One-step homogeneous immunoassay for small analytes

We have developed a one-step, homogeneous non-competitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. Small analytes e.g. abused drugs are an important and increasingly growing area of analytes. Competitive immunoassays performed in microtiter wells or dipsticks are relatively simple methods for detection of drugs of abuse. However, due to the unspecific nature of competitive immunoassays, these tests generate many false positives. Commercial immunoassay tests for morphine and heroin crossreact with e.g. codeine which is a widely used substance in cough medicines. Thus those tests are considered as opiate specific and confirmation of the morphine abuse must always be confirmed by other techniques. This is not only a technical issue but raises also ethical concerns. In our assay format a highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a na¿ve scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunisation. 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 labelled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within two minutes, 5 ng/ml of 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 such as pharmaceuticals, toxins, steroids etc