Validation of an Endopep-suspension immunoassay for the diagnostics of human botulism.

Botulism is a potentially life-threatening disease caused by botulinum neurotoxin (BoNT)-producing bacteria of the genus Clostridium. Laboratory detection of BoNTs in patients' samples is essential to confirm clinical diagnoses and to identify the causative BoNT serotype. The current 'gold standard' method for BoNT detection is the mouse bioassay (MBA), a highly stressful animal experiment. A viable animal experiment replacement method must demonstrate high sensitivity, specificity, reproducibility and robustness, as well as comprehensive BoNT subtype detection, and be widely accepted in the field, necessitating rigorous validation. Here, we report on the validation of a previously established in vitro endopeptidase suspension immunoassay (Endopep-SIA) for the simultaneous detection, differentiation and quantification of BoNT serotypes A and B, the most frequent serotypes associated with human botulism. This assay uses monoclonal antibodies for BoNT extraction, followed by detection of the catalytic activity using neoepitope-specific monoclonal antibodies and suspension array technology. The Endopep-SIA showed high reproducibility with intra- and inter-assay variabilities between 7 and 22%, it demonstrated a sensitivity two- to twenty-fold higher than the MBA for BoNT in buffer samples and was equally sensitive for human serum samples with a limit of detection of 0.4 MLD/mL for BoNT/A and 1.0 MLD/mL for BoNT/B. Importantly, it reliably detected all six BoNT/A and six BoNT/B subtypes tested, including clinically relevant and bivalent strains, hereby proving high diagnostic safety. Based on the results obtained, we expect the Endopep-SIA to be instrumental in markedly reducing the number of animals used in botulism diagnostics.