Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

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.

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…3), indicating that both methods represent excellent alternatives to the MBA as animal replacement methods [41]. On the other hand, only Endopep-ELISA and Endopep-MS provided acceptable quantitative results in this first international PT [41]. However, quantitative results substantially Table 4 and Table 5 in [21].…”

“…In the BoNT PT, analysis of success rates by grouping corresponding methods together provided the interesting result that Endopep-MS and MPN assays were as successful as the classic MBA (>97.3% correct qualitative results, Fig. 3), indicating that both methods represent excellent alternatives to the MBA as animal replacement methods [41]. On the other hand, only Endopep-ELISA and Endopep-MS provided acceptable quantitative results in this first international PT [41].…”

“…Nevertheless, quantification of BoNT was performed by only half of the participating laboratories and yielded scattered results, presumably due to the lack of a suitable CRM for calibration [41]. Two classic plate-bound sandwich ELISAs and one lateral flow assay proved to be successful in detecting and quantifying BoNT/A, B, and E ( [42]; Table 2).…”

“…However, apart from the mouse bioassay [39] and one sandwich ELISA [40], none of the other methods had been evaluated in a PT or method performance study until recently. A BoNT PT organized by the EQuATox consortium including 23 laboratories allowed the comparison of various methods and their performance [41]. In this exercise, high purity BoNT/A-F materials were generated and [21,[24][25][26] In vitro cytotoxicity tests Measurement of cell death in vitro (endpoint and real-time assays) [21,27] BoNT/A, B and E Immunological assays Sandwich ELISA: Detection of antigen by a combination of mAb and/or pAb, i) four combinations of two pAb each from goat and/or horse targeting BoNT/A, B, E, or F; detection of holotoxin [40] ii) three combinations of two mAb targeting BoNT/A (mAb TA18 + mAb TA13), BoNT/B (mAb TB6 + mAb TB17), or BoNT/E (mAb TE81 + mAb TE49); detection of HC-domain [42,107] iii) three combinations of mAb and/or pAb targeting BoNT/A (mAb A1688 + mAb HcA78), BoNT/B (mAb B279 + horse trivalent pAb), or BoNT/E (rabbit pAb KE97 + mAb E136); detection of holotoxin [41,42,106] Lateral Flow assay: Sandwich ELISA on a paper strip i) three combinations of two mAb targeting A (mAb TA12 + mAb TA9), B (mAb TB6 + mAb TB17), or E (mAb TE81 + mAb TE49); detection of HC-domain [41,42] (continued on next page) Chromatographic methods…”

On-site detection of bioterrorism-relevant agents

“…2a und b) [43,44]. Der größte Vorteil der neu entwickelten Methoden liegt in der deutlich größeren Anzahl an Erregern und Toxinen, die simultan aus einer Probe nachgewiesen werden kön-nen, wobei die Sensitivität vergleichbar oder besser zu LFAs ist [26,45]. Interessanterweise sind ausgewählte MultiplexVerfahren in der Lage, neben immunologischen Multiplex-Tests auch nukleinsäurebasierte Verfahren automatisiert durchzuführen [43,46].…”