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.

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.

Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

Jae-Wha Kim(김재화),Mi-Young Han(한미영),Hee-Gu Lee(이희구),Eun-Young Song(송은영),Tai-Wha Chung(정태화),Kyung-Soo Nam(남경수),In-Seong Choe(최인성),최용경 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.1

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할 수 있는 간편한 immunoassay 체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA 방법으로 분석하였으며 이들 클론 중 bis-indole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두 개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05 nM정도의 dimeric Vinca alkoloids까지도 측정할 수 있었다. Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

IL-1 수용체를 통한 T세포 활성화에 Genistein 과 Staurosporin이 미치는 영향

한미영,윤도영,오은숙,고우석,윤선영,정태화 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.4

Stimulation of T-cells through TCR/CD3 complex alone is known to be not enough but also require a costimulatory signal, such as IL-1 stimulation. to induce IL-2 production. In this report, we studied the differential effects of staurosporin (a PKC inhibitor) and genistein (a PTK inhibitor) on the IL-2 production by EL-4 and A2.5 cells, to determine which pathway is responsible for IL-1 receptor signal transduction. While staurosporin suppressed the IL-2 production induced in EL-4 cells upon PMA stimulation but not upon PHA plus IL-1, genistein inhibited the IL-2 production induced by PHA plus IL-1 but not by PMA. Genistein also completey abrogated the IL-2 production by A2.5 cell when stimulated with PHA and IL-1. These results suggest that the costimulatory signal by IL-1, which leads to IL-2 production and proliferation, is closely linked to the PTK activity in T cells.

Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

Song,Eun-Young,Chung,Tai-wha,Kim,Jae-Wha,Nam,Kyung-Soo,Han,Mi-Young,Choe,In-Seong,Lee,Hee-Gu THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할수 있는 간편한 immunoassay 체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로 부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA방법으로 분석하였으며 이들 클론 중 bis-indole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids 와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05nM정도의 dimeric Vinca alkaloids까지도 측정할 수 있었다. Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA((Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

IL-6 의존형 B 하이브리도마 세포의 신호전달에서 Grb2 단백질의 역할

윤선영,고우석,최인표,권병목,정태화,한미영 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.2

IL-6 is a pleiotropic cytokine generating multifunctional signals to several cell lines such as B cells, T cells, hepatocytes, myeloma, and leukemic cell lines. To understand the diverse signals generated by IL-6, it is essential to clarify the molecular and cellular events following IL-6 stimulation. Initially IL-6 binds to cells with its binding subunit of IL-6R(gp80). The binding triggers the association of gp80 with a signal transducer, gp130. Since neither gp80 nor gp130 have any protein tyrosine kinase domain, unknown protein kinases except Jak kinase might be involved in the early steps of IL-6 signal transduction. Growth factor receptor-bound protein 2 (Grb2) is a 25 kD adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. In order to identify phosphoproteins associated with Grb2 adaptor protein, we have constructed a GST-Grb2 fusion protein and isolated its associated tyrosine phosphoproteins, including 72, 70, 46 and 28 KD that were phospholyated with IL-6 stimulation in B 9-79 hybridoma cells. We also detected several protein kinases, including 50 KD and 44 KD protein by in vitro kinase assay. These results suggest that several phosphoproteins and kinases are involved in IL-6 signal transduction.