title: Hypersensitive detection and quantitation of BoNT/A by IgY antibody against substrate linear-peptide.
abstract: Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Kun
authors.initials: K
authors.lastname: Cai
authors.forename: Maoren
authors.initials: M
authors.lastname: Tian
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002021:Buffers; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology*; D051379:Mice; D008892:Milk / Q000737:chemistry; D010455:Peptides / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D002021:Buffers; C016512:IgY; D007136:Immunoglobulins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A
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