Development of aequorin-based mast cell nanosensor for rapid identification of botulinum neurotoxin type B.

Abstract An aequorin-based mast cell sensor was developed for rapid identification and detection of protein toxins. To monitor mast cell activation and to improve the sensitivity of detection, aequorin was stably expressed in the cells and used as an indicator of calcium flux and the stable cell line was created. The procedures for preparing cells were optimized to improve the quantum yield and sensitivity of detection. The cells sensitized with anti-DNP (dinitrophenyl) IgE were capable of detecting as little as 0.1 ng/mL DNP-BSA in less than 2 min. To demonstrate the utility of this mast cell sensor in detecting protein toxins, an IgE chimeric protein (p21-Fce) was created. This was achieved by fusing the Fc region of IgE antibody to a 21-amino acid peptide (p21) derived from residues 40-60 of synaptotagmin II (Syt II), the protein receptor of botulinum neurotoxin type B (BoNT/B). In addition, magnetic beads linked with anti-BoNT/B polyclonal antibody were prepared to capture BoNT/B molecules in solution to make targets multivalent and concentrated. The p21-Fce binds to FcsRI receptors on RBL (Rat basophilic leukemia) cells and can be cross-linked by BoNT/B captured by the magnetic beads. This led to cell activation and Ca2+ flux indicated by an increase of quantum yield. This assay can detect as little as 100 pM (15 ng/mL) BoNT/B in less than 1 hr, which included the initial sample preparation time. This study lays a foundation for detecting other protein toxins using mast cell sensors.