An internally referenced test strip immunoassay for morphine.

We describe an internally referenced immunochemical test-strip for use in the rapid detection of morphine. The method is based on the "enzyme-channelling" immunoassay technique, and a glucose oxidase-horseradish peroxidase enzyme pair is used to immunospecifically generate an insoluble, colored reaction product on the test-strip surface. Test strips are composed of two active surfaces, each of which contain co-immobilized glucose oxidase and antibody. The indicator pad contains antibody directed against the drug, and the color that develops on its surface is inhibited by the presence of drug in the sample. The reference pad contains anti-peroxidase and is used to set the assay detection limit and normalize for variations in temperature, timing, and sample interference. The 10-min assay protocol involves incubating the strip in sample, then incubating it in a developer solution containing glucose, a peroxidase chromogenic substrate, and a peroxidase conjugate of the analyte. The ratio of the color formed on the indicator pad to that formed on the reference pad is used to score the test as positive or negative for drug at a predetermined concentration.