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Rapid, low-sample-volume cholesterol and triglyceride assays

A technology for cholesterol and total cholesterol, which is applied in the determination/inspection of microorganisms, measuring devices, biological tests, etc., can solve the problems of increasing the cost of lipoprotein cholesterol sub-part analysis, etc.

Active Publication Date: 2015-10-21
THERANOS IP CO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the cost of analysis of lipoprotein cholesterol subfractions is increased by the need for several assays

Method used

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  • Rapid, low-sample-volume cholesterol and triglyceride assays
  • Rapid, low-sample-volume cholesterol and triglyceride assays
  • Rapid, low-sample-volume cholesterol and triglyceride assays

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0203] Embodiment 1 chemical method and reagent

[0204] Cholesterol Determination Chemistry:

[0205] The assays disclosed herein can be used to form colored products from reactions with cholesterol and cholesterol derivatives (eg, cholesteryl esters) found in blood samples. As shown in Figure 1, cholesteryl esters can react with cholesterol esterase to generate free cholesterol. Free cholesterol can react with cholesterol oxidase and oxygen to form hydrogen peroxide. The hydrogen peroxide formed in this reaction can be used to quantify the amount of cholesterol in the sample. Hydrogen peroxide can react with peroxidases to form colored products, for example in the presence of or through the use of colorants, and such reactions can be detected and quantified. For example, hydrogen peroxide in horseradish peroxidase and colorants such as aminoantipyrine compounds (eg 4-aminoantipyrine) and N-ethyl-N-(2-hydroxy-3-sulfopropyl )-3,5-dimethoxyaniline to form colored produc...

Embodiment 2

[0224] Example 2 - Cholesterol Determination

[0225] 1) Combine 30 μL of sample (neat plasma or serum diluted 1:30 with water or phosphate-buffered saline (PBS)) with 20 μL of reagent AC in the wells of a 384-well microplate reader (MTP) at 37°C.

[0226] 2) Shake the plate at 1800 rpm for 2 seconds.

[0227] 3) Incubate the plate at 37°C for 5 minutes.

[0228] 4) Add 20 μL of reagent BC. The plate was shaken at 1800 revolutions per minute (rpm) for 2 seconds.

[0229] 5) Incubate the plate at 37°C for 12 minutes in a spectrophotometer capable of reading absorbance at given time intervals.

[0230] 6) Record the absorbance (A) at 560 nm and 700 nm every 30 seconds (measured with a M5 spectrophotometer (Molecular Devices, Sunnyvale, CA)). Note that other intervals, including longer intervals, are also suitable.

[0231] sample:

[0232] figure 2 Lipid content of lipoproteins (results including triglycerides) measured in clinical serum samples used for the studies ...

Embodiment 4

[0273] Example 4 Combined Measurement of Blood Cholesterol and Triglycerides

[0274] Cholesterol (C) and triglycerides (TG) can be measured on aliquots of the same blood sample or on different blood samples taken from the same patient in order to compare with what would be achieved by measuring C alone or measuring D alone Provide more complete clinical information. Additionally, TG measurements can be used to provide a measure of VLDL in a blood sample. Therefore, the measurements of LDL-C, HDL-C, TC, VLDL-C and TG can be obtained by the methods of Examples 1, 2 and 3, and VLDL-C can be estimated by the following formula:

[0275] VLDL-C=TG / 5.

[0276] TC can be measured directly according to the methods and assays disclosed herein; similarly, HDL-C and LDL-C can be measured directly according to the methods and assays disclosed herein. Since TC is a measure of the total cholesterol in the sample, TC is the sum of the cholesterol in the lipoprotein fraction of the sampl...

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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.

Description

Background technique [0001] Lipids, especially cholesterol, play key roles in human health and disease. Cholesterol is an essential nutrient and a key component that forms the lipid membranes that surround the boundaries of cells and organelles. [0002] However, excess cholesterol is very dangerous because it can accumulate as "plaque" in blood vessels and can cause blood clots, stroke and other potentially fatal consequences in humans. To reduce these risks, lipids, especially cholesterol, are packaged into lipoproteins for transport through the body in the blood. [0003] Various heterogeneous forms of lipoproteins containing cholesterol (C) and triglycerides (TG) are known, including, for example, chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). The cholesterol content of these lipoproteins is called VLDL-cholesterol (VLDL-C), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C), respectively. [0004]...

Claims

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Application Information

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IPC IPC(8): C12Q1/28C12Q1/44C12Q1/60C12Q1/61G01N33/49G01N33/52
CPCC12Q1/61G01N33/92C12Q1/60G01N2333/904G01N2333/908G01N2333/92C12Q1/26C12Q1/44C12Q2326/00C12Q2326/90C12Q2326/96C12Q2523/115
Inventor D·迈特杰I·吉本斯P·帕特尔E·A·霍姆斯
Owner THERANOS IP CO LLC
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