In vitro evaluation of total venom–antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality based on size-exclusion high-performance liquid chromatography

Total venom–antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality can be evaluated using size-exclusion high-performance liquid chromatography (SE-HPLC). Simple integration of regions within SE-HPLC elution profiles was used to compare binding characteristics of Crotalidae Polyvalent Immune Fab (Ovine) antivenin (FabAV) and Crotalus atrox (western diamondback rattlesnake; C. atrox), C. varidis varidis (prairie rattlesnake; C. v. v.), Agkistrodon contortrix contortrix (southern copperhead; A. c. c.), and A. piscivorus leukostoma (western cottonmouth; A. p. l.) venom. Areas associated with bound venom and antivenin ({Areabnd}) were evaluated using a logistic dose–response equation to estimate EC50 and {Areabnd}max. The relative magnitudes of EC50, which inversely reflect venom–antivenin binding affinity, were C. atrox > C. v. v. > A. c. c. > A. p. l. Less than 50% of FabAV appeared to be reactive with each of the venoms based on {Areabnd}max. Data was also consistent with FabAV binding to multiple sites on polyvalent antigens within the venoms. Evaluation of immune complex formation using SE-HPLC was compared to neutralization of phospholipase A2 (PLA2) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA2 activity occurred, in general, prior to maximum immune complex formation. Venom–antivenin binding at EC50 determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD50 and ED50 reported by others. SE-HPLC analysis of venom–antivenin binding could provide a priori information, relevant to reducing the use of animals in evaluating antivenin protection against venom-induced toxicity and lethality.