Immunochromatographic assay for determination of botulinum neurotoxin type D

A highly sensitive combination of a membrane dipstick assay and a flatbed scanner for determination of botulinum toxin type D is described. The immunochromatographic assay is based on a sandwich format using two primary antibodies of distinct specificities and one secondary antibody. One of the primary antibodies was conjugated with colloidal gold (detector reagent), the secondary antibody (capture reagent) was immobilized within a defined detection zone (test line) on a diagnostic cellulose nitrate membrane. In combination with an effective sample pre-treatment, the gold conjugated antibody and the second distinct antibody formed a mobile sandwich complex with the toxin. Within the test line the mobile sandwich complex was immobilized and therefore concentrated by the secondary antibody resulting in a distinct red test line. The intensity of colour of the red test line (signal intensity), which correlated directly with the concentration of BoNT/D in the standard or spiked horse faecal sample, was assessed visually and by computer image analysis using a three-determination analysis. Thus, toxin concentrations down to 50 pg/mL were determined within 3.5 h. The mean relative error of the arithmetic mean of the signal intensity amounted to approximately 7%.