Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods.
Abstract: The correlation coefficients between in vivo neutralization of lethal toxicity (ED50), neutralization of the hemolytic activity (PLA2) and levels of antibodies measured by ELISA, was investigated to test the potency of horse anti-bothropic antivenom. Twenty six horses were hyperimmunized with Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, for neutralization of PLA2 activity and for determination of ED50 in Swiss mice, the whole Bothrops jararaca venom (reference venom for assessing the bothropic antivenom potency in Brazil) was used. The toxic fraction (purified from B. jararaca venom by Sephadex G-100 chromatography) was also used as antigen for ELISA. All antivenoms analyzed effectively neutralized the lethal activity in the range of 1.6 to 9.6 mg/ml of antivenom. The correlation coefficient between ED50 and ELISA antibody titers against the crude venom and toxic fraction was r = 0.65 (P < 0.001) and r = 0.85 (P < 0.0001), respectively. Correlation between ED50 and neutralization of PLA2 activity was r = 0.52 (P < 0.01), and the correlation between ELISA antibody titers and neutralization of PLA2 activity was r = 0.58 (P < 0.002). Thus, the ELISA which measures only the antibody against the major toxic fraction of the B. jararaca venom should be most suitable for use as an in vitro assay of bothropic antivenom potency.
Publication Date: 1998-09-02 PubMed ID: 9723841DOI: 10.1016/s0041-0101(98)00077-4Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the effectiveness of horse anti-bothropic antivenom in neutralizing the venom of the Bothrops snake species. The potency of the antivenom is tested via various methods, including lethality neutralization and hemolytic activity neutralization.
Research Methodology
- The study involved twenty-six horses that were hyperimmunized with Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. neuwiedii, and B. moojeni).
- An indirect ELISA (Enzyme-Linked Immunosorbent Assay), neutralization of Phospholipase A2 (PLA2) activity, and the determination of ED50 (the dose that was effective in 50% of the population) in Swiss mice were established using the whole venom of Bothrops jararaca.
- The toxic fraction of the B. jararaca venom, purified through Sephadex G-100 chromatography, was used as an antigen for the ELISA test.
Findings
- All the antivenoms analyzed effectively neutralized the lethal activity in the range of 1.6 to 9.6 mg/ml of antivenom.
- There was a significant correlation between the ED50 and ELISA antibody titers against the crude venom and toxic fraction, with a correlation coefficient of r = 0.65 (P < 0.001) and r = 0.85 (P < 0.0001), respectively.
- Correlation between ED50 and neutralization of PLA2 activity was r = 0.52 (P < 0.01), indicating a significant relationship.
- Moreover, a significant correlation of r = 0.58 (P < 0.002) was found between ELISA antibody titers and neutralization of PLA2 activity.
Conclusions
- The study suggests using ELISA to measure only the antibody against the major toxic fraction of the B. jararaca venom as the most suitable method for assessing the potency of bothropic antivenom in vitro.
The research is important as it provides a potentially effective method for testing the potency of antivenom in a controlled environment, which could pave the way for more efficient snakebite treatment protocols.
Cite This Article
APA
Maria WS, Cambuy MO, Costa JO, Velarde DT, Chávez-Olórtegui C.
(1998).
Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods.
Toxicon, 36(10), 1433-1439.
https://doi.org/10.1016/s0041-0101(98)00077-4 Publication
Researcher Affiliations
- Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil.
MeSH Terms
- Animals
- Antivenins / immunology
- Antivenins / pharmacology
- Biological Assay
- Bothrops / immunology
- Crotalid Venoms / immunology
- Crotalid Venoms / toxicity
- Enzyme-Linked Immunosorbent Assay
- Hemolysis / drug effects
- Horses / immunology
- In Vitro Techniques
- Lethal Dose 50
- Mice
- Neutralization Tests
- Phospholipases A / antagonists & inhibitors
- Phospholipases A2
Citations
This article has been cited 9 times.- Wu CJ, Liaw GW, Chen CK, Ouyang CH, Yang YX, Chu LC, Hsiao YC, Liu CH, Hsieh WC, Wang CY, Liou YS, Liu CC, Hsieh CH. Immunoprofiling of Equine Plasma against Deinagkistrodon acutus in Taiwan: Key to Understanding Differential Neutralization Potency in Immunized Horses. Trop Med Infect Dis 2023 Jan 9;8(1).
- Bhatia S, Blotra A, Vasudevan K. Evaluating Antivenom Efficacy against Echis carinatus Venoms-Screening for In Vitro Alternatives. Toxins (Basel) 2022 Jul 13;14(7).
- Sachetto ATA, Miyamoto JG, Tashima AK, de Souza AO, Santoro ML. The Bioflavonoids Rutin and Rutin Succinate Neutralize the Toxins of B. jararaca Venom and Inhibit its Lethality. Front Pharmacol 2022;13:828269.
- Liu CC, Hsiao YC, Chu LJ, Wang PJ, Liu CH, Hsieh WC, Yu JS. Development of Antibody Detection ELISA Based on Immunoreactive Toxins and Toxin-Derived Peptides to Evaluate the Neutralization Potency of Equine Plasma against Naja atra in Taiwan. Toxins (Basel) 2021 Nov 19;13(11).
- Gutiérrez JM, Vargas M, Segura Á, Herrera M, Villalta M, Solano G, Sánchez A, Herrera C, León G. In Vitro Tests for Assessing the Neutralizing Ability of Snake Antivenoms: Toward the 3Rs Principles. Front Immunol 2020;11:617429.
- Pruksaphon K, Tan KY, Tan CH, Simsiriwong P, Gutiérrez JM, Ratanabanangkoon K. 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. PLoS Negl Trop Dis 2020 Aug;14(8):e0008581.
- Ratanabanangkoon K, Simsiriwong P, Pruksaphon K, Tan KY, Chantrathonkul B, Eursakun S, Tan CH. An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja. Sci Rep 2018 Jun 26;8(1):9716.
- Liu BS, Wu WG, Lin MH, Li CH, Jiang BR, Wu SC, Leng CH, Sung WC. Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom. Toxins (Basel) 2017 Dec 25;10(1).
- Ratanabanangkoon K, Simsiriwong P, Pruksaphon K, Tan KY, Eursakun S, Tan CH, Chantrathonkul B, Wongwadhunyoo W, Youngchim S, Tan NH. A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding. Sci Rep 2017 Aug 17;7(1):8545.
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