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

One-step homogeneous immunoassay for small analytes

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One-step homogeneous immunoassay for small analytes

Timo Pulli et al. Anal Chem. .

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.

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