Composition for assaying glycated proteins

Abstract

Compositions for accurately assaying a glycated protein by: 1) avoiding effects of globulin and ascorbic acid components, 2) stabilizing proteases and at least enzymes acting on glycated amino acids; 3) accurately assaying albumin; and 4) assaying glycated albumin while avoiding the effects of glycated hemoglobin, and an assay method are provided. Thus, the contents of a glycated protein and glycated albumin can be more accurately determined.

1 . A method for calculating a ratio of glycated albumin to total albumin in a sample solution, comprising:
 dividing a sample solution into a first portion and a second portion;   contacting the first portion with a protease and a fructosyl amino acid oxidase, wherein the protease and the fructosyl amino acid oxidase are contacted with the first sample portion in any order;   determining the concentration of glycated albumin in said first portion;   contacting the second portion with bromocresol purple, wherein the second portion is treated with a protein denaturing agent, and/or a compound having an S—S bond, concurrently with bromocresol purple or before contact with bromocresol purple;   determining the concentration of total albumin in the second portion; and   dividing the concentration of glycated albumin as determined from the first portion by the concentration of total albumin as determined from the second portion, thereby calculating the ratio of glycated albumin to total albumin in the sample solution.   
     
     
         2 . The method according to  claim 1 , wherein the protein denaturing agent and/or the compound having an S—S bond is 2,2′-dithiodibenzoic acid, 4,4′-dithiodimorpholine, 2,2-dihydroxy-6,6′-dinaphthyl disulfide (DDD), 2,2′-dithiopyridine (2-PDS), 4,4′-dithiopyridine (4-PDS), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), or sodium lauryl sulfate. 
     
     
         3 . The method according to  claim 1 , wherein an ascorbic acid oxidase, and a buffer agent that does not have a 4-(2-hydroxyethyl)-1-piperazinyl group, are further contacted with the first portion along with said protease. 
     
     
         4 . The method according to  claim 3 , wherein the buffer agent that does not have a 4-(2-hydroxyethyl)-1-piperazinyl group, is tris-hydroxymethyl amino methane, or piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid). 
     
     
         5 . The method according to  claim 1 , wherein the fructosyl amino acid oxidase is a mutant fructosyl amino acid oxidase of which the reactivity with glycated valine has been decreased by replacing the lysine at the 372 position in the amino acid sequence of SEQ ID NO:2 with another amino acid. 
     
     
         6 . The method according to  claim 5 , wherein the lysine at the 372 position of the fructosyl amino acid oxidase amino acid sequence of SEQ ID NO:2 is replaced with tryptophan, methionine, or valine. 
     
     
         7 . The method according to  claim 1 , wherein the first portion is contacted with fructosyl amino acid oxidase before the protease is contacted with the first portion. 
     
     
         8 . The method according to  claim 1 , wherein the sample solution is a biological sample. 
     
     
         9 . The method according to  claim 8 , wherein the biological sample is selected from blood serum, blood plasma, blood cells, or whole blood. 
     
     
         10 . A method for calculating a ratio of glycated albumin to total albumin in a sample solution, comprising:
 dividing a sample solution into a first portion and a second portion;   contacting the first portion with a protease and a deglycation enzyme wherein the protease and the deglycation enzyme are contacted with the first sample portion in any order;   determining the concentration of glycated albumin in said first portion by measuring an amount of hydrogen peroxide produced from reacting the deglycation enzyme with the glycated amino acids or glycated peptides, wherein said glycated amino acids or glycated peptides are obtained from reacting the glycated albumin with the protease, wherein the presence or amount of hydrogen peroxide determines the presence or amount of glycated albumin in the sample;   contacting the second portion with bromocresol purple, wherein the second portion is treated with 2,2′-dithiodibenzoic acid, 4,4′-dithiodimorpholine, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), or sodium lauryl sulfate, and wherein the treatment is concurrent with the addition of bromocresol purple or before contact with bromocresol purple;   determining the concentration of total albumin in the second portion; and   dividing the concentration of glycated albumin as determined from the first portion by the concentration of total albumin as determined from the second portion, thereby calculating the ratio of glycated albumin to total albumin in the sample solution.   
     
     
         11 . The method according to  claim 10 , wherein an ascorbic acid oxidase, and a buffer agent that does not have a 4-(2-hydroxyethyl)-1-piperazinyl group, are further added to the first portion along with said protease. 
     
     
         12 . The method according to  claim 11 , wherein the buffer agent that does not have a 4-(2-hydroxyethyl)-1-piperazinyl group, is tris-hydroxymethyl amino methane, or piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid). 
     
     
         13 . The method according to  claim 10 , wherein the sample solution is a biological sample. 
     
     
         14 . The method according to  claim 13 , wherein the biological sample is selected from blood serum, blood plasma, blood cells, or whole blood. 
     
     
         15 . The method according to  claim 10 , wherein the deglycation enzyme is contacted with the first portion before the protease is contacted with the first portion, and wherein glycated amino acid or glycated peptide, if present before the protease treatment, is eliminated from the first portion of the sample. 
     
     
         16 . The method according to  claim 10 , wherein the protease is contacted with the first sample portion before the deglycation enzyme is contacted with the first portion. 
     
     
         17 . The method according to  claim 10 , wherein the deglycation enzyme catalyzes the oxidative degradation of at least a glycated amino acid. 
     
     
         18 . The method according to  claim 10 , wherein the deglycation enzyme catalyzes the oxidative degradation of at least a glycated peptide. 
     
     
         19 . The method according to  claim 10 , wherein the deglycation enzyme is ketoamine oxidase. 
     
     
         20 . The method according to  claim 19 , wherein the ketoamine oxidase is a mutant ketoamine oxidase of which the reactivity with glycated valine has been decreased by replacing the lysine at the 372 position in the amino acid sequence of SEQ ID NO:2 with another amino acid. 
     
     
         21 . The method according to  claim 20 , wherein the lysine at the 372 position of SEQ ID NO:2 is replaced with tryptophan, methionine, or valine. 
     
     
         22 . The method according to  claim 10 , wherein the deglycation enzyme catalyzes the oxidative degradation of a glycated α-amino acid or ε-amino acid amadori compound to produce glucosone, hydrogen peroxide, and a corresponding α-amino acid or ε-amino acid. 
     
     
         23 . A method for assaying glycated protein in a sample, wherein the sample is suspected to contain a glycated amino acid or a peptide, and wherein said glycated amino acid or said peptide is not the analyte of the assay, comprising the steps of:
 (i) adding a deglycation enzyme, to the sample to form a mixture of the sample and the deglycation enzyme;   (ii) incubating the mixture of (i) under conditions whereby the glycated amino acid or the peptide, if present in the sample, reacts with the deglycation enzyme, thereby eliminating said glycated amino acid or peptide from the sample;   (iii) adding a protease to the assay sample obtained in step (ii) and incubating the assay sample and the protease under conditions suitable to react the glycated protein and the protease, thereby obtaining glycated amino acids or peptides,   wherein the glycated amino acids or peptides obtained from the glycated protein subsequently react with the deglycation enzyme that was added to the sample in step (i) to produce hydrogen peroxide; and   (iv) measuring the amount of hydrogen peroxide produced in step (iii)   wherein the presence or amount of hydrogen peroxide determines the presence or amount of the glycated protein in the sample.   
     
     
         24 . The method according to  claim 23 , wherein the deglycation enzyme eliminates glycated amino acid if it is present in the sample. 
     
     
         25 . The method according to  claim 23 , wherein the deglycation enzyme catalyzes the oxidative degradation of at least a glycated amino acid. 
     
     
         26 . The method according to  claim 23 , wherein the deglycation enzyme catalyzes the oxidative degradation of at least a glycated peptide. 
     
     
         27 . The method according to  claim 23 , wherein the deglycation enzyme is ketoamine oxidase. 
     
     
         28 . The method according to  claim 23 , wherein the deglycation enzyme catalyzes the oxidative degradation of a glycated α-amino acid or ε-amino acid amadori compound to produce glucosone, hydrogen peroxide, and a corresponding α-amino acid or ε-amino acid.