(1) Centre de Recherche en Immunologie, Institut Armand-Frappier, Universite du Quebec, Laval, Canada.
(2) Rega Institute for Medical Research, Laboratory of Molecular Immunology and (3) University
Hospital, Division of Rheumatololgy, University of Leuven,  Leuven, Belgium.

Abstract

The contribution of the metalloproteinases (MMPs) in the degradation of matrix components in degenerative diseases is well established. Various members of this family have bveen found in excessive concentration in serum and synovial fluids of patients suffering from these diseases, suggesting that degradation of extracellular matrix proteins results from a positive balance toward excessive proteolytic activity. The measurement of excess activity in serum and synovial fluids, however, has not been documented to date since most conventional assays that monitor the presence of proteases are unsuitable for measuring the net proteolytic activity in biological fluids. We describe here the development of the fluorescent activated-substrate conversion (FASC) assay (patent pending 2,189,486) based on the degradation of fluorochrome-labeled substrates coated on microspheres. Our approach could be used to determine the repertoire of proteolytic activities in synovial fluids during the treatment of joint diseases and to monitor the efficacy of treatments that are directed toward diminishing MMP activity.
 

Figure Legends

Figure 1: In figures 1a and 1b, the principles of the FASC assay is depicted. The methodology has been describe previously by us (St-Pierre et al. (1996), Cytometry  25:374-380).




Figure 2:  We used a member of the matrix metalloproteinase, gelatinase B (MMP-9) to construct a dose-response curve for two incubation periods (90min and 18h). The total reaction volume was 100µl. For each concentration (done in triplicates) the residual fluorescence of the beads was evaluated with an Coulter XL-MCL. The fluorescence intensity (in log scale)  for the different concentration of gelatinase B after an incubation of 18h is shown.



Figure 3: We evaluated the potential of our technique to determine the net proteolytic activity contained in a biological fluids. We obtained synovial fluids (SF's) from patients suffering from inflammatory joint diseases, with known levels of MMP-9. We could evaluate the titer of the net proteolytic activtities of the SF's by performing a FASC assay on serial dilutions of the samples and the fluorescence evaluated after an incubation of 18h.


 
 

Figure 4: We performed a kinetics experiment to determined the minimum time required to obtained a significant response with a biological fluid (ex. SF's). With most SF's a response could be obtained within 30 min.



Figure 5: We compare our assay with the zymography, which is the usual assay for gelatinases. This assay is based of the degradation of the substrate (eg. gelatin) which is copolimerized in the seperating gel of a SDS-PAGE. The sample are run, renatured and the reaction proceed for 18h. Subsequently the gel is stained, and the presence of enzyme, and its levels of activity, is revealed by a clear zone on a blue background. When we compare the SF's with known levels of gelatinase B determined by zymography and the FASC titer for the same sample, we determined that no correlation exist between the presence of the enzyme and the net proteolytic activity.