A one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases-2 using monoclonal antibodies.
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.
Fujimoto
N
N
Department of Biochemistry, Fuji Chemical Industries, Ltd., Toyama, Japan.
Zhang
J
J
Iwata
K
K
Shinya
T
T
Okada
Y
Y
Hayakawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2
D000911:Antibodies, Monoclonal; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D008666:Metalloendopeptidases