心肌营养素-1 ELISA测定法的建立及初步应用

A biotin-streptavidin-enzyme-linked immunosorbent assay for detection of human cardiotrophin-1

目的应用生物素链霉亲和素酶联免疫吸附法(BSA ELISA)建立血浆心肌营养素1(CT1)测定法,并初步应用于冠心病患者血浆CT1水平的检测。方法用鼠抗重组人CT1单抗包被酶标板,加入待测抗原,并依次加入生物素化羊抗重组人CT1多克隆抗体、辣根过氧化物酶标记链霉亲合素,用H2O2邻苯二胺显色。检测35名冠心病患者及对照组血浆CT1含量的变化。结果方法的线性范围为10～2000pg/ml,检测下限为10pg/ml,批内变异系数为1.76%～7.64%,批间变异系数为5.96%～9.04%。结论该方法简便、快速,特异性、敏感性、重复性均在临床检测可接受范围内,标准曲线线性良好,适用于基础研究与临床检验。

Objective To develop a biotin-streptavidin-enzyme-linked immunosorbent assay (BSA-ELISA) for detection of human cardiotrophin-1 (CT-1) in plasma and investigate its preliminary application in the patients with coronary heart disease.Methods The main steps of the BSA-ELISA were as follows:96 well plates were coated with specific mouse IgG1 antibodies to human CT-1.After adding CT-1 standards or plasma samples,biotinylated specific goat IgG antibodies to human CT-1 were used as the second antibody.Streptavidin-HRP-IgG solution was then added and the colour was revealed with OPD.According to the standard curves,concentration of CT-1 in sample was determined.The plasma CT-1 levels in 35 patients with coronary heart disease and controls were measured.Results The working range of standard curve was 10～2 000 pg/ml,and the minimum detectable limit of the assay was 10 pg/ml.Variance coefficient within group was 1.01%～7.64% and variance coefficient between groups was 5.96%～9.04%.Conclusion The BSA-ELISA for plasma CT-1 developed in this study is a specific,sensitive and convenient method which should be suitable for clinical measurement and basal research.