Studies on Arachidonate Oxygenases of Human Platelets

制备了抗人血小板脂肪酸环加氧酶和花生四烯酸12-脂加氧酶单克隆抗体。用这些酶的部分纯化制剂作为抗原腹腔免疫小鼠。克隆产生单克隆抗体的杂交瘤罐，在小鼠腹腔培养，从苦行液中纯化抗体为IgG。利用结合环加氧酶蛋白不同表位的两种单克隆抗体，建立了环加氧酶的酶免疫测定方法。其中一种抗体经胃蛋白酶酶切，其Fab'片段与辣根过氧化物酶结合。在金黄色葡萄球菌表面与蛋白A结合的另一抗体沉淀出环加氧酶和过氧化物酶标记的Fab'复合物。免疫沉淀物中的过氧化物酶活性与测定混合物中环氧化酶的含量呈线性相关。用放射性花生四烯酸转化前列腺素（PG） H2的酶法测定环加氧酶的灵敏度要高得多。基本上相同的酶免疫分析法开发了花生四烯酸12-脂氧合酶的人血小板。本文报道了一例环加氧酶异常的临床病例。血小板环加氧酶活性（花生四烯酸=> PGH_2）明显降低，血栓素合成酶活性（血小板四烯酸=>血栓素A_2）维持正常水平。将上述建立的酶免疫分析法应用于本病例，测定血小板环加氧酶水平，检测到患者血小板环加氧酶水平正常。结果表明，患者血小板中存在异常的环加氧酶蛋白，该蛋白催化活性不强，但在免疫上与正常酶难以区分。

Monocloral antibodies against fatty acid cyclooxygenase and arachidonate 12-lipoxygenase of human platelets were prepared. Mice were immunized intraperitoneally with the partially purified preparations of these enzymes as antigens. The hybridoma cans producing monoclonal antibodies were cloned and grown in the abdominal cavity of mice, and the antibodies were purified to IgG from the ascetic fluid.An enzyme immurioassay for cyclooxygenase was established utilizing two species of monoclonal antibody which bound to different epitopes of the enzyme protein. One of the antibodies was digested with pepsin, and its Fab' fragment was conjugated to horseradish peroxidase. The complex of cyclooxygenase and peroxidase-labeled Fab' was precipitated with the other antibody which had been bound to protein A on the surface of S. aureus. The peroxidase activity in the immunoprecipitate was correlated lineally to the amount of cyclooxygenase present in the assay mixture. This enzyme immunoassay for cyclooxygenase was much more sensitive than the enzyme assay as assessed by the conversion of radioactive arachidonic acid to prostaglandin (PG) H2. Essentially the same enzyme immunoassay was developed for arachidonate 12-lipoxygenase of human platelets.A clinical case of cyclooxygenase abnormality was reported previously. The cyclooxygenase activity (arachidonic acid => PGH_2) in platelets was markedly reduced, whereas thromboxane syntilase activity (PGF_2 => thromboxane A_2) was at normal level. The enzyme immunoassay established as above was applied to this case to determine the platelet cyclooxygenase level, and detected a normal amount of cyclooxygenase in the patient's platelets. The result indicated that the anomalous cyclooxygenase protein was present in patient's platelets, which was catalytically inactive but immunologically indistinguishable from the normal enzyme.