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최상훈,강혜경,윤도영,최규삼,최명자,최인성,정태화 ( S . Choi,H . Kang,D . Yoon,K . Choi,M . Choi,I . Choe,T . Chung ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2
Pure subunits of human chorionic gonadotropin (hCG) were obtained by chromatographic separation using DEAE-Sephadex A-25 and Sephadex G-75 for the immunization. The spleen cells obtained after immunization of either w-hCG or β-subunits were fused with myeloma cells and twelve hybridoma cell lines were established. The results showed that the fusion efficiency and antibody producibility varied depending on the myeloma cell lines. The cell line P3×63-Ag8.653 was found to be a better fusion partner than SP2/0 for the production of antibodies from hybridoma cells. For the formation of ascitic fluid in mice, Incomplete Freund`s Adjuvant(IFA) was a better priming agent than pristane. The yield of specific monoclonal antibodies by the immuno-affinity purification method was 1.6-3.4 ㎎/㎖ ascitic fluid. Seven clones were studied for the specificity to w-hCG and its subunits using ^(125)I-labeled tracer and the clones were classified into 3 groups. The equilibrium affinity constants of the seven antibodies ranged from 5×10^7 M^(-1) to 8×10^8 M^(-1).
최상훈,강혜경,윤도영,최규삼,최명자,최인성,정태화,Choi, S.,Kang, H.,Yoon, D.,Choi, K.,Choi, M.,Choe, I.,Chung, T. 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2
hCG subunit를 순수 분리정제하여 w-hCG와 ${\beta}$-subunit를 각각 Balb/c 생쥐에 면역 주사하여 얻은 비장세포를 myeloma 세포와 융합하여 12개의 하이브리도마 세포주를 얻었다. Myeloma 세포주에 따른 세포융합의 효율과 hCG 항체 생성율을 조사했을 때 $P3{\times}63$-Ag 8.653이 SP2/0 보다 우수했으며, priming agent에 따른 복수생성율을 조사했을 때 IFA가 pristane 보다 우수함을 알았다. 하이브라도마 클론에서 유발된 복수액을 immuno-affinity 정제했을 때 특이 항체의 양은 클론에 따라 복수 ml 당 1.6-3.4 mg을 얻을 수 있었으며 모두 $IgG_1$으로 나타났다. 이 중 7개의 단일클론 항체클론에 대해 w-hCG 및 ${\alpha}$와 ${\beta}$-subunit에 대한 친화항수를 조사했을 때 $5{\times}10^7/M$에서 $8{\times}10^8/M$로 나타났으며 이 특이 친화성에 의해 항체를 3개의 그룹으로 분류할 수 있었다. Pure subunits of human chorionic gonadotropin (hCG) were obtained by chromatographic separation using DEAE-Sephadex A-25 and Sephadex G-75 for the immunization. The spleen cells obtained after immunization of either w-hCG or ${\beta}$-subunits were fused with myeloma cells and twelve hybridoma cell lines were established. The results showed that the fusion efficiency and antibody producibility varied depending on the myeloma cell lines. The cell line $P3{\times}63$-Ag8.653 was found to be a better fusion partner than SP2/0 for the production of antibodies from hybridoma cells. For the formation of ascitic fluid in mice, Incomplete Freund's Adjuvant(IFA) was a better priming agent than pristane. The yield of specific monoclonal antibodies by the immuno-affinity purification method was 1.6-3.4 mg/ml ascitic fluid. Seven clones were studied for the specificity to w-hCG and its subunits using $^{125}I$I-labeled tracer and the clones were classified into 3 groups. The equilibrium affinity constants of the seven antibodies ranged from $5{\times}10^7M^{-1}$ to $8{\times}10^8M^{-1}$.