Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Quantitative proteomics to reveal the composition of Southern India spectacled cobra (Naja naja) venom and its immunological cross-reactivity towards commercial antivenom.

Indian cobra (Naja naja) envenomation is frequently reported across Indian subcontinent. Geographical differences in the venom composition of a particular species of snake often leads to inconsistencies in the antivenom neutralization. Consequently, determining the venom proteome from every locale is necessary for the production of effective antivenom. In this study, we deciphered the proteome composition of N. naja venom (NnV) from southern India (SI) by label-free quantitative proteomics that identified 45 proteins (toxins) belonging to 14 venom protein families when searched against Elapidae (taxid: 8602) protein entries in the non-redundant NCBI database. Low molecular mass (<15 kDa) toxins such as PLA2 (18.2%) and 3FTx (37.4%) are the most abundant enzymatic and non-enzymatic proteins, respectively, in SI NnV. Nevertheless, the relative abundance of 3FTxs in SI NnV was found to be lower than the relative abundance of these toxins in previously determined eastern and western India NnV samples. Immuno-recognition and in vitro neutralization of some enzymatic activities and pharmacological properties of SI NnV by commercial polyvalent antivenom evidently demonstrated poor recognition of the most abundant low molecular mass toxins of SI NnV. This finding points to the need for new strategies for antivenom production for the successful treatment of cobra bite.

A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding.

Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER50s of 12 batches of antisera showed correlation (R 2) of 0.9809 (p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.