Development of protective agent against Hottentotta saulcyi venom using camelid single-domain antibody

• Successful immunization of camel with Iranian Hottentotta saulcyi scorpion venom.
• Venom fractionation using Sephadex G-50 column chromatography to obtain major toxic fraction.
• Preparation of a library from camel heavy chain antibodies using phage display technology.
• Selection a Nanobody (Nb12) capable of recognizing the venom components by phage ELISA.
• Evaluation of protective effect of Nb12 against whole soluble venom injected in mice at 2 LD values in pre-incubated and challenge rescue experiments.

Hottentotta saulcyi, medically important scorpion species, causes some of harmful toxic exposure in Iran. Administrated, conventional antivenom-based immunotherapy is still limited and hardly meet ideal characteristic of effective treatment for scorpion envenomation. In this study we aimed to develop a neutralizing agent directed against scorpion venom based on VHH, variable domain of the Camelidae heavy chain antibody or Nanobody. This promising biomolecule is well-established as an advantageous tool for therapeutic purposes due to its small size, stability, monomeric performance and less immunogenicity. In this study, a large Nb library was constructed and phage displayed after successful camel immunization using H. saulcyi scorpion crude venom. After a series of biopanning rounds on Sephadex G50 purified venom fraction and screening by monoclonal phage ELISA, the best reactive Nb was retrieved and designated Nb12. The selected Nb was then expressed as soluble protein in Escherichia coli, purified and confirmed by SDS-PAGE analysis and western blotting. The lead candidate Nb12 bound scorpion venom with Kaff value of 5 × 107 M−1. Nb12 was shown to be capable of neutralizing 2 LD50 of whole venom of scorpion toxin when injected in the ratio of the Nb/toxin of 1.4:1 into C57BL/6 mice. In challenge experiment, Nb succeeded to rescue all i.p. lethal dose injected mice even when administrated i.v., 20 min after envenoming. These results with ease of production and superior neutralizing activity make Nb a suitable anti-toxin candidate for treatment of scorpion envenoming.