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本文用自己提纯的K型丙酮酸激酶(K-Pyk)和兔抗K-Pyk血清(Ab_1),设计与建立了K-Pyk的放射免疫测定法(RIA)。用乳汁过氧化物酶法标记K-Pyk,经SePhadexG-25柱层析制备高比放射性~(125)I-K-Pyk(K-Pyk),免疫活性良好。双抗体法分离抗体结合的和游离的K-Pyk。其反应体系:牛血清白蛋白(BSA)稀释度为6％,最终工作浓度6/500;Ab_1的稀释度为1/2000,最终工作浓度为1:10000;选择总计数为2000CPm/分左右的K-Pyk;最佳孵育pH7.0;孵育时间为24小时;反应体系中加入不稀释的第二抗体(Ab_2)和1:10的兔正常抗血清,沉淀反应良好。竞争抑制曲线的测定范围为40～500ng。本法是灵敏、可靠和特异的。
In this paper, K-Pyk radioimmunoassay (RIA) was designed and established by using purified K-Pyk and rabbit anti-K-Pyk serum (Ab_1). K-Pyk was labeled with milk peroxidase (K-Pyk) and the radioactive 125-I-K-Pyk (K-Pyk) was prepared by Sephadex G-25 column chromatography. Antibody bound and free K-Pyk was isolated by the diabody method. The reaction system: bovine serum albumin (BSA) dilution of 6%, the final working concentration of 6/500; Ab_1 dilution of 1/2000, the final working concentration of 1: 10000; select a total count of about 2000CPm / min K-Pyk; optimal incubation pH7.0; Incubation time was 24 hours; reaction system was added with undiluted secondary antibody (Ab_2) and 1:10 rabbit normal antiserum, the precipitation reaction was good. The competitive inhibition curve ranged from 40 to 500 ng. This Law is Sensitive, Reliable and Specific.