单克隆抗体双抗体夹心ELISA快速检测肝炎患者血清中TIMP-2  

ELISA for rapid detection of TIMP-2 from serum samples in hepatitis patients with monoclonal antibody

作　　者：聂青和[1] 邵彬[1] 王平忠[1] 谢玉梅[1] 张亚飞[1] 苟艳子[1] 

机构地区：[1]第四军医大学唐都医院传染病科全军感染病诊疗中心,西安710038

出　　处：《胃肠病学和肝病学杂志》2005年第6期577-580,共4页Chinese Journal of Gastroenterology and Hepatology

基　　金：陕西省科技攻关项目(社发攻关No:2003K10G63)

摘　　要：目的建立一种检测人血清中基质金属蛋白酶组织抑制剂-2(TIMP-2)的双抗体夹心ELISA法,探讨血清TIMP-2的水平与肝纤维化病程进展程度的关系.方法确定包被TIMP-2单克隆抗体(McAb)和辣根过氧化物酶(HRP)标记的抗TIMP-2 McAb的最佳工作浓度,建立双抗体夹心ELISA方法.并用于临床初步检测408例肝炎血清TIMP-2水平.结果包被TIMP-2 McAb的最佳工作浓度为2μg/ml,HRP标抗体的最佳工作浓度1:400.该法具有良好的重复性,灵敏度达5 ng/ml,中和抑制率在80%以上,稳定性能好,与国外TIMP-2 ELISA试剂盒进行对比试验,表明两种检测方法有良好的相关性(Y=1.2752X-4.7465,r=0.976).临床检测表明TIMP-2随着病损纤维化程度加重而升高,与正常人血清TIMP-2水平相比差异显著(P＜0.01～0.001).结论该法能够快速检测人血清中TIMP-2浓度,血清TIMP-2可作为定量诊断肝纤维化指标之一.本试验为国内TIMP-2诊断试剂盒的研制和开发提供了科学实验依据.Objective To establish an enzyme-linked immunosorbent assay （ELISA） for tissue inhibitor of metalloproteinases-2（TIMP-2） in human serum, applied to the serological diagnosis of the progression of liver fibrosis. Methods Precoatingmicroplate with anti-TIMP-2 McAb, confining liquid with 15% calf serum, detecting method includes anti-TIMP-2 McAb with HRP, and was used in the detection of 408 hepatitis patients. Result Experiment showed that the optimal concentration of the coating antibody, diluted ratio of serum and McAb with HRP were 2μg/ml, 1：100 and 1：400, respectively. This assay had good reproducibility and steadiness, the sensitivity reached 5ng/ml, and neutralizing inhibition ratio over 80%. Compared with foreign TIMP-2 ELISA kit, the relative coefficient（r） was r = 0.976, Y = 1. 2752X-4.7465. Clinical detecting showed, the severer the hepatic fibrosis in the injured liver is, the higher the levels of TIMP-2 are. The serum TIMP-2 level in hepatitis was significantly higher than that in healthy adults（ P 〈 0.01 -0.001 ）. Conclusion The ELISA can be used for the quantitative serological diagnosis of the progression of liver fibrosis, and was detected TIMP-2 of serum in the nation, and explored the assessment of the affecting factors. The result presented in the assay are reliable and will provide scientific evidence for designing and developing national TIMP-2 kit.

关 键 词：肝纤维化 基质金属蛋白酶组织抑制剂 酶联免疫吸附法 血清学诊断 快速检测 

分 类 号：R575.1[医药卫生—消化系统]