Abstract
Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.
Highlights
Botulinum neurotoxins (BoNTs) are produced by the anaerobic spore-forming bacteriaClostridium (C.) botulinum and some strains of C. butyricum and C. baratii
Please note that the numbering of enzyme-linked immuno-sorbent assays (ELISA), lateral flow assay (LFA), and Endopep-ELISA formats follows the numbering in Worbs et al [35]
BoNT/B was detected in samples S6 and S8, BoNT/E was only found in sample S4
Summary
Botulinum neurotoxins (BoNTs) are produced by the anaerobic spore-forming bacteria. Clostridium (C.) botulinum and some strains of C. butyricum and C. baratii. EQuATox 2013 international BoNT PT panel consisted of thirteen blinded liquid samples (1 mL each) spiked with BoNT/A, B or E into buffer (0.1% BSA in PBS), cow’s milk, meat (minced pork and beef) extract or human serum at various concentrations, as described in detail by Worbs et al [35] These three immunological approaches were used to detect, differentiate and quantify the different serotypes in the proficiency panel samples and are presented here as examples of recommended strategies that could be further developed into recommended operating procedures based on their performance in the PT
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