α2-巨球蛋白活性检测方法的建立与优化

Establishment and optimization of α2-macroglobulin activity detection assay

目的建立α2-巨球蛋白(α2-macroglobulin,α2-M)活性检测方法,检测Cohn组分Ⅳ中α2-M的活性。方法α2-M可与胰蛋白酶相互作用形成"α2-M-胰蛋白酶复合物",利用酶标仪检测出"α2-M-胰蛋白酶复合物"与小分子底物Na-苯甲酰-DL-精氨酸-对硝基酰胺盐酸盐(Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride,BAPNA)显色反应后的光密度值(410 nm),根据建立的血浆α2-M活性标准曲线,定量计算出Cohn组分Ⅳ浓缩液中α2-M的活性。结果通过对实验体系中几个关键实验条件进行优化,建立了血浆α2-M活性标准曲线,并计算出Cohn组分Ⅳ浓缩液中α2-M活性的平均值为1.578 PU/ml。结论以正常人混合血浆作为参考标准品,用发色底物法检测Cohn组分Ⅳ浓缩液中α2-M的活性,方法简便易行,为α2-M制备过程中活性测定提供了实验基础。

Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction IV. Methods α2-M reacted with trypsin to form ＂ α2-M-trypsin complex＂. After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride（BAPNA） was added, absorption at 410 nm was detected with the microplate reader, α2-M activity in Cohn fraction IV was quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized. A standard curve of plasma α2-M activity was established. According to this standard curve, α2-M activity in Cohn fraction IV sample was detected to be 1. 578 PU/ml. Conclusion Using normal human plasma as the reference material, the α2-M activity in Cohn fractioniv can be detected through chromogenic substrate assay. This study provides a simple method to detect α2-M activity during the purification process of α2- M from Cohn fraction Ⅳ.