Vol. 30, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1617-1619 0095-1137/92/061617-03$02.00/0
Stability of the Recombinant Hepatitis B Core Antigen NRAPENDRA NATH,t* KEITH HICKMAN, SEAN NOWLAN,t DINESH SHAH, JACK PHILLIPS, AND SCOTT BABLER Pandex Division, Baxter Diagnostics Inc., Mundelein, Illinois 60060 Received 13 January 1992/Accepted 20 March 1992
The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70°C for 60 min but was inactivated at 85°C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacilus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50%o of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
use. Stock solutions of purified JM83/pSN502 were stored frozen at below -20°C. MELA. A microparticle enzyme immunoassay (MEIA) for HBcAg was developed by using paramagnetic particles (4- to 6-urm diameter) as the solid phase to which mouse monoclonal antibodies (RF HBc13) raised against HBcAg (12) were covalently linked. Anti-HBc RF HBc13 immunoglobulin G linked to p-galactosidase was used as the conjugate. Various dilutions of test sample containing recombinant HBcAg were incubated with RF HBc13-coated paramagnetic particles. HBcAg captured on the particle surface was detected with RF HBc13-enzyme conjugate. Particles were magnetically separated and washed, and then a solution containing 4-methylumbelliferyl-13-galactopyranoside as the enzyme substrate was added. The amount of fluorescence generated was determined by reading each well two times approximately 12 min apart. Assay values are expressed in nanomoles of coumarin on a 1-to-5,000 scale. Assay results were linear between 25 and 125 ng of HBcAg per ml (nanogram values were derived from an in-house standard). Heating. Aliquots of recombinant HBcAg (200 ,ug/ml) in screw-cap plastic vials were placed in a water bath set at various temperatures. A control vial was kept at 2 to 8°C. Heating at 70°C for 60 min did not significantly alter the immunoreactivity of recombinant HBcAg, but heating at 85°C inactivated it in 10 min (Table 1). Up to three cycles of freezing (-20°C) and thawing (2 to 8°C) did not significantly reduce HBcAg activity (data not shown). pH. One volume of recombinant HBcAg (200 ,ug/ml) was
Large-scale production of hepatitis B core antigen (HBcAg) resulting from successful cloning of its gene in Escherichia coli (10, 11) has greatly facilitated the study and understanding of its role in hepatitis B virus infection in both its clinical and subclinical manifestations. Antibodies to HBcAg appear first following hepatitis B virus infection (2, 3) and have been used to cover the "window" between infection and appearance of antibodies to hepatitis B surface antigen (HBsAg) (3). The presence of antibodies to HBcAg has been used until recently as a surrogate marker for non-A, non-B hepatitis infection to help identify and thus eliminate blood donations from possible carriers (4). HBcAg from patient liver has been purified and studied for its immunological, physical, and biochemical properties (1, 8, 9); however, because of liver tissue contaminants, it is difficult to determine which of these properties is specific to HBcAg. We tested the effects of heat, cycles of freezing and thawing, various pHs, and alcohols or other chemical denaturants on recombinant HBcAg. The full-length gene, 573 bp long, coding for hepatitis B core was cloned from a patient's serum (a gift of Howard Fields) by polymerase chain reaction amplification using primers (5'-GCTGTGCCTITGGGTGGCTETTGGGGC-3' and 5'-CCCACCTTATGAGTCCAAGGAATA-3') that allowed the use of two StyI restriction sites located upstream from the HBcAg initiating ATG codon and downstream from the termination codon TAG. The cloned gene was expressed in JM83 strains of E. coli (ATCC 35607). Recombinant HBcAg was purified from bacterial culture by a combination of ammonium sulfate precipitation and ion-exchange chromatography. Purified recombinant HBcAg (JM83/pSN502) gives a major band equivalent to a 23,000-molecular-weight peptide on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; it was confirmed to be HBcAg by N-terminal amino acid analysis and Western blotting (immunoblotting). A solution of recombinant HBcAg in isotonic buffered saline (IBS) solution (0.14 M NaCl, 0.005 M KCI, and 0.01 M NaN3) adjusted to 200 ,ug/ml was stored at 2 to 8°C prior to *
TABLE 1. Effect of heat on recombinant HBcAg Time
Corresponding author.
(min)
70°C
0 10 20 30 60
2,777 2,681 3,051 2,786 3,006
Assay value' at: 80°C (%) 85°C
2,950 (100) ND
2,010 (68.1) 2,179 (73.8) ND
2,611 < 10
JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1617-1619 0095-1137/92/061617-03$02.00/0
Stability of the Recombinant Hepatitis B Core Antigen NRAPENDRA NATH,t* KEITH HICKMAN, SEAN NOWLAN,t DINESH SHAH, JACK PHILLIPS, AND SCOTT BABLER Pandex Division, Baxter Diagnostics Inc., Mundelein, Illinois 60060 Received 13 January 1992/Accepted 20 March 1992
The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70°C for 60 min but was inactivated at 85°C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacilus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50%o of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
use. Stock solutions of purified JM83/pSN502 were stored frozen at below -20°C. MELA. A microparticle enzyme immunoassay (MEIA) for HBcAg was developed by using paramagnetic particles (4- to 6-urm diameter) as the solid phase to which mouse monoclonal antibodies (RF HBc13) raised against HBcAg (12) were covalently linked. Anti-HBc RF HBc13 immunoglobulin G linked to p-galactosidase was used as the conjugate. Various dilutions of test sample containing recombinant HBcAg were incubated with RF HBc13-coated paramagnetic particles. HBcAg captured on the particle surface was detected with RF HBc13-enzyme conjugate. Particles were magnetically separated and washed, and then a solution containing 4-methylumbelliferyl-13-galactopyranoside as the enzyme substrate was added. The amount of fluorescence generated was determined by reading each well two times approximately 12 min apart. Assay values are expressed in nanomoles of coumarin on a 1-to-5,000 scale. Assay results were linear between 25 and 125 ng of HBcAg per ml (nanogram values were derived from an in-house standard). Heating. Aliquots of recombinant HBcAg (200 ,ug/ml) in screw-cap plastic vials were placed in a water bath set at various temperatures. A control vial was kept at 2 to 8°C. Heating at 70°C for 60 min did not significantly alter the immunoreactivity of recombinant HBcAg, but heating at 85°C inactivated it in 10 min (Table 1). Up to three cycles of freezing (-20°C) and thawing (2 to 8°C) did not significantly reduce HBcAg activity (data not shown). pH. One volume of recombinant HBcAg (200 ,ug/ml) was
Large-scale production of hepatitis B core antigen (HBcAg) resulting from successful cloning of its gene in Escherichia coli (10, 11) has greatly facilitated the study and understanding of its role in hepatitis B virus infection in both its clinical and subclinical manifestations. Antibodies to HBcAg appear first following hepatitis B virus infection (2, 3) and have been used to cover the "window" between infection and appearance of antibodies to hepatitis B surface antigen (HBsAg) (3). The presence of antibodies to HBcAg has been used until recently as a surrogate marker for non-A, non-B hepatitis infection to help identify and thus eliminate blood donations from possible carriers (4). HBcAg from patient liver has been purified and studied for its immunological, physical, and biochemical properties (1, 8, 9); however, because of liver tissue contaminants, it is difficult to determine which of these properties is specific to HBcAg. We tested the effects of heat, cycles of freezing and thawing, various pHs, and alcohols or other chemical denaturants on recombinant HBcAg. The full-length gene, 573 bp long, coding for hepatitis B core was cloned from a patient's serum (a gift of Howard Fields) by polymerase chain reaction amplification using primers (5'-GCTGTGCCTITGGGTGGCTETTGGGGC-3' and 5'-CCCACCTTATGAGTCCAAGGAATA-3') that allowed the use of two StyI restriction sites located upstream from the HBcAg initiating ATG codon and downstream from the termination codon TAG. The cloned gene was expressed in JM83 strains of E. coli (ATCC 35607). Recombinant HBcAg was purified from bacterial culture by a combination of ammonium sulfate precipitation and ion-exchange chromatography. Purified recombinant HBcAg (JM83/pSN502) gives a major band equivalent to a 23,000-molecular-weight peptide on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; it was confirmed to be HBcAg by N-terminal amino acid analysis and Western blotting (immunoblotting). A solution of recombinant HBcAg in isotonic buffered saline (IBS) solution (0.14 M NaCl, 0.005 M KCI, and 0.01 M NaN3) adjusted to 200 ,ug/ml was stored at 2 to 8°C prior to *
TABLE 1. Effect of heat on recombinant HBcAg Time
Corresponding author.
(min)
70°C
0 10 20 30 60
2,777 2,681 3,051 2,786 3,006
Assay value' at: 80°C (%) 85°C
2,950 (100) ND
2,010 (68.1) 2,179 (73.8) ND
2,611 < 10
