Studies on Arachidonate Oxygenases of Human Platelets

制备了抗人血小板脂肪酸环氧合酶和花生四烯酸12-脂氧合酶的单克隆抗体。用这些酶的部分纯化制剂作为抗原腹腔内免疫小鼠。克隆产生抗环氧合酶单克隆抗体的杂交瘤细胞株，在小鼠腹腔内培养，从腹水中纯化抗体，制备IgG，利用两种单克隆抗体分别与环氧合酶蛋白的不同表位结合，建立了环氧合酶的酶免疫分析法。其中一种抗体用胃蛋白酶消化，其Fab'片段与辣根过氧化物酶缀合。环氧合酶和过氧化物酶标记的Fab'复合物与另一种抗体结合在S.金黄色。免疫沉淀物中的过氧化物酶活性与测定混合物中存在的环氧合酶的量线性相关。环氧合酶的这种酶免疫测定比通过放射性花生四烯酸转化为前列腺素（PG）H2评估的酶测定灵敏得多。本文报道了1例环氧化酶异常的临床病例，并建立了人血小板花生四烯酸12-脂氧合酶的酶免疫分析方法。血小板环氧合酶活性（花生四烯酸=> PGH_2）明显降低，而血栓素合成酶活性（PGF_2 =>血栓素A_2）则处于正常水平。应用上述建立的酶免疫法测定血小板环氧合酶水平，检测到患者血小板中环氧合酶的正常量。结果表明，异常环氧合酶蛋白存在于患者的血小板，这是催化失活，但免疫学上无法区分的正常酶。

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.