On-line version ISSN 1678-4936
J. Venom. Anim. Toxins vol. 2 n. 2 Botucatu 1996
DISSERTATION: R. A de Paula submitted this dissertation for the degree of Master of Science in Nuclear Technology publicly examined at the Department of Bioengineering of the Nuclear Energy Research Institute of the University of São Paulo, São Paulo, Brazil in 1995.
Advisor: Professor José Roberto Rogero
ABSTRACT. Snakebite is a major public health problem in Brazil. Envenoming by snakes of the genus Crotalus is the most severe. About 11% of the victims die without receiving serotherapy. The antivenoms are obtained from hyperimmune horse plasma. During antivenom production, the horses present signs of envenoming that result in a decrease of their organic resistance. Besides, horses are expensive both to purchase and to maintain, which restricts production of sera. A number of techniques to reduce venom toxicity and increase serum production using chemical and physical agents have been studied. Gamma rays are an excellent tool to detoxify venoms and toxins. They are also able to modify protein structures that decrease lethality and toxic and enzymatic activities without altering immunogenicity. Thus, it is important to evaluate production of sera in rabbits using gamma-ray detoxified venom and crotoxin as immunogens and their action as reagents in immunoassays. To obtain the antisera, either Crotalus durissus terrificus whole venom or isolated crotoxin was irradiated with 2,000 Gy in a Co source in a 150mM NaCl solution and inoculated in rabbits. Production of sera was screened by immunoprecipitation, immunoenzymatic (ELISA) and immunoradiometric (IRMA) assays. Specificity was studied by immunoelectrophoresis, ELISA and Western blot techniques. The neutralizing action was evaluated through the neutralization of phospholipase A2 activity of crotoxin in vitro. The antisera were used as reagents in antigen capture assays ELISA and IRMA immunoassays to detect circulating antigens in sera of mice experimentally inoculated with crotalic venom or crotoxin. The results showed that both the detoxified venom and the crotoxin were good immunogens. They were able to induce the production of antibodies that could recognize either non-irradiated venom or isolated crotoxin. These data suggest that the above-mentioned antibodies present more specificity and higher in vitro neutralizing action than the commercial anticrotalic antisera. Thus, they could be used in immunoassays as a tool to distinguish crotalic from bothropic envenomations.
J. R. ROGERO - Departamento de Bioengenharia, TBR, Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP, Travessa R, 400, Cidade Universitária, CEP 05508-900, São Paulo, SP, Brasil.