人血清中cTnI的化学发光酶免疫分析研究──实验条件的优化

Chemiluminescent Enzyme Immunoassay for Cardiac Troponin I Detection──Optimization of Experimental Parameters

采用高效化学发光试剂3-（2＇-螺旋金刚烷）-4-甲氧基-4-（3＇-羟基）苯-1，2-二氧杂环丁烷磷酸（AMPPD）作为检测底物，并将传统的ELISA两步双抗夹心法改为一步法，得到了高灵敏测定人血清中心肌肌钙蛋白I（cTnI）的化学发光酶免疫分析优化条件．采用单因素变化法和方阵滴定法得到的最佳实验条件为：捕获抗体包被浓度为10．00μg/ml，以pH=7．0的PBS作为免疫反应缓冲底液，以含质量分数为1．0％的BSApH=9．6的碳酸盐溶液缓冲液，于4℃封闭过夜，生物素-检测抗体（Biotin—IsG2）以及碱性磷酸酶．亲和素（AIJP—Avidin）结合物均采用1：2000稀释度，免疫反应条件为37℃，孵育时间60min，以去离子水作为洗涤剂，以1：100稀释的AMPPD作为发光反应底物，发光反应时间10min（37℃）．检出限为0．02ng／mL，比现行ELISA法灵敏度提高一个数量级；测定周期约75min，比两步法ELISA快得多；线性范围（0．04-36．20ng／mL）比ELISA法扩宽了两个数量级；加标回收率97．5％-102．8％，对标准样品的测定结果与用ELISA法的测定结果吻合；重复性好，3个样品批内变异系数均小于8．5％（n=12）．

Chemiluminescent immunoassays（CLIA） were intensively studied in antigen/antibody detection, due to the extremely sensitive chemiluminescent detection techniques. Here we present a sensitive immunoassay for cTnI based on this concept. The assay was executed as the traditional ELISA except that two major modifications were made to obtain a rapid test and a high sensitivity. The first modification is that the two-step dual monoclonal antibody ＂sandwich＂ principle of traditional ELISA was substituted by a one-step immunoreaction mode which decreased the turnaround time greatly. The second modification is that a chemiluminescent substrate （AMPPD） and chemiluminescent detection techniques, which elevated the sensitivity remarkably, were used to replace the colorimetric substrate and colorimetric detection method in ELISA. The detection procedure of the assay could be fulfilled within 75 min. A linear range between the luminescent signal current and the concentration of cTnI from 0. 04 to 36. 2 ng/mL and a detection limit of 0. 02 ng/mL were obtained. The established method was tested by determining cTnI in real samples by using ELISA for comparison analysis, and good results were obtained.