B 임파구의 항체생산을 위한 T 임파구의 역할 및 상호작용

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

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.

재조합 preS2 peptide 에 대한 단일 클론항체 생산과 그를 이용한 면역 친화성 column 제조에 관한 연구

한미영,최상훈,최인성,최명자,정태화 ( Mi Young Han,Sang Hoon Choi,In Seong Choe,Myung Ja Choi,Tai Wha Chung ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3

Three monoclonal antibodies specific to the recombinant preS₂ peptide were prepared using preS₂-β-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 preS₂ 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 preS₂ peptide was determined using preS₂-ABEI-H conjugate.

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.

고등식물 형질 전환용 벡터

홍주봉(Choo Bong Hong),최인성(In Seong Choe) 생화학분자생물학회(구 한국생화학분자생물학회) 1988 생화학총설집 Vol.2 No.-

인터루킨 - 2 정량을 위한 효소면역 측정법

최규삼,김길현,최명자,최인성,정태화 ( Kyu Usam Choi,Kil Hyoun Kim,Myung Ja Choi,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3

To establish an enzyme-linked immunosorbent assay technique to quantitate interleukin-2 (IL-2), five different monoclonal antibodies against human recombinant IL-2 were prepared. A pair of monoclonal antibodies recognizing different epitopes on the IL-2 molecule were selected for sandwich ELISA. The procedure involved utilization of one specific antibody immobilized on a microtiter plate and the other labelled with alkaline phosphatase. A linear relationship was achieved, using concentrations of IL-2 up to 100 ng/ml, when the absorbance of the reaction mixture was measured on a spectrophotometer. The whole assay procedure can be completed within five h. This assay technique for IL-2 would provide a simple and rapid alternative to other assays such as bioassay using IL-2 dependent CTLL cells.

Production, Purification and Characterization of Monoclonal Antibodies Specific to Progesterone

윤도영,최상훈,최명자,최인성,정태화,Yoon, Do-Young,Choi, Sang-Hoon,Choi, Myung-Ja,Choe, In-Seong,Chung, Tai-Wha Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.1

Progesterone-$11{\alpha}$-hemisuccinate-BSA를 제조하여 Balb/c 생쥐에 주사하여 얻은 비장세포를 myeloma 세포와 융합하여 여러 가지 subtype 형태($IgG_1$, $IgG_{2a}$, $IgG_{2b}$, IgM)를 가진 항체를 분비하는 7개의 하이브리도마 세포주를 얻었다. 이들이 분비하는 항체들을 간단히 ammonium sulfate 침전법, Protein A-Sepharose 4B 등으로 분리정제하여 그 특성을 조사하였다. 그 결과 단일클론 항체가 18-44% 정도의 수률로 SDS-PAGE상에서 순수분리 되었음을 확인하였다. 이들 항체들의 affinity constant는 $5{\times}10^7-4{\times}10^8M^{-1}$ 정도였으며, 이들 항체들은 Progesterone-3-(O-carboxymethyloxime)-BSA를 면역원으로 하여 얻은 항체와 비교했을 때, 특이성과 다른 steroid 호르몬과 교차반응성 등이 클론에 따라 특이하게 나타났다. 특히 이들 항체 중 051-01 clone은 다른 clone에 비해 매우 빠른 성장을 보였으며, 항체의 양이 복수액의 전체 단백질의 38% 정도나 되어 titer level이 매우 높았으며, 방사면역측정법이나 효소면역측정법으로 반응성을 측정했을 때 그 감도가 매우 좋았다. Seven hybridoma cell lines, 051-01 ($IgG_1$), 051-02 ($IgG_{2b}$), 051-03 ($IgG_1$), 051-04 (IgM), 051-05 ($IgG_{2a}$), 051-06 (IgM), and 051-07 ($IgG_{2a}$) prodcing monoclonal antibodies against progesterone were established. Progesterone $11{\alpha}$-hemisuccinate-bovine serum albumin conjugate was prepared using carbodiimide and then used as an immunogen. Spleen cells of Balb/c mice immunized with the immunogen were fused with mouse myeloma (P3-X63-Ag8.653) cells. Hybridoma cells were screened by radioimmunoassay using $^3H$-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE. The progesterone binding capacity of the 5 monoclonal antibodies ranged from 7 to 72 pmole/mg IgG and the affinity constant ranged from $5.0{\times}10^7\;M^{-1}$ to $4{\times}10^8\;M^{-1}$ by the Scatchard analysis method. Cross-reactivities of the monoclonal antibodies, 051-01 and 051-02 with other steroid hormones were compared. These antibody reactions were very specific to progesterone and sensitive in a competitive immunoassay.

A Simplified Procedure for C-reactive Protein Purification

이희구,김용호,이홍수,최명자,최인성,정태화,Lee, Hee-Gu,Kim, Yong-Ho,Lee, Hong-Soo,Choi, Myung-Ja,Choe, In-Seong,Chung, Tae-Wha 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4

환자의 혈장이나 복수로부터 C-reactive protein을 높은 수율로서 순수하게 분리정제할 수 있는 간이 정제법을 개발하였다. DPPC-Affinity chromatography와 Hydroxylapatite chromatography 분리과정을 거쳐 간단하고 신속한 방법으로 분리된 CRP는 단일확산 침전법과 폐렴균의 세포막인 C-polysaccharide를 이용한 EIA 방법으로 활성도를 측정하였다. 분리한 CRP는 SDS-PAGE에서 단일밴드로 나타났으며 immunoelectrophoresis에서도 단일 침전 arc로 나타나 순수분리 되었음을 확인하였다. 또한, Sephacryl S-200 gel filtration과 SDS-polyacrylamide gel 전기영동 분석결과에 의하면 분자량은 118,000으로서 5개의 동일한 subunit로 구성되어 있으며 그 subunit의외 분자량은 23,600이었다. CRP의 isoelectric point는 4.82였으며 500 ml의 복수로부터 분리된 CRP의 회수율은 약 67%로서 4.7 mg이었다. A simplified procedure for obtaining high purity and high yield of C-reactive protein (CRP) was established. The 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 hydroxylapatite chromatography. The active fractions of CRP during the purification procedure were determined by EIA using pneumococcal C-polysaccharide. The final recovery of CRP was 67% of the initial protein amount as quantitated by single radial immunodiffusion (SRID), and the purification fold was 3,600. The purified CRP was shown to be homogeneous by electrophoresis and immunoelectrophoresis. The molecular weight of CRP was approximately 118KD as determined by Sephacryl S-200 gel filtration and the molecular weight of a subunit was approximately 23.6 KD by SDS-polyacrylamide gel electrophoresis. The isoelectric point of CRP was found to range about 4.82.

프로제스테론 단일클론 항체의 생산과 정제 및 특성연구

윤도영,최상훈,최명자,최인성,정태화 ( Do Young Yoon,Sang Hoon Choi,Myung Ja Choi,In Seong Choe,Tai Wha Chung ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.1

Seven hybridoma cell lines, 051-01 (IgG₁), 051-02 (IgG_(2b)), 051-03 (IgG₁,), 051-04 (IgM), 051-05 (IgG_(2a)), 051-06 (IgM), and 051-07 (IgG_(2a)) prodcing monoclonal antibodies against progesterone were established. Progesterone llα-hemisuccinate-bovine serum albumin conjugate was prepared using carbodiimide and then used as an immunogen. Spleen cells of Balb/c mice immunized with the immunogen were fused with mouse myeloma (P3-X63-Ag8.653) cells. Hybridoma cells were screened by radioimmunoassay using ³H-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE. The progesterone binding capacity of the 5 monoclonal antibodies ranged from 7 to 72 pmole/㎎ IgG and the affinity constant ranged from 5.0×10^7 M^(-1) to 4×10^8 M^(-1) by the Scatchard analysis method. Cross-reactivities of the monoclonal antibodies, 051-01 and 051-02 with other steroid hormones were compared. These antibody reactions were very specific to progesterone and sensitive in a competitive immunoassay.

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.