Razvoj nove metode za ugotavljanje aktivnosti hialuronidaz na osnovi testa ELISA

As part of my master's thesis, we wanted to develop an efficient method for determining hyaluronidase activity. We developed an ELISA method by testing different primary detection reagents (primary detection reagents 1, 2, 3 and 4), as our hypothesis related to efficient primary detection using these reagents. In order to find an appropriate buffer solution for binding HA to the microtiter plate, we tested different buffer solutions. Since the hypothesis stated the success of determining hyaluronidase activity, we evaluated the hyaluronidase calibration standard in the 50% - 200% range of relative potency (relative biological activity) to verify the ability to determine the degradation rate. The results were analyzed with the mathematical models 4PL and PLA. We found that detection reagents 1, 2 and 3 bind non-specifically to the surface of the microtiter plate. Detection was established only with detection reagent 4, which proved to be suitable for detecting hyaluronic acid bound to the plate. The measured signal in this case showed the expected dose-dependent response. It was found that PBS buffer is the most suitable for binding HA to the microtiter plate. The measured signal after degradation of HA with hyaluronidase standard showed the expected dose-dependent response.