DIFFERENTIAL ELISA FOR Bothrops atrox AND Lachesis muta muta ACCIDENTS USING MONOCLONAL ANTIBODIES

COLOMBINI, M.; CARDOSO, D.F.; FERNANDES, I.; MOURA-DA-SILVA, A.M.

Laboratório de Imunopatologia, Instituto Butantan, SP

B. atrox and L. m. muta snakes are responsible for most accidents occurring in Brazilian Amazon. The clinical features of the accidents are similar, and there are still controversies about efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the efficacy of B. atrox and L. m. muta experimental antivenoms for cross-neutralisation of haemorrhagic and coagulant activities of the venoms, which were preferentially neutralised by homologous antivenoms. B. atrox antivenom failed completely in neutralising L. m. muta-induced coagulophaty, thus indicating the necessity of a differential diagnosis and the use of specific therapy. The differential diagnosis was accessed by ELISA, using polyclonal antibodies raised in rabbits (reference method) and a new assay using biotin-labelled monoclonal antibodies. Monoclonal antibodies were obtained using as antigens the non-variable specific 50 kDaB. atrox venom band and 24 kDa specific antigen for L. m. mutavenom. MoAbs were screened using homologous or heterologous venoms and 4 clones were selected for B. atrox and 6 clones for L. m. muta venoms. ELISA standardisation was carried out using venom standard curves diluted in normal human serum. Sensitivity was similar in both methods reaching venom concentrations of 0.1 ng/ml. Specificity was much higher in MoAbs assays since no reaction was detected with normal sera used as control, while cut-off levels of reference assay was around 10 ng/ml venom. The MoAb assay is under evaluation using serum samples of patients from Pará State. This test will be important both for diagnosis and epidemiological surveys, providing a rational for production and distribution of antibothropic, antilachetic or polyvalent antivenoms.

Supported by FAPESP 00/02396-9

CORRESPONDENCE TO:

Mônica Colombini, Rua Júlio de Castilhos, 1126, São Paulo, SP, CEP: 03059-000, Brasil, Email: colombi@usp.br