Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E

Abstract

Botulinum neurotoxins (BoNTs) are bacterial proteins that cause botulism, a life-threatening disease. The Endopep-MS assay permits rapid detection and serotypic differential diagnosis of BoNTs. The serotype-specific nature of this assay requires that each serum sample be aliquoted and individually tested, which in addition to the limited volume of clinical samples, especially in infants, points to the need for a multiplex assay. However, previous attempts to develop such an assay have been challenging, mainly due to inhibition of BoNT/A activity by the BoNT/E peptide substrate. BoNT/A and BoNT/E share the same native target protein as their substrate. We hypothesized that the steric interference between the BoNT/A and BoNT/E substrate peptides is responsible for the difficulty in simultaneously assaying these two toxins. To explore the basis for steric interference, we used the reported structure of BoNT/A in complex with SNAP-25 and modelled the structure of BoNT/E with SNAP-25. Following this thorough structural analysis, we designed a new peptide substrate for BoNT/A that maintained the assay sensitivity and allowed, for the first time, simultaneous detection of the three most abundant human botulinum serotypes. Adopting the multiplex assay will minimize the required sample volume and assay time for botulinum detection while maintaining the superior Endopep-MS assay performance.

Identification of foodborne botulism with mixed serotypes A, B and E and their in vivo profiles in four types of clinical specimens

“…S8-9 (Additional le 1). As for peptides BP and EP, they have been validated in Rosen's work [14,29] thus mainly the LODs were demonstrated here. Their sensitivities under the presence of corresponding BoNT/B and BoNT/E in serum were 1.5 U/mL and 1.8 U/mL (Additional le 1: Fig.…”

“…Here, we employed the BoNT speci c antibody-coated beads to extract BoNTs from clinical samples. In Rosen's work, serotypes A, B and E were extracted separately using the mono-serotype-speci c antibodies [29]. In our work, the usage of antibody mAb013, a monoclonal antibody simultaneously speci c to BoNT/A, B and E, can simplify the enrichment procedure and thus shorten the analysis time.…”

Rapid Detection of Botulinum Neurotoxins—A Review

“…The procedure takes around 4–8 h; while this is relatively lengthy, the ability to automate some of the steps in the detection method allow it to be adapted to process hundreds of separate samples in this time range. Rosen et al have developed a multiplex platform to allow the assay to simultaneously detect BoNT/A, B and E [108]. This adaptation will further improve assay usability, particularly in the human diagnostic and food testing fields, by minimising the sample volume required and reducing the assay time [108].…”

“…Rosen et al have developed a multiplex platform to allow the assay to simultaneously detect BoNT/A, B and E [108]. This adaptation will further improve assay usability, particularly in the human diagnostic and food testing fields, by minimising the sample volume required and reducing the assay time [108]. The sensitivity achieved is good with a detectable range across 100 fg/mL to 1 ng/mL, depending on the sample matrix [109].…”

“…There are, however, problems with this assay; mass spectrometers are very expensive, and not many laboratories have access to one. It requires a trained technician to not only run the instrument, but also to interpret the data, which again narrows its wide scale adoption [108]. The length of time required to run a sample from preparation through to final analysis is also too long when compared with other techniques, which hinders the need for a rapid diagnostic or food testing detection method due to the quick progression of botulism [108].…”