Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities.
A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.
Hallis
B
B
Centre for Applied Microbiology and Research, Salisbury, Wiltshire, United Kingdom.
James
B A
BA
Shone
C C
CC
D001905:Botulinum Toxins / Q000032:analysis*; D019274:Botulinum Toxins, Type A / Q000032:analysis*; D010450:Endopeptidases / Q000032:analysis*; D006735:Horseradish Peroxidase; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism; D009465:Neuromuscular Agents / Q000032:analysis*; D009498:Neurotoxins / Q000032:analysis*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D050683:R-SNARE Proteins; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D009498:Neurotoxins; D010446:Peptide Fragments; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; C096323:rimabotulinumtoxinB; D006735:Horseradish Peroxidase; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A