2009 Annual Report 1a.Objectives (from AD-416) 1. We will determine enteric dose-response relationships for crude preparations of botulinum neurotoxin (BoNT)in food using rodent models. 2. We will optimize sample preparation and develop rapid immunological and biochemical tests for BoNT. 1b.Approach (from AD-416) 1. We will administer BoNT in buffer and BoNT spiked food to mice, testing various time and temperature storage conditions (including heating) for effects on toxicity. We will compare commercially available purified BoNT to crude toxin (sterile culture fractions). 2. We will evaluate BoNT assays currently available commercially or through collaborators. We will develop new monoclonal antibodies to BoNT toxin and toxoid. Initial evaluations will be made in ELISA and other standard formats, and selected assays will be ported onto new assay platforms. Examples of new platforms include microbead assays read via microfluorimetry, paramagnetic fluorescent nanosphere assays, microarray analyses employing glass or nitrocellulose surfaces, "dip-stick" format rapid ELISAs, and "black-box" turnkey assay systems. Biochemical assays will include measurements of protease activity using fluorescence polarization and/or fluorescence resonance energy transfer. Documents SCA with the University of Wisconsin-Madison. 3.Progress Report The original goal of the research was collaborative development of sample preparation and assay technologies for detection of botulinum neurotoxin in foods. However, Cooperator also provides advice on administration of Select Agent research, and regular delivery of critical crude and purified botulinum neurotoxin reagents to the ADODR. These services are valuable and cost-effective. All material shipments were documented and contents confirmed upon arrival. The ADODR monitors this project through occasional telephone conversations and frequent email and fax exchanges with the Cooperator. The ADODR’s staff and the Cooperator occasionally meet at national scientific meetings for updates and additional coordination. In FY09 Cooperator and USDA SYs were coauthors of a published paper on characterizing high-affinity monoclonal antibodies to botulinum neurotoxin.