Development of an in-house ELISA for hyaluronidase activity testing

Mesarič, Tara (author), Rajčević, Uroš (mentor)

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.

• Language: Slovenian
• Keywords: hyaluronic acid, hyaluronidase, ELISA, 4PL, PLA
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2025
• PID: 20.500.12556/RUL-171518
• COBISS.SI-ID: 246957827
• Publication date in RUL: 28.08.2025

Secondary language

• Language: English
• Title: Razvoj nove metode za ugotavljanje aktivnosti hialuronidaz na osnovi testa ELISA

Abstract

V sklopu magistrske naloge smo želeli razviti učinkovito metodo za določanje hialuronidazne aktivnosti. Razvili smo metodo ELISA s preizkušanjem različnih primarnih detekcijskih reagentov (primarni detekcijski reagent 1, 2, 3 in 4), saj se je naša hipoteza nanašala na učinkovito primarno detekcijo z uporabo teh reagentov. Da bi našli ustrezno pufersko raztopino za vezavo HA na mikrotitrsko ploščo, smo preizkusili tudi različne puferne raztopine. Ker je hipoteza navajala uspešnost določanja hialuronidazne aktivnosti, smo testirali hialuronidazni kalibracijski standard v 50% - 200% razponu relativne potentnosti (relativne biološke aktivnosti), da smo preverili sposobnost določanja stopnje razgradnje. Rezultate smo analizirali z matematičnima modeloma 4PL in PLA. Ugotovili smo, da se detekcijski reagenti 1, 2 in 3 nespecifično vežejo na površino mikrotitrske plošče. Detekcija je bila ugotovljena zgolj z detekcijskim reagentom 4, ki se je izkazal za ustreznega pri zaznavanju hialuronske kisline, vezane na ploščo. Izmerjeni signal je namreč v tem primeru pokazal pričakovan od odmerka odvisni odziv. Ugotovljeno je bilo, da je pufer PBS najprimernejši za vezavo HA na mikrotitrsko ploščo. Izmerjeni signal po razgradnji HA s hialuronidaznim standardom je pokazal pričakovani, od odmerka odvisni odziv.

• Keywords: hialuronska kislina, hialuronidaza, ELISA, 4PL, PLA