9
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus.

      Journal of Immunological Methods

      Antibodies, Monoclonal, chemistry, genetics, immunology, Antibodies, Viral, Biosensing Techniques, Biotinylation, Encephalitis Virus, Venezuelan Equine, isolation & purification, Enzyme-Linked Immunosorbent Assay, Filtration, Fluorescent Antibody Technique, Immunoglobulin Variable Region, Light, Potentiometry, methods, Recombinant Proteins, Sensitivity and Specificity

      Read this article at

      ScienceOpenPublisherPubMed
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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 (LOD) of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody (mAb) 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 than 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbent assay (ELISA) 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 chemical biotinylation of antibody with resultant possible impairment of the antigen-binding site. Furthermore, the potential for batch-to-batch variability resulting from inequality in the number of biotin molecules labeled per antibody molecule is eliminated.

          Related collections

          Author and article information

          Journal
          15251409
          10.1016/j.jim.2004.03.007

          Comments

          Comment on this article