Self-corrected assay device

• US 5,171,688 A
• Filed: 08/23/1989
• Issued: 12/15/1992
• Est. Priority Date: 08/30/1988
• Status: Expired due to Term

• Current Assignee: Cholestech Corporation (Abbott Laboratories Incorporated)
• Original Assignee: Cholestech Corporation (Abbott Laboratories Incorporated)
• Inventors: Hewett, Gary E., Blunt, Judith A., Mielke, Steven T.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Chan, William
• Application Number:
  US07/396,326
• Time in Patent Office: 1,210 Days
• Field of Search: 435/7.91, 435/289, 435/28, 435/301, 435/24, 435/11, 435/170, 436/510, 436/164, 436/13, 422/55, 422/56
• US Class Current: 435/287.8
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: G01N 33/525 Multi-layer analytical elements
• CPC Class Codes: Y10T 436/104165 Lipid, cholesterol, or triglyceride standard or control

Abstract

An assay device and method for determining the concentration of an analyte in a fluid sample. The device includes a porous-matrix reservoir which receives the sample to be assayed, and at least two reaction zones into which sample fluid is drawn from the reservoir. In an embodiment containing two reaction zones, a single-point standard curve which corrects for reduced-signal effects related to loss of reagent activity and linear inhibition effects, is used to determine analyte concentration. In an embodiment containing three reaction zones, a two-point standard curve which corrects for reduced-signal effects related loss of reagent activity and non-linear inhibition effects, is used to determine analyte concentration. Also disclosed is an apparatus which uses the device for automated or semi-automated analyte assay.

Claims

1. An assay device for determining the concentration of an analyte in an aqueous fluid sample containing the analyte and other solutes, comprising

a first reaction zone containing analyte-specific component means effective, when an analyte-containing fluid sample is added to the zone, to utilize the analyte to produce a concentration of H₂ O₂ which is dependent on the concentration of analyte present in the fluid sample, when the analyte concentration is below a concentration which is saturating with respect to the analyte-specific component,

a second reaction zone containing (a) a known quantity of a reference compound which is not present in such aqueous fluid sample, and (b) reference compound-specific component means effective, when an analyte-containing fluid sample is added to the zone, to utilize the reference compound to produce a concentration of H₂ O₂ which is dependent on the concentration of reference compound present in the second reaction zone,

distributing means for delivering such fluid sample to each of the reaction zones, such that substantially the same analyte concentration is present in each reaction zone, when the fluid sample is applied to a single region of the distributing means, and

contained in each of said first and said second reaction zones, a peroxidase enzyme and a dye which can be oxidized in the presence of said enzyme and H₂ O₂ to a detectable product, at a concentration which is dependent on the concentration of H₂ O₂ in the associated reaction zone,

whereby the analyte-dependent signal generated by additional of sample to the first reaction zone can be corrected for (i) variations in signal level produced by the presence of such other solutes in the reaction zones and (ii) for the kinetics of the peroxidase-catalyzed oxidation of said dye from serum inhibition effects calculated from the second reaction zone.

2. The device of claim 1, wherein the reaction zones are porous-matrix zones designed to draw the fluid sample into the zones by surface wetting, and said distribution means includes a reservoir in contact with the two zones, for receiving and distributing the fluid sample to the reaction zones.

3. The device of claim 1, wherein said second reaction zone is composed of two layers, one containing the reference compound, and the other, said compound-specific component means.

4. The device of claim 1, which further includes a third reaction zone which contains said reference compound, in an amount which is different from that present in the said second zone, said compound-specific component means said peroxidase enzyme and said dye.

5. The device of claim 1, wherein said reference compound is a D-amino acid, and said compound-specific component means includes D-amino acid oxidase.

6. The device of claim 1, for determination of serum cholesterol, wherein said analyte-specific component means includes cholesterol ester hydrolase and cholesterol oxidase.

7. The device of claim 6, wherein said said reference compound is a D-amino acid, and said compound-specific component means includes D-amino acid oxidase.

8. The device of claim 6, for determination of total and free serum cholesterol only, which further includes a third reaction zone containing cholesterol oxidase, but not cholesterol esterase, and said compound-specific component means.

9. An apparatus for determining the amount of an analyte in an aqueous fluid sample containing the analyte and other solutes, comprising

an assay device for determining the concentration of an analyte in an aqueous fluid sample, comprising (a) a first reaction zone containing analyte-specific component means effective, when an analyte-containing fluid sample is added to the zone, to utilize the analyte to produce a concentration of H₂ O₂ which is dependent on the concentration of analyte present in the fluid sample, when the analyte concentration si below a concentration which is saturating with respect to the analyte-specific component, (b) a second reaction zone containing (i) a known quantity of a reference compound which is not present in such aqueous fluid sample, and (ii) reference compound-specific component means effective, when an analyte-containing fluid sample is added to the zone, to utilize the reference compound to produce a concentration of H₂ O₂ which is dependent on the concentration of reference compound present in the second reaction zone, (c) distributing means for delivering such fluid sample to each of the reaction zones, such that substantially the same analyte concentration is present in each reaction zone, when the fluid sample is applied to a single region of the distributing means, and (d) contained in each of said first and said second reaction zones, a peroxidase enzyme and a dye which can be oxidized in the presence of said enzyme and H₂ O₂ to a detectable product, at a concentration which is dependent on the concentration of H₂ O₂ in the associated reaction zone, whereby an analyte-dependent signal generated by addition of sample to the first reaction zone can be corrected (i) for variations in signal level produced by the presence of such solutes in the reaction zones and for (ii) the kinetics of the peroxidase-catalyzed oxidation of said dye from serum inhibition effects calculated form the second reaction zone;

a signal detector for measuring such signal value in each of the reaction zones; and

processor means for determining form the signal values of the reaction zones (a) the relationship between reference compound concentration and measured signal value, and (b) the concentration of analyte in the first reaction zone, using such determined relationship.

10. The apparatus of claim 9, wherein said the reaction zones are porous-matrix zones designed to draw the fluid sample into the zones by surface wetting, and said distributing means includes a reservoir in contact with the three zones, for receiving and distributing the fluid sample to the reaction zones.

11. The apparatus of claim 9, wherein said signal detector includes a light source for illuminating each zone with a light beam, and a beam detector for determining the intensity of reflectance of the beam from an illuminated zone.

12. The apparatus of claim 11, wherein said processor means is designed to determine (a) a corrected relationship between reference compound concentration and measured signal value, using the signal values measured in the second reaction zone, and (b) the concentration of analyte in the fluid sample, using the signal value measured in the first reaction zone and the corrected relationship between analyte concentration and measured signal value.

13. The apparatus of claim 10, for use in determining serum cholesterol, wherein said analyte-specific component means includes cholesterol ester hydrolase and cholesterol oxidase, for producing the intermediate reaction product H₂ O₂ in the presence of cholesterol analyte, said compound-specific component means is effective to react with the reference compound to produce H₂ O₂, and said signal-generating component means includes a peroxidase, and a dye which is converted by the peroxidase, in the presence of H₂ O₂, to a distinctively colored, signal reaction product.

14. The apparatus of claim 13, wherein said reference compound is a D-amino acid, and said compound-specific component means includes the corresponding D-amino acid oxidase.