摘要
目的建立定量检测人血清中金属蛋白酶抑制剂-I（TIMPI）的微孔板化学发光酶免疫分析方法。方法以辣根过氧化物酶标记TIMPI单抗，以过氧化氢（H2O2）一鲁米诺（1uminol）化学发光体系作为检测体系，采用双抗体夹心反应模式，优化筛选温育条件、抗体包被条件、酶标记物稀释度和发光反应时间等指标。同时进行了回收实验，热稳定性实验，并随机选取60例肝纤维化患者血清与进口试剂进行比对实验。结果所建立方法的线性范围为0．2—12rig／ml，相关系数0．996，检出限为0．12ng／ml，批内、批间变异均小于10％。检测TIMPI的临床高、中、低值血清回收率为分别为100．6％、96．5％和106．5％；在4℃和37℃条件下分别进行了3、5、7d的稳定性考察，线性相关系数均〉0．98，标准偏差〈6％；比对实验分析证明2种方法相关性具有统计学意义。结论成功建立定量TIMPI微孔板化学发光酶免疫分析方法，该方法具有较高的准确性、灵敏度及良好的重复性，并与进口试剂检测结果一致。
Objective To establish microplate chemiluminescence enzyme immunoassay （CLEIA） for quantitative analysis of tissue inhibitor of metalloproteinases I （TIMP I） in human serum. Methods A sandwich reaction was preformed with horseradish peroxidasc（HRP） labeled monoclonal antibody of TIMP I as the catalytic enzyme and the H202-1uminol as the luminescence reagent. Several physical and chemical parameters were studied and optimized such as immunoreaction conditions, the dilution ratio of TIMP I- HRP, luminescence reaction time and so on. In order to evaluate the method, recovery test, heat stabilization test and comparison test were carried out. Results The linear range was 0. 2-12 ng/ml with r =0. 996. The detection limit was 0. 12 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 100. 6% , 96. 5% and 106.5%. After stored at 4℃ and 37℃ for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0. 998 and RSD lower than 6%. The detected results with CLEIA closely corresponded to those with imported ELISA in 60 patients sera with liver fibrosis. Conclusion Established CLEIA for quantity determination of serum TIMP I has high accuracy, sensitivity and repeatability.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2013年第4期298-300,共3页
Chinese Journal of Experimental and Clinical Virology
基金
解放军三0二医院重点课题（YNKT2011010）
关键词
基质金属蛋白酶
化学发光
免疫酶技术
肝硬化
Matrix metalloproteinases
Chemiluminescence
Immunoenzyme techniques
Liver cirrhosis
