CroFab™ total anti-venom activity measured by SE-HPLC, and anti-PLA₂ activity assayed in vitro at physiological pH

One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab™ anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 °C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A₂ (PLA₂) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA₂ and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab™ anti-venin) inactivation of C. atrox PLA₂ activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.