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Methods and compositions for determination of glycated proteins

a technology of glycated proteins and compositions, applied in the direction of instruments, peptides, enzymology, etc., can solve problems such as the disfunction of target molecules

Inactive Publication Date: 2008-10-02
GENERAL ATOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an isolated chimeric protein that comprises a bacterial leader sequence and an amadoriase, which can be used to assay for glycated proteins in a sample. The chimeric protein oxidizes glycated peptides or amino acids generated from glycated proteins using a protease and an amadoriase. The invention also provides a nucleic acid encoding the chimeric protein and a kit for conducting the assay. The technical effect of the invention is to provide a reliable and accurate method for detecting glycated proteins in a sample.

Problems solved by technology

This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links.

Method used

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  • Methods and compositions for determination of glycated proteins
  • Methods and compositions for determination of glycated proteins
  • Methods and compositions for determination of glycated proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Glycated Serum Protein Enzymatic Assay Kit

[0099]Intended Use. The exemplary assay kit is for determination of glycated serum proteins (fructosamine) in human serum. Fructosamine is formed due to a non-enzymatic Maillard reaction between glucose and amino acid residues of proteins. In diabetic patients, elevated blood glucose levels correlate with increased fructosamine formation. Fructosamine is a medium term indicator of diabetic control (2-3 weeks).

[0100]Assay Principle. The exemplary enzymatic assay for glycated serum proteins (GSP) uses Proteinase K to digest GSP into low molecular weight glycated protein fragments (GPF), and uses Diazyme's specific Fructosaminase™, a microorganism originated amadoriase to catalyze the oxidative degradation of Amadori product GPF to yield PF or amino acids, glucosone and H2O2. The H2O2 released is measured by a colorimetric Trinder end-point reaction. The absorbance at 550 nm is proportional to the concentration of glycated serum proteins (GSP)....

example 2

Glycated Serum Protein Assay Precision and Linearity

[0132]Method Comparison

[0133]Determined by running 2 replicates of a set of random samples using both Diazyme GSP kit and Randox Fructosamine kit in one run. The analytical performance characteristics determined by Diazyme GSP kit were comparable to those observed with Randox Fructosamine kit when assays were performed under the conditions as described in the Example 1 (See also FIG. 1).

[0134]Assay Linearity

[0135]Determined by running 2 replicates of a set of series diluted serum samples in one run. The assay is linear from 40-856 umole / L (See FIG. 2).

[0136]Interference

[0137]Determined by running 3 replicates each of a control sample in the absence and presence of various potential interference substances at indicated concentrations (See the following Table 5).

TABLE 5Interference analysisInterfering substanceInterfering substancesconcentration% InterferenceAscorbic Acid 4 mg / dL−0.3Bilirubin 2 mg / dL−0.6Glucose1200 mg / dL −0.6Hemoglob...

example 3

Assay for Glycated Hemoglobin HbA1c

[0138]A. Glycated Valine Measurement:

[0139]1. Mix 10 ul 170 mM Glycated Valine (G-Valine) (This value was assumed all valine was converted to G-Valine in the cooking procedure.) with 300 ul R1 (80 mMCHES, 30 mmMOPS, 0.9% BRIJ). This mixture serves as sample stock solution (GVR1).

[0140]2. Set spectrometer wavelength at 726 nm, temperature at 37° C. Pipette 150 ul R2 (30 mMMES, 1 mMCaCl2, 2 mMWST-3, 1570 U / ml Proteinase K) to a cuvette, add 0 ul, 2.5 ul, 5 ul, 10 ul, 15 ul, 20 ul above GVR1 respectively for dose response, make up the sample volume to 20 ul with H2O in R1, incubate for 5 min, get the first O.D. reading, then add 30 ul R3 (0.08 mMDA-64, 240 mMTris, 180 U / ml HRP, 20 U / ml FAOD), incubate for 3 min, get the second O.D. reading. Calculate the O.D. difference between these two readings, using 20 ul H2O in R1 as control. FIGS. 3 and 4 show the dose-dependent reaction with fructosyl-valine.

[0141]B. Glycated Hemoglobin Measurement:

[0142]1. Mix...

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Abstract

This invention relates generally to the field of glycated protein detection. In particular, the invention provides chimeric proteins, nucleic acids encoding the chimeric proteins, methods and kits for assaying for a glycated protein in a sample, using inter alia, an amadoriase.

Description

BACKGROUND OF THE INVENTION[0001]A glycated protein is a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. See e.g., U.S. Pat. No. 6,127,138. Such a non-enzymatic and irreversible binding reaction is also called “Amadori rearrangement,” and therefore the above-mentioned glycated protein may also be called “Amadori compound” in some cases.[0002]Nonenzymatic glycation of proteins has been implicated in the development of certain diseases, e.g., diabetic complications and the aging process (Takahashi et al., J. Biol. Chem., 272(19):12505-7 (1997); and Baynes and Monnier, Prog. Clin. Biol. Res., 304:1-410 (1989)). This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links. Considerable interest has focused on the Amadori product that is the most important “early” modification during nonenzymatic gly...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/04C07H21/04C12N1/20C12N1/21C12N9/06C12Q1/26C12Q1/37G01N33/68G01N33/72
CPCC07K2319/40C07K2319/50C12N9/0032C12Q1/26C12Q1/37G01N33/6893G01N33/723G01N2400/00G01N2800/042
Inventor YUAN, CHONG-SHENGDATTA, ABHIJITWANG, YUPING
Owner GENERAL ATOMICS
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