Methods and compounds for increasing sensitivity of botulinum assays
Apparatus, systems and methods can provide improved detection of botulinum neurotoxins. In one aspect an isoquinolynyl compound can be used to enhance the sensitivity of both Förster resonance energy transfer (FRET) and non-FRET cell-based assays. In another aspect, non-FRET assays and constructs utilize a reporter that is not coupled with the second fluorophore in a manner that produces significant FRET. In that subject matter an environment cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. Where the environment is a cell, the cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.
1. A method of detecting the presence of a Botulinum toxin, comprising:
providing an assay with a cell having (a) a molecule with a cleavage sequence that is cleaved by a light chain of said Botulinum toxin and (b) a reporter that reports a signal responsive to cleavage of said molecule at said cleavage sequence induced by the light chain of said Botulinum toxin; and
incubating the cell in a culture media that contains a compound effective to increase sensitivity of the assay by at least 0.5 log;
treating the cell with said Botulinum toxin, then detecting the signal, and correlating the signal with the presence or absence of said Botulinum toxin,
wherein said compound is an isoquinolynyl compound that inhibits protein kinase C (PKC), and wherein said cleavage sequence comprises a SNARE protein, SNARE motif, or SNARE mutein.
2. The method of claim 1 , wherein said compound comprises 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7).
3. The method of claim 1 , wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.05% human serum albumin, and 15.4 mM sodium chloride.
4. The method of claim 1 , wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.0125% human serum albumin, and 0.25% lactose.
5. The method of claim 1 , wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.1% human serum albumin, and 0.47% sucrose.
6. The method of claim 1 , wherein said reporter is an artificial construct expressed in the cell.
7. The method of claim 1 , wherein said molecule includes a first fluorophore and the cleavage sequence.
8. The method of claim 7 , wherein said first fluorophore is selected from the group consisting of Yellow Fluorescent Protein (YFP), Citrine, Venus, and a YPet protein.
9. The method of claim 7 , wherein said first fluorophore comprises Yellow Fluorescent Protein (YFP).
10. The method of claim 7 , wherein said molecule comprises the reporter and a second fluorophore.
11. The method of claim 10 , wherein said reporter is not coupled with the second fluorophore in a manner that produces Forster resonance energy transfer (FRET).
12. The method of claim 1 , wherein said cell expresses an enzyme that facilitates degradation of the reporter at least two times faster after the cleavage than before the cleavage.
13. The method of claim 1 , wherein said cell expresses an enzyme that facilitates degradation of the reporter at least five times faster after the cleavage than before the cleavage.
14. The method of claim 1 , wherein presence of said Botulinum toxin correlates with reduction of the signal from a baseline signal.