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

Clin Chim Acta. 1993 Oct 29;220(1):31-45. doi: 10.1016/0009-8981(93)90004-n.

Abstract

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 horse-radish 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 microgram/l (16 pg/assay) and linearity was obtained between 6.3 and 50 micrograms/l (63-500 pg/assay). TIMP-2 levels in the sera of 20 patients with rheumatoid arthritis (68 +/- 25 micrograms/l, mean +/- S.D.) and 13 patients with hepatocellular carcinoma (76 +/- 46 micrograms/l) were significantly higher (P < 0.05) than those of 18 normal subjects (5.6 +/- 7.4 micrograms/l). In contrast, the levels in the sera of 10 patients with gastric cancer (45 +/- 18 micrograms/l) and 7 patients with cancer of the uterus (36 +/- 13 micrograms/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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal*
  • Cells, Cultured
  • Chromatography, Gel
  • Female
  • Humans
  • Immunoenzyme Techniques*
  • Metalloendopeptidases / antagonists & inhibitors*
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Placenta / chemistry
  • Pregnancy
  • Proteins / analysis*
  • Proteins / isolation & purification
  • Sensitivity and Specificity
  • Tissue Inhibitor of Metalloproteinase-2

Substances

  • Antibodies, Monoclonal
  • Proteins
  • Tissue Inhibitor of Metalloproteinase-2
  • Metalloendopeptidases