A ONE-STEP SANDWICH ENZYME-IMMUNOASSAY FOR HUMAN MATRIX METALLOPROTEINASE 2 (72-KDA GELATINASE TYPE-IV COLLAGENASE) USING MONOCLONAL-ANTIBODIES

A one-step sandwich enzyme immunoassay (EIA) for human matrix metallop roteinase 2 (MMP-2, 72-kDa gelatinase/type IV collagenase, EC 3.4.24.2 4) was established with a pair of monoclonal antibodies prepared again st the precursor form of MMP-2 (proMMP-2) purified from the conditione d medium of human skin fibroblasts or against a synthetic peptide corr esponding to the N-terminal domain of proMMP-2. ProMMP-2 in samples wa s allowed to simultaneously react with both solid-phase and peroxidase -labeled antibodies. Sensitivity of this EIA system was 2.4 pg/assay ( 0.24 mu g/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 mu g/l). The EIA system recognized both the free form of proM MP-2 and its complex form with TIMP-2 with the same degree of immunore activity. ProMMP-2 levels in human sera from patients in various disea se states were analyzed. In sera from patients with hyperthyroidism (1 2), primary biliary cirrhosis (8) and hepatocellular carcinoma (11), 7 49 +/- 166, 716 +/- 135 and 686 +/- 236 mu g/l of proMMP-2 were detect ed, respectively and these were significantly higher than that observe d in 213 normal human sera (570 +/- 118 mu g/l). In contrast, the leve ls in sera from 33 patients with osteoarthritis (449 +/- 72 mu g/l), 4 5 with rheumatoid arthritis (408 +/- 139 mu g/l), 13 with stomach canc er (427 +/- 103 mu g/l) and 10 with pancreatic cancer (422 +/- 130 mu g/l) were significantly lower than that found in normal sera. Immunobl ot and gel filtration analyses showed that human sera contain several MMP-2 species in addition to proMMP-2 which exist in a complex form wi th TIMP-2.