t e d in ELISA, y e t

d e t e c t a b l e u s i n g immunob l o t t i ng t e c h n i q u e s . P o l y c l o n a l r a b b i t

a n t i b o d i e s p r e p a r e d a g a i n s t t h e 105 KD p r o t e i n , which have

n e u t r a l i z i n g a c t i v i t y , have been shown t o b ind t o l o g a r i t h m i c growth1 fs

phase P. h a e m o l y t i ca c e l l s u s i n g i n d i r e c t immunof l uores cence . These

a n t i b o d i e s a l s o bound t o a 95 KD p r ima r y p r o t e o l y t i c breakdown p r od u c t

i d e n t i f i e d by i mmunob l o t t i ng and i t was u n c l e a r which o f t h e s e

p r o t e i n s was l a b e l e d in i mmunof l uor e sce nce . Othe r lower m o l e c u l a r

14 15we i g h t forms have a l s o been d e t e c t e d . s P r o t e o l y t i c d e g r a d a t i o n o r

d e n a t u r a t i o n o f t h e c y t o t o x i n e p i t o p e r e s p o n s i b l e f o r n e u t r a l i z a t i o n

c ou l d be r e s p o n s i b l e f o r t h e l a ck o f nMcAb r e a c t i v i t y in ELISA.

Cy t o t ox i n a c t i v i t y was a l s o n e u t r a l i z e d by s e r a f rom mice

immunized wi t h e i t h e r P. h a e m o l y t i ca s e r o t y p e 1 c a p s u l a r m a t e r i a l o r

c y t o t o x i n (Tab l e 1 ) . T h e r e f o r e , monoclonal a n t i b o d i e s deve l oped

a g a i n s t c a p s u l a r m a t e r i a l , s p e c i f i c f o r 1i p o p o l y s a c c h a r i d e ,

p a n - p a s t e u r e l l a c a r b o h y d r a t e , c a p s u l a r p o l y s a c c h a r i d e , and a 29 KD

p r o t e i n ( Ch a p t e r 1) were e v a l u a t e d in t h e MTT c o l o r i m e t r i c a s s a y .

a Maheswaran SK, U n i v e r s i t y o f Mi n ne s o t a , S t . Pau l : Pe r s ona l

communica t ion 1987.

67

These a n t i b o d i e s were i n c a p a b l e o f c y t o t o x i n n e u t r a l i z a t i o n and d i d not

a f f e c t n e u t r o p h i l r e d u c t i o n o f MTT dye in r e s po n s e t o a n t i g e n s in t h e

c y t o t o x i n p r e p a r a t i o n ( F i g u r e 2 ) . A p p a r e n t l y , t h e component a n t i g e n s

i d e n t i f i e d o r t h e i r a n t i g e n - a n t i b o d y complexes have l i t t l e e f f e c t on

t h e r e s p o n s e o f n e u t r o p h i l s in t h e a s s a y .

The main a d v a n t a g e s o f t h e MTT c o l o r i m e t r i c a s s a y were speed and

s e n s i t i v i t y , a l l o w i n g a l a r g e number o f samples t o be e v a l u a t e d f o r

c y t o t o x i n n e u t r a l i z a t i o n . These f e a t u r e s a l l owed e f f i c i e n t s c r e e n i n g

of hybridoma c u l t u r e d s u p e r n a t e s n e c e s s a r y t o d e t e c t monoclonal

a n t i b o d y c a p a b l e o f n e u t r a l i z i n g P. ha e mol y t i ca c y t o t o x i n . The use o f

t h e a s s a y was f u r t h e r ex t ende d t o d e t e r mi n e t h e e x t e n t o f c y t o t o x i n

n e u t r a l i z a t i o n by p e r i t o n e a l a s c i t i c f l u i d s and t h e e f f e c t o f d i f f e r e n t

a n t i b o d i e s and combi na t i ons on n e u t r o p h i l r e s p o n s e . The r e s u l t s o f t he

a s s ay were m i c r o s c o p i c a l l y and g r o s s l y a p p a r e n t , a i d i n g in r a p i d

q u a l i t a t i v e a s s e s s m e n t . The MTT c o l o r i m e t r i c a s s a y i s a r a p i d ,

s e n s i t i v e , and v e r s i t i l e means o f s t u d y i n g bovine n e u t r o p h i l

i n t e r a c t i o n w i t h b a c t e r i a l t o x i n s , a n t i g e n s , and s p e c i f i c a n t i b o d i e s .

BIBLIOGRAPHY

1. J ensen R, P i e r s o n RE, Braddy PM, e t a l . Sh ipp ing f e v e r pneumonia i n y e a r l i n g f e e d l o t c a t t l e . J Am Vet Med Assoc 1976;169: 500-506 .

2. Rehmutul l a AJ, Thompson RG. A revi ew o f t h e l e s i o n s in s h i p p i n g f e v e r o f c a t t l e . Can Vet J 1981; 2 2 : 1 - 8 .

3. C o l l i e r JR. S i g n i f i c a n c e o f b a c t e r i a in bov ine r e s p i r a t o r yd i s e a s e . J Am Vet Med Assoc 1968;153:1645-1651.

4. Benson ML, Thomson RG, V a l l i VEO. The bovine a l v e o l a r macrophageI I . In v i t r o s t u d i e s w i t h P a s t e u r e l l a h a e m o l y t i ca . Can J Comp Med 1978;42 : 368-369 .

5. Ba l uyn t CS, Simonson RR, Bemrick WJ, Maheswaran SK. I n t e r a c t i o n o f P a s t e u r e l l a h a e m o l y t i c a w i t h bov ine n e u t r o p h i l s :I d e n t i f i c a t i o n and p a r t i a l c h a r a c t e r i z a t i o n o f a c y t o t o x i n . Am J Vet Res 1981;42 : 1920-1926 .

6. Kaeh le r KL, Markham RJF, Muscopla t CC, Johnson DW. Evidence f o r t h e s p e c i e s s p e c i f i c i t y in t h e c y t o c i d a l e f f e c t s o f P a s t e u r e l l a h a e m o l y t i c a . I n f e c t Immun 1980 ;30 : 615-616 .

7. Slocombe RF, Derksen FJ , Robinson NE, e t a l . I n t e r a c t i o n o f co l ds t r e s s and P a s t e u r e l l a ha e mol y t i ca in t h e p a t h o g e n e s i s o f pneumonic p a s t e u r e l l o s i s in c a l v e s : Method o f i n d u c t i o n andhema t o l og i c and p a t h o l o g i c c hange s . Am J Vet Res 1984;45 : 1757-1763 .

8. Walker RD, Hopkins FM, S h u l t z TW, e t a l . Changes in l e u k o c y t e p o p u l a t i o n s in pulmonary l a vage f l u i d s o f c a l v e s a f t e r i n h a l a t i o n o f P a s t e u r e l l a h a e m o l y t i c a . Am J Vet Res 1985;46:2429-2433.

9. Slocombe RF, Malark J , I n g e r s o l R, Derkson FJ , e t a l . Impor t ance o f n e u t r o p h i l s i n t h e p a t h o g e n e s i s o f a c u t e pneumonic p a s t e u r e l l o s i s in c a l v e s . Am J Vet Res 1985; 46: 2253-2257.

10. B r i e d e r MA, Walker RD, Hopkins FM, e t a l . Pulmonary l e s i o n sinduced by P a s t e u r e l l a h a e m o l y t i ca in n e u t r o p h i l s u f f i c i e n t andn e u t r o p h i l d e f i c i e n t c a l v e s . Can J Vet Res 1988 ;52 : 205-209 .

11. Cho HJ, Bohae JG, Yates WDG, Ohmann HB. A n t i c y t o t o x i n a c t i v i t y o f bovine s e r a and body f l u i d s a g a i n s t P a s t e u r e l l a haemol y t i ca A- l c y t o t o x i n . Can J comp Med 1984;48 : 151-155 .

12. Gent ry MJ, Confer AW, P a n c i e r a RJ. Serum n e u t r a l i z a t i o n o f c y t o t o x i n from P a s t e u r e l l a h a e m o l y t i c a , s e r o t y p e 1 and r e s i s t a n c e t o e x p e r i m e n t a l bov i ne pneumonic p a s t e u r e l l o s i s . Vet Immunol Immunopathol 1985 ; 9 : 239 - 250 .

13. Shewen PE, Wi l k i e BN. P a s t e u r e l l a haemol y t i ca c y t o t o x i nn e u t r a l i z i n g a c t i v i t y in s e r a from O n t a r i o b e e f c a t t l e . Can JComp Med 1983 ;47 : 497 - 498 .

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69

14. Himmel ME, Yates MD, Lauerman LH, S q u i r e PG. P u r i f i c a t i o n and p a r t i a l c h a r a c t e r i z a t i o n o f a macrophage c y t o t o x i n from P a s t e u r e l l a h a e m o l y t i c a . Am J Vet Res 1982 ; 43 : 764-767 .

15. Mosier DA, L e s s l e y BA, Confer AW, e t a l . Chromatographic s e p a r a t i o n and c h a r a c t e r i z a t i o n o f P a s t e u r e l l a ha e mol y t i ca c y t o t o x i n . Am J Vet Res 1986;47 : 2233-2241 .

16. Chang YF, Young R, Pos t D, S t r uc k DK. I d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e P a s t e u r e l l a h a e m o l y t i ca l e u k o t o x i n . I n f e c t Immun 1987;55 : 2348-2354 .

CHAPTER 4

STRUCTURAL CHANGES IN BOVINE NEUTROPHILS EXPOSED TO

PASTEURELLA HAEMOLYTICA SEROTYPE 1 CYTOTOXIN

SUMMARY

P a s t e u r e l l a h a e m o l y t i c a s e r o t y p e 1 (Ph-1) c y t o t o x i n was produced

in a d e f i n e d medium, c o n c e n t r a t e d by u l t r a f i l t r a t i o n and used t o

c h a r a c t e r i z e s t r u c t u r a l changes in i s o l a t e d bovine p e r i p h e r a l blood

n e u t r o p h i l s . N e u t r o p h i l s were exposed t o Ph-1 c y t o t o x i n o ve r v a r i o u s

t ime i n t e r v a l s and c o n c e n t r a t i o n s f o r exa mi na t i on u s i n g l i g h t and

t r a n s m i s s i o n e l e c t r o n mi c r os copy . Ne u t r oph i l c y t o t o x i c i t y was

obs e r ved t o i n v o l v e c e l l p o l a r i z a t i o n wi t h uropod f o r m a t i o n , plasma

membrane d e f e c t s , and d i f f e r e n t i a l i n t r a c e l l u l a r d e g r a n u l a t i o n

p r o g r e s s i n g t o compl e t e d e g e n e r a t i o n and l y s i s w i t h i n 20 min.

I d e n t i c a l c y t o t o x i c changes were noted t h r o u g ho u t bo th p a r am e t e r s

s u g g e s t i n g c y t o t o x i c i t y i s both t ime and c o n c e n t r a t i o n depende n t .

INTRODUCTION

Bovine pneumonic p a s t e u r e l l o s i s o r s h i p p i n g f e v e r i s a s e v e r e

f i b r i n o u s pneumonia c a u s i n g ma jor economic l o s s t o t h e f e e d l o t c a t t l e

1 2i n d u s t r y . ’ Al though numerous p r e d i s p o s i n g f a c t o r s a r e t h o ug h t t o

i n f l u e n c e t h e p a t h o g e n e s i s o f t h i s d i s e a s e , i n f e c t i o n o f t h e lower

r e s p i r a t o r y t r a c t w i t h P a s t e u r e l l a h a emol y t i ca s e r o t y p e 1 (Ph-1) i s

t h e ma jo r caus e o f pneumonia and de a t h a s s o c i a t e d wi t h bov ine

1-4r e s p i r a t o r y d i s e a s e . P. h a emol y t i ca has been shown t o produce a

p r o t e i n c y t o t o x i n ( l e u k o t o x i n ) which i s t o x i c f o r bov ine a l v e o l a r

5-7ma crophages , p e r i p h e r a l b lood mononuc lear c e l l s , and n e u t r o p h i l s .

70

71

Thi s c y t o t o x i n may c o n t r i b u t e t o t h e p a t h o g e n e s i s o f pneumonic

p a s t e u r e l l o s i s by a l t e r i n g c e l l u l a r lung d e f e n s e mechanisms and

c a u s i n g n e u t r o p h i l l y s i s t h a t r e l e a s e s i n f l ammat ory m e d i a t o r s .

The c y t o t o x i n o f P. h a e m o l y t i c a was i n i t i a l l y c h a r a c t e r i z e d as a

high m o l e c u l a r w e i g h t , h e a t s e n s i t i v e p r o t e i n t h a t was l i b e r a t e d

8 9d u r i n g l o g a r t h mi c phase g r owt h . * Cy to t o x i n was l a t e r shown t o cause

c y t o c i d a l e f f e c t s in r umi nan t l e u k o c y t e s , b u t d i d no t a d v e r s e l y a f f e c t

nonrumi nan t c e l l s . A l l s e r o t y p e s o f P. h a e m o l y t i ca produce

c y t o t o x i n which i s immunogenic and n e u t r a l i z a b l e by a n t i b o d y from a

12-15v a r i e t y o f s o u r c e s . High serum a n t i b o d y c y t o t o x i n n e u t r a l i z i n g

t i t e r s have been c o r r e l a t e d wi t h r e s i s t a n c e t o e x p e r i m e n t a l l y induced

15-17pneumonic p a s t e u r e l l o s i s . R e c e n t l y , t h e c y t o t o x i n has been

c l on e d and e x p r e s s e d in E s c h e r i c h i a c o l i a l l o w i n g i t s i d e n t i f i c a t i o n ,

g e n e t i c , and b i oc he mi c a l p r o p e r t i e s t o be more e x t e n s i v e l y

19-21c h a r a c t e r i z e d . However, l i t t l e i s known a bou t t h e e f f e c t o f

c y t o t o x i n a t t h e c e l l u l a r l e v e l . Seve r a l methods have been deve l oped

t o q u a n t i t a t e c y t o t o x i n a c t i v i t y , u s i n g rumi nan t c e l l s , which i n c l u d e s

51v i t a l d y e s , chromium r e l e a s e , l u mi n o l - d e p e n de n t

c h e m i l u m i n e s c e n c e - i n h i b i t i o n , and m i c r o s c o p y . ^ " * 7 These s t u d i e s have

i n d i c a t e d t h a t t h e d e g r e e o f s t r u c t u r a l , f u n c t i o n a l and m e t a b o l i c

e f f e c t s a r e r e l a t e d t o c y t o t o x i n dose and ex p o s u r e t i me .

The s e v e r e f i b r i n o u s bronchopneumonia produced by P. h a e m o l y t i ca

i s c h a r a c t e r i z e d by t h e a c c u m u l a t i o n o f p r e d o m i n a t e l y n e u t r o p h i l s in

22 23a l v e o l a r s p a c e s . * N e u t r o p h i l s a r e c o n s i d e r e d i m p o r t a n t in t h e

p a t h o g e n e s i s no t o n ly b e ca us e o f t h e i r i n t e n s e pulmonary a c c u m u l a t i o n ,

b u t a l s o be c a use o f t h e i r a b i l i t y t o c l e a r b a c t e r i a and r e l e a s e

i n f l ammat o r y m e d i a t o r s . C y t o t o x i n i s r e c o g n i z e d t o d e s t r o y t h e

7 ftp h a g o c y t i c c a p a b i l i t i e s o f n e u t r o p h i l s and l e ad t o c e l l l y s i s . *

Upon u n c o n t r o l l e d d e g r a n u l a t i o n o r l y s i s , n e u t r o p h i l s r e l e a s e p o t e n t

oxygen depende n t and i n d e p e n d e n t m i c r o b i o c i d a l f a c t o r s which can

24-27f u r t h e r damage pulmonary t i s s u e and e x a c e r b a t e d i s e a s e .

L i k e wi s e , s t i m u l a t e d n e u t r o p h i l s m o b i l i z e and m e t a b o l i z e a r a c h a d o n i c

2fta c i d t o form many i m p o r t a n t i n f l ammat ory m e d i a t o r s . The i n t e r a c t i o n

o f _P. h a e m o l y t i c a c y t o t o x i n wi t h bov ine n e u t r o p h i l s i s c l e a r l y an

i m p o r t a n t o c c u r r e n c e in t h e p a t h o g e n e s i s o f pneumonic p a s t e u r e l l o s i s

and t h e t o p i c o f many i n v e s t i g a t i o n s . However, t h e mechanism o f

c y t o t o x i n ' s c y t o c i d a l e f f e c t s on n e u t r o p h i l s has remained o bs cu r e .

To ga i n a b e t t e r u n d e r s t a n d i n g o f c y t o t o x i n ' s mechanism o f

n e u t r o p h i l c y t o t o x i c i t y and hence i t s r o l e in t h e p a t h o g e n e s i s o f

pneumonic p a s t e u r e l l o s i s a f u r t h e r c h a r a c t e r i z a t i o n o f t h e t o x i n s

i n t e r a c t i o n w i t h n e u t r o p h i l s i s needed. The pu r poses o f t h i s s t u d y

were t o m i c r o s c o p i c a l l y c h a r a c t e r i z e t h e s t r u c t u r a l changes in bov ine

n e u t r o p h i l s exposed t o P. ha e mol y t i ca c y t o t o x i n o ve r d i f f e r e n t t i me

i n t e r v a l s and c y t o t o x i n c o n c e n t r a t i o n s .


P. h a e m o l y t i c a c y t o t o x i n :

C y to t o x i n from P a s t e u r e l l a h a e mo l y t i ca b i o t y p e A, s e r o t y p e 1 was

p roduced in a d e f i n e d medium, c o n c e n t r a t e d by u l t r a f i l t r a t i o n ( 1 5 : 1 ) ,

and q u a n t i t a t e d u s i n g bov i ne n e u t r o p h i l s as t a r g e t c e l l s in a

c o l o r i m e t r i c a s s a y as p r e v i o u s l y d e s c r i b e d ( Chap t e r 2 ) . Cy t o t o x in

d i l u t i o n s were per formed i n D u l b e c c o ' s PBS (pH 7 . 4 ) .

73

Bovine n e u t r o p h i l s and ex p o s u r e t o c y t o t o x i n :

Bovine n e u t r o p h i l s were i s o l a t e d from p e r i p h e r a l b lood by double

d i s c o n t i n u o u s d e n s i t y g r a d i e n t c e n t r i f u g a t i o n ( Chap t e r 2 ) . I s o l a t e d

n e u t r o p h i l s were suspended in D u l b ec c o ' s PBS t o a f i n a l c o n c e n t r a t i o n

o f 5X106 c e l l s / m l . Appr ox i ma t e l y 5-10% o f t h i s c e l l p o p u l a t i o n was

mononuc lear c e l l s wich c o - p u r i f i e d wi t h t h e n e u t r o p h i l s . A l i q u o t s o f

t h e n e u t r o p h i l s u sp e n s i o n ( 2 . 0 mi s ) were exposed t o 1 . 0 ml volumes o f

a 1 :8 d i l u t i o n o f c o n c e n t r a t e d c y t o t o x i n f o r 5 min i n t e r v a l s t o 20

min in t h e t empora l s t u d y . In t h e dose s t u d y , 2 . 0 ml a l i q u o t s o f t h e

n e u t r o p h i l s u sp e n s i o n were exposed t o 1 . 0 ml volumes o f a 2 - f o l d

c y t o t o x i n d i l u t i o n s e r i e s f o r 20 min. To h a l t c y t o t o x i c i t y , c e l l s

were f i x e d o v e r n i g h t w i t h 3 . 0 mis 0.2M sodium c a c o d y l a t e b u f f e r

(pH7.2) c o n t a i n i n g 3% g l u t a r a l y d e h y d e . Cont ro l n e u t r o p h i l s were

exposed t o D u l b e c c o ' s PBS o r RPMI-1640 c o n t a i n i n g 10 mM sodium

b i c a r b o n a t e and L - g l u t a m i n e under i d e n t i c a l c o n d i t i o n s . D u p l i c a t e

samples were p r e p a r e d f o r l i g h t and t r a n s m i s s i o n e l e c t r o n mi c roscopy .

The t empora l and dose s t u d i e s were per formed as s e p a r a t e e x p e r i m e n t s .

Microscopy o f N e u t r o p h i l s :

L i gh t mi c roscopy was pe r fo rmed on wet mounted s l i d e s s t a i n e d wi t h

e i t h e r new me t hy l ene b l u e o r t r y p a n b l u e . S l i d e s were examined a t

400x and lOOOx m a g n i f i c a t i o n . P r e p a r a t i o n o f t h e n e u t r o p h i l s f o r

t r a n s m i s s i o n e l e c t r o n mi c ros copy was a d ap t ed from t h e e n c a p s u l a t i o n

29method o f K e l l e n b e r g e r , e t a l . B r i e f y , t h e c e l l s were g e n t l y washed

w i t h f i x a t i v e , s t a i n e d wi t h 1% OsO^, and r ewashed. The osmium f i x e d

p e l l e t was e n c a p s u l a t e d w i t h 3% a g a r o s e c o n t a i n i n g u r any l a c e t a t e and

g l u t a r a l d e h y d e . Fo l lowing e n c a p s u l a t i o n , samples were a l co h o l

74

d e hy d r a t e d and i n f i l t r a t e d wi t h e p o n / a r a l d i t e and a l l owed t o

po l ymer i ze o v e r n i g h t a t 60C. Samples were s e c t i o n e d a t 60-80 nm and

p i c ke d up on 200-300 mesh coppe r g r i d s . The g r i d s were t he n s t a i n e d

30 31wi t h u r any l a c e t a t e and l e a d n i t r a t e s o l u t i o n s . ’ Gr ids were

examined u s i n g a Ze i s s EM 10-A t r a n s m i s s i o n e l e c t r o n mi c r os c ope .

RESULTS

Li gh t Microscopy:

L i g h t mi c roscopy o f wet mounted c o n t r o l n e u t r o p h i l s , no t exposed

t o c y t o t o x i n , d e m o n s t r a t e d b a s i c a l l y round and h i g h l y l i g h t r e t r a c t i l e

c e l l s . The m u l t i l o b e d n u c l e i o f t h e s e c e l l s were c e n t r a l l y l o c a t e d

and t h e c y t o p l a s mi c g r a n u l e s appea r ed e v e n l y d i s t r i b u t e d . C e l l u l a r

a g g r e g a t i o n was no t e v i d e n t g r o s s l y o r m i c r o s c o p i c a l l y . Cont ro l

n e u t r o p h i l s r e a d i l y e xc l ud e d t r y p a n b l u e dye.

Fo l lowing a f i v e min e xpos u r e t o c y t o t o x i n , t h e m a j o r i t y o f c e l l s

l o s t t h e i r r e t r a c t i l i t y and round morphology becoming oval t o

e l o n g a t e d . P rominan t c y t o p l a s m i c p r o j e c t i o n s deve l oped and

c y t o p l a s mi c g r a n u l e s were obs e r ve d t o be p o l a r i z e d t o t h e o p p o s i t e

s i d e f rom t h e s e p r o j e c t i o n s . Plasma membrane damage was conf i rmed by

t h e up t a ke o f t r y p a n b l u e dye . C e l l u l a r a g g r e g a t i o n was a p p a r e n t , bu t

no t pronounced .

A f t e r t e n min ex p o s u r e t o c y t o t o x i n , c e l l morphology became more

oval t o e l o n g a t e d . Large c y t o p l a s mi c p r o j e c t i o n s ex t ende d from c e l l

s u r f a c e s . Many c e l l s were r e c o gn i z e d t o have plasma membrane d e f e c t s

l a r g e enough f o r g r a n u l e s and n u c l e i t o e x i t t h e c y t op l a sm th r ough

t h e s e p r o j e c t i o n s . C e l l u l a r a g g r e g a t i o n was more prounounced and most

c e l l s r e a d i l y took up t r y p a n b l u e dye.

75

By 15 t o 20 min n e u t r o p h i l c y t o t o x i c i t y was j udge d as c o m p l e t e ,

on l y a g g r e g a t e d g h o s t c e l l s and c e l l u l a r d e b r i s r emained . Al l

n e u t r o p h i l s were s e v e r e l y a f f e c t e d , h i g h l y i r r e g u l a r c e l l shapes

c o n t a i n i n g few i n t r a c y t o p l a s m i c g r a n u l e s were o b s e r v a b l e . No

d i f f e r e n c e s were no t e d between t h e t empora l and dose s t u d i e s , s i m i l a r

mor pho l og i a l changes were o bs e r ved in s h o r t e xpos u r e s t o h igh

c y t o t o x i n c o n c e n t r a t i o n s . Exposure o f c e l l s t o h igh c y t o t o x i n

c o n c e n t r a t i o n s (<1 : 8 d i l u t i o n ) f o r p ro l onged t i mes (>20 min) caused

compl e t e n e u t r o p h i l l y s i s . L i g h t mi c r os co p i c ex a mi n a t i o n o f t h e

n e u t r o p h i l d e b r i s r e v e a l e d low numbers o f i n t a c t mononuc lear c e l l s

which were a p p a r e n t l y r e s i s t a n t t o c y t o t o x i n a t t h e d e s c r i b e d t i mes

and c o n c e n t r a t i o n s .

Tr an s mi s s i on e l e c t r o n mi c roscopy :

T r a n s mi s s i on e l e c t r o n mi c roscopy o f normal n e u t r o p h i l morphology

r e v e a l e d c e l l s w i t h a round shape c o n t a i n i n g a c e n t r a l l y l o c a t e d

m u l t i l o b e d n u c l e u s , c o n f i r m i n g l i g h t mi croscopy o b s e r v a t i o n s . The

c e l l s u r f a c e s were u n i f o r m l y i n v o l u t e d and c y t o p l a s mi c g r a n u l e s were

a b u nd an t , e v e n l y d i s t r i b u t e d , and d i s p l a y e d e l l i p s o i d a l t o round

p r o f i l e s , i n d i c a t i n g rod t o s p h e r i c a l s h a p e s . D i f f e r e n c e s in s i z e and

e l e c t r o n d e n s i t y o f t h e g r a n u l e s were a l s o e v i d e n t . Mi toc hondr i a were

e a s i l y d i f f e r e n t i a t e d from t h e g r a n u l e s based on t h e s e c h a r a c t e r i s t i c s

( F i g u r e I V . 1) .

76

Fi g u r e I V . 1. T r a n s mi s s i on e l e c t r o n mi c r ogr aph o f a normal bov ine

n e u t r o p h i l . Note round s h a p e , c e n t r a l l y l o c a t e d n u c l e u s , and wel l

d e f i n e d , e v e n l y d i s t r i b u t e d g r a n u l e s o f v a r i o u s s i z e s , s h a p e s , and

d e n s i t i e s . X10.000.

Fo l lowing a 5 min ex p o s u r e t o c y t o t o x i n ( 1 : 8 d i l u t i o n )

c o n s i d e r a b l e s t r u c t u r a l changes o c c u r e d . Prominant uropods deve l oped

from c e l l s u r f a c e s ( F i g u r e I V . 2 ) .

77

psSiip''

Fi g u r e I V . 2. E l e c t r o n mi c r og r aph o f a bov i ne n e u t r o p h i l exposed t o

c y t o t o x i n ( 1 : 8 d i l u t i o n ) f o r 5 min. Note e l o n g a t e d s h a p e , e c c e n t r i c

n u c l e u s , p o l a r i z e d g r a n u l e s , and p r omi nan t c y t o p l a s m i c p r o j e c t i o n

( u r op od ) . Granu l es a p p e a r l a r g e r , l e s s d i s t i n c t and more e l e c t r o n

d e n s e . X10,000.

Membranes l i m i t i n g t h e s e uropods had a moth e a t e n a p p e a r e n c e ,

i n d i c a t i n g damage which was conf i rmed by t h e i n a b i l i t y t o e x c l u d e

t r y p a n b lu e dye in l i g h t mi c r os c o p y . The c e l l s became p o l a r i z e d , in

t h a t n u c l e i were s h i f t e d t o an e c c e n t r i c s i t e and g r a n u l e s were

78

m o b i l i z e d t o t h e o p p o s i t e s i d e from t h e uropods l i m i t e d by damaged

membrane ( F i g u r e I V . 2 ) . Al l g r a n u l e s appe a r e d l a r g e r , more e l e c t r o n

d e n s e , and t h e i r s h a r p n e s s became l e s s d i s t i n c t . Higher m a g n i f i c a t i o n

o f t h e s e e n l a r g e d f uz z y g r a n u l e s r e v e a l e d a l t e r a t i o n o f t h e i r

i n t e g r i t y c h a r a c t e r i z e d by changes r e m i n i s c e n t o f smal l v a c u o l a r o r

t u b u l a r f o r ma t i o n s ( F i g u r e IV. 3 ) .

F i g u r e I V . 3. A l t e r a t i o n o f bov ine n e u t r o p h i l g r a n u l e i n t e g r i t y

o c c u r r i n g a t 5 min c y t o t o x i n ( 1 : 8 d i l u t i o n ) e xpos u r e s u g g e s t i v e o f

smal l v a c u o l a r o r t u b u l a r f o r m a t i o n s . X60,000.

79

A f t e r 10 min e xpos u r e t o c y t o t o x i n l a r g e r and b r o a d e r uropods

l i m i t e d by i n c o mp l e t e membranes were commonly obs e rved ( F i g u r e I V . 4 ) .

F i g u r e I V . 4. Bovine n e u t r o p h i l w i t h 2 l a r g e , broad c y t o p l a s m i c

p r o j e c t i o n s (u ropods ) t y p i c a l o f c e l l s f o l l o w i n g a 10 min c y t o t o x i n

( 1 : 8 d i l u t i o n ) e x p o s u r e . The plasma membrane l i m i t i n g t h e uropods has

a moth e a t e n a pp e a r a n c e and l a r g e dense p e r s i s t e n t g r a n u l e s c o n t a i n i n g

f o l d e d c o r d l i k e s t r u c t u r e s domina te in t h e cy t op l a s m. X10.000.

80

A d r a m a t i c d e c r e a s e in g r a n u l e numbers was obs e r ved and t h e r ema i n i ng

g r a n u l e s were much l a r g e r and more e l e c t r o n de ns e . The r emain ing

l a r g e g r a n u l e s c o n t a i n e d e l e c t r o n dense f o l d e d c o r d - l i k e s t r u c t u r e s

which e x h i b i t e d a s t r i k i n g p e r i o d i c i t y a t h i g h e r m a g n i f i c a t i o n s

( F i g u r e IV.5A and B).

‘ . -

'J .

Fi g u r e I V . 5. T r a n s mi s s i on e l e c t r o n mi c r og r aphs showing g r a n u l e s i n

n e u t r o p h i l s exposed t o c y t o t o x i n ( 1 : 8 d i l u t i o n ) f o r 10 min. (A)

f o l d e d c o r d - l i k e i n t r a g r a n u l a r s t r u c t u r e s o f i n c r e a s e d e l e c t r o n

d e n s i t y . X31.500. (B) Higher m a g n i f i c a t i o n o f F i g . IV.5A

d e m o n s t r a t i n g p e r i o d i c i t y . X63.000.

81

At 15 min, t h e l a r g e broad uropods commonly p r e s e n t in t h e 10 min

e x p o s u r e s , were r a r e l y o b s e r v e d . Round t o oval c e l l shapes wi t h a

h i g h l y i r r e g u l a r s u r f a c e and i nc ompl e t e membranes p r edomi na ted ( F i g u r e

I V . 6 ) .

F i g u re I V . 6. Ne u t r o p h i l f o l l o w i n g a 15 min c y t o t o x i n ( 1 : 8 d i l u t i o n )

e x p o s u r e . Note l a ck o f a p romi nan t uropod and plasma membrane

i n t e g r i t y . There i s a d e c r e a s e in t he number o f p e r s i s t e n t g r a n u l e s

which a r e forming c o n c e n t r i c a l l y l a mi n a t ed s t r u c t u r e s ( a r r o w ) .

X10.000.

82

The number o f l a r g e , d e n s e , p e r s i s t e n t g r a n u l e s d e c r e a s e d f u r t h e r and

many were in t h e p ro c e s s o f forming mye l in f i g u r e s . These

c o n c e n t r i c a l l y l a mi n a t ed s t r u c t u r e s w i t h a c e n t r a l e l e c t r o n d e n s i t y

were obs e r ve d t o r e p l a c e t h e l a r g e dense g r a n u l e s p e r s i s t i n g a t 10

min c y t o t o x i n e xpos u r e ( F i g u r e I V . 7 ) .

fL*

F i g u r e I V . 7. C o n c e n t r i c a l l y l a mi n a t ed mye l in f i g u r e formed from t h e

l a r g e p e r s i s t e n t g r a n u l e s . Note l a ck o f i n t e g r i t y and moth e a t e n

a pp e a r a n c e o f plasma membrane.

83

At 20 min, c e l l shape remained round t o oval wi th an i r r e g u l a r

s u r f a c e , r e s e mbl ing t h e 15 min e x p o s u r e . However, many c y t o p l a s mi c

v a c u o l e s were p r e s e n t t h a t c o n t a i n e d a f l o c c u l a n t m a t e r i a l and few t o

no g r a n u l e s remained ( F i g u r e I V . 8 ) .

F i g u r e IV. 8. Typi ca l n e u t r o p h i l a t 20 min p o s t c y t o t o x i n ( 1 : 8

d i l u t i o n ) e x p os u r e . There i s a l a ck o f membrane i n t e g r i t y and

c y t o p l a s m i c g r a n u l e s . Numerous vac u o l e s c o n t a i n i n g a f l o c c u l a n t

m a t e r i a l a r e p r e s e n t in t h e c y t o p l a s m. X10,000.

84

The n u c l e i o f t h e r e ma i n i ng c e l l s l o s t t h e i r t i g h t l y wound form and

became s wo l l e n .

At a low c y t o t o x i n c o n c e n t r a t i o n (1 :512 d i l u t i o n ) f o r 20 min,

mos t n e u t r o p h i l s formed uropods and became p o l a r i z e d . Nucle i s h i f t e d

t o an e c c e n t r i c l o c a t i o n and g r a n u l e s were a lways p r e s e n t a t a s i t e

o p p o s i t e f rom t h e c y t o p l a s mi c e x t e n s i o n s . Granule morphology appea r ed

normal i n t h a t s i z e s h a p e , and d e n s i t y remained unchanged ( F i g u r e

I V . 9 ) .

F i g u re I V . 9. T r a n s mi s s i on e l e c t r o n mi c rograph o f a bov ine n e u t r o p h i l

exposed t o a 1:512 c y t o t o x i n d i l u t i o n f o r 20 min. The c e l l s d i s p l a y s

a p o l a r i z e d morphology and 2 uropods a t t h e p o s t e r i o r a s p e c t . Plasma

membrane i n t e g r i t y and g r a n u l e morphology remain normal .

85

Plasma membranes were no t obse r ved t o be damaged us i ng e l e c t r o n

mi c roscopy and ve ry few n e u t r o p h i l s took up t r y pa n b l u e a t t h i s

c y t o t o x i n c o n c e n t r a t i o n i n l i g h t mi c r oscopy .

Doubl ing t h e dose o f c y t o t o x i n (1 :256 d i l u t i o n ) produced

n o t i c e a b l e changes in t h e plasma membrane and g r a n u l e s . Prominant

uropods d e v e l o p e d , i n d e n t i c a l t o t h o s e o c c u r r i n g in t h e t empora l

s t u d y , which were l i m i t e d by a membrane wi t h a moth e a t e n a p pe a r a n c e .

Granu l es appea r ed e n l a r g e d , l e s s c l e a r l y d e m a r ca t e d , and more e l e c t r o n

d en s e . These s t r u c t u r a l changes were i n d i s t i n g u i s h a b l e from t h o s e

o c c u r r i n g a t 5 min in t h e t empora l s t u d y ( F i g u r e I V . 10) .

F i g u r e I V . 10. N e u t r o p h i l exposed t o a 1 :2 5 6 c y t o t o x i n d i l u t i o n f o r 20

m in . Note i d e n t i t y w i t h F i g u r e 2 . X 1 0 . 0 0 0 .

86

Higher c o n c e n t r a t i o n s o f c y t o t o x i n ( 1 : 6 4 d i l u t i o n ) produced l a r g e

broad uropods i n c o m p l e t e l y l i m i t e d by a plasma membrane. Granule

number d e c r e a s e d and t h e p e r s i s t e n t ones became s w o l l e n , more e l e c t r o n

d e n s e , and c o n t a i n e d t h e f o l d e d c o r d - l i k e s t r u c t u r e s ( F i g u r e I V . 11) .

F ig u r e I V . 11. N e u t r o p h i l exposed t o a 1 :6 4 c y t o t o x i n d i l u t i o n f o r 20

m in . Note i d e n t i t y w i t h F i g u r e 4 . X 1 0 , 0 0 0 .

87

These s t r u c t u r a l changes were i d e n t i c a l t o t h o s e o c c u r i n g a t 10

min e xpos u r e t o a 1:8 c y t o t o x i n d i l u t i o n .

The l a r g e broad uropods o c c u r r i n g d u r in g exposu r e t o a 1:64

c y t o t o x i n d i l u t i o n were no t e v i d e n t i n n e u t r o p h i l s exposed t o 1:32

d i l u t i o n . I n c r e a s i n g c y t o t o x i n c o n c e n t r a t i o n produced round t o oval

shaped c e l l s w i t h an i r r e g u l a r ma rg i n , p a r a l l e l i n g t h o s e changes

t y p i f y i n g a 15 min e xpos u r e t o a 1:8 c y t o t o x i n d i l u t i o n ( F i g u r e

I V . 12) .

F i g u r e I V . 12. N e u t r o p h i l exposed t o a 1 :3 2 c y t o t o x i n d i l u t i o n f o r 20

m in . Note s i m i l a r i t i e s w i t h F ig u r e 6 . X 1 0 , 0 0 0 .

88

No d i f f e r e n c e s were obs e r ve d between a 1:16 and 1:8 c y t o t o x i n d i l u t i o n

in t h e dose s t u d y which were t y p i c a l o f c e l l s in F i gu r e 8. S t r u c t u r a l

changes were r ema r kab l y s i m i l a r t h r o u g h o u t t h e t ime and dose s t u d i e s .

DISCUSSION

Fol lowing s t i m u l a t i o n wi t h c h e m o t a t i c f a c t o r s n e u t r o p h i l s

commonly undergo s t r u c t u r a l changes c h a r a c t e r i z e d by an e l o n g a t e d

32-36p o l a r i z e d form. " P o l a r i z e d n e u t r o p h i l s deve l op l amel l apod ium o r

pseudopods towards t h e l e a d i n g edge o f s t i m u l a t i o n . P o s t e r i o r

c y t o p l a s mi c p r o j e c t i o n s o r uropods form t h e t a i l o f p o l a r i z e d c e l l s

j u s t behi nd t h e n u c l e u s . Dur ing p o l a r i z a t i o n c y t o p l a s mi c g r a n u l e s a r e

m o b i l i z e d a n t e r i o r l y , t owards t h e ps eudopods , f o r d e g r a n u l a t i o n w i t h i n

phagosomes. C e l l u l a r p o l a r i z a t i o n o f membrane r e c e p t o r s and

c o n t r a c t i l e f i l a m e n t s has a l s o been obse r ved t o o c c u r f o l l o w i n g

34 35 37-39c h e m o t a c t i c f a c t o r s t i m u l a t i o n . 5 * Membrane r e c e p t o r s a r e

d i f f u s e l y d i s t r i b u t e d o ve r t h e s u r f a c e o f u n s t i m u l a t e d c e l l s , bu t a r ep C p p p Q

found p r e d om i n a t e l y in t h e uropod o f p o l a r i z e d c e l l s . ’ * A f t e r

c y t o t o x i n e x p o s u r e , n e u t r o p h i l s in t h i s s t u d y were obse r ved t o

e l o n g a t e and assume a p o l a r i z e d o r i e n t a t i o n , s u g g e s t i n g s t i m u l a t o r y

c h e m o t a t i c f a c t o r s a r e p r e s e n t in t h e c y t o t o x i n p r e p a r a t i o n . The

p r e s e n c e o f c h e m o t a c t i c f a c t o r s in c y t o t o x i n p r e p a r a t i o n s cou l d be

r e l a t e d t o t h e e a r l y and i n t e n s e pulmonary n e u t r o p h i l a cc u mu l a t i o n

f o l l o w i n g e x p e r i me n t a l c h a l l e n g e . The c y t o p l a s mi c p r o j e c t i o n s

obs e r ved h e r e i n deve l oped p o s t e r i o r l y wi t h r e s p e c t t o t h e p o l a r i z e d

o r i e n t a t i o n , t e n t a t i v e l y i d e n t i f y i n g t h e s e s t r u c t u r e s as u ropods .

These s t r u c t u r e s b e a r r e ma r ka b l e s i m i l a r i t y t o uropods i nduced by

32-34c h e m o t a c t i c f a c t o r s in o t h e r s t u d i e s . These uropods i n c r e a s e d in

89

s i z e and l o s t membrane i n t e g r i t y as c y t o t o x i n dose and exposu r e t ime

i n c r e a s e d . The d i s r u p t i o n o f t h e uropod plasma membrane i n t e g r i t y

s u g g e s t s a r e c e p t o r me d i a t e d mechanism of c y t o t o x i c i t y s i n c e r e c e p t o r s

a r e known t o be c o n c e n t r a t e d in t h i s a r e a f o l l o w i n g s t i m u l a t i o n . The

c o n t e n t i o n o f a r e c e p t o r me d i a t e d mechanism o f c y t o t o x i c i t y may

e x p l a i n t h e rumi nan t c e l l s p e c i f i c a c t i o n o f c y t o t o x i n .

I n v e s t i g a t i o n s i n t o t h e d e g r a n u l a t i v e p ro c e s s o f human bovine

n e u t r o p h i l s has r e v e a l e d a complex e v e n t in which more t h a n one s i g n a l

i s i n v o l v e d . Calcium i onophor es (A23187) , phorbol m y r i s t a t e a c e t a t e ,

and immune complexes caus e a t i me and dose dependen t r e l e a s e o f

e nzyma t i c a c t i v i t y a s s o c i a t e d wi t h t h e s p e c i f i c g r a n u l e s wh i l e

40-42a z u r o p h i l g r a n u l e s remain c e l l a s s o c i a t e d . Fo l lowing

p h a g o c y t o s i s o r e xpos u r e t o s o l u b l e s t i m u l i , bov ine n e u t r o p h i l s

d i s c h a r g e by t r u e s e c r e t i o n bo th t h e i r l a r g e and s p e c i f i c g r a n u l e s .

43Azurophi l g r a n u l e s a r e f u l l y r e t a i n e d . These f i n d i n g s have

s u p p o r t e d a s e l e c t i v e i n d e p e n d e n t m o b i l i z a t i o n o f c y t o p l a s mi c g r a n u l e s

upon ex p o s u r e t o p a r t i c u l a t e as wel l as s o l u b l e s t i m u l i . This

d i f f e r e n t i a l d e g r a n u l a t i o n was a l s o not ed in n e u t r o p h i l s exposed t o

c y t o t o x i n by t h e p e r s i s t e n c e o f t h e l a r g e dense g r a n u l e s c o n t a i n i n g

t h e c o r d - l i k e s t r u c t u r e s e x h i b i t i n g p e r i o d i c i t y . The d i f f e r e n t i a l

i n t r a c e l l u l a r d e g r a n u l a t i o n o f bov ine n e u t r o p h i l s exposed t o c y t o t o x i n

c ou l d be r e l a t e d t o t h e mechanism o f c y t o t o x i c i t y .

Human n e u t r o p h i l g r a n u l e s have been r e p o r t e d t o c o n t a i n a c e n t r a l

44-46c r y s t a l o f wel l d e f i n e d p e r i o d i c i t y . A d d i t i o n a l l y , bovine

n e u t r o p h i l l a r g e ( t h i r d ) g r a n u l e s a r e a c c e p t e d t o c o n t a i n a c e n t r a l

47c o r e o f m a t r i x m a t e r i a l . The p e r s i s t e n t l a r g e g r a n u l e s d e s c r i b e d in

90

t h i s s t u d y a l s o c o n t a i n e d s u b g r a n u l a r s t r u c t u r e s e x h i b i t i n g

p e r i o d i c i t y . These g r a n u l e s were l a t e r obs e r ved t o form mye l in

f i g u r e s . However, t h e s i g n i f i c a n c e and f u n c t i o n of t h e s e s t r u c t u r e s

remain un d e t e r mi n e d . The number and morphology o f t h e p e r s i s t e n t

g r a n u l e s a r e c o n s i s t a n t w i t h d e s c r i p t i o n s o f t h e l a r g e ( t h i r d )

g r a n u l e s i n bov i ne n e u t r o p h i l s . 4 3 ’47 Thi s l a r g e g r a n u l e i s r e p o r t e d

t o c o n t a i n p r i m a r i l y l a c t o f e r r i n and o t h e r l e s s p romi nan t c a t i o n i c

43p r o t e i n s . I t i s i n t e r e s t i n g t o n o t e t h a t l a c t o f e r r i n and o t h e r i r o n

b i n d i n g compounds may s up p l y a u s a b l e s ou r c e o f i r o n f o r t o x i n o g e n e s i s

48by P. h a e m o l y t i c a . L a c t o f e r r i n r e l e a s e by n e u t r o p h i l s exposed t o

c y t o t o x i n may i n c r e a s e c y t o t o x i n p r o d u c t i o n in v i vo and e x a c e r b a t e

d i s e a s e .

Al l r umi nan t l e u k o c y t e s a r e r e p o r t e d t o be s e n s i t i v e t o

49-52P a s t e u r e l l a c y t o t o x i n . The d i f f e r e n t i a l s u s c e p t i b i l i t y o f bov ine

l e u k o c y t e s t o c y t o t o x i n has been p r e v i o u s l y r e p o r t e d and n e u t r o p h i l s

53were found t o be t h e most s e n s i t i v e . In t h i s s t u d y , a n e u t r o p h i l

p o p u l a t i o n c o n t a i n i n g 5-10% mononuc lear c e l l s was exposed t o an exce s s

o f c y t o t o x i n a c t i v i t y (15 : 1 c o n c e n t r a t e ) f o r p r o l onge d t i mes (>20

min) and r e s i s t a n t c e l l s were i d e n t i f i e d as mononuc lear u s i n g l i g h t

mi c r oscopy ( no t shown) . Our r e s u l t s s u g g e s t t h a t n e u t r o p h i l s a r e more

s e n s i t i v e t o c y t o t o x i n t h a n mononuc lear c e l l s and s u p p o r t s t h e

c o n t e n t i o n o f d i f f e r e n t i a l l e u k o c y t e s u s c e p t i b i l i t y .

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38. Ryan GB, Borysenko J Z , Karnovski MJ. F a c t o r s a f f e c t i n g t h er e d i s t r i b u t i o n o f s u r f a c e bound c o n c a n a v a l i n A on human po l ymor phonuc l ea r l e u k o c y t e s . J Cel l Biol 1974 ; 62 : 351-365 .

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43. Gennaro R, Donald B, H o r i s b e r g e r U, e t a l . A novel t yp e o f c y t o p l a s mi c g r a n u l e in bov ine n e u t r o p h i l s . J Cel l Biol 1983;96 : 1651-1661 .

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45. B r e t o n -G o r i u s J , Reyes F. U l t r a s t r u c t u r e o f human bone marrow c e l l m a t u r a t i o n I n t Rev Cytol 1976; 46 : 251 - 259 .

46. Daems WT. On t h e f i n e s t r u c t u r e o f human n e u t r o p h i l i c l e u ko cy te g r a n u l e s . J U l t r a s t r u c t Res 1968;24 : 343-352 .

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48. Gent ry MJ, Confer AW, Weinberg ED, Homer JT. Cy t o t ox i n ( l e u k o t o x i n ) p r o d u c t i o n by P a s t e u r e l l a h a e m o l y t i c a : Requi rement f o r an i r o n c o n t a i n i n g compound. Am J Vet Res 1986;47 : 1919-1923 .

49. Shewen PE, Wi l k i e BN. Cy t o t ox i n o f P a s t e u r e l l a h a emol y t i ca a c t i n g on bovine l e u k o c y t e s . I n f e c t Immun 1 9 82 ; 35 : 91 - 94 .

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51. S u t h e r l a n d AD. E f f e c t s o f P a s t e u r e l l a ha e mol y t i ca c y t o t o x i n on o v i ne p e r i p h e r a l b lood l e u k o c y t e s and lymphocytes o b t a i n e d from g a s t r i c lymph. Vet Mi c r ob i o l 1985 ; 10 : 431-438 .

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52. Chang YF, Renshaw HW, Mar tens RJ, L i v i n g s t o n CW. P a s t e u r e l l a ha e mol y t i ca l e u k o t o x i n : Chemi luminescen t r e s p o n s e s o f p e r i p h e r a l b lood l e ukocy t e s f rom s e v e r a l d i f f e r e n t mammalian s p e c i e s t o l e u k o t o x i n and o p s o n i n - t r e a t e d l i v i n g and k i l l e d P a s t e u r e l l a ha e mol y t i ca and S t r a p h y l o c o c c u s a u r e u s . Am J Vet Res 1 986 ; 47 : 67 - 74 .

53. O ' B r i en JK, Duffus WPH. P a s t e u r e l l a h a e m o l y t i c a c y t o t o x i n : R e l a t i v e s u s c e p t i b i l i t y o f bov ine l e u k o c y t e s . Vet Mic rob i o l 1987;13 : 321-334 .

CHAPTER 5

PURIFICATION OF THE SEROTYPE 1 SPECIFIC EPITOPE FROM

PASTEURELLA HAEMOLYTICA USING MONOCLONAL ANTIBODY IMMUNOAFFINITY

SUMMARY

A mur ine monoclonal IgM a g g l u t i n a t i n g a n t i b o d y was used in

i mm u n o a f f i n i t y t o p u r i f y t h e s e r o t y p e 1 s p e c i f i c e p i t o p e from

P a s t e u r e l l a h a e m o l y t i ca ( P h - 1 ) . The Ph-1 s p e c i f i c a n t i b o d y was

p r e c i p i t a t e d from p e r i t o n e a l a s c i t i c f l u i d , d i a l y z e d , e v a l u a t e d f o r

a c t i v i t y , and c o v a l e n t l y a t t a c h e d t o CNBr a c t i v a t e d Sepharos e 4B a t

a l k a l i n e pH. R e t e n t i o n o f p u r i f i e d a n t i b o d y a c t i v i t y and c o up l i n g

e f f i c i e n c y t o t h e s u p p o r t m a t e r i a l were g r e a t e r t han 99% when e v a l u a t e d

by ELISA, a g g l u t i n a t i o n , and p r o t e i n d e t e r m i n a t i o n s . KSCN was s e c l e c t e d

as an e l u a n t based upon r e v e r s i b l e d i s s o c i a t i o n o f b a c t e r i a l

a g g l u t i n a t i o n and t i t r a t e d f o r t h e l owes t e f f e c t i v e c o n c e n t r a t i o n .

Immunobead a c t i v i t y was o b s e r ve d m i c r o s c o p i c a l l y by i m m o b i l i z a t i o n o f

e n c a p s u l a t e d Ph-1 and r e v e r s i b l e d i s s o c i a t i o n a f t e r e l u t i o n wi t h 0.4M

KSCN. S p e c i f i c i t y o f i m m o b i l i z a t i o n was conf i rmed u s i n g P. h a e mo l y t i ca

s e r o t y p e s 2 and 5 which were no t bound and by b l o c k i n g Ph-1 b i n d i n g w i t h

homologous c a p s u l a r m a t e r i a l . S a l i n e e x t r a c t a b l e c a p s u l a r m a t e r i a l

from Ph-1 was used as an a n t i g e n s o u r ce and r e a c t e d w i t h an equal

volume o f immunobeads. The immobi l i zed a n t i g e n was s e p a r a t e d and

washed by c e n t r i f u g a t i o n . Fo l lowing e l u t i o n o f t h e Ph-1 s p e c i f i c

e p i t o p e , t h e p r o d u c t was d i a l y z e d and a n a l yz e d u s i n g chemica l and

immunologic methods . The p u r i f i e d Ph-1 s p e c i f i c e p i t o p e c o n t a i n e d no

d e t e c t a b l e p r o t e i n and a t l e a s t o n e - h a l f t h e o r i g i n a l hexosamine

96

97

c o n t e n t . Using d e f i n e d monoclonal a n t i b o d i e s i n ELISA, t i t r a t i o n of

t h e i mmunoa f f i n i t y p r od u c t and o r i g i n a l c a p s u l a r m a t e r i a l r ev e a l e d

s p e c i f i c r e t e n t i o n o f 1i p o p o l y s a c c h a r i d e , p a n - p a s t e u r e l l a c a r b o h y d r a t e ,

and s e r o t y p e 1 c a p s u l a r p o l y s a c c h a r i d e , s u g g e s t i n g e p i t o p e s h a r i n g

among hexosamine a n t i g e n s o f Ph-1.

INTRODUCTION

P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e 1 (Ph-1) i s r e c o g n i ze d as t he

major cause o f s e v e r e f i b r i n o u s pneumonia in c a t t l e t ermed pneumonic

1 2p a s t e u r e l l o s i s o r s h i p p i n g f e v e r . * Al though g r e a t e r t ha n 12 s e r o t y p e s

o f P. h a emol y t i ca have been d e f i n e d , i t i s p r e d o mi n a t e l y s e r o t y p e 1

and l e s s f r e q u e n t l y s e r o t y p e 2 o r P. mu l to c i da which a r e a s s o c i a t e d wi th

t h i s d i s e a s e .

The s e r o t y p e s o f P. h a e m o l y t i ca have been d e f i n e d by e i t h e r

i n d i r e c t h e a m a g g l u t i n a t i o n o r r a p i d p l a t e a g g l u t i n a t i o n u s i n g d i f f u s a b l e

4 5 6s u r f a c e a n t i g e n s or e n c a p s u l a t e d b a c t e r i a r e s p e c t i v e l y . * ’ An

a g e - de p e n d e n t c a p s u l a r m a t e r i a l has been d em o n s t r a t e d on e a r l y

l o g a r i t h m i c g rowt h-phase b a c t e r i a . ® Thi s c a p s u l a r m a t e r i a l i s

r e a d i l y d i f f u s a b l e and e a s i l y removed w i t h o u t a d v e r s e l y a f f e c t i n g

b a c t e r i a l v i a b i l i t y . 7 P o l y s a c c h a r i d e s p r e s e n t in c a p s u l a r m a t e r i a l a r e

8-10t h o u g h t t o c o n f e r s e r o t y p e s p e c i f i c i t y among t h e P a s t e u r e l l a e .

Because b a c t e r i a l c a p s u l a r p o l y s a c c h a r i d e s a r e r ec o g n i ze d as

p a t h o l o g i c a l d e t e r m i n a n t s i n numerous d i s e a s e s , t h e r o l e of

c a p s u l a r m a t e r i a l in pneumonic p a s t e u r e l l o s i s has been t h e s u b j e c t of

many i n v e s t i g a t i o n s . Immuniza t ion wi t h l i v e e n c a p s u l a t e d Ph-1 o r

c a p s u l a r e x t r a c t s has r e s u l t e d in enhanced r e s i s t a n c e t o expe r i me n t a l

1 5 -1 9c h a l l e n g e s . Large amounts o f c a p s u l a r m a t e r i a l have been

a s s o c i a t e d wi t h Ph-1 i s o l a t e d from c a t t l e wi t h pneumonic

p a s t e u r e l l o s i s ; l i t t l e t o no c a p s u l e e x i s t s on t h e o rgan i sm i s o l a t e d

from n o n d i s ea s ed a n i m a l s . 3 F u r t he r more , h igh a n t i b o d y t i t e r s t o

p o l y s a c c h a r i d e a n t i g e n s , d e r i v e d from s a l i n e e x t r a c t a b l e c a p s u l a r

m a t e r i a l o f Ph-1 , has been c o r r e l a t e d wi t h d i s e a s e r e s i s t a n c e . ^ These

f i n d i n g s s i g n i f y t h e impor t ance o f c a p s u l a r m a t e r i a l and e s p e c i a l l y t h e

Ph-1 s p e c i f i c a n t i g e n in t h e p a t h o g e n e s i s o f pneumonic p a s t e u r e l l o s i s .

However, c a p s u l a r m a t e r i a l e x t r a c t e d from Ph-1 i s known t o c o n t a i n

1i p o p o l y s a c c h a r i d e s and o u t e r membrane p r o t e i n s in a d d i t i o n t o c a p s u l a r

7 20-22p o l y s a c c h a r i d e s . ’ The r e l a t i v e c o n t r i b u t i o n o f t h e s e component

a n t i g e n s t o t h e impor t ance a t t r i b u t e d t o c a p s u l a r m a t e r i a l r emains

un d e t e r mi n e d . V e r s a t i l e p r o ce d u r e s a r e needed t o p u r i f y c a p s u l a r

a n t i g e n s in o r d e r t o d e t e r mi n e t h e i r i mpor t a nc e . Se ve r a l p r o c e d u r e s

9 21 23 24have been a dop t ed o r deve l oped t o s e p a r a t e c a p s u l a r a n t i g e n s . * 5 ’

The purposes o f t h i s s t u d y were t o e s t a b l i s h an o p t i mi z e d monoclonal

a n t i b o d y i mmu n o a f f i n i t y p r oc e d u r e t o p u r i f y t h e s e r o t y p e 1 s p e c i f i c

e p i t o p e from P. h a e m o l y t i ca and i d e n t i f y t h e p r od uc t a n t i g e n ( s ) . We

b e l i e v e t h i s i s t he f i r s t immunological p u r i f i c a t i o n o f b a c t e r i a l

p o l y s a c c h a r i d e a n t i g e n s .

a C o r s t v e t , RE, u n p ub l i sh e d o b s e r v a t i o n .

99


Monoclonal a n t i b o d i e s :

The development and c h a r a c t e r i z a t i o n o f mur ine monoclonal

a n t i b o d i e s has been d e s c r i b e d (Chap t e r 1) . A Ph-1 s p e c i f i c IgM

a g g l u t i n a t i n g a n t i b o d y was used as t h e l i g a n d in i m mu n o a f f i n i t y . Other

d e f i n e d monoclonal a n t i b o d i e s t o a n t i g e n s o f P. ha e mol y t i ca were

used t o e v a l u a t e t h e i m mu n o a f f i n i t y p r o d u c t . Hybridomas were i n j e c t e d

i n t o t h e p e r i t o n e a l c a v i t y o f p r i s t a n e pr imed BALB/c mice f o r a s c i t i c

f l u i d p r o d u c t i o n . Monoclonal a n t i b o d i e s were f r a c t i o n a t e d from a s c i t i c

f l u i d by p r e c i p i t a t i o n in 50% s a t u r a t e d ammonium s u l f a t e a t 4C. The

p r e c i p i t a t e d a n t i b o d i e s were c o l l e c t e d by c e n t r i f u g a t i o n and

r e s us pended in PBS (pH7.2) t o one h a l f t h e o r i g i n a l a s c i t i c f l u i d

volume. Res idua l ammonium s u l f a t e was removed by d i a l y s i s a g a i n s t PBS

(0.1M, pH 7 . 2 ) a t 4C f o r 48 h r . P u r i f i e d monoclonal a n t i b o d i e s were

f i l t e r s t e r i l i z e d ( 0 . 22 ym) and s t o r e d a t -70C p r i o r t o u s e . The

r e t e n t i o n o f p u r i f i e d a n t i b o d y a c t i v i t y r e l a t i v e to p e r i t o n e a l a s c i t i c

f l u i d was e v a l u a t e d u s i n g a n t i b o d y ELISA and a g g l u t i n a t i o n t i t e r sor

( Chap t e r I ) and Brad f o r d p r o t e i n d e t e r m i n a t i o n s . All ELISA t i t e r s

were e x p r e s s e d as t h e i n v e r s e o f t h e h i g h e s t d i l u t i o n p roduc i ng an

a bs o r ba nc e va l ue 1.5 t i mes g r e a t e r t han normal mouse serum (1 :100

d i l u t i o n ) used as a c o n t r o l .

An t igen s o u r ce :

Ca p s u l a r m a t e r i a l f rom Ph-1 s e r o t y p e 1 was e x t r a c t e d from 6 hourQ

c u l t u r e s a t 41C f o r 1 hour in PBS us i ng t h e method o f Gent ry .

E x t r a c t e d c a p s u l a r a n t i g e n s were e x t e n s i v e l y d i a l y z e d a g a i n s t 4 changes

100

of PBS o ve r 48 h r a t 4C and f i l t e r s t e r i l i z e d ( 0 . 2 2 urn). Capsu l a r

m a t e r i a l was c h a r a c t e r i z e d p r i o r t o i m mu n o a f f i n i t y u s i n g p r o t e i n and

26hexosamine d e t e r m i n a t i o n s . Ant i gens p r e s e n t in c a p s u l a r m a t e r i a l

were q u a n t i t a t e d u s i n g d e f i n e d monoclonal a n t i b o d i e s in ELISA (Chap t e r

1).

E l u a n t s e l e c t i o n :

E l u a n t s e l e c t i o n was based on d i s s o c i a t i o n o f b a c t e r i a l

a g g l u t i n a t i o n and r e v e r s i b i 1i t y upon d i l u t i o n wi t h d i s t i l l e d w a t e r .

The s e l e c t e d e l u a n t was t he n t i t r a t e d f o r t h e l owes t c o n c e n t r a t i o n

c a u s i n g d i s s o c i a t i o n p r i o r t o use .

Suppor t m a t e r i a l and c o u p l i n g c o n d i t i o n s :

CNBr a c t i v a t e d Sepharose 4B was used as t h e s u pp o r t m a t e r i a l and

t h e monoclonal a n t i bo dy was c o v a l e n t l y a t t a c h e d a t 10 mg/ml p r e s wo l l e n

beads a t pH8.3 in NaHCO^ (0.1M) b u f f e r c o n t a i n i n g 0.5M NaCl f o r 2 hr a t

room t e m p e r a t u r e . The p o s t - c o u p l i n g s u p e r n a t e was r e t a i n e d f o r

s ubs e que n t a n a l y s i s . Remaining b i n d i ng groups on t h e s u p p o r t m a t e r i a l

were b locked wi t h 0.1M T r i s b u f f e r pH 8 . 5 f o r 2 a d d i t i o n a l h r . Unbound

p r o t e i n was deso r bed from t h e immunobeads by r e p e a t e d a l t e r n a t e washes

o f a c e t a t e b u f f e r (0.1M, pH 4 . 0 ) and t h e c o u p l i n g b u f f e r , each

c o n t a i n i n g 0 . 5 M NaCl . The immunobeads were s t o r e d a t 4C and

r e p e a t e d l y washed wi t h PBS p r i o r t o u s e . Ant ibody c o u p l i ng e f f i c i e n c y

was de t e r mi n e d by compar ing t h e p u r i f i e d a n t i b o d y t o t h e p o s t - c o u p l i n g

25s u p e r n a t e u s i n g ELISA and a g g l u t i n a t i o n t i t e r s and p r o t e i n

d e t e r m i n a t i o n s . Immunobead a c t i v i t y was d e t e r mi n e d m i c r o s c o p i c a l l y by

o bs e r v i n g f o r immunobi1i z a t i o n o f e n c a p s u l a t e d Ph-1. The e f f i c a c y o f

101

t h e s e l e c t e d e l u a n t was conf i r med by o b s e r v i n g b a c t e r i a l e l u t i o n from

t h e immunobead s u r f a c e . S p e c i f i c i t y o f b a c t e r i a l i m m o b i l i z a t i o n was

e v a l u a t e d u s i n g e i t h e r e n c a p s u l a t e d P. ha e mol y t i ca s e r o t y p e 2 o r 5 and

b l o c k i n g Ph-1 b i n d i n g wi t h homologus c a p s u l a r m a t e r i a l .

An t i gen s e p a r a t i o n and a n a l y s i s :

C a p s u l a r m a t e r i a l was r e a c t e d wi t h an equal volume o f immunobeads,

in a p o l y p r o p y l e n e t e s t t u b e , f o r 30 min a t 37C w h i l e g e n t l y r o c k i n g .

The s e r o t y p e 1 s p e c i f i c e p i t o p e was s e p a r a t e d and washed 3 t i me s wi t h

PBS-Tween (pH 7 . 2 , 0.05% Tween 20) by c e n t r i f u g a t i o n a t 50 Xg f o r 2

min a t 22C. Al l s u p e r n a t e s from t h e s e washes were saved f o r f u r t h e r

a n a l y s i s . The s e r o t y p e 1 s p e c i f i c e p i t o p e was e l u t e d from t h e

immunobeads wi t h an equal volume o f 0 . 4 M KSCN f o r 15 min a t 22C w h i l e

g e n t l y r o c k i n g . The e l u a n t was removed from t h e s e r o t y p e 1 s p e c i f i c

e p i t o p e by d i a l y s i s a g a i n s t PBS a t 4C S e p a r a t i o n e f f i c i e n c y d u r i n gpc pp.

i m m u n o a f f i n i t y was d e t e r mi n e d by compar ing p r o t e i n and hexosamine

d e t e r m i n a t i o n s o f c a p s u l a r m a t e r i a l t o t h e PBS-Tween washes . The

d i a l y z e d i mm u n o a f f i n i t y p r o d u c t was a n a l y z e d u s i n g p r o t e i n and

hexosamine d e t e r m i n a t i o n s and d e f i n e d monoclonal a n t i b o d i e s in ELISA

( Ch a p t e r 1) .

RESULTS

Fol lowing p r e c i p i t a t i o n , d i a l y s i s , and f i l t e r s t e r i l i z a t i o n o f t h e

Ph-1 s p e c i f i c monoclonal a n t i b o d y , ELISA and a g g l u t i n a t i o n t i t e r s were

o bs e r ved t o i n c r e a s e r e l a t i v e t o p e r i t o n e a l a s c i t i c f l u i d (Tab l e V . l ) .

102

Table V. l - R e t e n t i o n o f Ant ibody A c t i v i t y and P r o t e i n C o n c e n t r a t i o n

o f P u r i f i e d Monoclonal Ant ibody .

A s c i t i c P u r i f i e d

Assay F lu i d Ant ibody

ELISA t i t e r 107 > i o n★

A g g l u t i n a t i o n t i t e r 32 64

P r o t e i n c o n c e n t r a t i o n NT 11.43 mg/ml

T i t e r s e x p r e s s e d as i n v e r s e o f h i g h e s t d i l u t i o n i m p a r t i n g a p o s i t i v e

r e s u l t .

NT = no t t e s t e d

ELISA a n t i b o d y t i t e r i n c r e a s e d more t ha n t h e a g g l u t i n a t i o n t i t e r .

P r o t e i n c o n c e n t r a t i o n o f t h e p u r i f i e d a n t i b o d y was d e t e r mi ne d f o r

op t i ma l c o u p l i n g t o CNBr a c t i v a t e d Sepharose 4B and subse quen t

e v a l u a t i o n s .

S a l i n e e x t r a c t a b l e c a p s u l a r m a t e r i a l f rom Ph-1 used as an a n t i g e n

s o u r c e in i m m u n o a f f i n i t y , was d e t e r mi n e d t o c o n t a i n 60 wg/ml p r o t e i n

and 0 . 20 pM/ml hexosamine . T i t r a t i o n o f c a p s u l a r m a t e r i a l in ELISA

us i n g d e f i n e d monoclonal a n t i b o d i e s , a t c o n s t a n t c o n c e n t r a t i o n ,

a l l owed q u a n t i f i c a t i o n o f s p e c i f i c a n t i g e n s f o r s ub se q u e n t compar i son

wi t h t h e i mm u n o a f f i n i t y p r o d u c t (Tab l e V.2 ) .

103

Table V.2 - C h a r a c t e r i z a t i o n o f S a l i n e E x t r a c t a b l e C a p s u l a r Ma t e r i a l

o f F\ h a e m o l y t i ca s e r o t y p e 1.

Assay R e s u l t

P r o t e i n (ug/ml ) 60

Hexosamine (uM/ml) 0 .2 0★

McAbs in ELISA

L i p o p o l y s a c c h a r i d e 32

P a n - P a s t e u r e l l a CH^O >512

Ph-1 s p e c i f i c Ag 16

29 KD P r o t e i n 8

★Ca p s u l a r a n t i g e n s were t i t r a t e d in ELISA and d e f i n e d monoclonal

a n t i b o d i e s (McAbs) were used t o i d e n t i f y t h e t i t e r e x p r e s s e d as t h e

i n v e r s e o f h i g h e s t c a p s u l a r a n t i g e n d i l u t i o n g i v i n g a p o s i t i v e r e s u l t .

CH^O = c a r b o h y d r a t e , Ag = a n t i g e n .

KSCN was s e l e c t e d as an i mmu n o a f f i n i t y e l u a n t based on r e v e r s i b l e

d i s s o c i a t i o n o f e n c a p s u l a t e d Ph-1 a g g l u t i n a t i o n by t h e Ph-1 s p e c i f i c

monoclonal a n t i b o d y . D i l u t i o n o f t h e KSCN d i s s o c i a t e d a g g l u t i n a t i o n

r e a c t i o n wi t h d i s t i l l e d w a t e r conf i rmed r e v e r s i b i l i t y o f t h e e f f e c t by

c a u s i n g a g g l u t i n a t i o n t o r e o c c u r . However, t h e de g r e e o f b a c t e r i a l

a g g l u t i n a t i o n was v i s u a l l y reduced upon d i l u t i o n . T i t r a t i o n o f KSCN

f o r t h e l o w e s t e f f e c t i v e c o n c e n t r a t i o n c a u s i n g d i s s o c i a t i o n o f

a g g l u t i n a t i o n r e v e a l e d 0.4M t o be t h e op t i mal c o n c e n t r a t i o n f o r

a n t i g e n e l u t i o n . R e v e r s i b i 1i t y o f a g g l u t i n a t i o n was e a s i l y a c h i eve d

104

wi t h minimum d i l u t i o n o f t h e 0.4M KSCN d i s s o c i a t e d a g g l u t i n a t i o n .

Na2^2^3 was found t 0 cause d i s s o c i a t i o n o f a g g l u t i n a t i o n , bu t t h i s

e f f e c t was n o t as comple t e as w i t h KSCN and cou l d no t be r e v e r s e d upon

d i l u t i o n . Glycine-HCL (0.2M,pH 2 . 5 ) d i d no t cause d i s s o c i a t i o n o f

a g g l u t i n a t i o n (Table V.3) .

Table V.3 - S e l e c t i o n o f an Optimal E l u a n t f o r Immunoaf f i n i t y Based on

D i s s o c i a t i o n o f B a c t e r i a l A g g l u t i n a t i o n .

E l uan t D i s s o c i a t i o n Re v e r s i b i 1 i t y C o n c e n t r a t i o n

KSCN +4 +2 0.4M

Na2S2°3 +4 Neg NT

Glycine-HCL Neg NT NT

(pH 2 . 5 0.2M)

NT = not t e s t e d

Ant ibody c o u p l i ng e f f i c i e n c y t o CNBr a c t i v a t e d Sepharose 4B was

de t e r mi ne d by compar ing t h e p u r i f i e d a n t i b o d y t o t h e p o s t - c o u p l i n g

s u p e r n a t e (Tab l e V. 4 ) . The l ack o f d e m o n s t r a b l e ELISA and

a g g l u t i n a t i o n t i t e r s and n e g l i g i b l e amount o f p r o t e i n in t h e

p o s t - c o u p l i n g s u p e r n a t e i n d i c a t e d g r e a t e r t han 99% o f t he Ph-1

s p e c i f i c a n t i b o d y was c o v a l e n t l y a t t a c h e d t o t h e s u pp o r t m a t e r i a l .

105

Table V.4 - Monoclonal Ant ibody Coupl ing E f f i c i e n c y t o CNBr A c t i v a t e d

Sepharose 4B.

P u r i f i e d P o s t - c o u p l i n g

Assay Ant ibody s u p e r n a t e

ELISA t i t e r >10U 0

A g g l u t i n a t i o n t i t e r 64 0

P r o t e i n 11.43 mg/ml <5.0ug/ml

Fi gu r e V. l - Phase c o n t r a s t mi c rograph o f e n c a p s u l a t e d P. h ae m o l y t i c a

s e r o t y p e 1 immobl ized on immunobead s u r f a c e . X400.

107

Phase c o n t r a s t mi c r oscopy d e m o n s t r a t e d immunobead a c t i v i t y by

i m m o b i l i z a t i o n o f e n c a p s u l a t e d Ph-1 on t h e s u r f a c e ( F i g u r e V . l ) . The

number o f b a c t e r i a bound was found t o be r e l a t e d t o t h e amount o f

c a p s u l a r m a t e r i a l in s o l u t i o n s i n c e t h i s i m m o b i l i z a t i o n cou l d be

c o m p l e t e l y b locked by p r e i n c u b a t i o n o f t h e immunobeads w i t h c a p s u l a r

m a t e r i a l . S p e c i f i c i t y o f b a c t e r i a l i m m o b i l i z a t i o n was conf i r med us i ng

e i t h e r P. h ae m o l y t i c a s e r o t y p e s 2 o r 5 which were no t bound ( F i g u r e

V . 2 ) .

108

F i g u r e V.2 - Phase c o n t r a s t mi c rograph o f e n c a p s u l a t e d P. ha e mol y t i ca

s e r o t y p e 2 and immunobeads d e m o n s t r a t i n g s p e c i f i c i t y by a l a ck o f

i m m o b i l i z a t i o n . P. h a e m o l y t i c a s e r o t y p e 5 was no t immobi l i zed and

mi c r ogr aph i s t y p i c a l f o l l o w i n g e l u t i o n wi t h 0.4M KSCN. X400.

109

E l u t i o n o f e n c a p s u l a t e d Ph-1 from t h e immunobead s u r f a c e wi t h 0.4M

KSCN was co mp l e t e , t y p i c a l o f F i g u r e V.2, and r e v e r s i b l e upon d i l u t i o n

w i t h d i s t i l l e d w a t e r , however , on l y a smal l number o f b a c t e r i a would

r e a s s o c i a t e ( n o t shown) . Immunobeads r e g e n e r a t e d by a n t i g e n e l u t i o n

wi t h 0.4M KSCN and e x t e n s i v e washing wi t h PBS c o n s i s t e n t l y bound

numerous e n c a p s u l a t e d s e r o t y p e 1 b a c t e r i a r e p r e s e n t a t i v e o f F i gu r e 1.

S e p a r a t i o n o f Ph-1 c a p s u l a r a n t i g e n s d u r i n g i mmunoa f f i n i t y was

e v a l u a t e d us i ng chemica l d e t e r m i n a t i o n s o f t h e washes r e l a t i v e t o t he

o r i g i n a l c a p s u l a r e x t r a c t (Tab l e V.5) .

Table V.5 - Chemical D e t e r m i n a t i on o f I mmunoa f f i n i t y Ant igen

S e p a r a t i o n .

Washes

Assay o r i g i n a l s t a r t 1 2 3

P r o t e i n (ug/ml ) 60 36 <18 <10 <4

Hexosamine (yM/ml) 0 .2 0 0 .1 0 ND ND ND

ND = not de t e rmined beca use below t e s t l i m i t s •

P r o t e i n and hexosamine c o n c e n t r a t i o n s d e c r e a s e d c o n s i s t e n t l y

t h r ou g ho u t washing i n d i c a t i n g h igh f i d e l i t y in s e p a r a t i o n . P r o t e i n

c o n c e n t r a t i o n approached z e r o wi t h r e p e a t e d washing whi l e one h a l f of

t h e o r i g i n a l c a p s u l a r hexosamines were r e t a i n e d by t h e immunobeads.

110

Chemical a n a l y s i s o f t h e i mmunoa f f i n i t y p r od uc t r e v e a l e d a l a ck

o f d e t e c t a b l e p r o t e i n and a p p r o x i m a t e l y one h a l f t h e hexosamine

c o n t e n t as t h e o r i g i n a l c a p s u l a r e x t r a c t (Tab l e V.6) .

Table V.6 - A na l y s i s o f t h e I mmunoa f f i n i t y P r oduc t .

Assay R e s u l t

P r o t e i n (yg/ml ) z e r o

Hexosamine (uM/ml) 0 .1 2

McAbs in ELISA

L i p o p o l y s a c c h a r i d e 16

P a n - p a s t e u r e l l a CH^O >512

Ph-1 s p e c i f i c Ag 16

29 KD p r o t e i n ze r o

★I mmunoa f f i n i t y p r o d u c t was t i t r a t e d in ELISA and d e f i n e d monoclonal

a n t i b o d i e s (McAbs) were used t o d e t e r mi n e t he t i t e r e x p r e s s e d as t h e

i n v e r s e o f o f t h e h i g h e s t d i l u t i o n p roduc i ng an abs o r ba nc e va l ue 1.5

t i mes g r e a t e r t han normal mouse serum used as a c o n t r o l . CH^O =

c a r b o h y d r a t e , Ag = a n t i g e n .

T i t r a t i o n o f t h e i mm u n o a f f i n i t y p r o d u c t in ELISA, us i ng d e f i n e d

monoclonal a n t i b o d i e s a t c o n s t a n t c o n c e n t r a t i o n s , r e v e a l e d 3 mo l ecu l e s

were s e l e c t i v e l y p u r i f i e d by t h e p r oc e du r e (Tab l e V.6) .

L i p o p o l y s a c c h a r i d e and p a n - p a s t e u r e l l a c a r b o h y d r a t e c o - p u r i f i e d wi t h

s e r o t y p e 1 s p e c i f i c a n t i g e n . One h a l f o f t h e o r i g i n a l c a p s u l a r

I l l

e x t r a c t 1i p o p o l y s a c c a r i d e was r e c o v e r e d u s i ng t h e s e methods . Al though

t h e t i t e r o f t he p a n - p a s t e u r e l l a c a r b o h y d r a t e was g r e a t e r than

a s s a y e d , in both t h e o r i g i n a l c a p s u l a r e x t r a c t and i mmunoa f f i n i t y

p r o d u c t , t h e o v e r a l l a b s o r b a n c e v a l ue s were g r e a t e r in t he

immunoa f f i n i t y p r od uc t i n d i c a t i n g s e l e c t i v e r e t e n t i o n o f t h i s mol ecu l e

( n o t shown) . The t i t e r o f t h e Ph-1 s p e c i f i c a n t i g e n was t h e same in

t h e o r i g i n a l c a p s u l a r e x t r a c t and t h e i mmunoa f f i n i t y p r o d u c t . The 29

KD p r o t e i n was a b s e n t , as e x p e c t e d s i n c e a l l p r o t e i n was removed by

wash ing , from t he p r o d u c t .

DISCUSSION

R e v e r s i b l e d i s s o c i a t i o n o f b a c t e r i a l a g g l u t i n a t i o n was used in

t h i s s t udy t o s e l e c t an op t i ma l e l u a n t f o r i mmu n o a f f i n i t y . Glyc i ne -

HCL (0.1M, pH 2 . 5 ) and Na2S203 were found t o be u n s u i t a b l e as e l u a n t s

u s i n g t h i s c r i t e r i a . Gl yc i ne - HCL, o f low pH has been used97 n

s u c c e s s f u l l y as an IgG e l u a n t in a v a r i e t y o f p r ev io us s t u d i e s .

A p p a r e n t l y , a s h i f t in pH had no e f f e c t on t he IgM - Ph-1 complex as i t

does wi t h IgG a n t i g e n complexes and a g g l u t i n a t i o n was not d i s s o c i a t e d .

N02S2O3 caused d i s s o c i a t i o n o f a g g l u t i n a t i o n , however , r e v e r s i b i 1i t y

cou l d no t be a c h i eve d upon d i l u t i o n wi t h d i s t i l l e d w a t e r . The r eason

f o r i r r e v e r s i b i l i t y e n c o u n t e r e d wi t h Na2S20g remains unknown, bu t

permanent a n t i g e n o r a n t i b o d y d e n a t u r a t i o n a r e p o s s i b i l i t i e s .

Cha o t rop i c t h i o c y a n a t e s have been p r e v i o u s l y used as s u c c e s s f u l

30-34i mmu n o a f f i n i t y e l u a n t s in a v a r i e t y o f a p p l i c a t i o n s . KSCN was

found t o s a t i s f y ou r c r i t e r i a in t h e s e l e c t i o n o f an e l u a n t and was

t i t r a t e d f o r t he l owes t c o n c e n t r a t i o n c a u s i n g d i s s o c i a t i o n . The l owes t

d i s s o c i a t i v e c o n c e n t r a t i o n was c o n s i d e r e d opt imal because i t produced

112

mi l d e l u t i o n c o n d i t i o n s which were e a s i l y r e v e r s i b l e . These r e s u l t s

i n d i c a t e p r o p e r t i e s o f i mmunoglobul ins , p a r t i c u l a r l y a g g l u t i n a t i o n and

p o s s i b l y p r e c i p i t a t i o n , can be used in t h e s e l e c t i o n and o p t i m i z a t i o n

o f i mm u n o a f f i n i t y e l u a n t s .

Immunobead a c t i v i t y was a s s e s s e d by m i c r o s c o p i c o b s e r v a t i o n o f

e n c a p s u l a t e d b a c t e r i a l i m m o b i l i z a t i o n . The a g g l u t i n a t i n g a c t i v i t y of

t h e Ph-1 s p e c i f i c IgM monoclonal a n t i b o d y was u n d oub t e d l y r e s p o n s i b l e

f o r t h i s phenomena. Fo l lowing e s t a b l i s h m e n t o f immunobead a c t i v i t y ,

t h e s p e c i f i c i t y o f b a c t e r i a l i m m o b i l i z a t i o n was e v a l u a t e d u s i n g P.

h ae m o l y t i c a s e r o t y p e s 2 and 5 and by b l o c k i n g Ph-1 b i n d i ng wi th

homologous c a p s u l a r m a t e r i a l . E n c a p s u l a t e d Ph-1 c ou l d be r e v e r s i b l y

d i s s o c i a t e d from t h e bead s u r f a c e u s i n g t h e o p t i mi z e d e l u a n t . These

f i n d i n g s s u pp o r t e d p r ev i o u s r a t i o n a l and c o n c l u s i o n s and p r ov i de d a

f a s t , e a s y , and p r a c t i c a l means o f a s s e s s i n g immunobead a c t i v i t y p r i o r

t o u s e . Ot he r i m mu n oa f f i n i t y p r oc e d u r e s have on l y t o r e l y on p r od uc t

e v a l u a t i o n , somet imes days t o weeks l a t e r , as an a s s e s s m e n t o f

a c t i v i t y . Thus , a g g l u t i n a t i o n by immunoglobul ins can be a p p l i e d t o

t e s t t h e f e a s i b i l i t y and s p e c i f i c i t y o f u s i n g d i f f e r e n t a n t i b o d y

c o u p l i n g p r o c e d u r e s , s u p p o r t m a t e r i a l s , and e l u a n t s in i m mu n oa f f i n i t y .

S e p a r a t i o n and washing o f t h e immunobead a n t i g e n complex was

per formed by low speed c e n t r i f u g a t i o n . Thi s proved t o be a f a s t , e a sy

means o f s e p a r a t i n g and washing t h e bound complex. Another adva n t age

of t h i s method was t h a t volumes o f wash and e l u a n t s o l u t i o n s were

e a s i l y c o n t r o l a b l e and he l d c o n s t a n t f o r s u b s e qu e n t e v a l u a t i o n s .

Common problems e n c o u n t e r e d wi t h a f f i n i t y columns a r e d i l u t i o n o f t h e

p r o d u c t upon e l u t i o n , pH n e u t r a l i z a t i o n , and i d e n t i f i c a t i o n o f t h e

p r o d u c t in numerous f r a c t i o n s . In t h i s s t u d y , volumes o f wash and

113

e l u a n t s o l u t i o n s e q u i v a l e n t w i t h t h e a n t i g e n volume were used a l l o w i n g

e a s y t r a c k i n g and e v a l u a t i o n o f t h e component s . Thi s method a l s o

d e l e t e d t h e need f o r s p e c i a l , e x p e n s i v e , and somet imes cumbersome

mechanica l equ i qmen t .

The p r oc e d u r e s used in t h i s s t u d y were e v a l u a t e d by chemica l and

immunological me thods . An t ibody a c t i v i t y was a s s e s s e d u s i n g ELISA and

a g g l u t i n a t i o n t i t e r s which were ob s e r ve d t o i n c r e a s e f o l l o w i n g

p u r i f i c a t i o n and c o n c e n t r a t i o n o f monoclonal a n t i b o d y from p e r i t o n e a l

a s c i t i c f l u i d . Thi s may have been due t o removal o f i n h i b i t o r y

s u b s t a n c e s , such as p r i s t a n e , l i p i d s , o r p r o t e o l y t i c enzymes d u r i n g

p u r i f i c a t i o n . The a g g l u t i n a t i o n t i t e r d i d no t i n c r e a s e as much as t h e

ELISA t i t e r and was a t t r i b u t e d t o s o l u b i l i z a t i o n o f c a p s u l a r a n t i g e n s ,

s i n c e Ph-1 b i n d i n g c ou l d be b locked wi t h c a p s u l a r m a t e r i a l . These

t i t e r s w i t h p r o t e i n d e t e r m i n a t i o n s were a l s o used t o e v a l u a t e c o v a l e n t

a n t i b o d y a t t a c h m e n t t o t h e s u p p o r t m a t e r i a l and i n d i c a t e d t h e d e s c r i b e d

c o n d i t i o n s removed a l l a n t i b o d y a c t i v i t y from s o l u t i o n .

C a p s u l a r m a t e r i a l from Ph-1 has been shown t o c o n t a i n bo th p r o t e i n

and hexosamine a n t i g e n s q u a n t i f i a b l e by chemica l and immunological7 q i n i c

methods . » » » » Chemical d e t e r m i n a t i o n s o f t h e c a p s u l a r

m a t e r i a l , wash s o l u t i o n s , and i mm u n o a f f i n i t y p r o du c t a l l owed e v a l u a t i o n

o f t h e s e p a r a t i o n p r o c e d u r e s . P r o t e i n c o n c e n t r a t i o n s d e c r e a s e d

c o n s i s t e n t l y and o n e - h a l f t h e hexosamines were removed d u r i n g wash i ng ,

i n d i c a t i n g t h e Ph-1 s p e c i f i c a n t i g e n was hexosamine in n a t u r e . Thi s

c o n c l u s i o n s u p p o r t s t h e work o f o t h e r i n v e s t i g a t o r s who have i d e n t i f i e d

9 10t h e Ph-1 s p e c i f i c a n t i g e n as hexosamine . ’

Fo l l owi ng a n t i g e n e l u t i o n and d i a l y s i s , chemica l a n a l y s i s o f t h e

i mm u n oa f f i n i t y p r o d u c t r e v e a l e d a l a c k o f d e t e c t a b l e p r o t e i n and

o n e - h a l f t h e o r i g i n a l hexosamine c o n t e n t . ELISA i d e n t i f i c a t i o n and

t i t r a t i o n o f t h e p r od u c t a n t i g e n s d e m o ns t r a t e d s e l e c t i v e r e t e n t i o n o f

1i p o p o l y s a c c h a r i d e , p a n - p a s t e u r e l l a c a r b o h y d r a t e and t h e Ph-1 s p e c i f i c

c a p s u l a r p o l y s a c c h a r i d e ; a l l hexosamine c o n t a i n i n g a n t i g e n s . The LPS

ELISA t i t e r , i n t he p r o d u c t , was d e c r e a s e d by one h a l f and t h e

p a n - p a s t e u r e l l a c a r b o h y d r a t e t i t e r r emained g r e a t e r t han a s s ay e d .

However, t h e a bs o r ba nc e v a l u e s ( no t shown) o f t h e p a n - p a s t e u r e l l a

c a r b o h y d r a t e were h i g h e r in t h e p r od u c t t han t h e o r i g i n a l c a p s u l a r

m a t e r i a l . The Ph-1 s p e c i f i c c a p s u l a r p o l y s a c c h a r i d e t i t e r remained t he

same f o l l o w i n g i m mu no a f f i n i t y . The r e a s o n f o r t h e s e l e c t i v e r e t e n t i o n

o f t h e 2 unexpec t ed a n t i g e n s remains u n de t e r mi n e d , however s e v e r a l

p o s s i b i l i t i e s e x i s t .

N o n - s p e c i f i c r e t e n t i o n o f a n t i g e n s t h r ough i m mu n o a f f i n i t y

35p r oc e d u r e s i s a common problem. Suppor t m a t e r i a l s may

n o n - s p e c i f i c a l l y a bs o r b a n t i g e n s by h y d ro ph ob i c , h y d r o p h i l i c , o r i o n i c

i n t e r a c t i o n s . These i n t e r a c t i o n s have been e x p l o i t e d in many a f f i n i t y

35p r oc e d u r e s t o p u r i f y v a r i o u s m o l e c u l e s . L i k e wi s e , i t i s no t

i n c o n c i e v a b l e t h a t , b l o c k i n g r e a g e n t s can modi fy s up p o r t s u r f a c e s

l e a d i n g t o n o n - s p e c i f i c a b s o r b t i o n . The r e l a t i v e l y mi ld washing

c o n d i t i o n s used h e r e i n may no t have been s t r i n g e n t enough t o remove

1i p o p o l y s a c c h a r i d e and p a n - p a s t e u r e l l a c a r b o h y d r a t e n o n - s p e c i f i c a l l y

a bs o r be d t o t h e s u pp o r t m a t e r i a l . Ano t he r p o s s i b i l i t y i n c l u d e s t h e

p r e s e n c e o f o t h e r immunog lobu l ins , in p e r i t o n e a l a s c i t i c f l u i d , which

bind 1i p o p o l y s a c c h a r i d e and p a n - p a s t e u r e l l a c a r b o h y d r a t e . However, one

would e x p e c t n o n - s p e c i f i c a b s o r b t i o n t o o c c u r on l y i n t r a c e amounts ,

l e a d i n g t o low, but d e t e c t a b l e l e v e l s o f c o n t a m i n a n t s . The p r o b a b i l i t y

o f n o n - s p e c i f i c r e t e n t i o n seems low beca use o f t h e l a r g e and n e a r l y

115

e q u i v a l e n t amount o f a n t i g e n s in t h e p r od u c t r e l a t i v e t o t h e o r i g i n a l

c a p s u l a r m a t e r i a l .

In s u p p o r t o f t h e s p e c i f i c r e t e n t i o n o f t h e two unexpec t ed

a n t i g e n s , t h e Ph-1 s p e c i f i c e p i t o p e cou l d be p r e s e n t on s e v e r a l

m o l e c u l e s . Using monoclonal a n t i b o d i e s , E s c h e r i c h i a c o l i K5 c a p s u l a r

p o l y s a c c h a r i d e has been shown t o s h a r e an a n t i g e n i c d e t e r m i n a n t w i t h

t h e e n t e r o b a c t e r i a l common a n t i g e n . S i m i l a r l y , l i p i d has been

i d e n t i f i e d on t h e c a p s u l a r p o l y s a c c h a r i d e s o f many Gram-nega t ive

37b a c t e r i a and common l i p i d c a r r i e r s have been i m p l i c a t e d in t he

s y n t h e s i s o f 1i p o p o l y s a c c h a r i d e s , p e p t i d o g l y c a n , and c a p s u l a r

38p o l y s a c c h a r i d e s . L i k e w i s e , k e t o - d e o x y - o c t o n o i c m o i e t i e s , known t o

compr i se t h e c o r e o f 1 i p o p o l y s a c c h a r i d e s , have been l o c a t e d on c a p s u l a r

39-42p o l y s a c c h a r i d e s . These s t u d i e s i n d i c a t e t h a t p o l y s a c c h a r i d e

e p i t o p e s can be s h a r e d by a v a r i e t y o f d i f f e r e n t c a r b o h y d r a t e

c o n t a i n i n g m o l e c u l e s . R e c e n t l y , i t was s u g ge s t e d t h a t s e r o t y p e

s p e c i f i c a n t i g e n s o f P. h a e m o l y t i ca a r e a p a r t o f t he

431i p o p o l y s a c c h a r i d e complex, s p e c i f i c a l l y t h e 0 - a n t i g e n s i d e c h a i n .

Othe r i n v e s t i g a t o r s have r e p o r t e d s e r o t y p e s p e c i f i c i t y r e s i d e s in

c a p s u l a r p o l y s a c c h a r i d e s . Our r e s u l t s s u g g e s t t h a t t h e Ph-1 s p e c i f i c

e p i t o p e i s p r e s e n t on c a p s u l a r p o l y s a c c h a r i d e , 1i p o p o l y s a c c h a r i d e , and

t h e p a n - p a s t e u r e l l a c a r b o h y d r a t e . Ep i t ope s h a r i n g among hexosamine

a n t i g e n s c ou l d have i m p o r t a n t i m p l i c a t i o n s i n p o l y s a c c h a r i d e

i mmunogeni c i t y .

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I

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43. Tas i LH, C o l l i n s MT, Hdibg N. A n t i g e n i c a n a l y s i s o f P a s t e u r e l l a ha e mol y t i ca s e r o u a r s 1 t r hough 15 by c r o s s e d i m mu n o e l e c t r o p h o r e s i s . Am J Vet Res 1988;49 : 213-222 .

CHAPTER 6

CONCLUSIONS AND FUTURE PERSPECTIVES

Bas ic r e s e a r c h c o n s i d e r e d n e c e s s a r y f o r a b e t t e r u n d e r s t a n d i n g o f

t h e p a t h o g e n e s i s in pneumonic p a s t e u r e l l o s i s was c o n d uc t e d . The

r a t i o n a l c o n s t r u c t i o n o f a component v a c c i n e i s dependen t on an

a d eq ua t e comprehens ion o f t h e p a t h o g e n e s i s . T h e r e f o r e , a

c h a r a c t e r i z a t i o n o f monoclonal a n t i b o d y and bovine n e u t r o p h i l

r e a c t i v i t y t o P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e 1 (Ph-1) a n t i g e n s was

per formed t o a i d in a c h i e v i n g t h e s e g o a l s .

The monoclonal a n t i b o d i e s (McAbs) p r e p a r e d and c h a r a c t e r i z e d

a g a i n s t Ph-1 c a p s u l a r m a t e r i a l p r ov i d e s some b a s i c m a t e r i a l s t o

i d e n t i f y , q u a n t i t a t e , and p u r i f y a n t i g e n s . I n f o r m a t i on c o n c e r n i n g t he

p a t h o g e n e s i s may be o b t a i n e d by i d e n t i f y i n g t h e amount o f Ph-1 a n t i g e n s

p r e s e n t in inoculums and b r o n c h o a l v e o l a r l a vage f l u i d s . The

i d e n t i f i c a t i o n and d i s t r i b u t i o n o f Ph-1 a n t i g e n s in pneumonic lung

t i s s u e u s i n g t h e McAbs and immunohis tochemical t e c h n i q u e s may a l s o a i d

in u n d e r s t a n d i n g t h e p a t h o g e n e s i s . I d e n t i f i c a t i o n and l a r g e s c a l e

p r u i f i c a t i o n o f s p e c i f i c Ph-1 a n t i g e n s i m p o r t a n t i n t h e p a t h o g e n e s i s

w i l l be r e q u i r e d t o c o n s t r u c t an e f f e c t i v e component v a c c i n e . The

McAbs d eve l oped and c h a r a c t e r i z e d in t h i s r e s e a r c h can a i d in

u n d e r s t a n d i n g t h e p a t h o g e n e s i s and in d e v e l o p i n g a v a c c i n e .

An u n d e r s t a n d i n g o f n e u t r o p h i l i n t e r a c t i o n wi t h Ph-1 a n t i g e n s i s

i m p o r t a n t i n t h e comprehens ion of t h e p a t h o g e n e s i s in pneumonic

p a s t e u r e l l o s i s . The l o g i c a l deve lopment o f a component v a c c i n e s hou l d

a d d r e s s t h e i n t e r a c t i o n o f n e u t r o p h i l s w i t h Ph-1 a n t i g e n s . The MTT

c o l o r i m e t r i c a s s a y a d a p t ed h e r e i n p r ov i d es a means o f measur i ng

120

121

n e u t r o p h i l c y t o t o x i c i t y and s t i m u l a t i o n . The a c t i v a t i o n o f n e u t r o p h i l s

by Ph-1 a n t i g e n s may e x p l a i n t h e i r r a p i d and i n t e n s e acc umul a t i on in

pulmonary t i s s u e f o l l o w i n g e x p e r i m e n t a l c h a l l e n g e . An e f f e c t i v e

component v a c c i n e shou ld r e s u l t in n e u t r a l i z a t i o n o f c y t o t o x i c a c t i v i t y

and b lock a c t i v a t i o n o f n e u t r o p h i l s . The Ph-1 a n t i g e n ( s ) r e s p o n s i b l e

f o r n e t u r o p h i l a c t i v a t i o n needs t o be i d e n t i f i e d and e f f e c t i v e l y

b l oc ke d .

The c y t o t o x i n - n e u t r a l i z i n g monclonal a n t i bo dy (nMcAb) p r e p a r e d and

p a r t i a l l y c h a r a c t e r i z e d h e r e i n m e r i t s f u r t h e r s t u d y . A l t e r n a t i v e s t o

de m o n s t r a t e nMcAb b i nd i ng t o c y t o t o x i n shou ld be sought t o i d e n t i f y and

q u a n t i t a t e c y t o t o x i n in inoculums and c l i n i c a l s ampl es . Because nMcAb

b i nd i ng t o c y t o t o x i n was d e m o ns t r a b l e by immunoblo t t ing i t i s p r o b ab l e

t h a t t h e a n t i b o d y can be used t o p u r i f y c y t o t o x i n u s i n g i m mu n o a f f i n i t y .

The a b s o l u t e c o n t r i b u t i o n o f c y t o t o x i n t o t h e p a t h o g e n e s i s c ou l d then

be de t e r mi n e d and pure c y t o t o x i n coul d be used i n a component v a c c i n e .

S t r u c t u r a l changes in n e u t r o p h i l s exposed t o c y t o t o x i n a r e

i m p o r t a n t t o comprehend t h e p a t h o g e n e s i s in pneumonic p a s t e u r e l l o s i s .

Neu t r op h i l p o l a r i z a t i o n wi t h uropod f o r ma t io n i s s u g g e s t i v e o f

a c t i v a t i o n by a c h e m o t a c t i c f a c t o r . V i s u a l i z a t i o n o f s t r u c t u r a l

changes in n e u t r o p h i l s o f f e r s an a l t e r n a t i v e t o t he MTT c o l o r i m e t r i c

a s s a y as a means o f i d e n t i f y i n g t h e Ph-1 component ( s ) c a u s in g

a c t i v a t i o n . A c t i v a t i o n o f n e u t r o p h i l s shou l d be i n h i b i t e d by an

e f f e c t i v e v a c c i n e .

The l a r g e s c a l e p u r i f i c a t i o n o f Ph-1 a n t i g e n s i s n e c e s s a r y f o r

d e t e r m i n i n g t h e i r c o n t r i b u t i o n t o t h e p a t h o g e n e s i s and f o r t h e

c o n s t r u c t i o n o f a v a c c i n e . I mmunoaf f i n i t y p r o c e d u r e s deve l oped in t h i s

122

r e s e a r c h p r o v i d e s a means o f e p i t o p e p u r i f i c a t i o n . P u r i f i e d e p i t o p e s

can t h e n be s t u d i e d t o d e t e r mi n e t h e i r c o n t r i b u t i o n t o n e u t r o p h i l

a c t i v a t i o n and t h e p a t h o g e n e s i s i n v i v o . P u r i f i e d e p i t o p e s i d e n t i f i e d

as i m p o r t a n t i n t h e p a t h o g e n e s i s can t h e n be i n c l u d e d i n a component

v a c c i n e .

Thi s r e s e a r c h s u p p l i e s some m a t e r i a l s , methods and r e s u l t s f o r a

g r e a t e r u n d e r s t a n d i n g o f t h e p a t h o g e n e s i s i n pneumonic p a s t e u r e l l o s i s

and c o n s t r u c t i o n o f a component v a c c i n e . F u r t h e r r e s e a r c h i s n e c e s s a r y

u s i n g monoclonal a n t i b o d i e s and n e u t r o p h i l s t o more f u l l y u n d e r s t a n d

t h e p a t h o g e n e s i s in pneumonic p a s t e u r e l l o s i s and c o n s t r u c t an e f f e c t i v e

component v a c c i n e .

VITAE

Name; Frank Wi l l i am A u s t i n

P r e s e n t A d d r e s s : 3350 Tula S t . #3Baton Rouge, LA 70802

Permanent A d d r e s s : 5708 Ra i nc r eek ParkwayA u s t i n , Texas 78750 (512) 345-5577

S . S . # : 553-84-6079

Date and P l a c e o f B i r t h : 11-24-57 A u s t i n Texas

Ma r i t a l S t a t u s : S i n g l e

Secondary E d u c a t i o n : Memorial High Schoo l , T u l s a , OK

U n i v e r s i t y E d u c a t i o n :Oklahoma S t a t e U n i v e r s i t y 1975-79, M i c r o b i o l o g y , B.S.Oklahoma S t a t e U n i v e r s i t y 1979-83, V e t e r i n a r y Medi c i ne , D.V.M. Lo u i s i a n a S t a t e U n i v e r s i t y 1984-1988, V e t e r i n a r y M i c r o b i o l o g y ,

Ph.D. (Minor : V e t e r i n a r y P a t ho l ogy ) (August 1988)

P r o f e s s i o n a l Employment :Resea rch A s s i s t a n t , Dept , o f M i c r o b i o l o g y , Oklahoma S t a t e

U n i v e r s i t y , 1978-1979.P r a c t i c i n g V e t e r i n a r i a n , S t a t e Line Animal C l i n i c , Poco l a ,

Oklahoma, 1983-1984.Gradua te Research A s s i s t a n t , Dept , o f V e t e r i n a r y Mi c r ob i o l ogy

and P a r a s i t o l o g y , L o u i s i an a S t a t e U n i v e r s i t y , 1984-1988.

P r o f e s s i o n a l A f f i l i a t i o n s :Oklahoma V e t e r i n a r y Medical A s s o c i a t i o n American V e t e r i n a r y Medical A s s o c i a t i o n American A s s o c i a t i o n f o r Advancement o f Sc i ence American A s s o c i a t i o n o f V e t e r i n a r y Immunologi s t s

P r o f e s s i o n a l L i c e n s e s :Oklahoma Board o f V e t e r i n a r y Medical Examiners (L i ce ns e #2458)USDA A c c r e d i t a t i o n f o r S t a t e o f Oklahoma

P r o f e s s i o n a l P r e s e n t a t i o n s :A u s t i n , F.W. , C o r s t v e t , R . E . , S c h n o r r , K.L. In v i t r o s t u d i e s on t h e

n e u t r a l i z a t i o n o f c y t o t o x i n from P a s t e u r e l l a ha e mol y t i ca t y p e 1. Animal D i sease Resea r ch Workers in t h e Sou t he rn S t a t e s , Mar. 1985, At he ns , GA, p. 34.

Nob l es , D. , C o r s t v e t , R . E . , S c h n o r r , K . L . , Aus t i n F.W. P r od uc t i o n and s p e c i f i c i t y o f monoclonal a n t i b o d i e s t o P a s t e u r e l l a h a e m o l y t i c a . Animal Di s e a s e Resea r ch Workers in t h e Sou t he rn S t a t e s , Mar. 1985, At he ns , GA, p. 71.

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A u s t i n , F.W., C o r s t v e t , R . E . , S c h n o r r , K . L . , Nobles , D. R e a c t i v i t y o f monoclonal a n t i b o d i e s p r e p a r e d a g a i n s t P a s t e u r e l l a h a emol y t i ca s e r o t y p e 1. Confe r ence o f Research Workers i n Animal D i s e a s e ,Nov. 1985, Chicago , 1 1 1 . , ( # 2 3 3 ) :43.

S ch n or r , K . L . , Todd, W . J . , C o r s t v e t , R . E . , A u s t i n , F.W. , Nob l es , D. I d e n t i f i c a t i o n and l o c a l i z a t i o n o f P a s t e u r e l l a h a e m o l y t i ca a n t i g e n s by monoclonal a n t i b o d i e s . The F i r s t I n t e r n a t i o n a l V e t e r i n a r y Immunology Symposium pg. 133.

A u s t i n , F.W. , C o r s t v e t , R . E . , S c h n o r r , K.L. C h a r a c t e r i z a t i o n o f a n e u t r a l i z i n g monoclonal a n t i b o d y p r e p a r e d a g a i n s t P a s t e u r e l l a ha e mol y t i ca s e r o t y p e 1 c y t o t o x i n . Animal Di s e a s e Research Workers in t h e Sou t he r n S t a t e s , Mar. 1986, G a i n e s v i l l e , FL. , ( # 1 4 ) : 6 .

A u s t i n , F.W., Lozano, F . , C o r s t v e t , R.E. A t empora l and dose a n a l y s i s o f s t r u c t u r a l changes in bov ine n e u t r o p h i l s exposed t o P a s t e u r e l l a h a e mo l y t i ca s e r o t y p e 1 c y t o t o x i n . Animal D i s ea s e Research Workers in t h e Sou t he r n S t a t e s , Mar . , 1987, Co l l ege S t a t i o n , TX, ( # 3 ) : 2 2 .

B u r g e r m e i s t e r , D . , C o r s t v e t , R . E . , A u s t i n , F . , Gaunt , S. E h r l i c h i a r i s t i c i i - P r o p a g a t i o n ; s e r o l o g i c s t u d i e s ; p roduc t i on" ! ) ? monoclonal a n t i b o d i e s . Animal D i s ea s e Research Workers i n t h e Sou t he r n S t a t e s , Mar. 1987, C o l l eg e S t a t i o n , TX, ( # 3 ) : 2 3 .

A u s t i n , F.W. , C o r s t v e t , R.E. I mmunoa f f i n i t y p u r i f i c a t i o n o f t heP a s t e u r e l l a h a e m o l y t i c a s e r o t y p e 1 s p e c i f i c a n t i g e n . Conference o f Research Workers i n Animal D i s e a s e , No. 1987, Ch i cago , 11 1 . , ( # 3 0 ) : 5 .

G o s s e t t , K.A. , A u s t i n , F.W. , C o r s t v e t , R . E . , e t . a l . B r o n c h o a l v e o l a r l a vage c y t o l o g y and hematology in c a l v e s f o l l o w i n g i n t r a b r o n c h i a l i n o c u l a t i o n wi t h P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e 1 ( P h - 1 ) , dead Ph-1 wi t h c a p s u l e removed, s e m i - p u r i f i e d Ph-1 c y t o t o x i n , o r Ph-1 c a p s u l a r m a t e r i a l . Confe r ence o f Resea r ch Workers in Animal D i s e a s e , No. 1987, Ch i cago , 1 1 1 . , ( # 3 9 ) : 7 .

A u s t i n , F.W. , C o r s t v e t , R.E. E v a l u a t i o n o f bov ine n e u t r o p h i l r e s p o n s e t o P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e 1 c y t o t o x i n . Confe r ence o f Research Workers in Animal D i s e a s e , Nov. 1987, Ch i cago , 111 . ,( # 4 0 ) : 7 .

Schmi dt , S . P . , C o r s t v e t , R . E . , A u s t i n , F.W. , e t a l . A s t u d y o f l e s i o n s and l e s i o n volumes ca us e d by i n t r a b r o n c h i a l i n o c u l a t i o n o f P a s t e u r e l l a h a e m o l y t i ca s e r o t y p e 1 component s . Animal Di s e a s e Research Workers in t h e Sou t he rn S t a t e s , Mar. 1988, Baton Rouge, LA, ( # 3 5 ) :9 .

P r o f e s s i o n a l M e e t i n g s :NC-107 Te c h n i c a l Commit tee on Bovine R e s p i r a t o r y D i s e a s e , Baton

Rouge, LA. , 1987.

Research G r a n t s :G o s s e t t , K.A. , A u s t i n , F.W. , B e a d l e , R . E . , F l o r y , W. Mechanisms o f

lung i n j u r y in t h e h o r s e : N e u t r op h i l p r o t e o l y t i c enzymes.Lo u i s i a n a S t a t e U n i v e r s i t y , School o f V e t e r i n a r y Med i c i ne , C o mp e t i t i v e I n d i v i d u a l G r a n t s , $ 3 , 9 6 0 . 0 0 funded .

125

Honors and Awards :Omega Tau Sigma Phi Zeta Sigma XiEx c e l l e n c e in r e s e a r c h in m i c r o b i o l o g y o f animal d i s e a s e s awarded a t Animal Di s e a s e Resea r ch Workers in Sou t he r n S t a t e s , Mar. 1986.

Research E x p e r i e n c e :I s o l a t i o n and i d e n t i f i c a t i o n o f b a c t e r i a f o r m i c r o b i a l enhanced

o i l r e c o v e r y .P ro d u c t i o n and c h a r a c t e r i z a t i o n o f P a s t e u r e l l a a n t i g e n s and

c y t o t o x i n . Development and a p p l i c a t i o n o f ELISA and b i o l o g i c a l a s s a y s f o r P a s t e u r e l l a a n t i g e n s . I n t e r a c t i o n o f P a s t e u r e l l a a n t i g e n s and c y t o t o x i n w i t h bov ine l e u k o c y t e s . Enzymat ic a n a l y s i s o f bov i ne n e u t r o p h i l s .

P r o d u c t i on and c h a r a c t e r i z a t i o n o f monoclonal a n t i b o d i e s .Monoclonal a n t i b o d y i mm u n o a f f in i t y p u r i f i c a t i o n s .

Animal models o f pulmonary d i s e a s e . B r o nc h o a l v e o l a r l avage t e c h n i q u e s and a n a l y s i s . Gross and h i s t o p a t h o l o g i c d i a g n o s i s .

Teaching E x p e r i e n c e :P r i v a t e t u t o r ; u n d e r g r a d u a t e m i c r o b i o l o g y and c h e m i s t r y . Mi n i -Cour s e V e t e r i n a r y I n t e r n s h i p Program a t LSU, summer 1984. Mi n i - Cour se V e t e r i n a r y I n t e r n s h i p Program a t LSU, summer 1985. Speaker f o r t h e Gr adua t e S t u d e n t s o f V e t e r i n a r y Mi c r ob i o l ogy and

P a r a s i t o l o g y , LSU, 1985-86.S t u d e n t Summer Resea r ch A p p r e n t i c e Program a t LSU, summer 1986. Gr adua t e A s s i s t a n t in S e n i o r V e t e r i n a r y S t u d e n t Necropsy

L a b o r a t o r y .S t u d e n t Summer Research A p p r e n t i c e Program a t LSU, summer 1987. Le c t u r e d t o s e n i o r v e t e r i n a r y s t u d e n t s i n C l i n i c a l V i r o l o g y , 1987. L e c t u r e s t o g r a d u a t e s t u d e n t s i n C e l l u l a r Immunology, 1987.

Community S e r v i c e :LeF l o re County 4-H Club Group Leader .Roland, Oklahoma High School C a r e e r Day.N o n - p r o f i t v a c c i n a t i o n c l i n i c t o b e n e f i t Pocola High School

marching band.N o n - p r o f i t v a c c i n a t i o n c l i n i c t o b e n e f i t L e f l o r e County 4-H Club.

P r o f e s s i o n a l I n t e r e s t :Hos t - Pa t hogen i n t e r a c t i o n s Monoclonal a n t i b o d y t e c h n o l o g y Novel d i a g n o s t i c t e c h n i q u e s Animal models o f pulmonary d i s e a s e Mechanisms o f b a c t e r i a l p a t h o g e n e s i s

DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Frank W. Aus t i n

Major Field: V e t e r i n a r y Mi c r ob i o l ogy

Title of Dissertation: C h a r a c t e r i z a t i o n and A p p l i c a t i o n o f Monoclonal Ant ibody andBovine N e u t r op h i l R e a c t i v i t y t o P a s t e u r e l l a h a e m o l y t i ca A n t i g e n s .

Approved:

M a j o r P r o f e s s o r a n d C h a i r m a n

D e a n o f t h e G r a d u a t e S c | j 6 d l '

EXAM INING COMMITTEE:

/ Kc

Date of Examination:

characterization and application of monoclonal antibody

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