Detection And Quantification Of Botulinum Neurotoxin Type A By A Novel Rapid In Vitro Fluorigenic Assay

Botulinum neurotoxin A (BoNT/A), the most poisonous substance to humans is a potential bioterrorist agent.

The light chain protein induces a flaccid paralysis through cleavage of SNAP-25 involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles.

The toxin is used at very low doses which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed.

The Nop and Pya were respectively introduced in positions 197 and 200 of the cleavable fragment (187-203) SNAP-25 (Nle(202)), acetylated at its N-terminus and amidated at its C-terminus.

Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25 and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the previous substrate was lengthened to account for exosite binding to BoNT/A.

The peptide PL50 Ac-156-203 SNAP-25-NH2 (Nop(197), Pya(200), Nle(202)) and its analogue PL51, in which all the methionines were replaced by non oxidizable Nle, were synthesized.

Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 10(6) M(-1)s(-1) and 8.85 10(6) M(-1)s(-1) respectively and behave as the best fluorigenic substrates of BoNT/A reported to date.

Under optimized assay conditions, they allow simple quantification of as low as 100 and 60 pg of BoNT/A respectively within 2 hours with a classical fluorimeter. Calibration of the method against the MLD50 assay unequivocally validates the enzymatic assay.


"Detection And Quantification Of Botulinum Neurotoxin Type A By A Novel Rapid In Vitro Fluorigenic Assay"
Appl Environ Microbiol. 2009 May 8; Poras H, Ouimet T, Orng SV, Fournié-Zaluski MC, Popoff MR, Roques BP (Hubmed.org)