• Archives of Pharmacal Research
• /
• 제21권5호
• /
• pp.543-548
• /
• 1998

We examined the immunological properties of the recombinant hepatitis B surface antigen (r-HBsAg) which was expressed in mammalian cell (C127). The cross-immunity of r-HBsAg and plasma-derived hepatitis B surface antigen (p-HBsAg) were tested using Western blotting and ELISA with guinea pig polyclonal antibody and naturally infected human-derived antibody and the both antigens show the same results in their response pattern and intensity, which indicate they have a good cross-immunity. from the measurement of $ED_{50}$ after formalin- or heat-inactivation, both r-HBsAg and p-HBsAg and p-HBsAg showed $ED_{50}$ of 0.2-0.3 in formalin-inactivaton, while r-HBsAg was 0.05-0.09 and p-HBsAg was 0.03-0.07 in heat-inactivation, which means heat-inactivation method is 3-4 times superior in immunogenicity. In the immunopersistency test performed in guinea pig for the period of 3 months with two different adjuvants, antibody titer was 34.2 with muramyl dipeptide adjuvant, which was 1.8 times greater than the antibody titer of 18.9 with $AIPO_{4}$ adjuvant. the mutagenicity of r-HBsAg has the same cross-immunity with p-HBsAg, and heat-inactivation method and muramyl dipeptide adjuvant allow development of r-HBsAg vaccine with excellent immunogenicity.

• Archives of Pharmacal Research
• /
• 제10권1호
• /
• pp.18-24
• /
• 1987

Mouse monocolonal antibodies to Hepatitis B surface antien (HBsAg) were prepared and their functional capabilities tested by the method of solid phase enzyme linked immuno sorbent assay (ELISA). HBsAg binding studies inicated that one monoclonal antibody 6E-1-1 bound more HBsAg at a faster rate than the other monoclonal antibodies. Also, for the binding inhibition studies with the selected monoclonal antibody 6E-1-1, one monoclonal antibody 8D-3-6 didn't exhibit binding inhibition for HBsAg. Then, a simultaneous ELISA method was developed for the immunodiagnosis of HBsAg. Different combinations of two monoclonal antibodies as solid phase and horseradish peroxidase (HRPO) labeled phase were studied. The combination of monoclonal antibody of higher affinity constant (6E-1-1) immobilized in a solid phase and monoclonal antibody of lower affinity constant (8D-3-6) as a HRPO laeled phase was more sensitive when two monoclonal antibodies of different affinity constants for HBsAg were prepared.

• KSBB Journal
• /
• 제15권2호
• /
• pp.214-218
• /
• 2000

면역크로마토그래피 진단방법을 이용하는 B형간염 스크리닝 kit를 개발하기 위하여 두가지의 항체를 이용하였다. 표식자항체로 사용된 것은 단세포군항체 anti-HBs이고 포획항체는 goat anti-HBs 인데 포획항체는 니트로셀룰로즈 막에 고정되고 표식자항체는 금 입자에 결합된다. 혈청 검체를 well에 가하면 유리섬유 표면에 건조상태로 침착되어 있던 conjugate가 활성화되어 검체중의 HBsAg와 결합한다. 검체를 가한 지 5분 후 검사결과가 나타나는데 HBsAg와 conjugate가 결합된 복합체가 니트로셀롤로즈 막의 하단부에 붉은 색 선으로 나타난다. 본 kit의 검출한계는 표준 HBs-Ag 용액을 사용하여 시험하였을 때 2 ng/ml이었다.

• Archives of Pharmacal Research
• /
• 제15권4호
• /
• pp.347-355
• /
• 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
• /
• 제17권1호
• /
• pp.20-25
• /
• 2002

We performed a basic experiment for rapid, on-line, real-time measurement of HBsAg by using a surface plasmon resonance biosensor to quantify the recognition and interaction of biomolecules. We immobilized the anti-HBsAg polyclonal antibody to the dextran layer on a CM5 chip surface which was pre-activated by N-hydroxysuccinimide for amine coupling. The binding of the HBsAg to the immobilized antibody was measured by the mass increase detected by the change in the SPR signal. The binding characteristics between HBsAg and its antibody followed typical monolayer adsorption isotherm. When the entire immobilized antibody was interacted, there was no additional, non-specific binding observed, which suggested the biointeraction was very specific as expected and independent of the ligand density. No significant steric hindrance was observed at 17.6 nm/$mm^2$ immobilization density. The relationship between the HBsAg concentration in the sample solution and the antigen bound to the chip surface was linear up to ca. $40\mu\textrm{g}$/mL, which is much wider than that of the ELISA method. It appeared the antigen-antibody binding was increased as the immobilized ligand density increased, but verification is warranted. This study showed the potential of this biosensor-based method as a rapid, simple, multi-sample, on-line assay. Once properly validated, it can serve as a more powerful method for HBsAg quantification replacing the current ELISA method.

• 약학회지
• /
• 제45권6호
• /
• pp.708-714
• /
• 2001

Many studies have provided evidences that hepatitis B surface antigen (HBsAg) including preS region could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. We established CHO cell lines, IY-CHO-2 and IY-CHO-11 expressing high levels of HBsAg containing preS2 and S protein by stable transfection method. These cell lines expressed the correct size (about 1 kb in length) of HBsAg mRNA as expected. The purified protein from the culture supernatants of the clones showed the same sizes as those expressed in native hepatitis B virus (24 kDa, 27 kDa, 34 kDa and 36 kDa). Antibody productivity of CHO-derived HBsAg protein at lower dose challenge was higher than the protein containing S region alone expressed in yeast system. These results indicate that CHO-derived HBsAg protein containing preS2 and S region can be effectively used for a better immune response as a HBV vaccine.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.231-234
• /
• 2001

Biacore 바이오센서를 이용하여 재조합 HBsAg 를 정량분석하기 위해 CM5 칩 위에 anti-HBsAg polyclonal antibody를 저밀도 (2,304.2 RU)와 고밀도(17635.9 RU)로 고정화한 후 다양한 농도의 HBsAg를 흘려주어 각 농도별 sensorgram을 얻었다. 각 sensorgram으로부터 결합된 HBsAg의 질량을 구하여 액체 시료 내의 HBsAg의 농도 대 칩에 결합된 HBsAg의 질량 사이의 상관관계를 구한 결과 Langmuir 단일층 흡착 등온선과 매우 유사한 형태의 calibration curve를 얻었다. 약 40 ${\mu}g/m{\ell}$ 까지 선형 관계가 유지되었다. 이 calibration curve를 double-reciprocal plotting 하여 $RU_{max}$ 값을 각각 구한 뒤, 단위 질량의 고정화된 항체 당 결합된 HBsAg의 질량을 구한 결과 저밀도 칩에서 0.024, 고밀도 칩에서 0.023 으로 매우 유사한 결과를 나타내었다.

• Annals of Laboratory Medicine
• /
• 제38권6호
• /
• pp.578-584
• /
• 2018

Background: Accurate, rapid, and cost-effective screening tests for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection may be useful in laboratories that cannot afford automated chemiluminescent immunoassays (CLIAs). We evaluated the diagnostic performance of a novel rapid automated fluorescent lateral flow immunoassay (LFIA). Methods: A fluorescent LFIA using a small bench-top fluorescence reader, Automated Fluorescent Immunoassay System (AFIAS; Boditech Med Inc., Chuncheon, Korea), was developed for qualitative detection of hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), and antibody to HCV (anti-HCV) within 20 minutes. We compared the diagnostic performance of AFIAS with that of automated CLIAs-Elecsys (Roche Diagnostics GmbH, Penzberg, Germany) and ARCHITECT (Abbott Laboratories, Abbott Park, IL, USA)-using 20 seroconversion panels and 3,500 clinical serum samples. Results: Evaluation with the seroconversion panels demonstrated that AFIAS had adequate sensitivity for HBsAg and anti-HCV detection. From the clinical samples, AFIAS sensitivity and specificity were 99.8% and 99.3% for the HBsAg test, 100.0% and 100.0% for the anti-HBs test, and 98.8% and 99.1% for the anti-HCV test, respectively. Its agreement rates with the Elecsys HBsAg, anti-HBs, and anti-HCV detection assays were 99.4%, 100.0%, and 99.0%, respectively. AFIAS detected all samples with HBsAg genotypes A-F and H and anti-HCV genotypes 1, 1a, 1b, 2a, 2b, 4, and 6. Cross-reactivity with other infections was not observed. Conclusions: The AFIAS HBsAg, anti-HBs, and anti-HCV tests demonstrated diagnostic performance equivalent to current automated CLIAs. AFIAS could be used for a large-scale HBV or HCV screening in low-resource laboratories or low-to middle-income areas.

• 대한임상검사과학회지
• /
• 제47권2호
• /
• pp.78-82
• /
• 2015

우리나라는 1995년부터 영유아를 대상으로 국가차원의 B형 간염백신 예방접종을 시작하였다. 현재 우리나라의 20대 대학생들은 신생아 정기 예방접종 시작 전후에 태어난 세대이며 영유아기에 접종을 받고 20여년이 경과되었다. 영유아를 대상으로 B형 간염 정기예방 접종 시작 후 장시간이 경과한 현 시점에서 B형 항체 보유율과 역가에 대한 확인은 매우 중요하다. 본 연구에서는 20대 대학생들의 B형 간염항체 양성률과 역가를 조사하였으며 연구대상은 2014년 4월부터 2014년 10월까지 경남 소재 대학의 대학생 262명을 대상으로 하였다. 추가접종자는 제외하였으며 간염항체의 양성판정은 10 mIU/mL이상으로 정하였다. 연구결과, B형 간염 항체의 양성률은 55.3% (145명)이었고 음성률은 44.7% (117명)이었다. 성별에 따라 양성률을 분류했을 때 여자는 57.9% (88명), 남자는 51.8% (57명)로 여자가 남자보다 높았으나 통계적으로 유의한 차이는 없었다. 연령에 따라 역가 차이를 비교했을 때 19~20세 연령군이 21세 이상의 연령군 보다 낮은 역가를 나타냈다. 이는 본 연구의 연구대상자들이 연령군간에 나이 차이가 적고 각 연령군의 측정 대상자수가 일정하지 않았으며 인원수가 적은 군에서 역가가 높게 나오는 경우가 발생하면 해당 연령군이 상대적으로 역가가 높은 분포에 속하게 되는 것으로 생각되어 추후 고려해야할 사항으로 사료된다. 양성으로 판명된 연구대상자의 62%가 역가의 하한선인 10-99.9 mIU/mL의 낮은 역가를 나타냈다. 소아기에 접종 후 일정기간이 지나면 항체 확인검사를 하고 국가차원에서 추가접종을 추진해야 할 것으로 사료된다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권4호
• /
• pp.309-314
• /
• 2005

We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface plasmon resonance biosensor. We immobilized anti­HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to the dextran layer on a CM5 chip surface that had previously been activated by N-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately $17.6 ng/mm^2$. The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up to ca. $40{\mu}g$/mL. This linearity was much higher than that of the ELISA method. It appeared the anti­gen-antibody binding increased as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method as a rapid, simple and multi­sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg quantification for replacing the ELISA.