Journal of Virological Methods, 28 (1990) 165-170
165
Elsevier VIRMET 01011
Monoclonal antibodies against hepatitis. B e antigen: production, characterization, and use for diagnosis E. Korec’, V. DostBlovB’, J. Korco&, P. Ma&al*, J. Kiinig, G. Borisova4, V. Cibinogen4, P. Pumpen4, E. Gren4 and I. HloBhnekl ‘Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague, 21nstitute of Sera and Vaccines, Prague, ‘Institute of Hygiene and Epidemiology, Prague, Czechoslovakia and %stitute of Organic Synthesis, Latvian Academy of Sciences, Riga, U.S.S.R.
(Accepted 16 January 1990)
Summary Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SPY0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples. Recombinant
HBeAg; ELISA; Hybridoma
Introduction Rapid, sensitive, and specific detection methods are indispensable for the diagnosis, monitoring, and possible eradication of Hepatitis B. At present it is generally accepted that the most important markers to be tested in human sera are HBsAg, anti-HBcAg antibody, HBeAg, and HBV DNA. Monoclonal antibodies Correspondence to: S. Korec, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovo n&m. 2, 166 37 Prague 6, Czechoslovakia.
0166-0934/90/$03.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
recognizing definite epitopes and enabling more specific and sensitive detection than routine polyclonal antibodies should be employed in these tests. Earlier, we developed a monoclonal-antibody-based assay for detection of HBsAg (Hloianek et al., 1986) and an assay for detection of anti-HBcAg antibody using recombinant HBcAg (Korec et al., 1989). The aim of the present work was to prepare hybridomas producing monoclonal antibodies against HBeAg, using a recombinant antigen for the immunization, and to develop a monoclonal assay detecting HBeAg in human sera.
Materials and Methods HBeAg
HBeAg for immunization and for enzyme immuno assays was prepared by cleaving purified recombinant HBcAg (Borisova et al., 1984), with 1% SDS for 2 h at 37°C (Ferns and Tedder, 1984) and by dialysing against phosphate-buffered saline for 24 h. Immunization
of mice
BALB/c mice were immunized subcutaneously on days 0,21, and 42 with 50 pg of recombinant HBeAg, first in complete and then incomplete Freund’s adjuvant. The mice were then immunized intrasplenically with 50 kg of HBeAg 4 days before removal of their spleens using the method of Gearing (1987). The fusion protocols and the selection and cloning of the hybridomas have been described elsewhere (HloLanek et al., 1986). Detection of anti-HBeAg
antibodies by ELISA
Wells of microtitre plates were coated with HBeAg in PBS (1 l&well) at 4°C for 16 h, washed with PBS, any free surface was blocked with 1% BSA in PBS, the wells were washed again, and test samples or positive or negative control samples were added. The plates were incubated overnight at 4°C washed, and the conjugate of goat anti-mouse immunoglobulin with horseradish peroxidase (Sigma) diluted l/3000 in 10% calf serum was added. The assay was completed in the standard way (Korec et al., 1986). The monoclonal antibodies against HBeAg (HBeAga and I-IBeAgB) as described by Ferns and Tedder (1984) were used as positive and normal mouse sera as negative controls. Monoclonal antibody specificity was tested by ELISA in which wells were coated with Escherichia coli K 802 lysates or recombinant HBcAg and the procedure was otherwise the same. Those hybridomas that produced antibodies strongly reacting with recombinant HBeAg, weakly with recombinant HBcAg, and not reacting with E. coli K 802 lysate were used for further cloning. Preparation of ascitic fluid, purification of monoclonal antibodies and isoelectric focusing were carried out using routine methods (Hloianek et al., 1986).
167
Isotype characterization of monoclonal antibody by ELISA Wells of microtitre plates were coated with swine anti-mouse antibody (2 &well) overnight at 4”C, washed, and test samples were added. Following overnight incubation at 4°C and washing, rabbit anti-isotype antibody (Miles) was added, the plates were incubated at 37°C for 1 h, washed, conjugate of swine anti-rabbit immunoglobulin with horseradish peroxidase was added, and the assay was completed in standard manner.
Results Splenocytes obtained from immunized mice were fused with cells of mouse myeloma cell line SP2/0 and the cultivation media were tested for reactivity with HBeAg, HBcAg, and E. coli K 802 lysate. Hybridomas producing monoclonals that gave a strong reaction with HBeAg, weak with HBcAg, and none with E. coli lysate were used for further cloning. Five different hybridoma clones were established and the monodonals were immunologically characterized (Table 1). Assay for HBeAg in human sera Monoclonal antibody A&, was used in the direct monoclonal assay for HBeAg. Peak sensitivity was achieved using 10 pg/ml antibody for well coating. Test samples were added after washing the coated wells and the plates were incubated overnight at 4°C. They were then washed, polyclonal goat anti-HBeAg antibody labelled with horseradish peroxidase diluted to a concentration of 1 ug/ml in 10% calf serum in PBS was added, the plates were incubated at 37°C for 1 h, and the assay was completed in standard manner. Samples were considered positive when their absorbance at AJg2 exceeded that of negative control at least 2.5fold. The negative control were human sera from healthy donors (absorbance Adg2 = 0.1-0.18). This assay permitted the detection of 3 U HBeAg/ml according to the standard of the Paul Ehrlich Institute, Frankfurt, F.R.G., i.e. it proved to be at least 30 times more sensitive than when human
TABLE 1 Characterization of hybridomas producing anti-HBeAg antibody Hybridoma clone
Isoelectric point
Isotype
A&tic fluid titre, ELISA
HBe HBe HBe HBe HBe
6.7 - 7.3 NT 7.6 - 8 7.5 - 8.1 NT
&?SB IgM IgG2A IgG2A IgG2A
10-a 10-7 10-B 10-a 10-6
A& B,G, 34E, 35H, 32A7
168 TABLE 2 Detection of significant hepatitis B markers in human sera
HBeAg HBeAg HBeAg HBeAg
negative negative positive positive
anti-HBeAg anti-HBeAg anti-HBeAg anti-HBeAg
Total
negative positive negative positive
HBsAg positive
HBsAg negative
No.
No.
20 39 96 2 157
% 12.7 24.8 61.2 1.3 100
93 18 1 0 112
% 83 16.1 0.9 0 100
polyclonal anti-HBeAg antibody was used for coating of the wells. Specificity of this monoclonal assay was checked by testing 150 clinical human serum samples - 85 of them HBeAg positive, 65 negative - of which the HBeAg status had been determined using the Abbott commercial reagents. The same results were obtained with the monoclonal assay. Assay for anti-HBeAg
antibody in human sera
A blocking assay was developed for detection of anti-HBeAg antibody. One tenth of a mil~litre of recombinant HBeAg ~ntaining 50 U HBeA~ml (Paul Ehrlich Institute, HBeAg standard) was mixed with 0.1 ml serum and the mixture was incubated overnight at 4°C. HBeAg was detected using the monoclonal HBeAg assay described above. Samples were considered positive when absorbance was at least 50% lower than in the negative control (negative human sera). The assay enabled quantitative determination of anti-HBeAg antibody down to 1 U/ml (Paul Ehrhch Institute standard). Specificity of the assay was verified by testing 95 human clinical sera (50 anti-HBeAg positive, 45 negative) of which the anti-HBeAg status had been determined using the Abbott reagents: our blocking test gave the same results. Detection of sign~~cant he~utii~ B markers in human clinical sera
A total of 269 sera obtained from patients with acute, chronic, or suspect hepatitis B or from convalenscents were tested. Detection of HBsAg was done with the Sevatest Micro kit (SEVAC, Prague, Czechoslovakia) based on monoclonal antibody lC&,, (Hlolanek et al., 1986). The above monoclonal assays were used for the detection of HBeAg and anti-HBeAg antibody. The results are summarized in Table 2. HBsAg was detected in 157 sera and was negative in 112. Of the HBsAg-negative sera, 99.1% were free of HBeAg, but 0.9% of these sera did contain HBeAg.
169
Discussion The discovery by Kohler and Milstein (1975) has led to new immunological assays: monoclonal antibodies recognizing definite epitopes make possible more specific, sensitive, and cheaper detection of antigens as compared with standard polyclonal antibodies. HBeAg appears in the blood of hepatitis B patients during the viraemic phase of the infection and thus HBeAg represents a valuable marker for diagnosing and especially monitoring hepatitis B. It has been the aim of this work to develop a monoclonal assay for the detection of HBeAg and to compare this assay with the test based on polyclonal antibodies. A number of hybridomas producing antibodies were prepared against HBeAg and one of these, produced by clone A#&, was employed for the monoclonal assay for HBeAg detection. This test permitted the detection of as little as 3 U HBeAg/ml (Paul Ehrlich Institute standard), i.e. it was shown to be at least 30 times as sensitive than human polyclonal anti-HBeAg antibody. In a comparison of the monoclonal assay with the Abbott commercial kit, identical results were obtained. We applied the assays for HBeAg and anti-HBeAg antibody and a commercial kit for HBsAg detection to a set of human clinical sera obtained from patients with acute or chronic hepatitis B and convalescents. The results were in agreement with the clinical status of the patients. Only one HBsAgnegative serum contained HBeAg. The serum giving this uncommon result is under investigation at present. References Borisova, G.P., Pumpen, P.P., Bichko, V.V., Pushko, P.M., Kalis, A.V., Dishler, A.V., Gren, E.J. (1984) Structure and expression of gene coding core antigen of human hepatitis B virus in E. coli. Dokl. Akad. Nauk. SSSR 279, 1245-1248. Ferns, R.B., Tedder, R.S. (1984) Monoclonal antibodies to hepatitis B e antigen (HBeAg) derived from. hepatitis B core antigen (HBcAg): their use in characterization and detection of HBeAg. J. Gen. Virol. 65, 899-908. Gearing, A.J.H., Thorpe, R., Spitz, L., Spitz, M. (1985) Use of ‘single shot’ intrasplenic immunization for production of monoclonal antibodies specific for human IgM. J. Immunol. Methods 76,337-343. HloZanek, I., Dost&lova, V., Korec, E., Zeleny, V., K6nig, J., NCmeEek, V. (1986) Monoclonal antibodies to hepatitis B surface antigen: production and characterization. Folia biol. (Praha) 32, 167-177. Korec, E., Korcova, J., Konig, J., HloZdnek, I. (1989) Detection of antibodies against hepatitis B core antigen using the avidin-biotin system. J. Virol. Methods 24, 321-326.
165
Elsevier VIRMET 01011
Monoclonal antibodies against hepatitis. B e antigen: production, characterization, and use for diagnosis E. Korec’, V. DostBlovB’, J. Korco&, P. Ma&al*, J. Kiinig, G. Borisova4, V. Cibinogen4, P. Pumpen4, E. Gren4 and I. HloBhnekl ‘Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague, 21nstitute of Sera and Vaccines, Prague, ‘Institute of Hygiene and Epidemiology, Prague, Czechoslovakia and %stitute of Organic Synthesis, Latvian Academy of Sciences, Riga, U.S.S.R.
(Accepted 16 January 1990)
Summary Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SPY0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples. Recombinant
HBeAg; ELISA; Hybridoma
Introduction Rapid, sensitive, and specific detection methods are indispensable for the diagnosis, monitoring, and possible eradication of Hepatitis B. At present it is generally accepted that the most important markers to be tested in human sera are HBsAg, anti-HBcAg antibody, HBeAg, and HBV DNA. Monoclonal antibodies Correspondence to: S. Korec, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovo n&m. 2, 166 37 Prague 6, Czechoslovakia.
0166-0934/90/$03.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
recognizing definite epitopes and enabling more specific and sensitive detection than routine polyclonal antibodies should be employed in these tests. Earlier, we developed a monoclonal-antibody-based assay for detection of HBsAg (Hloianek et al., 1986) and an assay for detection of anti-HBcAg antibody using recombinant HBcAg (Korec et al., 1989). The aim of the present work was to prepare hybridomas producing monoclonal antibodies against HBeAg, using a recombinant antigen for the immunization, and to develop a monoclonal assay detecting HBeAg in human sera.
Materials and Methods HBeAg
HBeAg for immunization and for enzyme immuno assays was prepared by cleaving purified recombinant HBcAg (Borisova et al., 1984), with 1% SDS for 2 h at 37°C (Ferns and Tedder, 1984) and by dialysing against phosphate-buffered saline for 24 h. Immunization
of mice
BALB/c mice were immunized subcutaneously on days 0,21, and 42 with 50 pg of recombinant HBeAg, first in complete and then incomplete Freund’s adjuvant. The mice were then immunized intrasplenically with 50 kg of HBeAg 4 days before removal of their spleens using the method of Gearing (1987). The fusion protocols and the selection and cloning of the hybridomas have been described elsewhere (HloLanek et al., 1986). Detection of anti-HBeAg
antibodies by ELISA
Wells of microtitre plates were coated with HBeAg in PBS (1 l&well) at 4°C for 16 h, washed with PBS, any free surface was blocked with 1% BSA in PBS, the wells were washed again, and test samples or positive or negative control samples were added. The plates were incubated overnight at 4°C washed, and the conjugate of goat anti-mouse immunoglobulin with horseradish peroxidase (Sigma) diluted l/3000 in 10% calf serum was added. The assay was completed in the standard way (Korec et al., 1986). The monoclonal antibodies against HBeAg (HBeAga and I-IBeAgB) as described by Ferns and Tedder (1984) were used as positive and normal mouse sera as negative controls. Monoclonal antibody specificity was tested by ELISA in which wells were coated with Escherichia coli K 802 lysates or recombinant HBcAg and the procedure was otherwise the same. Those hybridomas that produced antibodies strongly reacting with recombinant HBeAg, weakly with recombinant HBcAg, and not reacting with E. coli K 802 lysate were used for further cloning. Preparation of ascitic fluid, purification of monoclonal antibodies and isoelectric focusing were carried out using routine methods (Hloianek et al., 1986).
167
Isotype characterization of monoclonal antibody by ELISA Wells of microtitre plates were coated with swine anti-mouse antibody (2 &well) overnight at 4”C, washed, and test samples were added. Following overnight incubation at 4°C and washing, rabbit anti-isotype antibody (Miles) was added, the plates were incubated at 37°C for 1 h, washed, conjugate of swine anti-rabbit immunoglobulin with horseradish peroxidase was added, and the assay was completed in standard manner.
Results Splenocytes obtained from immunized mice were fused with cells of mouse myeloma cell line SP2/0 and the cultivation media were tested for reactivity with HBeAg, HBcAg, and E. coli K 802 lysate. Hybridomas producing monoclonals that gave a strong reaction with HBeAg, weak with HBcAg, and none with E. coli lysate were used for further cloning. Five different hybridoma clones were established and the monodonals were immunologically characterized (Table 1). Assay for HBeAg in human sera Monoclonal antibody A&, was used in the direct monoclonal assay for HBeAg. Peak sensitivity was achieved using 10 pg/ml antibody for well coating. Test samples were added after washing the coated wells and the plates were incubated overnight at 4°C. They were then washed, polyclonal goat anti-HBeAg antibody labelled with horseradish peroxidase diluted to a concentration of 1 ug/ml in 10% calf serum in PBS was added, the plates were incubated at 37°C for 1 h, and the assay was completed in standard manner. Samples were considered positive when their absorbance at AJg2 exceeded that of negative control at least 2.5fold. The negative control were human sera from healthy donors (absorbance Adg2 = 0.1-0.18). This assay permitted the detection of 3 U HBeAg/ml according to the standard of the Paul Ehrlich Institute, Frankfurt, F.R.G., i.e. it proved to be at least 30 times more sensitive than when human
TABLE 1 Characterization of hybridomas producing anti-HBeAg antibody Hybridoma clone
Isoelectric point
Isotype
A&tic fluid titre, ELISA
HBe HBe HBe HBe HBe
6.7 - 7.3 NT 7.6 - 8 7.5 - 8.1 NT
&?SB IgM IgG2A IgG2A IgG2A
10-a 10-7 10-B 10-a 10-6
A& B,G, 34E, 35H, 32A7
168 TABLE 2 Detection of significant hepatitis B markers in human sera
HBeAg HBeAg HBeAg HBeAg
negative negative positive positive
anti-HBeAg anti-HBeAg anti-HBeAg anti-HBeAg
Total
negative positive negative positive
HBsAg positive
HBsAg negative
No.
No.
20 39 96 2 157
% 12.7 24.8 61.2 1.3 100
93 18 1 0 112
% 83 16.1 0.9 0 100
polyclonal anti-HBeAg antibody was used for coating of the wells. Specificity of this monoclonal assay was checked by testing 150 clinical human serum samples - 85 of them HBeAg positive, 65 negative - of which the HBeAg status had been determined using the Abbott commercial reagents. The same results were obtained with the monoclonal assay. Assay for anti-HBeAg
antibody in human sera
A blocking assay was developed for detection of anti-HBeAg antibody. One tenth of a mil~litre of recombinant HBeAg ~ntaining 50 U HBeA~ml (Paul Ehrlich Institute, HBeAg standard) was mixed with 0.1 ml serum and the mixture was incubated overnight at 4°C. HBeAg was detected using the monoclonal HBeAg assay described above. Samples were considered positive when absorbance was at least 50% lower than in the negative control (negative human sera). The assay enabled quantitative determination of anti-HBeAg antibody down to 1 U/ml (Paul Ehrhch Institute standard). Specificity of the assay was verified by testing 95 human clinical sera (50 anti-HBeAg positive, 45 negative) of which the anti-HBeAg status had been determined using the Abbott reagents: our blocking test gave the same results. Detection of sign~~cant he~utii~ B markers in human clinical sera
A total of 269 sera obtained from patients with acute, chronic, or suspect hepatitis B or from convalenscents were tested. Detection of HBsAg was done with the Sevatest Micro kit (SEVAC, Prague, Czechoslovakia) based on monoclonal antibody lC&,, (Hlolanek et al., 1986). The above monoclonal assays were used for the detection of HBeAg and anti-HBeAg antibody. The results are summarized in Table 2. HBsAg was detected in 157 sera and was negative in 112. Of the HBsAg-negative sera, 99.1% were free of HBeAg, but 0.9% of these sera did contain HBeAg.
169
Discussion The discovery by Kohler and Milstein (1975) has led to new immunological assays: monoclonal antibodies recognizing definite epitopes make possible more specific, sensitive, and cheaper detection of antigens as compared with standard polyclonal antibodies. HBeAg appears in the blood of hepatitis B patients during the viraemic phase of the infection and thus HBeAg represents a valuable marker for diagnosing and especially monitoring hepatitis B. It has been the aim of this work to develop a monoclonal assay for the detection of HBeAg and to compare this assay with the test based on polyclonal antibodies. A number of hybridomas producing antibodies were prepared against HBeAg and one of these, produced by clone A#&, was employed for the monoclonal assay for HBeAg detection. This test permitted the detection of as little as 3 U HBeAg/ml (Paul Ehrlich Institute standard), i.e. it was shown to be at least 30 times as sensitive than human polyclonal anti-HBeAg antibody. In a comparison of the monoclonal assay with the Abbott commercial kit, identical results were obtained. We applied the assays for HBeAg and anti-HBeAg antibody and a commercial kit for HBsAg detection to a set of human clinical sera obtained from patients with acute or chronic hepatitis B and convalescents. The results were in agreement with the clinical status of the patients. Only one HBsAgnegative serum contained HBeAg. The serum giving this uncommon result is under investigation at present. References Borisova, G.P., Pumpen, P.P., Bichko, V.V., Pushko, P.M., Kalis, A.V., Dishler, A.V., Gren, E.J. (1984) Structure and expression of gene coding core antigen of human hepatitis B virus in E. coli. Dokl. Akad. Nauk. SSSR 279, 1245-1248. Ferns, R.B., Tedder, R.S. (1984) Monoclonal antibodies to hepatitis B e antigen (HBeAg) derived from. hepatitis B core antigen (HBcAg): their use in characterization and detection of HBeAg. J. Gen. Virol. 65, 899-908. Gearing, A.J.H., Thorpe, R., Spitz, L., Spitz, M. (1985) Use of ‘single shot’ intrasplenic immunization for production of monoclonal antibodies specific for human IgM. J. Immunol. Methods 76,337-343. HloZanek, I., Dost&lova, V., Korec, E., Zeleny, V., K6nig, J., NCmeEek, V. (1986) Monoclonal antibodies to hepatitis B surface antigen: production and characterization. Folia biol. (Praha) 32, 167-177. Korec, E., Korcova, J., Konig, J., HloZdnek, I. (1989) Detection of antibodies against hepatitis B core antigen using the avidin-biotin system. J. Virol. Methods 24, 321-326.
