血小板活化因子單株抗體之製備與血小板活化因子乙醯水解酵素的純化

The preparation of monoclonal antibodies against Platelet Ativating Factor (PAF) and the purification of PAF- Aetylhydrolase

黃正財(Jeng Chai Hwang)

指導教授 ： 陳朝洋

臺北醫學大學/藥學院/藥學研究所/博士(1994年)

摘要

以1-O-carboxyl-nonyl-2-O-acetyl-sn-glycerol-3-phospho- choline- Bovine Serum Albumin(C10 acetyl F1-BSA)等抗原誘使BALB/c小白鼠產 生抗體, 再以瘤細胞和免疫小白鼠之脾細胞製備融合瘤細胞.以改良之直 接鍵結型ELISA法, 可檢測抗體認識PAF和Lyso PAF的差異, 其最低檢測靈 敏度為100pg, 以此分析法選得二株可釋放抗PAF抗體之細胞株, 其中細胞 株4D2G4A10所分泌之抗體可檢測天然型PAF至300ng。天竺鼠的血小板活化 因子乙醯水解酵素(PAF-AH), 經各種分離管分離純化後, 得一單離的活性 PAF-AH(分子量63 KDa)。天竺鼠之PAF-AH和其磷酸水解酵素A2(PLA2; MW. 16.7 KDa)不同, 該 PAF-AH可被10mM鈣離子所活化, 但不為10mM的 EDTA抑制活性。　以[14碳]膽固醇當標準品和天竺鼠PAF-AH以密度梯度離 心後,發現此 PAF-AH不與低密度脂蛋白結合, 此性質和已知之其它PAF- AH 不同。

關鍵字

抗體 ； 血小板活化因子 ； 血小板活化因子乙醯水解酵素 ； 純化

並列摘要

1-O-Carboxyl-nonyl-2-O-acetyl-sn-glycerol-3-phosphocho- line- Bovine Serum Albumin(C10 acetyl F1-BSA) was used as an antigen to raise antibody to Platelet-Activating Factor (PAF) on BALB/c mice, and prepared hybridoma by fusing lympho- cyte cells with myeloma. A modified ELISA method enable the screening of antibody to PAF but lyso PAF, the low detection limit of this ELISA is 100ng. There were two hybridoma cell lines selected by this novel ELISA method. Cell line 4D2G4A10 secreted antibody was confirmed by TLC-immunostaining method with native PAF, the secreted antibody could detect native PAF up to 300ng. Guinea pig PAF-Acetylhydrolase(PAF-AH) was purified by columns separation to a near homogenity enzyme(M.W. 63 KDa). This purified PAF-AH is different to phospholipase A2 (PLA2; M.W. 16.7 KDa), this PAF-AH can be activated by 10mM calcium ion but not inhibited by 10 mM EDTA. An ultraspeed centrifuge method by [14C]cholesterol as standard, this PAF-AH shows its another different character to the other known PAF-AH, by not conjugated with low density lipoprotein.

並列關鍵字

Antibody ； Platelet-Activating Factor ； Platelet-Activating