Attomolar detection of botulinum toxin type A in complex biological matrices

Abstract

Background: A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs.

Methodology/principal findings: Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity.

Conclusions/significance: The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.

Figure 1. The ALISSA principle.

(A) Synthesis scheme for the immuno-affinity matrix for BoNT-enrichment. Protein A-agarose beads are coupled to affinity-purified anti-BoNT antibodies. Disuccinimidyl suberate (DSS) is used to crosslink the FC domain of the antibodies to protein A. Non-crosslinked antibodies are removed by stringent washing. (B) Immobilized BoNT/A cleaves a quenched FRET pair, provided in form of a fluorogenic peptide substrate, thereby releasing the fluorophore from the quencher, which restores fluorescence.

Figure 2. Optimization of the BoNT/A ALISSA in 10% fetal bovine serum.

Effect of (A) SNAPtide concentration of SNAPtide after a 1 hr reaction time, (B) SNAPtide cleavage time, (C) number of beads, (D) BoNT enrichment time, (E) temperature during BoNT/A enrichment and (F) temperature on cleavage of the SNAPtide. The following parameters were kept constant unless when varied as indicated: BoNT/A binding and cleavage reaction (25°C); SNAPtide concentration (1 µM); BoNT/A enrichment time and SNAPtide cleavage time (1 hr each); number of beads (120,000).

Figure 3. Sensitivities of BoNT/A ALISSA.

BoNT/A detection in (A) 10% fetal bovine serum (FBS) to various forms and sources of the toxin; ^a Metabiologics (Madison, WI) and ^b List Biological Laboratories (Campbell, CA); (B) in undiluted human serum, 50% carrot juice (with binding buffer), gelatin phosphate diluent (GPD), reconstituted powdered milk (fat free) and fresh whole milk (2% fat). Pre-act. = toxin preactivated with 5 mM DTT; all reactions with SNAPtide were carried out with 1.25 mM DTT.

Figure 4. BoNT/A ALISSA versus bead-free SNAPtide cleavage.

Comparison between (A, C) the bead-based assay and (B, D) the bead-free reaction in 10% FBS. (C, D) Lineweaver-Burk (double reciprocal) plots of the BoNT/A reaction rate as a function of the substrate (SNAPtide) concentration. Note different y-axis scales. The solid lines represent linear regression fits.