Unitary sandwich enzyme immunoassay cassette, device and method of use
First Claim
1. A method for performing a non-separation, enzyme sandwich immunoassay for at least one predetermined analyte comprising:
- (a) providing a reaction vessel divided into first and second chambers by a microporous membrane support, such that(i) said fist chamber is capable of containing at least a first enzyme-labeled antibody, wherein said first enzyme-labeled antibody specifically binds to said first analyte and said labeling enzyme is capable of reacting with a substrate to produce an electrochemically detectable product,(ii) said second chamber is capable of containing said substrate, and(iii) a side of said separating microporous membrane support facing said first chamber is coated with(1) a conductive metal layer and(2) at least a first capture antibody layer immobilized over said conductive metal layer in at least a first spatially distinct area of said microporous membrane support,wherein said conductive metal layer functions as an electrode to detect, directly or indirectly, said electrochemically detectable product and said microporous membrane support is permeable to said substrate;
(b) adding a sample to be tested to said first chamber to contact said capture antibody layer with any said at least one predetermined analyte in said sample;
(c) adding said at least a first enzyme-labeled antibody to said first chamber to bind to any said at least one predetermined analyte bound to said capture antibody layer;
(d) adding said substrate to said second chamber such that it diffuses through said microporous membrane support and react with said labeling enzyme to produce said electrochemically detectable product,(e) determining said electrochemically detectable product at said conductive metal layer to determine the presence or amount of said at least one predetermined analyte in said test sample; and
wherein the pH of the analyte-containing sample in the first chamber is different from the pH of the substrate solution added to the second chamber, such that a pH gradient is created near the conductive metal layer, and wherein either the activity of said labeling enzyme, or the activity of said electrochemically detectable product, is enhanced at said conductive metal layer relative to the bulk solution of said first chamber.
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Accused Products
Abstract
An enzyme sandwich immunoassay, device and cassette therefor for determining a target analyte are described. The enzyme sandwich immunoassay cassette (B) comprises a microporous membrane support (m) having coated thereon a conductive metal layer overlaid with a capture antibody layer (e). The electochemical device (A) comprises a first chamber (b) into which sample and an enzyme labelled antibody capable of specifically binding to the target analyte are placed, wherein the enzyme is capable of catalyzing the production of an electrochemically detectable product; a second chamber (a) adjoining the first chamber (b) into which a substrate for the enzyme is placed, wherein the substrate is capable of producing, when catalyzed by the enzyme, the electrochemically detectable product; and, separating the first (b) and second (a) chambers, a microporous membrane support (m) as described above, wherein the conductive metal layer (e) of the microporous membrane support (m) acts as an electrode to detect, directly or indirectly, the presence of the electrochemically detectable product.
40 Citations
14 Claims
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1. A method for performing a non-separation, enzyme sandwich immunoassay for at least one predetermined analyte comprising:
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(a) providing a reaction vessel divided into first and second chambers by a microporous membrane support, such that (i) said fist chamber is capable of containing at least a first enzyme-labeled antibody, wherein said first enzyme-labeled antibody specifically binds to said first analyte and said labeling enzyme is capable of reacting with a substrate to produce an electrochemically detectable product, (ii) said second chamber is capable of containing said substrate, and (iii) a side of said separating microporous membrane support facing said first chamber is coated with (1) a conductive metal layer and (2) at least a first capture antibody layer immobilized over said conductive metal layer in at least a first spatially distinct area of said microporous membrane support, wherein said conductive metal layer functions as an electrode to detect, directly or indirectly, said electrochemically detectable product and said microporous membrane support is permeable to said substrate; (b) adding a sample to be tested to said first chamber to contact said capture antibody layer with any said at least one predetermined analyte in said sample; (c) adding said at least a first enzyme-labeled antibody to said first chamber to bind to any said at least one predetermined analyte bound to said capture antibody layer; (d) adding said substrate to said second chamber such that it diffuses through said microporous membrane support and react with said labeling enzyme to produce said electrochemically detectable product, (e) determining said electrochemically detectable product at said conductive metal layer to determine the presence or amount of said at least one predetermined analyte in said test sample; and wherein the pH of the analyte-containing sample in the first chamber is different from the pH of the substrate solution added to the second chamber, such that a pH gradient is created near the conductive metal layer, and wherein either the activity of said labeling enzyme, or the activity of said electrochemically detectable product, is enhanced at said conductive metal layer relative to the bulk solution of said first chamber. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification