Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.