Journal of Immunological Methods, 22 (1978) 143--148
143
© Elsevier/North-Holland Biomedical Press
RADIOIMMUNOASSAY FO R H E PA T I T I S B SURFACE A N T I G E N AND ANTIBODY USING A TWO-COMPONENT IMMUNOADSORBENT
SEAN P. O'NEILL, THOMAS ROBB and PAUL SHENVI Electro-Nucleonics Laboratories, Inc., 4809 Auburn Avenue, Bethesda, MD 20014, U.S.A.
(Received 5 August 1977, accepted 10 February 1978)
A solid-phase radioimmunoassay is described for detection of hepatitis B surface antigen and antibody. The assay uses a two-site (antigen and antibody) immunoadsorbent capable of binding antigen and antibody. Antigen is detected by enhanced binding of [ 12si ]antibody through sandwich formations of antibody--antigens--[ 12si ]antibody on the immunoadsorbent. Antibody presence is revealed by inhibition of [12SI]antibody binding.
INTRODUCTION Investigation of antigen--antibody systems frequently requires sensitive m eth o d s for d et ect i on of each c o m p o n e n t . In following the course of an infectious disease, for example, useful i nf o rm at i on is obtained from identification o f a virus (or virus-associated antigenic particles) or its ant i body in b o d y fluids. Sensitive d e t e c t i o n of antigenic substance is conveniently carried out by a solid-phase competitive radioimmunoassay (Catt and Tregear, 1967). This p ro ced u r e c a n n o t be used, however, if the possibility exists that a test sample contains a n t i b o d y since this will interfere with the radioimmunoassay. To avoid this difficulty we have investigated a solid-phase procedure which will d e t e c t either antigen or ant i body in a single assay, while clearly distinguishing which c o m p o n e n t is present. This p r o ced u re is applied here to detection of hepatitis B surface antigen (HBsAg) and its a n t i b o d y (anti-HB~). There are several published radioimmunoassay procedures for detection o f HBsAg (Aach et al., 1971; Coller et al., 1971; Hollinger et al., 1971) and anti-HB~ ant i body (Langer et al., 1971; Patil and Pert, 1975). PRINCIPLE OF THE METHOD A solid-phase i m m u n o a d s o r b e n t containing both antigen and ant i body sites is used as a binding agent. The i m m u n o a d s o r b e n t is initially capable of binding a defined a m o u n t of labeled antibody.
144 As the immunoadsorbent is incubated with a test sample its antibody binding capacity will be altered if the test sample contains either antigen or antibody. Antigen in the test sample attaches to antibody sites on the immunoadsorbent, increasing its capacity to bind labeled antibody. Conversely, antibody in the test sample will block antigen sites on the immunoadsorbent, decreasing its capacity to bind labeled antibody. The presence of antigen or antibody is then revealed by enhanced or depressed binding of labeled antibody to the solid-phase immunoadsorbent. MATERIALS AND METHODS Immunoadsorbent A solid-phase reagent consisting of goat anti-HB s attached to controlledpore glass particles was obtained from a commercial kit for radioimmunoassay of HB~Ag (Electro-Nucleonics Laboratories, Inc., Bethesda, MD). This immunoadsorbent was modified by incubation with a human serum sample containing HBsAg particles. The resulting two-component immunoadsorbent did not display antibody activity towards normal human serum (NHS) as indicated by a lack of reactivity towards labeled [12SI]NHS and [12SI]human IgG. Both of these tracers gave only a small a m o u n t of non-specific binding (100--300 cpm) when incubated with the HBsAg/anti-HB~ immunoadsorbent. The level of HBsAg in the serum sample was chosen so that approximately 50% of the solid-phase antibody sites were complexed with HB~Ag, as judged by uptake of [12SI]anti-HBs after controlled complexing with cold HB~Ag compared with the radioactive uptake achieved after saturation of the immunoadsorbent with cold HBsAg. The variance in tube-to-tube concentration of HBsAg on the immunoadsorbent particles was less than +5%, again based on measurements of [12SI]anti-HBs binding. With different lots of HBsAg-positive sera used for coating immunoadsorbents, the mean level of attached HBsAg sites varied within limits of 30--50% of the total antibody sites. Controls were run with each assay to establish the radioactive binding level in the absence of HB~Ag and anti-HB~ antibody. This level was the basis for establishing criteria for identifying positive HB~Ag and anti-HB~ samples from negative samples in each assay. This two-site immunoadsorbent was found to be stable for at least one year when stored at 4°C either in a dry state or as a slurry, as shown by assay performance. Other reagents Radioactively labeled [12SI]anti-HBs antibody (Electro-Nucleonics Laboratories, Inc.) was used as tracer. This reagent is dissolved in a buffer solution of 15% normal goat serum in 0.01 M Tris, pH 7.2. A human serum sample free of HBsAg and anti-HB s was used as a negative control. The presence of HB~Ag was tested for by a solid-phase radioimmunoassay procedure (RIAUSURE, Electro-Nucleonics Laboratories, Inc.); anti-
145 HBs was tested for by a passive hemagglutination test (Electro-Nucleonics Laboratories, Inc.). All wash steps were carried out using phosphate-buffered saline (PBS), 0.05 M, pH 7.2.
Radioimmunoassay procedure A test sample (0.2 ml) was added to a reaction t ube containing the i m m u n o a d s o r b e n t . A small magnet was added and the slurry of test sample and glass particles was mixed by applying a magnetic field. After an incubation o f 4 h at r o o m t e m p e r a t u r e , the glass particles were washed free of test sample with 0.5 ml of PBS. After washing, the glass particles were allowed to settle for 90 sec before aspirating of f the wash buffer above the settled glass. Labeled [12SI]antibody (0.2 ml; 100,000 cpm) was added and incubated with the i m m u n o a d s o r b e n t by magnetic mixing for 1 h. The reaction tube was then washed free of u n b o u n d radioactivity using 4 washes of 0.5 ml PBS. Each assay t ube was t hen c o u n t e d for 1 min. Ten negative control samples were assayed with each lot of u n k n o w n sera. RESULTS A h u man serum sample containing a high c o n c e n t r a t i o n of anti-HB s antib o d y was progressively diluted with a sample containing a high concentration of HBsAg. The results (Fig. 1) display a typical curve for the assay procedure. Dilutions containing anti-HB s ant i body alone or in excess over HBsAg give results in a region below the negative control line. The equivalence point of HBsAg/anti-HB s a n t i b o d y is given by the intersection of the assay curve and the horizontal line representing the negative control value. Bound radioactive counts above this line indicate the presence of HBsAg alone or in excess over anti-HB s an tib o d y. The slight decrease in the curve at high HBsAg concentrations is due to an inhibition effect. At high c o n c e n t r a t i o n not all of the free HB~Ag particles are washed f r o m the assay tube, the remainder causes some inhibition o f the binding o f the radioactive tracer. The inhibition causes only the slight decline in binding shown in Fig. 1 even at concentrations of HB~Ag as high as 1014 particles/ml. The steep slope of the assay curve as it crosses the negative control line demonstrates the sensitivity of this assay procedure for det ect i on of an antigen--antibody equivalence point. The sensitivity of det ect i on of anti-HB s ant i body is compared in Table 1 with two o t h e r procedures. Test samples with low concentrations of antiHB~ were assayed by each m e t h o d . The levels of detectability are similar in each case. In the passive hemagglutination assay, the presence of a low concentration o f anti-HB~ a n t i b o d y results in a two-dilution increase in cell
146
12000I 1000C 8000 CPM 6000 4000 /
2000 2
4
/
16
64 256 DILUTION
1024
Fig. 1. R a d i o a c t i v e c o u n t s (CPM) b o u n d t o the i m m u n o a d s o r b e n t as a f u n c t i o n o f H B s A g and anti-HB s a n t i b o d y c o n c e n t r a t i o n in the assay sample. A h u m a n serum sample containing a n t i - H B s A g a n t i b o d y was progressively diluted with a s a m p l e c o n t a i n i n g a high c o n c e n t r a t i o n o f HBsAg.
a g g l u t i n a t i o n . T h e c o m p a r i s o n o f b o u n d r a d i o a c t i v e c o u n t s f o r t h e positive s a m p l e w i t h t h o s e f o r t h e n e g a t i v e c o n t r o l i n d i c a t e t h e degree o f discriminat i o n b e t w e e n positive a n d negative w i t h t h e r a d i o i m m u n o a s s a y s y s t e m s . T h e s a m e a n t i b o d y s a m p l e displays an e n h a n c e m e n t o f 6 9 6 c p m in b o u n d radioactivity with a s a n d w i c h R I A p r o c e d u r e a n d an i n h i b i t i o n in b i n d i n g o f 1 2 9 6 cpm with the two-site RIA described here. T h e sensitivity o f this R I A f o r d e t e c t i o n o f b o t h H B s A g a n d its a n t i b o d y ,
TABLE 1 RELATIVE SENSITIVITY OF DETECTION ENT ASSAY PROCEDURES
OF ANTI-HB s ANTIBODY
BY DIFFER-
Procedure
Negative control
Low concentration anti-HBs antibody sample
Passive hemagglutination a Sandwich RIA b Present RIA
1 : 4 titer 156 cpm 5265 cpm
1 : 16 titer 852 cpm 3969 cpm
a Electro-Nucleonics Laboratories, Inc. Total incubation time 45 rain. b Abbott Laboratories. Total incubation time 22 h.
147 TABLE 2 TYPICAL RESULTS OBTAINED FOR RADIOIMMUNOASSAY OF HUMAN SERUM SAMPLES CONTAINING ANTI-HBs ANTIBODY, HBsAg OR NEITHER OF THESE COMPONENTS Sample number
Sample type a
Relative concentration of either HBsAG or anti-HBs
Bound cpm
104 103 102 101 105
anti-HBs
very high b high b intermediate b low b --
164 637 2566 3205 4372 4504 5131 10324 9822
Negative
311
315 304 306
--
HBsAg
Negative control mean:
low c intermediate d high e 4602
a Samples are graded according to their degree of reaction with other assay procedures as indicated. b Graded by reaction with passive hemagglutination or sandwhich RIA results: "low" rating represents samples near the limits of detection by these procedures. c Low level of HBsAg , approximately 107 particles per test sample (0.2 ml), at the limits of detectability by RIA procedures. d Positive by RIA; negative by counterimmunoelectrophoresis (CEP) or agar gel diffusion techniques (AGD). e Positive by RIA, CEP and AGD.
is d e m o n s t r a t e d i n T a b l e 2. R e s u l t s are s h o w n f o r s a m p l e s c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f H B s A g o r a n t i - H B s. I n t h i s a s s a y t h e m e a n o f t h e 1 0 n e g a t i v e c o n t r o l s a m p l e s was 4 6 0 2 c p m . The criteria chosen for establishing cut-off values which would identify samples c o n t a i n i n g H B s A g o r a n t i - H B s a n t i b o d y , was t o c o n s i d e r v a l u e s w i t h i n a r a n g e o f + 3 0 % o f t h e n e g a t i v e c o n t r o l m e a n , as c o v e r i n g n e g a t i v e s e r u m s . S a m p l e s w i t h c o u n t s less t h a n 0.7 X n e g a t i v e c o n t r o l m e a n ( 3 2 2 1 c p m in T a b l e 2) are p o s i t i v e f o r a n t i - H B ~ a n t i b o d y . S a m p l e s w i t h c o u n t s g r e a t e r t h a n 1.3 X n e g a t i v e c o n t r o l m e a n ( 5 9 8 3 c p m i n T a b l e 2) are p o s i t i v e f o r HB~Ag. T h i s c r i t e r i o n was b a s e d o n r e s u l t s f o r 4 2 0 n e g a t i v e sera t e s t e d , i n w h i c h r e s u l t s f o r o n l y 3 s a m p l e s fell o u t s i d e t h e r a n g e o f +30% o f t h e c o n t r o l m e a n . These deviations were n o t r e p r o d u c i b l e w h e n the 3 samples were r e - t e s t e d a n d c a n b e a t t r i b u t e d t o e r r o r s in assay t e c h n i q u e . N o b i o l o g i c a l false p o s i t i v e r e s u l t s w e r e o b t a i n e d .
148 DISCUSSION T h e p o t e n t i a l usefulness o f the RIA described, rests o n (1) the convenience o f using a single solid-phase p r o c e d u r e to d e t e r m i n e t w o c o m p o n e n t s , an antigen and a n t i b o d y ; and (2) the a v o i d a n c e o f the necessity for having available purified antigen f o r radioactive labelings. T h e avoidance o f the prerequisite f o r a labeled antigen is i m p o r t a n t in instances w h e r e a virus is n o t readily available ( H u t c h i n s o n and Ziegler, 1 9 7 2 ) . This m e t h o d also allows the a p p l i c a t i o n o f a solid-phase RIA t o situations where a n t i b o d y interferes with the usual c o m p e t i t i v e assay ( E n l a n d e r et al., 1976). T h e sensitivity o f d e t e c t i o n o f e i t h e r c o m p o n e n t , in t h e assay, is high because o f the steep slope o f the b o u n d r a d i o a c t i v i t y in the region o f the negative c o n t r o l m e a n (Fig. 1). T h e limits o n t h e sensitivity c o m e f r o m the r e p r o d u c i b i l i t y o b t a i n a b l e for the negative c o n t r o l values. Variability introd u c e d at this p o i n t , is largely a f u n c t i o n o f the solid-phase material used and operator technique. The p r o c e d u r e is based on the use o f a solid phase c o n t a i n i n g b o t h antib o d y and antigen binding sites. A n t i b o d y was c h e m i c a l l y linked t o the solid' phase, so it is irreversibly b o u n d u n d e r the assay c o n d i t i o n s . Antigen was a t t a c h e d b y c o m p l e x i n g with t h e a n t i b o d y sites. It is possible t h a t during assay o f a sample c o n t a i n i n g a n t i b o d y , s o m e o f the biologically a t t a c h e d antigen is r e m o v e d f r o m the solid phase and c o m p l e x e d with soluble antib o d y . This will n o t i n t e r f e r e with the assay, since r e m o v a l o f antigen f r o m the solid phase will m e r e l y decrease t h e binding o f the labeled a n t i b o d y p r o b e , adding t o the n e t i n h i b i t i o n o f b o u n d c o u n t s , due to the presence of a n t i b o d y in t h e test sample. REFERENCES Aach, R.D., J.W. Grisham and C.W. Parker, 1971, Proc. Natl. Acad. Sci. U.S.A. 68, 1056. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Coller, J.A., I. Millman, T.C. Halbherr and B.S. Blumberg, 1971, Proc. Soc. Exp. Biol. Med. 138,249. Enlander, D., L.V. Dos Remedios, P.M. Weber and L. Drew, 1976, J. Immunol. Methods 10, 357. Hollinger, B.F., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hutchinson, H.D. and D.W. Ziegler, 1972, Appl. Microbiol. 24, 742. Lander, J.J., H.J. Alter and R.H. Purcell, 1971, J. Immunol. 106, 1166. Patil, J.R. and J.H. Pert, 1975, J. Immunol. Methods 7,169.
143
© Elsevier/North-Holland Biomedical Press
RADIOIMMUNOASSAY FO R H E PA T I T I S B SURFACE A N T I G E N AND ANTIBODY USING A TWO-COMPONENT IMMUNOADSORBENT
SEAN P. O'NEILL, THOMAS ROBB and PAUL SHENVI Electro-Nucleonics Laboratories, Inc., 4809 Auburn Avenue, Bethesda, MD 20014, U.S.A.
(Received 5 August 1977, accepted 10 February 1978)
A solid-phase radioimmunoassay is described for detection of hepatitis B surface antigen and antibody. The assay uses a two-site (antigen and antibody) immunoadsorbent capable of binding antigen and antibody. Antigen is detected by enhanced binding of [ 12si ]antibody through sandwich formations of antibody--antigens--[ 12si ]antibody on the immunoadsorbent. Antibody presence is revealed by inhibition of [12SI]antibody binding.
INTRODUCTION Investigation of antigen--antibody systems frequently requires sensitive m eth o d s for d et ect i on of each c o m p o n e n t . In following the course of an infectious disease, for example, useful i nf o rm at i on is obtained from identification o f a virus (or virus-associated antigenic particles) or its ant i body in b o d y fluids. Sensitive d e t e c t i o n of antigenic substance is conveniently carried out by a solid-phase competitive radioimmunoassay (Catt and Tregear, 1967). This p ro ced u r e c a n n o t be used, however, if the possibility exists that a test sample contains a n t i b o d y since this will interfere with the radioimmunoassay. To avoid this difficulty we have investigated a solid-phase procedure which will d e t e c t either antigen or ant i body in a single assay, while clearly distinguishing which c o m p o n e n t is present. This p r o ced u re is applied here to detection of hepatitis B surface antigen (HBsAg) and its a n t i b o d y (anti-HB~). There are several published radioimmunoassay procedures for detection o f HBsAg (Aach et al., 1971; Coller et al., 1971; Hollinger et al., 1971) and anti-HB~ ant i body (Langer et al., 1971; Patil and Pert, 1975). PRINCIPLE OF THE METHOD A solid-phase i m m u n o a d s o r b e n t containing both antigen and ant i body sites is used as a binding agent. The i m m u n o a d s o r b e n t is initially capable of binding a defined a m o u n t of labeled antibody.
144 As the immunoadsorbent is incubated with a test sample its antibody binding capacity will be altered if the test sample contains either antigen or antibody. Antigen in the test sample attaches to antibody sites on the immunoadsorbent, increasing its capacity to bind labeled antibody. Conversely, antibody in the test sample will block antigen sites on the immunoadsorbent, decreasing its capacity to bind labeled antibody. The presence of antigen or antibody is then revealed by enhanced or depressed binding of labeled antibody to the solid-phase immunoadsorbent. MATERIALS AND METHODS Immunoadsorbent A solid-phase reagent consisting of goat anti-HB s attached to controlledpore glass particles was obtained from a commercial kit for radioimmunoassay of HB~Ag (Electro-Nucleonics Laboratories, Inc., Bethesda, MD). This immunoadsorbent was modified by incubation with a human serum sample containing HBsAg particles. The resulting two-component immunoadsorbent did not display antibody activity towards normal human serum (NHS) as indicated by a lack of reactivity towards labeled [12SI]NHS and [12SI]human IgG. Both of these tracers gave only a small a m o u n t of non-specific binding (100--300 cpm) when incubated with the HBsAg/anti-HB~ immunoadsorbent. The level of HBsAg in the serum sample was chosen so that approximately 50% of the solid-phase antibody sites were complexed with HB~Ag, as judged by uptake of [12SI]anti-HBs after controlled complexing with cold HB~Ag compared with the radioactive uptake achieved after saturation of the immunoadsorbent with cold HBsAg. The variance in tube-to-tube concentration of HBsAg on the immunoadsorbent particles was less than +5%, again based on measurements of [12SI]anti-HBs binding. With different lots of HBsAg-positive sera used for coating immunoadsorbents, the mean level of attached HBsAg sites varied within limits of 30--50% of the total antibody sites. Controls were run with each assay to establish the radioactive binding level in the absence of HB~Ag and anti-HB~ antibody. This level was the basis for establishing criteria for identifying positive HB~Ag and anti-HB~ samples from negative samples in each assay. This two-site immunoadsorbent was found to be stable for at least one year when stored at 4°C either in a dry state or as a slurry, as shown by assay performance. Other reagents Radioactively labeled [12SI]anti-HBs antibody (Electro-Nucleonics Laboratories, Inc.) was used as tracer. This reagent is dissolved in a buffer solution of 15% normal goat serum in 0.01 M Tris, pH 7.2. A human serum sample free of HBsAg and anti-HB s was used as a negative control. The presence of HB~Ag was tested for by a solid-phase radioimmunoassay procedure (RIAUSURE, Electro-Nucleonics Laboratories, Inc.); anti-
145 HBs was tested for by a passive hemagglutination test (Electro-Nucleonics Laboratories, Inc.). All wash steps were carried out using phosphate-buffered saline (PBS), 0.05 M, pH 7.2.
Radioimmunoassay procedure A test sample (0.2 ml) was added to a reaction t ube containing the i m m u n o a d s o r b e n t . A small magnet was added and the slurry of test sample and glass particles was mixed by applying a magnetic field. After an incubation o f 4 h at r o o m t e m p e r a t u r e , the glass particles were washed free of test sample with 0.5 ml of PBS. After washing, the glass particles were allowed to settle for 90 sec before aspirating of f the wash buffer above the settled glass. Labeled [12SI]antibody (0.2 ml; 100,000 cpm) was added and incubated with the i m m u n o a d s o r b e n t by magnetic mixing for 1 h. The reaction tube was then washed free of u n b o u n d radioactivity using 4 washes of 0.5 ml PBS. Each assay t ube was t hen c o u n t e d for 1 min. Ten negative control samples were assayed with each lot of u n k n o w n sera. RESULTS A h u man serum sample containing a high c o n c e n t r a t i o n of anti-HB s antib o d y was progressively diluted with a sample containing a high concentration of HBsAg. The results (Fig. 1) display a typical curve for the assay procedure. Dilutions containing anti-HB s ant i body alone or in excess over HBsAg give results in a region below the negative control line. The equivalence point of HBsAg/anti-HB s a n t i b o d y is given by the intersection of the assay curve and the horizontal line representing the negative control value. Bound radioactive counts above this line indicate the presence of HBsAg alone or in excess over anti-HB s an tib o d y. The slight decrease in the curve at high HBsAg concentrations is due to an inhibition effect. At high c o n c e n t r a t i o n not all of the free HB~Ag particles are washed f r o m the assay tube, the remainder causes some inhibition o f the binding o f the radioactive tracer. The inhibition causes only the slight decline in binding shown in Fig. 1 even at concentrations of HB~Ag as high as 1014 particles/ml. The steep slope of the assay curve as it crosses the negative control line demonstrates the sensitivity of this assay procedure for det ect i on of an antigen--antibody equivalence point. The sensitivity of det ect i on of anti-HB s ant i body is compared in Table 1 with two o t h e r procedures. Test samples with low concentrations of antiHB~ were assayed by each m e t h o d . The levels of detectability are similar in each case. In the passive hemagglutination assay, the presence of a low concentration o f anti-HB~ a n t i b o d y results in a two-dilution increase in cell
146
12000I 1000C 8000 CPM 6000 4000 /
2000 2
4
/
16
64 256 DILUTION
1024
Fig. 1. R a d i o a c t i v e c o u n t s (CPM) b o u n d t o the i m m u n o a d s o r b e n t as a f u n c t i o n o f H B s A g and anti-HB s a n t i b o d y c o n c e n t r a t i o n in the assay sample. A h u m a n serum sample containing a n t i - H B s A g a n t i b o d y was progressively diluted with a s a m p l e c o n t a i n i n g a high c o n c e n t r a t i o n o f HBsAg.
a g g l u t i n a t i o n . T h e c o m p a r i s o n o f b o u n d r a d i o a c t i v e c o u n t s f o r t h e positive s a m p l e w i t h t h o s e f o r t h e n e g a t i v e c o n t r o l i n d i c a t e t h e degree o f discriminat i o n b e t w e e n positive a n d negative w i t h t h e r a d i o i m m u n o a s s a y s y s t e m s . T h e s a m e a n t i b o d y s a m p l e displays an e n h a n c e m e n t o f 6 9 6 c p m in b o u n d radioactivity with a s a n d w i c h R I A p r o c e d u r e a n d an i n h i b i t i o n in b i n d i n g o f 1 2 9 6 cpm with the two-site RIA described here. T h e sensitivity o f this R I A f o r d e t e c t i o n o f b o t h H B s A g a n d its a n t i b o d y ,
TABLE 1 RELATIVE SENSITIVITY OF DETECTION ENT ASSAY PROCEDURES
OF ANTI-HB s ANTIBODY
BY DIFFER-
Procedure
Negative control
Low concentration anti-HBs antibody sample
Passive hemagglutination a Sandwich RIA b Present RIA
1 : 4 titer 156 cpm 5265 cpm
1 : 16 titer 852 cpm 3969 cpm
a Electro-Nucleonics Laboratories, Inc. Total incubation time 45 rain. b Abbott Laboratories. Total incubation time 22 h.
147 TABLE 2 TYPICAL RESULTS OBTAINED FOR RADIOIMMUNOASSAY OF HUMAN SERUM SAMPLES CONTAINING ANTI-HBs ANTIBODY, HBsAg OR NEITHER OF THESE COMPONENTS Sample number
Sample type a
Relative concentration of either HBsAG or anti-HBs
Bound cpm
104 103 102 101 105
anti-HBs
very high b high b intermediate b low b --
164 637 2566 3205 4372 4504 5131 10324 9822
Negative
311
315 304 306
--
HBsAg
Negative control mean:
low c intermediate d high e 4602
a Samples are graded according to their degree of reaction with other assay procedures as indicated. b Graded by reaction with passive hemagglutination or sandwhich RIA results: "low" rating represents samples near the limits of detection by these procedures. c Low level of HBsAg , approximately 107 particles per test sample (0.2 ml), at the limits of detectability by RIA procedures. d Positive by RIA; negative by counterimmunoelectrophoresis (CEP) or agar gel diffusion techniques (AGD). e Positive by RIA, CEP and AGD.
is d e m o n s t r a t e d i n T a b l e 2. R e s u l t s are s h o w n f o r s a m p l e s c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f H B s A g o r a n t i - H B s. I n t h i s a s s a y t h e m e a n o f t h e 1 0 n e g a t i v e c o n t r o l s a m p l e s was 4 6 0 2 c p m . The criteria chosen for establishing cut-off values which would identify samples c o n t a i n i n g H B s A g o r a n t i - H B s a n t i b o d y , was t o c o n s i d e r v a l u e s w i t h i n a r a n g e o f + 3 0 % o f t h e n e g a t i v e c o n t r o l m e a n , as c o v e r i n g n e g a t i v e s e r u m s . S a m p l e s w i t h c o u n t s less t h a n 0.7 X n e g a t i v e c o n t r o l m e a n ( 3 2 2 1 c p m in T a b l e 2) are p o s i t i v e f o r a n t i - H B ~ a n t i b o d y . S a m p l e s w i t h c o u n t s g r e a t e r t h a n 1.3 X n e g a t i v e c o n t r o l m e a n ( 5 9 8 3 c p m i n T a b l e 2) are p o s i t i v e f o r HB~Ag. T h i s c r i t e r i o n was b a s e d o n r e s u l t s f o r 4 2 0 n e g a t i v e sera t e s t e d , i n w h i c h r e s u l t s f o r o n l y 3 s a m p l e s fell o u t s i d e t h e r a n g e o f +30% o f t h e c o n t r o l m e a n . These deviations were n o t r e p r o d u c i b l e w h e n the 3 samples were r e - t e s t e d a n d c a n b e a t t r i b u t e d t o e r r o r s in assay t e c h n i q u e . N o b i o l o g i c a l false p o s i t i v e r e s u l t s w e r e o b t a i n e d .
148 DISCUSSION T h e p o t e n t i a l usefulness o f the RIA described, rests o n (1) the convenience o f using a single solid-phase p r o c e d u r e to d e t e r m i n e t w o c o m p o n e n t s , an antigen and a n t i b o d y ; and (2) the a v o i d a n c e o f the necessity for having available purified antigen f o r radioactive labelings. T h e avoidance o f the prerequisite f o r a labeled antigen is i m p o r t a n t in instances w h e r e a virus is n o t readily available ( H u t c h i n s o n and Ziegler, 1 9 7 2 ) . This m e t h o d also allows the a p p l i c a t i o n o f a solid-phase RIA t o situations where a n t i b o d y interferes with the usual c o m p e t i t i v e assay ( E n l a n d e r et al., 1976). T h e sensitivity o f d e t e c t i o n o f e i t h e r c o m p o n e n t , in t h e assay, is high because o f the steep slope o f the b o u n d r a d i o a c t i v i t y in the region o f the negative c o n t r o l m e a n (Fig. 1). T h e limits o n t h e sensitivity c o m e f r o m the r e p r o d u c i b i l i t y o b t a i n a b l e for the negative c o n t r o l values. Variability introd u c e d at this p o i n t , is largely a f u n c t i o n o f the solid-phase material used and operator technique. The p r o c e d u r e is based on the use o f a solid phase c o n t a i n i n g b o t h antib o d y and antigen binding sites. A n t i b o d y was c h e m i c a l l y linked t o the solid' phase, so it is irreversibly b o u n d u n d e r the assay c o n d i t i o n s . Antigen was a t t a c h e d b y c o m p l e x i n g with t h e a n t i b o d y sites. It is possible t h a t during assay o f a sample c o n t a i n i n g a n t i b o d y , s o m e o f the biologically a t t a c h e d antigen is r e m o v e d f r o m the solid phase and c o m p l e x e d with soluble antib o d y . This will n o t i n t e r f e r e with the assay, since r e m o v a l o f antigen f r o m the solid phase will m e r e l y decrease t h e binding o f the labeled a n t i b o d y p r o b e , adding t o the n e t i n h i b i t i o n o f b o u n d c o u n t s , due to the presence of a n t i b o d y in t h e test sample. REFERENCES Aach, R.D., J.W. Grisham and C.W. Parker, 1971, Proc. Natl. Acad. Sci. U.S.A. 68, 1056. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Coller, J.A., I. Millman, T.C. Halbherr and B.S. Blumberg, 1971, Proc. Soc. Exp. Biol. Med. 138,249. Enlander, D., L.V. Dos Remedios, P.M. Weber and L. Drew, 1976, J. Immunol. Methods 10, 357. Hollinger, B.F., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hutchinson, H.D. and D.W. Ziegler, 1972, Appl. Microbiol. 24, 742. Lander, J.J., H.J. Alter and R.H. Purcell, 1971, J. Immunol. 106, 1166. Patil, J.R. and J.H. Pert, 1975, J. Immunol. Methods 7,169.
