Recombinant synthesis and immunological characterization of single-domain antibodies against botulinum neurotoxin serotype A and B

Nanobodies which are single-domain antibodies derived from the Variable Heavy domain of Heavy-chain-only antibodies (VHHs) found in camels and sharks, represent a novel approach for enhancing the detection and neutralization of food toxins. Botulinum neurotoxin (BoNT), a potent neurotoxin produced by Clostridium botulinum, can cause severe poisoning even in minimal amounts (approximately 1 - 2 ng/kg), necessitating the development of highly sensitive detection methods. In this study, we recombinantly synthesized the nanobodies ALc-B8 (A8) and JNE-B10 (J10), which specifically bind to the light chains of BoNT type A (BoNT/A) and BoNT type B (BoNT/B), respectively. Optimal conditions for expressing nanobodies A8 (33 kDa) and J10 (32 kDa) were identified using the pET28a vector in Escherichia coli  BL21 (DE3) RIL bacteria, with induction by 0.5 mM IPTG at an OD600 of approximately 0.6 - 0.8 for 16 - 18 hours at 16℃. The purified nanobodies A8 and J10, yielding 10 - 20 mg/L of culture, were able to specifically recognize the recombinant BoNT/A and BoNT/B light chains at a concentration of 100 μg/mL in Western blotting. These findings provide a foundation for the application of these nanobodies in the detection and neutralization of botulinum toxins.