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 enzyme-linked 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 alpha (hrIL-1 alpha). Total TIMP-1 was also measured in serum samples with known C-reactive protein (CRP) (n = 100) and alpha 1 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.
Subject(s)
C-Reactive Protein/analysis , Glycoproteins/analysis , Metalloendopeptidases/analysis , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/biosynthesis , Biotin , C-Reactive Protein/cerebrospinal fluid , Cells, Cultured , Cross Reactions , Culture Techniques , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix Proteins/analysis , Fibroblasts/enzymology , Glycoproteins/blood , Glycoproteins/cerebrospinal fluid , Humans , Immunoblotting , Metalloendopeptidases/blood , Metalloendopeptidases/cerebrospinal fluid , Synovial Fluid/enzymology , Tissue Inhibitor of Metalloproteinases , Tumor Cells, Cultured , alpha 1-Antichymotrypsin/analysis , alpha 1-Antichymotrypsin/bloodABSTRACT
IL1-stimulated pig articular cartilage fragments were cultured in the and absence of various metalloproteinase inhibitors. Tissue inhibitor of metalloproteinases (TIMP) was unable to stop the release of proteoglycan from the cartilage. Incubation of cartilage with a potent synthetic metalloproteinase inhibitor inhibited the release of proteoglycan in a dose-dependent fashion. The results suggest that low-M(r) metalloproteinase inhibitors may have therapeutic potential in limiting connective tissue breakdown in conditions such as rheumatoid arthritis.
Subject(s)
Cartilage, Articular/drug effects , Glycoproteins/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Proteoglycans/metabolism , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , In Vitro Techniques , Interleukin-1/pharmacology , Protease Inhibitors/pharmacology , Swine , Tissue Inhibitor of MetalloproteinasesABSTRACT
The production of collagenase by human articular chondrocytes in response to interleukin-1 beta is inhibited in a dose-dependent manner by interferon-gamma (1-1,000 units/ml). The analysis of culture medium samples by Western blotting and the measurement of levels of tissue inhibitor of metalloproteinases suggest that the decrease in measurable collagenase activity is primarily due to the inhibition of procollagenase production. These results provide evidence of a role for interferon-gamma in limiting connective tissue degradation.
