A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: Saliva from patients with Sjogren'ssyndrome contain increased latent and active gelatinase-B levels

Here we describe a new principle for accessing the activity of the differen t members of the human matrix-metalloproteinases (MMPs) by a colorimetric a ssay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys down arrow IleIleGlyGly), was replaced by a sequence that is specifically recog nized by MMPs (ArgProLeuCly down arrow IleIleClyGly). 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 t issue culture media, and MMP-activity of both active and latent MMP-9 can b e analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from pati ents with Sjogren's syndrome. Using a general gelatinase assay with radioac tively-labeled gelatinated collagen it was observed that gelatinase activit y was slightly, though not significantly, increased in patients: general ge latinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 ( 4.0 vs 36.1 +/- 1.9 x 10(4) c pm/ml (p > 0.05), for active and latent gelatinase, respectively. However u sing the immunocapture activity assay (using modified urokinase) specifical ly MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patien ts 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus act ive 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 subs trate can easily be adapted for the specific detection of the various membe rs of the MMP-family or other difficult to measure proteases, in a format t hat can be used for high throughput screening of compounds or samples.