1. DETECTION OF RICIN IN FOOD MATRICES USING ELECTROCHEMILUMINESCENCE (ECL)-BASED TECHNOLOGY Eric A.E. Garber 1, Thomas W. O’Brien 2, George Sigal 3 1 Division of Natural Products, OPDF, Center for Food Safety and Applied Nutrition, U.S.-Food and Drug Administration, College Park, MD 20740 2 Tetracore Inc., Gaithersburg, MD 20878 3 Meso Scale Diagnostics, LLC, Gaithersburg, MD 20877 ABSTRACT Background: Ricin is a potent ribosome inactivating protein (RIP-2) present in beans of the castor plant, Ricinus communis. Recent events of food tampering have made the detection of ricin a serious concern. Methods: Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using the 96-well format, Sector PRTM 100 electrochemiluminescence (ECL) detector manufactured by Meso Scale Diagnostics (MSD). Assay configurations including monoclonal or a polyclonal antibodies, and whether the samples and detector antibody were added sequentially or in combination in the capture step, were compared. Results: The ECL-based detector was able to detect ricin in all samples at concentrations orders of magnitude less than what would be considered a health concern. Using a polyclonal antibody preparation, 0.04 ng/ml ricin was detected in analytical samples prepared from several foods. The simultaneous incubation of the samples with the detector antibody decreased the assay time considerably while still enabling the detection of ricin at levels considerably less than that which might pose a health concern. Conclusions: ECL-based detection of ricin provides a sensitive, rapid one-hour alternative to ELISA technology for the detection of ricin in food. MATERIALS & METHODS Ricin was obtained from Vector Labs. All food samples were prepared by spiking with ricin dissolved in PBS and allowed to absorb into the food. Extraction Beverages were diluted 5-fold with 200 mM sodium phosphate, pH 6.8. Solid food samples (2g) were washed with 20 ml NaPi. Assays ECL analyses were conducted using biotinylated anti-ricin monoclonal antibody 19A-2C6 immobilized onto MSD Multi-Array TM streptavidin coated 96 well plates. Sequential analysis entailed incubating 100  L of sample for one hour at 22 °C with periodic shaking followed by washing and incubation of detector antibody (either monoclonal 30-2C9 or polyclonal T271003-01) for one hour. Simultaneous analysis entailed mixing the detector antibody with the sample for a single incubation. The plates were read using MSD T Read Buffer immediately after washing to remove the detector antibody. The concentration of detector, unless otherwise specified, was either 1.4.E-3 mg/ml monoclonal antibody or 8.2E-4 mg/ml polyclonal antibody. CONCLUSIONS Electrochemiluminescence enabled the detection of ricin in foods at concentrations many orders of magnitude less than that associated with health concerns. Sequential incubation of the sample and detector antibody enabled the detection of ricin at concentrations as low as 0.04 ppb in the analytical sample. Simultaneous incubation of the samples with the detector antibody for 20 minutes enabled the analysis of 96 samples in less than one hour with a limit of detection of 10 ppb in the analytical sample. The polyclonal antibody T271003-01 was more effective than the monoclonal antibody 30-2C9 in detecting ricin. LOD 0.04 ppb in Analytical Sample Sequential Incubation, Detector 3.3E-3 mg/mL polyclonal antibody. Background Typically < 1E+03 The limit of detection for ricin in soda increased upon dilution with buffer Data presented are actual responses not corrected for dilution. Sequential Incubation, Processing ~ 3 hoursSimultaneous Incubation, Processing < 2 hours Processing < 1 Hour, LODs ~ 10 ppb Single 20 min incubation, Detector 1.6E-3 mg/mL polyclonal antibody or 1.4E-3 mg/mL monoclonal antibody Mono- vs Polyclonal Ab Detection