Electrochemical enzymatic complementation immunoassay
First Claim
1. A diagnostic kit useful for an immunoassay of an analyte in a fluid sample, wherein the immunoassay utilizes an electrochemical measurement, the kit comprising:
- (a) an enzyme donor reagent, which includes1) an enzyme-donor polypeptide conjugate, and2) a labeled substrate, wherein the labeled substrate is 4(1,4,7,10-tetraoxadecyl)-1-naphthyl-β
-D-galactopyranoside or 4-methoxyl-1-naphthyl-β
-D-galactopyranoside; and
(b) an enzyme acceptor reagent, which includes1) an enzyme-acceptor polypeptide capable of combining with the enzyme-donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the substrate label from the substrate, and2) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate.
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Accused Products
Abstract
An immunoassay diagnostic kit, method, and apparatus for electrochemically determining the concentration of an analyte in a sample. A mixture is formed which includes the sample, an enzyme-acceptor polypeptide, an enzyme-donor polypeptide linked to an analyte analog (enzyme-donor polypeptide conjugate), a labeled substrate, and an antibody specific for the analyte to be measured. The analyte and the enzyme-donor polypeptide conjugate competitively bind to the antibody. When the enzyme-donor polypeptide conjugate is not bound to antibody, it will spontaneously combine with the enzyme acceptor polypeptide to form an active enzyme complex. The active enzyme hydrolyzes the labeled substrate, resulting in the generation of an electroactive label, which can then be oxidized at the surface of an electrode. A current resulting from the oxidation of the electroactive compound can be measured and correlated to the concentration of the analyte in the sample.
53 Citations
54 Claims
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1. A diagnostic kit useful for an immunoassay of an analyte in a fluid sample, wherein the immunoassay utilizes an electrochemical measurement, the kit comprising:
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(a) an enzyme donor reagent, which includes 1) an enzyme-donor polypeptide conjugate, and 2) a labeled substrate, wherein the labeled substrate is 4(1,4,7,10-tetraoxadecyl)-1-naphthyl-β
-D-galactopyranoside or 4-methoxyl-1-naphthyl-β
-D-galactopyranoside; and(b) an enzyme acceptor reagent, which includes 1) an enzyme-acceptor polypeptide capable of combining with the enzyme-donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the substrate label from the substrate, and 2) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. An immunoassay method for determining the concentration of an analyte in a fluid sample, wherein the immunoassay utilizes an electrochemical measurement, the method comprising:
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(a) preparing a mixture which includes 1) the fluid sample, 2) an enzyme acceptor polypeptide, 3) a labeled substrate, wherein the labeled substrate is 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β
-D-galactopyranoside or 4-methoxy-1-naphthyl-β
-D-galactopyranoside,4) an enzyme-donor polypeptide conjugate, capable of combining with the enzyme acceptor polypeptide to form an active enzyme complex capable of catalyzing the cleavage of the label from the substrate, the label being electroactive at the potential used for the electrochemical measurement when cleaved from the substrate, and 5) a first antibody, capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate; (b) applying the mixture to an electrochemical cell having working and reference electrodes; (c) applying, after incubation of the mixture, a potential difference between the working and reference electrodes sufficient to oxidize the label that has been cleaved from the substrate, thereby generating a current; and (d) measuring the current and correlating the current to analyte concentration. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25)
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26. An immunoassay method for determining the presence of an analyte in a fluid sample, wherein the immunoassay utilizes an electrochemical measurement, the method comprising:
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(a) preparing a mixture which includes 1) the fluid sample, 2) an enzyme acceptor polypeptide, 3) a labeled substrate, wherein the labeled substrate is 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β
-D-galactopyranoside or 4-methoxy-1-naphthyl-β
-D-galactopyranoside,4) an enzyme-donor polypeptide conjugate, capable of combining with the enzyme acceptor polypeptide to form an active enzyme complex capable of catalyzing the cleavage of the label from the substrate, the label being electroactive at the potential used for the electrochemical measurement when cleaved from the substrate, and 5) a first antibody, capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate; (b) applying the mixture to an electrochemical cell having working and counter electrodes; (c) applying a potential difference between the working and counter electrodes sufficient to oxidize the label that has been cleaved from the substrate, thereby generating a current; and (d) measuring the current and correlating the current to the presence of the analyte. - View Dependent Claims (27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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33. An immunosensor useful for an electrochemical immunoassay of an analyte in an fluid sample, comprising:
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(a) a first insulating substrate; (b) working and counter electrodes affixed to the first insulating substrate; (c) a second insulating substrate, which overlays the working and counter electrodes, has a window for exposing substantially equal surface areas of the working and counter electrodes, and has a cut out portion at one end to allow contact between the electrodes and a meter and a power source; (d) a porous substrate, which is impregnated with an enzyme donor reagent, overlays the window, and is spatially displaced from the working and counter electrodes, the enzyme donor reagent including 1) an enzyme-donor polypeptide conjugate, and 2) a labeled substrate, the label being substantially non-electroactive at the potential used for the electrochemical measurement when attached to the substrate, and being electroactive at the potential used for electrochemical measurement when cleaved from the substrate; (e) an enzyme acceptor reagent, which is placed on the second insulating substrate, the enzyme acceptor reagent including 1) an enzyme-acceptor polypeptide capable of combining with the enzyme-donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the substrate label from the substrate, and 2) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate; (f) a third insulating substrate, which overlays the second insulating substrate and has a cutout portion for exposing the enzyme-donor reagent, the enzyme-acceptor reagent, and the window in the second insulating substrate; and (g) a fourth insulating substrate, which overlays the third insulating substrate such that a capillary space is formed within the cutout portion of the third insulating substrate, has a window for exposing a portion of the enzyme acceptor reagent, and has a vent hole.
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45. An immunosensor useful for an electrochemical immunoassay of an analyte in a fluid sample, comprising:
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(a) a first insulating substrate; (b) working and reference electrodes affixed to the first insulating substrate; (c) a second insulating substrate, which overlays the working and reference electrodes, has a window for exposing substantially equal surface areas of the working and reference electrodes, and has a cut out portion at one end to allow contact between the electrodes and a meter and a power source; (d) an enzyme donor reagent, which is placed on the second insulating substrate, the enzyme donor reagent including 1) an enzyme-donor polypeptide conjugate, and 2) a labeled substrate, the label being substantially non-electroactive at the potential used for the electrochemical measurement when attached to the substrate, and being electroactive at the potential used for electrochemical measurement when cleaved from the substrate; (e) an enzyme acceptor reagent, which is placed on the second insulating substrate, the enzyme acceptor reagent including 1) an enzyme-acceptor polypeptide capable of combining with the enzyme-donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the substrate label from the substrate, and 2) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme-donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme-donor polypeptide conjugate; (f) a polymer, which is placed on the second insulating substrate between the enzyme acceptor reagent and the enzyme donor reagent; (g) a third insulating substrate, which overlays the second insulating substrate and has a cutout portion for exposing the enzyme-donor reagent, the enzyme-acceptor reagent, the polymer, and the window in the second insulating substrate; and (h) a fourth insulating substrate, which overlays the third insulating substrate such that a capillary space is formed within the cutout portion of the third insulating substrate, has a window for exposing a portion of the enzyme acceptor reagent, and has a vent hole. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54)
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Specification