Development and selection of candidate monoclonal antibodies for the detection of Clostridium botulinum neurotoxin serotype B light chain

Botulinum neurotoxin, produced by the bacterium Clostridium botulinum, causes botulism, a severe, rapidly progressing, and potentially fatal condition. Swift detection of the toxin and timely administration of antitoxin antibodies are critical for effective treatment. The current standard for Botulinum toxin testing is the mouse lethality assay, but this method is time-consuming and requires live animals. Consequently, a key focus of research is the development of antibodies for both diagnostic purposes and toxin neutralization. Botulinum neurotoxin serotype B (BoNT/B), one of the most dangerous and prevalent serotypes, is commonly involved in poisoning cases. Like other botulinum toxins, BoNT/B consists of heavy and light chains. In this study, we generated mouse monoclonal antibodies targeting the BoNT/B light chain (BoNT/B-LC) through hybridoma cell line development. Two monoclonal hybridomas (3B7 and 3C6) were selected from a pool of 18 polyclonal hybridomas and used to produce anti-BoNT/B-LC antibodies through the ascites fluid production. The antibodies were utilized for indirect ELISA detection of recombinant BoNT/B-LC. Notably, the assay with 3B7 demonstrated higher sensitivity, allowing for the detection of TrxA-fused BoNT/B-LC (68.9 kDa) at concentrations as low as 4 ng/mL. These results highlight the potential of the generated antibodies for rapid BoNT/B detection, offering a promising alternative to animal-based testing.