A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids : Saliva from patients with Sjögren's syndrome contain increased latent and active gelatinase-B levels
article
Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys ↓ IlelleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly ↓ IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 ± 4.9 vs 12.2 ± 2.5 x 104 cpm/ml (p > 0.05, and 44.0 ( 4.0 vs 36.1 ± 1.9 x 104 cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 ± 2.5 U/mg, controls 1.0 ± 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 ± 9.8 U/mg, controls 16.5 ± 2.6 U/mg (p = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.
Topics
Activity assayGelatinase BMatrix metalloproteinases (MMP)Sjogren's syndromeUrokinaseAnimalsAntibodies, MonoclonalAntibody SpecificityCollagenasesFemaleHumansImmunoassayMatrix Metalloproteinase 9MiceMice, Inbred BALB CSalivaSensitivity and SpecificitySjogren's SyndromeTissue Inhibitor of Metalloproteinase-2Animalia
TNO Identifier
234719
ISSN
0945053X
Source
Matrix Biology, 17(8-9), pp. 657-665.
Pages
657-665
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