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 metalloproteinase 2 (MMP-2, 72-kDa gelatinase/type IV collagenase, EC 3.4.24.24) was established with a pair of monoclonal antibodies prepared against the precursor form of MMP-2 (proMMP-2) purified from the conditioned medium of human skin fibroblasts or against a synthetic peptide corresponding to the N-terminal domain of proMMP-2. ProMMP-2 in samples was allowed to simultaneously react with both solid-phase and peroxidase-labeled antibodies. Sensitivity of this EIA system was 2.4 pg/assay (0.24 microgram/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 micrograms/l). The EIA system recognized both the free form of proMMP-2 and its complex form with TIMP-2 with the same degree of immunoreactivity. ProMMP-2 levels in human sera from patients in various disease states were analyzed. In sera from patients with hyperthyroidism (12), primary biliary cirrhosis (8) and hepatocellular carcinoma (11), 749 +/- 166, 716 +/- 135 and 686 +/- 236 micrograms/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 +/- 118 micrograms/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 +/- 72 micrograms/l), 45 with rheumatoid arthritis (408 +/- 139 micrograms/l), 13 with stomach cancer (427 +/- 103 micrograms/l) and 10 with pancreatic cancer (422 +/- 130 micrograms/l) were significantly lower than that found in normal sera. Immunoblot and gel filtration analyses showed that human sera contain several MMP-2 species in addition to proMMP-2 which exist in a complex form with TIMP-2.
Fujimoto
N
N
Department of Biochemistry, Fuji Chemical Industries, Ltd., Toyama, Japan.
Mouri
N
N
Iwata
K
K
Ohuchi
E
E
Okada
Y
Y
Hayakawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000201:enzymology; D005069:Evaluation Studies as Topic; D005260:Female; D018093:Gelatinases / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D006980:Hyperthyroidism / Q000201:enzymology; D007124:Immunoenzyme Techniques; D008105:Liver Cirrhosis, Biliary / Q000201:enzymology; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000097:blood* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000201:enzymology; D011498:Protein Precursors / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000276:immunology; D012015:Reference Standards; D019716:Tissue Inhibitor of Metalloproteinase-2
D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2