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Identification and characterisation of the main protective components of complex antigens is a necessary prerequisite for improvement of the production of immunobiologicals, as well as of the methods for their quality control. The venom of the most poisonous European snake, Vipera a. ammodytes, is a complex mixture of over hundred mostly protein components that has been used as antigen for immunisation of production animals in order to produce effective antivenoms. Two biological activities have been considered crucial for the toxicity of the long-nosed viper venom, neurotoxic and hemorrhagic. Members of the secretory phospholipase A2 group of enzymes are responsible for the neurotoxicity (ammodytoxins, Atxs). Hemorrhagic action of the venom is due to particular metalloproteinases (hemorrhagins, H). In contrast to the detailed knowledge on their structure and function, almost nothing has been known about their immunogenicity. The aim of the study was to prepare the rabbit antisera specific to both classes of these toxic venom compounds and to study the involvement of prepared antibodies in neutralisation of the whole venom toxicity. The only regulatory approved assay for the evaluation of the lethal toxicity neutralisation potential of the venom-specific antisera is still in vivo assay in mice. Our results showed that anti-Atx antibodies participate in neutralisation of the venom's toxicity, while anti-H antibodies are not protective. Such finding becomes extremely important when knowing that the prevailing clinical manifestation of envenomation in human is hemorrhage, but the only approved assay for the quality of antivenoms doesn't measure anti-H antibodies at all.

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity– neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity–neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity–neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.

Long-nosed viper (Vipera ammodytes ammodytes) venom (Vamam venom) has been used as an antigen for immunisation of animals (horses) in the process of antivenom (venom-specific antisera) production. Recently we have shown that the level of antibodies specific to Vamam venom's most toxic component - ammodytoxin A (AtxA) (anti-AtxA IgG) in Vamam venom immunised rabbit sera was highly correlated to their venom-toxicity neutralization potential (Halassy et al. Comp Biochem Physiol C 2008 ; 148, 178). Since all ammodytoxins are highly immuno-crossreactive, antibodies measured by anti-AtxA IgG ELISA might be considered as anti-Atx antibodies in total. Thus allows to hypothesize that the total Atx content of venom used for immunisation might be one of the critical factors determining the outcome of immunisation procedure. The aim of this study was to investigate the outcome of immunisation procedure for antivenom production in rabbits, when antigens for immunisation were venom batches with high or low ammodytoxin content, respectively. The venom batches used for immunisation were selected on the basis of previously noticed differences in Atx content by recently developed HPLC method (Halassy et al. J Liq Chromatogr RT 2008 ; 31, 38). The new sandwich ELISA was developed for precise determination of Atx content in Vamam venom and Atx quantified in venoms used for immunisation of rabbits. Results showed that indeed animals immunised with the venom containing lower amounts of Atx developed the significantly lower level of venom toxicity neutralising antibodies. The level of anti-AtxA IgG was again highly correlated to the venom-toxicity neutralising potential of immunised rabbit sera.

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.