Simultaneous multiplex detection and confirmation of the proteinaceous toxins abrin, ricin, botulinum toxins, and Staphylococcus enterotoxins a, B, and C in food.

Detection of proteinaceous toxins in complex heterogeneous mixtures requires highly specific and sensitive methods. Multiplex technology employing multiple antibodies that recognize different epitopes on a toxin provides built-in confirmatory analysis as part of the initial screen and thereby increases the reliability associated with both presumptive positive and negative results. Polyclonal and monoclonal antibodies were obtained for abrin, botulinum toxins, ricin, and Staphylococcus enterotoxins A, B, and C (SEA, SEB, and SEC). Food samples were spiked with the toxins either individually or mixed and analyzed following 40-fold dilution. Abrin, botulinum toxin A complex, ricin, and SEB displayed limits of detection in the original food samples ranging from 0.03 to 1.3 microg/mL, from 0.03 to 0.07 microg/mL, from 0.01 to 0.1 microg/mL, and from <0.01 to 0.03 microg/mL, respectively. Redundancy, that is, multiple antibodies for each toxin, some recognizing different epitopes or displaying different binding affinities, provided a "fingerprint" for the presence of the toxins and built-in confirmation, thus reducing the likelihood of false-positive and false-negative results. Inclusion of internal controls, including a unique protein, helped control for variations in dilution. Paramagnetic microspheres facilitated the detection of analyte in foods containing particulate matter incompatible with the use of filter plates normally used in the wash steps of assays employing standard polystyrene microspheres.