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An in vitro α-neurotoxin—nAChR binding assay correlates with lethality and in vivo neutralization of a large number of elapid neurotoxic snake venoms from four continents
- Source :
- PLoS Neglected Tropical Diseases, PLoS Neglected Tropical Diseases, Vol 14, Iss 8, p e0008581 (2020)
- Publication Year :
- 2020
- Publisher :
- Public Library of Science (PLoS), 2020.
-
Abstract
- The aim of this study was to develop an in vitro assay for use in place of in vivo assays of snake venom lethality and antivenom neutralizing potency. A novel in vitro assay has been developed based on the binding of post-synaptically acting α-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with the in vivo murine lethality tests (Median Lethal Dose, LD50), and the neutralization of lethality assays (Median Effective Dose, ED50) by antisera against Naja kaouthia, Naja naja and Bungarus candidus venoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, the in vitro median inhibitory concentrations (IC50s) for α-neurotoxin binding to purified nAChR correlated well with the in vivo LD50s of the venoms (R2 = 0.8526, p < 0.001). Furthermore, using this assay, the in vitro ED50s of a horse pan-specific antiserum against these venoms correlated significantly with the corresponding in vivo murine ED50s, with R2 = 0.6896 (p < 0.01). In the case of four elapid venoms devoid or having a very low concentration of α-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, the in vitro α-neurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which α-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variable in vitro assay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world.<br />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 an in vitro assay 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.
- Subjects :
- 0301 basic medicine
Nicotinic Acetylcholine Receptors
Physiology
RC955-962
Antivenom
Snake Bites
Receptors, Nicotinic
Pharmacology
Toxicology
Pathology and Laboratory Medicine
Biochemistry
Median lethal dose
Binding Analysis
Mice
0302 clinical medicine
Arctic medicine. Tropical medicine
Medicine and Health Sciences
Toxins
Elapidae
biology
Chemistry
Elapid Venoms
Eukaryota
Snakes
Squamates
Body Fluids
Blood
Infectious Diseases
Snake venom
Vertebrates
Biological Assay
Public aspects of medicine
RA1-1270
Anatomy
Research Article
Signal Transduction
Asia
Transmembrane Receptors
Toxic Agents
Neurotoxins
030231 tropical medicine
Research and Analysis Methods
complex mixtures
03 medical and health sciences
Neutralization Tests
In vivo
Bungarus candidus
Animals
Humans
Horses
Receptor Binding Assays
Chemical Characterization
Antiserum
Toxicity
Venoms
Immune Sera
Ligand binding assay
Organisms
Australia
Public Health, Environmental and Occupational Health
Biology and Life Sciences
Proteins
Reptiles
Cell Biology
Blood Serum
biology.organism_classification
030104 developmental biology
Acetylcholine Receptors
Amniotes
Africa
Americas
Immune Serum
Zoology
Subjects
Details
- ISSN :
- 19352735
- Volume :
- 14
- Database :
- OpenAIRE
- Journal :
- PLOS Neglected Tropical Diseases
- Accession number :
- edsair.doi.dedup.....4951ba1335d2a6829a86d73a7a398455