title: Neutralisation of the pharmacological activities of Bothrops alternatus venom by anti-PLA2 IgGs.
abstract: Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.
authors.affiliation: Laboratorio de Química de Proteinas, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Argentina; Laboratorio de Farmacología y Toxicología, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral 2139, CP 3400, Corrientes, Argentina. Electronic address: garciadenegri@gmail.com.
authors.forename: María E
authors.initials: ME
authors.lastname: Garcia Denegri
authors.affiliation: Laboratorio de Farmacología y Toxicología, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral 2139, CP 3400, Corrientes, Argentina.
authors.forename: Silvana
authors.initials: S
authors.lastname: Maruñak
authors.affiliation: Laboratorio de Bioquímica, Facultad de Medicina, Universidad Nacional del Nordeste (UNNE), Corrientes, Argentina.
authors.forename: Juan S
authors.initials: JS
authors.lastname: Todaro
authors.affiliation: Laboratório de Química de Proteínas, Departamento de Bioquímica, Instituto de Biología, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Ponce-Soto
authors.affiliation: Laboratorio de Farmacología y Toxicología, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral 2139, CP 3400, Corrientes, Argentina.
authors.forename: Ofelia
authors.initials: O
authors.lastname: Acosta
authors.affiliation: Laboratorio de Química de Proteinas, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Leiva
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis*; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000302:isolation & purification; D000918:Antibody Specificity; D000997:Antivenins / Q000737:chemistry*; D017837:Bothrops; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000302:isolation & purification; D008297:Male; D051379:Mice; D009500:Neutralization Tests; D064801:Phospholipase A2 Inhibitors / Q000737:chemistry*; D054467:Phospholipases A2 / Q000737:chemistry*; D011817:Rabbits
chemical_list: D000888:Antibodies, Anti-Idiotypic; D057134:Antibodies, Neutralizing; D000997:Antivenins; D003435:Crotalid Venoms; D007074:Immunoglobulin G; D064801:Phospholipase A2 Inhibitors; C048844:anti-IgG; D054467:Phospholipases A2
keywords: Anti-PLA(2) immunoglobulins; Bothrops alternatus; Envenomation; Non-myotoxic PLA(2)