SPADA

Detection techniques for botulinum neurotoxins Limit of Detection     Multiplex Format   Sample‐Matrix   Automation  (ml ‐1 )                                                        Interference   

Method                                

1. Mouse Bioassay *                             20 pg  ELISA*                                                 2.

No                             Limited                  No

                    Li it d                    M bl         Li it d m e anagea e m e 3. ECL*                                                 5 pg ‐50 ng                   Limited                    Manageable         Limited 4. Lateral flow assay*                         5‐50 ng                    No                             High                      N/A 5. Column flow assay*                        1‐50 ng                     No                            High                       N/A 6. Flow cytometry assay                     50 pg‐20 ng                 Yes                            Manageable         Yes 7. Immuno‐PCR                                     1 pg‐ 5 pg                    Limited                    Manageable          No 8. L‐PCR                                                 0.02 fg                        Limited                    N/A                         No 9. BDG assay                                          100 ng                          Yes                           Low                        Yes                             10. Array biosensor assay                   40‐200 ng                   Yes                           Low                         Yes 11. Aptamer electrochemical assay          40 pg                     Yes                           Low                         Yes 12. Peptide array assay                               3 pg                        Yes                        High                        Yes 13. ALISSA                                             0.5 fg                       No                           Low                          No                        14. Endopep‐MS assay*                          0.4 ‐6 pg                    Yes                          High                         Yes 15. Cell based assays                               1‐10 ng                      No                           High                         No  5 pg ‐ 2 ng

(* assay used for food analysis); Enzyme-linked immunosorbent assay (ELISA); Electro-chemiluminiscent (ECL) assay; Liposomal-PCR (L-PCR): Bidiffractive grating (BDG) assay; Assay with a large immunosorbent surface area (ALISSA)

Fitness for Purpose To validate a rapid multi plex Clostridium botulinum - neurotoxin detection assay that has the ability to: • Detect and differentiate serotypes A through G • Detect bivalent toxins effectively and also determine the dominant type • Differentiate complex toxin with associated proteins from purified toxin (di-chain) and toxoid

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