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 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.
Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge, UK.
T A
TA
Plumpton
I M
IM
Clark
C
C
Plumpton
J
J
Calvin
T E
TE
Cawston
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis; D001710:Biotin; D002097:C-Reactive Protein / Q000032:analysis* / Q000134:cerebrospinal fluid; D002478:Cells, Cultured; D003429:Cross Reactions; D046508:Culture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D006801:Humans; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D013582:Synovial Fluid / Q000201:enzymology; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured; D000514:alpha 1-Antichymotrypsin / Q000032:analysis / Q000097:blood
D000911:Antibodies, Monoclonal; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D000514:alpha 1-Antichymotrypsin; D001710:Biotin; D002097:C-Reactive Protein; D008666:Metalloendopeptidases