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‘Aspria’: A new sensitive assay for the detection of hepatitis B surface antigen
Journal o f Immunological Methods, 31 (1979) 365--371 © Elsevier/North-Holland Biomedical Press
' A S P R I A ' 1: A N E W S E N S I T I V E A S S A Y F O R T H E D E T E C T I O N HEPATITIS B SURFACE ANTIGEN :
365
OF
IRVING MILLMAN and MADELYN SMITH 3 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, U.S.A.
(Received 21 June 1979, accepted 10 August 1979)
A sensitive method for the detection of hepatitis B surface antigen (HBsAg) is described whereby the separation of specific antibody by affinity chromatography is incorporated into one of the assay reagents. HBsAg, which may be obtained from tissue culture fluids of a human hepatoma cell line, is first reacted with polystyrene beads. Unoccupied spaces on the plastic are next covered with a non-specific protein (e.g. albumin). The plastic is then reacted with either whole antiserum or an IgG fraction of antiHBs. Care must be taken to cover all of the reacting sites of the first layer of HBsAg with sufficient anti-HBs. This results in a reagent capable of detecting antigen. No affinity purified antibody was necessary because the reagent, at this stage, contains only specific antibody on the surface. Antigen reacting with this reagent can be detected by the addition of radioactively labeled IgG fraction of anti-HBs, which will complete the 'sandwich'. This ,assay has been evaluated and found to be as sensitive as those commercially available.
INTRODUCTION The most sensitive assays currently favored for the detection of hepatitis B s u r f a c e a n t i g e n ( H B s A g ) axe s o l i d - p h a s e r a d i o i m m u n o a s s a y s ( H o l l i n g e r e t al., 1 9 7 1 ; L i n g a n d O v e r b y , 1 9 7 2 ; R o s e n t h a l e t al., 1 9 7 2 a , b; P u r c e l l e t a l . , 1973). The current commercially available tests make use of a substrate, u s u a l l y a p l a s t i c b e a d o r t u b e , t o w h i c h is a t t a c h e d t h e I g G f r a c t i o n o f a n antiserum containing anti-HBs. These antisera are from either human donors or hyperimmunized a n i m a l s . T h e H B s A g in t h e t e s t s e r u m r e a c t s w i t h a t t a c h e d a n t i b o d y a n d is d e t e c t e d b y t h e a d d i t i o n o f a n a n t i - H B s 12sIlabeled IgG fraction. A sandwich of antibody, antigen and radioactive antib o d y is f o r m e d . T h e m e t h o d is e x t r e m e l y s e n s i t i v e b e c a u s e w i t h i n c r e a s e in a n t i g e n c o n c e n t r a t i o n t h e r e is a n i n c r e a s e in r a d i o a c t i v i t y . S e n s i t i v i t y o f t h e assay can be increased by substituting an affinity purified antibody for the 1 U.S. Patent Application Serial No. 903,238. 2 This work was supported by USPHS Grants CA-06551, RR-05539 and CA-06927 from the National Institutes of Health and by an appropriation from the Commonwealth of Pennsylvania. 3 Student of Drexel University (Philadelphia, PA) on work-study program.
366 IgG fraction containing anti-HBs. This substitution can be made for the labeled or unlabeled antibody (at either end of the sandwich). Such a change is effective since even in hyperimmune globulin fractions only a small percentage of the a n t i b o d y is specific anti-HBs. The production of affinity purified antibody in sufficient amounts is difficult and expensive and contributes to the high cost of the presently available assay kits. The favored procedure for producing specific antibody involves affinity chromatography with subsequent dissociation of complexes by a variety of means. Such procedures often result in low yields because of incomplete dissociation or unsatisfactory yields because of alterations (denaturation) or antibody molecules. This report describes a novel procedure which allows the separation of specific antibody without prior affinity chromatography. Affinity chromatography, currently used to manufacture specific antibody for either end of the sandwich (either to coat a solid substrate or for radioactive labeling), is incorporated within the assay for HBsAg itself. A reagent is produced which we call 'Aspria' (for affinity solid-phase radioimmunoassay), which can be used to detect HBsAg with sensitivity equal to assays currently available. An added advantage is that the reagent is less costly to manufacture. MATERIALS AND METHODS
(1) Supporting material Polystyrene beads, 0.25 in. diameter, were purchased from United States Plastic Corporation, Lima, OH and The Superior Ball Co., Hartford, CT. Disposable microtiter plates were purchased from Cooke Engineering Co., Alexandria, VA, and disposable polystyrene tubes were purchased from Falcon, Oxnard, CA.
(2) HBsAg HBsAg from human donors was purified according to the procedure described earlier (Millman et al., 1970). Less purified material (containing traces of normal h u m a n proteins) was also used. The extent of HBsAg purification required for the assay depended on the source of anti-HBs used and will be described in more detail in Results. HBsAg was also harvested from a human h e p a t o m a cell line originally isolated by Alexander et al. (1976). A 6--8 day culture fluid was concentrated 100-fold by pressure dialysis using an Amicon XM 300 filter (Amicon Corp., Lexington, MA). The concentration of HBsAg in all preparations was determined by radioimmunoassay using A b b o t t 'Ausria II' kits. All antigen preparations were adjusted so that a 1--106 dilution (in normal human serum) would still produce a positive assay.
(3) Antisera All antisera were purified by 50% saturated a m m o n i u m sulfate precipita-
367 tion followed by passage through a DEAE cellulose col um n using 0.01 M p h o s p h ate b u f f er pH 8.0 as eluant. The IgG fractions were c o n c e n t r a t e d by Amicon pressure dialysis using a PM 30 filter.
(4) Radioactive antibody The IgG fraction with anti-HBs activity was labeled with ~2sI by the p ro ced u r e o f H u n t e r and G r e e n w o o d (1962). Both high titered h u m a n and animal anti-HBs(+) antisera were used. Prior to labeling, the IgG fraction was c o n c e n t r a t e d to 0.1 the original volume by Amicon pressure dialysis using a PM 30 filter. RESULTS Fig. 1A and B show a diagrammatic representation of reagent at 2 stages o f m a n u f a c t u r e and Fig. 1C and D, the pr o cedure for completing the assay. P o l y s ty r en e beads, 0.25 in. in diameter, are reacted with HBsAg (diluted in 0.05 M Tris-HC1, pH 9.0) for 18 h at r o o m t em perat ure. HBsAg obtained f r o m h u man d o n o r sera or tissue culture fluids (from the Alexander h u m a n h e p a t o m a cell line) was used. The dilution of antigen for this initial coating (Fig. 1A) is empirical and is chosen so that all antigen reactive sites can be covered by a second coat of anti-HBs (Fig. 1B). If antigen sites are exposed, t h e y will react with radioactively labeled ant i body. This can be d e t e r m i n e d by taking a representative n u m b e r (1 or 2%) o f washed antigen-covered beads, reacting t h e m first with a non-specific protein such as 10% albumin to block exposed sites on t he bead surfaces, washing t hem , reacting t h e m with anti-HBs and determining w he t he r t hey react with labeled anti-HBs to an e x t e n t greater than would be e x p e c t e d by background c o u n t alone. If an appreciable a m o u n t of radioactivity results, then either a more c o n c e n t r a t e d anti-HBs must be used for the second coat or a f u r t h e r dilution of HBsAg must be made for the first coat. The first coat (HBsAg) must be c o m p l e t e l y b o u n d by the second coat (anti-HBs). We prefer to det erm i ne the suitability o f IgG concentrates as a second coat before resorting to the use of greater dilutions of antigen as a first coat. The sensitivity of the assay can be increased by using t he m a x i m u m a m o u n t of first coat antigen which can be covered by second coat a n t i b o d y . The aim here is to avoid having to resort to isolating a n t i b o d y by prior affinity chr o m at ographi c procedures. Acceptable reagents have been made with the stock concent rat es of antigen described in Materials and Methods, diluted 1 : 10 to 1 : 20 and covered by a second c o at of high titered (1 : 128 by cross-over electrophoresis (CEP)) anti-HBs IgG c o n c e n t r a t e d 10-fold. The e x t e n t of antigen purification necessary for t h e first coat will d e p e n d on the source o f anti-HBs. If h u m a n anti-HBs is used, then the first coat of antigen need n o t be purified to any great e x t e n t provided th at the h u m a n anti-HBs does n o t contain antibodies which react with h u m a n proteins. With tissue culture fluid concentrates, we f o u n d t hat purification was unnecessary since our anti-HBs antisera contained no reacting
368
(A)
(B)
(C)
(D)
Fig. 1. A: an enlarged diagrammatic r e p r e s e n t a t i o n of a cross-section of a suitable bead showing attached purified HBsAg and a non-specific protein to cover any spaces n o t covered by the HBsAg. B: a similar representation, but including the further a t t a c h m e n t of anti-HBs. Serum containing such a n t i b o d y is b r o u g h t into c o n t a c t for sufficient time to completely cover the HBsAg. Excess a n t i b o d y reacting groups remain free and following washing, this particular bead is ready to be used for testing. C: a similar representation, b u t after the particular test bead in question has been exposed to serum to be tested for HBsAg. This serum includes HBsAg which has a t t a c h e d itself to the layer of anti-HBs which forms the outer layer of the test bead. T h e n the reminder of the test serum is removed. D: a similar representation, b u t at a stage where the bead of C has been exposed to radioactively labeled anti-HBs. Some of this a n t i b o d y has a t t a c h e d itself to the HBsAg from the test serum. Following the removal of the u n a t t a c h e d radioactively labeled a n t i b o d y , one is enabled to d e t e r m i n e the presence or absence of HBsAg in the test serum, within the limits of sensitivity of the test, by the presence or absence of significant radioactivity in the test bead. antibodies for other components o f t h e tissue c u l t u r e c o n c e n t r a t e . Fig. 1B represents the reagent and shows that antibody sites in sufficient number remain available for reaction with HBsAg in test sera (Fig. 1C). To assay for
369 TABLE 1 'ASPRIA' USING POLYSTYRENE BEADS The source of anti-HBs for second coating is as indicated. See text for details of reagents and assay procedure. Ratio -- cpm test/cpm mean of NHS (7) Dilution of ttBsAg 'adw' normal human sera containing HBsAg
1 1 1 1 1 1 1 I 1 1 1 1
: 10 : 100 : 1000 : 2000 : 3000 : 4000 : 5000 : 6000 : 7000 : 8000 : 10,000 : 100,000
Polystyrene beads first coated with 1 : 10 dilution HBsAg
Polystyrene beads first coated with 1 : 20 dilution HBsAg
1 2nd coating source : goat and human 1--1
2 2nd coating source: human
3 2nd coating source : goat and human 1--1
4 2nd coating source : human
23.6 13.6 9.4 7.6 6.8 5.0 3.7 3.9 3.0 3.0 2.5 1.1
25.1 22.1 14.3 8.9 8.3 4.5 3.9 3.7 4.3 2.6 2.0 1.2
17.7 18.8 9.6 7.1 6.2 5.1 4.3 3.5 3.3 2.7 2.6 0.95
25.8 20.8 15.5 16.3 11.8 8.9 9.3 7.3 5.5 5.2 3.9 1.2
H B s A g t h e b e a d is c o v e r e d w i t h t e s t s e r u m ( 0 . 2 m l ) a n d i n c u b a t e d e i t h e r f o r 4 h a t 4 5 ° C o r o v e r n i g h t a t r o o m t e m p e r a t u r e . T h e b e a d s are w a s h e d w i t h d i s t i l l e d w a t e r ( 2 . 5 - - 5 m l p e r b e a d ) a n d are n e x t r e a c t e d w i t h a 12hi-labeled IgG fraction of serum containing anti-HBs. After a further incubation for 2 h a t r o o m t e m p e r a t u r e , t h e b e a d s are w a s h e d as p r e v i o u s l y d e s c r i b e d a n d t h e i r r a d i o a c t i v e c o u n t d e t e r m i n e d . A l l t e s t c o u n t s s h o u l d b e c o m p a r e d w i t h a set of negative serum controls (7--10) run simultaneously. A radioactive ratio ( t e s t s e r a / m e a n o f n o r m a l sera} o f 2 o r m o r e is c o n s i d e r e d p o s i t i v e . T h e p r o c e d u r e w i t h m i c r o t i t e r p l a t e s a n d p o l y s t y r e n e t e s t t u b e s is a p p r o x i m a t e l y t h e s a m e as d e s c r i b e d f o r b e a d s . B o t h flat- a n d r o u n d - b o t t o m p l a t e s w e r e u s e d . 0.1 m l o f a n t i g e n (first c o a t ) was c a r e f u l l y a d d e d t o t h e b o t t o m of each well or t u b e . With test t u b e s , 0.2 ml of a n t i g e n m a y be used for the first coat. Table 1 shows the p e r f o r m a n c e of assays using 'Aspria'. A n adw test s e r u m c o u l d b e d i l u t e d t o 1 : 1 0 , 0 0 0 a n d still b e d e t e c t e d as ' p o s i t i v e ' ( r a t i o ~>2). T h e ' A s p r i a ' b e a d s u s e d f o r t h e c o m p i l a t i o n o f t h e s e d a t a w e r e m a d e b y u s i n g e i t h e r a 1 : 1 0 o r 1 : 2 0 d i l u t i o n o f h e p a t o m a cell l i n e t i s s u e c u l t u r e f l u i d s t o c k as t h e p r i m a r y c o a t . T h e s e c o n d c o a t o f a n t i - H B s w a s e i t h e r a
370 m i x tu r e of goat and human anti-HBs globulin, c o n c e n t r a t e d from material eluted from a DEAE-cellulose column, or hum an globulin fraction alone. The sensitivity of the reagent was judged equal to that p r o d u c e d by the test kits of 'Ausria II'. A p p r o x i m a t e l y 100 sera from a collection of Philadelphia renal dialysis patients and staff and HBsAg carriers from Senegal and Korea were coded and assayed by 'Aspria' and 'Ausria II' ( A b b o t t Labs, N. Chicago, IL) simultaneously. An analysis of the results showed that the 75% positive sera picked up by one pr oc e dur e were positive by the other. No discrepancies were found. Positives were confirmed by mixing these sera with an equal volume of human anti-HBs. A re-assay, by bot h procedures, showed either com pl e t e neutralization or a drop in radioactive c o u n t of 50% or more. DISCUSSION Specific a n t i b o d y is an i m p o r t a n t ingredient in very sensitive solid-phase assays detecting HBsAg. An IgG fraction does n o t contain sufficient anti-HBs a n t i b o d y for an acceptable reagent if it is used on both ends of the sandwich. It is d o u b t f u l th at any m a n u f a c t u r e r of HBsAg kits uses dissociated a n t i b o d y for both ends of the sandwich technique. To do so would result in a very sensitive assay indeed, but with our present t echnol ogy it would prove impractical f r o m a cost standpoint. 'Aspria' does n o t require affinity purified a n t i b o d y but instead incorporates the affinity chromatographic separation step into the assay itself. For the first coat of the 'Aspria' reagent either HBsAg from h u m a n h e p a t o m a cell line tissue culture fluids or fractions of h u man HBsAg(+) carrier sera may be used. With hum an carrier sera the source of anti-HBs should be of human origin. While our data show that the sensitivity of several batches of 'Aspria' was equal in sensitivity to 'Ausria II', the potential is present for making 'Aspria' even mo r e sensitive. The primary coat containing HBsAg can be increased, t h e r e b y allowing an increased a m o u n t of specific anti-HBs a n t i b o d y to form the i m p o r t a n t reacting second coat. The limit of sensitivity, therefore, is the p o t e n c y of c o n c e n t r a t e d IgG fractions of anti-HBs; we have used 10-fold c o n c e n t r a t e d IgG fractions of anti-HBs. It appears reasonable to assume that a c o n c e n t r a t i o n of antisera of higher titer than used here would produce an assay of even greater sensitivity w i t h o u t resorting to separate affinity chromatographic purifications. The simplicity of 'Aspria' should permit an even greater n u m b e r of assays for the presence of HBsAg than are currently being used t o d a y . REFERENCES Alexander, J.J., E.M. Bey, E.W. Geddes and G. Lecastas, 1976, S. Afr. Med. J. 50, 2124. Hollinger, F.B., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hunter, W.M. and F.L. Greenwood, 1962, Nature 194,495.
371 Ling, C.M. and L.R. Overby, 1972, J. Immunol. 109,834. Millman, I., L.A. Loeb, M.E. Bayer and B.S. Blumberg, 1970, J. Exp. Med. 131, 1190. Purcell, R.H,, D.C. Wong, H.J. Alter and P.V. Holland, 1973, Appl. Microbiol. 26, 478. Rosenthal, J.D., K. Hayashi and A.L. Notkin, 1972a, App|. Microbiol. 25,567. Rosenthal, J.D., K. Hayashi and A.L. Notkin, 1972b, J. Immunol. 100,586.
' A S P R I A ' 1: A N E W S E N S I T I V E A S S A Y F O R T H E D E T E C T I O N HEPATITIS B SURFACE ANTIGEN :
365
OF
IRVING MILLMAN and MADELYN SMITH 3 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, U.S.A.
(Received 21 June 1979, accepted 10 August 1979)
A sensitive method for the detection of hepatitis B surface antigen (HBsAg) is described whereby the separation of specific antibody by affinity chromatography is incorporated into one of the assay reagents. HBsAg, which may be obtained from tissue culture fluids of a human hepatoma cell line, is first reacted with polystyrene beads. Unoccupied spaces on the plastic are next covered with a non-specific protein (e.g. albumin). The plastic is then reacted with either whole antiserum or an IgG fraction of antiHBs. Care must be taken to cover all of the reacting sites of the first layer of HBsAg with sufficient anti-HBs. This results in a reagent capable of detecting antigen. No affinity purified antibody was necessary because the reagent, at this stage, contains only specific antibody on the surface. Antigen reacting with this reagent can be detected by the addition of radioactively labeled IgG fraction of anti-HBs, which will complete the 'sandwich'. This ,assay has been evaluated and found to be as sensitive as those commercially available.
INTRODUCTION The most sensitive assays currently favored for the detection of hepatitis B s u r f a c e a n t i g e n ( H B s A g ) axe s o l i d - p h a s e r a d i o i m m u n o a s s a y s ( H o l l i n g e r e t al., 1 9 7 1 ; L i n g a n d O v e r b y , 1 9 7 2 ; R o s e n t h a l e t al., 1 9 7 2 a , b; P u r c e l l e t a l . , 1973). The current commercially available tests make use of a substrate, u s u a l l y a p l a s t i c b e a d o r t u b e , t o w h i c h is a t t a c h e d t h e I g G f r a c t i o n o f a n antiserum containing anti-HBs. These antisera are from either human donors or hyperimmunized a n i m a l s . T h e H B s A g in t h e t e s t s e r u m r e a c t s w i t h a t t a c h e d a n t i b o d y a n d is d e t e c t e d b y t h e a d d i t i o n o f a n a n t i - H B s 12sIlabeled IgG fraction. A sandwich of antibody, antigen and radioactive antib o d y is f o r m e d . T h e m e t h o d is e x t r e m e l y s e n s i t i v e b e c a u s e w i t h i n c r e a s e in a n t i g e n c o n c e n t r a t i o n t h e r e is a n i n c r e a s e in r a d i o a c t i v i t y . S e n s i t i v i t y o f t h e assay can be increased by substituting an affinity purified antibody for the 1 U.S. Patent Application Serial No. 903,238. 2 This work was supported by USPHS Grants CA-06551, RR-05539 and CA-06927 from the National Institutes of Health and by an appropriation from the Commonwealth of Pennsylvania. 3 Student of Drexel University (Philadelphia, PA) on work-study program.
366 IgG fraction containing anti-HBs. This substitution can be made for the labeled or unlabeled antibody (at either end of the sandwich). Such a change is effective since even in hyperimmune globulin fractions only a small percentage of the a n t i b o d y is specific anti-HBs. The production of affinity purified antibody in sufficient amounts is difficult and expensive and contributes to the high cost of the presently available assay kits. The favored procedure for producing specific antibody involves affinity chromatography with subsequent dissociation of complexes by a variety of means. Such procedures often result in low yields because of incomplete dissociation or unsatisfactory yields because of alterations (denaturation) or antibody molecules. This report describes a novel procedure which allows the separation of specific antibody without prior affinity chromatography. Affinity chromatography, currently used to manufacture specific antibody for either end of the sandwich (either to coat a solid substrate or for radioactive labeling), is incorporated within the assay for HBsAg itself. A reagent is produced which we call 'Aspria' (for affinity solid-phase radioimmunoassay), which can be used to detect HBsAg with sensitivity equal to assays currently available. An added advantage is that the reagent is less costly to manufacture. MATERIALS AND METHODS
(1) Supporting material Polystyrene beads, 0.25 in. diameter, were purchased from United States Plastic Corporation, Lima, OH and The Superior Ball Co., Hartford, CT. Disposable microtiter plates were purchased from Cooke Engineering Co., Alexandria, VA, and disposable polystyrene tubes were purchased from Falcon, Oxnard, CA.
(2) HBsAg HBsAg from human donors was purified according to the procedure described earlier (Millman et al., 1970). Less purified material (containing traces of normal h u m a n proteins) was also used. The extent of HBsAg purification required for the assay depended on the source of anti-HBs used and will be described in more detail in Results. HBsAg was also harvested from a human h e p a t o m a cell line originally isolated by Alexander et al. (1976). A 6--8 day culture fluid was concentrated 100-fold by pressure dialysis using an Amicon XM 300 filter (Amicon Corp., Lexington, MA). The concentration of HBsAg in all preparations was determined by radioimmunoassay using A b b o t t 'Ausria II' kits. All antigen preparations were adjusted so that a 1--106 dilution (in normal human serum) would still produce a positive assay.
(3) Antisera All antisera were purified by 50% saturated a m m o n i u m sulfate precipita-
367 tion followed by passage through a DEAE cellulose col um n using 0.01 M p h o s p h ate b u f f er pH 8.0 as eluant. The IgG fractions were c o n c e n t r a t e d by Amicon pressure dialysis using a PM 30 filter.
(4) Radioactive antibody The IgG fraction with anti-HBs activity was labeled with ~2sI by the p ro ced u r e o f H u n t e r and G r e e n w o o d (1962). Both high titered h u m a n and animal anti-HBs(+) antisera were used. Prior to labeling, the IgG fraction was c o n c e n t r a t e d to 0.1 the original volume by Amicon pressure dialysis using a PM 30 filter. RESULTS Fig. 1A and B show a diagrammatic representation of reagent at 2 stages o f m a n u f a c t u r e and Fig. 1C and D, the pr o cedure for completing the assay. P o l y s ty r en e beads, 0.25 in. in diameter, are reacted with HBsAg (diluted in 0.05 M Tris-HC1, pH 9.0) for 18 h at r o o m t em perat ure. HBsAg obtained f r o m h u man d o n o r sera or tissue culture fluids (from the Alexander h u m a n h e p a t o m a cell line) was used. The dilution of antigen for this initial coating (Fig. 1A) is empirical and is chosen so that all antigen reactive sites can be covered by a second coat of anti-HBs (Fig. 1B). If antigen sites are exposed, t h e y will react with radioactively labeled ant i body. This can be d e t e r m i n e d by taking a representative n u m b e r (1 or 2%) o f washed antigen-covered beads, reacting t h e m first with a non-specific protein such as 10% albumin to block exposed sites on t he bead surfaces, washing t hem , reacting t h e m with anti-HBs and determining w he t he r t hey react with labeled anti-HBs to an e x t e n t greater than would be e x p e c t e d by background c o u n t alone. If an appreciable a m o u n t of radioactivity results, then either a more c o n c e n t r a t e d anti-HBs must be used for the second coat or a f u r t h e r dilution of HBsAg must be made for the first coat. The first coat (HBsAg) must be c o m p l e t e l y b o u n d by the second coat (anti-HBs). We prefer to det erm i ne the suitability o f IgG concentrates as a second coat before resorting to the use of greater dilutions of antigen as a first coat. The sensitivity of the assay can be increased by using t he m a x i m u m a m o u n t of first coat antigen which can be covered by second coat a n t i b o d y . The aim here is to avoid having to resort to isolating a n t i b o d y by prior affinity chr o m at ographi c procedures. Acceptable reagents have been made with the stock concent rat es of antigen described in Materials and Methods, diluted 1 : 10 to 1 : 20 and covered by a second c o at of high titered (1 : 128 by cross-over electrophoresis (CEP)) anti-HBs IgG c o n c e n t r a t e d 10-fold. The e x t e n t of antigen purification necessary for t h e first coat will d e p e n d on the source o f anti-HBs. If h u m a n anti-HBs is used, then the first coat of antigen need n o t be purified to any great e x t e n t provided th at the h u m a n anti-HBs does n o t contain antibodies which react with h u m a n proteins. With tissue culture fluid concentrates, we f o u n d t hat purification was unnecessary since our anti-HBs antisera contained no reacting
368
(A)
(B)
(C)
(D)
Fig. 1. A: an enlarged diagrammatic r e p r e s e n t a t i o n of a cross-section of a suitable bead showing attached purified HBsAg and a non-specific protein to cover any spaces n o t covered by the HBsAg. B: a similar representation, but including the further a t t a c h m e n t of anti-HBs. Serum containing such a n t i b o d y is b r o u g h t into c o n t a c t for sufficient time to completely cover the HBsAg. Excess a n t i b o d y reacting groups remain free and following washing, this particular bead is ready to be used for testing. C: a similar representation, b u t after the particular test bead in question has been exposed to serum to be tested for HBsAg. This serum includes HBsAg which has a t t a c h e d itself to the layer of anti-HBs which forms the outer layer of the test bead. T h e n the reminder of the test serum is removed. D: a similar representation, b u t at a stage where the bead of C has been exposed to radioactively labeled anti-HBs. Some of this a n t i b o d y has a t t a c h e d itself to the HBsAg from the test serum. Following the removal of the u n a t t a c h e d radioactively labeled a n t i b o d y , one is enabled to d e t e r m i n e the presence or absence of HBsAg in the test serum, within the limits of sensitivity of the test, by the presence or absence of significant radioactivity in the test bead. antibodies for other components o f t h e tissue c u l t u r e c o n c e n t r a t e . Fig. 1B represents the reagent and shows that antibody sites in sufficient number remain available for reaction with HBsAg in test sera (Fig. 1C). To assay for
369 TABLE 1 'ASPRIA' USING POLYSTYRENE BEADS The source of anti-HBs for second coating is as indicated. See text for details of reagents and assay procedure. Ratio -- cpm test/cpm mean of NHS (7) Dilution of ttBsAg 'adw' normal human sera containing HBsAg
1 1 1 1 1 1 1 I 1 1 1 1
: 10 : 100 : 1000 : 2000 : 3000 : 4000 : 5000 : 6000 : 7000 : 8000 : 10,000 : 100,000
Polystyrene beads first coated with 1 : 10 dilution HBsAg
Polystyrene beads first coated with 1 : 20 dilution HBsAg
1 2nd coating source : goat and human 1--1
2 2nd coating source: human
3 2nd coating source : goat and human 1--1
4 2nd coating source : human
23.6 13.6 9.4 7.6 6.8 5.0 3.7 3.9 3.0 3.0 2.5 1.1
25.1 22.1 14.3 8.9 8.3 4.5 3.9 3.7 4.3 2.6 2.0 1.2
17.7 18.8 9.6 7.1 6.2 5.1 4.3 3.5 3.3 2.7 2.6 0.95
25.8 20.8 15.5 16.3 11.8 8.9 9.3 7.3 5.5 5.2 3.9 1.2
H B s A g t h e b e a d is c o v e r e d w i t h t e s t s e r u m ( 0 . 2 m l ) a n d i n c u b a t e d e i t h e r f o r 4 h a t 4 5 ° C o r o v e r n i g h t a t r o o m t e m p e r a t u r e . T h e b e a d s are w a s h e d w i t h d i s t i l l e d w a t e r ( 2 . 5 - - 5 m l p e r b e a d ) a n d are n e x t r e a c t e d w i t h a 12hi-labeled IgG fraction of serum containing anti-HBs. After a further incubation for 2 h a t r o o m t e m p e r a t u r e , t h e b e a d s are w a s h e d as p r e v i o u s l y d e s c r i b e d a n d t h e i r r a d i o a c t i v e c o u n t d e t e r m i n e d . A l l t e s t c o u n t s s h o u l d b e c o m p a r e d w i t h a set of negative serum controls (7--10) run simultaneously. A radioactive ratio ( t e s t s e r a / m e a n o f n o r m a l sera} o f 2 o r m o r e is c o n s i d e r e d p o s i t i v e . T h e p r o c e d u r e w i t h m i c r o t i t e r p l a t e s a n d p o l y s t y r e n e t e s t t u b e s is a p p r o x i m a t e l y t h e s a m e as d e s c r i b e d f o r b e a d s . B o t h flat- a n d r o u n d - b o t t o m p l a t e s w e r e u s e d . 0.1 m l o f a n t i g e n (first c o a t ) was c a r e f u l l y a d d e d t o t h e b o t t o m of each well or t u b e . With test t u b e s , 0.2 ml of a n t i g e n m a y be used for the first coat. Table 1 shows the p e r f o r m a n c e of assays using 'Aspria'. A n adw test s e r u m c o u l d b e d i l u t e d t o 1 : 1 0 , 0 0 0 a n d still b e d e t e c t e d as ' p o s i t i v e ' ( r a t i o ~>2). T h e ' A s p r i a ' b e a d s u s e d f o r t h e c o m p i l a t i o n o f t h e s e d a t a w e r e m a d e b y u s i n g e i t h e r a 1 : 1 0 o r 1 : 2 0 d i l u t i o n o f h e p a t o m a cell l i n e t i s s u e c u l t u r e f l u i d s t o c k as t h e p r i m a r y c o a t . T h e s e c o n d c o a t o f a n t i - H B s w a s e i t h e r a
370 m i x tu r e of goat and human anti-HBs globulin, c o n c e n t r a t e d from material eluted from a DEAE-cellulose column, or hum an globulin fraction alone. The sensitivity of the reagent was judged equal to that p r o d u c e d by the test kits of 'Ausria II'. A p p r o x i m a t e l y 100 sera from a collection of Philadelphia renal dialysis patients and staff and HBsAg carriers from Senegal and Korea were coded and assayed by 'Aspria' and 'Ausria II' ( A b b o t t Labs, N. Chicago, IL) simultaneously. An analysis of the results showed that the 75% positive sera picked up by one pr oc e dur e were positive by the other. No discrepancies were found. Positives were confirmed by mixing these sera with an equal volume of human anti-HBs. A re-assay, by bot h procedures, showed either com pl e t e neutralization or a drop in radioactive c o u n t of 50% or more. DISCUSSION Specific a n t i b o d y is an i m p o r t a n t ingredient in very sensitive solid-phase assays detecting HBsAg. An IgG fraction does n o t contain sufficient anti-HBs a n t i b o d y for an acceptable reagent if it is used on both ends of the sandwich. It is d o u b t f u l th at any m a n u f a c t u r e r of HBsAg kits uses dissociated a n t i b o d y for both ends of the sandwich technique. To do so would result in a very sensitive assay indeed, but with our present t echnol ogy it would prove impractical f r o m a cost standpoint. 'Aspria' does n o t require affinity purified a n t i b o d y but instead incorporates the affinity chromatographic separation step into the assay itself. For the first coat of the 'Aspria' reagent either HBsAg from h u m a n h e p a t o m a cell line tissue culture fluids or fractions of h u man HBsAg(+) carrier sera may be used. With hum an carrier sera the source of anti-HBs should be of human origin. While our data show that the sensitivity of several batches of 'Aspria' was equal in sensitivity to 'Ausria II', the potential is present for making 'Aspria' even mo r e sensitive. The primary coat containing HBsAg can be increased, t h e r e b y allowing an increased a m o u n t of specific anti-HBs a n t i b o d y to form the i m p o r t a n t reacting second coat. The limit of sensitivity, therefore, is the p o t e n c y of c o n c e n t r a t e d IgG fractions of anti-HBs; we have used 10-fold c o n c e n t r a t e d IgG fractions of anti-HBs. It appears reasonable to assume that a c o n c e n t r a t i o n of antisera of higher titer than used here would produce an assay of even greater sensitivity w i t h o u t resorting to separate affinity chromatographic purifications. The simplicity of 'Aspria' should permit an even greater n u m b e r of assays for the presence of HBsAg than are currently being used t o d a y . REFERENCES Alexander, J.J., E.M. Bey, E.W. Geddes and G. Lecastas, 1976, S. Afr. Med. J. 50, 2124. Hollinger, F.B., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hunter, W.M. and F.L. Greenwood, 1962, Nature 194,495.
371 Ling, C.M. and L.R. Overby, 1972, J. Immunol. 109,834. Millman, I., L.A. Loeb, M.E. Bayer and B.S. Blumberg, 1970, J. Exp. Med. 131, 1190. Purcell, R.H,, D.C. Wong, H.J. Alter and P.V. Holland, 1973, Appl. Microbiol. 26, 478. Rosenthal, J.D., K. Hayashi and A.L. Notkin, 1972a, App|. Microbiol. 25,567. Rosenthal, J.D., K. Hayashi and A.L. Notkin, 1972b, J. Immunol. 100,586.