Monoclonal antibodies neutralizing the neurotoxin from the venom of the snake Naja kaouthia

Abstract

Monoclonal antibodies were produced against the principal neurotoxin (siamensis toxin 3) purified from venom of the Thai cobra, Naja kaouthia (Naja naja siamensis), as part of a study aimed at enhancing the neutralization capacity of commercial horse antivenins. The rationale is to assess the range of neutralization capacities within a population of mouse anti-toxin antibodies by sampling individual specificities of varying degrees of neutralization. In order to overcome the lethal toxicity and poor immunogenicity of the native toxin, several modified forms of the low MW toxin were made to be used as immunogen including reaction with glutaraldehyde or formaldehyde and crosslinking of toxin to higher MW carrier proteins such as thyroglobulin, using a water-soluble carbodiimide. These modifications of toxin enable higher doses for immunization, thus enhancing the antibody responses. The hybridomas were produced using splenocytes from mice immuniaed with toxin-thyroglobulin conjugates or glutaraldehyde toxoid. Ten hybridomas secreting monoclonal antibodies against the purified native toxin were selected by ELISA for further analysis. Eight of 10 monoclonal antibodies concentrated from hybridoma-induced ascites fluids by half-saturated ammonium sulfate fractionation, were mixed in approximately a 17-fold excess (i.e., 1 mg Ig) of IgG paratope with an amount of purified toxin equivalent to two mouse 50% lethal doses (2 x LD(,50)). All clones tested exhibited a degree of protection reflected by a prolongation of survival time of mice injected with neurotoxin ranging from 2.6-41.8 times that of the control group. None, however, showed absolute protection. A solid-phase antibody binding competition assay showed some relationship in terms of recognition of the same or overlapping epitopes among all the tested clones in at least 1 direction, but not in both directions for half of them. This suggests that the epitopes recognized by these monoclonal antibodies are members of a large immunodominant region on the toxin molecule which was not destroyed by the modification. The data clearly indicate heterogeneity among anti-toxin antibodies; not all antibodies are equivalent in neutralizing capacity. It is not clear from these results whether additional epitope variation differentiates the best neutralizing antibodies, not the extent to which antibody affinities and even precipitation reactions contribute to neutralization.