Recombinant C. botulinum Neurotoxin Type B Heavy chain

Free-energy terms that contribute to complex formation between the catalytic domain of botulinum neurotoxin type B (BoNT/B-L-C) and a 36-residue synaptobrevin fragment were estimated by using a combination of microscopic simulations and continuum methods. The complex for a non-hydrolyzed substrate was calculated by optimizing an energy function applied to the X-ray co-crystal structure of BoNT/B-L-C bound with reaction products from a cleaved synaptobrevin peptide, refined to high crystallographic thermal factors. The estimated absolute binding affinity of the simulation structure is in good qualitative agreement with the experimental free energy of Michaelis complex formation, given the approximations of the model calculations. The simulation structure revealed significant complex stabilization from the hydrophobic effect, while the electrostatic cost of releasing water molecules from the interface determined to be highly unfavorable. By partitioning the total electrostatic and hydrophobic terms into residue free-energy contributions, a binding-affinity 'signature' for synaptobrevin was developed from the optimized conformation. The results demonstrate the effect of substrate length on complex formation and identify a peripheral high-affinity binding site near the N-terminal region that might initiate cooperative activation responsible for the large minimal substrate length requirement. The so-called SNARE motif is observed to contribute negligible free energy of binding.

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%. (c) 2005 Elsevier B.V. All rights reserved.