The Taiwanese cobra, Naja atra, is a clinically significant species of snake observed in the wild in Taiwan. Victims bitten by N. atra usually experience severe pain and local tissue necrosis. Although antivenom is available for treatment of cobra envenomation, its neutralization potency against cobra-induced necrosis is weak, with more than 60% of cobra envenoming patients developing tissue necrosis after antivenom administration. The present study found that cytotoxin (CTX) is a key component of N. atra venom responsible for cytotoxicity against myoblast cells. Anti-CTX IgY was generated in hens, and the spleens of these hens were used to construct libraries for the development of single chain variable fragments (scFv). Two anti-CTX scFv, S1 and 2S7, were selected using phage display technology and biopanning. Both polyclonal IgY and monoclonal scFv S1 reacted specifically with CTX in cobra venom. In a cell model assay, the CTX-induced cytolytic effect was inhibited only by monoclonal scFv S1, not by polyclonal IgY. Moreover, the neutralization potency of scFv S1 was about 3.8 mg/mg, approximately three times higher than that of conventional freeze-dried neurotoxic antivenom (FNAV). Collectively, these results suggest that scFv S1 can effectively neutralize CTX-induced cytotoxicity and, when combined with currently available antivenom, can improve the potency of the latter, thereby preventing tissue damage induced by cobra envenoming.

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.