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.
Highlights
IntroductionSnakebite is a relatively neglected tropical disease affecting more than 1.8 million people worldwide annually [1]
Key Contribution: Here, we have developed an antibody detection ELISA system as an in vitro alternative to evaluate whether the neutralization potency of hyperimmunized equines is adequate for large-scale harvesting
During the freeze-dried neurotoxic antivenom (FNAV) production process, 60 Tanaka units/mL for N. atra venom was defined as the acceptable potency of hyperimmunized equine plasma for large-scale harvesting
Summary
Snakebite is a relatively neglected tropical disease affecting more than 1.8 million people worldwide annually [1]. In Taiwan, approximately 1000 snakebite cases occur each year predominantly by the six major venomous species, Naja atra, Bungarus multicinctus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Deinagkistrodon acutus, and Daboia russelii siamensis [2,3]. N. atra, known as Taiwanese cobra, has been classified as a category 1 World Health Organization (WHO) group in the guidelines for antivenom production [4], and accounts for 23.5–36% of envenoming cases in Taiwan [2,3]. In Taiwan, freeze-dried neurotoxic antivenom (FNAV) is available for clinical treatment of N. atra envenoming [5,6]. Taiwan Centers for Disease Control (CDC) have developed FNAV against N. atra and B. multicinctus, greatly facilitating control of snakebiterelated mortality in Taiwan. During the process of antivenom production, horses are repeatedly immunized with snake venom and an in vivo murine assay is conducted to monitor the neutralization potency of hyperimmune horse plasma at different time-points
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
