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Method and devices for quantitation of glycated protein

A protein and saccharification technology, which can be used in measurement devices, color/spectral property measurement, instruments, etc., and can solve problems such as changes in results

Inactive Publication Date: 2010-04-28
无锡博慧斯生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it appears that the results of this method may vary depending on the time of co-incubation of the glycosylated protein with the borated carrier

Method used

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  • Method and devices for quantitation of glycated protein
  • Method and devices for quantitation of glycated protein
  • Method and devices for quantitation of glycated protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Preparation of borated solid support matrix

[0092] According to a preferred aspect of the detection method of the present invention, a solid support matrix suitable for strip construction can be obtained by covalently attaching a boronate derivative, m-aminophenylboronate, to a carboxycellulose-based material using conventional attachment chemistries. .

[0093] Material

[0094] 1. Solid carrier matrix: cellulose-based solid (Sartobind C membrane Sartoricus) to which carboxylic acid groups are covalently attached.

[0095] 2. EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (Pierce)

[0096] 3. Borate derivative meta-aminoboronic acid (Sigma-Aldrich)

[0097] 4. Buffer: 0.1M MES buffer, pH6.5

[0098] method

[0099] 46.7 mg of m-aminophenylboronic acid was dissolved in 25 ml of 100 mM MES buffer. The pH was readjusted to 6.5 after the boric acid was dissolved. 28.9 mg of EDC was dissolved in MES-boric acid solution. Immerse the solid ...

Embodiment 2

[0103] Effect of variable incubation time

[0104] A single measurement test with the borated support of Example 1 ("Single Measurement") was compared with the method of the present invention to determine the effect of variable incubation times on the concentration results obtained by each method.

[0105] The following tests were performed to compare the two methods. In the single assay test, blood lysates from non-diabetic and diabetic individuals were treated with 50 mM Mg ++ 500 mM ammonium acetate buffer (pH=9.5) was diluted 1:1, and then the samples were contacted with the borated solid support matrix prepared in Example 1 for different time periods. After incubation, the borated solid support was rinsed with ammonium acetate buffer (pH=9). Glycated hemoglobin was eluted with tris buffer (pH=8.0) containing 200 mM sorbitol. The absorbance of the eluate was measured at 415 nm.

[0106] In a second test corresponding to the method of the present invention, blood lysa...

Embodiment 3

[0111] Effect of pH on Binding

[0112] The pH of the buffer affects the amount of hemoglobin bound to the borated carrier. If the pH is low, both glycated and non-glycated hemoglobin are bound, and if the pH is high, only glycated hemoglobin will be bound. Figure 4 Binding of hemoglobin to carboxycellulose membranes (absence of boronic acid groups) was shown following the test procedure described in Example 2. In the absence of boric acid, the membrane loses its ion-binding capacity for hemoglobin between pH 6 and 7.

[0113] Figure 5 The hemoglobin and human serum albumin binding properties of carboxycellulose supports with added boronic acid groups prepared as described in Example 1 are shown. Binding of proteins occurs at lower pH due to the negatively charged carboxyl groups. The proteins bound at higher pH due to phenylboronic acid were glycated proteins in the sample.

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Abstract

The present invention provides methods for quantitation of glycated in a biological sample using a solid support matrix by making a first bound protein measurement total bound protein under conditionswhere both glycated and non-glycated protein bind to the support and making a second bound protein measurement under conditions where glycated protein is bound to the support and non-glycated proteinis not substantially bound. Diagnostic devices and kits comprising the methods of the present invention are also provided.

Description

[0001] priority information [0002] This application claims priority to US Patent Application Serial No. 60 / 265,229 (filed January 31, 2001). field of invention [0003] The present invention relates to a method for the quantitative determination of the content of glycated proteins in a biological sample suitable for use, for example, in reflectometers used by diabetics for self-monitoring of blood glucose concentrations. Background of the invention [0004] Control of blood glucose concentrations in diabetic patients has been shown to reduce the frequency and severity of the chronic microvascular and neurological complications of the disease. Measurements of glycated hemoglobin and protein in the blood can be used to determine how well blood sugar levels are being controlled over an extended period of time. [0005] The rate of formation of glycated hemoglobin is directly related to the concentration of glucose in the blood. The average lifespan of red blood cells is 120...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/72G01N21/27G01N33/483
CPCG01N33/6803G01N33/6842G01N33/68G01N33/723G01N2400/00
Inventor C·E·梅尔顿R·P·迈克克罗斯基
Owner 无锡博慧斯生物医药科技有限公司
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