Archives of Virology
Archives of Virology 62, 131--136 (1979)
~) by Springer-V~rlag 1979
Comparison of Hemafglutination-Inhibition~ Complement-Fixation and Enzyme-Linked Immunosorbent Assay for Ouantitation of Human Rotavirus Antibodies By M. L. M~R~IN, G. W. G~Y~ J~., and E. L. PALMER Virologs~ Division, Bureau of Laboratories, U.S. Department of Health, Education, and Welfare, Public l-Iealth Service, Center for Disease Control, Atlanta, Georgia, U.S.A.
With 3 Figures Accepted May 22, 1979 Summary The hemaggtutination-inhibition test for detecting rot~virus antibody was evaluated b y using simian rotavirus S A d 1 as hemagglutinating antigen. Results show t h a t the test is as sensitive as either complement fixation or the enzymelinked immunosorbent assay for detecting antibody to rotavirus in human sera.
Introduction Recent studies show that some strains of bovine rotavirus and simian rotavirus SA-11 hemagglutinate (HA) various types of erythrocytes (1, 4, 6). Because H A activity is associated with double-shelled particles, it has been suggested t h a t H A inhibition (ItAI) m a y be a useful method for defining rotavirus types (4, 6). Limited studies have indicated t h a t bovine rotavirus H A antigen is less efficient in t t A I tests t h a n the complement-fixation (CF) method for detecting seroconversion with human acute and convalescent-phase sera (1). The H A I test with simian rotavirus SA-11 has not been evaluated. I n a seroepidemiological study of an epidemic of human rotavirus in islands of the mid-Pacific (FOSTER et al., in preparation), we obtained convalescent phase sera and sera from non-ill inhabitants of other mid-Pacific islands which were not affected b y the rotavirus epidemic. These sera were tested b y HAI, with simian rotavirus SA-11 used as antigen, and antibody levels were compared with those obtained when the same sera were tested by CF and the enzyme-linked immunosorbent assay (ELISA). The results presented show comparative antibody tilers obtained b y these three tests in sera with either very high or low levels of antihuman rotavirus antibody.
0304-S60S/79/0062/0131 / $ 01.20
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M . L . MAaTI~, G. W . GARY, J]~., a n d E. L. PALMER:
Materials and Methods H A and H A I A n t i g e n c o n s i s t e d of cell-associated s i m i a n r o t a v i r u s S A - 1 I , p r o p a g a t e d i n CV-1 cells o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e Collection as CCL 70 a n d f u r t h e r subc u l t u r e d i n roller b o t t l e s a t t h e C e n t e r for Disease C o n t r o l (CDC). W h e n cell m o n o l a y e r s w e r e c o n f l u e n t , t r a c e s of calf s e r u m u s e d in ceil p r o p a g a t i o n were r e m o v e d b y w a s h i n g e a c h b o t t l e t h r e e t i m e s w i t h M 199 c o n t a i n i n g n o s e r u m . B o t t l e s were t h e n i n o c u l a t e d w i t h a b o u t 5 v i r u s p a r t i c l e s p e r cell; t h e v i r u s w a s allowed t o a d s o r b for 30 m i n u t e s , a n d 50 m l of M 199 w i t h o u t s e r u m or a l b u m i n was a d d e d t o e a c h b o t t l e as m a i n t e n a n c e m e d i u m . A f t e r a b o u t 4 d a y s of i n c u b a t i o n w h e n 50 t o 75 p e r c e n t of t h e cells d e m o n s t r a t e d c y t o p a t h i c effect, t h e cells f r o m t h e s u p e r n a t a n t m e d i u m were h a r v e s t e d b y cent r i f u g a t i o n ; t h e a t t a c h e d cells were h a r v e s t e d b y s c r a p i n g w i t h a r u b b e r p o l i c e m a n . T h e cells were c o m b i n e d , t h e s u p e r n a t a n t was discarded, a n d t h e cells were t h e n l y s c d w i t h sterile distilled w a t e r a n d e x t r a c t e d w i t h f l u o r o c a r b o n . T h e final v o l u m e of v i r u s a n d d i l u e n t was a p p r o x i m a t e l y 10 m l for e a c h roller b o t t l e used. E l e c t r o n microscopic e x a m i n a t i o n of t h e a n t i g e n s h o w e d i t to c o n t a i n p r e d o m i n a t e l y d o u b l e - s h e l l e d particles a l o n g w i t h some free-lying c a p s o m e r e s a n d a smM1 a m o u n t of fine cell debris. T h e SA-11 v i r u s was p r o v i d e d b y H . H, MALtIEI~BE a n d h a s b e e n p a s s e d t w i c e i n p r i m a r y A f r i c a n g r e e n m o n k e y cells a n d twice in CV-1 cells a t t h e CDC. T e s t s for s i m i a n r o t a v i r u s H A a n d I.IAI were d o n e in m i c r o t i t e r p l a t e s b y a m o d i f i c a t i o n of t h e m e t h o d of INABA et al. (3). H A was t i t t e r e d b y serial t w o f o l d d i l u t i o n of 0.05-ml a m o u n t s of a n t i g e n i n p h o s p h a t e b u f f e r e d saline, p t t 7.4. T h e s e were m i x e d w i t h 0.05-ml a m o u n t s of 0.5 p e r c e n t h u m a n t y p e " 0 " e r y t h r o c y t e s i n t h e s a m e buffer. P l a t e s were i n c u b a t e d a t 37 ° C for 1 h o u r , a n d t i t e r s were e x p r e s s e d as t h e reciprocal of t h e h i g h e s t d i l u t i o n s h o w i n g H A . F o r H A I , 0.5 m l of a 1 : 5 d i l u t i o n of s e r u m was first a d s o r b e d for 30 m i n u t e s w i t h 0.5 m l of a 25 p e r c e n t s o l u t i o n of a c i d - w a s h e d kaolin. K a o l i n w a s r e m o v e d b y e e n t r i f u g a t i o n a n d t h e s e r u m t a k e n as a 1 : 10 s t a r t i n g dilution. H u m a n sera were n o t a d s o r b e d w i t h e r y t h r o c y t e s b e c a u s e t h e y d i d n o t c o n t a i n nonspecific h u m a n " 0 " e r y t h r o c y t e a g g l u t i n i n s . E i g h t H A u n i t s i n 0.025 m l were u s e d i n t h e I-IAI test. T h e y were a d d e d to 0.025 m l of serially d i l u t e d sere. S e r e a n d a n t i g e n were i n c u b a t e d for 1 h o u r a t r o o m t e m p e r a t u r e a n d t h e n m i x e d w i t h 0.05 m l of a 0.5 p e r c e n t s u s p e n s i o n of e r y t h r o c y t e s . T e s t s were i n c u b a t e d for I h o u r a.t 37 ° C, a n d tigers were e x p r e s s e d as t h e reciprocal of t h e h i g h e s t s e r u m d i l u t i o n s h o w i n g c o m p l e t e H A I . S e r u m c o n t r o l s to t e s t for nonspeeifie a g g l u t i n a t i o n c o n s i s t e d of sere d i l u t e d as i n t h e H A I test, b u t w i t h b u f f e r a d d e d t o e a c h well i n s t e a d of a n t i g e n .
Complement -Fixation A n t i g e n for t h e t e s t c o n s i s t e d of h u m a n r o t a v i r u s o b t a i n e d f r o m a pool of fecal e x t r a c t s f r o m c h i l d r e n h o s p i t a l i z e d i n t 9 7 6 i n Atlanta,, Georgia, for t r e a t m e n t of g a s t r o e n t e r i t i s . V i r u s w a s purified b y c o m b i n a t i o n g l y c e r o l - p o t a s s i u m t a r t r a t e d e n s i t y g r a d i e n t e e n t r i f u g a t i o n as d e s c r i b e d (5). E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n of t h e a n t i g e n s h o w e d i t t o c o n t a i n p r e d o m i n a n t l y d o u b l e - s h e l l e d r o t a v i r u s . S e r e were t e s t e d b y t h e L a b o r a t o r y B r a n c h C o m p l e m e n t F i x a t i o n ( L B C F ) (7) t e s t a d a p t e d t o m i c r o t i t e r . All sera were n o t t e s t e d b y C F b e c a u s e sufficient h u m a n r o t a v i r u s a n t i g e n w a s n o t available.
Enzyme-Linked Immunosorbent Assay ( E L I S A ) E L I S A t e s t s were p e r f o r m e d b y a m i c r o m e t h o d w i t h a calf r o t a v i r u s , s t r a i n C 486 ( p r o v i d e d b y L. BABIUK, S a s k a t o o n , C a n a d a ) u s e d as a n t i g e n . A n t i g e n w a s p r e p a r e d b y i n f e c t i n g M A t 0 4 cells i n roller flasks b y t h e m e t h o d d e s c r i b e d a b o v e for t h e H A a n t i g e n . T h e M A 104 cells were k i n d l y p r o v i d e d b y L. McLAREN, A l b u q u e r q u e , N e w Mexico. T h e o p t i m a l d i l u t i o n of a n t i g e n w a s d e t e r m i n e d b y c h e c k e r b o a r d t i t r a t i o n , d i l u t e d i n c a r b o n a t e b u f f e r ( p H 9.6) a n d a d d e d in 0.2-ml v o l u m e s p e r well to p o l y s t y r e n e m i c r o t i t e r p l a t e s ( M I C R O E L I S A * , Cooke L a b o r a t o r y P r o d u c t s ) . P l a t e s were h e l d * U s e of t r a d e n a m e s is for i d e n t i f i c a t i o n o n l y a n d does n o t c o n s t i t u t e e n d o r s e m e n t b y t h e P u b l i c H e a l t h Service or b y t h e U.S. D e p a r t m e n t of H e a l t h , E d u c a t i o n , a n d Welfare.
Rotavirus Hemagglutination-Inhibition
133
16 hours at 4 ° C and then washed three times with phosphate-buffered saline, p H 7.4, containing 0.05 per cent Tween 20 (PBST). Test sera were serially twofold diluted in the antigen-coated wells of the plates and incubated 75 minutes at 37 ° C. Plates were washed as before, 0.2 ml/well of alkaline phosphatase-labelled goat antihuman IgG conjugate (Miles Research Products, Elkhart, IN) (1:1600 in PBST, dilution as determined by checkerboard titration) was added, and plates were incubated 90 minutes at 37 ° C. Plates were then washed three times with PBST, and 0.2 ml of Sigma 104 substrate (p-nitrophenyl phosphate, disodium) at a concentration of l rag/rot in 10 per cent diethanolamine buffer was added to each well; plates were protected from light and incubated at room temperature. After 30 minutes, 0.05 ml of 3 N N a O H was added to each well to stop the reaction, and color was read at 400 nm on a spectrophotometer with a microsampling cuvette (Stasar I I , Gilford Instruments, Oberlin, OH). E n d points were read as the highest dilution which had an absorbanee reading of 0.75 or greater, a value chosen because it most closely related to visual endpoint determinations. The starting serum dilution, 1 : 64, was also tested against, a normal antigen prepared from uninfected MA 104 cell cultures to detect nonspecific reactions.
Results
Comparison o/ H A I and ELISA /or Detection o] Antihuman Rotavirus Antibody F i g u r e 1 shows r e l a t i v e l y high levels of a n t i b o d y were d e t e c t e d b y b o t h H A I a n d E L I S A in 100 of 102 convalescent p a t i e n t sera. N e i t h e r was m o r e sensitive t h a n t h e o t h e r since all sera h a d d e t e c t a b l e a n t i b o d y . E L I S A titers were, however, a p p r o x i m a t e l y 10 t i m e s higher t h a n H A I titers. T w o sera w i t h titers of 20 a n d 40 b y H A I also h a d low E L I S A titers (256). Most convalescent p a t i e n t sera h a d titers of 80 a n d a b o v e b y H A I (97 of 102) a n d 1024 or a b o v e b y E L I S A (97 of 102).
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Archives of Virology 62, 131--136 (1979)
~) by Springer-V~rlag 1979
Comparison of Hemafglutination-Inhibition~ Complement-Fixation and Enzyme-Linked Immunosorbent Assay for Ouantitation of Human Rotavirus Antibodies By M. L. M~R~IN, G. W. G~Y~ J~., and E. L. PALMER Virologs~ Division, Bureau of Laboratories, U.S. Department of Health, Education, and Welfare, Public l-Iealth Service, Center for Disease Control, Atlanta, Georgia, U.S.A.
With 3 Figures Accepted May 22, 1979 Summary The hemaggtutination-inhibition test for detecting rot~virus antibody was evaluated b y using simian rotavirus S A d 1 as hemagglutinating antigen. Results show t h a t the test is as sensitive as either complement fixation or the enzymelinked immunosorbent assay for detecting antibody to rotavirus in human sera.
Introduction Recent studies show that some strains of bovine rotavirus and simian rotavirus SA-11 hemagglutinate (HA) various types of erythrocytes (1, 4, 6). Because H A activity is associated with double-shelled particles, it has been suggested t h a t H A inhibition (ItAI) m a y be a useful method for defining rotavirus types (4, 6). Limited studies have indicated t h a t bovine rotavirus H A antigen is less efficient in t t A I tests t h a n the complement-fixation (CF) method for detecting seroconversion with human acute and convalescent-phase sera (1). The H A I test with simian rotavirus SA-11 has not been evaluated. I n a seroepidemiological study of an epidemic of human rotavirus in islands of the mid-Pacific (FOSTER et al., in preparation), we obtained convalescent phase sera and sera from non-ill inhabitants of other mid-Pacific islands which were not affected b y the rotavirus epidemic. These sera were tested b y HAI, with simian rotavirus SA-11 used as antigen, and antibody levels were compared with those obtained when the same sera were tested by CF and the enzyme-linked immunosorbent assay (ELISA). The results presented show comparative antibody tilers obtained b y these three tests in sera with either very high or low levels of antihuman rotavirus antibody.
0304-S60S/79/0062/0131 / $ 01.20
132
M . L . MAaTI~, G. W . GARY, J]~., a n d E. L. PALMER:
Materials and Methods H A and H A I A n t i g e n c o n s i s t e d of cell-associated s i m i a n r o t a v i r u s S A - 1 I , p r o p a g a t e d i n CV-1 cells o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e Collection as CCL 70 a n d f u r t h e r subc u l t u r e d i n roller b o t t l e s a t t h e C e n t e r for Disease C o n t r o l (CDC). W h e n cell m o n o l a y e r s w e r e c o n f l u e n t , t r a c e s of calf s e r u m u s e d in ceil p r o p a g a t i o n were r e m o v e d b y w a s h i n g e a c h b o t t l e t h r e e t i m e s w i t h M 199 c o n t a i n i n g n o s e r u m . B o t t l e s were t h e n i n o c u l a t e d w i t h a b o u t 5 v i r u s p a r t i c l e s p e r cell; t h e v i r u s w a s allowed t o a d s o r b for 30 m i n u t e s , a n d 50 m l of M 199 w i t h o u t s e r u m or a l b u m i n was a d d e d t o e a c h b o t t l e as m a i n t e n a n c e m e d i u m . A f t e r a b o u t 4 d a y s of i n c u b a t i o n w h e n 50 t o 75 p e r c e n t of t h e cells d e m o n s t r a t e d c y t o p a t h i c effect, t h e cells f r o m t h e s u p e r n a t a n t m e d i u m were h a r v e s t e d b y cent r i f u g a t i o n ; t h e a t t a c h e d cells were h a r v e s t e d b y s c r a p i n g w i t h a r u b b e r p o l i c e m a n . T h e cells were c o m b i n e d , t h e s u p e r n a t a n t was discarded, a n d t h e cells were t h e n l y s c d w i t h sterile distilled w a t e r a n d e x t r a c t e d w i t h f l u o r o c a r b o n . T h e final v o l u m e of v i r u s a n d d i l u e n t was a p p r o x i m a t e l y 10 m l for e a c h roller b o t t l e used. E l e c t r o n microscopic e x a m i n a t i o n of t h e a n t i g e n s h o w e d i t to c o n t a i n p r e d o m i n a t e l y d o u b l e - s h e l l e d particles a l o n g w i t h some free-lying c a p s o m e r e s a n d a smM1 a m o u n t of fine cell debris. T h e SA-11 v i r u s was p r o v i d e d b y H . H, MALtIEI~BE a n d h a s b e e n p a s s e d t w i c e i n p r i m a r y A f r i c a n g r e e n m o n k e y cells a n d twice in CV-1 cells a t t h e CDC. T e s t s for s i m i a n r o t a v i r u s H A a n d I.IAI were d o n e in m i c r o t i t e r p l a t e s b y a m o d i f i c a t i o n of t h e m e t h o d of INABA et al. (3). H A was t i t t e r e d b y serial t w o f o l d d i l u t i o n of 0.05-ml a m o u n t s of a n t i g e n i n p h o s p h a t e b u f f e r e d saline, p t t 7.4. T h e s e were m i x e d w i t h 0.05-ml a m o u n t s of 0.5 p e r c e n t h u m a n t y p e " 0 " e r y t h r o c y t e s i n t h e s a m e buffer. P l a t e s were i n c u b a t e d a t 37 ° C for 1 h o u r , a n d t i t e r s were e x p r e s s e d as t h e reciprocal of t h e h i g h e s t d i l u t i o n s h o w i n g H A . F o r H A I , 0.5 m l of a 1 : 5 d i l u t i o n of s e r u m was first a d s o r b e d for 30 m i n u t e s w i t h 0.5 m l of a 25 p e r c e n t s o l u t i o n of a c i d - w a s h e d kaolin. K a o l i n w a s r e m o v e d b y e e n t r i f u g a t i o n a n d t h e s e r u m t a k e n as a 1 : 10 s t a r t i n g dilution. H u m a n sera were n o t a d s o r b e d w i t h e r y t h r o c y t e s b e c a u s e t h e y d i d n o t c o n t a i n nonspecific h u m a n " 0 " e r y t h r o c y t e a g g l u t i n i n s . E i g h t H A u n i t s i n 0.025 m l were u s e d i n t h e I-IAI test. T h e y were a d d e d to 0.025 m l of serially d i l u t e d sere. S e r e a n d a n t i g e n were i n c u b a t e d for 1 h o u r a t r o o m t e m p e r a t u r e a n d t h e n m i x e d w i t h 0.05 m l of a 0.5 p e r c e n t s u s p e n s i o n of e r y t h r o c y t e s . T e s t s were i n c u b a t e d for I h o u r a.t 37 ° C, a n d tigers were e x p r e s s e d as t h e reciprocal of t h e h i g h e s t s e r u m d i l u t i o n s h o w i n g c o m p l e t e H A I . S e r u m c o n t r o l s to t e s t for nonspeeifie a g g l u t i n a t i o n c o n s i s t e d of sere d i l u t e d as i n t h e H A I test, b u t w i t h b u f f e r a d d e d t o e a c h well i n s t e a d of a n t i g e n .
Complement -Fixation A n t i g e n for t h e t e s t c o n s i s t e d of h u m a n r o t a v i r u s o b t a i n e d f r o m a pool of fecal e x t r a c t s f r o m c h i l d r e n h o s p i t a l i z e d i n t 9 7 6 i n Atlanta,, Georgia, for t r e a t m e n t of g a s t r o e n t e r i t i s . V i r u s w a s purified b y c o m b i n a t i o n g l y c e r o l - p o t a s s i u m t a r t r a t e d e n s i t y g r a d i e n t e e n t r i f u g a t i o n as d e s c r i b e d (5). E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n of t h e a n t i g e n s h o w e d i t t o c o n t a i n p r e d o m i n a n t l y d o u b l e - s h e l l e d r o t a v i r u s . S e r e were t e s t e d b y t h e L a b o r a t o r y B r a n c h C o m p l e m e n t F i x a t i o n ( L B C F ) (7) t e s t a d a p t e d t o m i c r o t i t e r . All sera were n o t t e s t e d b y C F b e c a u s e sufficient h u m a n r o t a v i r u s a n t i g e n w a s n o t available.
Enzyme-Linked Immunosorbent Assay ( E L I S A ) E L I S A t e s t s were p e r f o r m e d b y a m i c r o m e t h o d w i t h a calf r o t a v i r u s , s t r a i n C 486 ( p r o v i d e d b y L. BABIUK, S a s k a t o o n , C a n a d a ) u s e d as a n t i g e n . A n t i g e n w a s p r e p a r e d b y i n f e c t i n g M A t 0 4 cells i n roller flasks b y t h e m e t h o d d e s c r i b e d a b o v e for t h e H A a n t i g e n . T h e M A 104 cells were k i n d l y p r o v i d e d b y L. McLAREN, A l b u q u e r q u e , N e w Mexico. T h e o p t i m a l d i l u t i o n of a n t i g e n w a s d e t e r m i n e d b y c h e c k e r b o a r d t i t r a t i o n , d i l u t e d i n c a r b o n a t e b u f f e r ( p H 9.6) a n d a d d e d in 0.2-ml v o l u m e s p e r well to p o l y s t y r e n e m i c r o t i t e r p l a t e s ( M I C R O E L I S A * , Cooke L a b o r a t o r y P r o d u c t s ) . P l a t e s were h e l d * U s e of t r a d e n a m e s is for i d e n t i f i c a t i o n o n l y a n d does n o t c o n s t i t u t e e n d o r s e m e n t b y t h e P u b l i c H e a l t h Service or b y t h e U.S. D e p a r t m e n t of H e a l t h , E d u c a t i o n , a n d Welfare.
Rotavirus Hemagglutination-Inhibition
133
16 hours at 4 ° C and then washed three times with phosphate-buffered saline, p H 7.4, containing 0.05 per cent Tween 20 (PBST). Test sera were serially twofold diluted in the antigen-coated wells of the plates and incubated 75 minutes at 37 ° C. Plates were washed as before, 0.2 ml/well of alkaline phosphatase-labelled goat antihuman IgG conjugate (Miles Research Products, Elkhart, IN) (1:1600 in PBST, dilution as determined by checkerboard titration) was added, and plates were incubated 90 minutes at 37 ° C. Plates were then washed three times with PBST, and 0.2 ml of Sigma 104 substrate (p-nitrophenyl phosphate, disodium) at a concentration of l rag/rot in 10 per cent diethanolamine buffer was added to each well; plates were protected from light and incubated at room temperature. After 30 minutes, 0.05 ml of 3 N N a O H was added to each well to stop the reaction, and color was read at 400 nm on a spectrophotometer with a microsampling cuvette (Stasar I I , Gilford Instruments, Oberlin, OH). E n d points were read as the highest dilution which had an absorbanee reading of 0.75 or greater, a value chosen because it most closely related to visual endpoint determinations. The starting serum dilution, 1 : 64, was also tested against, a normal antigen prepared from uninfected MA 104 cell cultures to detect nonspecific reactions.
Results
Comparison o/ H A I and ELISA /or Detection o] Antihuman Rotavirus Antibody F i g u r e 1 shows r e l a t i v e l y high levels of a n t i b o d y were d e t e c t e d b y b o t h H A I a n d E L I S A in 100 of 102 convalescent p a t i e n t sera. N e i t h e r was m o r e sensitive t h a n t h e o t h e r since all sera h a d d e t e c t a b l e a n t i b o d y . E L I S A titers were, however, a p p r o x i m a t e l y 10 t i m e s higher t h a n H A I titers. T w o sera w i t h titers of 20 a n d 40 b y H A I also h a d low E L I S A titers (256). Most convalescent p a t i e n t sera h a d titers of 80 a n d a b o v e b y H A I (97 of 102) a n d 1024 or a b o v e b y E L I S A (97 of 102).
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