Detection and quantification of botulinum neurotoxin type a by a novel rapid in vitro fluorimetric assay

Abstract

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (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. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle(202)]), which is 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 peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH(2) acetylated at positions 156 to 203 [Nop(197), Pya(200), Nle(202)]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10(6) M(-1) s(-1) and 8.85 x 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 little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.

FIG. 1.

SDS-PAGE analysis of purified BoNT/A. A BoNT/A sample (5 μl) was electrophoresed under nonreducing conditions on a 10% polyacrylamide gel in parallel with BSA standards. After Coomassie brilliant blue staining, purified BoNT/A migrating as a single 150-kDa band was quantified by densitometric analysis against the BSA standards. Lane 1 shows the molecular mass standards, while lane 2 contains BoNT/A. Lanes 3 to 5 were loaded with the BSA standards (2, 4, and 6 μg, respectively).

FIG. 2.

Comparative hydrolysis of peptides 7 and 8. BoNT/A (150 kDa; 50 ng/ml) was added to peptide substrates 7 and 8 (20 μM) in 100 μl of reaction buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; DTT, 5 mM; BSA 1 mg/ml) and incubated at 37°C for 90 and 180 min, times at which the fluorescent signal was monitored. The bar graph shows the mean deltas of fluorescence (total fluorescence − fluorescence of the peptide substrate alone in buffer) measured with peptide 7 (gray bars) and peptide 8 (black bars).

FIG. 3.

Comparison of the emission fluorescent spectra (λ_ex = 343 nm) of peptide substrate 10 (Ac-IIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEAN-Nop-RA-Pya-K-Nle-L-NH₂) and of its fluorescent metabolite RA-Pya-K-Nle-L-NH₂ (peptide 12) at the same concentration (0.25 μM).

FIG. 4.

HPLC analysis of the PL50 cleavage products. BoNT/A (10 ng/ml) was incubated for 5 h at 37°C with PL50 (15 μM) in standard assay buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; DTT, 5 mM; BSA, 1 mg/ml) in a final reaction volume of 100 μl. The reaction products were then separated by HPLC on a Kromasil C₁₈ column using a gradient of 10 to 90% CH₃CN (0.1% TFA) in 30 min, and the resulting chromatograms are shown. (A) Peptide 12 metabolite in assay buffer (1 μM), with an Rt of 13.03 min; (B) PL50 (15 μM) incubated in assay buffer without toxin (Rt = 15.96 min); (C) result of the hydrolysis of PL50 (15 μM) by BoNT/A. At 343 nm, the wavelength at which Pya emits, the only observed peaks are those corresponding to PL50 and to peptide 12.

FIG. 5.

Characterization of the BoNT/A enzymatic assay using PL50. (A) There was a linear correlation (R² = 0.97) between the fluorescent response (Δfluorescence = endpoint fluorescence − fluorescence of PL50 in assay buffer) produced by 10 ng/ml of the 150-kDa BoNT/A toxin incubated for 60 min at 37°C in assay buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; DTT, 5 mM; BSA, 1 mg/ml) and the PL50 concentration between 1 and 10 μM. (B) With the highest concentration of PL50 (10 μM), the BoNT/A (10 ng/ml) fluorescent response was linear (R² = 0.99) over 180 min. Under these conditions, 10 ng/ml of BoNT/A toxin was detected in about 30 min.

FIG. 6.

Sensitivity of the BoNT/A-PL50 enzymatic assay and MLD₅₀ calibration. Serial dilutions of a BoNT/A (150 kDa) stock solution (325 μg/ml) were used either in an MLD₅₀ assay (Table 3) or in a BoNT/A-PL50 enzymatic assay. Decreasing concentrations of BoNT/A (150 kDa) were incubated for 5 h at 37°C with 10 μM PL50 in 100 μl of reaction buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; DTT, 5 mM; BSA, 1 mg/ml), and the fluorescence was read every 15 min. (A) Deltas of fluorescence (total fluorescence − fluorescence of the peptide in buffer) were measured over 5 h for the six studied dilutions (ranging from 0.5 × 10⁻³ to 10⁻⁶). Based on the toxin concentration of the starting stock solution, toxin concentrations used were 162.5 (□), 32.5 (•), 16.2 (⧫), 3.2 (▾), 1.6 (▴), and 0.3 (▪) ng/ml. (B) Linearity (R² = 0.99) of the detection at 120 min (▴) as a function of BoNT/A concentration in the assay in nanograms or in the corresponding MLD₅₀ (using 1 MLD₅₀ or 1.9 pg). The inset graph zooms in on the lower BoNT/A concentrations, which allowed us to determine the assay detection limit to be 115 MLD₅₀s.

FIG. 7.

BoNT/A enzymatic assay optimization using PL50 and PL51. BoNT/A was incubated at increasing concentrations at 37°C with either 10 μM PL50 (▪) or 10 μM PL51 (▾) under optimized conditions. Substrate stock solutions were prepared so as to obtain a final DMF concentration of 1.5% in 100 μl of the optimized reaction buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; TCEP, 2.5 mM). The inset represents a zoom of the lower BoNT/A concentrations, which allowed us to determine the detection limits, namely, 100 pg (53 MLD₅₀s) or 60 pg (32 MLD₅₀s) in 120 min using PL50 or PL51, respectively. Mean values of endpoint measurements (120 min) from two independent experiments performed in duplicate are represented.