FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Toxins (Basel) / Development of Antibody Detection ELISA Based on Immunoreact...
Toxins2021; 13(11); 818; doi: 10.3390/toxins13110818

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.

Abstract: , also known as Taiwanese cobra, is one of the most prevalent venomous snakes in Taiwan. Clinically, freeze-dried neurotoxic antivenom (FNAV) produced from horses by Taiwan Centers for Disease Control (CDC) has been the only approved treatment for envenoming for the last few decades. During antivenom production, large numbers of mice are used in the in vivo assay to determine whether the neutralization potency of hyperimmunized equines is satisfactory for large-scale harvesting. However, this in vivo assay is extremely laborious, expensive, and significantly impairs animal welfare. In the present study, we aimed to develop an in vitro ELISA-based system that could serve as an alternative assay to evaluate the neutralization potency of plasma from hyperimmunized equines. We initially obtained 51 plasma samples with known (high or low) neutralization potency assessed in vivo from 9 hyperimmunized equines and subsequently determined their antibody titers against the five major protein components of venom (neurotoxin (NTX), phospholipase A2 (PLA), cytotoxin (CTX), cysteine-rich secretory protein (CRISP), and snake venom metalloproteinase (SVMP)) via ELISA. The antibody titer against NTX was the most effective in discriminating between high and low potency plasma samples. To identify the specific epitope(s) of NTX recognized by neutralization potency-related antibodies, 17 consecutive NTX-derived pentadecapeptides were synthesized and used as antigens to probe the 51 equine plasma samples. Among the 17 peptides, immunoreactive signals for three consecutive peptides (NTX1-8, NTX1-9, and NTX1-10) were significantly higher in the high potency relative to low potency equine plasma groups ( < 0.0001). Our ELISA system based on NTX1-10 peptide (RWRDHRGYRTERGCG) encompassing residues 28-42 of NTX displayed optimal sensitivity (96.88%) and specificity (89.47%) for differentiating between high- and low-potency plasma samples (area under the receiver operating characteristic curve (AUC) = 0.95). The collective data clearly indicate that the antibody titer against NTX protein or derived peptides can be used to efficiently discriminate between high and low neutralization potency of plasma samples from venom-immunized horses. This newly developed antibody detection ELISA based on NTX or its peptide derivatives has good potential to complement or replace the in vivo rodent assay for determining whether the neutralization potency of equine plasma is satisfactory for large-scale harvesting in the antivenom production process against
Get Full Text
Publication Date: 2021-11-19 PubMed ID: 34822602PubMed Central: PMC8622849DOI: 10.3390/toxins13110818Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study discusses the development of a lab-based method (ELISA) to gauge the effectiveness of antivenom from hyperimmunised horses against the venom of Naja atra, a Taiwanese cobra. This method aims to replace the current procedure that uses mice, which is manpower intensive, costly, and raises animal welfare concerns.

Background

  • The Taiwanese cobra (Naja atra), a venomous snake prevalent in Taiwan, needs a specific antivenom for effective treatment after an encounter.
  • Currently, this antivenom is produced from horse plasma by the Taiwan Centers for Disease Control, and its potency (i.e., effectiveness) is tested using live mice.
  • This in vivo testing method is not only time-consuming and costly but also adds to animal suffering.

Objective of the Study

  • The researchers aim to find an alternative method for checking the serum’s anti-venom potency developed from hyperimmunized horses.
  • They propose an in vitro (lab-based) enzyme-linked immunosorbent assay (ELISA) system which determines the level of antibodies (that could neutralize the venom) in the horse plasma.

Method and Findings

  • For this study, they collected 51 plasma samples from nine hyperimmunized horses that were known to have high or low neutralization potency.
  • They then checked the antibody levels in these samples against five main elements of cobra venom using ELISA.
  • They found that the antibody against one particular toxin (neurotoxin) was best at distinguishing between high- and low-potency samples.
  • Also, they found specific regions of this neurotoxin (known as epitopes) that the neutralizing antibodies would recognize.
  • Initial findings show that their ELISA system, based on this neurotoxin or its derivatives, has high sensitivity and specificity for classifying high- and low-potency plasma samples.

Conclusion

  • This research paves the way for a new method of assessing the neutralizing potential of horse-plasma-based antivenom against Naja atra.
  • This method, rooted in ELISA systems, works by identifying the presence and extent of antibodies against the neurotoxin component of the venom.
  • As it moves away from the use of mice for testing, it represents an improvement in both cost and animal welfare, while retaining accuracy and efficacy.
  • This new method has the promise to augment or even replace in vivo testing in the large-scale production of antivenom.

Cite This Article

APA
Liu CC, Hsiao YC, Chu LJ, Wang PJ, Liu CH, Hsieh WC, Yu JS. (2021). 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), 13(11), 818. https://doi.org/10.3390/toxins13110818

Publication

Toxins
ISSN: 2072-6651
NlmUniqueID: 101530765
Country: Switzerland
Language: English
Volume: 13
Issue: 11
PII: 818

Researcher Affiliations

Liu, Chien-Chun
  • Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.
Hsiao, Yung-Chin
  • Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Liver Research Center, Chang Gung Memorial Hospital, Linkou, Tao-Yuan 33305, Taiwan.
Chu, Lichieh Julie
  • Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Liver Research Center, Chang Gung Memorial Hospital, Linkou, Tao-Yuan 33305, Taiwan.
Wang, Po-Jung
  • Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.
Liu, Chien-Hsin
  • Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei 11561, Taiwan.
Hsieh, Wen-Chin
  • Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei 11561, Taiwan.
Yu, Jau-Song
  • Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan.
  • Liver Research Center, Chang Gung Memorial Hospital, Linkou, Tao-Yuan 33305, Taiwan.
  • Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, Tao-Yuan 33303, Taiwan.

MeSH Terms

  • Animals
  • Antibodies, Neutralizing / immunology
  • Antivenins / immunology
  • Cobra Neurotoxin Proteins / immunology
  • Elapid Venoms / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Horses
  • Male
  • Mice
  • Mice, Inbred ICR
  • Naja naja
  • Peptides / immunology

Conflict of Interest Statement

The authors have declared that no competing interests exist.

References

This article includes 34 references
  1. Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming.. Nat Rev Dis Primers 2017 Oct 5;3:17079.
    doi: 10.1038/nrdp.2017.79pubmed: 28980622google scholar: lookup
  2. Chang KP, Lai CS, Lin SD. Management of poisonous snake bites in southern Taiwan.. Kaohsiung J Med Sci 2007 Oct;23(10):511-8.
    doi: 10.1016/S1607-551X(08)70009-3pubmed: 18055297google scholar: lookup
  3. Hung DZ. Taiwan's venomous snakebite: epidemiological, evolution and geographic differences.. Trans R Soc Trop Med Hyg 2004 Feb;98(2):96-101.
    doi: 10.1016/S0035-9203(03)00013-0pubmed: 14964809google scholar: lookup
  4. Cichutek K, Epstein J, Griffiths E, Hindawi S, Jivapaisarnpong T, Klein H, Minor P, Moftah F, Reddy VR, Slamet LS. WHO Expert Committee on Biological Standardization Sixty-seventh report. WHO Tech. Rep. Ser. 2017;1004:1–591.
  5. Chieh-Fan C, Tzeng-Jih L, Wen-Chi H, Hua-Wei Y. Appropriate Antivenom Doses for Six Types of Envenomations Caused by Snakes in Taiwan. J. Venom. Anim. Toxins 2009;15:479–490.
    doi: 10.1590/S1678-91992009000300009google scholar: lookup
  6. Liau MY, Huang RJ. Toxoids and antivenoms of venomous snakes in Taiwan. J. Toxicol.-Toxin Rev. 1997;16:163–175.
  7. Sells PG. Animal experimentation in snake venom research and in vitro alternatives.. Toxicon 2003 Aug;42(2):115-33.
    doi: 10.1016/S0041-0101(03)00125-9pubmed: 12906883google scholar: lookup
  8. Gutiérrez JM, Solano G, Pla D, Herrera M, Segura Á, Vargas M, Villalta M, Sánchez A, Sanz L, Lomonte B, León G, Calvete JJ. Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.. Toxins (Basel) 2017 May 13;9(5).
    doi: 10.3390/toxins9050163pmc: PMC5450711pubmed: 28505100google scholar: lookup
  9. Milovanovic V, Dimitrijevic L, Petrusic V, Kadric J, Minic R, Zivkovic I. Application of the 3r Concept in the Production of European Antiviperinum on Horses—Multisite, Low Volumes Immunization Protocol and Elisa. Acta Vet.-Beograd 2018;68:401–419.
    doi: 10.2478/acve-2018-0033google scholar: lookup
  10. Theakston RD, Reid HA. Enzyme-linked immunosorbent assay (ELISA) in assessing antivenom potency.. Toxicon 1979;17(5):511-5.
    doi: 10.1016/0041-0101(79)90284-8pubmed: 516083google scholar: lookup
  11. Rungsiwongse J, Ratanabanangkoon K. Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom.. J Immunol Methods 1991 Jan 24;136(1):37-43.
    doi: 10.1016/0022-1759(91)90247-Dpubmed: 1995711google scholar: lookup
  12. Maria WS, Cambuy MO, Costa JO, Velarde DT, Chávez-Olórtegui C. Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods.. Toxicon 1998 Oct;36(10):1433-9.
    doi: 10.1016/S0041-0101(98)00077-4pubmed: 9723841google scholar: lookup
  13. Heneine LG, Carvalho AD Jr, Barbosa CF, Arávjo dos Santos MR. Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom.. Toxicon 1998 Oct;36(10):1363-70.
    doi: 10.1016/S0041-0101(98)00014-2pubmed: 9723835google scholar: lookup
  14. Ramada JS, Becker-Finco A, Minozzo JC, Felicori LF, Machado de Avila RA, Molina F, Nguyen C, de Moura J, Chávez-Olórtegui C, Alvarenga LM. Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.. Toxicon 2013 Oct;73:47-55.
    doi: 10.1016/j.toxicon.2013.07.007pubmed: 23856101google scholar: lookup
  15. Khaing EM, Hurtado PR, Hurtado E, Zaw A, White J, Warrell DA, Alfred S, Mahmood MA, Peh CA. Development of an ELISA assay to determine neutralising capacity of horse serum following immunisation with Daboia siamensis venom in Myanmar.. Toxicon 2018 Sep 1;151:163-168.
    doi: 10.1016/j.toxicon.2018.07.012pubmed: 30017790google scholar: lookup
  16. Liu CC, Chou YS, Chen CY, Liu KL, Huang GJ, Yu JS, Wu CJ, Liaw GW, Hsieh CH, Chen CK. Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models.. PLoS Negl Trop Dis 2020 Feb;14(2):e0008054.
    doi: 10.1371/journal.pntd.0008054pmc: PMC7032728pubmed: 32032357google scholar: lookup
  17. Mandrekar JN. Receiver operating characteristic curve in diagnostic test assessment.. J Thorac Oncol 2010 Sep;5(9):1315-6.
    doi: 10.1097/JTO.0b013e3181ec173dpubmed: 20736804google scholar: lookup
  18. Yu C, Bhaskaran R, Chuang LC, Yang CC. Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study.. Biochemistry 1993 Mar 9;32(9):2131-6.
    doi: 10.1021/bi00060a002pubmed: 8443154google scholar: lookup
  19. Connolly PJ, Stern AS, Hoch JC. Solution structure of LSIII, a long neurotoxin from the venom of Laticauda semifasciata.. Biochemistry 1996 Jan 16;35(2):418-26.
    doi: 10.1021/bi9520287pubmed: 8555211google scholar: lookup
  20. Kini RM, Doley R. Structure, function and evolution of three-finger toxins: mini proteins with multiple targets.. Toxicon 2010 Nov;56(6):855-67.
    doi: 10.1016/j.toxicon.2010.07.010pubmed: 20670641google scholar: lookup
  21. Rial A, Morais V, Rossi S, Massaldi H. A new ELISA for determination of potency in snake antivenoms.. Toxicon 2006 Sep 15;48(4):462-6.
    doi: 10.1016/j.toxicon.2006.07.004pubmed: 16893558google scholar: lookup
  22. Chander R, Batra M, Singh D, Kumar Y, Rawat S, Kumar S. A new in-vitro agglutination technique for potency estimation of antisnake venom serum (ASVS).. Toxicon 2006 Dec 15;48(8):1011-7.
    doi: 10.1016/j.toxicon.2006.08.007pubmed: 16982078google scholar: lookup
  23. 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.
    doi: 10.1038/s41598-017-08962-3pmc: PMC5561211pubmed: 28819275google scholar: lookup
  24. 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.
    doi: 10.1038/s41598-018-27794-3pmc: PMC6018763pubmed: 29946111google scholar: lookup
  25. Alape-Girón A, Miranda-Arrieta K, Cortes-Bratti X, Stiles BG, Gutiérrez JM. A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus.. Toxicon 1997 Apr;35(4):573-81.
    doi: 10.1016/S0041-0101(96)00150-Xpubmed: 9133712google scholar: lookup
  26. Steward MW, Lew AM. The importance of antibody affinity in the performance of immunoassays for antibody.. J Immunol Methods 1985 Apr 22;78(2):173-90.
    doi: 10.1016/0022-1759(85)90074-2pubmed: 2580911google scholar: lookup
  27. Chang LS, Chou YC, Lin SR, Wu BN, Lin J, Hong E, Sun YJ, Hsiao CD. A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: purification, characterization, and gene organization.. J Biochem 1997 Dec;122(6):1252-9.
    doi: 10.1093/oxfordjournals.jbchem.a021889pubmed: 9498573google scholar: lookup
  28. Fruchart-Gaillard C, Gilquin B, Antil-Delbeke S, Le Novère N, Tamiya T, Corringer PJ, Changeux JP, Ménez A, Servent D. Experimentally based model of a complex between a snake toxin and the alpha 7 nicotinic receptor.. Proc Natl Acad Sci U S A 2002 Mar 5;99(5):3216-21.
    doi: 10.1073/pnas.042699899pmc: PMC122499pubmed: 11867717google scholar: lookup
  29. Teixeira-Clerc F, Ménez A, Kessler P. How do short neurotoxins bind to a muscular-type nicotinic acetylcholine receptor?. J Biol Chem 2002 Jul 12;277(28):25741-7.
    doi: 10.1074/jbc.M200534200pubmed: 12006581google scholar: lookup
  30. Sunagar K, Jackson TN, Undheim EA, Ali SA, Antunes A, Fry BG. Three-fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of snake venom toxins.. Toxins (Basel) 2013 Nov 18;5(11):2172-208.
    doi: 10.3390/toxins5112172pmc: PMC3847720pubmed: 24253238google scholar: lookup
  31. 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).
    doi: 10.3390/toxins10010010pmc: PMC5793097pubmed: 29295601google scholar: lookup
  32. Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid.. Arch Biochem Biophys 1969 Nov;134(2):279-84.
    doi: 10.1016/0003-9861(69)90285-9pubmed: 4982185google scholar: lookup
  33. Villalta M, Pla D, Yang SL, Sanz L, Segura A, Vargas M, Chen PY, Herrera M, Estrada R, Cheng YF, Lee CD, Cerdas M, Chiang JR, Angulo Y, León G, Calvete JJ, Gutiérrez JM. Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: keys to understand the variable immune response in horses.. J Proteomics 2012 Oct 22;75(18):5628-45.
    doi: 10.1016/j.jprot.2012.08.008pubmed: 22906718google scholar: lookup
  34. Howard-Jones N. A CIOMS ethical code for animal experimentation.. WHO Chron 1985;39(2):51-6.
    pubmed: 4090462

Citations

This article has been cited 3 times.
  1. 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).
    doi: 10.3390/tropicalmed8010051pubmed: 36668958google scholar: lookup
  2. Liu CC, Chou YS, Yang YX, Hsiao YC, Chu LJ, Yang C, Yan Z, Peng R, Chao HY, Li MX, Wu CJ. Enhancing Deinagkistrodon acutus antivenom potency through acutolysin A-targeted antibody supplementation. PLoS Negl Trop Dis 2025 Dec;19(12):e0013847.
    doi: 10.1371/journal.pntd.0013847pubmed: 41385578google scholar: lookup
  3. 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.
    doi: 10.1016/j.toxcx.2024.100195pubmed: 38606385google scholar: lookup
Subscribe to our newsletter
Join 99,359 horse owners like you who receive our email updates on horse health and nutrition.