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. a trox), 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 ({Area(bnd)}) were evaluated using a logistic dose-response equation to estimate EC50 and {Area(bnd)}(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 {Area(bnd)}(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 A(2) (PLA(2)) activity of C atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA(2) 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. (C) 2011 Elsevier Ltd. All rights reserved.