An in vitro alpha-neurotoxin-nAChR binding assay correlates with lethality and in vivo neutralization of a large number of elapid neurotoxic snake venoms from four continents

• Main Authors: Pruksaphon, Kritsada, Tan, Kae Yi, Tan, Choo Hock, Simsiriwong, Pavinee, Gutierrez, Jose Maria, Ratanabanangkoon, Kavi
• Format: Article
• Published: Public Library of Science 2020
• Subjects: R Medicine
• Online Access: http://eprints.um.edu.my/36525/
• Institution: Universiti Malaya

The aim of this study was to develop anin vitroassay for use in place ofin vivoassays of snake venom lethality and antivenom neutralizing potency. A novelin vitroassay has been developed based on the binding of post-synaptically acting alpha-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with thein vivomurine lethality tests (Median Lethal Dose, LD50), and the neutralization of lethality assays (Median Effective Dose, ED50) by antisera againstNaja kaouthia,Naja najaandBungarus candidusvenoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, thein vitromedian inhibitory concentrations (IC50s) for alpha-neurotoxin binding to purified nAChR correlated well with thein vivoLD(50s)of the venoms (R-2= 0.8526,p< 0.001). Furthermore, using this assay, thein vitroED(50s)of a horse pan-specific antiserum against these venoms correlated significantly with the correspondingin vivomurine ED(50)s, with R-2= 0.6896 (p< 0.01). In the case of four elapid venoms devoid or having a very low concentration of alpha-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, thein vitro alpha-neurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which alpha-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variablein vitroassay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world. Author summary Snakebite envenomation is an important public health problem recognized by the World Health Organization (WHO) as a neglected tropical disease affecting about 2 million of poor people of the tropical world. The most effective therapy is the timely administration of efficacious antivenoms which are usually produced in horses. The serum/plasma of horse immunized with snake venoms is purified and tested for its efficacies in neutralizing the target venoms. The neutralization is assayed using mice injected with the venom together with the antivenom. This assay requires about 60 mice for each pair of venom and antivenom. The assay is expensive, laborious, giving highly variable results and is objected on ethical and religious grounds. The present study involves the development of anin vitroassay involving the binding of a snake neurotoxin to a soluble receptor protein called nicotinic acetylcholine receptor. It is shown here that this receptor binding assay gave good correlation with the assay using mice. The test tube assay is simpler, faster, cheaper and less variable when compared with the mouse assay and thus could reduce or even replace the use of life animal. Furthermore, it could expedite the development of effective antivenoms against various venomous snakes in many parts of the world.