PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST BOTHROPSTOXIN-1, A K49 PHOSPHOLIPASE A2

Campos, L.A.(1), Smith, T.J.(2), Nascimento, N.(1), Smith, L.A.(2), Spencer, P.J.(1)

(1)Grupo de Química de Proteínas, Laboratório de Biologia Molecular, IPEN, CNEN, SP. (2)Toxinology Division, US Army Medical Research Institute of Infectious Diseases, USA.

Myotoxins are components of venom of many snakes from different genera. These toxins belong to two major groups, one that includes short chains peptides like crotamine and myotoxin A, while the other group is composed of molecules with a phospholipase A2 structure. Many of the latter myotoxins display critical mutations within the calcium-binding loop and, therefore, are practically inactive when assayed in the usual mixed micelles enzyme tests. These mutations include Q4/E, Y28/Q and D49/K. Toxins bearing these features are commonly referred to as K49 myotoxins. Although enzymatically inactive, the K49 toxins induce severe myonecrosis by a mechanism not yet fully understood. Evidences indicate a role for the cationic region of the c-terminal domain. Also, site directed mutagenesis studies allowed a good mapping of the protein surface. However, it is not clear whether these described regions are involved in binding domain recognition or in toxicity. Our goal was to produce and characterize monoclonal antibodies against non-overlapping epitopes of bothropstoxin-1, a K 49  myotoxin. The antibodies were screened by surface plasmon resonance aiming to isolate antibodies against non overlapping epitopes.  In our primary screening, 11 clones were selected for ascitic fluid production and subsequent IgG purification . The ascitic fluids were tested in vitro, after a pre-incubation step, against differentiated  C2C12myotubes.  Our data suggest that 4 of our antibodies are able to neutralize the cytolytic effect of the toxin. Whether these antibodies bind to topologically close sites or to different sites involved in the toxic activity has yet to be investigated .

CORRESPONDENCE TO:

Spencer, P.J., Laboratório de Imunopatologia, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo, 05503-900, SP, Brasil, Email: pspencer@net.ipen.br