Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities
Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities
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DOI：
10.1128/jcm.34.8.1934-1938.1996
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发表时间：
1996-08-01
影响因子：
9.4
通讯作者：
Shone, CC
中科院分区：
文献类型：
--
作者：
Hallis, B;James, BAF;Shone, CC
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 del eloped 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, Trpsin 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.
