Elsevier

Clinica Chimica Acta

Volume 240, Issue 2, 15 September 1995, Pages 137-154
Clinica Chimica Acta

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

https://doi.org/10.1016/0009-8981(95)06137-7Get rights and content

Abstract

A panel of six monoclonal antibodies (MAbs) was raised against purified human fibroblast tissue inhibitor of metalloproteinase-1 (TIMP-1) and characterised. All possible antibody pairs were tested for their suitability as capture and revealing antibodies in a two-site enzymelinked immunosorbent assay (ELISA) to measure total TIMP-1 (both free TIMP-1 and TIMP-1 together with matrix metalloproteinases (MMPs)). Using the best combination of MAbs the assay was optimised. The sensitivity of detection of the assay was 1.4 ng/ml, and inter- and intra-assay coefficients of variation were between 10.4–13.7% and 8.8–9.7%, respectively. Dilution series of human cerebrospinal and synovial fluids, plasma and sera paralleled those of the TIMP-1 standard curve indicating that 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 and collagenase were measured in conditioned medium from A2058 human melanoma cells cultured in the absence or presence of human recombinant interleukin-1 α (hrlL-lα). Total TIMP-1 was also measured in serum samples with known C-reactive protein (CRP) (n = 100) and α1 antichymotrypsin (ACT) (n = 52) concentrations; no correlation was found between TIMP-1 levels and either of these acute phase reactants although the levels of TIMP-1 were raised when compared to normal sera. This ELISA provides a rapid and convenient procedure for the quantitation of total TIMP-1 in human biological fluids and supernatants from cultured cell lines.

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