Dispersed Ascorbyl palmitate (ASC16) as an additive in adjuvant formulations

Abstract

Different aspects determine the choice of an adjuvant to generate optimal antibody productionwhile ensuring the safety of the producing animal. In this context, ASC16 that can form safehydrogels or act as a toxin inhibitor depending on whether it is present at high or low concentrations, respectively. For these reasons, it was proposed to evaluate the effect of dispersed ASC16 (low concentration) as an additive adjuvant in the production of experimental antivenom. BALB/c mice were immunised with Freund’s adjuvant in the presence or absence of additive (AFMo and AF) and hydrogel with or without additive (Pa40Mo and Pa40). Afterwards, ELISA, avidity andimmunoblotting tests were performed to determine the titre, binding strength and specificity ofthe antibodies. Finally, we determine the effective dose 50 (ED50) that neutralises PLA2 activityof Crotalus durissus terrificus venom. We used the t-tests for two independent samples to verifyif there were significant differences. Our results show that AFMo induced the highest titres (4.07),followed by AF and Pa40Mo (both 3.75), although without significant differences (p>0.05), and finally Pa40 (3.27). Notably, the additive ASC16 also improved antibody avidity, with AFMo (4.35 MKSCN) standing out with significant differences compared to the rest (p≤0.05), followed by AFand Pa40Mo (3.21 and 3.17 M KSCN) with no significant differences (p>0.05), and finally Pa40(1.61 M KSCN). In addition, the antibodies recognised the main antigenic components of thevenom. The sera produced by formulations with additive presented a better ED50 for PLA2 activity, highlighting Pa40Mo (7.04 µL), followed by AFMo (9.02 µL) with significant differences (p≤0.05) compared to Pa40 (12.20 µL) and AF (12.23 µL). This study shows that formulationswith additive ASC16 as an enhancer induce higher antibody titres, improve avidity, have a greaterspecificity and neutralising effect on PLA2 activity than formulations without the additive.