• KSBB Journal
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• v.15 no.2
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• pp.214-218
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• 2000

A hepatitis B Surface Antigen(HBsAg)-screening kit using immunochromatographic assay(ICA) method was developed by e employing two kinds of antibodies. One is mouse monoclonal anti-HBs for tracer antibody and the other is goat p이yclonal a anti-HBs for capture antibody. This capture antibody was immobilized on the surface of nitroceliulose(NC) membrane and the t tracer antibody was conjugated with g미d particles. When serum sample was added to the sample well, the $\infty$njugates d deposited in a dry state on the surface of glass fiber filter were reconstituted and then combined with HBsAg in serum. In 5 5 min after adding, the assay result was visible through the window, that is, the complexes composed of HBsAg and the c conjugates appeared as maroon line on the lower part of the NC membrane. The detection limit of the ICA kit was 2 ng/ml w when being tested with the reference HBsAg.

• Archives of Pharmacal Research
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• v.15 no.4
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• pp.347-355
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• 1992

To elucidate structure of group "a" epitope, mouse antibodies that express idiotype monoclonal antibody and anti-idiotype monoclonal antibody against the group specific "a" determinant were purified by hydroxyapatite column. To obtain hepatitis B surface antigens (HBsAg). HBsAg positive blood was sequencially purified by ammonium sulfate precipitation, hydroxyapatite, sepharose 4B column chromatography and ultracentrifugation. The major protein (p25) and glycoprotein (gp30) of HBsAg were isolated by concanavalin-A-sepharose 4B. The ability of p25-gp30 among the HBsAg to inhibit the idiotype-anti-idiotype reaction was dependent on conformation, since reduced and alkylated p25-gp30 virtualy lost their inhibitory capacity when compared to native HBsAg. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of p25-gp30.

• KSBB Journal
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• v.5 no.4
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• pp.365-371
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• 1990

To improve the growth of mouse hybridoma 2c3.1 secreting anti-Hepatitis B surface antigen monoclonal antibody (anti-HBsAg MAb), we had constructed an enriched medium and observed the effects of fetal bovine serum and serum-free supplements including human serum albumin, 'insulin and transferrin', and monoethanolamine. For further enhancement of growth, the concentrations of two major energy sources, glucose and glutamine, were strengthened with various ratios in the enriched medium. Maximum cell growth and monoclonal antibody production obtained in various ratios of glucose/glutamine with an inoculation concentration of 2$\times$105 cells/ml were 0.73$\times$106-4.62$\times$106 cells/ml and 65.1-422.6 $\mu\textrm{g}$/ml, respectively. Glutamine was round to be a major energy source and a limiting nutrient in comparison to glucose for 2c3.1 cell cultivation in enriched media with low serum.

• IMMUNE NETWORK
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• v.3 no.4
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• pp.281-286
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• 2003

Background: Hepatitis B virus (HBV) infection is one of the worldwide public health problem affecting about 300 million people. The envelope protein of HBV consists of three components known as preS1, preS2, and S antigen. According to the recent study, anti-HBs Ab showed effective neutralization ability against HBV from chronic hepatitis B and liver transplant patients, suggesting the possible development of therapeutic antibody. Methods: Spleen cells immunized with S antigen of HBV were fused with myeloma cell line to obtain HBsAg specific monoclonal antibodies. High affinity antibodies against HBsAg (adr, ad and ay type) were selected by competitive ELISA method. Nucleotide sequence of the variable regions of monoclonal antibodies was analyzed by RT-PCR followed by conventional sequencing method. Results: We produced 14 murine monoclonal antibodies which recognize S antigen of HBV. Two of them, A9-11 and C6-9 showed the highest affinity. The sequence analysis of A9-11 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain I (B) and light chain lambda 1, respectively. Likewise, the sequence analysis of C6-9 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain II (B) and light chain kappa 1, respectively. Neutralization assay showed that A9-11 and C6-9 effectively neutralize the HBV infection. Conclusion: These results suggest that A9-11 and C6-9 mouse monoclonal antibodies can be used for the development of therapeutic antibody for HBV infection.

• Bulletin of the Korean Chemical Society
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• v.32 no.6
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• pp.1975-1979
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• 2011

The modification of a gas diffusion layer (GDL), a vital component in polymer electrolyte fuel cells, is described here for use in the electrochemical detection of antibody-antigen biosensors. Compared to other substrates (gold foil and graphite), mouse anti-rHBsAg monoclonal antibody immobilized on gold-coated GDL (G-GDL) detected analytes of goat anti-mouse IgG antibody-ALP using a relatively low potential (-0.0021 V vs. Ag/AgCl 3 M NaCl), indicating that undesired by-reactions during electrochemical sensing should be avoided with G-GDL. The dependency of the signal against the concentration of analytes was observed, demonstrating the possibility of quantitative electrochemical biosensors based on G-GDL substrates. When a sandwich method was employed, target antigens of rHBsAg with a concentration as low as 500 ng/mL were clearly measured. The detection limit of rHBsAg was significantly improved to 10 ng/mL when higher concentrations of the 4-aminophenylphosphate monosodium salt (APP) acting on substrates were used for generating a redox-active product. Additionally, it was shown that a BSA blocking layer was essential in improving the detection limit in the G-GDL biosensor.

• Journal of Yeungnam Medical Science
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• v.13 no.2
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• pp.272-278
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• 1996

Four subtypes of hepatitis B surface antigen are useful in the epidemiologic studies of the route of virus transmission and clinical significance of simultaneous occurance of hepatitis B surface antigen and antibody to hepatitis B surface antigen in the same serum as well as useful marker for population migration. The sera were obtained from 214 HBs Ag positive patients who are diagnosed as chronic liver disease and following up in the Yeungnam university hospital. The subtypes were determined by solid-phase sandwich EIA using monoclonal antibodies. Among 214 specimens, the subtype adr was 93.9%, adw was 2.8%, ayr was 0.9%, ar was 0.9%, adwr was 1.4% and ayw was not detected. There were no correlation between subtype pattern and disease. In summary, the subtype adr was prominent in our study and the difference of subtype pattern by severity of disease was not significant. However, to determine the prognostic value of HBs Ag subtype and relationship between subtype and disease progression, long-term follow up will be needed.

• BMB Reports
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• v.38 no.6
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• KSBB Journal
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• v.9 no.3
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• pp.253-265
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• 1994

We have studied the effects of serum concentration and initial cell density on hybridoma cell growth and monoclonal antibody (MAb) production at various media supplemented with different types of serum. The types of serum were fetal bovine sera, newborn bovine calf sera, calf sera including supplemented calf sera, horse serum, and goat serum. The concentrations of each serum were 0.5, 1.25, 2.5, and 5% (v/v) and the inoculum densities were $5{\times}10^4, 1{\times}10^5, 2{\times}10^5,$ cells/ml. The hybridoma cell growth and anti-Hepatitis B surface antigen (anti-HBsAg) MAb production were found to be enhanced by increasing the serum concentration and by increasing inoculum density regardless of serum type. We found that test sera purchased from different companies show different effects on cell growth and MAb production, although they are the same type of serum.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.82-82
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• 1997

The hepatitis B virus(HBV) is a small, enveloped virus with a circular, double-stranded DNA genome. HBV causes active and chronic hepatitis worldwide, including Korea, and is considered to be a major factor for liver cirrhosis and hepatocellular carcinoma. In contrast to the wealth of knowledge on the gene structure and expressional regulation, immunological and pathological mechanisms for HBV-induced hepatocellular injury are not well known. In the present study, vaccinia virus which has been demonstrated to be a useful eukaryotic expression vector was used to clone the gene for HBV surface antigen, L(S+preS2+preS1). The recombinant vaccinia virus vector, pMJ-L, which contains L surface antigen gene of adr-type HBV was constructed, and subseouently used for making recombinant vaccinia virus VV-$\textrm{HBV}_{L}$. Expression of the HBV antigen was examined by immunofluorescent antibody (IFA) test using mouse monoclonal anti-hepatitis B surface antigen. HBsAg was detected in the recombinant virus indicating that the VV-$\textrm{HBV}_{L}$ expressed S antigen successfully. The HBV-Vaccinia Virus recombinant obtained in this study is currently being used for studying the immunological aspects of HBV infection.

• KSBB Journal
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• v.5 no.1
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• pp.87-94
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• 1990

Enhancement of hybridoma cell growth and monoclonal antibody(MAb) production by the addition of a small amount of serum into both serum-free medium and enriched medium was studied. The enriched medium was constructed by mixing a basal serum-free medium and a nutrient-fortified RPMI 1640 medium. It was supplemented with human serum albumin, insulin, transferrin, and monoethanolamine. It was found that addition of low concentration of serum with other serum-free supplements was favorable for growth of a mouse hybridoma 2c3.1 cells. The concentration of serum was determined to 0.5%. The maximum cell concentration obtained in this enriched medium supplemented with 0.5% fetal bovine serum (FBS) was $3.06{\times}10^6$ cells/ml and the concentration of secreted anti-Hepatitis surface antigen (antiHBsAg) MAb was $159.7{\mu\textrm{g}}\;/\;ml$ compared to $43{\mu\textrm{g}}\;/\;ml$ in RPMI 1640 medium with 10% FBS and $50{\mu\textrm{g}}\;/\;ml$ in previously-developed serum-free medium. The 2c3.1 cell growth and MAb production could be enhanced considerably by using the enriched medium supplemented with 0.5% FBS and serum-free supplements instead of RPMI 1640 medium or serum-free medium. The enhancement in MAb production in the enriched medium was more noticeable.