Rapid, low-sample-volume cholesterol and triglyceride assays

Patent metadata

Field	Value
Publication number	US-9546394-B2
Application number	US-201514604538-A
Country	US
Kind code	B2
Filing date	Jan 23, 2015
Priority date	Dec 10, 2012
Publication date	Jan 17, 2017
Grant date	Jan 17, 2017

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 reagent for use in an assay for the simultaneous, rapid measurement of at least two of total cholesterol, low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in lipoproteins in a sample of blood from a subject without substantial precipitation of said lipoproteins, consisting of a sodium phosphate buffer, between about 0.01% and about 1% Triton X-100, between about 0.5 g/L and about 20 g/L pluronic L64, between about 0.5 mM and about 20 mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (ALPS), horse-radish peroxidase (HRP), cholesterol esterase from Pseudomonas sp. , and cholesterol oxidase from Pseudomonas sp.

2. A kit comprising a reagent and instructions for the use of said reagent in a cholesterol assay, the reagent consisting of a sodium phosphate buffer, between about 0.01% and about 1% Triton X-100, between about 0.5 g/L and about 20 g/L pluronic L64, between about 0.5 mM and about 20 mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)- 3 , 5 -dimethoxyaniline (ALPS), horse-radish peroxidase (HRP), cholesterol esterase from Pseudomonas sp. , and cholesterol oxidase from Pseudomonas sp.

3. The kit of claim 2 , comprising a first reagent, a second reagent, and instructions for the use of said reagents in a cholesterol assay, wherein the first reagent comprises α-cyclodextrin sulfate, dextran sulfate, magnesium chloride, between about 1 mM and about 5 mM 4-aminoantipyrene and a sodium phosphate buffer, and the second reagent consists of a sodium phosphate buffer, between about 0.01% and about 1% Triton X-100, between about 0.5 g/L and about 20 g/L pluronic L64, between about 0.5 mM and about 20 mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (ALPS), horse-radish peroxidase (HRP), cholesterol esterase from Pseudomonas sp. , and cholesterol oxidase from Pseudomonas sp.

Assignees

• Theranos Inc

Inventors

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

Classifications

• C12Q1/61 Primary

  involving triglycerides · CPC title

• G01N2333/904

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

• C12Q2326/00

  Chromogens for determinations of oxidoreductase enzymes · CPC title

• C12Q2326/90

  Developer · CPC title

• G01N2333/908

  acting on hydrogen peroxide as acceptor (1.11) · CPC title