Using a low toxic phospholipases A2 (P9-Cdt) as a tool for antivenom production

FUSCO LUCIANO; RODRIGUEZ JUAN PABLO; TORRES-HUACO, FANK; MARANGONI SERGIO; PONCE-SOTO LUIS; ACOSTA OFELIA; LEIVA LAURA CRISTINA

Rio de Janeiro

Congreso; I encontro internacional vital para o brasil sobre animais peçonhentos; 2013

Up to now, heterologous antibody administration has been the treatment of choice for snakebite victims and so far it is the only procedure endorsed by the WHO. This practice requires the injection of large amounts of toxins engaging horse, sheep, goat or rabbit immunization, usually causing great host impairment and suffering. We have previously isolated and purified two PLA2 isoforms from the Crotalus durissus terrificus (C.d.t.) venom, called P9(Cdt-PLA2) and P10(Cdt-PLA2). The biological profile of P10(Cdt-PLA2) isoform was more edematogenic (10 fold), myotoxic (1.4 fold) and lethal (2 fold) compared with the biological effect of P9(Cdt-PLA2). In this work, we investigated the immunogenic properties of both isoforms in order to use one of them, in the production process of crotalic antivenom. P9(Cdt-PLA2) and P10(Cdt-PLA2) were isolated in one purification step using a RP- HPLC on a C-8 Discovery® Bio Wide column. Chromatographic profile and mass recovery data showned that P9(Cdt-PLA2) is 2.5 times more abundant than P10(Cdt-PLA2). The PLA2 enzymatic activity was measured employing the chromogenic substrate 4-nitro-3-(octanoyloxy) benzoic. Both antisera were obtained according to previously set and tested inoculation protocols, based on successive intramuscular and subcutaneous injections of different amount (2-15 μg) of PLA2 isoforms per mice. We used ELISA tests in order to evaluate the rising titer of antibodies produced in animals and their specificity was observed by western blott. The effective amount of each antiserum was determined using the indirect hemolytic activity neutralization assay against whole C.d.t. venom. Then, the effective dose 100 (DE100) of both antisera was used to investigate the lethal activity neutralization of the whole venom. We observed that both isoforms were able to generate antibodies with very similar DE100, and a high level of protection against the lethal activity of the whole venom, however, the high toxicity of P10(Cdt-PLA2) and its poor recovery from whole venom complicates the development of immunization plan. The above indicates that P9(Cdt-PLA2) may be an useful tool to be used as immunogen since that it can be obtained in adequate quantities and it does not involve a great direct damage that may compromise the health of the producing animals.