engleski

The novel chromatographic method for analysis of long-nosed viper venom basic components

Long-nosed viper (Vipera a. ammodytes) venom has been used for the immunobiological production of antivenoms against the European poisonous snakes’ bites. Toxicity of the venom is one of its intrinsic characteristics and it seems also to determine the quality of produced antivenoms. The method for assessment the toxicity of the venom requires the use of large number of experimental animals and although painful for the animals, is legally approved, since there has been no in vitro alternative yet. Here we present the novel method that might be useful for predicting the long nosed viper venom toxicity and consequently its potential to induce a high-quality antiserum in immunized animals. The method is based on rapid HPLC separation of the venom using Convective Interaction Media (CIM) and the fact that the content of ammodytoxins (Atx), basic neurotoxic phospholipases A2 (sPLA2), in the venom has been correlated to the ability of the venom to produce highly protective antiserum in rabbits. Selective retention of the venom basic proteins on carboxymethyl (CM) CIM was achieved in 50 mM Tris/HCl buffer, pH 9.0. Bound proteins were separated into five fractions (EF1-5) using discontinuous gradient of NaCl in the range from 0 to 0.5 M. Pure AtxA and AtxC were used to establish their precise elution positions. Chromatographic fractions of the venom were assayed for protein composition by SDS-PAGE, lethality in mice and the presence of Atx by ELISA using anti-AtxA antibodies. AtxA was found concentrated in fraction EF2, in agreement with the retention time of the pure AtxA. Two batches of the long-nosed viper venom different in their biological properties related to AtxA content, i.e. lethality and anti-AtxA-antibody-inducing potential in rabbits, were comparatively analyzed under described experimental conditions. Significantly different height and shape of respective peak EF2 qualify the method for rapid prediction of the potential of a particular venom sample to produce a high-quality antiserum in immunized animals.

long-nosed viper venom; toxicity; CIM; HPLC

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