Tags

Type your tag names separated by a space and hit enter

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 11 19; 13(11)T

Abstract

Naja atra, 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 N. atra 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 N. atra venom (neurotoxin (NTX), phospholipase A2 (PLA2), 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 (p < 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 N. atra.

Authors+Show Affiliations

Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.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.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.Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 33302, Taiwan.Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei 11561, Taiwan.Center for Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei 11561, Taiwan.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.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

34822602

Citation

Liu, Chien-Chun, et al. "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, vol. 13, no. 11, 2021.
Liu CC, Hsiao YC, Chu LJ, et al. 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;13(11).
Liu, C. C., Hsiao, Y. C., Chu, L. J., Wang, P. J., Liu, C. H., Hsieh, W. C., & Yu, J. S. (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, 13(11). https://doi.org/10.3390/toxins13110818
Liu CC, et al. 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 11 19;13(11) PubMed PMID: 34822602.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Liu,Chien-Chun, AU - Hsiao,Yung-Chin, AU - Chu,Lichieh Julie, AU - Wang,Po-Jung, AU - Liu,Chien-Hsin, AU - Hsieh,Wen-Chin, AU - Yu,Jau-Song, Y1 - 2021/11/19/ PY - 2021/10/15/received PY - 2021/11/11/revised PY - 2021/11/16/accepted PY - 2021/11/25/entrez PY - 2021/11/26/pubmed PY - 2022/2/23/medline KW - Naja atra KW - epitope KW - freeze-dried neurotoxic antivenom KW - neurotoxin KW - neutralization potency KW - peptide-based ELISA JF - Toxins JO - Toxins (Basel) VL - 13 IS - 11 N2 - Naja atra, 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 N. atra 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 N. atra venom (neurotoxin (NTX), phospholipase A2 (PLA2), 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 (p < 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 N. atra. SN - 2072-6651 UR - https://www.unboundmedicine.com/medline/citation/34822602/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_ L2 - https://www.mdpi.com/resolver?pii=toxins13110818 DB - PRIME DP - Unbound Medicine ER -