Development of a Novel Immuno-PCR Assay for Detection of Ricin in Ground Beef, Liquid Chicken Egg, and Milk

Abstract

Reliable, sensitive, and high-throughput methods are essential for food defense, to detect foodborne contaminants and to facilitate remediation and recovery from potential toxin-related incidents. Ricin is a protein toxin that has been used for intentional contamination of foods in the past. In this study, we developed procedures for quantification of ricin in foods using immuno-PCR (IPCR). The direct adsorption of ricin onto the wells of a microtitration plate was compared with indirect immobilization via a capture antibody (sandwich IPCR). The latter procedure provided much greater sensitivity. We also compared a protocol with the immunoassay and PCR conducted in a single plate to a two-step procedure in which the PCR was conducted in a second plate, following release and transfer of the DNA marker. The two-step procedure proved 1,000-fold more sensitive for ricin detection, so this format was used to detect ricin in spiked samples of ground beef, chicken egg, and milk, and the results were compared with those obtained from enzyme-linked immunosorbent assay (ELISA). The IPCR had a limit of detection of 10 pg/ml in chicken egg and milk samples and 100 pg/ml in ground beef extracts. Comparable ELISA results were in the 1 to 10 ng/ml range. Thus, IPCR affords sensitivity that is 10-fold greater in the ground beef matrix, 100-fold greater in the milk, and 1,000-fold greater in the egg matrix than the sensitivity obtained by ELISA. Further optimization of the sandwich IPCR was performed by comparing various antibody combinations. Among the four formats investigated, the pAb-pAb combination yielded the lowest limit of detection (10 fg/ml).