Rational design and pairing of single-domain antibodies for developing rapid detection methods for botulinum neurotoxins A and B.

[BACKGROUND] Botulinum neurotoxin (BoNT) is a toxin produced by Clostridium botulinum that can cause neurological dysfunction in humans and animals, with BoNT/A and BoNT/B being responsible for the majority of poisoning cases. Due to its extremely high toxicity, BoNT is considered a potential biological warfare and bioterrorism agent. Currently, there is a lack of rapid and highly sensitive detection methods for botulinum toxin, making the development of fast and effective detection methods for BoNT essential. Here, we developed rapid detection methods for BoNT/A and BoNT/B based on nanobodies, including two ELISA methods and Time-resolved fluorescence immunochromatography assay (TRFICA).

[RESULTS] By analyzing the binding epitopes of BoNT/A, BoNT/B, and nanobodies, we rationally designed and prepared six nanobodies, adding a Trx tag at the N-terminus, a 6 × His tag at the C-terminus, and fusing them with HRP, for use in developing two ELISA methods for BoNT/A and BoNT/B detection. Additionally, TRFICA was developed in combination with lanthanide microspheres. These three methods were validated using simulated samples containing BoNT in milk, sand, and human serum. The limits of detection for ELISA 1 and 2 were 0.17 and 4.5 ng/mL, respectively, while the TRFICA had a detection limit of 0.05 ng/mL and provided results in approximately 15 min. TRFICA also successfully detected clinical samples, with a sensitivity of 98 % and a specificity of 96.7 %.

[SIGNIFICANCE] Overall, the TRFICA method we established has a short detection time, is easy to operate, and effectively addresses the challenge of rapid BoNT detection in clinical diagnostics, with significant clinical application value. Furthermore, based on the sequences and binding epitopes of nanobodies, we rationally designed six nanobodies and successfully applied them in the development of various BoNT detection methods. This approach and pipeline provide valuable guidance for the future development of rapid, low-cost detection methods.