One-step homogeneous immunoassay for small analytes

Anal Chem. 2005 Apr 15;77(8):2637-42. doi: 10.1021/ac048379l.

Abstract

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / genetics
  • Antibodies / immunology
  • Antibody Specificity
  • Codeine / analysis
  • Codeine / immunology
  • Cross Reactions
  • Female
  • Fluorescence Resonance Energy Transfer / methods*
  • Heroin / analysis
  • Heroin / immunology
  • Humans
  • Immunoassay / methods*
  • Immunoglobulin Variable Region / genetics
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Morphine / analysis*
  • Morphine / immunology
  • Peptide Library

Substances

  • Antibodies
  • Immunoglobulin Variable Region
  • Peptide Library
  • Heroin
  • Morphine
  • Codeine