blood imp 4

8/3/2019 Blood Imp 4

1/12

I M M U N O C H E M I C A L S T U D I E S O N B L O O D G R O U P SLI V. CLASSIFICATION OF AN TI-I AND ANTI-i SERA INTO GROUPS BASE D ON

REACTIVITY PATTERN S WITH VARIOUS ANTIGENS RELATED TOTHE BLOO D GRO UP A, B, H, LE a, LE b AND PRECU RSOR

SUBSTANCES*BY T EN FEIZI:~ Am) ELVIN A. KA BA T

(From the Departm ents of Microbiology, Neurolo gy, and Hum an Genetics and Develop-ment, College of Physicians and S urgeons, Colum bia University; the NeurologicalInstitute, P resbyterian Hospita l, New Fork 1 0032; and the Rocke/ellerUniversity, New York 10021)(Received for publication 18 February 1972)

E ar l i e r s t ud i es (1 , 2) o f t he I - a n t i - I co l d agg l u t i n i n sys t em have i nd i ca t edt ha t t he I an t i gen ic de t e rmi n an t s a r e compl ex and t ha t t h ey a r e p r esen t once r t a i n p r ecur sor s o f A , B , H , L e a , and L e b subs t ances . I n hem aggl u t i na t i on-i nh i b i t i on as says seve ra l an t i - I s e r a were i nh i b it ed by t h e hu ma n b l ood groupprecu r sor subs t ance OG f rom o var i an cys t f l ui d (3, 4 ) and t wo , M a and O r t ,r eac t ed wi t h p ro duc t s o f one s t age o f pe r i oda t e ox i da t i on and S mi t h d egrada t i on( f ir s t 104 s t age ) o f b l ood group A and B subs t ances . Qua n t i t a t i ve p r ec i p i t i nas says w i t h t h r ee an t i - I s e r a , M a , Or t , and S t ep , showed t hem t o be d i s t i nc t l yd i f f e ren t i n spec i fi c i ty a s r evea l ed b y t he i r r eac t i ons w i t h f r ac t i ons o f OG, 1 andwi t h a huma n mi l k f r ac t i on , w i t h cow b l ood group subs t ances , and w i t h t hef i rs t IO4 s t ages o f A , B , and H subs t ances . T h e p r ec i p i t in r eac t i on be t we en an t i -I s e rum M a and precur sor subs t ance OG was i nh i b i t ed bes t by o l i gosacchar i deswi t h t e rmi na l nonreduc ing / ~ DGaI (1--+4) /3DGl cNAc(1- -+6) - s t r uc t u r e . T h i ss t ruc t u r e was found (4 ) i n OG subs t ance . T h e de t e rm i nan t s i nvo l ved i n t heother I speci f ic i t ies have not been es tabl i shed.

E i gh t o t he r an t i - I s e r a and f ive an ti - i se r a have now been s t ud i ed b y qua n t i t a -t i ve p r ec ip i t i n a s says . T h ey a ll r eac t s t r ong l y wi t h t he 20% 2X f r ac t i on o f OGi ndi ca t i ng t ha t i t con t a ins bo t h I and i de t e rmi nan t s . Based on t he i r r eac t ionswi th var ious O G fract ions , the f i rs t 104 s tages of A, B, and H substanc es , w i thcow subs t ances , and wi t h c rude hyd a t i d cys t fl u id , t he e l even an t i - I s e r a canbe divided in to at leas t s ix groups . Fou r of the f ive ant i- i sera resembled one

* Aided by a gra nt from the N ational Science Foundation GB-25686 and a Gen eral Re-search Support grant from the US Public Health Service to Columbia University.:~ Fellow of the Arthritis Founda tion 1969-197 2.1 Fractions obtained after peptic digestion and ethanol precipitation of cyst fluid, extrac-tion with 90% phenol, and precipitation by different concentrations of ethanol from thephenol (3).THE JOURNAL OF EXPERIMENTAL MEDICINE VOLUME 135, 1972 1247

Published


2/12

124 8 IMMUNOCHEMICAL STUDIES ON BLOO D GROUPS. LIV

a n o t h e r i n t h e i r s t r o n g r e a c t i o n w i t h O G 2 0 % 2 X w h i l e r e a c t i n g m i n i m a l l y o rn o t a t a ll w i t h t h e o t h e r s u b s t a n c e s, b u t t h e y m a y n o t r e p r e s e n t a s i ng l e a n t i -b o d y s p e c i f i c i ty . T h e f i f t h d i f f er s i n t h a t i t r e a c t s w i t h h y d a t i d c y s t f l u i d a n dw i t h m i l k .

M a t e r i a l s a n d M e t h o d sThe cold agglutin ins were typed as anti-I or anti- i (5, 6) on the basis of their re la t ive reac-

tions with group OI, Oi cord, and Oi adult cells.Anti-I sera: All but one were from patients w ith the chronic cold agglutin in d isease . Case

Win (7) was a patie nt w ith h igh t i ter cold agglutin ins associa ted with Mycoplasma pneumonia~infection . The remaining sera have been previously described (1 , 2 , 8); sera Ma, Oft , an d S tepare included for comparison with the o ther cold agglutin in sera .

Anti4 sera: All f ive were from patient s with chronic cold agglutin in d isease . Sera Ho andTho have been previously described (2). Sera Den and M cDald were g if ts from Mrs. M arieCrookston of the Univers i ty of Toronto , and case Nic is newly d iagnosed.

Ano ther serum (Ro) w ith a ~?A cold agglutinin of so-called P rl specificity (9) was prov idedby D r. F. Mullinax of The Medical College of Virgin ia , Richmond. T his cold agglutin in isconsidered to have neither I nor i specif ic i ty ; i t agglutinates a l l human erythrocytes andthose of the ra t and guinea p ig .

Blood Group and Other Substances.--These have been described earlier (1) and consisted oftwo I ac tive fractions of human milk (fraction C and 20% 2X) ; human ovarian cyst precursorsubstances OG (3) and F1 (10); human ovarian cyst A (MSS) (11), B (Beach) (12), H (JS)(11), and Le a (N-I) (13) substan ces; hog A + H and cow substan ces (cf. reference 1). Inaddit ion , the P1 fraction2 of B subst ance (12, 14) and th e first IO4 stages (15) of huma novarian cyst A, B, and H substances and of hog A + H substances were tes ted (1) . A crudeconcentrate of sheep hydatid cyst fluid was also used. This consisted of pooled lyophilizedhydati d cyst f lu id obta ined from sheep l ivers in New Zealand and was a g if t f rom Dr. J . IV[.Stavely , Auckland, New Zealand. The cyst f lu id was dissolved in d is t i lled water to achieve atenfold concentra t ion .

Oligosaccharicles.--3nGal(1 ~ 3)DGlcNAc and/~DGal(1 --* 4)DGlcNAc were obtained fromDr. F. ZilUken (16), ~3nGal(1 -~ 6)DGNAc and ethyl ~DGIcNAc from Dr. R. Kuhn (17).A te trasaccharlde

~DGlcNAc(1 --~ 6) "~~DGal(1 --~ 4)Dsorbitol and a pentas accha rlde

~DGlc NAc(1 --~ 3) /z/~DGIcNAc(1 --~ 6) "-~

3DGaI(1 --~ 4)Dsor bitol~DGal (1 --* 3)/3DGlcNAc(1 ~ 3 ) /z

were prepared from hum an milk by Drs . A. Kobata and V. Ginsburg of the NIH . N-1 R L0.41, 0.71b, and 1.1 are oligosaccharides produc ed by degr adati on of blood group Le a sub-stance with NaOD-NaBD4 (13, 2) . OG RL 0 .44 and 1 .1 are o ligosaccharides produced bydegradation of precursor substance OG with N aOD -- NaBD 4 (4, 2) . Thei r s tructures are asfollows:OG RL 1.1 and N-1 RE 1.1 ~I)Gal(1 --* 4)/39GlcNAc(1 --* 6?)-3-hexenetetrol(s)N-1 R L 0 .71b/3DGal(1 --~ 4)~l)GlcNAc(1 ~ 6)-hexa ne-l ,2 ,4 ,5 ,6-pento l(s)OG RL 0.44 an d N-1 R L 0.41

2 The nondialyzable fraction of B substance af ter heating a t 100C for 2 hr a t pH 1 .5-1 .8 .

Published


3/12

TEN I~ EIZI AND ELV IN A. KABAT 1249#DG aI(1 ---*4)/3DGlcNAc

1

6D-galactitol3T1~DGal(1 ~ 3)13DGlcNAc

Quantitative Precipitin and Quantitative Inhibition Assays.--A ssays were carried out at0-4C as previously described (2).RESULTS

Quantitative Precipitin Studi es.--Ea ch se r um was te s ted wi th f ive fr ac t ions ofOG and w i th F1 subs tance a l l of which lacked A, B , H, L ea, and Le b act ivi t iesand were thus considered as precursors of these blood group substances (3, 10) .As seen f rom Figs . 1-3, a l l ant i- I and anti- i sera reacted well with OG 20% 2X.The i r r eac t ions wi th the o the r OG f r ac tions and wi th F1 wer e e i the r weaker orabsen t excep t for anti- I M a which (cf. reference 2) reacted equally well with al lof these substances .

Ant i - I s e r a M a and O r t had been shown to d i f fe r f r om a ll o the r s e r a te s ted inreact ing w ith mil k f ract ion C, with the f ir s t IO4 s tages of A and B substan cesand wi th the P1 f r ac t ion of hum an B subs tance ( 1 , 2) . Howe ver , each has beenass igned to a dif ferent group (Table I , groups 1 and 2 respectively) s ince theirrelat ive react ivi t ies with these substances were dif ferent . The remaining anti- Isera and al l ant i- i sera gave weak or no react ions with these substances , andthe r e f ore M a and O r t wer e the on ly spec imens in gr oups 1 and 2 , re spec t ive ly .

Quant i t a t ive pr ec ip i f in a s says wi th the hyda t id cys t f lu id and wi th two cowsubs tances 21 and 18, r evea led f ur the r d i s t inc t pa t te r n s of r eac t ion among theremaining anti- I sera enabling their divis ion into four addit ional groups (Figs .1 and 2 and Table I ) . Gr oup 3 , S tep , Sch, W in , and N ay, showed s t rong r eac -t ions wi th hyd a t id cys t f luid and wi th cow 21 .

An ti- I sera Gre and D a w ere ass igned to groups 4 and 5 respectively, s ince thef or mer r eac ted we l l wi th hyd a t id cys t f lu id and ha r d ly a t a ll wi th cow 21 andthe la t ter reacted well with cow 21 but not a t a l l with hydatid cyst f luid.

Ser a Per , Ph i , and Too cons t i tu ted gr oup 6 and r eac ted poor ly or no t a t a l lwi th cow 21 and wi th hy da t id cys t f lu id.

The an t i - i s e r a , wi th the except ion of McDald , d id no t r eac t wi th the cowsubs tances or wi th hyda t id cys t f lu id. McDa ld , however , p r ec ip i ta ted moder -a te ly wi th hyda t id cys t f lu id and wi th mi lk f r ac t ion C .

Two of the an t i - I s e r a in gr oup 3 ( S tep and W in) r eac ted mo der a te ly wi th thef ir s t IO4 s tage of hu ma n H substan ce JS; S tep (cf. reference 2), however , reactedwith hog A + H f ir s t IO4 s tage while Win did not . Th e othe r two group 3 sera ,Sch and Nay, gave negl ig ib le r eac t ions wi th bo th subs tances sugges t ing tha t

Published


4/12

Group 1Mo 15/zl

I Tota lvol. 400 u.I

4 .I6 4 a o - -o - - .J k x . x ,

40 80

Group 2Ort 400 ff lTotal vol 1 ml

16-_ , l f ' l X2 - / ` i

o 4O 80

Group 3Step 20,u.I Win 75 u,I

2 l Total volume 50 0 ff,[ o . Total volume ,500 MIL6-&

O~ ' - - oi &"

, , , E i i i i i ~/ 140 80 120 160 200 40 80 t20 !60 20024q Nay 50 /zl

I Sch 50/z l ] / To ta lvo l . 300 f f l

So /_!/! ? o

40 80 20 160 200 500 40 80 i20 160 200 279.u-g ant ige n or /~1 hyda t id cyst f l u id

FIG. 1. Q uan t i t a t iv e p r ec ip i t in cu rves fo r an t i - I s e r a , g roups 1, 2, and 3.** Cer ta in s ubs tanc es w h ich gave l i t t le o r no s pec i fi c p r ec ip i t a t ion a r e no t s how n . T hes e

are : H um an A (M S S ) , B (Beach ) , H ( J S ) , and hog A + H o r ig inal s ubs tances . I n add i t ion ,cow 18 i s no t s how n w i th s e r a W in , N ay , G re , P e r , and T oo ; Beach P 1 i s no t s how n w i th s e r aW in and T oo . N o te tha t the am o un t s of c rude hyda t i d cys t f lu id u s ed as an t igen a r e exp res s edas m ic ro l i t e r s and no t a s m ic rog ram s , the quan t i ty o f ac t ive s ubs tance be ing unde te rm ined .

1250

Published


5/12

TEN FEIZI AND ELVIN A. KABAT 1251th is g r oup ma y eventua l ly be f ur the r subdiv ided . Th e an t i - i s e ra d id no t r eac twith the first IO4 stages of J.S. or of hog A + H.Two f r ac t ions ( phenol inso lub le and 10% f r om 20%) of hmn an Le a subs tance( N- I ) wer e te s ted which had ea rl i e r been f ound to c r os s- r eact appr ec iab ly wi thant i - I Ma and to a le s se r ex ten t wi th S tep ( 2) ; wi th the o the r an t i - I s e r a the i rreact ions were much weaker or negative. However , when a discernible react ionwas seen, the 10 % f rom 20 % f ract ion reacted m ore s trongly th an did the pheno l-insoluble f ract ion in ag reem ent w ith ear l ier f indings . No s ignif icant react ion withthe anti- i sera was found.

The an t i - P r l s e r um Ro d id no t p r ec ip i ta te wi th OG 20 % 2X over a wide r angeof concentrat ion s , nor did i t react w ith 46 58 ~g of : OG 10% from 20% , OG10% 2N, mi lk 20% 2N , cow 21 , hum an B ( Beach) P1 , and the f i rs t IO4 s tagesof hu ma n A (MSS) , B (Beach) , H (JS) , and of hog A + H.

Relative Proportions of I and i An tigenic Determin ants on OG 20 % 2 X . - - F r o mFigs. 1 - 3 i t i s appar en t tha t the amo unts o f OG 20 % 2) 4 r equi r ed to pr ec ip i ta tea g iven amo unt o f spec if ic n i t r ogen va r y wide ly f r om one a n t i - I o r an t i - i s e r umto another . This pres uma bly ref lects dif ferences in the numb ers of I a nd idete rmina nts of var ious specif ici t ies recognized by the sera . To mak e thef indings compar ab le , the da ta f r om pr ec ip i t in cur ves wer e ad jus ted to a un i f or mva lue of 6 gg of max imum pr ec ip i tab le a n t ibody N and f r o m these cur ves ,the qu ant i t i e s o f OG 20% 2) 4 r equi r ed to pr ec ip i ta te 3 pg of N ( 50% of themaxim um) w er e read . These da ta a r e g iven in the la s t co lumn of Table I I . I t i sev ident tha t OG 20% 2X i s ex t r emely ac t ive in pr ec ip i ta t ing an t i - I Ma andOrt , only ab out 3 gg being required for 50 % precipi ta t io n, while with al l of theother gro ups of anti- I an d w ith al l ant i- i sera f rom 4 to 16 t imes as much O G20% 2X was r equi r ed. Gr oups 3 and 6 appear ed r a the r un i f or m whi le the an t i -igroup showed considerable spread and might be considered to fal l into four

Symbols:OG fract ions , 20% 2X (O)10% from 20% (e)10% 2X (ID)20% from 10% ((1)

Milk fraction C (Q)Milk 20% 2X (~-)~1 ()Cow 21 (&)Cow 28 (~X)Human A (MSS) first IO4 stage (~7)Human B (Beach) first 104 stage stage (~)Beach P1 (~)Human Lea (N-I) phenol insoluble (A)X-1 10% of 1st 20% (m)Hydatid cyst fluid (V)Hog A -t- H first 104 stage (3)Human H (JS) first 104 stage (~)

Published


6/12

1252 IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS. LIVgroups. T he single examples in the anti-I groups 4 an d 5 differed from eachother and from groups 3 and 6.i Quantitative Inhibition Assays with Oligosaccharides.--Exploratory assayswere carried out with anti-I Step and anti-i Tho using OG 20% 2 X as antigen.

I0 -

6 ~

4 -

2 -

0

Group 4Gre 250 F.ITotal volume 450 p.I

40 80 120 160 20 0

Group 5Do 37.5 ,u.ITotal volume 250ff l

{

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o ~ , + , - ~ , ~ , , ' , , ,4 0 8 0 I 2 0 1 6 0 2 0 0

o 8 -5 .",5~J~_ 6-g -o 4-

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Group 6Per 150 ffl Phi 20 /zlTotal volume 40 0f fl Total volume 500,u.I

of\ ]4 ! /

40 80 120 160 200 40 80 120 160Too 10 .,~1

i T o t a l v o l u m e 4 0 0 f f l

8i o

I / , ~ t ~ - O i .~ ~ .~,0 l 'l ;' t- l~ i r i i i i i I I ~ " ~ - -4 0 8 0 i 2 0 I 6 0 2 0 0 2 4 0 2 8 0

F-g ant igen or ,u . I hydot id cys t f l u id

f2O O

FIo. 2. Q u a n t i t a t i v e p r e c i p i t i n c u r v e s for anti-I sera , groups 4, 5, and 6. For symbols se eFig. 1.

Published


7/12

TEN :FEIZI AND ELVIN A. KABAT 1253There was no signif icant inhibi t ion of precipi ta t ion wi th any of the ol igosac-charides a t the doses tested (Table III) .

DISCUSSIONT h e c l a s si f i ca t i o n of th e e l e v e n a n t i - I s e r a b a s e d o n t h e i r r e a c t i v i t y p a t t e r n s

in quan ti ta t ive precipit in assays with se lected blood group substances and with2 0 -

12

I 0 -

8 -6 -

4 -

2 -

0

(3"3) 6-~' 4-E 2-::L -

0

~o

15FITotal volume 300 ffl 40-

52-/ 24 -16 -< ,8 -

~ x 0 a 0 ~. ~40 80 120 160 200

Den diluted 1:16, 20 /zlTotal volume 5 00 ffl

o/ t,,~.~ , 9 - - + ,40 80 120

1 6 -

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le-~l 4 0160 200Ha 15p.ITotal vol, 5 0 ~ / ~

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Tho 7.5 fflTotal volume 5 00 ffl

Of40 80 120 160 200

Nic d i luted F16 20pI o/ /Total

oo/o

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40 so ~2o ,60 200 240

, oi40 80 120 160 200

fig antigen or/xl hydalid cyst fluidFro. 3. Quantit ative precipitin curves for anti-i sera. For symbols see Fig. 1.

Published


8/12

1254 I M M U N O C H E M I C A L S T U D I E S O N B L O O D G R O U P S . L IVhydatid cyst fluid (Table I) is consistent with the computation of the relativenumbers of antigenic determinants on OG 20% 2X reacting with the variousantisera (Table II). Further subdivisions of group 3 are, however, likely sincethey show differences in their reactions with th e first IO4 stage of hum an H (JS)and of hog A + H sub stances (Fig. 1 and Table I). The anti-I serum Win ap-peared transiently during convalescence in a patient with Mycoplasma pneu-

TABLE IClassification of Anti -I Sera on the Basis of their Reaction with OG 20% 2X ,

Cow 21, Milk Fraction C, Hydatid Cyst Fluid, and First 104 Stagesof A and B Substances

Gr oup S e r a OG 20% Cow 21 Hyda t i d Mi lk A and B2 X cyst f luid f ractio n C f ir s t IO4 Comm entss tages1 Ma S t r ong S t r ong \ : e r y weak S t r ong S t r ong Al l OG f r ac t ions r eac ted

s trongly as did F1 (1, 2)2 Or t S t r ong S t r ong n .d .* Mod . Mod . Reac t ion wi th m i lk and

with the f ir s t 104 s tages ofA and B s ubs tances washa l f a s s t r ong a s wi th OG20% 2X. Othe r OG f r ac -t ions and F I r eac ted inan in te r m edia te m anne r(1, 2)

3 S tep , S ch , S t r ong S t r ong S t r ong Neg . o r Neg . o rWin, N a y w e a k w e a k Var ia t ions wi th in g r oup inr e la t ive r eac t ions wi thOG f r ac tions and Mth thef ir s t IO4 s tages of humanI t and hog A + H sub-s tances

4 Gr e S t long Ver y weak S t r ong Neg . Neg . Othe r OG f r ac t ions ve r yweak o r nega t ive5 Da S t r cng S t r ong Neg . Ve r y weak Neg . Othe r OG f r ac t ions ve r y

weak: or negative6 P e r , P h i , S t r ong Neg . o r Neg . o r Neg . o r Neg . Othe r OG f r ac t ions , nod-

T oo weak weak weak e r a te o r weak* N o t t e s t e d .

moniae infect ion. I t fe ll into group 3 together w ith three anti- I sera f rom patie ntswith chronic cold agglutinin disease. These la t ter ant ibodies are W alde nstr6 mmacr oglobul ins . Ant i - I W in r esembled the W aldens t r 6m macr oglobulins inhaving on ly kapp a cha ins of r e s tr ic ted mobi l i ty in ac id ur ea s ta r ch ge l e lec t ro-phores is (7) . The computat ions in Table I I for anti- i sera suggest that there areat least four kinds of anti- i specificities.The nega t ive r eac t ion of the an t i - P r l s e r um Ro wi th OG 20% 2X ind ica testha t th i s g lycopr o te in though complex conta ins on ly a l im i ted n umb er of de -te r minants .

Published


9/12

TEN FEIZI AND ELVIN A. KABAT 1255The differences in the reactions of the various blood group glycoproteins with

the anti-I and anti-i sera are now clearly attributable to differences in theirrelative proportions of minor populations of incomplete chains with various Iand i specificities as has been emphasized previously (2). Thus while the firstIO4 stages of A and B substances create I determinants from complete A and Bdetermi nants by exposing receptors reacting with anti -I Ma a nd Ort, they m ay

TABLE IIEstimate of Reh tire Numbers of Antigenic Deierminants on OG 20~o 2~( Reacting wi th Various

Anti-I and Anti-i SeraMaximum AmountOG 20% 2X needed o AmountOG 20% 2X needed oGroup Ant ibo dy antibody precipitate0% of the precipitate #g N, when otalprecipitated antibody antibody s converted o 6 #g N

Anti-I tzg Ng izgMa 6 3 3Ort 7 3 2.6Step 19 52 16Win 6 20 20Sch 8 24 18Nay 20 76 23Gre 10 58 35Da 8 57 43Per 7.5 26 21Too 15 63 25Phi 6 18 18

Anti-iMcDald 20 40 12Tho 48 115 14Den 5.5 20 22Ho 19 90 28Nic 8.8 73 50

also uncover or destroy receptors reacting with the other anti-I or anti-i seradepending on the heterogeneity with respect to incomplete A and B deter-min an ts of the original A and B substance s.

The differences in Table II in the amounts of OG 20% 2X required to pre-cipitate 50 % of the a ntibody from the various anti- I and anti-i sera indicatethat it contains the different I and i determinants in appreciable propomons.This might be due to incomplete biosynthesis or might also be the result ofdegradation in the cyst cavity. It becomes of importance to establish whethersimilar variations in the numbers of receptors with these distinct specificities

Published


10/12

1256 IMMUNOC ttEMICA L STUDIES ON BLOOD GROUPS. LIVc a n b e f o u n d o n e r y t h r o c y t e s o r on f r e s h l y s e c r e t e d s o l u b l e b l o o d g r o u p s u b -s t a n c e s .I n h i b i t i o n a s s a y s wi t h t h e a v a i l a b l e o l i g o s a c c h a r i d e s ( Ta b l e I I I ) we r e e n t i r e l yn e g a t i ve . T h u s i n f o r m a t i o n o n t h e s t r u c t u r e s o f t h e d e t e r m i n a n t s r e a c t i n g w i t ht h e a n t i - I a n d a n t i - i o t h e r t h a n a n t i - I M a ( 2 ) a re c o m p l e t e l y la c k i n g . Th el i m i t e d a m o u n t s o f o l i g o s a c c h a r i d e s f r o m N- 1 a n d OG c y s t s p r e c l u d e d a s s a y s

TABLE I I IMaximum Amounts of Oligosaccharides Used in Quantitative Inhibition Assays with Ant i-I

Serum Step and Anti4 Serum Tho*Oligosaccharide Maximum amountSerum Step Serum Tho

IzmolesEthyl-~-GlcNA c 19.3 5.9,SDGal(1 --* 3)DGlcNA c 5.0 5.1BDGal(1 --* 4)9GlcN Ac 5.2 5.0~DGal(1 ~ 6)DGlcNAc 3.7 5.3N-1 Re 1.1 0.9 n.d.OG RL 1,1 n.d.:~ 0. 94N-1 RL 0.71b 0.3 0.3N-1 RL 0.41 0.6 n.d.OG RL 0.44 n.d. 0.4BoGlcNA c(1 --~ 6)

~DGal 1 --* 4)DSorbitol 0. 9 n.d.7~DGal(1 --* 3)BDGIcN Ac(1 --* 3)flDGlcNAc(1 --~ 6)

~DGal(1 --* 4)DS orbitol 0. 9 n.d .7/~DGlcNAc(1 --~ 3)* None of these oligosaccharides inhibited th e pre cipita tion of the two a ntisera wi thOG 20% 2X.~; No t tested.

a t h i g h e r c o n c e n t r a t i o n s o f i n h i b i t o r . Th u s o n e c a n n o t b e s u r e wh e t h e r t h eo t h e r a n t i - I a n d a n t i - i s e r a h a v e s p e c i f i c i t y i n v o l v i n g d i f f e r e n t d e t e r m i n a n t s o rwh e t h e r t h e s p e c i f i c i t y i s d i r e c t e d t o w a r d d e t e r m i n a n t s o f l a r g e r s iz e ( c f.r e f e r e n c e 1 8 ), a n d t h e s m a l l e r o l i g o s a c c h a r id e s t e s t e d we r e n o t u s e d i n s u f f i c ie n tc o n c e n t r a t i o n t o i n h i b i t .

SUMMARYQu a n t i t a t i v e p r e c i p i t i n a s s a y s we r e c a r r i e d o u t wi t h e l e v e n a n t i - I a n d f i v e

a n t i - i c o l d a g g l u t i n i n s e r a . Th e i r r e a c t i o n p a t t e r n s wi t h p r e c u r s o r s u b s t a n c e sa n d wi t h c e r t a i n a n t i g e n s r e l a t e d t o t h e A , B , H , Le% a n d L e b s u b s t a n c e s r e -

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TEN FEIZ I AND ELVIN A. KABAT 1257v e a l e d a t l e a s t s i x t y p e s o f I a n d f o u r t y p e s o f i s p e c i f ic i t y . A l l o f t h e I a n d id e t e r m i n a n t s a p p e a r t o b e r e p r e s e n t e d i n v a r y i n g a m o u n t s o n t h a t f r a c t io n o ft h e o v a r i a n c y s t p r e c u r s o r s u b s t a n c e O G w h i c h w a s p r e c i p i t a t e d b y 2 0 %e t h a n o l ( O G 2 0 % 2 X ) . O n l y s o m e of t h e s e d e t e r m i n a n t s c a n b e d e t e c t e d in t h ef i r st p e r i o d a t e o x i d a t i o n s t a g e s o f A , B , a n d H s u b s t a n c e s , i n c o w s u b s t a n c e s ,a n d i n h y d a t i d c y s t f lu i d.

REFERENCES1. Feizi , T. , E . A. Kaba t , G . Vicari, B. Anderson, and W. L. Ma rsh. 1971. Imm uno -

chemica l s tudies on b lood groups . XLVII . The I an t igen complex-precursorsin the A, B, H, Le a , and Le b b lood group sys tem-h emagglut ina t ion- inhib i t ionstudies. J . Exp. Med. 133:39.

2. Feizi , T. , E. A. Kaba t , G. Vicari, B. Anderson, and W. L. Marsh. 1971. Imm uno-chemical studies on blood groups. XLI X. The I an t igen complex: specif ici tydifferences among ant i-I se ra revealed by quanti tat ive prec ipi t in studies. Part ia lstructure of the I d eterm inant specif ic for one ant i-I serum. J . Immunol . 106:1578.

3. Vicari , G., and E. A. Kab at . 1969. Immun ochem ical studies on blood g roups.XL II . I so la t ion and charac ter iza t ion f rom ovar ian cys t f luid of a b lood groupsubstance lacking A, B, H, Le a and L eb specificity. J . [mmunol . 102:821.

4. Vicari , G., and E. A. Ka bat , 1970. Immun ochem ical studies on blood groups. XLV.Structure s and ac t ivi t ies of ol igosaccharides prod uced by alkal ine degrad at ionof a blood group substance lack ing A, B, H, Le~ and Leb specificities. Biochemis-t ry. 9:3414.

5. Wiener, A. S. , L. J . Unger, L. Cohen, an d J . F eldma n. 1956. Type-specif ic coldautoant ibodies as a cause of acquired hemolyt ic anemia and hemolyt ic trans-fusion react ions: biologic test w ith bovine red cel ls . Ann. In tern . Med. 44:221.

6. Marsh , W. L. 1961. Anti- i : a cold ant ib ody defining the I i relat ionship in huma nred cells. Brit. J. Haematol. 7:200.7. Feizi , T. , and M. S chumacher. 1968. Ligh t chain homoge neity of post- infect ivecold agglut inins. Clin. Exp. Immunol. 2:923.

8. Rosse, W. F., T. Borsos, and H. J . Rap p. 1968. Cold-react ing ant ibod ies: the en-hancement of ant ibody f ixa t ion by the f i rs t component of complement , C ' l a . J .Immunol . 100:259.

9. Roelcke, D., and G. Uhlenbruck. 1970. Let t er to Editor . Vox Sang. 18:478.10. M organ, W. T. J . 1960. The Croonian lecture: a contr ibut ion to huma n biochemi-

cal genet ics; the chemical basis of blood-group specif ici ty. Proc. Roy. Soc. Ser.B. Biol. Sci. 151:308.

11. Schiffman, G., E. A. Ka bat , and W. Thomp son. 1964. Immu noche mical studies onblood groups . XXX . Cleavage of A, B and H blood group subs tances by a lka l i .Biochemistry. 3:113.

12. Allen, P. Z., and E. A. Kab at . 1959. Immunoch emical studies on blood groups.XX II . Immunochemica l s tudies on the non-dia lyzable res idue f rom par t i a l lyhydrolyzed b lood group A, B and O(H) subs tances (P1 f rac t ions) . J . Immunol .82:340.13. Lloyd , K. O., E. A. Kaba t , and E. Licerio. 1968. Immunoch emical studies on blood

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1258 IMMUNOCFIEMICAL STUDIES ON BLOOD GROUPS. LIVgroups. X XX VI I I . S t ruc tures and ac t iv i t i es of o l igosacchar ides produced byalkal ine degrad at ion of blood group Lew isa substance. P roposed struc ture of thecarbohydra te cha ins of human blood group A, B, H , Le ~ and Le b subs tances .Biochemistry. 7:2976.

14. Leskowitz, S., and E. A . Kaba t . 1954. Immu noche mical studies on blood groups.XV. The effect of mild acid hydrolysis on the glucosamine and galaetosam ine inblood group substances. J. A mer. Chem. Soc. 76:5060.

15. L loyd, K. O., and E. A. Kab at . 1968. Immu nochem ical studies on blood groups.XLI . Proposed s t ruc tures for the carbohydra te por t ions of b lood group A, B,H, Lewis~ and Lewis~' substan ces. Proc. Nat. Acad. Sci. U.S.A. 61:1470.

16. Kabat , E. A. 1962. Immunochemical studies on blood groups. XXIX. Action ofvarious ol igosaccharides from hum an milk in inhibi t ing cross-react ions of typeXIV ant ipneumococca l sera wi th par t i a l ly hydrolyzed b lood group subs tances(P1 fract ions) . Arch. Biochem. Biophys. (Suppl. 1):181.

17. Beychok, S. , and E. A. Ka bat . 1965. Optical act ivi t y and conform ation of carbo-hydra tes . I . Opt ica l ro ta tory d i spers ion s tudies on immunochemica l ly reac t iveamino sugars and their glycosides, milk ol igosaccharides, ol igosaccharides ofglucose, and bloo d group substances. Biochemistry. 4:2565.

18. K abat , E. A. 1968. St ruc tura l Concepts in Immunology and Imm unochemis t ry .Hol t , Rinehar t and W ins ton Inc ., New York.

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