Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom.
Abstract: The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further chromatographed in a Sephadex G-75 column and again tested for the correlation. Two fractions with haemorrhagic activity displayed a correlation of r = 0.77 and r = 0.8 against the individual horse antivenom sera and of r = 0.79 and r = 0.8 for the ampouled antivenom. For all results p < 0.001. Two other fractions with phospholipase A2 activity showed a correlation of r = 0.66 (p < 0.01) and r = 0.56 (p < 0.03) against the individual horse antivenom sera. Electrophoresis results show a similar composition for both antigens with haemorrhagic activity. Results indicate that the fractions purified would be suitable for the desired objective of this study.
Publication Date: 1998-09-02 PubMed ID: 9723835DOI: 10.1016/s0041-0101(98)00014-2Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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The research article is about the development of a test (enzyme-linked immunosorbent assay or ELISA) for assessing the effectiveness of a variety of horse-derived antivenoms against snake venom.
Objective and Method of the Study
- The main goal of the research was to find a suitable venom antigen that can be used as the basis for an alternative in vitro (in lab condition) immunoassay, a test used to measure the concentration of antibodies in the blood.
- Instead of the standard in vivo (within living organisms) antivenom neutralization assay that involves mice, the researchers wanted to implement an in vitro method, which is more ethical and efficient.
- The researchers used venom from the Bothrops jararaca snake, separated its components using a technique called chromatography, and then tested the different parts to see which ones were able to stimulate the production of antibodies (measured using ELISA) that could neutralize the venom. They examined if there was any correlation between the concentrations of antibodies and the venom potency.
Results of the Study
- The researchers used antivenoms from 14 different horses and 15 separate antivenoms manufactured by FUNED.
- They discovered that two particular fractions of the venom, which have haemorrhagic (causing bleeding) activity, had the highest correlation (r = 0.77 and r = 0.8) with the concentrations of antibodies in the horse antivenom serum.
- These two fractions also showed high correlation (r = 0.79 and r = 0.8) with the ampouled antivenom, indicating that these venom components were effectively neutralized by both types of antivenom.
- Two other venom fractions with an action called phospholipase A2 activity also showed correlation but it was lower (r = 0.66 and r = 0.56).
- A technique called electrophoresis (used for analyzing complex mixtures of protein or DNA) showed that the two venom fractions with haemorrhagic activity had similar compositions.
Implication of the Results
- With high correlation results, the study suggests that the selected fractions (haemorrhagic fractions) can be effectively used in an ELISA procedure to assess the potency of horse therapeutic polyvalent antibothropic antivenom.
- This in vitro method could be an alternative to the standard in vivo method, potentially reducing the need for testing in living animals.
Cite This Article
APA
Heneine LG, Carvalho AD, Barbosa CF, Arávjo dos Santos MR.
(1998).
Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom.
Toxicon, 36(10), 1363-1370.
https://doi.org/10.1016/s0041-0101(98)00014-2 Publication
Researcher Affiliations
- Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Brazil.
MeSH Terms
- Animals
- Antivenins / immunology
- Blood Coagulation / drug effects
- Bothrops
- Chemical Fractionation
- Chromatography, Ion Exchange
- Crotalid Venoms / chemistry
- Crotalid Venoms / immunology
- Crotalid Venoms / toxicity
- Electrophoresis, Polyacrylamide Gel
- Enzyme-Linked Immunosorbent Assay / methods
- Horses
- Lethal Dose 50
- Mice
- Neutralization Tests
- Phospholipases A / metabolism
- Phospholipases A2
Citations
This article has been cited 4 times.- Araya X, Okumu M, Durán G, Gómez A, Gutiérrez JM, León G. Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom. Toxicon X 2024 Jun;22:100195.
- 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.
- 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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