Development of Rapid Tools for Detection of Clostridium Botulinum Neurotoxoin

Two rapid immunoassays, BoNT/A colloidal gold immunochromatographic assay and BoNT/A magnetic bead based enzyme immunoassay, were developed to detect botulinum neurotoxins type A (BoNT/A). Both assays were based on the sandwich format using monoclonal antibody for detection of BoNT/A. For BoNT/A colloidal gold immunochromatographic assay, monoclonal antibody (150-3) which had the best capacity to capture BoNT/A was immobilized to a defined detection zone on a porous nitrocellulose membrane while another monoclonal antibody (44.1A) was conjugated to colloidal gold particles which served as detection reagent. The BoNT/A-containing sample was added to the membrane and allowed to react with 44.1A-coated particles. The mixture was then passed along the porous membrane by capillary action past the 150-3 in the detection zone, which would bind the particles that had BoNT/A bound to the surface, giving a red color within this detection zone. The sensitivity of immunochromatographic method reached to 50 ng/ml of BoNT/A, and detection time was less than 10 min. The developed assay showed no cross reaction to type B neurotoxin (BoNT/B) and type E neurotoxin (BoNT/E). As for BoNT/A magnetic bead based enzyme immunoassay, the same pair of monoclonal antibodies was utilized to develop a magnetic bead based enzyme immunoassay for detection of BoNT/A. With this method, 500 pg/ml of BoNT/A was detected in 30 min. In simulated sample test, most of the 1:10 diluted samples do not interfere the sensitivity of these assays.