TIMP-1 ELISA检测方法的建立及临床初步应用

Development and application of enzyme-linked immunosorbent assay for tissue inhibitor of metalloproteinases-1(TIMP-1)

目的建立一种检测人血清中基质金属蛋白酶组织抑制剂-1(TIMP-1)的酶联免疫吸附方法(ELISA),用于血清学诊断人肝纤维化病程进展。方法以抗人TIMP-1单克隆抗体(McAb)为包被抗体,15%小牛血清为封闭液,和辣根过氧化物酶(HRP)标记的抗TIMP-1 McAb作为标记抗体建立检测人血清TIMP-1 ELISA法。并用于临床初步检测408例肝病患者血清。结果实验理想的包被抗体浓度为1μg/ml,血清稀释倍数1∶100,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3 ng/ml,中和抑制率在85%以上,稳定性能好,与国外TIMP-1 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=0.0085+0.9892X,r=0.988)。临床检测表明TIMP-1随着病损纤维化程度加重而升高,与正常人血清TIMP-1水平相比差异显著(P<0.01)。结论该法可用于肝纤维化进展定量血清学诊断。同时对影响因素的判定进行了初步的摸索研究,结果可靠。为国内TIMPs诊断试剂盒的研制和开发提供了科学实验依据。

Obhective To establish an enzyme-linked immunosorbent assay （ELISA） for tissue inhibitor of metalloprotelnases-1 （TIMP-1） in human serum,applied to the serological diagnosis of the progression of liver fibmsis.Mefltods Preeoating microphte with anfi-TIMP-1 McAb, confining liquid with 15 % calf serum, detecting method includes anfi-TIMP-1 McAb with HRP, and was used in the detection of 408 hepatitis patients. Results Experiment showed that the optimal concentration of the coating antibody, diluted ratio of serum and McAb with HRP were 1 μg/ml, 1 ： 100 and 1：400,respecfivdy.This assay had good reproducibility and steadiness, the sensitivity reached 3 ng/ml, and neutralizing inhibition ratio over 85%. Compared with foreign TIMP-1 ELISA kit, the relative coefficient（r） was 0.988, Y = 0.0085 ＋ 0.9892X. Clinical detecting showed, the severer the hepatic fibrosis in the injured liver is, the higher the levels of TIMP-1 are. The serum TIMP-1 level in hepatitis was significantly higher that in healthy adults（P 〈 0.01）. Condusion The ELISA can be used for the quantitative serological diagnosis of the progression of liver fibrosis,and was detected TIMP- 1 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-1 kit.