Abstract
In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.
Highlights
Botulinum neurotoxins (BoNTs) comprise a family of high molecular weight bacterial toxins which are produced by the anaerobic Gram-positive bacteria Clostridium (C.) botulinum, C. butyricum and C. baratii
To set up a proper proficiency test (PT) test plan, 13 samples were selected for further preparatory analysis taking into account the following: (i) The samples were planned to comprise the most important BoNT serotypes pathogenic to humans, i.e., BoNT/A, BoNT/B and BoNT/E, to test the laboratories’ ability to detect and to differentiate the different neurotoxins
Since one major goal of the BoNT PT was to define good analytical strategies, the PT was open with respect to the methods applied by the PT participants
Summary
Botulinum neurotoxins (BoNTs) comprise a family of high molecular weight bacterial toxins which are produced by the anaerobic Gram-positive bacteria Clostridium (C.) botulinum, C. butyricum and C. baratii. Independent of the aforementioned approaches different cell-based assays have been developed which are currently in use for precise potency determination of highly pure pharmacological preparations and for BoNT inhibitor screening [45,46] Their applicability for routine diagnostic purposes has to be evaluated in the future. In planning an appropriate exercise, it had to be considered that the current PT was the very first international activity testing the laboratories’ diagnostic capabilities, so it had to cover a broad range of potential technical approaches and capabilities Along this line, the very first international exercise on BoNT detection and quantification was a “confidence building exercise” offering the possibility to discuss on potential difficulties and limitations with experts from different countries. The results set a basis for further steps in quality assurance and highlight priorities in the process of harmonization and standardization of analytical approaches
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