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Human-Mouse Heterohybridoma를 이용한 사람 항암세포 항체(Monoclonal Antibody)의 생산
하윤문,박재경,남상윤,최규철,최용묵 大韓免疫學會 1987 大韓免疫學會誌 Vol.9 No.1
Human lymphocytes from peripheral blood, lymph node, spleen, and ascite of cancer patients were fused with mouse P3x 63Ag 8.653 myeloma cells. With 8 patients, 13 fusions were performed. Human-mouse heterohybridomas usually appeared between day 7 and 14, and 613 clones(average; 13 clones per 10' lymphocytes) were successfully propagated. Human immunoglobulins(Ig) were detected from 7 hybridoma culture supernatants by ELISA, and levels of Ig synthesis ranged from 0.1 to 1.5pg/ml. Pretreatment of lymphocytes with mitogen(pokeweed mitogen, or lipopolysacchar ide) increase neither fusion frequency nor yield of Ig-secreting hybridomas. Most of hybridomas were unstable for Ig secretion and only I of the 7 clones produced Ig over 3 month period. We observed the loss of human chromosome in the hybrid clones which stopped secreting lg. Monoclonal antibodies, HDB7 was demonstrated to react with autologus tumor cells, but not stroma, lymphocytes, or allogeneic tumor cells. Our results suggest that human monoclonal antibodies specific against autologous tumor cells can be generated by human-mouse hybridization.
하윤문,Ha, Youn-Mun The Korea Society for Microbiology 1977 大韓微生物學會誌 Vol.12 No.1
계난백(鷄卵白) Lysozyme N-와 C 말단(末端) 항원결정기(抗原決定基)($P_{17}$: sequence $Lys^1-{cys-}^6-Asn^{27},\;{Trp^{12}}_2-Cys^{127}-Leu^{129}$)의 특이성(特異性)에 대(對)하여 연구(硏究)했다. $^{14}C-acetyl$ Lysozyme 과 정제(精製)한 guinea pig 항(抗)-$P_{17}$ 항체(抗體)와의 결합(結合)을 Scatchard plot 상(上)에서 나타낸 결과(結果) 그 실험치(實驗値)가 거의 r=1였다. 이것은 Lysozyme에 대(對)하여 각각(各各) 다른 친화성(親和性)을 가진 2개(個)의 항체군(抗體群)의 존재(存在)나 그렇잖으면 제(第)1의 항체결합부위(抗體結合部位)에 최초(最初)의 Lysozyme 분자(分子)가 결합(結合)함으로 인(因)하여 항체분자(抗體分子)의 제(第)2의 결합부위(結合部位)에 다른 Lysozyme 분자(分子)의 결합(結合)을 방해하는 즉 steric hindrance에 의한 가능성(可能性)을 시사(示唆)한다. 여러가지 peptides의 항원활성(抗原活性)을 $^{14}C-acetyl-P_{17}$과 항(抗)-$P_{17}$ 항체(抗體)와의 결합저해(結合沮害)시험에서 측정(測定)했다. 그 결과(結果) 단지 $P_{17}$과 $P_{17}t$(sequence $Lys^1-cys^5-Homoser^{12},\;Trp^{123}-cys^{127}-Leu^{128})$)만이 억제되었고 그 $K_1$치(値)가 각각(各各) $2.0{\times}10^4$과 $8.1{\times}10^3$이였다. 이들 결과(結果)를 종합(綜合)하면 $P_{17}$의 항(抗)-$P_{17}$ 항체(抗體)와의 직접적 결합부위(結合部位)는 $P_{17}$의 말단부위(末端部位)에 국재(局在)해 있는 것을 암시해 준다. 한편 $P_{17}$의 나머지 부분(部分)은 이 항원결정기(抗原決定基) 구조(構造)를 유지(維持)하는데 중요(重要)한 역할(役割)을 할 것으로 생각되며 또한 이 결정기내(決定基內)의 한 개의 disulphide 결합(結合)은 면역학적(免疫學的) 활성(活性)을 나타내는데 필수적(必須的)인 것으로 믿어진다. The specificity of the N- and C-terminal antigenic determinant($P_{17}$: sequence $Lys^1-{cys-}^6-Asn^{27},\;{Trp^{12}}_2-Cys^{127}-Leu^{129}$) of hen egg-white lysozyme(HL) was studied in more detail. In a Scatchard plot of the binding of $^{14}C$-acetyl HL with guinea pig purified anti-$P_{17}$ antibody experimental values bent sharply aear r=1. This suggests of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of $^{14}C-acetyl-P_{17}$ with the antibody, Only $P_{17}$ and $P_{17}t$(sequence $Lys^1-cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{128})$) were inhibitory, with $K_1$ values of $2.0{\times}10^4$ and $8.1{\times}10^3$, respectively. These results indicate that the direct binding site of $P_{17}$ to anti-$P_{17}$ antibody may be located in the terminal portion of $P_{17}$ (sequence $Lys^1-Cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{129})$) while the rest of $P_{17}$ may be important in maintaining the conformation of this determinant. The single disulphide bond involved in this determinant is essential for manifestation of immunological activity.
林壽德,全茂炯,尹在一,韓乙男,夏潤文 WHO COLLABORATING CENTRE FOR TRADITIONAL MEDICINE 1981 東西醫學硏究所 論文集 Vol.1 No.1
A human fibroblast cell strain, GM 2767, selected to use for human interferon bioassay was investigated on the in vitro biological properties; morphology, growth cycle under various culture conditions, cryopreservation and sensitivity to vesicular stomatitis virus. The results obtained are summarized as follows; 1. The uninactivated fetal bovine serum(FBS) supplemented in the medium resulted in higher growth rate of GM 2767 than the inactivated FBS. The higher concentration of the uninactivated FBS in the medium induced the more stimulated growth rate. Cell growth rate was lower in pH 6.8 and 7.8 than in pH 7.3 of medium. 2. Growth rate was gradually decreased as the passage level was increased. It took more than 7 days to form full monolayer at the passage levles after the 11th. 3. In comparison of two cryopreservative agents of glycerol and dimethyl sulfoxide(DMSO), no difference was found in preservation of cell viability. However, the plating efficiency of the preserved cells was higher in DMSO than in glycerol. 4. GM 2767 cells was highly susceptible to vesicular stomatitis virus(VSV) challenge, showing typical cytopathic effects of VSV at 12-36 hours postinoculation.