A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies

Noboru Fujimoto, Nobuko Mouri, Kazushi Iwata, Eiko Ohuchi, Yasunori Okada, Taro Hayakawa

Research output: Contribution to journalArticle

170 Citations (Scopus)

Abstract

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 μg/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 μg/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 ± 236gmg/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 ± 118 gmg/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 ± 72 gmg/l), 45 with rheumatoid arthritis (408 ± 139 gmg/l), 13 with stomach cancer (427 ± 103 gmg/l) and 10 with pancreatic cancer (422 ± 130 gmg/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.

Original languageEnglish
Pages (from-to)91-103
Number of pages13
JournalClinica Chimica Acta
Volume221
Issue number1-2
DOIs
Publication statusPublished - 1993 Nov 30

Fingerprint

Matrix Metalloproteinase 2
Collagenases
Matrix Metalloproteinases
Immunoenzyme Techniques
Monoclonal Antibodies
Enzymes
Serum
Tissue Inhibitor of Metalloproteinase-2
Assays
Biliary Liver Cirrhosis
Hyperthyroidism
Fibroblasts
Conditioned Culture Medium
human MMP2 protein
Pancreatic Neoplasms
Osteoarthritis
Peroxidase
Stomach Neoplasms
Gel Chromatography
Hepatocellular Carcinoma

Keywords

  • Matrix metalloproteinase 2
  • Monoclonal antibody
  • Sandwich enzyme immunoassay
  • Tissue inhibitor of metalloproteinase 2

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry

Cite this

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies. / Fujimoto, Noboru; Mouri, Nobuko; Iwata, Kazushi; Ohuchi, Eiko; Okada, Yasunori; Hayakawa, Taro.

In: Clinica Chimica Acta, Vol. 221, No. 1-2, 30.11.1993, p. 91-103.

Research output: Contribution to journalArticle

Fujimoto, Noboru ; Mouri, Nobuko ; Iwata, Kazushi ; Ohuchi, Eiko ; Okada, Yasunori ; Hayakawa, Taro. / A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies. In: Clinica Chimica Acta. 1993 ; Vol. 221, No. 1-2. pp. 91-103.
@article{b65a79539bbf46bba4f27b0dcc3db30f,
title = "A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies",
abstract = "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 μg/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 μg/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 ± 236gmg/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 ± 118 gmg/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 ± 72 gmg/l), 45 with rheumatoid arthritis (408 ± 139 gmg/l), 13 with stomach cancer (427 ± 103 gmg/l) and 10 with pancreatic cancer (422 ± 130 gmg/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.",
keywords = "Matrix metalloproteinase 2, Monoclonal antibody, Sandwich enzyme immunoassay, Tissue inhibitor of metalloproteinase 2",
author = "Noboru Fujimoto and Nobuko Mouri and Kazushi Iwata and Eiko Ohuchi and Yasunori Okada and Taro Hayakawa",
year = "1993",
month = "11",
day = "30",
doi = "10.1016/0009-8981(93)90024-X",
language = "English",
volume = "221",
pages = "91--103",
journal = "Clinica Chimica Acta",
issn = "0009-8981",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies

AU - Fujimoto, Noboru

AU - Mouri, Nobuko

AU - Iwata, Kazushi

AU - Ohuchi, Eiko

AU - Okada, Yasunori

AU - Hayakawa, Taro

PY - 1993/11/30

Y1 - 1993/11/30

N2 - 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 μg/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 μg/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 ± 236gmg/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 ± 118 gmg/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 ± 72 gmg/l), 45 with rheumatoid arthritis (408 ± 139 gmg/l), 13 with stomach cancer (427 ± 103 gmg/l) and 10 with pancreatic cancer (422 ± 130 gmg/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.

AB - 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 μg/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 μg/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 ± 236gmg/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 ± 118 gmg/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 ± 72 gmg/l), 45 with rheumatoid arthritis (408 ± 139 gmg/l), 13 with stomach cancer (427 ± 103 gmg/l) and 10 with pancreatic cancer (422 ± 130 gmg/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.

KW - Matrix metalloproteinase 2

KW - Monoclonal antibody

KW - Sandwich enzyme immunoassay

KW - Tissue inhibitor of metalloproteinase 2

UR - http://www.scopus.com/inward/record.url?scp=0027723123&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027723123&partnerID=8YFLogxK

U2 - 10.1016/0009-8981(93)90024-X

DO - 10.1016/0009-8981(93)90024-X

M3 - Article

C2 - 8149645

AN - SCOPUS:0027723123

VL - 221

SP - 91

EP - 103

JO - Clinica Chimica Acta

JF - Clinica Chimica Acta

SN - 0009-8981

IS - 1-2

ER -