Molecular Detection of Ricin

Abstract

Conflicting analytical results observed during recent biological attacks against the Homeland using ricin, exposed inadequacies of the current rapid detection technologies. Future errors may be prevented by combining immunological identification with detection of ricin's enzymatic activity. Ricin, by way of N-glycosidase, depurinates ribosomes causing cessation of protein synthesis, followed by cellular death. It was hypothesized that ricin activity could be identified by measuring N-glycosidase depurination of an oligonucleotide using a novel PCR-based abasic site assay. Also, it was hypothesized that the sensitivity of DoD ricin immunological assays can be increased by utilizing I-PCR. The DoD's immunological reagents were successfully incorporated into an immuno-PCR assay with improved sensitivity when tested with ricin toxoid. This was followed by development of the technique to identify toxin activity. Ricin and apurinic/apyrimidinic endonuclease (APE) both demonstrated their respective enzymatic activities on the oligonucleotide substrate; however, the assay was unsuccessful because ricin depurination could not be coupled to detection of the abasic substrate in the APE assay. Future research shall focus on techniques for improving the sensitivity of the I-PCR assay by reducing intrinsic background, and incorporating stable isotopes into oligonucleotide substrates to detect ricin activity using spectrophotometric techniques.