REFLECTANCE METHOD FOR SIMPLE DETERMINATION OF PROTEINASE ACTIVITY IN MICROLITER SAMPLES OF A COMPLEX SERUM-LIKE FLUID

A technique using an optical instrument, a reflectometer, for quantitative determination of proteinase activity in microliter samples of complex serum-like fluids, e.g., crevicular exudate from single sites, was developed. The technique allowed the use of various proteins as enzyme substrate. The reflectometer measures the mass of a layer, such as protein, adsorbed to a reflecting surface. This is done by measuring the reflected light intensity of the p-polarized light beam on a surface. We used methylized silicon surfaces that were coated with fibrinogen, alpha-2-macroglobulin, or hemoglobin as enzyme substrates. The test solution was incubated overnight in a basin made in an agar gel applied on the top of the protein-coated surface. In 82 exudates from periodontitis sites, with pocket depths greater-than-or-equal-to 6 mm, fibrinogenolytic activity corresponding to 1-mu-g ml-1 of trypsin and pronase P was found in 20% of the samples.