A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies.
Interstitial collagenase (EC 3.4.24.7, matrix metalloproteinase-1, MMP-1) is synthesized and secreted by many cells, and plays an important role in a wide variety of pathophysiological degradation processes of extracellular matrices. The activity of MMP-1 is regulated by tissue inhibitors of metalloproteinases, TIMP-1 or TIMP-2, which form a non-covalent complex with the active enzyme. We raised monoclonal antibodies against zymogen of MMP-1, proMMP-1 purified from human skin fibroblasts. The antibodies recognized both precursor and active forms of MMP-1, but did not cross-react with 72-kDa and 92-kDa gelatinase/type IV collagenases or stromelysin-1. A specific and sensitive one-step sandwich enzyme immunoassay for human MMP-1 was developed using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The assay can be completed within 1 h (30 min for immunoreaction and 15 min for color development) and the sensitivity is 0.12 microgram/l with the linearity between 0.12 and 10 micrograms/l. Active MMP-1 shows 1.3-fold higher absorption at 492 nm than proMMP-1. However, the recognition rate of MMP-1 is decreased to approximately 50% and < 3% for the MMP-1-TIMP-1 and MMP-1-TIMP-2 complex forms, respectively. The MMP-1 levels in human sera from 120 healthy subjects are shown to be in the range of 8.5 +/- 5.2 micrograms/l (mean +/- S.D.) and the levels of 95% of the samples range from 0 to 20 micrograms/l.
Zhang
J
J
Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Japan.
Fujimoto
N
N
Iwata
K
K
Sakai
T
T
Okada
Y
Y
Hayakawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D005109:Extracellular Matrix / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology; D008666:Metalloendopeptidases / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D009842:Oligopeptides; D008666:Metalloendopeptidases