Membrane-based immunoassay method
First Claim
1. A method for determining the presence or amount of an analyte of interest that has at least a first and a second sterically separate antigenic sites, said method comprising:
- (a) providing a reactive membrane comprising a calibration zone and a test zone, wherein said calibration zone is provided by(i) immobilizing a predetermined amount of a first antibody on a plurality of microparticles, wherein said first antibody is an antibody or immunoreactive fragment thereof capable of specifically binding to said first antigenic site on said analyte, in order to form a first immobilized antibody;
(ii) contacting a first portion of said first immobilized antibody with a predetermined amount of said analyte to form a plurality of first immobilized specific binding pairs;
(iii) reacting said first immobilized specific binding pairs with an amount of a covalent cross-linking agent effective to cross-kink said first immobilized antibody such that any remaining specific 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, wherein said labelled second antibody is a directly or indirectly labelled antibody or immunoreactive fragment thereof capable of specifically binding to said second antigenic site; and
,(iv) immobilizing said cross-linked immobilized specific binding pairs of step (a) (iii) in said calibration zone;
and wherein said test zone is provided by immobilizing a second portion of said first immobilized antibody in said test zone;
(b) contacting said reactive membrane with a test sample in order to specifically bind any said analyte of interest in said test sample to said immobilized first antibody in said test zone;
(c) contacting said reactive membrane of step (b) with said labelled second antibody; and
(d) determining the presence or amount of said analyte in said test sample by comparing the amount of labelled second antibody specifically bound in said test zone with the amount of labelled second antibody specifically bound in said calibration zone.
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Accused Products
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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Citations
20 Claims
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1. A method for determining the presence or amount of an analyte of interest that has at least a first and a second sterically separate antigenic sites, said method comprising:
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(a) providing a reactive membrane comprising a calibration zone and a test zone, wherein said calibration zone is provided by (i) immobilizing a predetermined amount of a first antibody on a plurality of microparticles, wherein said first antibody is an antibody or immunoreactive fragment thereof capable of specifically binding to said first antigenic site on said analyte, in order to form a first immobilized antibody; (ii) contacting a first portion of said first immobilized antibody with a predetermined amount of said analyte to form a plurality of first immobilized specific binding pairs; (iii) reacting said first immobilized specific binding pairs with an amount of a covalent cross-linking agent effective to cross-kink said first immobilized antibody such that any remaining specific 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, wherein said labelled second antibody is a directly or indirectly labelled antibody or immunoreactive fragment thereof capable of specifically binding to said second antigenic site; and
,(iv) immobilizing said cross-linked immobilized specific binding pairs of step (a) (iii) in said calibration zone; and wherein said test zone is provided by immobilizing a second portion of said first immobilized antibody in said test zone; (b) contacting said reactive membrane with a test sample in order to specifically bind any said analyte of interest in said test sample to said immobilized first antibody in said test zone; (c) contacting said reactive membrane of step (b) with said labelled second antibody; and (d) determining the presence or amount of said analyte in said test sample by comparing the amount of labelled second antibody specifically bound in said test zone with the amount of labelled second antibody specifically bound in said calibration zone. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification