Microplate chemiluminescence enzyme immunoassay for the quantitative analysis of tissue inhibitor of metalloproteinases I

Abstract

To establish microplate chemiluminescence enzyme immunoassay (CLEIA) for quantitative analysis of tissue inhibitor of metalloproteinases I (TIMP I) in human serum. A sandwich reaction was preformed with horseradish peroxidase(HRP) labeled monoclonal antibody of TIMP I as the catalytic enzyme and the H2O2-luminol 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. 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 degrees C and 37 degrees C 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. Established CLEIA for quantity determination of serum TIMP I has high accuracy, sensitivity and repeatability.