Rapid, low-sample-volume cholesterol and triglyceride assays

Patent metadata

Field	Value
Publication number	US-11268130-B2
Application number	US-201916530322-A
Country	US
Kind code	B2
Filing date	Aug 2, 2019
Priority date	Dec 10, 2012
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

Reagents, assays, methods, kits, devices, and systems for rapid measurement of cholesterol and cholesterol sub-fractions from a blood sample are provided. Total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol can be measured in a single assay using kinetic measurements, under conditions in which cholesterol sub-species are converted to a detectable product at distinct rates. The detectable product is measured at different times after assay initiation. A lipase, cholesterol esterase, cholesterol oxidase and a peroxidase may be used together to produce colored product in amounts directly proportional to the quantity of cholesterol converted. Methods for calculating very-low density lipoprotein cholesterol levels by further including triglyceride measurements are disclosed. Assays may be performed in a single reaction mixture, allowing more accurate and precise cholesterol determinations, including ratios of cholesterol sub-fractions to total cholesterol, at less expense, than would be expected by performing several different assays in different reaction mixtures.

First claim

The invention claimed is:

1. A method for performing a cholesterol assay, the method comprising:
using a reagent comprising a lipoprotein solubilization agent, lipoprotein interactant, and a buffer, wherein said lipoprotein interactant is present at a concentration level that provides for conversion of high density lipoprotein cholesterol (HDL-C) to a measureable colored product at a substantially different rate than for conversion of low density lipoprotein cholesterol (LDL-C) to a measureable colored product and at a substantially different rate than for conversion of very low density lipoprotein cholesterol (VLDL-C) to a measureable colored product when said reagent is used in a cholesterol assay
wherein said reagent comprising magnesium ions and dextran sulfate and the ratio of dextran sulfate to magnesium ions in the reagent is between about 0.002 to about 0.02; and
measuring HDL-C, LDL-C, and VLDL-C in a single solution, wherein HDL-C is measured at a time different from when LDL-C is measured and different from when VLDL-C is measured.

2. The method of claim 1 , comprising using α-cyclodextrin sulfate, dextran sulfate, magnesium chloride, 4-aminoantipyrene and a sodium phosphate buffer.

3. The method of claim 2 , wherein said ratio of dextran sulfate to magnesium chloride is between about 0.002 to about 0.02.

4. The method of claim 1 , said reagent comprising magnesium and dextran sulfate in a ratio of dextran sulfate to magnesium ions effective that there is no substantial precipitation of low-density lipoprotein (LDL), or that there is no substantial precipitation of very low-density lipoprotein (VLDL), or both, when a blood sample containing LDL or VLDL is put in contact with said reagent.

5. The method of claim 4 , wherein said ratio of dextran sulfate to magnesium ions is between about 0.002 to about 0.02.

6. The method of claim 1 , wherein said lipoprotein solubilization agent is selected from the group consisting of: a surfactant, and a negatively charged α-cyclodextrin derivative; and
said lipoprotein interactant comprises a low molecular weight negatively charged dextran derivative.

7. The method of claim 1 , wherein said lipoprotein interactant is a dextran derivative.

8. A method for performing a cholesterol assay, the method comprising:
using a reagent comprising a lipoprotein solubilization agent, lipoprotein interactant, and a buffer, wherein said lipoprotein interactant is present at a concentration level that provides for conversion of high density lipoprotein cholesterol (HDL-C) to a measureable colored product at a substantially different rate than for conversion of low density lipoprotein cholesterol (LDL-C) to a measureable colored product and at a substantially different rate than for conversion of very low density lipoprotein cholesterol (VLDL-C) to a measureable colored product when said reagent is used in a cholesterol assay
wherein said reagent comprising magnesium ions and dextran sulfate and the ratio of dextran sulfate to magnesium ions in the reagent is between about 0 . 002 to about 0 . 02 ; and
measuring HDL-C, LDL-C, and VLDL-C in a single solution, wherein HDL-C is measured at a time different from when LDL-C is measured and different from when VLDL-C is measured,
wherein said lipoprotein solubilization agent is a nonionic surfactant.

9. A method for performing a cholesterol assay, the method comprising:
using a reagent comprising a lipoprotein solubilization agent, lipoprotein interactant, and a buffer, wherein said lipoprotein interactant is present at a concentration level that provides for conversion of high density lipoprotein cholesterol (HDL-C) to a measureable colored product at a substantially different rate than for conversion of low density lipoprotein cholesterol (LDL-C) to a measureable colored product and at a substantially different rate than for conversion of very low density lipoprotein cholesterol (VLDL-C) to a measureable colored product when said reagent is used in a cholesterol assay
wherein said reagent comprising magnesium ions and dextran sulfate and the ratio of dextran sulfate to magnesium ions in the reagent is between about 0 . 002 to about 0 . 02 ; and
measuring HDL-C, LDL-C, and VLDL-C in a single solution, wherein HDL-C is measured at a time different from when LDL-C is measured and different from when VLDL-C is measured,
wherein said lipoprotein solubilization agent is a zwitterionic surfactant.

Assignees

• Labrador Diagnostics Llc

Inventors

• Matje Douglas
• Gibbons Ian
• Patel Paul
• Holmes Elizabeth A

Classifications

• C12Q1/61 Primary

  involving triglycerides · CPC title

• C12Q1/44

  involving esterase · CPC title

• C12Q2326/00

  Chromogens for determinations of oxidoreductase enzymes · CPC title

• G01N33/92

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title

• G01N2333/904

  acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1) · CPC title