An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence.
Synthetic biotinylated RNA substrates were cleaved by the combined actions of ricin holotoxin and a chemical agent, N,N'-dimethylethylenediamine. The annealing of the product with a ruthenylated oligodeoxynucleotide resulted in the capture of ruthenium chelate onto magnetic beads, enabling the electrochemiluminescence (ECL)-based detection of RNA N-glycosidase activities of toxins. ECL immunoassays and the activity assay exhibited similar limits of detection just below signals with 0.1 ng/ml of ricin; the ECL response was linear as the ricin concentration increased by two orders of magnitude. Activities were detected with other adenine-specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin, and abrin II. The substrate that provided the greatest sensitivity was composed of a four-residue loop, GdAGA, in a hairpin structure. When the 2'-deoxyadenosine (dA) was substituted with adenosine (A), 2'-deoxyinosine, or 2'-deoxyuridine, toxin-dependent signals were abolished. Placing the GdAGA motif in a six-residue loop or replacing it with GdAdGA or GdAAA resulted in measurable activities and signal patterns that were reproducible for a given toxin. Data indicated that saporin and abrin II shared one pattern, while ricin and RCA shared a distinct pattern. A monoclonal antibody that enhanced the activities of ricin, RCA, and abrin II to different extents, thus improving the diagnostic potential of the assay, was identified .
Keener
William K
WK
United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA. william.keener@amedd.army.mil
Rivera
Victor R
VR
Young
Charles C
CC
Poli
Mark A
MA
D004563:Electrochemistry; D007118:Immunoassay; D008163:Luminescent Measurements / Q000379:methods*; D009699:N-Glycosyl Hydrolases / Q000032:analysis* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010947:Plants, Toxic; D054788:Ribosome Inactivating Proteins; D012276:Ricin / Q000032:analysis*; D012278:Ricinus / Q000378:metabolism
D012276:Ricin; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins