a new method in criminology

8/9/2019 A New Method in Criminology

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Z Rechtsmed (1984) 93:117-121Ze i t sch r i f t fo rRechtsmedizin Springer-Verlag 1984

A New Met ho d in Cr imino lo g y :U s e o f E L I S A t o D e t e c t A F P o n D i f fe r e n t M a te r ia lsw ith M o n o c lo n a l Anti- -fetoproteinE. Ku rucz 1, F. K4sa 1, l~. Mon osto ri 2, and I. And o 21 Dept. of Forensic Medicine, University of Szeged, Kossuth Lajos sgt. 40, H-6724 Szeged,Hungary2 Institute of Genetics, Biologic Research Cen ter, H ungarian A cademy of Sciences,

H-6701 Szeged, Hung ary

S u m m ary . A n ew m e t h o d h a s b een i n t ro d u ced t o d i s t i n g u i s h n o rm a l ad u l ts e ru m s t a i n s f ro m fe t a l o r n ew b o rn s e ru m an d am n i o t i c f l u i d s t a i n s w i t hEL IS A i n ca se s o f c r i m in a l ab o r t i o n an d i n fan ti c i d e. Th e m e t h o d i s b a s ed o nt h e s en si t iv e d e t ec t i o n o f a - f e t o p ro t e i n (A F P ) b y a tw o - s i te en zy m e i m m u n o -a s s ay (E I A ) fo l lo w i n g it s e l u t i o n w i th h i g h e f f i c i en cy f ro m d i f f e r en t m a t e -r ia ls ( e . g . , c o t t o n , p a p e r , s y n t h e t i c f ab r i c , o r g la ss ) b y p h o s p h a t e -b u f f e r ed0.5 M NaC1 solu t io nKey words: M o n o c l o n a l a n t i b o d y - E L I S A t e c h n i q u e - F e t a l um b i li c al c o r db lood-s ta in - Am nio t ic f lu id s ta inZusammenfassung: Es w u r d e e i n e M e t h o d e e r a rb e i t e t , u m i n F f il len v o nA b t r e i b u n g b z w . K i n d e s t 6 t u n g ( T 6 t u n g d e s N e u g e b o r e n e n ) z w i s c h e n d e nv o m N e u g e b o r e n e n h e r r t i h r e n d e n b i o l o g i s c h e n S p u r e n ( B l u t - u n d F r u c h t -w a s s e r f l e c k e ) u n d d e n v o n a n d e r e n I n d i v i d u e n s t a m m e n d e n B l u t f l e c k e nu n t e r s ch e i d en zu k 6 n n en . V o n d e r O b e r f l f i ch e d e r v e r s ch i ed en en S p u ren -t r f i g e r (B au m w o l l s t o f f , P ap i e r , K u n s t f a s e r u n d G l a s ) w u rd en d i e e i n g e -t ro ck n e t en F l eck e m i t 0 ,5 M N aC 1 en t h a l t en d em P h o s p h a t p u f f e r e l u i e r t u n dd a n n a u s d e n E l u a t e n d a s A l p h a - F e t o p r o t e i n m i t m o n o k l o n a l e n A n t i -k 6 r p e r n m i t H i l f e d e r E L I S A - T e c h n i k na c h g e w i es e n .Schliisselw6rter: M o n o k l o n a l e A n t i k 6 r p e r - E L I S A - T e c h n i k - N a b e l b lu t -f l e ck e - F ru ch t w as s e r f l e ck e

In spec ia l cases o f hom ic ide , i . e . , c r im ina l abor t i on and in fan t ic ide , the dem on-s t ra t ion o f f ind ings re la t ing to the fe tus o r in fan t a t the s i t e o f the c r imina l ac t iso f g rea t i m p o r t a n ce i n m ed i co l eg a l l ab o ra t o ry i n v es ti g a t io n s . I f t h e o r ig i n o f ab l o o d s t a i n i s u n k n o w n , t h e d i s t i n c t i o n b e t w een f e t a l u m b i l i c a l co rd s e ru m o ram n i o t i c f l u i d an d ad u l t s e ru m w o u l d p ro v i d e an d ad d i t i o n a l p o s s i b i l i t y fo rev i d en ce i n m ed i co l eg a l p r ac t i c e .Offprint requests to: F. K6sa, M .D., Associate Professor (address see above)


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118 E. Kurucz et al.AFP is a glycoprotein that has been detected at very low concentration

(10ng/ml) in normal adult serum and in higher concentrations [1, 4, 5, 8] inmatern al and fetal sera and amniotic fluid. It is synthesized by the yolk sac andfetal liver. After birth it decreases gradually, reaching the level of normalhealthy adults with in 1 month [2, 6, 9]. In standard clinical practice measure-ment of the AFP level in maternal serum and in amniotic fluid is used for thedetection o f neural tube defects [3, 11].

Fetal and adult blood stains may be distinguished via the detection of AFP.To detect AFP from dried and eluted samples on different trace supports (e.g.,cotton, paper, synthetic fabric or glass), we have used a sandwich ELISA tech-nique [2, 10].

R e a g e n t sThe two monoclonal antibodies used for ELISA were described by Monostoriet al. [7]. Anti-AFP-1 antibody was used to coat the plate. Anti-AFP-33 anti-body was conjugated with horseradish peroxidase according to Wilson andNakane [12]. The AFP standard was obtained from DAKO.

Mater ia l a nd Meth odsPreparation and processing of samples: 50, 100, 500, and 1,000 gl fetal umbilical cord serumand blood, amniotic fluid, normal adult serum and whole blood were applied to different solidsupports, such as cotton, paper, synthetic fabric, and glass. The samples were eluted for I h atroom temperature with 1 ml phosphate-buffered 0.5 M NaC1 at pH 7.2. The protein concen-trations of the eluates were measured at 280nm with a Spectromom 195 spectrophotometer.The extinction coefficient w a s E I ~ g/rnl 1.65. Sandwich ELISA is based on the method ofUotila et al. [10]. Briefly, the wells of polystyrene microtiter plates (DYNATECH) were coat-ed with 100 ~tl anti-AFP-1 monoclonal antibody solution (2.5 gg/ml) diluted with carbonatebuffer of pH 9.6. The plates were incubated at 4 C overnight. After incubation, the wells werewashed three times with phosphate-buffered saline of pH 7.2 containing 0.05% Tween 20(PBS-Tween). One hundred microliters standard AFP solutions or eluate samples were addedto the wells. The standards and sample dilutions were made in PBS-Tween buffer containing0.5% bovine serum albumin (Sigma). After three washing steps and the addition of 100glanti-AFP-33 HRPO, a dilution of 1:2,500 was incubated in each well at 37C for 1 h. Afterthree washing steps, 200 gl substrate solution (20 mg o-phenylenediamine and 10 ~t130%H202in 60 ml phosphate-citric acid buffer of pH 6.0) was added to each well. After a 30-min incuba-tion at 37C, the reaction was stopped with 50 gl 4 N H2SO4,and the absorbance was meas-ured at 492 nm with a Gilford spectrophotometer.

R e s u l t sThe dried samples could be stored at room temperature for at least 1 weekwithout change. The concentrations of the recovered proteins were measuredspectr ophoto metrica lly at 280 nm.

Figure 1 shows the amount of total protein in fetal umbilical cord serum(Fig. la) , amniotic fluid (Fig. lb ) and adult serum (Fig. lc) , and in their eluatesfrom cotton, paper, synthetic fabric, and glass supports. Of the total protein


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A New Method in Criminology 119[mg]

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CORD S E R U M- - - A F P C O N T E N T I N O ~ D I N A L S A M P L E S

- - - A F P C O N T E N T I N O ~ O I N AL S A M P L E S - - A F P C O N T E N T I N E L UA T ES F R O M- - AF P CONTENT N ELUATES FROM SOLID SUPPORTS "COTTON x SYNTHETrC FABRIC

SOLID SUPPORTS "[OTTON * SYNTNETI[ FABRIC o PAPE R ~OLASSo PAPER oOLASS

F i g . 3 a , b . T h e A F P c o n t e n t s o f t h e o ri g in a l a n d e lu t e d s a m p l e s o f f e t a l u m b i l ic a l co r d s e r u ma n d a m n i o t i c fl u id

e x t e n t o f A F P r e c o v e r y f r o m t h e so l id s u p p o r t s w a s 1 0 % - 4 0 % . S i m i l a r r e s u lt sw e r e o b t a i n e d w h e n w h o l e b l o o d w a s u s e d i n s te a d o f s e r u m ( d a t a n o t s h o w n ) .T h e r e w a s n o d e t e c t a b l e A F P i n e i t h e r t h e o r i g in a l o r t h e e lu t e d n o r m a l a d u l ts e r u m a n d w h o l e b l o o d .

D i s c u s s i o nI n m e d i c o l e g a l p r a c t i c e , c a s e s o f c r im i n a l a b o r t i o n a n d i n f a n t i c i d e o f t e n n e c e s s i -t a t e t h e d e t e c t i o n o f t r a c e s r e la t i n g t o t h e f e t u s o r t h e i n f a n t a t t h e s i t e o f t h ec r i m i n a l a ct a n d t h e d i s t i n c t i o n b e t w e e n f e ta l a n d a d u l t b lo o d - s t a i n s . W e h a v ed e v e l o p e d a t e c h n i q u e f o r t h e d i f f e r e n t ia t i o n o f f e t a l a n d a d u l t d r i e d s e r u m a n d


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A N e w M e t h o d i n C r i m i n o l o g y 12 1a m n i o t i c f l u i d s a m p l e s m i m i c k i n g t r a c e s o f a cr i m i n a l e v e n t . T h e d i s t in c t i o n isb a s e d o n t h e d e t e c t io n o f A F P . T h i s is p e r f o r m e d w i t h tw o m o n o c l o n a l a n t i -A F P a n t i b o d i e s , w h i c h re a c t w i t h tw o d i s t i n c t a n t i g e n ic d e t e r m i n a n t s o n t h em o l e c u l e . T h e a d v a n t a g e s o f u s i n g m o n o c l o n a l a n t i b o d i e s a r e th e i r s t ri c t s p e ci -f i ci t y a n d h i g h a f f i n i t y , a n d t h e r e a d y a v a i l a b i l i ty o f s t a n d a r d a n t i b o d i e s , t h e s ep r o p e r t i e s c o m p l y w i t h t h e r e q u i r e m e n t s o f m e d i c o l e g a l in v e s t ig a t i o n s . T h eb l o o d , s e r u m a n d a m n i o t i c fl u id s a m p l e s w e r e d r i e d o n d i f fe r e n t s o li d s u p p o r t s .O f t h e t o ta l p r o t e i n c o n t e n t 3 0 % - 9 0 % a n d 1 0 % - 4 0 % o f t h e t o t a l A F P w e r ee l u t e d f r o m t h e s o l i d s u p p o r t s .

T h e s e n s i t iv i ty o f t h e a p p l i e d s a n d w i c h E L I S A t e c h n i q u e a l lo w s th e q u a n t i -t a t i v e d e t e r m i n a t i o n o f A F P f r o m a s l it tl e a s 5 0 ~tl f e t a l b l o o d a n d 1 0 0 g l a m n i o -t ic f l u id o n a s u p p o r t , b u t t h e r e i s n o d e t e c t a b l e A F P i n t h e s t a i n s o f a d u l t b l o o do r s e r u m .Acknowledgment. W e a r e g r a t e fu l t o M . D . J . Sz a b 6 , H o s p i t a l I I , Sz e g e d (H u n g a r y ) f o r co l-lec t ing the fe ta l umbil ica l cord b loo d and m atern al b lo od samples .AbbreviationsAFP, a - f e t o p r o t e i n ; E I A , e n z y m e i m m u n o a s s a y ; E L I SA , e n z y m e - l i n k e d i m m u n o s o r b e n tassay; PBS-Tween, phosphate-buffered sa l ine conta ining 0 .05% Tw een 20; H R PO , horseradishpe rox idase ; an t i -AFP-33 HRPO, an t ibody con juga ted wi th ho rse rad i sh pe rox idase

Re f e r enc e s

1 . A b e l e v G L ( 19 65 ) A n t i g e n i c s tr u c tu r e o f c h e m i ca l ly in d u c e d h e p a t o m a s . P r o g E x p T u m o rR e s 7 : 1 0 4 - 1 5 72 . B e l a n g e r L , H a m e l D , D u f o u r D e t a l. ( 19 76 ) D o u b l e - a n t i b o d y en z y m e i m m u n o a s s a ya p p li e d t o h u m a n a l p h a - l -f e t o p r o te i n . C li n C h e m 2 2 : 1 9 8 - 2 0 43 . Bro ck DJ , W ald NJ , Cuck e H (1977) Organ isa t ion o f ma te rna l se rum a lpha fe top ro te insc reen ing fo r f e ta l neu ra l - tube de fec ts . Lan ce t I : 7004 . Caba l le ro C , Vakamans M, Lopez KJG, Rob in C (1977) Se rum a lpha - fe top ro te in inadu l t s, in wom en du r ing p reg nancy , in ch i ld ren a t b i r th and du r ing the f i r s t wee k o f l if e:A sex d i f ference . Am J O bs te t G yneco l 127 : 384 -3895 . Gi t l in D , Bo esm an M (1966) Se rum a lph a - fe top ro te in , a lbumin and gamm a-G-g lobu l in inthe hu m an conce pt . J Cl in Inve st 45 : 1826-18386 . G i t lin D , Bo esm an M (1967) S i te s o f se rum a lpha - fe top ro te in synthesi s in the hum an an din th e ra t. J Clin Inv est 46 : 1010-10167 . M onos to r i E , Sz iics P , Veres G , A nd6 I (1984) The mea su rem en t o f ma te rna l se ruma lpha - fe top ro te in by ELISA sys tem us ing monoc lona l an tobod ie s . Orvos i he t i l ap ( sub -mi t ted )8 . Ruoslah t i E, Sep pala M (1971) Studies of carc ino-fe ta l pro te ins : physica l and chem icalp rope r t i e s o f huma n a lpha - fe top ro te in . In t J C ance r 7 : 218 -2259 . R uos lah t i E , Seppa la M (1972) Alpha - fe top ro te in in no rm a l se rum. N a tu re 235 :1 61 -16210 . Uo t i l a M , Engva l l E , Ruos lah t i E (1980) M onoc lona l an t ibod ie s to huma n a l fa - fe to -p ro te in . M ol Im mu no l 17 : 791 -79411 . Wald NJ , Cuck le H , Brock JH e t a l . (1977) Mate rna l se rum-a lpha - fe top ro te in measu re -me n t in an tena ta l s c reen ing fo r sp ina b if ida in ea r ly p regnancy . R epo r t o f UK co l labo ra -t ive s tudy on a lpha - fe top ro te in in re la t ion to neu ra l - tube de fec t s. L ance t I : 1323 -13321 2. W i ls o n MB , N a k a n e P K ( 19 78 ) R e c e n t d e v e l o p m e n t s i n t h e p e r i o d a te m e t h o d o f c on -juga t ing ho rse rad i sh pe rox idase (HR PO ) to an t ibod ie s . In : Kn app W e t a l. (eds)Imm unof luo rescence and re la ted s ta in ing techn iques . E l sev ie r , Nor th -H o l land , A ms te r -d a m , p p 2 1 5 - 2 2 4

Rece ived March 5 , 1984

a new method in criminology

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