In ddH2O, the residues at the cleavage sites may be more easily uncovered without BoNT/A or ALc cleavage, which led to a higher background value and influenced the sensitivity. 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 total, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis. Introduction Botulinum neurotoxin (BoNT), the most acutely harmful material to humans known, is produced by under anaerobic conditions [1], [2]. Seven types or serotypes (A to G) of botulinum toxin are currently known. Each serotype is composed of a heavy and a light chain linked by disulfide bonds [3], [4]. The heavy chain is responsible for binding to specific pre-synaptic neuronal cell receptors and facilitating internalization. The light chain is usually a zinc-dependent endopeptidase that specifically cleaves soluble SNARE proteins essential for docking and fusion neurotransmitters made up of vesicles at the nerve terminal. Types A, E, and C1 toxins cleave SNAP251-206 (synaptosomal associated protein with 25 kDa molecular mass) at the Q197CR198, R180CI181, and R198CA199 positions [5]. Types B, D, F, and G toxins cleave vesicle-associated membrane protein (VAMP) at the Q76CF77, K59CL60, Q58CK59, and A81CA82 positions [6]. Types C1 toxin is known to also cleave Syntaxin. Botulinum neurotoxins type A (BoNT/A) is the most harmful serotype to human. The 50% SPTAN1 lethal dose (LD50) of BoNT/A to humans is only 0.1C1 ng/kg [2]. Given its small intoxicating dose, short eclipse period, and simple production, BoNT/A is usually a potential bioterrorism agent. Thus, BoNT/A has Tubeimoside I become a research hotspot in medical shielding research in recent years. If botulism diagnosis is usually promptly made, a proper therapy method can be applied and significantly decrease fatality. Therefore, a swift, precise Tubeimoside I assay for botulinum neurotoxin analysis is usually important for BoNT prevention and remedy. The mouse bioassay has been the standard for screening BoNT-containing samples for the past 30 years [7]C[9]. However, this assay is usually time consuming, requires the use of many animals, and has poor repeatability because of numerous fluctuant parameters involved. Several in vitro assays have also been reported for the detection of BoNT/A, relying either on mass spectrometry [10]C[12], immunological detection [13]C[15], F?rster resonance energy transfer (FRET) [16]C[18], or endopeptidase activity [19]C[27]. The advantage of the endopeptidase assay is usually that it steps and quantitates the L-chain activity of the toxin, which is usually directly responsible for neurotransmission inhibition. However, many of these methods require a multi-step process or suffer from high variability, low sensitivity, or long reaction time. The residues of the substrate SNAP25 at cleavage sites, which are normally buried in a helix, are uncovered after BoNT cleavage, making the substrate become a linear peptide. Using the IgY antibody against this linear-peptide substrate, we improved a previous endopeptidase assay and developed a simple method for the detection and quantitation of BoNT/A. This method can be expanded to detect other types of botulinum toxins or specific enzymes in the future. Materials and Methods Bacterial strains, plasmids, and media type A expression vectors pET32a (+) and pET22b (+) were from in our laboratory. BL21 (DE3), DH5a, and pMD18-T cloning vectors were purchased from Beijing TransGen Biotech (China). Taq DNA polymerase, T4 DNA ligase, and restriction endonucleases were obtained Tubeimoside I from New England Biolabs (Beijing, China). PCR primers were synthesized by Beijing Sunbio Tech Co. Ltd. Plasmid mini-kits and gel extraction kits were from Beijing Biomed Co. Ltd. HisTrap FF columns (5 mL) were purchased from GE Healthcare (Beijing, China). All other chemicals and reagents were obtained from other commercial sources and were of the highest purity available. Animals and ethics statement All necessary permits.