DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY TO MEASURE TOTAL TIMP-1 (FREE TIMP-1 AND TIMP-1 IN COMBINATION WITH MATRIX-METALLOPROTEINASES) AND MEASUREMENT OF TIMP-1 AND CRP IN SERUM

A panel of six monoclonal antibodies (MAbs) was raised against purifie d human fibroblast tissue inhibitor of metalloproteinase-1 (TIMP-1) an d characterised. All possible antibody pairs were tested for their sui tability as capture and revealing antibodies in a two-site enzyme-link ed immunosorbent assay (ELISA) to measure total TIMP-1 (both free TIMP -1 and TIMP-1 together with matrix metalloproteinases (MMPs)). Using t he best combination of MAbs the assay was optimised, The sensitivity o f detection of the assay was 1.4 ng/ml, and inter- and intra-assay coe fficients of variation were between 10.4-13.7% and 8.8-9.7%, respectiv ely, Dilution series of human cerebrospinal and synovial fluids, plasm a and sera paralleled those of the TIMP-1 standard curve indicating th at the immunoreactivity detected in these samples was authentic TIMP-1 , TIMP-2 shows no detectable cross reactivity in this assay confirming that this ELISA is specific for TIMP-1. The levels of total TIMP-1 an d collagenase were measured in conditioned medium from A2058 human mel anoma cells cultured in the absence or presence of human recombinant i nterleukin-1 alpha (hrIL-1 alpha). Total TIMP-1 was also measured in s erum samples with known C-reactive protein (CRP) (n = 100) and alpha(1 ) antichymotrypsin (ACT) (n = 52) concentrations; no correlation was f ound between TIMP-1 levels and either of these acute phase reactants a lthough the levels of TIMP-1 were raised when compared to normal sera. This ELISA provides a rapid and convenient procedure for the quantita tion of total TIMP-1 in human biological fluids and supernatants from cultured cell lines.