Assay device and methods

a technology of assay device and method, which is applied in the field of amphipathic polymers, can solve the problems of adverse effect on the accuracy of the device, uncontrollable flow, and inability to collect a large sample, and achieve the effects of rapid and inexpensive point of care cholesterol assay system, high efficiency and/or speed of assay, and easy quantification

Inactive Publication Date: 2011-12-15
L3 TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]The Applicants have made the surprising discovery that by use of amphipathic polymers in an assay system, considerable gains in both the efficiency and/or speed of the assay can be achieved. The Applicant's methods are also applicable to rapidly combining hydrophobic reagents and compounds such as luminophores with analytes, for example lipopr

Problems solved by technology

However, when the test sample is blood or a component of blood, the collection of a large sample is not always possible, particularly at a point of care such as a doctor's surgery.
Unfortunately, variations associated with sample transfer and diffusion of the sample to the membrane result in a flow that is largely uncontrolled and uneven before reaching a test area.
Such a reliance on capillary action may have an adverse affect on the accuracy of the device because the amount of analyte and/or label captured across a test area is not consistent.
The use of capillary action alone also means that assays are slow because of unreliable fluid wicking.
The assays are also unsuitable for small fluid samples, such as in nucleic acid detection, where the membrane may dry before the assay is completed or there may be insufficient fluid to travel the length of the test device.
It is well known that the concentration of various lipoproteins in the blood is correlated with the risk of an individual developing atherosclerosis.
Thus, if the levels of HDL cholesterol are de

Method used

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  • Assay device and methods
  • Assay device and methods

Examples

Experimental program
Comparison scheme
Effect test

example 1

Total Cholesterol Assay

[0174]The assay utilizes a triple enzyme system capable of converting one molecule of cholesterol or cholesterol ester into a molecule of hydrogen peroxide (H2O2). The hydrogen peroxide generated is then used to oxidize the dye Amplex Red (non-fluorescent) to generate the highly fluorescent product Resorufin.

Stabilization of Enzymes onto Plastic

The total cholesterol assay uses the following enzymes and dye:

Cholesterol Esterase (3.1.1.13)

Cholesterol Oxidase (1.1.3.6)

Horseradish Peroxidase (1.11.1.7)

[0175]Amplex Red: 10-acetyl-3,7-dihydroxyphenoxazine

[0176]Enzymes were stabilized onto plastic using Gafquat as a stabilizing agent. The three enzymes were added to a solution of 0.01M potassium Phosphate buffer, pH 7.0. Final activity of each enzyme was measured at 200 U / ml of buffer. The solution was then diluted 1:1 with Gafquat (highly positively charged polymer) formulation and 5 ul of the resulting solution was deposited onto a plastic surface and dried at 30° ...

example 2

Total Lipid Assay

[0191]The K37 dye was dissolved in DMF to a final concentration of 1.0 mM. Next 5% w / v PEG 2000 was dissolved into the dye solution and 60 nanolitres of the resulting solution was deposited onto a plastic surface and dried by removing the solvent under vacuum for 1 hour at room temperature in the dark.

Assay Procedure—Utilising Diluted Sample

[0192]The sample to be assayed (plasma) was first diluted 1 part into 80 parts phosphate buffered saline containing 50 mM Sodium Octanoate, pH 7.4.

[0193]5 μl of diluted plasma sample was applied to the dried dye which spontaneously hydrated. The Total lipid content was measured by exciting the sample at 440 nm (10 nm bandpass) and measuring the resulting fluorescence passing through a 495 nm filter (10 nm bandpass). The total lipid content was determined empirically by reference to known standards.

Assay Procedure—Utilising Undiluted Sample

[0194]Neat biological samples are assayed by applying the sample to a borosilicate filter im...

example 3

HDL Cholesterol Assay

[0195]Nile Red was dissolved in DMF to a final concentration of 0.5 mM. Next 5% w / v PEG 2000 was dissolved into the dye solution and 60 nanolitres of the resulting solution was deposited onto a plastic surface and dried by removing the solvent under vacuum for 1 hour at room temperature in the dark.

Assay procedure—Utilising Diluted Sample

[0196]The sample to be assayed (plasma) was first diluted 1 part into 80 parts phosphate buffered saline containing 50 mM Sodium Octanoate, pH 7.4.

[0197]5 μl of diluted plasma sample was applied to the dried dye which spontaneously hydrated. The HDL cholesterol content was measured by exciting the sample at 580 nm (10 nm bandpass) and measuring the resulting fluorescence passing through a 610 nm filter (10 nm bandpass). HDL cholesterol content was calculated by use of the algorithm described in the main specification. An equivalent assay may also be performed utilising whole, undiluted blood.

Assay procedure—Utilising Undiluted S...

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Abstract

The present invention relates to the use of amphipathic polymers to enhance lateral flow and reagent mixing on assay devices. More specifically, the invention relates to use of an amphipathic polymer in assay methods including a device for determining the concentration of lipids in blood serum or plasma.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the use of amphipathic polymers to enhance lateral flow and reagent mixing on assay devices. More specifically, the invention relates to use of an amphipathic polymer in assay methods including a device for determining the concentration of lipids in blood serum or plasma.BACKGROUND TO THE INVENTION[0002]Lateral flow assay devices and methods are known in the art. Previously, such devices have been developed to test samples that are easily available in large quantities. However, when the test sample is blood or a component of blood, the collection of a large sample is not always possible, particularly at a point of care such as a doctor's surgery.[0003]Generally these devices comprise a lateral flow matrix, for example, nitrocellulose membranes and the like. A sample applied to the matrix flows along the matrix, and one or more analytes within the sample react with one or more reagents within the lateral flow matrix. Typica...

Claims

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Application Information

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IPC IPC(8): C12M1/34G01N33/53C07C43/10G01N21/64
CPCB01F13/0071B01F13/0084B01L3/502769G01N21/64B01L2400/0406B01L2400/088G01N33/92B01L2300/161Y02P20/582B01F33/3021B01F33/30351G01N33/4875G01N33/5306G01N33/54393
Inventor NICHOLLS, ANTHONYGARCIA, LAURAHUDSON, MARKJONES, GARETHCLARKE, DAVID
Owner L3 TECH
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