Publication

Abstract : We used a paramagnetic bead format to develop a rapid throughput electrochemiluminescence (ECL) assay for botulinum toxins A and B (BoNT A and B). The assay took advantage of the toxins’ endoproteolytic activity by incubating custom-made ruthenium/biotin-labeled substrates with the neurotoxin and estimating the extent of signal reduction after cleavage of the amino acid terminus of the substrates. The neurotoxin's specific requirements for substrate length and amino acid sequence made the assay specific for these particular serotypes (A, B). Magnetic beads were added as solid support and captured the reaction product. The optimal pH for the reaction was 7.0 for both A and B substrates. A concentration of 50 mM {HEPES} was optimal for substrate cleavage, while concentrations above 80 mM resulted in a significant reduction in cleavage. Adding bovine serum albumin (BSA) (0.25–1.0 mg/ml) to the assay buffer increased cleavage rate. As expected, the activity of the enzyme required the addition of ZnCl, with 0.25–0.5 mM giving the best cleavage while concentrations over 1 mM resulted in decreased hydrolysis. Adding {EDTA} completely inhibited the reaction, confirming the requirement of Zn2+ for enzymatic activity. A significant decrease in substrate cleavage was observed at a temperature of 100 °C for both substrates but minor differences in cleavage occurred between 250 and 370 °C. The reaction time course showed increased cleavage of substrate with increased incubation time. The described assay offers considerable potential for both the detection and confirmation of BoNT A and B. The {ECL} assay offers a new alternative for the determination of the enzymatic activity of these toxins. The stable non-isotopic nature of the Ru label allows liquid-phase reagent convenience. The enhanced sensitivity in combination with adjustable incubation times means fast result turnaround. The rapid throughput nature of the assay could be used in the search for novel therapies against intoxication and provide a platform that could be expanded towards analytes with similar activities.