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Sensitive and Specific Bifunctional Assay for Botulinum Neurotoxin Type A

Suryadi, K.; Christian, T.; Shine, N.

Background: The purpose of this study is to develop a highly specific and sensitive method to assess two of the key steps important for Botulinum Neurotoxin type A (BoNT/A) toxicity. This bifunctional assay measures both the integrity of the protein receptor site as well as the enzymatic function. To demonstrate specific binding of the toxin via its protein receptor site, BoNT/A is captured using magnetic beads coated with the luminal domain of SV2c. This binding is then quantitated through measurement of the endopeptidase activity using a specific substrate, SNAPtide, which is based on the native SNAP-25 protein. Methods: Recombinant GST-SV2c (List Prod #690) and recombinant GST Synaptobrevin-2 (List Prod #510A), a control protein, were attached to glutathione coated magnetic beads. Each set of coated beads was exposed to varying amounts of BoNT/A in buffer, washed, and incubated with SNAPtide (List Prod #520), a quenched fluorogenic (FRET) substrate. Detection of cleaved peptide was monitored using reverse phase HPLC with fluorescence detection. Results: Various conditions were tested to optimize the binding and to limit the non specific binding of BoNT/A to the beads. Conditions were established allowing the detection of as low as 1.25 pg of SV2c bound BoNT/A in an overnight digestion at room temperature. Conclusions: This highly specific and sensitive assay captured, on the native receptor, SV2c, and detected enzymatic activity using as little as 1.25 pg of BoNT/A, significantly less than one mouse LD50 (5 pg). This bifunctional assay of BoNT/A toxicity can be used to assess the integrity of the binding and catalytic domains of BoNT/A.