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
中文摘要
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。
英文摘要
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.
