A one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases-2 using monoclonal antibodies

A one-step sandwich enzyme immunoassay system was developed with a pair of monoclonal antibodies against two individual oligopeptides prepared from the amino acid sequence of the human tissue inhibitor of metalloproteinases-2 (TIMP-2). The assay system consisting of two simultaneous immunoreactions used a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody. The system detected a free form of TIMP-2 and that complexed with active forms of matrix metalloproteinases (MMPs) giving a different sensitivity for each MMP but not TIMP-2 complexed with the precursor of 72 kDa gelatinase/type IV collagenase (MMP-2). The sensitivity of the system was 1.6 μg/l (16 pg/assay) and linearity was obtained between 6.3 and 50 μg/l (63-500 pg/assay). TIMP-2 levels in the sera of 20 patients with rheumatoid arthritis (68 ± 25 μgl, mean ± S.D.) and 13 patients with hepatocellular carcinoma (76 ± 46 μg/l) were significantly higher (P < 0.05) than those of 18 normal subjects (5.6 ± 7.4 μg/l). In contrast, the levels in the sera of 10 patients with gastric cancer (45 ± 18 μg/l) and 7 patients with cancer of the uterus (36 ± 13 μg/l) were significantly lower (P < 0.05 or P< 0.01) than those of normal subjects. Immunoreactivity analyses suggested that the precursor of MMP-2 in normal sera exists in a complexed form with TIMP-2 by interacting with the C-terminal domain of TIMP-2.