A One‐Step Sandwich Enzyme Immunoassay for Human Matrix Metalloproteinase 9 Using Monoclonal Antibodies

Abstract

Matrix metalloproteinase 9 (MMP-9) has been implicated in various pathophysiologic conditions such as cancer invasion and and bone re~orption.~ To study the release and function of MMP-9 in body fluid and tissue, we established a one-step sandwich enzyme immunoassay (EIA) system for MMP-9 and applied it to measure MMP-9 in human plasma from normal subjects and patients with disease.ProMMP-9 was purified from the conditioned medium of HT1080 cells stimulated with tumor necrosis factor-alpha as previously described? Monoclonal antibodies for MMP-9 were prepared against the purified proMMP-9. A monoclonal antibody for TIMP-1 from bovine dental pulp is capable of cross-reacting with human TIMP-1.6 A one-step sandwich EIA system for MMP-9 was developed using a combination of monoclonal antibodies (clones 73-18B3 and 73-74E11) as a solid phase and a peroxidase conjugate, respectively.The sensitivity of this EIA for standard proMMP-9 was 2.4 pglassay (0.24 ng/mL), and linearity was obtained between 0.24 and 250 ng/mL (2.4-2,500 pg/ assay) (FIG. 1). The intra-assay coefficients of variation (CVs) of normal plasma having MMP-9 at a concentration of 35,104, and 109 ng/mL were 1.8-3.9% (n = 10). Inter-assay CVs were 1 . 8 4 2 % (n = 8) at plasma MMP-9 levels of 38, 47, and 114 ng/mL. Recovery of standard proMMP-9 added to human plasma having an MMP-9 concentration of 35,45, and 100 ng/mL over a range of 12.5-200 ng/mL was 103 C 7.6% (mean rf: SD). This EIA system recognized proMMP-9, the intermediate form with M , = 83 kDa and their complexes formed with TIMP-I.The concentration of MMP-9 in normal human sera by our ElA was 346 c 197 ng/mL. MMP-9 levels in human sera increased with time after venipuncture. Therefore, our one-step sandwich EIA technique was applied to evaluate MMP-9

Aspirin suppresses TNF‑α‑induced MMP‑9 expression via NF‑κB and MAPK signaling pathways in RAW264.7 cells

“…Increasing evidence indicates that the concentrations of MMPs are elevated not only in certain patients with cancer, liver cirrhosis or rheumatoid arthritis (7,8), but is also involved in the pathogenesis and progression of atherosclerotic plaque formation and rupture (9). Among the MMP family, MMP-9 (also known as gelatinase B) is the predominant MMP and can degrade type IV collagen, which is the major component of the basement membrane (10).…”

Elevation of Serum Levels of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-1 and Type IV Collagen, and Plasma Levels of Metalloproteinase-9 in Polycystic Kidney Disease

“…To measure the concentration of MMP-9 in plasma, a one-step sandwich enzyme immunoassay using two monoclonal antibodies (clone 73-74E11 and 73-18B3) was performed as previously reported [9,10]. The sensitivity of the assay was 0.24 ng/ml (2.2 pg/assay), and linearity was obtained from 0.24 to 250 ng/ml (2.2-2,280 pg/assay).…”

Quantification of matrix metalloproteinase activity in plasma of patients enrolled in a BAY 12-9566 phase I study

“…69,70 This may also explain why the MMP-9 activity in serum increases with time after venipuncture. 71 For the collection of blood we used lithium heparin tubes that have been shown to provide the most consistent results for MMP determination among the different tubes available. 72 No relationships between the plasma BAY 12-9566 concentration at day 15 of treatment and the percent change in MMP activity could be established.…”