An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja

Kavi Ratanabanangkoon,Choo Hock Tan,Sukanya Eursakun,Pavinee Simsiriwong,Kritsada Pruksaphon,Bunkuea Chantrathonkul,Kae Yi Tan

doi:10.1038/s41598-018-27794-3

Abstract

In order to facilitate/expedite the production of effective and affordable snake antivenoms, a novel in vitro potency assay was previously developed. The assay is based on an antiserum’s ability to bind to postsynaptic neurotoxin (PSNT) and thereby inhibit the PSNT binding to the nicotinic acetylcholine receptor (nAChR). The assay was shown to work well with antiserum against Thai Naja kaouthia which produces predominantly the lethal PSNTs. In this work, the assay is demonstrated to work well with antiserum/antivenom against Bungarus candidus (BC), which also produces lethal presynaptic neurotoxins, as well as antivenom against Sri Lankan Naja naja (NN), which produces an abundance of cytotoxins. The in vitro and in vivo median effective ratios (ER50s) for various batches of antisera against BC showed a correlation (R2) of 0.8922 (p < 0.001) while the corresponding value for the anti-NN antivenom was R2 = 0.7898 (p < 0.01). These results, together with the known toxin profiles of various genera of elapids, suggest that this in vitro assay could be used with antisera against other species of Bungarus and Naja and possibly other neurotoxic snake venoms worldwide. The assay should significantly save numerous lives of mice and accelerate production of life-saving antivenoms.

Highlights

Snake envenomation is an important yet neglected medical problem in various developing countries with an estimated annual envenomation world wide of about 5.5 million cases[1,2]
Inhibition of nicotinic acetylcholine receptor (nAChR) binding to Naja kaouthia postsynaptic toxin 3 (NK3)-coated plate by B. candidus or N. naja venom
Because ELISA involves the binding of antivenom antibodies to venom proteins whose toxicity might not be neutralized, results of these in vitro assays usually show poor correlation with results from the murine lethality neutralization assay

Summary

Introduction

Snake envenomation is an important yet neglected medical problem in various developing countries with an estimated annual envenomation world wide of about 5.5 million cases[1,2]. Snakes in the genus Bungarus (kraits) produce, in addition to PSNTs, the lethal presynaptic neurotoxins (β-neurotoxins) which in elapids, belong to the group 1 phospholipase A2 enzymes These toxins do not bind to nAChR but react with receptors on the membrane of the motor nerve terminals which contain the acetylcholine vesicles. Another interesting case is the Naja naja (Indian cobra) which is a WHO category 1 medically most important elapid in India, Pakistan and Sri Lanka Envenomation by this snake resulted in muscle weakness and death by respiratory failure which is likely the effect of the venom PSNTs. Interestingly, the venom of the Sri Lankan snake was shown by proteomics study to contain 71.55% of cytotoxins (cardiotoxins)[18]. It is interesting to investigate whether the developed in vitro nAChR binding assay could be used for potency assay of AV against this cobra

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Results

Conclusion