Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential
Introduction
Snake venoms are very heterogeneous biological systems showing undeniable interspecies and intraspecies variations (Chippaux et al., 1991) expressed by qualitative and quantitative diversity of biological activity and protein patterns (Serrano et al., 2005). Interspecies variations can be explained by the evolutionary aspects of venomous snakes (Sasa, 1999). Intraspecies variation has been attributed to age (Gutiérrez et al., 1980, Minton and Weinstein, 1986, Daltry et al., 1996b, Saravia et al., 2002, Alape-Girón et al., 2008), sex (Menezes et al., 2006), geographical location or climate (Minton and Weinstein, 1986, Saravia et al., 2002, Alape-Girón et al., 2008, Schenberg, 1959, Jayanthi and Veerabasappa Gowda, 1988, Mukherjee and Maity, 1998, Creer et al., 2003, Ferquel et al., 2007, Lanari et al., 2010, Aguilar et al., 2007), genetics (Daltry et al., 1996b, Menezes et al., 2006, Fry, 2005), alimentary habits (Sasa, 1999, Daltry et al., 1996a, Sanz et al., 2006, Li et al., 2005, Gibbs and Mackessy, 2009) and the complex, not well-understood interactions of some of these factors (Francischetti et al., 2000).
Such complex and variable mixtures have been used as antigens for the immunization of production animals (horses mostly or sheep) in the process of antivenom production (venom-specific antiserum or its derivatives - pure immunoglobulins, their F(ab')2 or Fab fragments). Awareness has been raised of possible implications of variations in the venom consistency on production process and on the quality of the final product (Chippaux et al., 1991, Aguilar et al., 2007, Chippaux and Goyffon, 1998), but detailed studies on the relationship between venom's composition variation and immunogenicity changes are scarce.
Vipera ammodytes (long-nosed viper) is the most poisonous (or venomous) European snake. Its venom has been used as an antigen for immunization of animals for production of monospecific and polyspecific antivenoms against European snakes' envenomation. The long-nosed viper subspecies Vipera ammodytes ammodytes (Vaa) inhabits western part of the Balkan Peninsula, including also Croatia. The variability in biochemical properties and biological activities of Vaa venoms collected from snakes originating in different geographical regions (Lang Balija et al., 2005) and from snakes milked in different seasons of the year has already been reported (Gubenšek et al., 1974). Recently we have demonstrated the positive correlation between the protective efficacy of venom-specific antiserum and the quantity of antibodies specific for one particular class of venom proteins – ammodytoxins (Halassy et al., 2008a). Ammodytoxins (Atxs) are potent neurotoxins and the most toxic compounds isolated so far from the Vaa venom (Pungerčar and Križaj, 2007). We have also demonstrated that venom with more Atxs is also able to induce the production of higher quantity of anti-Atxs antibodies, resulting in higher neutralization potential of respective serum (Halassy et al., 2010). That raised the hypothesis that analytical methods determining Atxs content in the venom might be more informative regarding the venom's suitability for immunization purposes, than methods conventionally used for venom characterization by different producers. The question that we tackled in the presented study was how informative and predictive are different methods on venom's suitability for antivenom production. To get the answer we analyzed eight different batches of Vaa venom of known immunogenicity by two recently developed methods for Atxs content estimation (HPLC method for extraction and separation of highly basic Vaa venom's components (CIM CM HPLC) (Halassy et al., 2008b) and ELISA for Atxs content determination (Halassy et al., 2010)) and capillary gel electrophoresis-on-a-chip, which has been shown to be a useful tool for quality control of snake venoms (Marchetti et al., 2007). Obtained results were compared to different conventionally and usually used assays, like lethal toxicity in mice, total protein content, phospholipase activity and SDS-PAGE.
Section snippets
Reagents, chemicals and instruments
Bovine serum albumin (BSA), Tween 20 and o-phenylenediamine dihydrochloride (OPD) were from Sigma-Aldrich, USA. Horseradish peroxidase-conjugated goat anti-rabbit IgG (HRP-anti rabbit IgG) was from Bio-Rad Laboratories, USA. Chemicals for buffers and solutions were from Kemika, Croatia, unless otherwise stated. Microplate reader Reader 530 and microplate washer Multiwash were from Organon Teknika, the Netherlands. Water for injection (WFI) was from the Institute of Immunology (Zagreb, Croatia).
Chromatographic media and instrumentation
Results and discussion
The empirical data on immunogenicity of the Vaa venom samples (Table 1) were collected from the Croatian producers (at the Institute of Immunology) of antivenoms. They observed that in periods when venom samples of low immunogenicity in Table 1 were used for horse immunization, the resulting horse antisera were of lower neutralizing potential against the venom toxicity than the antisera produced in periods when venom samples of high immunogenicity were used. The observed empirical
Conclusions
In conclusion, classical and conveniently used methods of snake venom analysis are useful and informative for venom species identification. However, for manufacturers who want to optimize their antivenom production, the intraspecies differences in the quantitative composition of venoms could be very relevant, as it was shown for Vaa venom. Methods determining ammodytoxin content in the Vaa venom, as hereby presented CIM CM HPLC of highly basic components or ELISA, could improve venom selection
Acknowledgements
This work was supported in part by the Croatian Ministry of Science, Education and Sports (grant 021-0212432-2033), the Bilateral Cooperation Grant Croatia–Austria (HR 13/2010), the Bilateral Cooperation Grants Croatia-Slovenia (HR-SI 25/2010) and through scholarship (to MB) given by the National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia. The CIM CM disks were kindly provided by Miloš Barut, PhD, from BIA Separations. Thanks to Andreas
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