高等学校化学学报

高等学校化学学报

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人血清中cTnI的化学发光酶免疫分析研究——实验条件的优化

何农跃1, 郭会时1, 顾春荣2, 杨笛2, 张寄南2   

    1. 东南大学生物电子学国家重点实验室, 南京210096;
    2. 南京医科大学第一临床医学院心血管病研究所, 南京 210029
  • 收稿日期:2006-06-14 修回日期:1900-01-01 出版日期:2007-02-10 发布日期:2007-02-10
  • 通讯作者: 何农跃

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

HE Nong-Yue1, GUO Hui-Shi1, GU Chun-Rong2, YANG Di2, ZHANG JI-Nan2   

    1. State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China;
    2. Institute of Cardiovascular Disease, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China
  • Received:2006-06-14 Revised:1900-01-01 Online:2007-02-10 Published:2007-02-10
  • Contact: HE Nong-Yue

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被引次数

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摘要： 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10.0 μg/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BSA pH=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-IgG2)以及碱性磷酸酶-亲和素(ALP-Avidin)结合物均采用1:2000稀释度, 免疫反应条件为37 ℃, 孵育时间60 min, 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10 min(37 ℃). 检出限为0.02 ng/mL, 比现行ELISA法灵敏度提高一个数量级; 测定周期约75 min, 比两步法ELISA快得多; 线性范围(0.04~36.20 ng/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).

关键词: 化学发光免疫分析, 急性心肌梗死, AMPPD, 心肌肌钙蛋白(cTnI)

Abstract: Chemiluminescent immunoassays(CLIA) were intensively studied in antigen/antibody detection, due to the extremely sensitive chemiluminescent detection techniques. Here we present a sensitive immuno-assay 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 immuno-reaction mode which decreased the turnaround time greatly. The second modification is that a chemiluminescent substrate(AMPPD) and chemiluminescent detection techniques, which elevated the sensitivity remar-kably, 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.

Key words: Chemiluminescent immunoassay, Acute myocardial infarction, AMPPD, Cardiac troponin I(cTnI)

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