Last data update: May 13, 2024. (Total: 46773 publications since 2009)
Records 1-4 (of 4 Records) |
Query Trace: Krilich J[original query] |
---|
Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates
Wang D , Krilich J , Baudys J , Barr JR , Kalb SR . Bioorg Med Chem 2015 23 (13) 3667-73 It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum neurotoxins, the most toxic substances and the causative agents of botulism. Type C botulinum neurotoxin (BoNT/C) represents one of the seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians. Here we report the development of optimized peptide substrates for improving the detection of BoNT/C and /CD mosaic toxins using an Endopep-MS assay, a mass spectrometry-based method that is able to rapidly and sensitively detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Based on the sequence of a short SNAP-25 peptide, we conducted optimization through a comprehensive process including length determination, terminal modification, single and multiple amino acid residue substitution, and incorporation of unnatural amino acid residues. Our data demonstrate that an optimal peptide provides a more than 200-fold improvement over the substrate currently used in the Endopep-MS assay for the detection of BoNT/C1 and /CD mosaic. Using the new substrate in a four-hour cleavage reaction, the limit of detection for the BoNT/C1 complex spiked in buffer, serum and milk samples was determined to be 0.5, 0.5 and 1mouseLD50/mL, respectively, representing a similar or higher sensitivity than that obtained by traditional mouse bioassay. |
Detection of botulinum toxins A, B, E, and F in foods by Endopep-MS
Kalb SR , Krilich JC , Dykes JK , Luquez C , Maslanka SE , Barr JR . J Agric Food Chem 2015 63 (4) 1133-1141 Botulism is caused by exposure to botulinum neurotoxins (BoNTs). BoNTs are proteins secreted by some species of clostridia; these neurotoxins are known to interfere with nerve impulse transmission, thus causing paralysis. Botulism may be contracted through consumption of food either naturally or intentionally contaminated with BoNT. The human lethal dose of BoNT is not known but is estimated to be between 0.1 and 70 mug; thus, it is important to be able to detect small amounts of this toxin in foods to ensure food safety and to identify the source of an outbreak. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric-based endopeptidase method for the detection and differentiation of BoNT. This method can detect BoNT at levels below the historic standard mouse bioassay in clinical samples such as serum, stool, and culture supernatants. We have now expanded this assay to detect BoNT in over 50 foods including representative products that were involved in actual botulism investigations. The foods tested by the Endopep-MS included those with various acidities, viscosities, and fat levels. Dairy and culturally diverse products were also included. This work demonstrates that the Endopep-MS method can be used to detect BoNT/A, /B, /E, and /F in foods at levels spiked below that of the limit of detection of the mouse bioassay. Furthermore, we successfully applied this method to investigate several foods associated with botulism outbreaks. |
A two-stage multiplex method for quantitative analysis of botulinum neurotoxins type A, B, E, and F by MALDI-TOF mass spectrometry
Wang D , Baudys J , Krilich J , Smith TJ , Barr JR , Kalb SR . Anal Chem 2014 86 (21) 10847-54 In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples. |
Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep-MS assay
Wang D , Krilich J , Baudys J , Barr JR , Kalb SR . Anal Biochem 2014 468 15-21 Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to mankind. It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A to G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep-MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared to the current substrate for the detection of both not trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouseLD50/mL was achieved using the novel peptide substrate in the assay to detect not trypsin-activated BoNT/E complex spiked in serum, stool and food samples. |
- Page last reviewed:Feb 1, 2024
- Page last updated:May 13, 2024
- Content source:
- Powered by CDC PHGKB Infrastructure