Assay for analysis of whole blood

• US 4,637,978 A
• Filed: 10/28/1983
• Issued: 01/20/1987
• Est. Priority Date: 10/28/1983
• Status: Expired due to Fees

• Current Assignee: Clinical Diagnostic Systems Inc.
• Original Assignee: Eastman Kodak Company
• Inventors: Dappen, Glen M.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/546,694
• Time in Patent Office: 1,180 Days
• Field of Search: 435/11, 435/10, 435/14, 435/28, 435/25, 435/805, 422/56, 422/57, 436/169, 436/170, 436/810
• US Class Current: 435/11
• CPC Class Codes: G01N 33/526 the element being adapted for a specific analyte
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

Disclosed herein is an assay useful for the determination of an analyte in whole blood. In particular, this assay is useful for the quantitative determination of peroxide-generating analytes, such as glucose or cholesterol, in whole blood. This assay utilizes a multizone element consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone. The reagent/spreading zone has a void volume and average pore size effective to accommodate whole blood, and contains an interactive composition necessary for the analysis. Such composition is capable of providing, upon interaction with the analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm.

Claims

1. A method for the determination of an analyte other than hemoglobin in whole blood comprising the steps of:

(A) physically contacting a sample of whole blood containing an analyte other than hemoglobin and a multizone element, said element consisting essentially of support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone,

provided said element does not contain a radiation-blocking zone,

said reagent/spreading zone having a void volume and average pore size effective to accommodate whole blood, an containing an interactive composition capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm; and

(B) detecting said dye at a wavelength greater than about 600 nm.

2. The method of claim 1 wherein said reagent/spreading zone comprises a particulate structure comprising a plurality of particles non-swellable in and impermeable to whole blood, said particles having a particle size of from about 1 to about 200 microns and being bonded together on surface areas of adjacent particles where said adjacent particles are in closest proximity to form a coherent, three-dimensional lattice which is non-swellable in whole blood.

3. The method of claim 2 wherein said particles are chemically bonded through reactive groups at the points of particle contact which reactive groups are incorporated in said particles.

4. The method of claim 2 wherein said particles are bonded with an adhesive material.

5. The method of claim 1 wherein said reagent/spreading zone comprises a fibrous material.

6. The method of claim 1 wherein said interactive composition comprises a substance having peroxidative activity.

7. The method of claim 1 wherein said void volume is from about 40 to about 60 percent and said average pore size is at least 5 microns.

8. A method for the determination of an analyte other than hemoglobin in whole blood comprising the steps of:

(A) physically contacting a sample of whole blood containing an analyte other than hemoglobin and a multizone element, said element consisting of a support having thereon, in order and in fluid contact, a registration zone, a subbing zone and a reagent/spreading zone,

said reagent/spreading zone having a void volume and average pore size effective to accommodate whole blood, and containing an interactive composition capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm; and

(B) quantitatively detecting said dye at a wavelength greater than about 600 nm.

9. A method for the determination of an analyte other than hemoglobin in whole blood comprising the steps of:

(A) physically contacting a sample of whole blood containing an analyte other than hemoglobin and a multizone element, said element consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone,

provided said element does not contain a radiation-blocking zone,

said reagent/spreading zone containing an interactive composition capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm,

said reagent/spreading zone also comprising a particulate structure having a void volume of from about 40 to about 60 percent and an average pore size of at least 5 microns, said particulate structure comprising;

(i) a plurality of heat-stable, organopolymeric particles non-swellable in and impermeable to whole blood, said particles having a particle size of from about 1 to about 200 microns, and

(ii) an adhesive, in an amount less than about 10 percent by weight of said particles, comprising an organic polymer different from that of said particles and insoluble in whole blood;

substantially all of said adhesive being concentrated on surface areas of adjacent particles where said adjacent particles are in closest proximity, and bonding said particles together with said adhesive into a coherent, three-dimensional lattice which is non-swellable in whole blood; and

(B) quantitatively detecting said dye at a wavelength greater than about 600 nm.

10. The method of claim 9 wherein said analyte is glucose.

11. The method of claim 9 wherein said analyte is cholesterol.

12. A method for the determination of glucose in whole blood comprising the steps of:

(A) physically contacting a sample of whole blood containing glucose and a multilayer element, said element consisting essentially of support having thereon, in order and in fluid contact, a registration layer and a reagent/spreading layer,

provided said element does not contain a radiation-blocking layer,

said reagent/spreading layer having a void volume and average pore size effective to accommodate whole blood, and containing a composition comprising glucose oxidase, a substance having peroxidative activity, an amino-antipyrine and a color-forming coupler which, upon the interaction of said composition with glucose, provides a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm; and

(B) quantitatively detecting said dye at a wavelength greater than about 600 nm.

13. The method of claim 12 wherein said peroxidative substance is peroxidase, and said aminoantipyrine is 4-aminoantipyrine.

14. The method of claim 12 carried out at a pH of from about 4 to about 7.

15. The method of claim 12 wherein said color-forming coupler is a toluidine compound or a substituted aniline compound.

16. A multizone element for the determination of an analyte other than hemoglobin in whole blood consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone,

provided said element does not contain a radiation-blocking zone,

said reagent/spreading zone having a void volume and average pore size effective to accommodate whole blood containing an anlyte other than hemoglobin and containing an interactive composition for said analyte,

said interactive composition comprising a substance having peroxidative activity, and capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm.

17. The element of claim 16 wherein said void volume of said reagent/spreading layer is from about 25 to about 80 percent and said average pore size is at least 5 microns.

18. The element of claim 16 wherein said analyte is glucose.

19. The element of claim 16 wherein said analyte is cholesterol.

20. The element of claim 16 wherein said reagent/spreading zone comprises a fibrous material.

21. A multizone element for the determination of an analyte other than hemoglobin in whole blood consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone,

provided said element does not container a radiation-blocking zone,

said reagent/spreading zone comprising a particulate structure having a void volume and average pore size effective to accommodate whole blood containing an analyte other than hemoglobin,

said particulate structure comprising a plurality of particles non-swellable in and impermeable to whole blood, said particles having a particle size of from about 1 to about 200 microns and being bonded together on surface areas of adjacent particles where said adjacent particles are in closest proximity to form a coherent, three-dimensional lattice which is non-swellable in whole blood,

said reagent/spreading zone containing an interactive composition necessary for said determination, said interactive composition comprising a substance having peroxidative activity, and capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm.

22. The element of claim 21 wherein said particles are chemically bonded through reactive groups at the points of particle contact which reactive groups are incorporated in said particles.

23. The element of claim 21 wherein said particles are bonded together with an adhesive material.

24. A multizone element for the determination of an analyte other than hemoglobin in whole blood consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone,

provided said element does not contain a radiation-blocking zone,

said reagent/spreading zone also comprising a particulate structure having a void volume of from about 40 to about 60 percent and an average pore size of at least from about 15 to about 65 microns, said particulate structure comprising:

(i) a plurality of heat-stable, organopolymeric particles non-swellable in and impermeable to whole blood containing an analyte other than hemoglobin, said particles having a particle size of from about 1 to about 200 microns, and

(ii) an adhesive, in an amount less than about 10 percent by weight of said particles, comprising an organic polymer different from that of said particles and insoluble in whole blood;

substantially all of said adhesive being concentrated on surface areas of adjacent particles where said adjacent particles are in closest proximity, and bonding said particles together with said adhesive into a coherent, three-dimensional lattice which is non-swellable in whole blood,

said reagent/spreading zone containing an interactive composition comprising a substance having peroxidative activity, and capable of providing, upon interaction with said analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm.

25. The element of claim 24 wherein said particles comprise an addition polymer formed from one or more of the following ethylenically unsaturated polymerizable monomers:

(a) up to 100 weight percent of an amino-substituent-free vinyl carbocyclic aromatic;

(b) up to about 25 weight percent of an acrylic acid ester;

(c) up to 100 weight percent of a methacrylic acid ester;

(d) up to about 30 weight percent of an ethyleically unsaturated carboxylic acid;

(e) up to about 75 weight percent of an ethylenically unsaturated nitrile;

(f) up to about 20 weight percent of an amino-substituted vinyl carbocyclic aromatic;

(g) up to about 20 weight percent of an ethylenically unsaturated crosslinkable monomer;

(h) up to about 20 weight percent of a tertiary aminoalkyl acrylate or methacrylate;

(i) up to 100 weight percent of a N-heterocyclic vinyl monomer; and

(j) up to about 20 weight percent of an acrylamide or methacrylamide,

and said adhesive comprises an addition polymer formed from a blend of ethylenically unsaturated polymerizable monomers selected from the following group;

A. a blend containing from about 1 to about 35 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics and from about 65 to about 99 weight percent of one or more alkyl acrylates or methacrylates;

B. a blend containing from about 20 to about 95 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics, acrylic or methacrylic acid esters and ethylenically unsaturated polymerizable crosslinkable monomers, and from about 5 to about 80 weight percent of one or more ethylenically unsaturated polymerizable monomers having an active hydrogen or salts thereof; and

C. a blend containing from about 15 to 100 weight percent of one or more ethylenically unsaturated monomers selected from the group consisting of 1-vinylimidazole, vinylbenzyl alcohol, ethyl acrylate or an acrylamide or methacrylamide, and up to 85 weight percent of one or more ethylenically unsaturated polymerizable crosslinkable monomers.

26. A multilayer element for the determination of glucose in whole blood consisting essentially of a support having thereon, in order and in fluid contact, a registration layer and a reagent/spreading layer,

provided said element does not contain a radiation-blocking layer,

said spreading/reagent layer having a void volume and average pore size effective to accommodate whole blood containing glucose, and containing an interactive composition comprising glucose oxidase, a substance having peroxidative activity, an aminoantipyrine and a color-forming coupler which, upon the interaction of said composition with glucose, provides a dye which can be spectrophotometrically detected at a wavelength greater than about 600 nm.

27. The element of claim 26 wherein said interactive composition comprises peroxidase, 4-aminoantipyrine and a buffer which maintains a pH of from about 4 to about 7.

28. The element of claim 26 wherein said color-forming coupler is a toluidine compound or a substituted aniline compound.

29. The element of claim 28 wherein said interactive composition comprises glucose oxidase, peroxidase, 4-aminoantipyrine, 3,3-dimethylglutaric acid and 8-anilino-1-naphthalenesulfonic acid;

and said reagent/spreading layer comprises a particulate structure comprising

(i) a plurality of poly(vinyltoluene-co-p-t-butylstyrene-co-methacrylic acid) particles having a substantially uniform particle size of from about 40 to about 60 microns; and

(ii) an adhesive, in an amount less than about 10 percent by weight of said particles, comprising poly(n-butyl acrylate-co-styrene-co-2-acrylamido-2-methylpropane sulfonic acid).

30. A multizone element for the determination of an analyte other than hemoglobin in whole blood consisting of a support having thereon, in order and in fluid contact, a registration zone, a subbing zone and a reagent/spreading zone,

said reagent/spreading zone having a void volume and average pore size effective to accommodate whole blood containing an analyte other than hemoglobin, and containing an interactive composition for said analyte,

said interactive composition comprising a substance having peroxidative activity, and capable of providing, upon interaction with said analyte, a dye which can be spectro-photometrically detected at a wavelength greater than about 600 nm.