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从人血浆和人尿中纯化了蛋白C系统各组分及抗凝血酶Ⅲ。免疫家兔产生了抗血清,氯胺T法,Iodogen法和Bolton-Hunter法制备了碘标记化合物。采用平衡法建立了放射免疫分析方法。所有方法的最低可测限均小于10μg/L,回收率在94.30％～105.22％之间,未见与因子Ⅱ和凝血酶的交叉反应。采用所建的放射免疫分析法和流式细胞技术分析了蛋白C系统在内皮细胞表面的功能表达和调节。建立了简单、可靠,适于分析APC耐性的APC-APTT方法和可明确因子Ⅴ纯合子和杂合子G1691A点突变的聚合酶链反应方法。结果表明,中国和其他地区的黄种人因子Ⅴ G1691A点突变发生率显著低于欧洲白种人,而APC耐性并不低。提示中国等黄种人群有独立于因子Ⅴ G1691A点突变以外的因子Ⅴ或因子Ⅷ突变点存在的可能或存在其他致APC耐性因素等。
Purification of components of protein C system and antithrombin III from human plasma and human urine. Antisera were produced in rabbits immunized with iodine-labeled compounds by chloramine T method, Iodogen method and Bolton-Hunter method. Balance method was used to establish a radioimmunoassay method. The minimum detectable limits of all the methods were less than 10μg / L and the recoveries ranged from 94.30% to 105.22%. No cross-reaction with factor Ⅱ and thrombin was found. Using the established radioimmunoassay and flow cytometry, we analyzed the functional expression and regulation of protein C system on the surface of endothelial cells. A simple and reliable APC-APTT method which is suitable for analyzing APC tolerance and a polymerase chain reaction method which can confirm the point mutation of factor Ⅴ homozygote and heterozygous G1691A were established. The results showed that in China and other regions, the incidence of point mutation of G1691A was significantly lower than that of European Caucasians, while APC tolerance was not low. This suggests that other yellow populations in China may have or may have other APC tolerance factors that are independent of factor V or factor VIII mutations beyond the point mutation of factor G1691A.