• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.173-177
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• 2006

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PH A-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.

• Journal of Preventive Medicine and Public Health
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• 제35권2호
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• pp.129-135
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• 2002

목적 : 1998년 한국인 성인에서의 혈청 HBsAg의 양성률을 추정하는 것을 일차적 목적으로 하며, HBsAg 양성률의 연령별로 분포, 지역적 차이, 과거 간질환력, 만성간질환 가족력 및 예방접종과의 관련성을 파악하며 또한 양성자를 6개월간 추적후 재검사 하여 B형 간염의 만성보균율을 파악하고자 하는 것을 이차적 목적으로 하였다. 방법 : 1998년 전국 10개 지역에서 의료보험관리공단의 정기건강검진 수진자를 대상으로 하여 HBsAg의 혈청유병률을 조사하였다. HBsAg 양성인 사람들을 대상으로 6개월 이상 추적조사하여 B형간염 만성보균율을 파악하였다. 총 1,816명에 대한 혈청과 설문서가 수집되었다. HBsAg는 RIA로 측정하였다. 결과 : HBsAg의 혈청유병률은 5.5%(95% CI-4.5-6.6)였으며 남자에서 7.4%(95% CI=5.8-9.4), 여자에서 3.6%(95% CI=2.5-5.0)로 나타났다. 급성간질환 과거력과 만성간질환 가족력을 HBsAg 혈청검사결과와 비교한 결과 유의한 관련성이 있는 것으로 나타났다. HBsAg 양성자에서 6개월 후에 음성으로 전환한 사람은 3.2%(95% CI=0.1-16.7)였으며 따라서 B형간염의 만성보균율은 5.3%(95% CI=3.7-6.6)로 추정되었다. 결론 : 본 연구결과에서는 HBsAg의 양성률이 1980년대의 연구결과들에 비하여 비교적 낮게 추정되었으며 이는 특히 여성과 젊은 연령층에서 두드러지게 나타났다. 그러나 우리나라에서의 간암 및 만성간질환의 공중보건학적 중요성을 고려하면 지속적인 간염발생의 예방대책이 필요하다고 할 수 있다.

• IMMUNE NETWORK
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• 제2권1호
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• pp.41-48
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• 2002

Background: Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8+ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/$K^b$ transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge. Methods: HBx-peptides encapsulated by pH-sensitive liposomes were prepared. $A2K^b$ transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with $1{\times}10^7pfu$/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK-143B cells. IFN-${\gamma}$ secretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A ($2{\mu}g/ml$), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with $20{\mu}g/ml$ of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard $^{51}Cr$-release assay and intracellular IFN-${\gamma}$ assay. Results: Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8+ T cells. Mice immunized with $50{\mu}g/head$ were much better protected against viral challenge compared to those immunized with $5{\mu}g$/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with $5{\mu}g/head$ than $50{\mu}g/head$ group. Increase of the number of cells that intracellular IFN-${\gamma}$ secreting cell among CD8+ T cells showed similar result. Conclusion: Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by $^{51}Cr$-release assay and the percentage of accumulated intracellular IFN-${\gamma}$ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of $^{51}Cr$-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of $50{\mu}g/head$, while in the in vitro restimulation, it showed more spectific response in $5{\mu}g$/head group.