Importance

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Abstract

The present invention is directed to a membrane-based immunoassay method for an analyte of interest having at least two sterically separate antigenic sites. The method comprises providing a reactive membrane having a calibration zone and a test zone, wherein the calibration zone is characterized by having a predetermined amount of the analyte of interest immobilized via a first antibody as a first specific binding pair to a solid phase, the immobilized first binding pair being covalently cross-linked such that any remaining binding sites on said first immobilized antibody are substantially incapable of further specifically binding to any additional analyte, but at least some of said analyte is capable of specifically binding to a preselected amount of a labelled second antibody. The method further includes the steps of contacting the reactive membrane with a predetermined amount of sample and allowing any analyte in the test sample to become specifically bound to immobilized first antibody in the test zone; contacting the immobilized analyte in the test and calibrator zones with a labelled second antibody capable of binding to a second antigen site on the immobilized analyte; and determining the presence or amount of analyte in the test sample by comparing the amount of labelled second antibody specifically bound in the test zone versus the amount of labelled second antibody specifically bound in the calibration zone.

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First Claim