[ODP-094] エンドぺプ-マス法によるボツリヌス毒素の検出
The mouse lethality bioassay (MLB) is the most widely accepted method for the detection of botulinum neurotoxins (BoNTs). However, alternatives to animal experiments are required for animal protection. Recently the endo-peptidase mass-spectrometry (Endopep-MS) assay was developed by CDC. We applied this method to samples in Japan.
Materials and methods: Twenty three strains of C. botulinum (BoNT/A,B,E,F), BoNT/E producing C. butyricum, 10 other Clostridium strains, 3 bottles of honey (2 commercial food and 1 causative food of infant botulism) and purified BoNT/A,B,E,F were tested for Endopep-MS assay.
The extraction and the Endopep-MS reaction were performed smaller volume than the original method and manually. Mass spectra were obtained by Bruker Biotyper and analyzed using FlexAnalysis.
Results and Conclusions: In the Endopep-MS assay, purified BoNT/A and BoNT/B were detected about 100 pg. These were almost the same as the MLB. All BoNT-producing strains were positive for BoNTs, and other Clostridium spp. were negative. The Honey of infant botulism was positive for BoNT/A produced by C. botulinum. Of the two commercial bottles of honey, one was positive by MLB, but both were positive by the Endopep-MS assay.
From the results it is considered that the Endopep-MS assay, which is rapid, easily and high sensitivity method, can be used as an alternative method to the MLB.
Materials and methods: Twenty three strains of C. botulinum (BoNT/A,B,E,F), BoNT/E producing C. butyricum, 10 other Clostridium strains, 3 bottles of honey (2 commercial food and 1 causative food of infant botulism) and purified BoNT/A,B,E,F were tested for Endopep-MS assay.
The extraction and the Endopep-MS reaction were performed smaller volume than the original method and manually. Mass spectra were obtained by Bruker Biotyper and analyzed using FlexAnalysis.
Results and Conclusions: In the Endopep-MS assay, purified BoNT/A and BoNT/B were detected about 100 pg. These were almost the same as the MLB. All BoNT-producing strains were positive for BoNTs, and other Clostridium spp. were negative. The Honey of infant botulism was positive for BoNT/A produced by C. botulinum. Of the two commercial bottles of honey, one was positive by MLB, but both were positive by the Endopep-MS assay.
From the results it is considered that the Endopep-MS assay, which is rapid, easily and high sensitivity method, can be used as an alternative method to the MLB.