B 임파구의 항체생산을 위한 T 임파구의 역할 및 상호작용

최인성 ( In Seong Choe ) 생화학분자생물학회 1992 생화학총설집 Vol.4 No.-

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

대장균군 검사용 간이 시험지 개발

이인애(In-Ae Lee),김재화(Jae-Wha Kim),이회구(Hee-Gu Lee),성창근(Chang-Keun Seong),최인성(In-Seong Choe),정태화(Tai-Wha Chung) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

대장균군 검사용 간이 시험지는 본 실험실에서 국내 최초로 고안, 개발하였으며 이 간이 시험지법은 현장 검사법의 하나로 대장균군이 내는 succinic acid dehydrogenase 때문에 tetrazolium salt가 환원되어 적색 반점을 형성하는 것을 이용한 방법으로서 이 간이 시험지의 제조는 대체로 종래의 표준 평판법과 거의 동일한 조성의 배지와 사약을 사용하여 여지에 흡착치킨 후, 건조시켜(60℃) 멸균한 것으로 표준 평판법과 어떤 상관관계가 있는가를 검토하였다. 이 간이 시험지의 제조에서는 bile salt No. 3를 deoxycholate로 대체하여 제조 원가를 절감하였고, 또한 일본에서 현재 시판되고 있는 제품과 품질 비교시험을 하여 더 좋은 결과를 얻었으며 종래의 표준 평판법과 비교하였을 때도 오히려 표준 평판법(24-48시간 배양)보다 빠른 시간(16-20시간 배양)내에 판정할 수 있는 이점이 있으며, 표준 평판법에서는 없어서는 안될 배지나 배양 접시, pipette등의 자료 및 기구가 일체 필요없고 언제 어디서나 현장에서 직접 시험할 수 있어 매우 간편하며 또한 저렴한 가격으로 제조할 수 있는 경제성이 높은 이점을 갖고 있다. The objective of this study was to develop a paper strip which could determine E. coli qualitatively and quantitatively in water, wastewater, drinks, or food. This paper strip method was a simple and rapid test method that determine E. coli by visual identification. In this study, nutrient culture media were formulated and characterized for optimum conditions. Paper strips were then prepared by impregnating into the media and dried at 60℃. The test procedure is quite simple to use. The paper strip was dipped into a sample, and excess sample was removed. The strip was then incubated at 37℃ for 16 to 20 hours and the number of colonies on the strip was counted. The color of the colony spots produced by microorganisms varied depending on the media formulation. Violet-red spots were produced by E. coli. The test method was simple, rapid and no special laboratory equipment was necessary for visual identification. Therefore, this test method is applicable to on-site tests such as field tests or home tests. The paper strip method was compared with the standard agar plate method and Japanese commercial product. The method of the economical preparation of test strips was studied for production on industrial scale.

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.

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.

과산화효소를 이용한 백혈구 측정용 뇨 검사지 제조에 관한 연구

송은영(Eun-Young Song),이홍수(Hong Soo Lee),김희정(Hee Jung Kim),김종완(Jong Wan Kim),최인성(In Seong Choe),변시명(Si Myung Byun),정태화(Tai-Wha Chung) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.2

백혈구에 존재하는 과산화 효소를 이용하여 뇨 중 백혈구의 수를 간접적으로 측정하는 새로운 방법의 뇨 검사지를 개발하였다. 과산화 수소원으로서 포도당 산화효소와 포도당을, 색원체로서 tetramethylbenzidine을 처리하여 백혈구 측정용 뇨 검사지를 제조하였으며 검출한계는 10 cells/㎕ (5 cells/hpf)이었다. 개발된 백혈구 측정용 뇨 검사지로 뇨를 분석하였을 때 뇨 ㎕당 10-25 cells의 백혈구가 존재할 경우에는 2분내에 연녹색을, 75-250 cells/㎕에서는 녹색을 보였고 500 cells/㎕ 이상에서는 녹청색을 보였다. 172명의 환자뇨를 대상으로 뇨중 백혈구 수치를 현미경 측정 결과와 본 연구에서 개발한 백혈구 측정용 뇨 검사지와 현재 수입 시판 중인 미국의 A사 제품과 B사 제품으로 분석 비교한 결과 좋은 상관관계를 보였다. 이와 같은 평가분석을 통하여 볼 때 본 연구에서 개발한 백혈구 측정용 뇨 검사지는 에스터라아제를 이용한 백혈구 측정용 뇨 검사지와 함께 뇨중 백혈구를 스크리닝 하는데 효과적임을 알 수 있었다. A new test strip to detect leukocytes using the myeloperoxidase in urine was developed. The reagent strip contains tetramethylbenzine, glucose and glucose oxidase. The detection limit was between 10 cells per 1㎕ urine(5 cells/hpf), showing greenish yellow color in the range of 10-25 cells/㎕, green color in the range of 75-250 cells/㎕, greenish blue color in the range of 500 cells/㎕. The result can be obtained within two minute. The performance of the new method was evaluated by comparing the results of microscopic examination and other commercial products. Good correlations were shown between the values obtained by our urine strip and those by other commercial products with 172 urine samples. The results were proven that new methods were useful as primary screening reagents to detect leukocytes in urine.

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.

In Vitro Induction of Human Antibody Against Hepatitis B Virus Surface Antigen

한미영,오재욱,남경수,최인성,정태화,Han, Mi-Young,Oh, Jae-Wook,Nam, Kyung-Soo,Choe, In-Seong,Chung, Tai-Wha 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

B형 간염 바이러스 표면항원에 대한 항체를 가지고 있는 사람이 peripheral blood lymphocyte를 Epstein-Barr virus로 transformation시켰을 때 strong positive clone이 나오는 확률은 최고 23%였으며, cloning과 항체분비 test를 계속했을 때 가장 오랫동안 항체를 분비하는 clone이 3개월간 양성반응을 보였다. Polyclonal B cell activator 중에서 pokeweed mitogen을 in vitro에서 처리했을 때 B형 간염 바이러스 표면항원에 대한 항체를 생산하였으며 $[^3H]$-thymidine incorporation도 증가하였다. Human peripheral blood lymphocytes (PBL) were isolated from the heparinized blood of antibody positive donors by centrifugation with Ficoll/Paque solution. The PBL were transformed with Epstein-Barr virus and the clones were screened for anti-HBsAg antibody production. Twenty-three percent of the total clones were HBsAg antibody positive. Among them, one clone showed stable antibody production. One of the polyclonal B cell activators, pokeweed mitogen, was effective for production of anti-HBsAg antibody and cell proliferation.

B 형 간염 바이러스 표면항원에 대한 항체의 유도

한미영,오재욱,남경수,최인성,정태화 ( Mi Young Han,Jae Wook Oh,Kyung Soo Nam,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

Human peripheral blood lymphocytes (PBL) were isolated from the heparinized blood of antibody positive donors by centrifugation with Ficoll/Paque solution. The PBL were transformed with Epstein-Barr virus and the clones were screened for anti-HBsAg antibody production. Twenty-three percent of the total clones were HBsAg antibody positive. Among them, one clone showed stable antibody production. One of the polyclonal B cell activators, pokeweed mitogen, was effective for production of anti-HBsAg antibody and cell proliferation.

Production of anti-$preS_2$, peptide monoclonal antibodies and preparation of anti-$preS_2$, immunoaffinity column

한미영,최상훈,최인성,최명자,정태화,Ha, Mi-Young,Choi, Sang-Hoon,Choe, In-Seong,Choi, Myung-Ja,Chung, Tai-Wha 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3

$PreS_2-{\beta}$-galactosidase 융합단백질을 면역원으로 하여 재조합 $preS_2$ peptide에 대한 단일 클론항체 3종류를 생산하였으며 모두 IgM class로 확인되었다. 그 중 040-01 단일 클론항체를 6% PEG로 처리하여 대장균에서 발현되는 재조합 $preS_2$ peptide를 정제시키기 위한 면역 친화성 column 제조에 이용하였다. Elution buffer로는 0.1M-glycine-HCl(pH 2.5)이 다른 종류의 buffer에 비해 IgM 항체 활성도를 가장 안정되게 유지시켰다. $preS_2$ peptide에 대한 면역 친화성 column의 정제력은 $preS_2$-ABEI.H conjugate를 이용하여 결정하였다. Three monoclonal antibodies specific to the recombinant $preS_2$ peptide were prepared using $preS_2-{\beta}$-galactosidase as an immunogen. All three were classified as immunoglobulin M. One of the three monoclonal antibodies (040-01) was precipitated by 6% PEG and used for the preparation of immunoadsorbent column. This immunoaffinity column was utilized to purify recombinant $preS_2$ peptide which was solubilized and extracted from sonicated E. coli cells. For the storage of this specific antibody, 0.1 M glycine-HCl buffer (pH 2.5) gave the best result. Other buffers tested in this study caused a significant decrease in antibody reactivity. The binding capacity of an immunoadsorbent column for $preS_2$ peptide was determined using $preS_2$-ABEI H conjugate.