Rapid, Detection of Active versus Inactive Botulinum Toxin

Clostridium botulinum neurotoxins (BoNT) are among the most poisonous substances known. Although production and dissemination of pure toxin is not trivial, it is highly feasible, making BoNT a potentially devastating bioweapon. Four serotypes A, B, E and F cause illness in humans. At present, the only method that is used with confidence to detect BoNT is the acute toxicity test performed with mice. This mouse bioassay has a number of drawbacks: it is expensive to perform, requires a large number of animals, takes four days to complete, and does not confirm toxin serotype unless carried out in parallel with antisera neutralization tests. Some immunoassays have demonstrated sensitivities similar to the mouse bioassay, but immunoassays alone do not indicate the toxin's proteolytic activity (the measure of its toxic action), which can lead to false positive results. CDC has demonstrated a sensitive BoNT detection method that determines proteolytic activity based on mass spectrometry, but its drawbacks are expensive instrumentation, the need for a highly trained operator, and 5-6 hours assay time. Dr. Cliff Shone of Health Protection Agency in the United Kingdom, has demonstrated two sensitive BoNT detection methods by immunoassay and by mass spectrometry, both of which require extensive manual operation and 5-6 hours assay time. We propose an assay, cartridge and an instrument to meet the critical need for a BoNT detection method. It will be better than the existing technologies because of the following characteristics:  Rapid - 2 to 3 hours  Sensitive - at least as sensitive as the mouse bioassay  Confirms toxin proteolytic activity  Portable, countertop instrument  Automated assay not requiring highly trained personnel Phase I of the proposal will demonstrate the part of the novel assay that demonstrates the activity of BoNT and sensitivity of the method.