One-step homogeneous immunoassay for small analytes.
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.
Pulli
Timo
T
VTT Biotechnology, Tietotie 2, P.O. Box 1500, FI-02044 VTT, Espoo, Finland. timo.pulli@vtt.fi
Höyhtyä
Matti
M
Söderlund
Hans
H
Takkinen
Kristiina
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D003061:Codeine / Q000032:analysis / Q000276:immunology; D003429:Cross Reactions; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D003932:Heroin / Q000032:analysis / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009020:Morphine / Q000032:analysis* / Q000276:immunology; D019151:Peptide Library
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D003932:Heroin; D009020:Morphine; D003061:Codeine