Accession Number:

ADA456993

Title:

An Activity-Dependent Assay for Ricin and Related RNA N-Glycosidases Based on Electrochemiluminescence

Descriptive Note:

Journal article

Corporate Author:

ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD

Report Date:

2006-01-01

Pagination or Media Count:

9.0

Abstract:

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 0.1ngml 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.

Subject Categories:

  • Biochemistry
  • Toxicology
  • Electricity and Magnetism

Distribution Statement:

APPROVED FOR PUBLIC RELEASE