α2－巨球蛋白活性检测方法的建立与优化

摘要

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.Methodsα2-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 fractionⅣwas 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 Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.