Cholesterol Oxidase 를 생성하는 토양 미생물의 분리 및 효소 생산에 관한 연구

이인애(In - Ae Lee),최용경(Yong - Kyung Choe),이홍수(Hong - Soo Lee),최인성(In - Seong Choe),정태화(Tai - Wha Chung) 한국미생물생명공학회 1992 한국미생물·생명공학회지 Vol.20 No.4

인체 S100A2 단백질에 특이적인 단일클론 항체

김재화,윤선영,김주헌,주종혁,김진숙,이영희,염영일,최용경,최인성,Kim, Jae Wha,Yoon, Sun Young,Kim, Joo Heon,Joo, Jong-Hyuck,Kim, Jin Sook,Lee, Younghee,Yeom, Young Il,Choe, Yong-Kyung,Choe, In Seong 대한면역학회 2003 Immune Network Vol.3 No.1

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

인체 S100A6 단백질에 특이한 단일클론 항체

김재화,윤선영,주종혁,강호범,이영희,최용경,최인성,Kim, Jae Wha,Yoon, Sun Young,Joo, Joung-Hyuck,Kang, Ho Bum,Lee, Younghee,Choe, Yong-Kyung,Choe, In Seong 대한면역학회 2002 Immune Network Vol.2 No.3

Background: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. Methods: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. Results: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti-S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. Conclusion: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.

미립자 응집반응을 이용한 C-reactive Protein의 면역측정법에 관한 연구

김재화(Jae-Wha Kim),송은영(Eun-Young Song),이희구(Hee-Gu Lee),최용경(Yong-Kyung Choe),최명자(Myung-Ja Choi),김용호(Yong Ho Kim),최인성(In-Seong Choe),정태화(Tai-Wha Chung) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

환자의 복수와 늑막액으로부터 p-diazonium phenylphosphorylcholine(DPPC) coupled Separose-4B affinity chromatography와 hydroxylapatite chromatography를 실시하여 C-reactive protein(CRP)를 분리, 제정하였다. 정제된 CRP를 토끼에게 면역화하여 항혈청을 얻고 affinity chromatography를 하여 면역항체(IgG)를 분리하였다. 분리된 면역항체를 미립자에 감작시킨 후 미립자 응집반응에 의하여 3분내에 CRP를 측정할 수 있는 간이 면역측정법을 개발하였다. 본 연구에서 개발된 CRP측정법의 검출범위는 0.5~20㎎/dl이며, 임상 시험 결과 0.7~2.9㎎/dl에서는 강한 응집반응을, 5.0~13.2㎎/dl에서는 약한 응집반응을 보였고 28㎎/dl이상에서는 항원 과잉으로 인한(zone of Ag excess phenomenon) 위음성을 나타냈다. 74명의 환자 혈청을 대상으로 CRP의 농도를 조사한 결과 평균치는 3.8㎎/dl이었으며 대부분의 환자에서는 10㎎/dl 이하의 농도로 존재하였다. 그러므로 1차판정시 음성을 나타낸 시료라도 혈청을 5~10배정도 희석하여 재분석한다면 오차없이 CRP를 검출할 수 있었다. 환자 혈청을 검체로 하여 본 연구에서 개발한 면역측정법과 현재 수입 시판중인 프랑스의 B사 제품과 일본의 I사 제품을 비교한 결과 좋은 상관관계를 보였다. 이와 같은 평가분석을 통하여 볼 때 본 연구에서 개발한 간이 면역측정법은 사용이 비교적 간편하며 신빙성이 있어 CRP를 스크리닝 하는데 효과적임을 알 수 있었다. The C-reactive protein(CRP) from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine(DPPC) and hydroxylapitite chromatography. Polyclonal antibody was prepared from rabbit by immunizing the purified CRP. Specific immunoglobulin G was isolated using affinity chromatography and coupled to microparticles. A sensitive microparticle-based immunoassay was developed to measure CRP within 3 mins. The detection range was between 0.5㎎/dl and 20㎎/dl in serum, showing strong response in the range of 0.7~2.9 ㎎/dl, weak response in 5.0~13.2 ㎎/dl and zone phenomenon over 28㎎/dl. The average value of CRP in 74 samples was 3.8㎎/dl and most of the values were lower than 10㎎/dl. The CRP values of serum samples were determined by our microparticle-based immunoassay, and were compared with those obtained using the other commercial products(B Co., France and I Co., Japan). Good correlations were shown between the values obtained by our developed mi-croparticle-based immunoassay system and those by other commercial products. All performance characteristics evaluated make our developed microparticles-based immunoassay suitable for a simple, rapid, and reliable screening of CRP in serum.

결핵균 감염에 의한 THP-1 세포에서의 Prothymosin alpha 유전자 발현증가

송호연(Ho Yeon Song),장광식(Kwang Sik Jang),변희선(Hee Sun Byoun),이신제(Shin Je Lee),김진구(Jin Koo Kim),최용경(Yong Kyung Choe),고광균(Kwang Kjune Ko) 大韓微生物學會 2000 大韓微生物學會誌 Vol.35 No.2

미립자 응집반응을 이용한 C-reactive Protein의 면역측정법에 관한 연구

최용경,정태화,최명자,김재화,최인성,김용호,송은영,이희구 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

환자의 복수와 늑막액으로부터 p-diazonium phenylphosphorylcholine(DPPC) coupled Separose-4B affinity chromatography와 hydroxylapatite chromatography를 실시하여 C-reactive protein (CRP)를 분리, 정제하였다. 정제된 CRP를 토끼에게 면역화하여 항혈청을 얻고 affinity chromatography를 하여 면역항체(IgG)를 분리하였다. 분리된 면역항체를 미립자에 감작시킨 후 미립자 응집반응에 의하여 3분내에 CRP를 측정할 수 있는 간이 면역측정법을 개발하였다. 본 연구에서 개발된 CRP측정법의 검출범위는 0.5∼20㎎/㎗이며, 임상 시험 결과 0.7∼2.9㎎/㎗에서는 강한 응집반응을, 5.0∼13.2㎎/㎗에서는 약한 응집반응을 보였고 28㎎/㎗이상에서는 항원 과잉으로 인한(zone of Ag excess phenomenon) 위음성을 나타냈다. 74명의 환자 혈청을 대상으로 CRP의 농도를 조사한 결과 평균치는 3.8㎎/㎗이었으며 대부분의 환자에서는 10㎎/㎗ 이하의 농도로 존재하였다. 그러므로 1차판정시 음성을 나타낸 시료라도 혈청을 5∼10배정도 희석하여 재분석한다면 오차없이 CRP를 검출할 수 있었다. 환자 혈청을 검체로 하여 본 연구에서 개발한 면역측정법과 현재 수입 시판중인 프랑스의 B사 제품과 일본의 I사 제품을 비교한 결과 좋은 상관관계를 보였다. 이와 같은 평가 분석을 통하여 볼 때 본 연구에서 개발한 간이 면역측정법은 사용이 비교적 간편하며 신빙성이 있어 CRP를 스크리닝 하는데 효과적임을 알 수 있었다. The C-reactive protein(CRP) from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine(DPPC) and hydroxylapitite chromatography. Polyclonal antibody was prepared from rabbit by immunizing the purified CRP. Specific immunoglobulin G was isolated using affinity chromatography and coupled to microparticles. A sensitive microparticle-based immunoassay was developed to measure CRP within 3 mins. The detection range was between 0.5㎎/㎗ and 20㎎/㎗ in serum, showing strong response in the range of 0.7∼2.9㎎/㎗, week response in 5.0∼13.2㎎/㎗ and zone phenomenon over 28㎎/㎗. The average value of CRP in 74 samples was 3.8㎎/㎗ and most of the values were lower than 10㎎/㎗. The CRP values of serum samples were determined by our microparticle-based immunoassay, and were compared with those obtained using the other commercial products(B Co., France and I Co., Japan). Good correlations were shown between the values obtained by our developed microparticle-based immunoassay system and those by other commercial products. All performance characteristics evaluated make our developed microparticles-based immunoassay suitable for a simple, rapid, and reliable screening of CRP in serum.

BCG를 이용한 복합백신 개발

최용경 한국생화학분자생물학회 1992 생화학분자생물학회 소식 Vol.12 No.2

Tuberculosis and leprosy, caused by Mycobacterium tuberculosis and M. leprae respectively, remain major problems in the developing world. Mycobacteria also causes the opportunistic infection of AIDS patients. BCG, an attenuated strain derived from virulent bovine tubercle bacilli(M bovis), is one of the most widely used human vaccine against tuberculosis and has been proposed as a host for the construction of recombinant live vaccines expressing novel protective epitopes. BCG offers some unique advantages for developing such a multivaccine vehicle, including a long record of use in humans with a low incidence of serious complications, stability in the field, long-lived immune responses with a single-dose usage and a low cost to produce. With the development of methods for introducing DNA into mycobacteria, and the propagation and expression of foreign genes, BCG may well take on a fresh release of life, the old stager re-emerging as a star performer.

합성 펩티드를 이용한 Interleukin-2수용체 β-Chain에 대한 단일클론항체의 제작

이희구,이홍수,이인애,최용경,최인성,정태화,김길현 梨花女子大學校 藥學硏究所 1995 藥學硏究論文集 Vol.- No.5

Hybridoma producing monoclonal antibodies reactive with the ß-chain of interleukin-2 receptor (IL-2R) were generated from mice immunized with a synthetic pepride of 19 amino acids. The synthetic peptide constitutes strongly hydrophilic region of extracellular domain of the IL-2R molecules(residues 32-50) as revealed by hydrophathy plot analysis. Hybridomas were screened by ELISA using Hut102 cells and the synthetic peptide as antigenic moiety, and by measuring the binding activity of antibodies to the Hut102 cell surface that was monitored by a flow cytometer. The antibodies were further screened by their inhibitory effect against the interaction between IL-2R and interleukin-2(IL-2). Antibodies were further characterized by immunoprecipitation followed by autoradiography to reveal that the antibodies recongnize the 75 KD protein molecules expressed on the Hut 102 cell surface and human peripheral blood mononuclear cells(PBMC) which confirms the antigenic moiety to be ß-chain of IL-2 recepter molecules.

합성 펩티드를 이용한 Interleukin-2수용체 β-Chain에 대한 단일클론항체의 제작

이희구,이홍수,이인애,최용경,최인성,정태화,김길현 이화여자대학교 생명과학연구소 1995 생명과학연구논문집 Vol.6 No.-

Hybridoma producing monoclonal antibodies reactive with the β-chain of interleukin-2 receptor (IL-2R) were generated from mice immunized with a synthetic peptide of 19 amino acids. The synthetic peptide constitutes strongly hydrophilic region of extracellular domain of the IL-2R molecules(residues 32-50) as revealed by hydrophathy plot analysis. Hybridomas were screened by ELISA using Hut102 cells and the synthetic peptide as antigenic moiety, and by miasuring the binding activity of antivodies to the Hut102 cell surface that was monitored by a flow cytometer. The antibodies were further screened by their inhibitory effect against the interaction between IL-2R and interleukin-2(IL-2). Antibodies were further characterized by immunoprecipitation followed by autoradiography to reveal that the antibodies recongnize the 75 KD protein molecules expressed on the Hut102 cell surface and human peripheral blood mononuclear cells(PBMC) shich confirms the antigenic moiety to be β-chain of IL-2 recepter molecules.

인체 S100A6 단백질에 특이한 단일클론 항체

김재화,윤선영,김주헌,주종혁,김진숙,이영희,염영일,최용경,최인성 Official Journal of the Korean society for immunol 2003 Immune Network Vol.3 No.1