Upsala J Med Sci 91: 17-27, 1986 Determination of Sialic Acid Residues in Tkansferrin by Oxidative-Reductive Immunoassay Erik Cervkn and Gunnar Ronquist Institute of Medical and Physiological Chemistry, Biomedical Center and Department of Clinical Chemistry, University Hospital, University of Uppsala, Sweden ABSTRACT A method i s d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f s i a l i c a c i d r e s i d u e s i n g l y c o p r o t e i n s d i s p l a y i n g m i c r o h e t e r o g e n e i t y i n t h e s u g a r r e s i d u e . The new method i s based o n c o m b i n i n g an o x i d a t i v e - r e d u c t i v e s t e p w i t h b i n d i n g o f t h e g l y c o p r o t e i n t o an immunoadsorbent. A f t e r a m i l d o x i d a t i o n w i t h sodium meta- p e r i o d a t e t h e s u g a r i s r e d u c e d w i t h l a b e l e d s o d i u m b o r o t r i t i d e . The c h e m i c a l m o d i f i c a t i o n o f t h e s u g a r r e s i d u e s does n o t seem t o i m p a i r ' t h e b i n d i n g o f g l y c o p r o t e i n t o t h e immunoadsorbent. The p r o c e d u r e , w h i c h has been e l a b o r a - t e d f o r human t r a n s f e r r i n , can be c a r r i e d o u t i n t h e p r e s e n c e o f o t h e r sub- s t a n c e s i n body f l u i d s . INTRODUCTION Combined p h y s i o c o c h e m i c a l s e p a r a t i o n t e c h n i q u e s 1 i k e i s o e l e c t r i c f o c u s s i n g and c h r o m a t o f o c u s s i n g a r e p r e c i s e b u t time-consuming methods t h a t a r e n o t s u i t a b l e f o r use on a l a r g e m u l t i - s a m p l e s c a l e b a s i s . Such methods a l s o r e q u i r e e x p e n s i v e equipment and c h e m i c a l s as w e l l a s s k i l l e d manual g u i d a n c e . When complex m i x t u r e s a r e a n a l y z e d , t h e two methods g e n e r a l l y have t o be complemented w i t h some k i n d o f i d e n t i f i c a t i o n o f t h e i n v e s t i g a t e d p r o t e i n . F o r t h e s e r e a s o n s such methods a r e n o t always s u i t a b l e f o r r o u t i n e a n a l y t i c a l use on a m i c r o s c a l e b a s i s . T h e r e f o r e , new methods a r e needed t h a t a r e c o m p a t i b l e w i t h r a p i d and s i m u l t a n e o u s p r o c e s s i n g o f a l a r g e number o f samples, and w h i c h p e r m i t q u a n t i t a t i v e measurements. F o r example, t h e m i c r o h e t e r o g e n e i t y o f g l y c o p r o t e i n s m o s t l y i n v o l v e s s i a l i c a c i d and i s a s t a t i s t i c a l phenomenon, where t h e a v e r a g e c o n t e n t o f s i a l i c a c i d u s u a l l y g i v e s more i n f o r m a t i o n t h a n t h e r e l a t i v e amount of one p a r t i c u l a r i s o e l e c t r i c component o u t o f s e v e r a l . I n such cases no u s e f u l i n f o r m a t i o n i s l o s t b y r e c o r d i n g average c o n c e n t r a t i o n s o f s i a l i c a c i d . R e c e n t l y , a method has been p r e s e n t e d h a v i n g t h e d e s i r e d p r o p e r t i e s , t h e r a t i o n a l name o f w h i c h i s R a d i o - L e c t i n Immunoassay (1). However, a m a j o r drawback o f methods i n v o l v i n g l e c t i n s i s t h a t m o s t l e c t i n s b i n d w i t h l o w 2 - 868571 17 a f f i n i t y t o t h e i r c o r r e s p o n d i n g sugars. T h i s means t h a t t h e c o n d i t i o n s have t o be s t a n d a r d i z e d w i t h r e s p e c t t o s e v e r a l p a r a m e t e r s t h a t a r e d i f f i c u l t t o c o n t r o l , such as t h e t u r b u l e n c e o c c u r r i n g when r e p l a c i n g t h e b u f f e r d u r i n g w a s h i n g t h e inimunoadsorbent. Hence, t h e r e i s an a c t u a l want o f r a p i d and r e l i a b l e methods f o r d e t e r m i n a t i o n o f s u g a r r e s i d u e s i n g l y c o p r n t e i n s . The p r e s e n t p a p e r d e s c r i b e s a s i m p l e and r a p i d method f o r d e t e r m i n a t i o n o f s i a l i c a c i d r e s i d u e s i n g l y c o p r o t e i n s d i s p l a y i n g m i c r o h e t e r o g e n e i t y . The method i s a t l e a s t s e m i q u a n t i t a t i v e and w i l l t h e r e f o r e r e p l a c e i n some i n s t a n c e s q u a l i t a t i v e p r o c e d u r e s l i k e i s o e l e c t r i c f o c u s s i n g . MATERIALS B u f f e r s a l t s , c i t r i c a c i d and u n l a b e l e d sodium b o r o h y d r i d e were r e a g e n t g r a d e from Merck AG, Darmstadt, W . Germany. T r i s , t r a n s f e r r i n , n e u r a m i n i - dase, a l b u m i n and N ’ N ’ - d i m e t h y l f o r m a m i d e were from Sigma, S t L o u i s , Miss. USA. A f f i - G e l 10 was f r o m B i o r a d L a b o r a t o r i e s , Richmond, C a l i f . USA, a n t i - t r a n s f e r r i n ( n e p h e l o m e t r i c t i t e r , 0.35 mg o f a n t i g e n h l ) f r o m K a l l e s t a d , Behringwerke, F r a n k f u r t am Main, W . Germany, t r i t i a t e d sodium b o r o h y d r i d e (350 mCi/mmol) and Aquasol f r o m NEN Chemicals, D r e i e i c h , W . Germany. B l u e D e x t r a n and Sephadex 6200 were f r o m Pharmacia AB, Uppsala, Sweden. METHODS P r e p a r a t i o n o f Immunoadsorbent. Two m l o f A f f i - G e l 10 was washed 5 t i m e s w i t h an i c e - c o l d s o l u t i o n c o n s i s t i n g o f 20 mM Na2HP04, 100 mM NaC1, pH a d j u s t e d t o 7.5 w i t h HC1 (PBS, pH 7.5). Twenty mg o f t r a n s f e r r i n were d i s s o l v e d i n 1 m l o f t h e same b u f f e r , t h e g e l suspended i n a t o t a l volume o f 3 m l and t r a n s f e r r e d t o t h e t r a n s f e r r i n s o l u t i o n . A f t e r i n c u b a t i o n f o r 30 min a t room t e m p e r a t u r e w i t h g e n t l e m a g n e t i c s t i r r i n g , CaC12 was added and t h e c o u p l i n g r e a c t i o n c o n t i n u e d f o r a n o t h e r 30 m i n a t room t e m p e r a t u r e . 0.2 m l of 1 M e t h a n o l a m i n e - HC1, pH 8.0 were t h e n added f o l l o w e d b y i n c u b a t i o n w i t h s t i r r i n g f o r 1 h a t room t e m p e r a t u r e , t h e g e l t r a n s f e r r e d t o a P a s t e u r p i p e t t e and washed o v e r n i g h t w i t h PBS, pH 7.5 (200 ml) and t h e n w i t h 100 m l o f 150 mM NaCl phosphate c i t r a t e b u f f e r p r e p a r e d b y a d d i n g s o l i d Na2HP04 t o 10 mM c i t r a t e u n t i l t h e pH r e a c h e d 2.8 ( p h o s p h a t e - c i t r a t e , pH 2.8). S u b s e q u e n t l y i t was washed w i t h 100 m l o f PBS, pH 7.5, c o n t a i n i n g 1 M NaC1, and t h e n a g a i n w i t h p h o s p h a t e - c i t r a t e . F i n a l l y i t was e q u i l i b r a t e d w i t h PBS, pH 7.5, and 20 m l o f a n t i t r a n s f e r r i n i n t h e same b u f f e r were r e c i r c u l a t e d f o r 3 h o r o v e r - n i g h t a t 4OC, t h e column washed w i t h PBS, pH 7.5 c o n t a i n i n g 1 M NaCl f o l l o w e d b y e l u t i o n o f t h e a n t i t r a n s f e r r i n b y t h e p h o s p h a t e - c i t r a t e b u f f e r . A p p r o x i m a t e l y 10 mg o f a n t i t r a n s f e r r i n were o b t a i n e d , j u d g i n g f r o m A280 and w i t h t r a n s f e r r i n a s a s t a n d a r d . The e l u t e d a n t i t r a n s f e r r i n was n e u t r a l i z e d and 18 c o n c e n t r a t e d t o a p p r o x i m a t e l y 10 mg/ml i n PBS, pH 7.5. One m l o f A f f i - G e l i n 2 ml o f PBS, pH 7.5 was t h e n added and t h e c o u p l i n g r e a c t i o n a l l o w e d t o t a k e p l a c e w i t h g e n t l e s t i r r i n g a t room t e m p e r a t u r e f o r 2h. T h e r e a f t e r , 0.2 ml o f 1M e t h a n o l a m i n e -HC1, pH 8.0 were added and t h e i n c u b a t i o n c o n t i n u e d f o r 1 h a t room t e m p e r a t u r e . F i n a l l y , t h e g e l was t r a n s f e r r e d t o PBS, pH 7.5 as a 50% packed g e l s u s p e n s i o n . Neuraminidase D i g e s t i o n o f Transfer& Twenty mg o f human t r a n s f e r r i n c o n t a i n i n g a p p r o x i m a t e l y 0.9 pmoles o f s i a l i c a c i d were d i s s o l v e d i n 1 ml o f 0.1 M sodium phosphate b u f f e r , pH 6.5 and i n c u b a t e d w i t h 0.5 U o f n e u r a m i - n i d a s e from D a c t y l i u m D e n d r o i d e s f o r 30 m i n a t 37OC. (One u n i t o f n e u r a m i n i - dase i s d e f i n e d a s t h e amount n e c e s s a r y t o l i b e r a t e 1.0 umole o f s i a l i c a c i d p e r m i n a t pH 5.0 and a t 37OC). A f t e r d i g e s t i o n , B l u e D e x t r a n was added as a m a r k e r f o r t h e v o i d volume and t h e sample f r a c t i o n a t e d i n PBS, pH 7.0 (20 mM Na2HP04, 150 mM NaC1, pH a d j u s t e d w i t h HC1) on a Sephadex 6-200 column w i t h a d i a m e t e r o f 1.2 cm and a l e n g t h o f 38 cm. The t r a n s f e r r i n was e l u t e d and l o c a t e d b y i t s a b s o r p t i o n a t 280 nm. The c o n c e n t r a t i o n o f t r a n s f e r r i n was a d j u s t e d t o 1 mg/ml w i t h n a t i v e t r a n s f e r r i n as a s t a n d a r d . M i x t u r e s o f t h e n e u r a m i n i d a s e - d i g e s t e d t r a n s f e r r i n and t r a n s f e r r i n were t h e n used f o r c o m p a r a t i v e a n a l y s i s o f s i a l i c a c i d u s i n g t h e d e s c r i b e d method. A c i d H y d r o l y s i s o f T r a n s f e r r i n . Ten mg o f t r a n s f e r r i n were h y d r o l y z e d i n 1 ml o f 1N H2S04 a t 8OoC f o r I h , t h e s o l u t i o n n e u t r a l i z e d w i t h K2HP04 and t h e a s i a l o t r a n s f e r r i n s e p a r a t e d on t h e Sephadex 6200 column d e s c r i b e d above. Comparative a n a l y s i s o f s i a l i c a c i d . The a n a l y s i s o f s i a l i c a c i d i n t r a n s - f e r r i n was c a r r i e d o u t b y c o m b i n i n g an o x i d a t i v e - r e d u c t i v e s t e p w i t h b i n d i n g t h e g l y c o p r o t e i n t o a n t i t r a n s f e r r i n immunoadsorbent. The volume and c o n c e n t r a - t i o n s o f t h e v a r i o u s s o l u t i o n s v a r i e d a c c o r d i n g t o t h e sequence i n w h i c h t h e s e s t e p s were p e r f o r m e d and t h e p r e s e n c e o r absence o f o t h e r g l y c o p r o t e i n components t h a n t r a n s f e r r i n . The f o l l o w i n g s o l u t i o n s were used: PBS, pH 7.0: 20 mM Na2HP04, 150 mM NaC1, pH a d j u s t e d w i t h HC1. T r i s - g l y c i n e - g l y c e r o l : 0.1 M T r i s - b a s e a d j u s t e d t o pH 8.0 w i t h 0.1 M g l y c i n e and g l y c e r o l added t o 0.1 M. NaOH/N’N:dimethylformamide (1: 1) t o a f i n a l c o n c e n t r a t i o n o f 1.78 m C i / m l . Gel p r o t e c t i n g r e a g e n t . T h i s m i x t u r e p r o t e c t e d t h e s o l i d phase g e l , b y i n h i - b i t i n g t h e r e a c t i o n o f l a b e l e d compounds w i t h t h e p a r t i c u l a r t y p e o f g e l used, and was p r e p a r e d as f o l l o w s : One volume o f PBS, pH 7.0 c o n t a i n i n g 2 mM sodium m e t a - p e r i o d a t e was i n c u b a t e d w i t h one volume o f T r i s - g l y c i n e - g l y c e r o l f o r 30 m i n a t room t e m p e r a t u r e , and t h e n w i t h one volume o f 50% 0.1 M NaOH and 50% N’N’-dimethylformamide c o n t a i n i n g 2mN sodium b o r o h y d r i d e . B o r o t r i t i d e s o l u t i o n : T r i t i a t e d sodium b o r o h y d r i d e d i s s o l v e d i n 0.1 M 19 Washing solution PBS, pH 7 . 5 , containing 10% N- N- -dimethyl-formamide. 0.1 mg of transferrin were routinely used in the assay. The oxidative step was always performed after the solutions had reached O°C in an ice-bath. Sodium meta-periodate (2mM) in PBS, pH 7.0 was added for oxidation which was allowed to proceed for 10 min. The reactions were quenched with Tris-gly- cine-glycerol and then either reduced with borotritide or washed and equi- librated with 20 ~1 of imnunoadsorbent suspension. The details are described in the Figure legends. After reduction, the gel was washed free of excess reagent, transferred t o a scintillation via7 and counted by liquid scintillation using 10 ml of Aquasol. Fig.1 0.1 mg of transferrin was dissolved in 0.1 ml of PBS, pH 7.0 and 0.1 m l of the same buffer containing 2 mM sodium metaperiodate was mixed at ice-temperature and incubated for 10 min. A mixture from 0.1 M glycine and 0.1 M Tris, pH 8.0 containing 0.1 M glycerol was then added followed by incubation with stirring at room temperature for 30 min. The mixture was kept in ic’e for 30 m.in and 0.1 m l sodium borotritide, 1.78 mCi/ml, in 50% 0.1M NaOH/50% N’N’-dimethylformamide added. After incubation on ice for 30 min the labeled transferrin was separated from excess reagent by passing the mixture over a Sephadex 6 - 2 5 column. The total fractions were mixed with Aquasol and counted for tritium, ( 0 ) transferrin, (0) neuraminidase-digested transferrin, (x) acid-hydrolyzed transferrin (0.1 N H2S04 for lh at 8 O o C ) . 20 RESULTS AND D I S C U S S I O N One hundred ug o f t r a n s f e r r i n o r d e s i a l y l a t e d t r a n s f e r r i n were l a b e l e d w i t h sodium meta-periodate-borotritide and t h e n s e p a r a t e d f r o m excess l a b e l e d b o r o t r i t i d e b y f r a c t i o n a t i o n on Sephadex 6-25 columns ( P D - 1 0 , Pharmacia A B ) ( F i g . 1). Subsequent e s t i m a t i o n o f t h e s u r f a c e a r e a u n d e r t h e v o i d peak c o n t a i n i n g t h e l a b e l e d p r o t e i n i n d i c a t e d t h a t s i a l o t r a n s f e r r i n i n c o r p o r a t e d a p p r o x i m a t e l y 3 t i m e s more l a b e l t h a n a s i a l o t r a n s f e r r i n . The a s i a l o t r a n s f e r r i n w h i c h was p r e p a r e d b y n e u r a m i n i d a s e d i g e s t i o n o r h y d r o l y s i s i n 0.1 N H2S04 a t 8 O o C f o r 1 h gave s i m i l a r r e s u l t s . The o x i d a t i o n w i t h p e r i o d a t e i s n o t e x c l u - s i v e l y s p e c i f i c f o r s i a l i c a c i d (2) and t h e r e f o r e , a c e r t a i n background a c t i v i t y i n t h e absence o f s i a l i c a c i d must be a n t i c i p a t e d . F i g . 2A E s t i m a t i o n o f t r a n s f e r r i n - b i n d i n g c a p a c i t y o f t h e a n t i t r a n s f e r r i n q e l . CPI 200 T r a n s f e r r i n was l a b e l e d - b y o x i d a t i o n w i t h 1 mM NaI04 a t pH 7.0, O°C f o r 10 m i n f o l l o w e d b y q u e n c h i n g w i t h 0.1 M g l y c i n e - T r i s c o n t a i - n i n g 0.1 M g l y c e r o l and t r i t i a t i o n w i t h sodium b o r o - t r i t i d e . The l a b e l e d s i a l o - t r a n s f e r r i n was t h e n t r a n s - f e r r e d t o PBS pH 7.5 b y g e l f i l t r a t i o n on Sephadex 6-25 (PD-10 column) and d i l u t e d t o 1 mg/ml and $ s p e c i f i c a c t i v i t y o f 5x10 cpm p e r mg. Samples o f 2-150 ug o f l a b e l e d t r a n s f e r r i n were t h e n d i l u t e d t o e q u a l volume and i n c u b a t e d w i t h 20 u1 o f 50% ( V / V ) a n t i t r a n s f e r r i n g e l suspension, o r c o n t r o l g e l suspension, t h e m i x t u r e d i l u t e d t o 3 m l w i t h PBS pH 7.5 and t h e g e l p e l l e t e d a f t e r w h i c h 0.1 m l o f t h e s u p e r n a t a n t was t r a n s f e r r e d t o a s c i n t i l l a t i o n v i a l and c o u n t e d w i t h Aquasol u s i n g t h e t r i t i u m c h a n n e l i n a l i q u i d s c i n t i l l a t i o n coun- t e r . The p o i n t s r e p r e s e n t t h e r a d i o a c t i v i t y c o n t a i n e d i n t h e s u p e r n a t a n t , i n ( x ) t e s t t u b e o n l y ; ( 0 ) : A f f i - -Gel -10, t r e a t e d as f o r b i n d i n g a n t i t r a n s f e r r i n ; (0): A f f i - G e l c o u p l e d t o a n t i t r a n s f e r r i n . 21 Labeled sialotransferrin was prepared and used to assess the antigen-binding capacity of the antitransferrin gel in relation to unspecific adsorption (Fig 2A,B). It is evident from Fig 2A, where a sample from the supernatant not reacting with the gel is analyzed, that virtually no background binding of labeled transferrin occurred, neither to the gel nor to the test tube. Ten p1 of the antitransferrin gel bound 10-25 pg of transferrin when equilibrated with a total amount of 15-150 pg in solution, judging from the control standard curve with no transferrin antibodies (Fig 2 A ) . When 100 pg of transferrin were added, approximately 22 pg bound to the gel. In Fig 28 where the amount of gel was varied, figures from 18-36 pg of bound transferrin per 10 u l of antitransferrin gel ( 2 0 P I o f gel suspension) were obtained. The higher values probably represented a saturation phenomenon in the equilibrium binding of the antigen-antibody during the short (30 min) incubation time used. The results clearly show that approximately 20 ug of transferrin bound to 10 p1 of gel. CPM 150C 1000 500 I I I I 20 40 60 80 GEL SUSPENSION ( p l ) :i,g. 2i’ Same experiment as except that the amount of gel suspension was varied and the amount of labeled transferrin kept constant, 10.0 vg: The radioactivity remaining in 0.1 ml of supernatant after incubation of the mixture, dilution to 3 ml, and pel- leting was measured. 22 Fig. 3 A Combination o f oxi d a t i a n - r e d u c t i on w i t h binding , of t r a n s f e r r i n followed by s e p a r a t i o n of the l a b e l e d immunocomplex i n a s i n g l e step. The procedure i n F i g 2 was followed, P B S , pH 7.5 was added t o a t o t a l volume o f 0.5 ml and 20 vl of t h e a n t i t r a n s f e r r i n gel added and t h e mixture incubated w i t h vigorous shaking f o r 30 m i n a t room temperature. A f t e r i n c u b a t i o n , the gel was allowed t o sediment, t h e s u p e r n a t a n t a s p i r a t e d and t h e gel washed 5 times w i t h 5-10 m l o f washing s o l u t i o n . The f i n a l s u p e r n a t a n t was checked t o a s s u r e c l o s e t o background-levels of r a d i o - a c t i v i t y : 0-0 T r a n s f e r r i n o n l y , 0-0 T r a n s f e r r i n with 25 u1 of serum. 0 20 40 60 80 100 SIALOTRANSFERRlN( PER CENTOF ADDED AMOUNT OF TRANSFERRIN INCLUDING ASIALOTRANSFERRIN ) 23 TOTAL C PM 1o.ooc 8000 6000 4 000 2000 1 1 I I F i g . 38 C o m b i n a t i o n o f o x i d a t i o n - r e d u c t i o n w i t h b i n d i n g o f t r a n s f e r r i n f o l l o w e d by s e p a r a t i o n o f t h e l a b e l e d immunocomplex'in a s i n g l e s t e p . The c o n c e n t r a t i o n o f T r i s g l y c i n e - g l y c e r o l was r e d u c e d t o h a l f b y d i l u t i n g w i t h w a t e r , o t h e r w i s e t h e o x i d a t i o n - q u e n c h i n g - r e d u c t i - on sequence f o l l o w e d . The a n t i t r a n s f e r r i n g e l was f i r s t i n c u b a t e d w i t h g & l - p r o t e c t i n g r e a g e n t a t 0 C. The c o m p o s i t i o n o f t h i s r e a g e n t i s d e s c r i b e d i n t h e Methods s e c t i o n . T h e r e a f t e r t h e t r a n s f e r r i n was t r a n s f e r r e d t o t h e g e l s u s p e n s i o n and t h e m i x t u r e i n c u b a t e d f o r 60 m i n a t room t e m p e r a t u r e f o l l o w e d b y washing. 0 20 40 60 80 100 SIALOTRANSFERRIN (PER CENTOF TRANSFERRIN ) 24 I n F i g 3A-C, t h e o x i d a t i o n - r e d u c t i o n o f t r a n s f e r r i n was combined w i t h b i n d i n g t h e g l y c o p r o t e i n t o t h e immunoadsorbent and s e p a r a t e d f r o m t h e m i x t u r e i n a s i n g l e s t e p . I n 3A, t h e p r o c e d u r e i n F i g 2 was f o l l o w e d and t h e g l y c o p r o t e i n i n c u b a t e d w i t h t h e g e l i n t h e p r e s e n c e o f excess r a d i o a c t i v e p r o d u c t s . The f i n a l s u p e r n a t a n t a f t e r w a s h i n g was checked t o a s s u r e c l o s e t o background l e v e l s o f r a d i o a c t i v i t y . The h i g h background a c t i v i t y w i t h a s i a l o t r a n s f e r r i n o n l y , was due t o c h e m i c a l r e a c t i o n o f t h e r a d i o a c t i v e p r o d u c t s w i t h t h e g e l m a t r i x , s i n c e i t was o b s e r v e d w i t h a s i m i l a r l y t r e a t e d g e l l a c k i n g a n t i t r a n s f e r r i n . The h i g h background a c t i v i t y was n o t r e d u c e d b y t r e a t i n g t h e g e l w i t h 1 mM sodium b o r o h y d r i d e p r i o r t o e x p o s u r e t o t h e r a d i o a c t i v e p r o d u c t s . The two c u r v e s i n F i g 3A r e p r e s e n t t r a n s f e r r i n o f v a r i o u s s i a l i c a c i d c o n t e n t w i t h o r w i t h o u t 25 u1 o f serum added b e f o r e t h e o x i d a t i v e s t e p . As e x p e c t e d t h e a d d i t i o n o f serum c o n t a i n i n g s i a l y l a t e d t r a n s f e r r i n i n c r e a s e d t h e l a b e l i n g o f t h e samples w i t h a l o w c o n t e n t o f t r a n s f e r r i n and a h i g h c o n t e n t o f a s i a l o t r a n s f e r r i n . The c o n c e n t r a t i o n o f t r a n s f e r r i n i n t h e serum sample can be e s t i m a t e d t o be 3 . 4 g / 1 f r o m t h e r a t i o o f d i l u t e d / u n d i l u t e d sample s i n c e t h e t o t a l amount o f t r a n s f e r r i n added exceeded t h e t r a n s f e r r i n - b i n d i n g c a p a c i t y o f t h e immunoadsorbent. T h i s was w i t h i n t h e normal r a n g e ( 4 ) , d e m o n s t r a t i n g t h a t t h e method was r e l i a b l e q u a n t i t a t i v e l y . I n F i g 3B, t h e g e l was f i r s t i n c u b a t e d w i t h t h e g e l - p r o t e c t i n g r e a g e n t b e f o r e i n c u b a t i o n w i t h t h e t r a n s f e r r i n m i x t u r e c o n t a i n i n g r a d i o a c t i v e p r o d u c t s . T h i s d e c r e a s e d t h e background r a d i o a c t i v i t y s i g n i f i c a n t l y . The o b s e r v e d background a c t i v i t y i n F i g 38, u s i n g a s i a l o t r a n s f e r r i n o n l y , was a p p r o x i m a t e l y 1/3 o f t h e i n c o r p o r a t i o n o f t r i t i u m i n t h e f u l l y s i a l y l a t e d t r a n s f e r r i n . T h i s p e r c e n t a g e was comparable t o t h e u n s p e c i f i c i n c o r p o r a t i o n i n t o a s i a l o t r a n s f e r r i n as d e m o n s t r a t e d i n F i g 1, w h i c h was most p r o b a b l y due t o i n c o r p o r a t i o n o f l a b e l i n t o o t h e r s u g a r r e s i d u e s t h a n s i a l i c a c i d . I n F i g 3C, t h e t r a n s f e r r i n was f i r s t o x i d i z e d , t h e n bound t o t h e a n t i t r a n s f e r r i n g e l , washed, and f i n a l l y i n c u b a t e d w i t h b o r o t r i t i d e and t h e a c t i v i t y i n c o r p o r a t e d i n t o t h e s o l i d phase t r a n s f e r r i n measured. The e x p e r i m e n t s i l l u s t r a t e d i n F i g 3 show t h a t t h e method p r e s e n t e d can be used t o assess s i a l i c a c i d c o n t e n t i n a g l y c o p r o t e i n and t h a t t h e sequence o f t h e v a r i o u s i n c u b a t i o n s i s n o t c r i t i c a l t o a c h i e v e a c o m p a r a t i v e a n a l y s i s o f t h e sugar. The c r i t i c a l s t e p i s t h e s e p a r a t i o n f r o m a m i x t u r e o f t h e immuno- complex c o n t a i n i n g t h e l a b e l e d s u g a r . The method p r e s e n t e d may be used as an a l t e r n a t i v e t o i s o e l e c t r i c f o c u s s i n g when a l a r g e number o f samples a r e p r o c e s s e d s i m u l t a n e o u s l y t o o b t a i n i n f o r m a t i o n on t h e a v e r a g e c o n t e n t o f s i a l i c a c i d i n a g l y c o p r o t e i n . T h e method does n o t p r e s e n t l y seem t o be s e n s i t i v e enough t o measure t h e p r e v i o u s l y r e p o r t e d m i c r o h e t e r o g e n e i t y o f serum t r a n s f e r r i n i n c o n n e c t i o n w i t h 25 alcohol abuse which a t b e s t involves a d i f f e r e n c e of about 10% of t h e amount of t r a n s f e r r i n and 5% of t h e t o t a l amount of t r a n s f e r r i n - b o u n d s i a l i c a c i d ( 3 ) . TOTAL CPM 20.000 15 OOC 10.000 500C I I I I 3 C Combination o f - oxi a ti on-reducti o n with binding of trans- f e r r i n followed by s e p a r a t i o n o f t h e l a b e l e d immunocomplex i n a s i n g l e s t e p . The t r a n s f e r r i n was f i r s t o x i d i z e d , then incubated with T r i s g l y - c i n e - g l y c e r o l f o r 60 min a t room temperature and then with 20 ,1 of gel suspension f o r a n o t h e r 60 min. There- a f t e r the gel was washed, f i r s t w i t h PBS c o n t a i n i n g 1% albumin and then with PBS pH 7.0 0.1 ml of b o r o t r i - t i d e s o l u t i o n was then added and t h e volume made 0.6 ml w i t h PBS, pH 7 . 0 followed by i n c u b a t i o n f o r 5 m i n a t room t e m p e r a t u r e , washing and measuring bound r a d i o a c t i v i t y . 0 20 40 60 80 100 SIALOTRANSFERRIN (PER CENT OF TRANSFERRIN) 26 REFERENCES 1. Cervgn, E., S t i b l e r , H. & Borg, S.: D e t e r m i n a t i o n o f t e r m i n a l s u g a r s i n t r a n s f e r r i n b y r a d i o - l e c t i n immunoassay ( R L I A ) - A new m i c r o a n a l y t i c a l p r o c e d u r e . U p s a l a J Med S c i 86: 39-53, 1981. I n : The C a r b o h y d r a t e s . C h e m i s t r y and B i o c h e m i s t r y , Vol I B . ( e d s . W . Pigman, D H o r t o n ) p 255-280. Academic P r e s s , New York and London, 1980. S t i b l e r , H., Borg, S. & A l l g u l a n d e r , C . : Abnormal m i c r o h e t e r o g e n e i t y o f t r a n s f e r r i n - A new m a r k e r o f a l c o h o l i s m ? S u b s t A l c o h o l A c t i o n s M i s u s e 1: 247-252, 1980. 2. P e r l i n , A.S.: 3 . 4. Weeke, B. & K r a s i l n i k o f f , P.A.: The c o n c e n t r a t i o n o f 2 1 serum p r o t e i n s i n normal c h i l d r e n and a d u l t s . A c t a Med Scand 192: 149-155, 1972. Address f o r r e p r i n t s : Gunnar R o n q u i s t Department o f C l i n i c a l C h e m i s t r y U n i v e r s i t y H o s p i t a l S-751 85 Uppsala 27