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US 4,791,068 AGranted
Diagnostic assay for inhibitor of tissue-type and urokinase-type plasminogen activators
AssigneeScripps Clinic and Research Foundation
InventorsDavid J. Loskutoff, Raymond R. Schleef
Life of this patent
Estimating the term…
Abstract
A biochemical reagent system and methods for preparing and using same, as well as diagnostics utilizing the reagent system are disclosed. The biochemical reagent system comprises a receptor raised in an animal host to plasminogen activator inhibitor, and indicating means. The receptor binds to a specific plasminogen activator inhibitor that itself binds to tissue-type or urokinase-type plasminogen activators.
Claims16
A diagnostic kit for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor in a sample, said kit comprising at least one package that contains:
- (1) as an effective amount of a biochemical reagent system comprised of (a) receptor protein that can bind to said inhibitor and (b) an indicating group, wherein said reagent system binds selectively with a specific plasminogen activator inhibitor that binds to and inhibits a plasminogen activator, and
- (2) a plasminogen activator selected from the group consisting of tissue-type plasminogen activator and urokinase-type plasminogen activator that can bind to plasminogen activator inhibitor.
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor, in a sample to be assayed, comprising he steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a binding reagent that binds to said inhibitor to form a solid phase support, said binding reagent being either (i) a polyclonal receptor to said inhibitor, or (ii) a plasminogen activator selected from the group consisting of tissue-type plasminogen activator and urokinase-type plasminogen activator;
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said binding reagent to bind to said inhibitor present in said sample to form a solid phase-bound inhibitor-reagent complex;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of a second binding reagent that binds to said inhibitor portion of said inhibitor-reagent complex bound on said solid support to form a second solid-liquid phase admixture, said second binding reagent being the other of the two of said first binding reagents not used in step (b);
- (g) maintaining said second solid-liquid phase admixture for a predetermined time sufficient for said second binding reagent to bind to said inhibitor present in said complex;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said second binding reagent that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogoen activator inhibitor in a sample to be assayed comprising the steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a binding reagent that binds to said inhibitor to form a solid phase support, said binding reagent being selected from the group consisting of tissue-type plasminogen activator and urokinase-type plasminogen activator;
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said binding reagent to bind to inhibitor present in said sample;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of a second binding reagent that binds to said inhibitor bound on said solid support with said separated solid phase, said second binding reagent being a receptor for said inhibitor;
- (g) maintaining said solid-liquid admixture for a predetermined time sufficient for said second binding reagent to bind to said inhibitor;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said second binding reagent that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor, in a sample to be assayed, comprising the steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a polyclonal receptor that binds to said inhibitor to form a solid phase support;
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said polyclonal receptor to bind to said inhibitor present in said sample to form a solid phase-bound inhibitor-reagent complex;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of tissue-type plasminogen activator that binds to said inhibitor portion of said inhibitor-reagent complex on said solid support to form a second solid-liquid phase admixture;
- (g) maintaining said second solid-liquid phase admixture for a predetermined time sufficient for said tissue-type plasminogen activator to bind to said inhibitor present in said complex;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said tissue-type plasminogen activator that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor, in a sample to be assayed, comprising the steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a polyclonal receptor that binds to said inhibitor to form a solid phase support,
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said polyclonal receptor to bind to said inhibitor present in said sample, to form a solid phase-bound inhibitor-reagent complex;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of a urokinase-type plasminogen activator that binds to said inhibitor portion of said inhibitor-reagent complex bound on said solid support to form a second solid-liquid phase admixture;
- (g) maintaining said second solid-liquid phase admixture for a predetermined time sufficient for said urokinase-type plasminogen activator to bind to said inhibitor present in said complex;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said urokinase-type plasminogen activator that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor, in a sample to be assayed, comprising the steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a tissue-type plasminogen activator that binds to said inhibitor to form a solid phase support;
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said tissue-type plasminogen activator to bind to said inhibitor present in said sample, to form a solid phase-bound inhibitor-reagent complex;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of a polyclonal receptor that binds to said inhibitor portion of said inhibitor-reagent complex bound on said solid support to form a second solid-liquid phase admixture;
- (g) maintaining said second solid-liquid phase admixture for a predetermined time sufficient for said polyclonal receptor to bind to said inhibitor present in said complex;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said polyclonal receptor that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
A solid phase assay method for detecting the presence and quantity of an endothelial cell plasminogen activator inhibitor, in a sample to be assayed, comprising the steps of:
- (a) providing a solid matrix on which to assay said sample;
- (b) affixing on said solid matrix a urokinase-type plasminogen activator that binds to said inhibitor to form a solid phase support;
- (c) admixing an aliquot of a liquid sample to be assayed with said solid phase support to form a solid-liquid phase admixture;
- (d) maintaining said admixture for a predetermined time sufficient for said urokinase-type plasminogen activator to bind to said inhibitor present in said sample, to form a solid phase-bound inhibitor-reagent complex;
- (e) separating said solid and liquid phases;
- (f) admixing an aqueous liquid solution of a polyclonal receptor that binds to said inhibitor portion of said inhibitor reagent complex bound on said solid support to form a second solid-liquid phase admixture;
- (g) maintaining said second solid-liquid phase admixture for a predetermined time sufficient for said polyclonal receptor to bind to said inhibitor present in said complex;
- (h) separating the solid and liquid phases of said second solid-liquid phase admixture;
- and
- (i) determining the quantity of said polyclonal receptor that bound to said inhibitor and thereby determining the quantity of inhibitor.
7 more elements
Dependencies are inferred from the claim text.