Proteomics of Naja kaouthia venom from North East India and assessment of Indian polyvalent antivenom by third generation antivenomics.

In the present study, venom composition, toxic effects, and immunological characteristics of Naja kaouthia venom from North East India has been studied. Using RP-HPLC, venom components were separated and proteins in the fractions were identified using ESI-LC MS/MS. Proteins identified belong to 9 different snake venom protein families. Three finger toxins and PLA2 were the most abundant protein families detected by mass spectrometry analysis. The other minor proteins families identified in the venom were kunitz-type serine inhibitors, waprin, L-amino acid oxidase, CRISP, vespyrn, nerve growth factor and metalloproteinase. This proteome composition correlated with the tested enzymatic and toxic activities of the venom. Western blot and third generation antivenomics analysis using Vins polyvalent antivenom revealed immunoreactivity towards Naja kaouthia venom of North East India. Concentration-dependent immunocapturing profile carried out using RP-HPLC displayed immunerecognition of majority of venom proteins of Naja kaouthia except few three-finger toxins. Presence of such non-immunodepleted toxins apparently may affect the performance of Vins polyvalent antivenom. Thus, inclusion of antibodies of most relevant non-immunorecognized toxins in antivenom might help to improve the quality of antivenom. BIOLOGICAL SIGNIFICANCE: Envenomings by genus Naja, represent a serious medical problem in Asian countries including North east India. In North East India, Naja kaouthia is most prevalent cobra species causing a large number of fatalities. To gain deeper insight into the spectrum of medically relevant toxins, we applied proteomics approach to unveil the proteome profile of Naja kaouthia venom. The proteomic analysis divulged the presence of two major protein families: three finger toxins and phospholipases A2. In general, polyvalent antivenom is administered for Naja kaouthia envenomings, however, this venom is not included in the immunization mixtures (only Indian Big Four venoms) for production of these polyvalent antivenoms. For the first time, third generation antivenomics approach was used to decipher maximal binding capacity of Indian polyvalent antivenom against Naja kaouthia venom. Although Vins polyvalent antivenom was effective in immunocapturing majority of venom components, however, large amount of antivenom was required to immunocapture the venom proteins. Moreover, the study revealed poor immunorecognition capacity of Vins antivenom towards four three finger toxin subtypes. This may have significant impact on antivenom efficacy in treating Naja kaouthia envenomings.