Journal o f Immunological Methods, 24 (1978) 371--375
371
© Elsevier/North-Holland Biomedical Press
A H I G H L Y SENSITIVE R A D I O I M M U N O A S S A Y TECHNIQUE FOR S U B T Y P I N G T H E A N T I B O D Y T O HEPATITIS B S U R F A C E A N T I G E N
C.T. FANG, N. NATH 1, H. BERBERIAN and R.Y. DODD American Red Cross, Blood Research Laboratory, Bethesda, MD 2001.I, U.S.A.
(Received 27 March 1978, accepted 26 June 1978)
A highly sensitive technique for determining the subtype specificity of antibody to hepatitis B surface antigpn (anti-HBs) is described, lmmunoadsorbent consisting of controlled pore glass coated with subtype specific HBsAg was used to remove homologous antibody from the test samples before testing them for residual antibody by a commercially available radioimmunoassay (RIA). A total of 73 anti-HBs-positive samples from asymptomatic blood donors were tested. In nearly 80% of these samples the subtype reactivity could be determined by this technique. Only 67% could be typed by conventional liquid phase absorption RIA and 22% by passive hemagglutination inhibition techniques. Among the samples with low anti-HBs liter, ad and ay subtypes were found with equal frequency; however, with the increase in anti-HBs tiler, considerably higher proportion of ad specificity was detected.
INTRODUCTION T h e h e p a t i t i s B s u r f a c e a n t i g e n ( H B s A g ) h a s 4 m a j o r s u b t y p e s - - a d w , adr, of the specific determinants a, d , y , w, a n d r. T h e s e s u b t y p e s b r e e d t r u e a n d a r e v i r a l l y e n c o d e d ( H o l l a n d , 1975). The subtype specificity of antibody to HBsAg (anti-HBs) from h u m a n o r a n i m a l s o u r c e s , t h e r e f o r e , will b e t h e s a m e as t h e s u b t y p e o f t h e eliciting antigen. T h e i m p o r t a n c e o f t h e s e s u b t y p e s h a s b e e n r e c o g n i z e d in s e r o - e p i d e m i o l o gic s t u d i e s o f t y p e B h e p a t i t i s . In t h e U n i t e d S t a t e s , 7 . 5 % o f v o l u n t e e r b l o o d d o n o r s a r e p o s i t i v e f o r a n t i - H B s ( P e t e r s o n e t al., 1 9 7 3 ) a n d o n l y 0 . 1 1 % a r e p o s i t i v e f o r H B s A g ( D o d d e t al., 1 9 7 5 ) . A s i m p l e a n d s e n s i t i v e t e c h n i q u e f o r d e t e r m i n i n g t h e s u b t y p e s p e c i f i c i t y o f a n t i - H B s w o u l d r e s u l t in a b e t t e r a y w , and ayr -- based on d i f f e r e n t c o m b i n a t i o n s
I Reprint requests to: N. Nath, Ph.D., American Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014, U.S.A. Contribution No. 412 from the American Red Cross. Abbreviations: HBsAg, hepatitis B surface antigen; anti-HBs, antibody to hepatitis B surface antigen; RIA, radioimmunoassay; CPG, controlled pore glass; PHA, passive hemagglutination; RIA-LPA, radioimmunoassay with liquid phase absorption; RIA-SPA, radioimmunoassay with solid-phase absorption.
372 epidemiologic evaluation of exposure to hepatitis B virus subtypes. tlere we describe a method for determining the subtype specificity of antitlBs; a modification of the technique described by Hoofnagle et al. (1977). The use of solid phase absorption increases the sensitivity at least 4 times. MATERIALS AND METHODS
Samples tested A total of 862 human plasma samples from the collection of the American Red Cross Blood Research Laboratory were tested for the presence of antiHBs by a commercial solid phase radioimmunoassay (Ausab ¢', Abbott Laboratories, North Chicago, IL). These samples were from volunteer blood donors and were known to be non-reactive for HBsAg by RIA (Ausria-I[ ~', Abbott Laboratories), and had been stored at --30°C for up to 6 years. A sample was considered positive for anti-HBs if it was reactive when tested by Ausab test kits. The titer of anti-HBs was determined by the passive hemagglutination technique (PHA) of Vyas and Shulman (1970) using HBsAg-coated human erythrocytes (Electro-Nucleonics Laboratories, Inc., Bethesda, MD).
Determination of subtype specificity of antibody to HBsAg Determination of subtype specificity of anti-HBs was then performed on the positive samples by 3 different techniques, as described below. (a) Passive hemagglutination inhibition (PHAI). The technique of Gold et al. {1974) was followed. In brief, sample was diluted to give a known standard titer, separately absorbed with partially purified HBsAg of ad or ay subtype specificity prepared by the technique described by Nath et al. (1976). Hemagglutination of human erythrocytes coated with HBsAg/ad or HBsAg/ay (Electro-Nucleonics, Inc.) was used to indicate the specificity of antibodies. (b) Radioimmunoassay with liquid phase absorption (RIA-LPA). The technique described by Hoofnagle et al. {1977) was used. The test sample was absorbed by the addition of HBsAg-positive serum of ad or ay specificity. RIA {Ausab, A b b o t t Laboratories) was used to detect the presence of residual anti-HBs activity. (c) Radioimmunoassay with solid-phase absorption (RIA-SPA). To prepare the controlled pore glass (CPG) immunoadsorbents, the technique of Dodd and Kobita (1978) was used. CPG (Bioglas ~;'~1500) was obtained from BioRad Laboratories, Richmond, CA. A 5 ml aliquot from HBsAg-positive human plasma (titer l 0 s by RIA) of known subtype (ad or ay) was used to coat 1 g of CPG at pH 3.0. The suspension of CPG in HBsAg plasma was mixed by rotation for 5 h at room temperature; then the CPG was centrifuged down and washed thoroughly with phosphate-buffered saline (PBS; NaC1, 0.073 M; KH2PO4, 0.018 M; Na~HPO4, 0.057 M, pH 7.2). The washed HBsAg coated CPG (CPG-HBsAg) was stored at 4°C in 10 ml of PBS with
373 s o d i u m azide (1 : 1000). T h u s 0.1 ml o f vigorously stirred suspension gave a p p r o x i m a t e l y 10 mg o f CPG. A sample for s u b t y p i n g was diluted, if necessary, to give a PHA titer o f less t h a n 100. A 0.25 ml a m o u n t o f diluted sample was added to a v i a l containing 10 mg o f HBsAg/ad c o a t e d CPG and a n o t h e r 0.25 ml was a d d e d to a s e c o n d vial c o n t a i n i n g 10 mg o f HBsAg/ay c o a t e d CPG. T h e vials were r o t a t e d at r o o m t e m p e r a t u r e for 2.5 h. T h e CPG was allowed to settle and the s u p e r n a t a n t was tested for u n a b s o r b e d a n t i b o d y . A s u b t y p e was assigned on the basis o f a significant r e d u c t i o n (over 50%) in the c o u n t s per m i n u t e ( c p m ) following a b s o r p t i o n with CPG-HBsAg/ad or CPG-HBsAg/ay. RESULTS T o c o m p a r e the sensitivity o f the 3 t e c h n i q u e s described above, t w o r a b b i t sera c o n t a i n i n g a n t i b o d y to HBsAg o f ad and ay s u b t y p e specificities were diluted serially. B o t h antisera had an anti-HBs titer o f 1 : 1 0 , 0 0 0 b y PHA. S u b t y p e specificity was d e t e r m i n e d o n each d i l u t i o n by all 3 techniques. Table 1 shows the highest d i l u t i o n at which the s u b t y p e c o u l d be determ i n e d b y each o f the 3 t e c h n i q u e s . Using PHAI t e c h n i q u e antisera c o u l d be diluted up to a m a x i m u m o f 1 : 50, higher d i l u t i o n resulted in failure to subt y p e . T h e R I A - L P A t e c h n i q u e for s u b t y p i n g was 40 times m o r e sensitive than PHAI for anti-HBs/ad; h o w e v e r , o n l y 10 times higher for anti-HBs/ay samples. On the o t h e r h a n d , RIA-SPA was the m o s t sensitive o f the 3 techniques. Its sensitivity was 4 times higher t h a n R I A - L P A for samples containing anti-HBs/ad or ay s u b t y p e specificities. A m o n g 862 h u m a n plasma samples tested for anti-HBs b y RIA, 73 (8.47%) were f o u n d reactive. Results o f a n t i b o d y s u b t y p e specificity d e t e r m i n a t i o n on these 73 samples by all 3 t e c h n i q u e s are p r e s e n t e d in Table 2. T h e n a t u r e o f
TABLE 1 RELATIVE SENSITIVITY OF 3 TECHNIQUES FOR DETERMINING THE SUBTYPE SPECIFICITY OF TWO RABBIT ANTISERA Technique a
PHAI RIA-LPA RIA-SPA
Anti-HBs/ad
Anti-HBs/ay
Highest dilution b
Relative sensitivity
Highest dilution b
Relative sensitivity
50 2000 8000
I 40 160
50 500 2000
1 10 40
a PHAI, passive hemagglutination inhibition; RIA-LPA, radioimmunoassay with liquid phase absorption; RIA-SPA, radioimmunoassay with solid-phase absorption. b Highest dilution of sample at which the subtype could be determined.
374 TABLE 2 COMPARISON OF 3 TECHNIQUES FOR Anti-HBs SUBTYPE DETERMINATION OF HUMAN PLASMA SAMPLES Number tested 17 36 20 Total 73
PHA titer 100
PHAI
RIA-LPA
ad
ay
%a
ad
0 0 15 15
0 0 1 1
0 0 80 21.9
3 10 17 39
ay
2 6 2 10
RIA-SPA %a
ad
29.4 69.4 95.0 67.1
5 22 17 44
ay
5 7 2 14
%a
58.8 80.6 95.0 79.5
a Percentage of samples that could be subtyped in this PHA titer range. s u b t y p e s p e c i f i c i t y d e t e r m i n e d was always identical b y t h e 3 t e c h n i q u e s . In no instance did R I A - S P A fail to d e t e r m i n e s u b t y p e w h e r e P H A I or R I A - L P A had s u c c e e d e d . S u b t y p e s c o u l d be d e t e r m i n e d b y R I A - S P A in m o r e s a m p l e s (80%) t h a n b y the o t h e r t w o t e c h n i q u e s (67% b y R I A - L P A and 22% b y P H A I ) . T h e r e were 9 s a m p l e s t h a t c o u l d be s u b t y p e d o n l y b y R I A - S P A . F i f t y - t h r e e s a m p l e s h a d anti-HBs titers l o w e r t h a n 100 b y P H A , and R I A SPA c o u l d d e t e c t s u b t y p e s p e c i f i c i t y in 39 (73.6%) o f t h e m . In c o n t r a s t , R I A - L P A d e t e c t e d s u b t y p e s p e c i f i c i t y in 30 {56.6%), while P H A I failed to s u b t y p e any. H o w e v e r , all 3 t e c h n i q u e s w e r e equally effective in determ i n i n g s u b t y p e s p e c i f i c i t y o f s a m p l e s w i t h P H A t i t e r higher t h a n 100. T a b l e 2 also shows t h a t at low anti-HBs t i t e r (~
371
© Elsevier/North-Holland Biomedical Press
A H I G H L Y SENSITIVE R A D I O I M M U N O A S S A Y TECHNIQUE FOR S U B T Y P I N G T H E A N T I B O D Y T O HEPATITIS B S U R F A C E A N T I G E N
C.T. FANG, N. NATH 1, H. BERBERIAN and R.Y. DODD American Red Cross, Blood Research Laboratory, Bethesda, MD 2001.I, U.S.A.
(Received 27 March 1978, accepted 26 June 1978)
A highly sensitive technique for determining the subtype specificity of antibody to hepatitis B surface antigpn (anti-HBs) is described, lmmunoadsorbent consisting of controlled pore glass coated with subtype specific HBsAg was used to remove homologous antibody from the test samples before testing them for residual antibody by a commercially available radioimmunoassay (RIA). A total of 73 anti-HBs-positive samples from asymptomatic blood donors were tested. In nearly 80% of these samples the subtype reactivity could be determined by this technique. Only 67% could be typed by conventional liquid phase absorption RIA and 22% by passive hemagglutination inhibition techniques. Among the samples with low anti-HBs liter, ad and ay subtypes were found with equal frequency; however, with the increase in anti-HBs tiler, considerably higher proportion of ad specificity was detected.
INTRODUCTION T h e h e p a t i t i s B s u r f a c e a n t i g e n ( H B s A g ) h a s 4 m a j o r s u b t y p e s - - a d w , adr, of the specific determinants a, d , y , w, a n d r. T h e s e s u b t y p e s b r e e d t r u e a n d a r e v i r a l l y e n c o d e d ( H o l l a n d , 1975). The subtype specificity of antibody to HBsAg (anti-HBs) from h u m a n o r a n i m a l s o u r c e s , t h e r e f o r e , will b e t h e s a m e as t h e s u b t y p e o f t h e eliciting antigen. T h e i m p o r t a n c e o f t h e s e s u b t y p e s h a s b e e n r e c o g n i z e d in s e r o - e p i d e m i o l o gic s t u d i e s o f t y p e B h e p a t i t i s . In t h e U n i t e d S t a t e s , 7 . 5 % o f v o l u n t e e r b l o o d d o n o r s a r e p o s i t i v e f o r a n t i - H B s ( P e t e r s o n e t al., 1 9 7 3 ) a n d o n l y 0 . 1 1 % a r e p o s i t i v e f o r H B s A g ( D o d d e t al., 1 9 7 5 ) . A s i m p l e a n d s e n s i t i v e t e c h n i q u e f o r d e t e r m i n i n g t h e s u b t y p e s p e c i f i c i t y o f a n t i - H B s w o u l d r e s u l t in a b e t t e r a y w , and ayr -- based on d i f f e r e n t c o m b i n a t i o n s
I Reprint requests to: N. Nath, Ph.D., American Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014, U.S.A. Contribution No. 412 from the American Red Cross. Abbreviations: HBsAg, hepatitis B surface antigen; anti-HBs, antibody to hepatitis B surface antigen; RIA, radioimmunoassay; CPG, controlled pore glass; PHA, passive hemagglutination; RIA-LPA, radioimmunoassay with liquid phase absorption; RIA-SPA, radioimmunoassay with solid-phase absorption.
372 epidemiologic evaluation of exposure to hepatitis B virus subtypes. tlere we describe a method for determining the subtype specificity of antitlBs; a modification of the technique described by Hoofnagle et al. (1977). The use of solid phase absorption increases the sensitivity at least 4 times. MATERIALS AND METHODS
Samples tested A total of 862 human plasma samples from the collection of the American Red Cross Blood Research Laboratory were tested for the presence of antiHBs by a commercial solid phase radioimmunoassay (Ausab ¢', Abbott Laboratories, North Chicago, IL). These samples were from volunteer blood donors and were known to be non-reactive for HBsAg by RIA (Ausria-I[ ~', Abbott Laboratories), and had been stored at --30°C for up to 6 years. A sample was considered positive for anti-HBs if it was reactive when tested by Ausab test kits. The titer of anti-HBs was determined by the passive hemagglutination technique (PHA) of Vyas and Shulman (1970) using HBsAg-coated human erythrocytes (Electro-Nucleonics Laboratories, Inc., Bethesda, MD).
Determination of subtype specificity of antibody to HBsAg Determination of subtype specificity of anti-HBs was then performed on the positive samples by 3 different techniques, as described below. (a) Passive hemagglutination inhibition (PHAI). The technique of Gold et al. {1974) was followed. In brief, sample was diluted to give a known standard titer, separately absorbed with partially purified HBsAg of ad or ay subtype specificity prepared by the technique described by Nath et al. (1976). Hemagglutination of human erythrocytes coated with HBsAg/ad or HBsAg/ay (Electro-Nucleonics, Inc.) was used to indicate the specificity of antibodies. (b) Radioimmunoassay with liquid phase absorption (RIA-LPA). The technique described by Hoofnagle et al. {1977) was used. The test sample was absorbed by the addition of HBsAg-positive serum of ad or ay specificity. RIA {Ausab, A b b o t t Laboratories) was used to detect the presence of residual anti-HBs activity. (c) Radioimmunoassay with solid-phase absorption (RIA-SPA). To prepare the controlled pore glass (CPG) immunoadsorbents, the technique of Dodd and Kobita (1978) was used. CPG (Bioglas ~;'~1500) was obtained from BioRad Laboratories, Richmond, CA. A 5 ml aliquot from HBsAg-positive human plasma (titer l 0 s by RIA) of known subtype (ad or ay) was used to coat 1 g of CPG at pH 3.0. The suspension of CPG in HBsAg plasma was mixed by rotation for 5 h at room temperature; then the CPG was centrifuged down and washed thoroughly with phosphate-buffered saline (PBS; NaC1, 0.073 M; KH2PO4, 0.018 M; Na~HPO4, 0.057 M, pH 7.2). The washed HBsAg coated CPG (CPG-HBsAg) was stored at 4°C in 10 ml of PBS with
373 s o d i u m azide (1 : 1000). T h u s 0.1 ml o f vigorously stirred suspension gave a p p r o x i m a t e l y 10 mg o f CPG. A sample for s u b t y p i n g was diluted, if necessary, to give a PHA titer o f less t h a n 100. A 0.25 ml a m o u n t o f diluted sample was added to a v i a l containing 10 mg o f HBsAg/ad c o a t e d CPG and a n o t h e r 0.25 ml was a d d e d to a s e c o n d vial c o n t a i n i n g 10 mg o f HBsAg/ay c o a t e d CPG. T h e vials were r o t a t e d at r o o m t e m p e r a t u r e for 2.5 h. T h e CPG was allowed to settle and the s u p e r n a t a n t was tested for u n a b s o r b e d a n t i b o d y . A s u b t y p e was assigned on the basis o f a significant r e d u c t i o n (over 50%) in the c o u n t s per m i n u t e ( c p m ) following a b s o r p t i o n with CPG-HBsAg/ad or CPG-HBsAg/ay. RESULTS T o c o m p a r e the sensitivity o f the 3 t e c h n i q u e s described above, t w o r a b b i t sera c o n t a i n i n g a n t i b o d y to HBsAg o f ad and ay s u b t y p e specificities were diluted serially. B o t h antisera had an anti-HBs titer o f 1 : 1 0 , 0 0 0 b y PHA. S u b t y p e specificity was d e t e r m i n e d o n each d i l u t i o n by all 3 techniques. Table 1 shows the highest d i l u t i o n at which the s u b t y p e c o u l d be determ i n e d b y each o f the 3 t e c h n i q u e s . Using PHAI t e c h n i q u e antisera c o u l d be diluted up to a m a x i m u m o f 1 : 50, higher d i l u t i o n resulted in failure to subt y p e . T h e R I A - L P A t e c h n i q u e for s u b t y p i n g was 40 times m o r e sensitive than PHAI for anti-HBs/ad; h o w e v e r , o n l y 10 times higher for anti-HBs/ay samples. On the o t h e r h a n d , RIA-SPA was the m o s t sensitive o f the 3 techniques. Its sensitivity was 4 times higher t h a n R I A - L P A for samples containing anti-HBs/ad or ay s u b t y p e specificities. A m o n g 862 h u m a n plasma samples tested for anti-HBs b y RIA, 73 (8.47%) were f o u n d reactive. Results o f a n t i b o d y s u b t y p e specificity d e t e r m i n a t i o n on these 73 samples by all 3 t e c h n i q u e s are p r e s e n t e d in Table 2. T h e n a t u r e o f
TABLE 1 RELATIVE SENSITIVITY OF 3 TECHNIQUES FOR DETERMINING THE SUBTYPE SPECIFICITY OF TWO RABBIT ANTISERA Technique a
PHAI RIA-LPA RIA-SPA
Anti-HBs/ad
Anti-HBs/ay
Highest dilution b
Relative sensitivity
Highest dilution b
Relative sensitivity
50 2000 8000
I 40 160
50 500 2000
1 10 40
a PHAI, passive hemagglutination inhibition; RIA-LPA, radioimmunoassay with liquid phase absorption; RIA-SPA, radioimmunoassay with solid-phase absorption. b Highest dilution of sample at which the subtype could be determined.
374 TABLE 2 COMPARISON OF 3 TECHNIQUES FOR Anti-HBs SUBTYPE DETERMINATION OF HUMAN PLASMA SAMPLES Number tested 17 36 20 Total 73
PHA titer 100
PHAI
RIA-LPA
ad
ay
%a
ad
0 0 15 15
0 0 1 1
0 0 80 21.9
3 10 17 39
ay
2 6 2 10
RIA-SPA %a
ad
29.4 69.4 95.0 67.1
5 22 17 44
ay
5 7 2 14
%a
58.8 80.6 95.0 79.5
a Percentage of samples that could be subtyped in this PHA titer range. s u b t y p e s p e c i f i c i t y d e t e r m i n e d was always identical b y t h e 3 t e c h n i q u e s . In no instance did R I A - S P A fail to d e t e r m i n e s u b t y p e w h e r e P H A I or R I A - L P A had s u c c e e d e d . S u b t y p e s c o u l d be d e t e r m i n e d b y R I A - S P A in m o r e s a m p l e s (80%) t h a n b y the o t h e r t w o t e c h n i q u e s (67% b y R I A - L P A and 22% b y P H A I ) . T h e r e were 9 s a m p l e s t h a t c o u l d be s u b t y p e d o n l y b y R I A - S P A . F i f t y - t h r e e s a m p l e s h a d anti-HBs titers l o w e r t h a n 100 b y P H A , and R I A SPA c o u l d d e t e c t s u b t y p e s p e c i f i c i t y in 39 (73.6%) o f t h e m . In c o n t r a s t , R I A - L P A d e t e c t e d s u b t y p e s p e c i f i c i t y in 30 {56.6%), while P H A I failed to s u b t y p e any. H o w e v e r , all 3 t e c h n i q u e s w e r e equally effective in determ i n i n g s u b t y p e s p e c i f i c i t y o f s a m p l e s w i t h P H A t i t e r higher t h a n 100. T a b l e 2 also shows t h a t at low anti-HBs t i t e r (~
