Both these mAbs are highly specific for their respective serotypes. Here, we describe the use of F1-2 and MCS-6-27 capture antibodies in combination with two novel detection antibodies developed in our laboratory, F1-51, a BoNT/A HC-specific mAb and BoB-92-32, a BoNT/B HC-specific MAb, in the development of a rapid BoNT LFD. Our LFD is capable of resolving BoNT/A and /B as two independent colorimetric lines on a single strip, with sensitivities > 10 ng/mL for purified toxins and 10–500 ng/mL in toxin fortified beverages. These results demonstrate the capability of these mAb pairs to simultaneously detect BoNT serotypes A and B on a simple and inexpensive immunochromatographic

test strip. These devices could be used to aid in BoNT

Selleckchem BIBF-1120 detection by first responders or as part of commercial food processing where natural contamination of C. botulinum bacteria is suspect. Botulinum toxins (BoNT) serotypes A and B where purchased from Metabiologics, Inc (Madison, WI). Colloidal gold (40 nm), PVC backing cards and plastic cassettes were purchased from Diagnostic Consulting Network (Carlsbad, CA). Immunopore SP membrane, CF6 absorbent sink, Standard14 conjugate release pad and Fusion5 membrane were purchased from GE Healthcare. Affinity purified find more donkey anti-mouse IgG was obtained from Jackson ImmunoResearch (West Grove, PA). The monoclonal antibodies F1-51 and BoB-92-32 were produced as previously described (Scotcher et al., 2010 and Stanker et al., 2008). F1-51 was demonstrated to bind the HC of BoNT/A while BoB-92-32 bound the HC of BoNT/B (unpublished observation, LHS). The lowest Montelukast Sodium possible concentration of mAb required for stabilizing colloidal gold particles was prepared according to previously published procedures with some modification (Yokota, 2010). Briefly, each mAb was diluted to 5, 15, 20, 25, 30 and 40 μg/mL in water and the pH was adjusted to 9 with 0.2 M K2CO3. Next, 0.5 mL of colloidal gold (pH 9) was added to 100 μL of each antibody dilution and incubated for 10 min

at room temperature. Next, 100 μL of 10% NaCl was added to each tube and the change in color was assessed. The lowest antibody concentration with no color change represented the optimal concentration for stabilizing the gold sol. Antibody-gold conjugates were prepared using the determined antibody concentration. Unconjugated antibody was removed by centrifugation at 15,000 ×g at 4 °C for 30 min. Conjugates were stored in buffer A (50 mM phosphate, pH 9, 0.1% tween-20, 1% BSA) at 4 °C. Capture antibodies were diluted in 10 mM phosphate buffer with 3% v/v methanol and applied to the membrane at 1 mg/mL using a BioJet Quanti Dispenser (BioDot, Irvine, CA), dried at 37 °C for 30 min, then blocked in 10 mM PBS, 0.1% fish gelatin, 1% BSA and 0.5% Triton X-100 for 1 h. The blocked membrane was dried for 30 min at 37 °C and assembled on to the backing card with a 2 mm overlap by the absorbent sink.