A microtiter-based assay for hyaluronidase activity not requiring specialized reagents

A sensitive, rapid microtiter-based assay for hyaluronidase activity is described that does not require highly specialized biological reagents, as required heretofore. The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtiter plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtiter plate, artifacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples with an interassay variation of less than 5%. Using this new assay, we measured the distribution profile of plasma hyaluronidase levels in normal human sera. A 1-microl sample of plasma was sufficient for assays in triplicate. Hyaluronidase activity in human foreskin primary keratinocyte cultures was also quantitated. A 25-fold increase in hyaluronidase activity was observed in keratinocyte cultures induced to differentiate in high calcium (1.5 mM), compared to levels in low calcium (0.05 mM) media. The microtiter-based assay may be used as a routine clinical laboratory procedure.