Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay.

Ilenia Drigo,Elena Tonon,Luca Bano,Fabrizio Anniballi,Suzanne R Kalb,Simone Pascoletti

doi:10.3390/toxins13010010

Abstract

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66–100%), 96.08% (95% CI: 86.54–99.52%), and 93.33% (95% CI: 78.25–98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15–99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

Highlights

Botulinum neurotoxins (BoNTs) are proteins produced by Gram-positive, rod-shaped, spore-forming, anaerobic bacteria belonging to Clostridium (Clostrisium botulinum, Clostrisium butyricum, Clostridium baratii, Clostrisium argentinense, and Clostridium sporogenes) [1]
The limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 per 500 μL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments
The tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins

Summary

Introduction

Botulinum neurotoxins (BoNTs) are proteins produced by Gram-positive, rod-shaped, spore-forming, anaerobic bacteria belonging to Clostridium (Clostrisium botulinum, Clostrisium butyricum, Clostridium baratii, Clostrisium argentinense, and Clostridium sporogenes) [1]. The test most widely used to confirm the presence of BoNTs and identification them is the mouse bioassay (MBA) [10] This lethality assay possesses high sensitivity, can measure toxin activity, and can detect toxin serotype by means of specific antitoxins. An EndoPep-MS method for BoNTs and serotype differentiation based on LC-ESI-MS/MS and MALDI-TOF MS, coupled with antibody purification and enrichment of toxins, was developed and has been successfully applied in various types of samples for detection of BoNT/A, B, C, D, E, F, and G [14,15,16]. In 2017, Perry and coworkers [11] implemented the EndoPep-MS method for the detection of BoNT/A, B, E, and F toxins using the Bruker MALDI Biotyper This is a lower performance instrument compared with other more costly ones, but is commonly found in microbiology laboratories, in both the human and veterinary fields, being routinely dedicated to bacterial identification. We compared the test performance of EndoPep-MS with the MBA test, considered the “gold standard” for botulinum neurotoxin detection

Methods

Results

Discussion

Conclusion