Assessment of a screening test for MMP-8 activity in the diagnosis of periodontal diseases

grantor: University of TorontoActive neutrophil collagenase (MMP-8) is associated with progressive periodontitis and the measurement of this enzyme in gingival crevicular fluid (GCF) can facilitate diagnosis. Assays with sufficient sensitivity to detect collagenase in GCF currently use radiolabelled substrates and can take several days to complete. To provide a more rapid analysis of collagenase activity that is better adapted to clinical studies, I validated the use of a novel assay (biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. The assay can detect $<$56 pg collagen degraded/hour, which is comparable to the most sensitive radioactive assay. The total assay time is 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF and peripheral blood neutrophils (PMNs) (n = 76) determined by the SBA and radioactive assays, a high correlation (r = 0.97) was obtained. To optimize the GCF collection protocol, the stability of MMP-8 activity in GCF samples was examined. Cross-sectional and longitudinal analyses of frozen GCF showed that MMP-8 activity increased with increasing severity of periodontitis and decreased following treatment. To examine the relationship between activation and binding to native or denatured collagen, MMP-8 from PMN (N = 52) and from GCF samples (N = 7) was incubated on plastic substrates coated with either type I collagen (fibrils or monomers) or gelatin. The results indicate that active MMP-8 can bind to fibrillar collagen, is localized in diseased tissue and can be detected in GCF by a novel assay that is specific, simple, rapid and reproducible and which can facilitate periodontal diagnosis. (Abstract shortened by UMI.)M.Sc