, 2006, Brunt et al., 2010 and Brooks et al., 2011). Still others have exploited the endopeptidase activity of the toxin for detection using in vitro assays ( Wictome et al., 1999 and Rasooly and Do, 2008). While many of these assays approach the sensitivity of the mouse bioassay 17-AAG solubility dmso they still require specialized equipment and trained personnel. The development of highly sensitive BoNT detection assays as part of an overall bio-defense strategy should also include inexpensive portable diagnostic devices with simple visual verification for use by minimally trained personnel.

A rapid colorimetric BoNT LFD would be of value to both emergency first responders in the assessment of possible contamination and to food processing facilities as part of routine quality assurance. A simple inexpensive BoNT LFD offers the potential to meet the need for rapid BoNT detection from a variety of substrates and settings. Here we report the design and use of a single lateral flow device capable of detecting and distinguishing between BoNT/A and /B. The LFD demonstrated the greatest sensitivity for BoNT/A, detecting as little as 5 ng/mL in 2%, defatted milk. BoNT/B could be detected down to 10 ng/mL

in spiked 1% and 2% defatted milk and undiluted apple juice. In contrast to currently available commercial LFDs, which utilize polyclonal antibodies that are cross reactive for BoNT/A and /B, our device can distinguish between BoNT/A and /B serotypes as it uses two sets of highly specific Z-VAD-FMK cell line monoclonal antibody pairs. Recently, Sharma et al. evaluated the Alexeter Technologies BoNT/A/B strip, which cannot distinguish between the two serotypes (Sharma et al., 2005). In these studies, the Alexeter strip demonstrated a Montelukast Sodium lower limit of detection of 100 ng/mL when spiked

milk products were diluted and 10 ng/mL when they were defatted. The device developed here achieved similar sensitivities in milk, but outperformed the Alexeter Technologies strip in spiked orange juice samples by four-fold, detecting both BoNT/A and /B in orange juice spiked at 25 ng/mL. Gessler et al. evaluated the BioThreat Alert BoNT/A/B test strip, available from Tetracore, with a number of spiked clinical samples (Gessler et al., 2007). Interestingly, the test could not detect purified toxin, suggesting that the antibodies used in the strip are likely specific for epitopes of the BoNT complex and not the actual toxin itself. Both capture antibodies used in our device, F1-2 and MCS-6-27, recognize specific epitopes on the heavy chains of BoNT/A and B, respectively (Scotcher et al., 2009 and Scotcher et al., 2010), and are thus capable of detecting purified toxin as well as crude toxin preparations. Colloidal gold labeling of antibodies is one of the most widely employed strategies for building lateral flow devices because it is relatively inexpensive and very stable in its dried form.