Application of an Engineered Streptavidin-Binding Single Chain Fragment Variable Antibody in Light Addressable Potentiometric Immunoassays for Identification of VEEV

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Authors
  1. Hu, W-G.
  2. Thompson, H.G.
  3. Alvi, A.Z.
  4. Nagata, L.P.
  5. Suresh, M.R.
  6. Fulton, R.E.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN);Alberta Univ, Edmonton ALTA (CAN) Faculty of Pharmacy and Pharmaceutical Sciences;SYNX Pharma Inc, Toronto Ont (CAN)
Abstract
A genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE) was applied in a system consisting of an immunofiltration enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE. The IFA involved formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capture of the sandwich by filtration on biotinylated membrane, and labeling of the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies was investigated and optimized. By the IFA/LAPS assay, the limit of detection of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody was applied. Total assay variance of the IFA/LAPS assay for both intra- and inter-assay precision was less than 20%. Assay accuracy was measured by comparing VEE concentrations estimated by IFA/LAPS standard curve to those obtained by conventional protein assay. VEE concentrations were found to differ by no more that 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzymelinked immunosorbant assay utilizing polystyrene plates and a chromogenic substrate, however, less time and effort were required for performance of the IFA/LAPS assay. More importantly, use of genetically biotinylated scFv in the IFA/LAPS assay obviates the need for
Keywords
Venezuelan equine encephalitis virus;Genetically biotinylated recombinant antibody;Immunofiltration assay;Light addressable potentiometric sensor
Report Number
DRDC-SUFFIELD-SL-2002-173 — Scientific Literature
Date of publication
08 Feb 2017
Number of Pages
25
DSTKIM No
CA044641
CANDIS No
804965
Format(s):
Electronic Document(PDF)

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