Device and method for measuring LDL-associated cholesterol

• US 7,824,879 B2
• Filed: 01/09/2008
• Issued: 11/02/2010
• Est. Priority Date: 01/09/2007
• Status: Expired due to Fees

• Current Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Cholestech Corporation (Abbott Laboratories Incorporated)
• Inventors: Gee, Eileen, Bennett, Greg, Chapman, William H. Jr., Schaal, Thomas D., Khan, Shireen, Shindelman, Jeff, Qian, Suyue, Withers, George E. III
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US12/008,394
• Publication Number:
  US 20080166745A1
• Time in Patent Office: 1,028 Days
• Field of Search: 435/11, 435/962, 422/58, 436/539, 436/671
• US Class Current: 435/11
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: G01N 33/526 the element being adapted for a specific analyte
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

An assay device and method for determining the concentration of low density lipoprotein (LDL)-associated cholesterol (LDL-C) in a body-fluid sample, such as a blood sample is described.

Claims

1. A cassette for determining an amount of low density lipoprotein associated cholesterol (LDL C) in a body fluid sample containing very low density lipoprotein (VLDL), chylomicrons, low density lipoprotein (LDL), high density lipoprotein (HDL), and triglycerides (TG) comprising:

(a) a body having a sample well for receiving a sample fluid;

(b) first and second detection zones mounted on said body for producing a detectable product related to the amount of HDL and an adjusted total cholesterol composed of HDL plus LDL without VLDL and chylomicrons, respectively, in said sample fluid,

(c) fluid flow pathways for transferring sample fluid from the sample well to said detection zones,

(d) at least a first reagent in one or more of said fluid flow pathways and optionally, in said sample well, for selectively removing VLDL and chylomicrons without removing LDL in sample fluid transferred from the sample well to the first and second detection zones, and at least a second reagent for selectively removing LDL in sample fluid transferred from the sample well to the first detection zone.

2. The cassette of claim 1, further comprising:

a third detection zone mounted on said body for producing a detectable product related to the amount of TG in said sample fluid; and

a fourth detection zone mounted on said body for producing a detectable product related to the amount of total cholesterol in said sample fluid.

3. The cassette of claim 1, which is in a dry-strip cassette format, wherein

(i) said fluid pathways include at least one dry-strip spreading layer for transferring sample fluid from the sample well to the first and second detection zones, and

(ii) said first and second detection zones include first and second reaction pads, respectively, carried on a reaction bar that is mounted on said body for movement toward and away from the body, to bring said pads into contact with said at least one spreading layer, to transfer sample fluid from the spreading layers to said pads.

4. The cassette of claim 2, wherein said third and fourth detection zones include third and fourth reaction pads, respectively, carried on a reaction bar that is mounted on said body for movement toward and away from the body, to bring said pads into contact with at least one spreading layer, to transfer sample fluid from the spreading layer to said pads.

5. The cassette of claim 3, wherein said first and second reaction pads contain reagents for producing a detectable product related to the amount of free and lipoprotein-associated cholesterol in said pads.

6. The cassette of claim 4, wherein said third reaction pad contains reagents for producing a detectable product related to the amount of triglycerides in said pad, and said fourth reaction pad includes reagents for producing a detectable product related to the amount of free and lipoprotein-associated cholesterol in said pad.

7. The cassette of claim 3, wherein the reagents for selectively removing VLDL and chylomicrons in sample fluid transferred from the sample well to the first and second reaction pads are contained in a dry-strip flap that provides fluid communication between the sample well and the at least one spreading layer, and the reagents for selectively removing LDL in sample fluid transferred from the sample well to the first reaction pad are contained in a filter pad carried on said reaction bar, immediately upstream of said first reaction pad.

8. The cassette of claim 3, wherein the reagents for selectively removing VLDL and chylomicrons in sample fluid transferred from the sample well to the second reaction pad are contained in a filter pad carried on said reaction bar, immediately upstream of said second reaction pad, and the reagents for selectively removing LDL in sample fluid transferred from the sample well to the first reaction pad are contained in a filter pad carried on said reaction bar, immediately upstream of said first reaction pad.

9. The cassette of claim 7, wherein said reaction bar is movable toward said body from a first position to a second position, to bring said flap into a position of fluid communication between the sample well and said at least one spreading layer, and from said second position to a third position, to bring said reaction pads into contact with said at least one spreading layer.

10. The cassette of claim 8, wherein said reaction bar is movable toward said body from a first position to a second position, to bring said reaction pads into contact with said at least one spreading layer.

11. The cassette of claim 1, which is in a microfluidics format, wherein said fluid pathways include a plurality of microchannels for transferring sample fluid from the sample well to the first and second detection zones, and said first and second reaction zones are formed in said body.

12. The cassette of claim 2, which is in a microfluidics format, wherein said fluid pathways include a plurality of microchannels for transferring sample fluid from the sample well to the third and fourth detection zones, and said third and fourth reaction zone is formed in said body.

13. The cassette of claim 11, wherein said first and second detection zones contain reagents for producing a detectable product related to the amount of free and lipoprotein-associated cholesterol.

14. The cassette of claim 12, wherein said third detection zone contains reagents for producing a detectable product related to the amount of triglycerides, and said fourth detection zone contains reagents for producing a detectable product related to the amount of free and lipoprotein-associated cholesterol.

15. The cassette of claim 11, wherein the reagents for selectively removing VLDL and chylomicrons in sample fluid transferred from the sample well to the first and second detection zones include affinity binding reagents in said first microchannel upstream of the first and second detection zones, and the reagents for selectively removing LDL in sample fluid transferred from the sample well to the second detection zone include affinity binding reagent contained in a region of the first microchannel between the first and second detection zones and downstream of the second detection zone.

16. Apparatus for determining the amount of low-density lipoprotein (LDL) in a body-fluid sample containing very low density lipoprotein (VLDL), chylomicrons, LDL and high-density lipoproteins (HDL), comprising:

(a) the cassette of claim 1, and

(b) a device reader having (i) a device holder for receiving said cassette, (ii) a sensor for measuring the amount of detectable product produced in said first and second zones of the device, and (iii) a processor operably connected to said sensor for determining sample amounts of HDL and adjusted total cholesterol and for calculating sample LDL level based on determined levels of HDL and adjusted total cholesterol.

17. The apparatus of claim 16, wherein said sensor is configured to measure the amount of detectable product produced in a third and fourth zone of the device, and the processor is operably connected to said sensor for determining sample amounts of triglycerides and total cholesterol, respectively.

18. The apparatus of claim 17, wherein said processor is operable to determine sample LDL level based on the difference between total adjusted cholesterol and HDL, when the detected level of triglycerides is above 250 mg/dl, and based on the difference between total cholesterol and HDL plus an adjusted triglyceride value, according to the Friedewald equation, when the detected level of triglycerides is below 250 mg/dl.

19. The apparatus of claim 16, wherein said processor is operable to determine sample LDL level from a lookup table whose LDL values are based on the difference between total adjusted cholesterol and HDL, when the detected level of triglycerides is above 250 mg/dl, and based on the difference between total cholesterol and HDL plus an adjusted triglyceride value;

according to the Friedewald equation, when the detected level of triglycerides is below 250 mg/dl.

20. The apparatus of claim 16, wherein said processor is operable to determine sample LDL level from an algorithm which calculates LDL values based on the difference between total adjusted cholesterol and HDL, when the detected level of triglycerides is above 250 mg/dl, and based on the difference between total cholesterol and HDL plus an adjusted triglyceride value, according to the Friedewald equation, when the detected level of triglycerides is below 250 mg/dl.

21. The cassette of claim 13, wherein said at least one of said first detection zone or said second detection zone includes an asymmetric polysulfone membrane.

22. The cassette of claim 21, wherein reagents contained in said first detection zone includes at least one of:

cholesterol oxidase, cholesterol esterase, peroxidase, 4-aminoantipyrine, and TOOS.

23. The cassette of claim 21, wherein reagents contained in said second detection zone includes at least one of:

cholesterol oxidase, cholesterol esterase, peroxidase, 4-aminoantipyrine, and TOOS.

24. The cassette of claim 22, wherein said membrane said first detection zone is in contact with a reagent membrane which includes a solution containing at least one of the following:

dextran sulfate, Mg(OAc)₂.

25. The cassette of claim 23, wherein said membrane said second detection zone is in contact with a reagent membrane which includes a solution containing at least one of the following:

dextran sulfate, MgSO₄.