A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies

Jian Zhang, Noboru Fujimoto, Kazushi Iwata, Tomoe Sakai, Yasunori Okada, Taro Hayakawa

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

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 0.12μg/1 with the linearity between 0.12 and 10μg/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 ∼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 μg/l (mean ± S.D.) and the levels of 95% of the samples range from 0 to 20 μg/l.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalClinica Chimica Acta
Volume219
Issue number1-2
DOIs
Publication statusPublished - 1993 Oct 15
Externally publishedYes

Keywords

  • Interstitial collagenase
  • Monoclonal antibody
  • Sandwich enzyme immunoassay
  • Tissue inhibitors of metalloproteinases

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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