Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells

Toxins (Basel). 2022 Jul 4;14(7):459. doi: 10.3390/toxins14070459.

Abstract

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.

Keywords: Naja atra; cobra venom; cytotoxicity; cytotoxin (CTX); necrosis; single-chain variable fragment (scFv).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antivenins / pharmacology
  • Chickens
  • Cytotoxins
  • Elapid Venoms / toxicity
  • Elapidae
  • Female
  • Myoblasts
  • Naja naja*
  • Necrosis
  • Single-Chain Antibodies* / pharmacology

Substances

  • Antivenins
  • Cytotoxins
  • Elapid Venoms
  • Single-Chain Antibodies

Grants and funding

This work was supported by the Ministry of Science and Technology, Taiwan (grant MOST 110-2314-B-182A-012 to C.-K.C. and MOST 109-2320-B-182-009-MY3 to J.-S.Y.); and by Chang Gung Memorial Hospital, Taiwan (grant CMRPG3L0651, CMRPG3M0241 to C.-K.C. and CMRPG3L1231 to C.-J.W.). This research was also supported by the “Molecular Medicine Research Center, Chang Gung University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.