Patent Document

TECHNICAL FIELD 
       [0001]    The present invention relates to a method for quantifying glycated hemoglobin contained in a sample. 
       BACKGROUND ART 
       [0002]    Glycated hemoglobin (hereinafter, referred to as “HbA 1 c”) is a kind of glycated protein formed by nonenzymatically binding a glucose to a hemoglobin A (hereinafter, referred to as “HbA”) included in a red blood cell. The HbA 1 c is used as a diabetes marker. 
         [0003]    Patent document 1 discloses a method for quantifying HbA 1 c by an enzyme assay method. More particularly, the method disclosed in patent document 1 comprises the following steps (a) and (b). 
         [0004]    (a) decomposing HbA 1 c with protease to produce fructosyl peptide; and 
         [0005]    (b) quantifying the fructosyl peptide produced in the step (a) with fructosyl peptide oxidase (hereinafter, referred to as “FPOX”). 
         [0006]    Patent document 2 also discloses a method for obtaining fructosyl peptide. This method is characterized by that a sample containing protein such as HbA 1 c is subjected to a protease treatment in the presence of a tetrazolium compound. Patent document 2 further discloses that not only the tetrazolium compound but also a surfactant promotes the protease treatment more. 
         [0007]    Patent document 3 also discloses a method for obtaining fructosyl peptide. In this method, HbA 1 c is subjected to a protease treatment in the presence of an isothiazoline derivative and a surfactant. 
       PRIOR ART DOCUMENTS 
     Patent Documents 
       [0008]    Patent Document 1: U.S. Patent Application Publication No. 2007/0037243 
         [0009]    Patent Document 2: U.S. Patent Application Publication No. 2003/0186346 
         [0010]    Patent Document 3: Canadian Patent Application Publication No. 2 806 261 A 
         [0011]    Patent Document 4: U.S. Pat. No. 7,235,378 
         [0012]    Patent Document 5: U.S. Pat. No. 7,855,079 
       SUMMARY OF THE INVENTION 
     Problems to be Solved by the Invention 
       [0013]    In the enzyme assay method, the step (a) is required to be performed rapidly. In greater detail, it is necessary to more increase the decomposition rate of HbA 1 c, which is caused by a protease. 
         [0014]    Furthermore, the chemical reagent used in the step (a) must not harm the step (b). In more detail, the chemical reagent used in the step (a) must not inactivate the FPOX. 
         [0015]    The purpose of the present invention is to provide a method for quantifying glycated hemoglobin rapidly. 
         [0016]    The present invention relates to a method for quantifying glycated hemoglobin (HbA 1 c) contained in a sample, the method comprising the steps of: 
         [0017]    (a) mixing the sample with a protease in the presence of a composition of a cationic surfactant and a tetrazolium salt to obtain a glycated-peptide aqueous solution containing a glycated peptide; 
         [0018]    (b) mixing the glycated-peptide aqueous solution obtained in the step (a) with FPOX (fructosyl peptide oxidase) to obtain hydrogen peroxide; wherein the glycated-peptide aqueous solution contains the composition; and 
         [0019]    (c) calculating a concentration of the glycated hemoglobin (HbA 1 c) on the basis of an amount of the hydrogen peroxide obtained in the step (b). 
         [0020]    The present invention relates to a method for quantifying glycated hemoglobin (HbA 1 c) contained in a sample, the method comprising steps of: 
         [0021]    (a) mixing the sample with a protease and FPOX (fructosyl peptide oxidase) in the presence of a composition of a cationic surfactant and a tetrazolium salt to obtain hydrogen peroxide; and 
         [0022]    (b) calculating a concentration of the glycated hemoglobin (HbA 1 c) on the basis of an amount of the hydrogen peroxide obtained in the step (a). 
         [0023]    The sprits of the present invention include a composition for activating a protease containing a cationic surfactant a tetrazolium salt. 
       EFFECTS OF THE INVENTION 
       [0024]    The present invention provides a method for quantifying glycated hemoglobin rapidly. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]      FIG. 1A  shows the results of the test example 1. 
           [0026]      FIG. 1B  shows the results of the test example 1. 
           [0027]      FIG. 2A  shows the results of the test example 2. 
           [0028]      FIG. 2B  shows the results of the test example 2. 
           [0029]      FIG. 3  shows the results of the test example 3. 
           [0030]      FIG. 4  shows the results of the test example 4. 
           [0031]      FIG. 5  shows the results of the test example 5. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0032]    The embodiments of the present invention are described below. 
         [0033]    (Embodiment 1) 
         [0034]    First, the embodiment 1 is described. 
         [0035]    (Step (a)) 
         [0036]    In the step (a), a sample containing HbA 1 c is mixed with a protease in the presence of a composition of a cationic surfactant and a tetrazolium salt. Preferably, the sample is an aqueous solution. 
         [0037]    The HbA 1 c is decomposed by the protease to produce a glycated peptide. An example of the glycated peptide is a fructosyl peptide. An example of the fructosyl peptide is fructosyl-valine-histidine (hereinafter, referred to as “Fru-Val-His”). In this way, obtained is an aqueous solution containing a glycated peptide. 
         [0038]    An example of the sample containing HbA 1 c is blood derived from a human. The dilution of the blood derived from a human is also included in the sample containing HbA 1 c. 
         [0039]    An example of the cationic surfactant is a quaternary ammonium salt, an alkylamine salt or a pyridine derivative. A preferable cationic surfactant is a quaternary ammonium salt, which is represented by the chemical formula R 1 R 2 R 3 R 4 N + X − . 
         [0040]    Preferably, R 1  is an alkyl group having a carbon number of not less than 8 and not more than 18. Preferably, R 2 , R 3  and R 4  are lower alkyl groups independently. More preferably, R 2 , R 3  and R 4  are a methyl group or an ethyl group independently. A methyl group is still more preferable. X represents halogens. Preferably, X represents chlorine or bromine. 
         [0041]    Examples of a preferable cationic surfactant are listed below. 
         [0042]    Trimethyl octyl ammonium chloride 
         [0043]    Trimethyl decyl ammonium chloride 
         [0044]    Trimethyl dodecil amonium chloride 
         [0045]    Trimethyl tetradecyl ammonium chloride 
         [0046]    Trimethyl cetyl ammonium chloride 
         [0047]    Trimethyl stearyl ammonium chloride 
         [0048]    Trimethyl octyl ammonium bromide 
         [0049]    Trimethyl decyl ammonium bromide 
         [0050]    Trimethyl dodecil amonium bromide 
         [0051]    Trimethyl tetradecyl ammonium bromide 
         [0052]    Trimethyl cetyl ammonium bromide 
         [0053]    Trimethyl stearyl ammonium bromide 
         [0054]    Examples of a preferable tetrazolium salt are listed below. 
         [0055]    2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST1) 
         [0056]    2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-3) 
         [0057]    2-(2-Benzothiazolyl)-3-(4-carboxy-2-methoxyphenyl) -5[4-[( 2 -sodiosulfoethyncarbamoyl]phenyl]-2H-tetrazol-3-ium (WST-4) 
         [0058]    2,2′-(3,3′-dimethoxy-4,4′-biphenylylene) bis[3-(2-benzothiazolyl) -5-[4-[N-[2-(sodiooxysulfonyl) ethyl]-N-(2-sulfoethyl) carbamoyl]phenyl]-2H-tetrazole -3-ium] (WST-5) 
         [0059]    5-[2,4-Bis(sodiooxysulfonyl)phenyl]-3-(2-methoxy-4-nitrophenyl) -2-(4-nitrophenyl)-2H-tetrazole-3-ium (WST-8) 
         [0060]    2-(4-Nitrophenyl)-5-phenyl-3[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium, monosodium salt (WST-9) 
         [0061]    2,5-Di(4-nitrophenyl)-3-[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium, monosodium salt (WST-10) 
         [0062]    2-(4-Nitrophenyl)-5-(2-sulfophenyl)-3-[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium, disodium salt (WST-11) 
         [0063]    An example of the protease is thermolysin, papain, chymotrypsin, subtilisin, caspase, pepsin, or cathepsin D. Thermolysin and papain are preferable. 
         [0064]    As demonstrated in the sample solutions A 8 -A 10  and B 8 -B 10  included in the test examples 1 and 2, which are described later, the combination of the cationic surfactant and the tetrazolium salt significantly improves a decomposition rate of HbA 1 c. In other words, the synergistic effect of the cationic surfactant and the tetrazolium salt significantly improves the decomposition rate of HbA 1 c. See  FIG. 1A ,  FIG. 1B ,  FIG. 2A  and  FIG. 2B . 
         [0065]    As demonstrated in the sample solutions A 1  and B 1  included respectively in the test examples 1 and 2, an anionic surfactant such as sodium dodecyl sulfate (hereinafter, referred to as “SDS”) also improves the decomposition rate of HbA 1 c. However, an anionic surfactant must not be used in the present invention. The reason is described in the description of the step (b). 
         [0066]    As demonstrated in the sample solutions A 2  and B 2  included respectively in the test examples 1 and 2, the decomposition rate of HbA 1 c is not improved when the cationic surfactant is used alone. Similarly, as demonstrated in the sample solutions A 5 -A 7  and B 5 -B 7  included in the test examples 1 and 2, the decomposition rate of HbA 1 c is not improved when the tetrazolium salt is used alone. 
         [0067]    As demonstrated in the sample solutions A 3 , A 4 , B 3  and B 4  included in the test examples 1 and 2, the decomposition rate of HbA 1 c is not improved when a nonionic surfactant is used instead of the cationic surfactant. As demonstrated in the sample solutions A 11 -A 16  and B 11 -B 16  included in the test examples 1 and 2, the decomposition rate of HbA 1 c is not improved, even when a nonionic surfactant is used together with the tetrazolium salt,. 
         [0068]    (Step (b)) 
         [0069]    The step (b) is performed after the step (a). 
         [0070]    The glycated peptide aqueous solution obtained in the step (a) is mixed with fluctosylpeptide oxidase (hereinafter, referred to as “FPOX”). As disclosed in patent document 2, the glycated peptide reacts with the FPOX to produce hydrogen peroxide. In the step (b), the glycated peptide aqueous solution contains the composition of the cationic surfactant and the tetrazolium salt, similarly to the step (a). 
         [0071]    As demonstrated in the sample solutions C 8 -C 10 , D 8 -D 10  and E 8 -E 10  included in the test examples 3-5, which are described later, the glycated peptide reacts with the FPOX more rapidly in a case where the composition of a cationic surfactant and the tetrazolium salt is used, compared to the case where the cationic surfactant is used alone (See sample solutions C 2 , D 2 , and E 2 ). 
         [0072]    An anionic surfactant improves the decomposition rate of HbA 1 c. However, the anionic surfactants inactivate the FPOX, as demonstrated in the sample solutions C 1 , D 1  and E 1 , which is included in the test example 3, 4 and 5, which are described later. For this reason, in case where the anionic surfactant is used, then the HbA 1 c failed to be quantified. 
         [0073]    (Step (c)) 
         [0074]    The step (c) is performed after the step (b). 
         [0075]    The amount of the hydrogen peroxide produced in the step (b) is proportional to the amount of the HbA 1 c contained in the sample, as disclosed in patent document 4 (see column  8  lines  6 - 37 ) and patent document  5  (see column  10  line  63 -column  11  line  7 ). These patent documents are incorporated herein by reference. The following home page discloses that the amount of the hydrogen peroxide produced in the step (b) is proportional to the amount of the HbA 1 c contained in the sample. 
         [0076]    http ://www.sekisuimedical.jp /english/business/diagnostics/biochemistry/hb a 1 c/index.html 
         [0077]    Accordingly, the amount of the HbA 1 c contained in the sample is calculated on the basis of the amount of the hydrogen peroxide with use of a standard curve obtained in advance. In this way, the HbA 1 c contained in the sample is quantified. In other words, the concentration of the HbA 1 c is measured. 
         [0078]    (Embodiment 2) 
         [0079]    Then, the embodiment 2 is described. 
         [0080]    (Step (d)) 
         [0081]    In the step (d), the sample containing HbA 1 c is mixed with the protease and the FPOX in the presence of the composition of the cationic surfactant and the tetrazolium salt to obtain hydrogen peroxide. Preferably, the sample is an aqueous solution. 
         [0082]    (Step (e)) 
         [0083]    The step (e) is performed after the step (d). In the step (e), HbA 1 c is quantified on the basis of the quantity of the hydrogen peroxide obtained in the step (d) 
         [0084]    In the step (d), the step (a) and the step (b) are performed simultaneously. The step (e) is identical to the step (c). Accordingly, the detailed descriptions of the step (d) and the step (e) are omitted. 
       TEST EXAMPLE 
       [0085]    (Preparation of the sample solution) 
         [0086]    Whole human blood (available from BIOPREDIC INTERNATIONAL) was diluted to make a ten-fold dilution thereof. In this way, a sample solution containing hemoglobin was prepared. Hereinafter, this sample solution is referred to as “Sample Solution P”. 
         [0087]    (Test example 1) 
         [0088]    (Preparation of the sample solution) 
         [0089]    The following sample solutions A 1 -A 16  were prepared. A comparative sample solution al was also prepared. The source of the used reagents is listed below. 
         [0090]    Thermolysin: Wako Pure Chemical Industries, Ltd. SDS: Wako Pure Chemical Industries, Ltd. TTAB (tetradecyl trimethyl ammonium bromide): Wako Pure Chemical Industries, Ltd. TritonX-100: Wako Pure Chemical Industries, Ltd. Tween20: Wako Pure Chemical Industries, Ltd. WST-3: Dojindo Laboratories WST-4: Dojindo Laboratories WST-5: Dojindo Laboratories 
         [0091]    (Sample Solution A 1 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL SDS PBS solution: 10% by weight 
         [0092]    (Sample Solution A 2 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight 
         [0093]    (Sample Solution A 3 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight 
         [0094]    (Sample Solution A 4 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight 
         [0095]    (Sample Solution A 5 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL WST-3 water solution: 2% by weight 
         [0096]    (Sample Solution A 6 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL WST-4 PBS solution: 0.5% by weight 
         [0097]    (Sample Solution A 7 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL WST-5 PBS solution: 2% by weight 
         [0098]    (Sample Solution A 8 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TTAB 10%: by weight WST-3 PBS solution: 2% by weight 
         [0099]    (Sample Solution A 9 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TTAB 10%: by weight WST-4 PBS solution: 0.5% by weight 
         [0100]    (Sample Solution A 10 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight WST-5 PBS solution: 2% by weight 
         [0101]    (Sample Solution A 11 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight 
         [0102]    (Sample Solution A 12 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight WST-4 PBS solution: 0.5% by weight 
         [0103]    (Sample Solution A 13 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight WST- 5  PBS solution: 2% by weight 
         [0104]    (Sample Solution A 14 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight 
         [0105]    (Sample Solution A 15 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight WST-4PBS solution 0.5%: by weight 
         [0106]    (Sample Solution A 16 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight WST-5 PBS solution: 2% by weight 
         [0107]    (Comparative Sample Solution a 1 ) Sample solution P Thermolysin PBS solution: 150,000 U/mL 
         [0108]    (HbA 1 c Decompsition Reaction Using Thermolysin) 
         [0109]    The temperature of the sample solutions A 1 -A 16  and the comparative sample solution al was maintained at 37 degrees Celsius for ten minutes and the reaction occurred. 
         [0110]    (Comparison of the Decomposition Reaction Progress) 
         [0111]    After the reaction, the sample solutions A 1 -A 16  and the comparative sample solution al were subjected to polyacrylamide gel electrophoresis (PAGE).  FIG. 1A  and  FIG. 1B  show the electrophoresis patterns. The character “M” depicted in  FIG. 1A  and  FIG. 1B  indicates a protein maker. 
         [0112]    The degree of the HbA 1 c decomposition is reflected in the color strength of the band near 64.5 KDa, which corresponds to hemoglobin molecular region included in the electrophoresis pattern. If the color strength of the band near 64.5 KDa decreases, the more amounts of HbA 1 c is decomposed. 
         [0113]    As is clear from the electrophoresis patterns shown in  FIG. 1A  and  FIG. 1B , the more amount of HbA 1 c was decomposed in the sample solutions A 1 , A 8 , A 9  and A 10 , compared to the comparative sample solution a 1 . The more amount of HbA 1 c was decomposed in the sample solutions A 8 , A 9  and A 10 , compared to the sample solutions A 2 , A 5 , A 6 , and A 7 . 
         [0114]    SDS (Sample solution A 1 ), the composition of TTAB and WST-3 (Sample solution A 8 ), the composition of TTAB and WST-4 (Sample solution A 9 ) and the composition of TTAB and WST-5 (Sample solution A 10 ) improve the decomposition rate of HbA 1 c using thermolysin. 
         [0115]    The composition of TTAB and WST-3 (Sample solution A 8 ) improves the decomposition rate of HbA 1 c using thermolysin, compared to the case where TTAB was used alone (Sample solution A 2 ) and the case where WST-3 was used alone (Sample solution A 5 ). 
         [0116]    The composition of TTAB and WST-4 (Sample solution A 9 ) improves the decomposition rate of HbA 1 c using thermolysin, compared to the case where TTAB was used alone (Sample solution A 2 ) and the case where WST-4 was used alone (Sample solution A 6 ). 
         [0117]    The composition of TTAB and WST-5 (Sample solution A 10 ) improves the decomposition rate of HbA 1 c using thermolysin, compared to the case where TTAB was used alone (Sample solution A 2 ) and the case where WST-5 was used alone (Sample solution A 7 ). 
         [0118]    (Test Example 2) 
         [0119]    (Preparation of the Sample Solution) 
         [0120]    The following sample solutions B 1 -B 16  were prepared. A comparative sample solution b 1  was also prepared. The source of the used reagents is listed below. 
         [0121]    Papain: Roche Diagnostics K.K. SDS: Wako Pure Chemical Industries, Ltd. TTAB (tetradecyl trimethyl ammonium bromide): Wako Pure Chemical Industries, Ltd. TritonX-100: Wako Pure Chemical Industries, Ltd. Tween20: Wako Pure Chemical Industries, Ltd. WST-3: Dojindo Laboratories WST-4: Dojindo Laboratories WST-5: Dojindo Laboratories 
         [0122]    (Sample Solution B 1 ) Sample solution P Papain PBS solution: 300 U/mL SDS PBS solution: 10% by weight 
         [0123]    (Sample Solution B 2 ) Sample solution P Papain PBS solution: 300 U/mL TTAB: 10% by weight 
         [0124]    (Sample Solution B 3 ) Sample solution P Papain PBS solution: 300 U/mL TritonX-100 PBS solution: 10% by weight 
         [0125]    (Sample Solution B 4 ) Sample solution P Papain PBS solution: 300 U/mL Tween20 PBS solution: 10% by weight 
         [0126]    (Sample Solution B 5 ) Sample solution P Papain PBS solution: 300 U/mL WST-3 water solution: 2% by weight 
         [0127]    (Sample Solution B 6 ) Sample solution P Papain PBS solution: 300 U/mL WST-4 PBS solution: 0.5% by weight 
         [0128]    (Sample Solution B 7 ) Sample solution P Papain PBS solution: 300 U/mL WST-5 PBS solution: 2% by weight 
         [0129]    (Sample Solution B 8 ) Sample solution P Papain PBS solution: 300 U/mL TTAB 10%: by weight WST-3 PBS solution: 2% by weight 
         [0130]    (Sample Solution B 9 ) Sample solution P Papain PBS solution: 300 U/mL TTAB 10%: by weight WST-4 PBS solution: 0.5% by weight 
         [0131]    (Sample Solution B 10 ) Sample solution P Papain PBS solution: 300 U/mL TTAB: 10% by weight WST-5 PBS solution: 2% by weight 
         [0132]    (Sample Solution B 11 ) Sample solution P Papain PBS solution: 300 U/mL TritonX-100 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight 
         [0133]    (Sample Solution B 12 ) Sample solution P Papain PBS solution: 300 U/mL TritonX-100 PBS solution: 10% by weight WST-4 PBS solution: 0.5% by weight 
         [0134]    (Sample Solution B 13 ) Sample solution P Papain PBS solution: 300 U/mL TritonX-100 PBS solution: 10% by weight WST- 5  PBS solution: 2% by weight 
         [0135]    (Sample Solution B 14 ) Sample solution P Papain PBS solution: 300 U/mL Tween 20  PBS solution: 10% by weight WST-3 PBS solution: 2% by weight 
         [0136]    (Sample Solution B 15 ) Sample solution P Papain PBS solution: 300 U/mL Tween 20  PBS solution: 10% by weight WST-4 PBS solution 0.5%: by weight 
         [0137]    (Sample Solution B 16 ) 
         [0138]    Sample solution P Papain PBS solution: 300 U/mL Tween20 PBS solution: 10% by weight WST-5 PBS solution: 2% by weight 
         [0139]    (Comparative Sample Solution b 1 ) Sample solution P Papain PBS solution: 300 U/mL 
         [0140]    (HbA 1 c Decompsition Reaction Using Papain) 
         [0141]    The temperature of the sample solutions B 1  - B 16  and the comparative sample solution b 1  was maintained at 37 degrees Celsius for ten minutes and the reaction occurred. 
         [0142]    (Comparison of the Decomposition Reaction Progress) 
         [0143]    After the reaction, the sample solutions B 1 -B 16  and the comparative sample solution b 1  were subjected to polyacrylamide gel electrophoresis (PAGE).  FIG. 2A  and  FIG. 2B  show the electrophoresis patterns. The character “M” depicted in  FIG. 2A  and  FIG. 2B  indicates a protein maker. 
         [0144]    The degree of the HbA 1 c decomposition is reflected in the color strength of the band near 64.5 KDa, which corresponds to hemoglobin molecular region included in the electrophoresis pattern. If the color strength of the band near 64.5 KDa decreases, the more amounts of HbA 1 c is decomposed. 
         [0145]    As is clear from the electrophoresis patterns shown in  FIG. 2A  and  FIG. 2B , the more amount of HbA 1 c was decomposed in the sample solutions B 1 , B 8 , B 9  and B 10 , compared to the comparative sample solution b 1 . The more amount of HbA 1 c was decomposed in the sample solutions B 8 , B 9  and B 10 , compared to the sample solutions B 2 , B 5 , B 6 , and B 7 . 
         [0146]    SDS (Sample solution B 1 ), the composition of TTAB and WST-3 (Sample solution B 8 ), the composition of TTAB and WST-4 (Sample solution B 9 ) and the composition of TTAB and WST-5 (Sample solution B 10 ) improve the decomposition rate of HbA 1 c using papain. 
         [0147]    The composition of TTAB and WST-3 (Sample solution B 8 ) improves the decomposition rate of HbA 1 c using papain, compared to the case where TTAB was used alone (Sample solution B 2 ) and the case where WST-3 was used alone (Sample solution B 5 ). 
         [0148]    The composition of TTAB and WST-4 (Sample solution B 9 ) improves the decomposition rate of HbA 1 c using papain, compared to the case where TTAB was used alone (Sample solution B 2 ) and the case where WST-4 was used alone (Sample solution B 6 ). 
         [0149]    The composition of TTAB and WST-5 (Sample solution B 10 ) improves the decomposition rate of HbA 1 c using papain, compared to the case where TTAB was used alone (Sample solution B 2 ) and the case where WST-5 was used alone (Sample solution B 7 ). 
         [0150]    (Test Example 3) (Preparation of the Sample Solution) 
         [0151]    The following sample solutions C 1 -C 12  were prepared. A comparative sample solution c 1  was also prepared. The source of the used reagents is listed below. 
         [0152]    FPOX-CE: Kikkoman Corporation SDS: Wako Pure Chemical Industries, Ltd. Peroxidase: Wako Pure Chemical Industries, Ltd. KN-111 (color forming dye): Dojindo Laboratories TTAB (tetradecyl trimethyl ammonium bromide): Wako Pure Chemical Industries, Ltd. TritonX-100: Wako Pure Chemical Industries, Ltd. Tween20: Wako Pure Chemical Industries, Ltd. WST-3: Dojindo Laboratories WST-4: Dojindo Laboratories WST- 5 : Dojindo Laboratories 
         [0153]    (Sample Solution C 1 ) SDS PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase 20 U/mL KN-111: 2 mM 
         [0154]    (Sample Solution C 2 ) TTAB: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase 20 U/mL KN-111: 2 mM 
         [0155]    (Sample Solution C 3 ) TritonX-100 PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0156]    (Sample Solution C 4 ) Tween 20  PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0157]    (Sample Solution C 5 ) WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0158]    (Sample Solution C 6 ) WST-4 PBS solution: 0.5% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase 20 U/mL KN-111: 2 mM 
         [0159]    (Sample Solution C 7 ) WST-5 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0160]    (Sample Solution C 8 ) TTAB: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0161]    (Sample Solution C 9 ) TTAB: 10% by weight WST-4 PBS solution: 0.5% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0162]    (Sample Solution C 10 ) TTAB: 10% by weight WST-5 PBS solution: 2% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0163]    (Sample Solution C 11 ) TritonX-100 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0164]    (Sample Solution C 12 ) Tween20 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0165]    (Comparative Sample Solution c 1 ) FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0166]    (Reaction of the Glycated Peptide with FPOX) 
         [0167]    The temperature of the sample solutions C 1 -C 12  and the comparative sample solution c 1  was maintained at  37  degrees Celsius for ten minutes and the reaction occurred. 
         [0168]    After the reaction, five milliliter of Fru-Val-His 1 mM PBS solution (Available from Peptide Institute Inc.) was each added to the sample solution C 1 -C 12  and comparative sample solution c 1  (95 microliter). In this way, mixture solutions C 1 -C 12  and a comparative mixture solution c 1  were obtained. 
         [0169]    (Evaluation of FPOX Activity) 
         [0170]    Absorbance of the mixture solutions C 1 -C 12  and the comparative mixture solution c 1  at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer (available from Tecan Group Inc., under the trade name of Infinite 200 Pro).  FIG. 3  shows the results. 
         [0171]    The glycated peptide Fru-Val-His reacts with the FPOX to generate hydrogen peroxide. Hydrogen peroxide reacts with the color forming dye KN-111 to change the color forming dye KN-111 into red. Accordingly, the activity of the FPOX is evaluated by measuring the absorbance of the color forming dye KN-111 at a wavelength of 660 nanometers (red). 
         [0172]    As shown in  FIG. 3 , an absorbance change was observed in all of the mixture solutions C 2 -C 12  other than the mixture solution C 1  and the comparative sample solution c 1 . Among them, it was observed that the absorbance changes were similar to that of the comparative mixture solution c 1 , other than the mixture solution C 2 . It was observed that the absorbance change of the mixture solution C 2  was more gradual than that of the comparative mixture solution c 1 . 
         [0173]    On the other hand, in the sample solution C 1 , the absorbance change was not observed at all. 
         [0174]    In this way, SDS, which is an anionic surfactant, inactivates FPOX (see the result of the mixture solution C 1 ). Therefore, SDS is not allowed to be used for the present quantification method of HbA 1 c using the FPOX. On the other hand, a cationic surfactant and a non-ionic surfactant do not inactivate FPOX (see the results of the mixture solutions C 2 -C 12  and the comparative mixture solution c 1 ). 
         [0175]    As is clear from  FIG. 3 , the FPOX reacts more rapidly with the glycated peptide in the case where the composition of TTAB and WST-3, the composition of TTAB and WST-4, or the composition of TTAB and WST- 5  is used (mixture solutions C 8 -C 10 ), compared to the case where TTAB is used alone (mixture solution C 2 ). 
         [0176]    (Test Example 4) 
         [0177]    (Preparation of the Sample Solution) 
         [0178]    The following sample solutions D 1 -D 12  were prepared. A comparative sample solution d 1  was also prepared. The source of the used reagents is listed below. FPOX-CE: Kikkoman Corporation Thermolysin: Wako Pure Chemical Industries, Ltd. SDS: Wako Pure Chemical Industries, Ltd. Peroxidase Wako Pure Chemical Industries, Ltd. KN-111 (color forming dye): Dojindo Laboratories TTAB (tetradecyl trimethyl ammonium bromide): Wako Pure Chemical Industries, Ltd. TritonX-100: Wako Pure Chemical Industries, Ltd. Tween20: Wako Pure Chemical Industries, Ltd. WST-3: Dojindo Laboratories WST-4: Dojindo Laboratories WST-5: Dojindo Laboratories 
         [0179]    (Sample Solution D 1 ) Thermolysin PBS solution: 150,000 U/mL SDS PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0180]    (Sample Solution D 2 ) Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0181]    (Sample Solution D 3 ) Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0182]    (Sample Solution D 4 ) Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0183]    (Sample Solution D 5 ) Thermolysin PBS solution: 150,000 U/mL WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0184]    (Sample Solution D 6 ) Thermolysin PBS solution: 150,000 U/mL WST-4 PBS solution: 0.5% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0185]    (Sample Solution D 7 ) Thermolysin PBS solution: 150,000 U/mL WST-5 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0186]    (Sample Solution D 8 ) Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0187]    (Sample Solution D 9 ) Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight WST-4 PBS solution: 0.5% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0188]    (Sample Solution D 10 ) Thermolysin PBS solution: 150,000 U/mL TTAB: 10% by weight WST-5 PBS solution: 2% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0189]    (Sample Solution D 11 ) Thermolysin PBS solution: 150,000 U/mL TritonX-100 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0190]    (Sample Solution D 12 ) Thermolysin PBS solution: 150,000 U/mL Tween20 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0191]    (Comparative Sample Solution c 1 ) Thermolysin PBS solution: 150,000 U/mL FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0192]    (Reaction of the Glycated Peptide with FPOX) 
         [0193]    The temperature of the sample solutions D 1 -D 12  and the comparative sample solution d 1  was maintained at 37 degrees Celsius for ten minutes and the reaction occurred. 
         [0194]    After the reaction, five milliliter of Fru-Val-His 1 mM PBS solution (Available from Peptide Institute Inc.) was each added to the sample solution D 1 -D 12  and comparative sample solution d 1  (95 microliters). In this way, mixture solutions D 1 -D 12  and a comparative mixture solution dl were obtained. 
         [0195]    (Evaluation of FPOX Activity) 
         [0196]    Absorbance of the mixture solutions D 1 -D 12  and the comparative mixture solution d 1  at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer (available from Tecan Group Inc., under the trade name of Infinite 200 Pro).  FIG. 4  shows the results. 
         [0197]    The glycated peptide Fru-Val-His reacts with the FPOX to generate hydrogen peroxide. Hydrogen peroxide reacts with the color forming dye KN-111 to change the color forming dye KN-111 into red. Accordingly, the activity of the FPOX is evaluated by measuring the absorbance of the color forming dye KN-111 at a wavelength of 660 nanometers (red). 
         [0198]    As shown in  FIG. 4 , an absorbance change was observed in all of the mixture solutions D 2 -D 12  other than the mixture solution D 1  and the comparative sample solution d 1 . Among them, it was observed that the absorbance changes were similar to that of the comparative mixture solution d 1 , other than the mixture solution D 2 . It was observed that the absorbance change of the mixture solution D 2  was more gradual than that of the comparative mixture solution d 1 . 
         [0199]    On the other hand, in the sample solution D 1 , the absorbance change was not observed at all. 
         [0200]    In this way, SDS, which is an anionic surfactant, inactivates FPOX (see the result of the mixture solution D 1 ). Therefore, SDS is not allowed to be used for a quantification method of HbA 1 c using the FPOX. On the other hand, a cationic surfactant and a non-ionic surfactant do not inactivate FPOX (see the results of the mixture solution D 2 -D 12  and the comparative mixture solution c 1 ). Thermolysin also does not inactivate the FPOX. 
         [0201]    As is clear from  FIG. 4 , the FPOX reacts more rapidly with the glycated peptide in the case where the composition of TTAB and WST-3, the composition of TTAB and WST-4, or the composition of TTAB and WST-5 is used (mixture solutions D 8 -D 10 ), compared to the case where TTAB is used alone (mixture solution D 2 ). 
         [0202]    (Test Example 5) 
         [0203]    (Preparation of the Sample Solution) 
         [0204]    The following sample solutions E 1 -E 12  were prepared. A comparative sample solution e 1  was also prepared. The source of the used reagents is listed below. 
         [0205]    FPOX-CE: Kikkoman Corporation Papain: Roche Diagnostics K.K. SDS: Wako Pure Chemical Industries, Ltd. Peroxidase: Wako Pure Chemical Industries, Ltd. KN-111 (color forming dye): Dojindo Laboratories TTAB (tetradecyl trimethyl ammonium bromide): Wako Pure Chemical Industries, Ltd. TritonX-100: Wako Pure Chemical Industries, Ltd. Tween20: Wako Pure Chemical Industries, Ltd. WST-3: Dojindo Laboratories WST-4: Dojindo Laboratories WST-5: Dojindo Laboratories 
         [0206]    (Sample Solution E 1 ) Papain 300 U/mL SDS PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase 20 U/mL KN-111: 2 mM 
         [0207]    (Sample Solution E 2 ) Papain: 300 U/mL TTAB: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0208]    (Sample Solution E 3 ) Papain: 300 U/mL TritonX-100 PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0209]    (Sample Solution E 4 ) Papain: 300 U/mL Tween20 PBS solution: 10% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0210]    (Sample Solution E 5 ) Papain: 300 U/mL WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0211]    (Sample Solution E 6 ) Papain: 300 U/mL WST-4 PBS solution: 0.5% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0212]    (Sample Solution E 7 ) Papain: 300 U/mL WST-5 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0213]    (Sample Solution E 8 ) Papain: 300 U/mL TTAB: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0214]    (Sample Solution E 9 ) Papain: 300 U/mL TTAB: 10% by weight WST-4 PBS solution: 0.5% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0215]    (Sample Solution E 10 ) Papain: 300 U/mL TTAB: 10% by weight WST-5 PBS solution: 2% by weigh FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0216]    (Sample Solution E 11 ) Papain: 300 U/mL TritonX-100 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0217]    (Sample Solution E 12 ) Papain: 300 U/mL Tween20 PBS solution: 10% by weight WST-3 PBS solution: 2% by weight FPOX-CE PBS solution: 28 U/mL Peroxidase 20 U/mL KN-111: 2 mM 
         [0218]    (Comparative Sample Solution c 1 ) Thermolysin PBS solution: 150,000 U/mL FPOX-CE PBS solution: 28 U/mL Peroxidase: 20 U/mL KN-111: 2 mM 
         [0219]    (Reaction of the Glycated Peptide with FPOX) 
         [0220]    The temperature of the sample solutions E 1 -E 12  and the comparative sample solution el was maintained at 37 degrees Celsius for ten minutes and the reaction occurred. 
         [0221]    After the reaction, five milliliter of Fru-Val-His 1 mM PBS solution (Available from Peptide Institute Inc.) was each added to the sample solution E 1 -E 12  and comparative sample solution e 1  (95 microliters). In this way, mixture solutions E 1 -E 12  and a comparative mixture solution e 1  were obtained. 
         [0222]    (Evaluation of FPOX Activity) 
         [0223]    Absorbance of the mixture solutions E 1 -E 12  and the comparative mixture solution e 1  at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer (available from Tecan Group Inc., under the trade name of Infinite 200 Pro).  FIG. 5  shows the results. 
         [0224]    The glycated peptide Fru-Val-His reacts with the FPOX to generate hydrogen peroxide. Hydrogen peroxide reacts with the color forming dye KN-111 to change the color forming dye KN-111 into red. Accordingly, the activity of the FPOX is evaluated by measuring the absorbance of the color forming dye KN-111 at a wavelength of 660 nanometers (red). 
         [0225]    As shown in  FIG. 5 , an absorbance change was observed in all of the mixture solutions E 2 -E 12  other than the mixture solution E 1  and the comparative sample solution e 1 . Among them, it was observed that the absorbance changes were similar to that of the comparative mixture solution e 1 , other than the mixture solution E 2 . It was observed that the absorbance change of the mixture solution E 2  was more gradual than that of the comparative mixture solution e 1 . 
         [0226]    On the other hand, in the sample solution E 1 , the absorbance change was not observed at all. 
         [0227]    In this way, SDS, which is an anionic surfactant, inactivates FPOX (see the result of the mixture solution E 1 ). Therefore, SDS is not allowed to be used for a quantification method of HbA 1 c using the FPOX. On the other hand, a cationic surfactant and a non-ionic surfactant do not inactivate FPOX (see the results of the mixture solution E 2 -E 12  and the comparative mixture solution c 1 ). Papain also does not inactivate the FPOX. 
         [0228]    As is clear from  FIG. 5 , the FPOX reacts more rapidly with the glycated peptide in the case where the composition of TTAB and WST-3, the composition of TTAB and WST-4, or the composition of TTAB and WST-5 is used (mixture solutions E 8 -E 10 ), compared to the case where TTAB is used alone (mixture solution E 2 ). 
       INDUSTRIAL APPLICABILITY 
       [0229]    The present invention is useful for diagnosis of diabetes.

Technology Category: g