Session: Parallel session 5 - Forensic and cultural heritage
Endopep-MS analysis for the detection of Botulinum Neurotoxin A at Ultra-trace level in human serum
Objective
Clostridium botulinum bacteria produce botulinum neurotoxins (BoNTs). Due to the low lethal dose and the lack of efficient therapy, BoNTs are classified as bioterrorism threats. Eight types of BoNTs (A to H) exist, of which types A, B, E and F are associated with human botulism. BoNTs are Zinc proteases (150 kDa) made of two polypeptide chains. The heavy chain binds to the neuronal receptor and allows the translocation of the light chain (LC). The LC then cleaves the SNARE proteins, inhibiting the neurotransmitter release resulting in muscle paralysis and respiratory failure. The aim of the project is to decrease the LOD of BoNTs below 10 pg/mL by an activity assay called Endopep-Mass Spectrometry (E-MS). E-MS consists of the detection of cleaved products (CPs) generated by BoNTs from SNARE peptide mimics by MALDI-TOF MS.
Methods
Tosylactivated beads were coated with an antibody against BoNT-A. Using the KingFisher robot, the beads were mixed with the human serum spiked with the toxin for immunoprecipitation (IP). The samples were then washed before incubation of the beads with the peptide substrate for 4 hours. Finally, CPs were analysed on an UltrafleXtreme MALDI-TOF instrument from Bruker using an HCCA matrix.
Results/Conclusion
A LOD at 10 pg/mL for BoNT-A was achieved previously by our group (Dorner MB et al, 2023), considering the detection of at least one CP with a S/N ratio above 3. In this project, IP and MALDI-TOF detection were further optimised, by carefully evaluating the conditions of beads washing and spotting onto the MALDI plate. The sensitivity of the optimised E-MS is now determined at 1 pg/mL based on 2 CPs detected after BoNT-A dilutions spiked in human serum, illustrating a ten-fold increase in sensitivity compared to the literature.
Other magnetic beads, e.g. protein A, streptavidin and anti-Fc, are now being evaluated to further improve the sensitivity. In the end, the final experimental conditions will be extended to BoNT B, E and F.
Clostridium botulinum bacteria produce botulinum neurotoxins (BoNTs). Due to the low lethal dose and the lack of efficient therapy, BoNTs are classified as bioterrorism threats. Eight types of BoNTs (A to H) exist, of which types A, B, E and F are associated with human botulism. BoNTs are Zinc proteases (150 kDa) made of two polypeptide chains. The heavy chain binds to the neuronal receptor and allows the translocation of the light chain (LC). The LC then cleaves the SNARE proteins, inhibiting the neurotransmitter release resulting in muscle paralysis and respiratory failure. The aim of the project is to decrease the LOD of BoNTs below 10 pg/mL by an activity assay called Endopep-Mass Spectrometry (E-MS). E-MS consists of the detection of cleaved products (CPs) generated by BoNTs from SNARE peptide mimics by MALDI-TOF MS.
Methods
Tosylactivated beads were coated with an antibody against BoNT-A. Using the KingFisher robot, the beads were mixed with the human serum spiked with the toxin for immunoprecipitation (IP). The samples were then washed before incubation of the beads with the peptide substrate for 4 hours. Finally, CPs were analysed on an UltrafleXtreme MALDI-TOF instrument from Bruker using an HCCA matrix.
Results/Conclusion
A LOD at 10 pg/mL for BoNT-A was achieved previously by our group (Dorner MB et al, 2023), considering the detection of at least one CP with a S/N ratio above 3. In this project, IP and MALDI-TOF detection were further optimised, by carefully evaluating the conditions of beads washing and spotting onto the MALDI plate. The sensitivity of the optimised E-MS is now determined at 1 pg/mL based on 2 CPs detected after BoNT-A dilutions spiked in human serum, illustrating a ten-fold increase in sensitivity compared to the literature.
Other magnetic beads, e.g. protein A, streptavidin and anti-Fc, are now being evaluated to further improve the sensitivity. In the end, the final experimental conditions will be extended to BoNT B, E and F.