中文摘要  以

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

 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 300 ng 。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 P AF-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。

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



英文摘要



1-O-Carboxyl-nonyl-2-O-acetyl-sn-glycerol-3-phosphocho- line-Bovine Serum Alb

umin(C10 acetyl F1-BSA) was used as an antigen to raise antibody to Platelet-Acti

vating Factor (PAF) on BALB/c mice, and prepared hybridoma by fusing lympho-

cyte cells with myeloma. A modified ELISA method enable the screening of antibo

dy to PAF but lyso PAF, the low detection limit of this ELISA is 100ng. There wer

e two hybridoma cell lines selected by this novel ELISA method. Cell line 4D2G4

A10 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 homogenit

y 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]cholester

ol as standard, this PAF-AH shows its another different character to the other know

n PAF-AH, by not conjugated with low density lipoprotein.