Vox Sang. 35: 137-142 (1978)
Radioimmunossay for the Detection of the Antibody against Hepatitis-B-Core Antigen (Anti-HBc) G . Hess and J . W.K. Shih Molecular Anatomy Program, Oak Kidgc National Laboratory, Rockeville, Md.
Abstract. In this paper we describe a new sensitive solid phase radioimmunoassay for the detection of anti-HBc. This test was shown to be more sensitive than the widely used immune adherence hemagglutination test (IAHA) and at least as sensitive as the radioimmunoassay using the blocking principle. The new test system appears to be very useful to screen larger groups of individuals (e.g. blood donors) for the presence of anti-HBc.
Introduction Three different morphological forms exposing hepatitis-B-surface antigen (HBsAg) determinants can be differentiated in sera of HBsAg carriers and patients with an acute virus B hepatitis: a spherical 22 nm particle, a filamentous particle which is 22 nm in diameter and of variable length and the spherical 42 nm Dane particle. The Dane particle is the only one of these three particles which consists of an inner core (hepatitis B core antigen (HBcAg)) and an outer shell (HBsAg). Two subpopulations of Dane particles have been demonstrated [3, 7, 151. One subpopulation contains a circular DNA and a DNA polymerase and is therefore believed to be the complete hepatitis B virus (HBV) [8,141. The second Dane particle population lacks DNA and DNA poly-
merase activity and is considered to be a defective HBV. Infectivity studies corroborate these assumptions [l, 12, 151. It is well established that individuals carrying an antibody against HBcAg (anti-HBc) have undergone or are undergoing an HBV infection [4, 5, 91. In this paper we describe a new radioimmunoassay for the detection of anti-HBc and compare its sensitivity with two other well established anti-HBc tests.
Material and Methods Assays
HBcAg was determined by solid phase radioimmunoassay as described by Purrell et al. [13]. HBV-specific DNA polymerase activity was measured by the method of Kaplan et 01. [8]. Sera
Hess/Sh i h
138
were tested for the presence of anti-HBc by three different mtthods: (a) Immune adherence hemagglutination test (IAHA): This test was carried out as described by Tsudn et (11. 1161 and modified as published by Moritscrgu er al. [lo]. The sera were screened at dilutions of l:lO, 1:lOO and 1:1,000 and starting from the highest dilution positive at twofold dilutions. The titer given is the highest serum dilution positive for anti-HBc. (b) Solid phase radioimmunoassay: This test using the blocking principle was done as described hy Purrell et al. [13]. A serum blocking the binding of lesl-anti-HBc to a standard HBcAg by more than 40% was considered to be anti-HBc positive. To give a titer, tenfold dilutions were prepared and a curve was drawn. Where this curve crossed the ‘40% inhibition line’ the titer was read on a linear scale. (c) The third procedure is specified in the section Results. Prepcrrntion of HBcAg
HBcAg was isolated from a serum containing a high quantity of Dane particles. Dane particles
in 200 ml of serum were concentrated by layering 36ml of serum on 2.51-111 of 65% (w/w) sucrose and centrifuging it in a SW 27 rotor at 27,000 RPM for h h at 4 “C. The interfaces were collected and diluted with an equal volume of 0.01 M phosphate buffered saline (PBS). The procedure was repeated once resulting in a Dane particle concentrate of 10 ml. This material was then dialyzed against 0.01 M PBS overnight. 5 ml each of the dialyzed Dane-rich material were layered on 6 ml of 20% (w/w) sucrose with a 65% (w/w) sucrose cushion and centrifuged at 40,000RPM for 4 h at 4OC. The interface was harvested and the procedure was repeated twice to remove residual anti-HBc. The final Dane particle concentrate of 2 ml was incubated with 2 ml H,O, 200 ! t l of 10% Nonidet P 4 0 (NP40) and 30rtl of 1 : l O diluted 2-mercaptoethanol. After an incubation period of I h at 37 C, 2 ml were layered on top of a cesium chloride gradient consisting of 1 ml of 1.40g/ml CsCI, 3.5 ml of 1.35 g/’ml CsCI, 3 ml of 1.30 g/ml CsCl and 2.5 ml of 1.25 g/ml CsCI. After centrifugation i n a SW 41 rotor at 30,000 RPM for 16 h at 4 O C , fractions were collected and assayed for HBcAg and DNA polymerase activity. Two peaks of
HBcAg activity were detected: one DNA polymerase activity positive HBcAg population banded at a density of 1.36g/ml. The major HBcAg peak, which was DNA polymerase activity negative, was found at a density of 1.30g/ml. This second peak of HBcAg served as an antigen pool for all radioimmunoassay anti-HBc tests. Source und Preporation o/ A n t i - H B c IgG fractions containing anti-HBc in a high titer were prepared from a HBeAg-positive serum of a chronic HBsAg carrier. The IgG was isolated on a DEAE column and labeled with 1261 using the chloramine T method as described by Hunter and Greenwood [6].
Results Test Procedure Serial tenfold dilutions were prepared from test sera using 0.01 M PBS containing 0.5% bovine serum albumin (BSA) as a diluent. Each well of a polyvinyl microtiter plate (Cooke Laboratory Products, Alexandria Va.) received 75 ,MI of the diluted test sera. After an incubation period of 4 h at room temperature the sera were removed from the wells and the wells were washed twice with PBS. Then each well was filled with 250p1 of PBS containing 0.5% BSA and the plate was incubated at 4 “C overnight. The removal of the PBS-BSA buffer from the wells was followed by five wash cycles with PBS. After adding 25 p l of purified and standardized HBcAg the plates were incubated at 4 ‘C for 24 h. The unbound antigen was removed and the wells were washed five times with PBS. Finally each well received 50 p l of 1 2 5 1 anti-HBc. Following incubation at 37 “C for 4 h the plates were again washed five times. Then the wells were cut apart with scissors, placed into 12 x 75 mm polystyrene tubes (Fal-
Radioimmunoassay for Anti-HBc
139
2 1 reciprocal LOGlo antibody dilution
2
3
4
5
6
7
8
reciprocal LOG10 antibody dilution
8
Fig. 1. Radioimmunoassay for the detection of anti-HBc ( 0 ) using the RIA described in this paper. In one experiment the antigen (HBcAg) was replaced by PBS (0). = PN/ 6.3 line. Fig. 2. Kadioimmunoassay for the detection of anti-HBc ( 0 )using the blocking principle. The same reagents as in figure 1 were used. -.-= 40% inhibition line. Fig. 3. Demonstration of the specificity of the RIA described in this paper: anti-HBc antiHBs (n), normal human serum .).(
-6
lii I v7
b,q E
3
(a),
con Plastics, Oxnard, Calif.) and counted in a Packard gammacounter. HBsAg-negative sera which were anti-HBc-negative in previous experiments served as negative controls for this test. A serum was considered to be positive for anti-HBc if its positivehegative (P/N) ratio exceeded 6.3. Figure 1 shows a typical curve for an anti-HBc-positive serum. The titer was read where the curve crossed the P/N 6.3 line. The same anti-HBc-positive serum was also tested with the radioimmunoassay blocking test (procedure see methods) and using identical reagents (fig. 2).
reciprocal L O G l o antibody dilution
Specificity In one experiment HBcAg was replaced by PBS. As shown in figure 1, P/N ratios between 1 and 2 were measured under these conditions. In addition normal human sera (free of HBsAg and anti-HBc) and antiHBs-positive sera (without detectable antiHBc in previous experiments) were tested. Figure 3 gives the results of these tests: P/N ratios between 1 and 2 were observed for all normal human sera and all anti-HBspositive sera tested. A typical dose-response curve was seen with the anti-HBc-positive sera.
I40
Hess/Sh i h
Table 1. Effect of the time of coating with the lest serum on the anti-HBc titer
Table 11. Effect of the HBcAg concentration on the anti-HBc titer
Time of coating with the test serum (at room temperature), h
Anti-HBc titer
Antigen concentration CPM
Anti-HBc tiler
I 2 4
I :200,000 I :400,000 I :400,000 I : 500,000
18,000 (3 x 1 6,000 ( 1 x ) 2,000 (1/3 x )
I :1 ,000.000 I : 800,000 I :90,000
8
Sensitivity
To obtain maximal sensitivity of the test system, the test procedure was modified. In a first experiment the time allowing the test serum to coat the well of the microtiter plate was varied. As shown in table I the amount of antibody bound to the microtiter plate was maximal after an incubation period of 2 h at room temperature, a longer incubation period produced no change in antiHBc titer. In a second experiment the amount of antigen (HBcAg) was modified. As demonstrated in table 11, low amounts of antigen, expressed as CPM as tested in a HBcAg assay, equalled a loss in sensitivity. There was, however, no significant difference in anti-HBc titer when medium or high antigen concentrations were chosen. Finally, the time of HBcAg incubation was varied. As demonstrated in table I11 the binding of anti-HBc to HBcAg was complete after an incubation period of 18 h at 4 " C . A longer incubation period did not affect anti-HBc titers. The sensitivity of the test described in this paper was compared with two well-established anti-HBc tests. Sera of ten asymptomatic HBsAg carriers were tested for antiHBc by IAHA, a radioimmunoassay blocking test and the radioimmunoassay procedure described in this paper. The results are
Table 111. Effect of the time of HBcAg incubation (4°C) on the anti-HBc titer Time of HBcAg incubation (at 4"C), h
Anti-H Bc titer
5
I :80,000
18
1 : 300,000
30 48 60 72 120
1 :400,OOo I : 300,000 I :400,000 I :600,000 1 :500,000
Table 1V. Relative sensitivity of three anti-HBc assays No.
Anti-HBc titer IAHA
I 2
3 4 5 6 7 8 9 10
blocking RIA
1:16,000 1 :60,000 1:8,000 1 :80,000
I :2,000 1:4,000 1:8,000 1:16,000 1:8.000 1:8,000
1:8,000 1:8,000
I :20,000 I :50,000 1 :50,000 1 :70,000 1 :90,000 1 :40,000 I :50,000 1 :40,000
described test (RIA) 1 : 500,000 I : 700,000 1 :90.000 I :500.000 1 :400,000 1 :800,000 1 :600,000 I : 500,000 I :400.000 I :200,000
Kadioimmunoassay for Anti-HBc
given in table IV. The least sensitive antiHBc test was the IAHA. The test described in this paper appeared to be more sensitive than the radioimmunoassay blocking test.
Discussion
In this paper we describe a new radioimmunoassay for the detection of anti-HBc in serum. As demonstrated, this test is more sensitive than the IAHA and seems also to be more sensitive than the radioimmunoassay blocking test. Although this point was not specifically studied, our test is probably less sensitive than the two radioimmunoprecipitation tests (RIP) previously described [2, 111. Both RIP work with labeled HBcAg. One test uses HBcAg whose DNA has been labeled with the DNA polymerase reaction as an antigen [ I l l . The second RIP utilizes r2sI labeled HBcAg [2]. Although very sensitive, both tests have significant disadvantages. The DNA containing Dane particles from which the DNA containing HBcAg is released represents only a minor fraction of all Dane particles [7]. Therefore the available antigen material is very limited. The second RIP using 1 2 5 1 labeled HBcAg can use HBcAg of both Dane particle subpopulations but requires a very pure and nearly protein-free HBcAg preparation which leads to huge losses during the preparation procedure. In addition, 1851 HBcAg shows a rather rapid degradation [ 2 ] . Both RIP fail to work in case of aggregation of the HBcAg material. For these reasons both RIP have never been used to determine anti-HBc in a larger population. Solid phase radioimmunoassays can be carried out with HBcAg derived from both Dane particle subpopulations. A
141
HBcAg preparation largely free from antiHBc is required, but contaminating proteins do not interfere with the test system. For this reason the radioimmunoassay appears to be useful to test larger groups of individuals. The only disadvantage of this test system is that completely comparable titers are only obtained if the sera are tested simultaneously. The IAHA does not have these problems and gives reliable and comparable results for years, its disadvantage being its lower sensitivity. The test described in this paper represents a very sensitive radioimmunoassay and appears to be useful to test larger groups of individuals (e.g. blood donors) for the presence of anti-HBc. Therefore, the assay may help to eliminate the rare, potentially infective blood units in which anti-HBc is the only marker of a HBV infection.
References 1 Alter, H . J.; Seef, L. B.; Kaplan, P. M.; McAu-
2
3
4
5
liffe, V. J.; Wright, E. C.; Gerin, L. J.; Purcell, R. H.; Holland, P. V., and Zimmermann, H. D.: Type B hepatitis: the infectivity of blood positive for e antigen and D N A polymerase after accidental needlestick. New Engl. J . Med. 294: 909-913 (1976). Budkowska, A.; Shih, J . W.-K., and Gerin, L. J.: Immunochemistry and polypeptide composition of hepatitis B core antigen (HBcAg). J. Immun. 118: 1300-1305 (1977). Gerin, L. J.; Ford, E.C., and Purcell, R. H.: Biochemical characterization of Australia antigen: evidence for defective particles of hepatitis-B-virus Am. J . Path. XI: 351-662 (1975). Hoofnagle, J. H.; Gerety, R. J., and Barker, L. F.: Antibody to hepatitis-B-virus core in man. Lancet ii: 869-873 (1973). Hoofnagle, J . H.; Gerety, R. J., and Barker, L. F.: Antibody to hepatitis B core antigen. Am. J . med. Sci. 270: 179-187 (1975).
142
6 Hunter, W. M. and Greenwood, F. C.: Preparation of 1811 labeled human growth hormone of high specific activity. Nature 194: 495498 (1962). 7 Kaplan, P. M.; Ford, E. C.; Purcell, R. H., and Gerin, J. L.: Demonstration of subpopulations of Dane particles. J. Virol. 17: 885-893 (1976). 8 Kaplan, P. M.; Greenman, R. L.; Gerin, L. J.; Purcell, R. H., and Robinson, W. S.: DNA polymerase associated with human hepatitis B antigen. J. Virol. 12: 995-1005 (1973). 9 Krugman, S.; Hoofnagle, J. H.; Gerety, R. J.; Kaplan, P. M., and Gerin, L. J.: DNA polymerase activity and antibody to hepatitis B core antigen. New Engl. J. Med. 290: 1331-1335 (1974). 10 Moritsugu, Y.; Dienstag, J. L.; Valdesuso, J.; Wong, D. C.; Wagner, J.; Routenberg, J. A., and Purcell, R. H.: Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination. Infec. Immunity 13: 898-908 (1976). I I Moritsugu, Y.; Gold, J . W. M.; Wagner, J.; Dodd, R. Y., and Purcell, R. H.: Detection of antibody by radioimmunoprecipitation. J. Immun. 114: 1792-1798 (1975). 12 Okada, K.; Kamiyama, I.; Inomata, M.; Imai, M.; Miyakawa, Y.,and Mayumi, M.: e Antigen and anti-e in the serum of asymptomatic carrier mothers as indicators or positive and neg-
Hess/Shih
13
14
15
16
ative transmission of hepatitis B virus to their infants. New Engl. J . Med. 294: 746-749 (1976). Purcell, R. H.; Gerin, L. J.; Almeida, J. B., and Holland, P. V.: Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2: 231-234 (19731 74). Robinson, W. S.; Clayton, D. A., and Greenman, R. L.: D N A of a human hepatitis B virus candidate. J. Virol. 14: 384-1236 (1973). Thomssen, R.; Gerlich, W.; Stamm, B.; Biswas, R.; Lorenz, P. R.; Majer, M; Weinmann, E; Arnold, W.; Hess, G.; Wepler, W. und Klinge, 0.: Atiologie der Hepatitis B - vorlaufige Ergebnisse einer kooperativen Studie. Zentbl. Bakt. Hyg. I Abtl. Orig. A 235: 242-252 (1976). Tsuda, F.; Takahashi, T.; Takahashi, K.; Miyakawa, Y., and Mayumi, M.: Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. J . Immun. 115: 834-837 (1975).
Received: September 20, 1977 Accepted: November 24, 1977 Dr. G . Hess, Free University of Berlin, Spandauer Damm 130, D-1000 Berlin 19 (FRG)
Radioimmunossay for the Detection of the Antibody against Hepatitis-B-Core Antigen (Anti-HBc) G . Hess and J . W.K. Shih Molecular Anatomy Program, Oak Kidgc National Laboratory, Rockeville, Md.
Abstract. In this paper we describe a new sensitive solid phase radioimmunoassay for the detection of anti-HBc. This test was shown to be more sensitive than the widely used immune adherence hemagglutination test (IAHA) and at least as sensitive as the radioimmunoassay using the blocking principle. The new test system appears to be very useful to screen larger groups of individuals (e.g. blood donors) for the presence of anti-HBc.
Introduction Three different morphological forms exposing hepatitis-B-surface antigen (HBsAg) determinants can be differentiated in sera of HBsAg carriers and patients with an acute virus B hepatitis: a spherical 22 nm particle, a filamentous particle which is 22 nm in diameter and of variable length and the spherical 42 nm Dane particle. The Dane particle is the only one of these three particles which consists of an inner core (hepatitis B core antigen (HBcAg)) and an outer shell (HBsAg). Two subpopulations of Dane particles have been demonstrated [3, 7, 151. One subpopulation contains a circular DNA and a DNA polymerase and is therefore believed to be the complete hepatitis B virus (HBV) [8,141. The second Dane particle population lacks DNA and DNA poly-
merase activity and is considered to be a defective HBV. Infectivity studies corroborate these assumptions [l, 12, 151. It is well established that individuals carrying an antibody against HBcAg (anti-HBc) have undergone or are undergoing an HBV infection [4, 5, 91. In this paper we describe a new radioimmunoassay for the detection of anti-HBc and compare its sensitivity with two other well established anti-HBc tests.
Material and Methods Assays
HBcAg was determined by solid phase radioimmunoassay as described by Purrell et al. [13]. HBV-specific DNA polymerase activity was measured by the method of Kaplan et 01. [8]. Sera
Hess/Sh i h
138
were tested for the presence of anti-HBc by three different mtthods: (a) Immune adherence hemagglutination test (IAHA): This test was carried out as described by Tsudn et (11. 1161 and modified as published by Moritscrgu er al. [lo]. The sera were screened at dilutions of l:lO, 1:lOO and 1:1,000 and starting from the highest dilution positive at twofold dilutions. The titer given is the highest serum dilution positive for anti-HBc. (b) Solid phase radioimmunoassay: This test using the blocking principle was done as described hy Purrell et al. [13]. A serum blocking the binding of lesl-anti-HBc to a standard HBcAg by more than 40% was considered to be anti-HBc positive. To give a titer, tenfold dilutions were prepared and a curve was drawn. Where this curve crossed the ‘40% inhibition line’ the titer was read on a linear scale. (c) The third procedure is specified in the section Results. Prepcrrntion of HBcAg
HBcAg was isolated from a serum containing a high quantity of Dane particles. Dane particles
in 200 ml of serum were concentrated by layering 36ml of serum on 2.51-111 of 65% (w/w) sucrose and centrifuging it in a SW 27 rotor at 27,000 RPM for h h at 4 “C. The interfaces were collected and diluted with an equal volume of 0.01 M phosphate buffered saline (PBS). The procedure was repeated once resulting in a Dane particle concentrate of 10 ml. This material was then dialyzed against 0.01 M PBS overnight. 5 ml each of the dialyzed Dane-rich material were layered on 6 ml of 20% (w/w) sucrose with a 65% (w/w) sucrose cushion and centrifuged at 40,000RPM for 4 h at 4OC. The interface was harvested and the procedure was repeated twice to remove residual anti-HBc. The final Dane particle concentrate of 2 ml was incubated with 2 ml H,O, 200 ! t l of 10% Nonidet P 4 0 (NP40) and 30rtl of 1 : l O diluted 2-mercaptoethanol. After an incubation period of I h at 37 C, 2 ml were layered on top of a cesium chloride gradient consisting of 1 ml of 1.40g/ml CsCI, 3.5 ml of 1.35 g/’ml CsCI, 3 ml of 1.30 g/ml CsCl and 2.5 ml of 1.25 g/ml CsCI. After centrifugation i n a SW 41 rotor at 30,000 RPM for 16 h at 4 O C , fractions were collected and assayed for HBcAg and DNA polymerase activity. Two peaks of
HBcAg activity were detected: one DNA polymerase activity positive HBcAg population banded at a density of 1.36g/ml. The major HBcAg peak, which was DNA polymerase activity negative, was found at a density of 1.30g/ml. This second peak of HBcAg served as an antigen pool for all radioimmunoassay anti-HBc tests. Source und Preporation o/ A n t i - H B c IgG fractions containing anti-HBc in a high titer were prepared from a HBeAg-positive serum of a chronic HBsAg carrier. The IgG was isolated on a DEAE column and labeled with 1261 using the chloramine T method as described by Hunter and Greenwood [6].
Results Test Procedure Serial tenfold dilutions were prepared from test sera using 0.01 M PBS containing 0.5% bovine serum albumin (BSA) as a diluent. Each well of a polyvinyl microtiter plate (Cooke Laboratory Products, Alexandria Va.) received 75 ,MI of the diluted test sera. After an incubation period of 4 h at room temperature the sera were removed from the wells and the wells were washed twice with PBS. Then each well was filled with 250p1 of PBS containing 0.5% BSA and the plate was incubated at 4 “C overnight. The removal of the PBS-BSA buffer from the wells was followed by five wash cycles with PBS. After adding 25 p l of purified and standardized HBcAg the plates were incubated at 4 ‘C for 24 h. The unbound antigen was removed and the wells were washed five times with PBS. Finally each well received 50 p l of 1 2 5 1 anti-HBc. Following incubation at 37 “C for 4 h the plates were again washed five times. Then the wells were cut apart with scissors, placed into 12 x 75 mm polystyrene tubes (Fal-
Radioimmunoassay for Anti-HBc
139
2 1 reciprocal LOGlo antibody dilution
2
3
4
5
6
7
8
reciprocal LOG10 antibody dilution
8
Fig. 1. Radioimmunoassay for the detection of anti-HBc ( 0 ) using the RIA described in this paper. In one experiment the antigen (HBcAg) was replaced by PBS (0). = PN/ 6.3 line. Fig. 2. Kadioimmunoassay for the detection of anti-HBc ( 0 )using the blocking principle. The same reagents as in figure 1 were used. -.-= 40% inhibition line. Fig. 3. Demonstration of the specificity of the RIA described in this paper: anti-HBc antiHBs (n), normal human serum .).(
-6
lii I v7
b,q E
3
(a),
con Plastics, Oxnard, Calif.) and counted in a Packard gammacounter. HBsAg-negative sera which were anti-HBc-negative in previous experiments served as negative controls for this test. A serum was considered to be positive for anti-HBc if its positivehegative (P/N) ratio exceeded 6.3. Figure 1 shows a typical curve for an anti-HBc-positive serum. The titer was read where the curve crossed the P/N 6.3 line. The same anti-HBc-positive serum was also tested with the radioimmunoassay blocking test (procedure see methods) and using identical reagents (fig. 2).
reciprocal L O G l o antibody dilution
Specificity In one experiment HBcAg was replaced by PBS. As shown in figure 1, P/N ratios between 1 and 2 were measured under these conditions. In addition normal human sera (free of HBsAg and anti-HBc) and antiHBs-positive sera (without detectable antiHBc in previous experiments) were tested. Figure 3 gives the results of these tests: P/N ratios between 1 and 2 were observed for all normal human sera and all anti-HBspositive sera tested. A typical dose-response curve was seen with the anti-HBc-positive sera.
I40
Hess/Sh i h
Table 1. Effect of the time of coating with the lest serum on the anti-HBc titer
Table 11. Effect of the HBcAg concentration on the anti-HBc titer
Time of coating with the test serum (at room temperature), h
Anti-HBc titer
Antigen concentration CPM
Anti-HBc tiler
I 2 4
I :200,000 I :400,000 I :400,000 I : 500,000
18,000 (3 x 1 6,000 ( 1 x ) 2,000 (1/3 x )
I :1 ,000.000 I : 800,000 I :90,000
8
Sensitivity
To obtain maximal sensitivity of the test system, the test procedure was modified. In a first experiment the time allowing the test serum to coat the well of the microtiter plate was varied. As shown in table I the amount of antibody bound to the microtiter plate was maximal after an incubation period of 2 h at room temperature, a longer incubation period produced no change in antiHBc titer. In a second experiment the amount of antigen (HBcAg) was modified. As demonstrated in table 11, low amounts of antigen, expressed as CPM as tested in a HBcAg assay, equalled a loss in sensitivity. There was, however, no significant difference in anti-HBc titer when medium or high antigen concentrations were chosen. Finally, the time of HBcAg incubation was varied. As demonstrated in table I11 the binding of anti-HBc to HBcAg was complete after an incubation period of 18 h at 4 " C . A longer incubation period did not affect anti-HBc titers. The sensitivity of the test described in this paper was compared with two well-established anti-HBc tests. Sera of ten asymptomatic HBsAg carriers were tested for antiHBc by IAHA, a radioimmunoassay blocking test and the radioimmunoassay procedure described in this paper. The results are
Table 111. Effect of the time of HBcAg incubation (4°C) on the anti-HBc titer Time of HBcAg incubation (at 4"C), h
Anti-H Bc titer
5
I :80,000
18
1 : 300,000
30 48 60 72 120
1 :400,OOo I : 300,000 I :400,000 I :600,000 1 :500,000
Table 1V. Relative sensitivity of three anti-HBc assays No.
Anti-HBc titer IAHA
I 2
3 4 5 6 7 8 9 10
blocking RIA
1:16,000 1 :60,000 1:8,000 1 :80,000
I :2,000 1:4,000 1:8,000 1:16,000 1:8.000 1:8,000
1:8,000 1:8,000
I :20,000 I :50,000 1 :50,000 1 :70,000 1 :90,000 1 :40,000 I :50,000 1 :40,000
described test (RIA) 1 : 500,000 I : 700,000 1 :90.000 I :500.000 1 :400,000 1 :800,000 1 :600,000 I : 500,000 I :400.000 I :200,000
Kadioimmunoassay for Anti-HBc
given in table IV. The least sensitive antiHBc test was the IAHA. The test described in this paper appeared to be more sensitive than the radioimmunoassay blocking test.
Discussion
In this paper we describe a new radioimmunoassay for the detection of anti-HBc in serum. As demonstrated, this test is more sensitive than the IAHA and seems also to be more sensitive than the radioimmunoassay blocking test. Although this point was not specifically studied, our test is probably less sensitive than the two radioimmunoprecipitation tests (RIP) previously described [2, 111. Both RIP work with labeled HBcAg. One test uses HBcAg whose DNA has been labeled with the DNA polymerase reaction as an antigen [ I l l . The second RIP utilizes r2sI labeled HBcAg [2]. Although very sensitive, both tests have significant disadvantages. The DNA containing Dane particles from which the DNA containing HBcAg is released represents only a minor fraction of all Dane particles [7]. Therefore the available antigen material is very limited. The second RIP using 1 2 5 1 labeled HBcAg can use HBcAg of both Dane particle subpopulations but requires a very pure and nearly protein-free HBcAg preparation which leads to huge losses during the preparation procedure. In addition, 1851 HBcAg shows a rather rapid degradation [ 2 ] . Both RIP fail to work in case of aggregation of the HBcAg material. For these reasons both RIP have never been used to determine anti-HBc in a larger population. Solid phase radioimmunoassays can be carried out with HBcAg derived from both Dane particle subpopulations. A
141
HBcAg preparation largely free from antiHBc is required, but contaminating proteins do not interfere with the test system. For this reason the radioimmunoassay appears to be useful to test larger groups of individuals. The only disadvantage of this test system is that completely comparable titers are only obtained if the sera are tested simultaneously. The IAHA does not have these problems and gives reliable and comparable results for years, its disadvantage being its lower sensitivity. The test described in this paper represents a very sensitive radioimmunoassay and appears to be useful to test larger groups of individuals (e.g. blood donors) for the presence of anti-HBc. Therefore, the assay may help to eliminate the rare, potentially infective blood units in which anti-HBc is the only marker of a HBV infection.
References 1 Alter, H . J.; Seef, L. B.; Kaplan, P. M.; McAu-
2
3
4
5
liffe, V. J.; Wright, E. C.; Gerin, L. J.; Purcell, R. H.; Holland, P. V., and Zimmermann, H. D.: Type B hepatitis: the infectivity of blood positive for e antigen and D N A polymerase after accidental needlestick. New Engl. J . Med. 294: 909-913 (1976). Budkowska, A.; Shih, J . W.-K., and Gerin, L. J.: Immunochemistry and polypeptide composition of hepatitis B core antigen (HBcAg). J. Immun. 118: 1300-1305 (1977). Gerin, L. J.; Ford, E.C., and Purcell, R. H.: Biochemical characterization of Australia antigen: evidence for defective particles of hepatitis-B-virus Am. J . Path. XI: 351-662 (1975). Hoofnagle, J. H.; Gerety, R. J., and Barker, L. F.: Antibody to hepatitis-B-virus core in man. Lancet ii: 869-873 (1973). Hoofnagle, J . H.; Gerety, R. J., and Barker, L. F.: Antibody to hepatitis B core antigen. Am. J . med. Sci. 270: 179-187 (1975).
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Received: September 20, 1977 Accepted: November 24, 1977 Dr. G . Hess, Free University of Berlin, Spandauer Damm 130, D-1000 Berlin 19 (FRG)
