Assay devices for concurrently detecting an analyte and confirming the test result

A self-performing confirmatory assay for an analyte in a test sample involving a sandwich assay format, comprising the steps of:

• a) providing a device comprising
• i) a solid phase having at least a first and a second separate fluid-flow pathways, wherein said first pathway contains an assay capture site, and wherein said second pathway contains both a mobile confirmatory reagent and a confirming assay site located downstream from said mobile confirmatory reagent;
• ii) an optional application pad in fluid contact with said first and second fluid flow pathways;
• iii) a specific binding member, which directly or indirectly specifically binds a labelled analyte complex formed by direct or indirect specific binding of a labelled reagent with an analyte in said test sample, wherein said labelled reagent is optionally incorporated either within said application pad or in said first and second pathways, and said specific binding member is immobilized both in said first pathway at said assay capture site and in said second pathway at said confirming assay site;
• iv) said mobile confirmatory reagent comprising an unlabelled specific binding member which specifically binds to said analyte and inhibits the binding of said analyte or said labelled analyte complex with said specific binding member immobilized in said confirming assay site;
• b) contacting said test sample with said labelled reagent if said labelled reagent is not incorporated in said device to form a reaction mixture and contacting said reaction mixture with the device, or contacting the test sample with said device if said labelled reagent is incorporated in said device;
• and
• c) detecting a signal producing label from said labelled reagent, wherein when said signal producing label is an enzyme, a substrate is subsequently allowed to contact said enzyme, and wherein said labelled analyte complex that is immobilized within said first pathway at said assay capture site is related to the presence or amount of said analyte in the test sample, and wherein said confirmatory reagent prevents the binding of said analyte or said labelled analyte complex with said specific binding member immobilized in said confirming assay site, thereby confirming that signal displayed by said assay capture site indicates a positive test sample.