Purification of F(ab’)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield.
Abstract: Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.
Publication Date: 1989-01-01 PubMed ID: 2728022DOI: 10.1016/0041-0101(89)90177-3Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research explored a rapid method to procure highly effective, pure, and substantial IgG fragment quantities using caprylic acid to treat horse plasma with antibodies against venom from the Crotalus durissus terrificus snake. The performance was compared with those purified using the prevalent ammonium sulphate precipitation method and the uncleaved IgG purified with caprylic acid.
Experiment Design
- The researchers first collected horse plasma enriched with antibodies against the Crotalus durissus terrificus venom.
- This pooled horse plasma was digested using pepsin using an enzyme-to-substrate ratio of 8:1 at pH 3.1 for 40 minutes.
- The digested plasma was treated with 8.7% caprylic acid in a solution of pH 5.0 to purify F(ab’)2M fragments.
- For comparison, F(ab’)2B fragments were purified by precipitation with ammonium sulphate and uncleaved IgG fragments were purified using caprylic acid.
- Finally, Fab’ fragments were obtained using reduction and alkylation of the F(ab’)2B fragments.
Numerical Findings
- The venom-antibody titers measured by Dot-Blot were 12,800 for native IgG, 6,400 for F(ab’)2B, 4,800 for F(ab’)2M, and 3,200 for Fab’B.
- The researchers found that 1 milligram of F(ab’)2B, F(ab’)2M, or Fab’B could neutralize 20.7 µg, 20.2 µg, or 13.8 µg of Crotalus durissus terrificus venom, respectively.
Immunochemical Analysis
- The treated plasma fragments were subjected to SDS gel electrophoresis and Western blot for molecular analysis. It was observed that F(ab’)2M samples were free from IgG and other plasma proteins, unlike F(ab’)2B.
- The double Immunodiffusion technique was also used to detect discrepancies in the molecular structure of raw and treated plasma antibodies.
Interpretation and Implications
- This study indicated that purification of plasma samples using caprylic acid yielded fragments with high neutralizing activity against snake venom.
- This method of purification offers a rapid, efficient, and reliable approach to obtaining large quantities of IgG fragments from horse plasma that can prove highly useful in antivenom production.
- While pure and efficient, the F(ab’)2M fragments displayed lower venom-neutralizing titers than IgG, showing that further research is needed to amplify their potency.
Cite This Article
APA
dos Santos MC, D'Império Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W.
(1989).
Purification of F(ab’)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield.
Toxicon, 27(3), 297-303.
https://doi.org/10.1016/0041-0101(89)90177-3 Publication
Researcher Affiliations
- Fundação Ezequiel Dias, Universidade Federal de Minas Gerais, Brazil.
MeSH Terms
- Animals
- Antibody Specificity
- Antivenins / isolation & purification
- Blotting, Western
- Caprylates / pharmacology
- Crotalid Venoms / immunology
- Electrophoresis, Polyacrylamide Gel
- Horses / immunology
- Immunoglobulin Fab Fragments / isolation & purification
- Immunoglobulin G / isolation & purification
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Neutralization Tests
Citations
This article has been cited 11 times.- Sánchez A, Cerdas M, Gutiérrez J, Vargas M, Segura Á, Herrera M, Chaves-Araya S, Sánchez R, Villalta M, Durán G, Sánchez A, Solano G, Cordero D, Sánchez P, Gutiérrez JM, León G. Pilot-scale evaluation of a dynamic body-feed filtration system for primary clarification of snake antivenoms produced by the caprylic acid method. Toxicon X 2024 Sep;23:100202.
- Bourel L, Bray F, Vivier S, Flament S, Guilbert L, Chepy A, Rolando C, Launay D, Dubucquoi S, Sobanski V. Comparative Analysis of Laboratory-Scale Immunoglobulin G Purification Methods from Human Serum. J Proteome Res 2024 Sep 6;23(9):3933-3943.
- Norouznejad N, Zolfagharian H, Babaie M, Ghobeh M. Purification of Therapeutic Serums of Snake Anti-Venom with Caprylic Acid. J Pharmacopuncture 2022 Jun 30;25(2):114-120.
- Alomran N, Alsolaiss J, Albulescu LO, Crittenden E, Harrison RA, Ainsworth S, Casewell NR. Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms. PLoS Negl Trop Dis 2021 Aug;15(8):e0009659.
- Kurtović T, Lang Balija M, Brgles M, Sviben D, Tunjić M, Cajner H, Marchetti-Deschmann M, Allmaier G, Halassy B. Refinement strategy for antivenom preparation of high yield and quality. PLoS Negl Trop Dis 2019 Jun;13(6):e0007431.
- Megale ÂAA, Magnoli FC, Kuniyoshi AK, Iwai LK, Tambourgi DV, Portaro FCV, da Silva WD. Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities. J Venom Anim Toxins Incl Trop Dis 2018;24:38.
- Guidolin FR, Caricati CP, Marcelino JR, da Silva WD. Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique. PLoS Negl Trop Dis 2016 Jan;10(1):e0004325.
- Khamehchian S, Zolfagharian H, Dounighi NM, Tebianian M, Madani R. Study on camel IgG purification: a new approach to prepare Naja Naja Oxiana antivenom as passive immunization for therapy. Hum Vaccin Immunother 2014;10(6):1633-8.
- Squaiella-Baptistão CC, Marcelino JR, Ribeiro da Cunha LE, Gutiérrez JM, Tambourgi DV. Anticomplementary activity of horse IgG and F(ab')2 antivenoms. Am J Trop Med Hyg 2014 Mar;90(3):574-84.
- Vargas M, Segura A, Herrera M, Villalta M, Estrada R, Cerdas M, Paiva O, Matainaho T, Jensen SD, Winkel KD, León G, Gutiérrez JM, Williams DJ. Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea. PLoS Negl Trop Dis 2011 May;5(5):e1144.
- Burnouf T, Griffiths E, Padilla A, Seddik S, Stephano MA, Gutiérrez JM. Assessment of the viral safety of antivenoms fractionated from equine plasma. Biologicals 2004 Sep;32(3):115-28.
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