The regulatory assay of antivenom quality (mouse assay of venom lethality neutralisation) does not detect antibodies neutralising the hemorrhagic activity of the Vipera ammodytes venom

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.