JP2015165827A - Method of measuring glycated hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To complete the measurement of glycated hemoglobin in a short time.SOLUTION: Disclosed is a method of measuring glycated hemoglobin, in which 1) a step of determining hemoglobin in a test liquid, 2) a step of previously eliminating endogenous glycated amino acids and glycated peptides by allowing a fructosyl amino acid oxidase to act on under specific conditions, and 3) a step of determining glycated hemoglobin by allowing a protease to act on under specific conditions are performed in the same reaction vessel.

Description

  The present invention relates to a method for measuring glycated hemoglobin, and more particularly to a method for measuring a ratio of glycated hemoglobin to hemoglobin. The present invention also relates to a reagent for measuring glycated hemoglobin, and more particularly to a reagent for measuring the ratio of glycated hemoglobin to hemoglobin.

A reagent for measuring glycated hemoglobin (for example, HbA1c) using a conventional enzyme method has been affected by glycated amino acids or glycated peptides other than the measurement target component present in the infusion solution. In order to eliminate glycated amino acids or glycated peptides other than those to be measured, various measuring systems that incorporate an erasing system are known.
However, in order to build an elimination system that can be applied to a general-purpose automatic analyzer, particularly a measurement system of about 10 minutes in total, which is about 5 minutes for the first reaction and about 5 minutes for the second reaction, which is most often used, (1) Cleavage of glycated amino acid or glycated peptide from glycated hemoglobin by protease, (2) generation of hydrogen peroxide by oxidation of the glycated amino acid or glycated peptide, and (3) quantification of generated hydrogen peroxide Although it must be completed in about 5 minutes of the second reaction, in the conventional method, the reaction of the protease is particularly rate-limiting, and the reaction is not completed in the second reaction (about 5 minutes).

Accordingly, various methods for adding a protease reaction accelerator have been devised in order to complete the reaction in a short time.
For example, Patent Documents 1 to 7 and Non-Patent Document 1 use a method of adding a tetrazolium compound, Patent Documents 8 and 9 use a sulfone compound and / or a nitro compound, and Patent Document 10 uses a sucrose ester. Protease reaction promoter containing at least one of a method of allowing protease to act, Patent Document 11 includes a compound containing an acetate group or a salt thereof, N-acyl taurine or a salt thereof, or polyoxyethylene alkyl ether sulfate or a salt thereof Are each disclosed.

WO2002 / 027012 WO2002 / 027330 WO2002 / 027331 JP 2000-93199 A JP 2001-292895 A JP2003-232789 WO2000 / 210100 WO2004 / 007760 WO2003 / 107011 WO2006 / 120976 WO2006 / 013921

Ikunosuke Sakurabayashit. Al., “New Enzymatic Assay for Glycated Hemoglobin, Vol. 3, Criminal Chem. 269-274

  In the methods described in Patent Documents 1 to 7 and Non-Patent Document 1, the tetrazolium salt reacts strongly with a reducing compound, for example, glycated protein or ascorbic acid, so that the color is high. It causes abnormal values in samples containing ascorbic acid.

  In the methods described in Patent Documents 8 and 9, many sulfone compounds are inhibitors of enzymes. Particularly frequently used sodium lauryl sulfate, sodium dodecylbenzenesulfonate, lithium lauryl sulfate, and the like are glycated amino acids and / or saccharified. It is an inhibitor that is powerful enough to be a reaction terminator for enzymes that act on peptides. Furthermore, the nitro compound is colored, causing the reagent blank to rise and the measurement accuracy to decrease.

  In the method described in Patent Document 10, when a sucrose ester is used, the stability of the coexisting enzyme is low compared to the reaction accelerator of this patent. Industrial sucrose esters are inexpensive, but their purity is low and leuco dyes may self-color. High-purity sucrose esters are more expensive than common surfactants.

In the method described in Patent Document 11, the additive used is generally expensive.
The “compound containing an acetic acid group or a salt thereof”, “N-acyl taurine or a salt thereof” and “polyoxyethylene alkyl ether sulfate or a salt thereof” disclosed in Patent Document 11 are all anionic surfactants. .
Among these, as described in Example 1, N-acylamino acids sarcosinate CN-30, sarcosinate MN, sarcosinate PN, alaninate LN-30, alkyl ether carboxylic acids ECTD-3NEX, ECTD- 6NEX, AKYPO-RLM100, N-acyl tauric acid LMT, SMT, polyoxyethylene alkyl ether sulfates SBL-2T-36, SBL-4T, SBL-203-27 actually have a protease reaction promoting effect It has been demonstrated that
However, in general, anionic surfactants are said to adsorb to protein molecules (enzymes) and suppress their activity, and in the form of coexistence with the enzyme to be measured, when actually stored for a long time rather than in an accelerated test Furthermore, there is a possibility that the enzyme activity itself is lowered and adversely affects the measurement.

  An object of the present invention is to solve the problems in the prior art.

The present inventors have found that the protease reaction is significantly promoted by treating the protein with protease in the presence of a compound represented by the following formula (I) of HLB12 or higher, and have reached the present invention.
R-O-X (I)
Here, R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue. The alkyl group of R has 9 or more carbon atoms, preferably 9 to 18, more preferably 12 to 16. Most preferably, the alkyl group of R has 12 carbon atoms.

That is, the present invention has the following configuration.
[Claim 1]
A method for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin, the method comprising the following (a) and (b):
(A) The following steps 1) to 3) are performed in the same reaction vessel.
(B) Steps 1) and 2) are performed in the same step, and step 3) is started in the next step.
1) quantifying hemoglobin in the test solution;
2) In the presence of a compound represented by the following formula (I) of HLB 12 or more (preferably HLB 14 or more, more preferably HLB 16 or more) in the test solution, the pH is within a range of 5.0 to 9.5. Adding ~ 100 U / mL fructosyl amino acid oxidase to pre-erase endogenous glycated amino acids and glycated peptides;
R-O-X (I)
(R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue.)
3) A step of quantifying glycated hemoglobin by allowing a protease of 500 to 500,000 U / mL to act on the reaction solution of 2) within a range of pH 5.0 to 9.5.
[Section 2]
In the method according to Item 1,
Method in which the protease is allowed to act in the presence of the compound represented by the above formula (I) of HLB 12 or more (preferably HLB 14 or more, more preferably HLB 16 or more) and a metal ion in the step 3) [Item 3]
In the method according to Item 1 or 2,
Method for allowing protease to act in the presence of potassium ferrocyanide and / or nitrite in the step 3) [Item 4]
A reagent for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin in the same reaction tank, characterized by the following (a) and (b):
(A) It is composed of two parts, a first reagent and a second reagent.
(B) a compound represented by the following formula (I) having HLB of 12 or more (preferably HLB of 14 or more, more preferably HLB of 16 or more), and a buffer having a buffer capacity in the range of pH 5.0 to 9.5; The solution contains 0.1 to 100 U / mL fructosyl amino acid oxidase, and the second reagent contains 500 to 500,000 U / mL protease.
R-O-X (I)
(R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue.)
[Section 5]
In the reagent according to Item 4,
A reagent containing a glycated hemoglobin quantitative dye in the first reagent.
[Claim 6]
In the reagent according to Item 4 or 5,
A reagent containing potassium ferrocyanide and / or nitrite in either the first reagent or the second reagent.

According to the present invention, the reaction of the protease is accelerated (for example, the entire reaction is completed in only 5 minutes of the second reaction), and as a result, an elimination system can be formed. False high values due to the influence of glycated amino acids or glycated peptides other than components are eliminated.
The selected protease promoter does not inhibit the stability of the main reaction enzymes (fructosyl amino acid oxidase (FAOD), peroxidase (PEO), protease) and other erasing enzymes (catalase (CAO)). The selected reaction accelerator has a surface active action and has an effect that it is not necessary to newly add a surface active agent that is essential for a diagnostic agent compatible with an automatic analyzer.

The data which examined the elimination ability of the endogenous glycated amino acid in this invention. The correlation of the method of this invention and HPLC method regarding the measuring method of HbA1c. The correlation of the method of this invention and HPLC method regarding the measuring method of HbA1c. The correlation of the method of this invention and HPLC method regarding the measuring method of HbA1c. The correlation of the method of this invention and HPLC method regarding the measuring method of HbA1c. The correlation of the method of this invention and HPLC method regarding the measuring method of HbA1c. The reaction time course example of HbA1c measurement by the method of this invention.

  An embodiment of the present invention will be described in detail as follows, but the present invention is not limited to this. In addition, all the nonpatent literatures and patent literatures described in this specification are used as reference in this specification. In addition, “˜” in the present specification means “above and below”. For example, if “★ to ☆” is described in the specification, it means “★ or more and ☆ below”. In the present specification, “and / or” means either one or both.

Characteristic configuration of the measurement method of the present invention A characteristic configuration of the present invention is a method for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin, which includes the following (a ) And (b).
(A) The following steps 1) to 3) are performed in the same reaction vessel.
(B) Steps 1) and 2) are performed in the same step, and step 3) is started in the next step.
1) quantifying hemoglobin in the test solution;
2) In the presence of a compound represented by the following formula (I) of HLB 12 or more (preferably HLB 14 or more, more preferably HLB 16 or more) in the test solution, the pH is within a range of 5.0 to 9.5. Adding ~ 100 U / mL fructosyl amino acid oxidase to pre-erase endogenous glycated amino acids and glycated peptides;
R-O-X (I)
(R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue.)
3) A step of quantifying glycated hemoglobin by allowing a protease of 500 to 500,000 U / mL to act on the reaction solution of 2) within a range of pH 5.0 to 9.5.

[Test solution]
The test solution is not particularly limited as long as it is a target for which at least one of hemoglobin, glycated hemoglobin, and the ratio of glycated hemoglobin to hemoglobin is to be measured. For example, whole blood, plasma, serum, In addition to blood cells and the like, the present invention can also be applied to biological samples such as urine and cerebrospinal fluid (that is, samples collected from the living body), drinking water such as juice, foods such as soy sauce and sauce.
Examples of glycated hemoglobin include HbA1c (glycated protein in which the β chain of hemoglobin is glycated).
Since the method of the present invention can be applied to diabetes diagnosis, it is particularly useful for whole blood samples and blood cell samples. Although not particularly limited, when measuring glycated hemoglobin in erythrocytes, whole blood is lysed as it is, or erythrocytes separated from whole blood are lysed, and this hemolyzed sample is used as a sample for measurement. And it is sufficient.

  The hemolysis method is not particularly limited, and for example, a method using a surfactant, a method using ultrasonic waves, a method using a difference in osmotic pressure, a method using freeze lysis and the like can be used. Among these, a method using a surfactant or a method using a difference in osmotic pressure is preferable for reasons such as ease of operation. In addition, in an automatic analyzer with an automatic hemolysis function (for example, JEOL BM9130 type automatic analyzer), since the stirring power at the time of automatic hemolysis is strong, the surfactant generates bubbles and interferes with sampling. A method using the difference in osmotic pressure is more preferable.

  Examples of the surfactant include polyoxyethylene-pt-octylphenyl ether (Triton surfactant, etc.), polyoxyethylene sorbitan alkyl ester (Tween surfactant, etc.), polyoxyethylene alkyl ether ( Nonionic surfactants such as Brij type surfactants can be used, and specific examples include trade name Triton X-100, trade name Tween-20, trade name Brij 35, and the like. The treatment conditions with the surfactant are usually such that when the blood cell concentration in the treatment solution is 1 to 10% by volume, the surfactant is added so that the concentration in the treatment solution is 0.1 to 1% by weight. And stirring at room temperature for about 5 seconds to 1 minute.

  Moreover, when utilizing the said osmotic pressure difference, for example, 2-100 times volume of purified water is added with respect to the volume of whole blood, and hemolysis is carried out.

  Note that the hemolysis treatment can be performed simultaneously with the reaction in the first step, not as a pretreatment.

[Measurement of glycated hemoglobin]
First, the measurement of glycated hemoglobin in the present invention will be described. Although the following description includes what illustrates the method of measuring HbA1c as glycated hemoglobin, the measurement of glycated hemoglobin is not limited to this.
The measurement of hemoglobin and the measurement of the ratio of glycated hemoglobin to hemoglobin will be described later.

Examples of the measurement of glycated hemoglobin in the present invention include a method of measuring HbA1c in a measurement sample (for example, whole blood or blood) by a method including the following reaction steps (1) and (2).
(1) Reaction of fragmenting a glycated protein into a glycated amino acid and / or glycated peptide by a protease having the ability to excise FVH (fructosylvalylhistidine) from the glycated β-chain N-terminus of glycated hemoglobin (2) Fragmentation FAOD (fructosyl amino acid oxidase) that reacts with the generated FVH and satisfies the characteristics shown in the following (a) and (b) performs an oxidation-reduction reaction with the FVH, and quantifies the generated hydrogen peroxide or oxygen. Reaction

Protease applied to the present invention The protease used in the method of the present invention is not particularly limited.
As these proteases, commercially available ones may be used as they are, or those produced by publicly known literature according to the description may be used.
Examples thereof include at least one protease selected from the group consisting of thermolysin, bromelain, papain, porcine pancreatic trypsin, metalloproteinase, and neutral protease.

The proteases used in the present invention are preferably the following from the viewpoints of stability, reaction rate (specific activity), availability, and the like.
Metalloprotease, serine protease, serine carboxypeptidase, proteinase K, bromelain, papain, porcine pancreatic trypsin, Bacillus subtilis-derived protease, Aspergillus oryzae-derived protease, Aspergillus saitoi-derived protease, Streptomyces grisease, etc. It is.
Examples of commercially available products include Protease A “Amano” G (Amano Enzyme), Protease M “Amano” G (Amano Enzyme), Protease S “Amano” G (Amano Enzyme), Peptidase R (Amano Enzyme), Papain M? 40 (Amano Enzyme), Thermolysin (Daiwa Kasei), Samoaase PC10 (Daiwa Kasei), Trade name Protease N (Furuka), Trade name Protease N “Amano” (Amano Pharmaceutical), Bacillus genus metalloproteinase (Toyobo: Trade name) Toyoteam), endoproteinase Glu-C (Roche) and the like. Among them, a Bacillus genus-derived metalloproteinase (Toyobo: trade name Toyoteam), thermolysin (Amano Enzyme), Samoaase PC10 (Amano Enzyme), protease (derived from Streptomyces grieseus, Fluka), and protease (Aspergillus saitoi) are preferred. .

Even if the protease used in the present invention is other than those described above, the protease whose purification method is known has been purified and the structure has been elucidated, or the partial amino acid sequence of the purified enzyme has been clarified, and the information can be obtained. It may be obtained by using and newly screening. Further, it may be obtained by newly screening using a gene or protein sequence estimated from information published in a database.
Furthermore, a protease whose gene or protein sequence is actually known, or a protease newly obtained by the above-mentioned method is further modified by genetic engineering or chemical methods. Also good.

FAOD applied to the present invention
The FAOD used in the method of the present invention is not particularly limited. For example, as a known one, enzymes derived from the genus Koniochaeta, Eupenicillium and variants thereof, or enzymes derived from Carbaria clavater YH923 or Neocosmospora vasinfecta 474 and variants thereof can be used.
In addition, a Cryptococcus neoformans-derived enzyme described in JP-A 2010-35469 and a modified product thereof, or an Aspergillus nidulans-derived enzyme described in JP-A 2010-57474, and a modified product thereof can be used.
In addition, commercially available products such as FPOX-301 (trademark) (Toyobo), FPOX-CE (trademark) (Kikkoman), FPOX-EE (trademark) (Kikkoman) and the like can be used.

Alternatively, examples of the FAOD used in the method of the present invention include FAOD selected from Phaeosphaeria nodorum-derived enzymes disclosed in PCT / JP2009 / 005173 and variants thereof.
Specifically, the protein which has a fructosyl valyl histidine oxidase activity in any one of the following (I) to (III) can be illustrated.
(I) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(II) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has fructosyl valyl histidine oxidase activity protein.
(III) A protein comprising an amino acid sequence having 86.0% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having fructosyl valyl histidine oxidase activity.

As such FAOD, among the proteins described in (II) or (III) above, among the 58th isoleucine from the amino terminus, the 110th glycine from the amino terminus, and the 282nd phenylalanine from the amino terminus A protein in which at least one site is substituted with another amino acid is preferable.
As such FAOD, a protein in which the 58th isoleucine from the amino terminus is substituted with methionine, threonine, alanine, asparagine, serine, valine, or leucine is preferable.
As such FAOD, the 110th glycine from the amino terminus is glutamine, methionine, glutamic acid, threonine, alanine, cysteine, histidine, lysine, asparagine, arginine, serine, valine, leucine, aspartic acid, isoleucine, tyrosine or Proteins substituted with phenylalanine are preferred.
Such FAOD is preferably a protein in which the 282nd phenylalanine from the amino terminus is substituted with tyrosine.
Alternatively, a combination of these mutations may be used.

For obtaining the FAOD, for example, a recombinant vector containing any of the following polynucleotides (IV) to (VII) is prepared, and a transformant obtained by transforming a host with the recombinant vector is used. Culture may be performed to produce a protein having fructosyl valyl histidine oxidase activity, and the protein may be collected.
(IV) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2.
(V) a protein having a fructosyl valyl histidine oxidase activity, comprising a base sequence in which one or more and 30 or less bases are substituted, deleted, inserted and / or added in the base sequence described in SEQ ID NO: 2 A polynucleotide encoding
(VI) A polynucleotide encoding any one of the proteins of the present invention.
(VII) A protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide of any one of (IV) to (VI) above and has fructosyl valyl histidine oxidase activity Polynucleotide.

  The FAOD that can be used in the present invention comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of the protein described above, and is a fructosylva. Including a protein having rilhistidine oxidase activity, the site where one or several amino acids are substituted, deleted, inserted and / or added is full of the protein after substitution, deletion, insertion and / or addition. Any site in the amino acid sequence may be used as long as it has kutosylvalylhistidine oxidase activity. Here, “1 or several amino acid residues” is specifically the number of amino acid residues in the range of 10 or less, preferably the amino acid residue in the range of 6 or less.

  The FAOD that can be used in the present invention is 86.0% or more, more preferably 90.0% of the amino acid sequence of the protein according to the present invention described above (shown in SEQ ID NO: 1). As described above, it includes a protein having an amino acid sequence having a homology of 95.0% or more and having fructosyl valyl histidine oxidase activity.

  In addition, the homology of an amino acid sequence can be calculated | required by a well-known method. Specifically, using GENETYX-WIN (trade name of GENETICS Co., Ltd.) according to the manual of the product, the amino acids that match by the homology search (homology search) between the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence to be compared Homology can be calculated as a percentage of the sequence. Homology can be determined by comparing two sequences that are optimally aligned over the entire region of the sequence being compared. Here, in order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed.

  The FAOD protein etc. that can be used in the present invention include, for example, those subjected to intermolecular crosslinking and / or intramolecular crosslinking (for example, disulfide bond), chemical modification (for example, sugar chain, phosphoric acid or other Functional group), label (for example, histidine tag, Myc tag, or Flag tag), or fusion protein (for example, streptavidin, cytochrome, GST or GFP) However, it is not limited to these. Furthermore, the FAOD protein that can be used in the present invention may also include a chimeric protein constituted by combining several protein fragments as long as the fructosyl valyl histidine oxidase activity is substantially maintained.

Even if FAOD cannot be used in the present invention at this time, it can be used in the present invention if it has been modified in the future by genetic engineering or chemical methods to have the above characteristics. .
Alternatively, FAOD newly obtained in the future and having the above characteristics can be used in the present invention.
And the measurement system of HbA1c constructed | assembled using such FAOD is also included by the technical idea of this invention.

Method for measuring glycated hemoglobin Hereinafter, a method for measuring HbA1c as glycated hemoglobin will be described as an example.
The specific aspect of the HbA1c measurement method is not particularly limited as long as it is designed to include the reaction steps (1) and (2) described in [0023], and a commercially available instrument or device is appropriately selected. However, it is convenient to use a general-purpose automatic analyzer (for example, Hitachi 7180 type automatic analyzer).
In order to apply the enzyme method to these automatic analyzers, typically, before the measurement, reagents for carrying out the enzyme method are used as liquid two agents (first reagent (hereinafter also referred to as R1), Prepare by dividing into two reagents (hereinafter also referred to as R2).
A typical measurement pattern is that after dispensing a sample into a measurement cell, the first reagent is added and kept at a constant temperature for several minutes (for example, about 5 minutes for the Hitachi 7180 automatic analyzer described above). (Hereinafter also referred to as the first reaction), and then the second reagent is added and kept at a constant temperature for several minutes (for example, about 5 minutes for the Hitachi 7180 automatic analyzer described above) (this process is referred to as the second reaction hereinafter). (Describe) During this time, the absorbance is continuously measured at a substantially constant time interval appropriately set based on the specifications of the equipment used.
The measurement wavelength, the amount of liquid added to the sample and each reagent (ratio of liquid amount), the time required for each step, and the like can be adjusted as appropriate based on the specifications of the equipment used.

For example, as a typical embodiment, a reagent in which FAOD is arranged in R1 and protease is arranged in R2 may be prepared, and measurement may be performed using an automatic analyzer.
When this reagent is applied to an automatic analyzer, in the first reaction, ROD is added to free (so-called endogenous) glycated amino acid and / or glycated peptide in which FAOD is present in the measurement object for some reason. Acts to produce hydrogen peroxide (“elimination reaction”).

However, this “free glycated amino acid and / or glycated peptide present for some reason in the measurement object” is not “an object that FAOD should act on” that was originally excised from HbA1c by a protease. It is a non-measurement target and gives a positive error to the measurement value.
Therefore, the hydrogen peroxide generated by the above reaction is, for example, in the first step by arranging catalase or the like in R1 when using a system that generates a quinone dye as a hydrogen peroxide quantitative system. Hydrogen peroxide derived from such non-measurement objects is decomposed into oxygen and water immediately (substantially at the same time) following the generation (ie, a quantitative reaction of hydrogen peroxide in the second step). (The “removal reaction”). In this case, the peroxidase is preferably arranged on R2.

Here, in R1, a compound represented by the following formula (I) of HLB12 or more is present. Preferably it is HLB14 or more, More preferably, it is HLB16 or more.
Compound represented by Formula (I) wherein R is an alkyl group having 9 or more carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue. The alkyl group of R has 9 or more carbon atoms, preferably 9 to 18, more preferably 12 to 16. Most preferably, the alkyl group of R has 12 carbon atoms.

Specific compound names and product names of the compounds represented by the above formula (I) are exemplified in Table 1.
Specifically, polyoxyethylene oleyl ether, polyoxyethylene lauryl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyalkylene alkyl ether, polyoxyethylene distyrenated phenyl ether, polyoxyethylene Sorbitan monococonut fatty acid ester, polyoxyethylene nonylphenyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene polyoxypropylene decyl ether, polyoxyethylene oleate, polyoxyethylene alkyl ether, polyoxyethylene (20) nonylphenyl Ether, polyoxyethylene (18) nonylphenyl ether, polyoxyethylene (15) nonylphenyl Ether, polyoxyethylene (30) octylphenyl ether, polyglycerol laurate, and the like are exemplified.
Among them, polyoxyethylene lauryl ether is preferable because it has a remarkable reaction promoting effect at HLB 12 or higher. More preferred is HLB14 or higher, and further preferred is HLB16 or higher.

[Measurement method of HLB value]
Whether or not a compound having a predetermined HLB value (or HLB value) is present is determined if the compound being added is clear, and if the compound is a commercial product, the manufacturer of the product Adopts the values described in the catalog. When the manufacturer does not disclose the information, the value described in the academic paper or patent document is adopted.
When the chemical formula is clear or when the critical micelle concentration (cmc) is clear, the following formula is used.
HLB value = 7 + Σ (the number of hydrophilic groups) −n (the number of CH ₂ groups)
Or HLB value = 7 + 4.02 log / cmc
If the structure and cmc of the compound being added are unknown, use an oil phase with a known HLB value and an active agent with a known HLB, and an active agent with an unknown HLB value and a known active agent. Can be stopped by mixing the mixture in various proportions, conducting an emulsification experiment under a certain condition using this mixed activator, and comparing it with the state of the emulsion.
For example, when determining the HLB of surfactant C (HLB is x) using oil phase A (HLB = 7.5) and surfactant B (HLB = 2.1), B is 32% When C and C are mixed by 68%, the best emulsion is obtained.
From 2.1 × 32% + xx68% = 7.5, x = 10 can be obtained.
Moreover, when mixing 2 or more types of surfactant with a known HLB value, the HLB value of a liquid mixture is represented by the weighted average of each single HLB value.

In the present invention, the concentration at which the compound represented by the formula (I) is present is not particularly limited, but in the case where the protease reaction is performed, the lower limit is 0.14% (W / W (wt%) )) The above is preferable. A more preferred lower limit is 0.3% or more, and a more preferred lower limit is 0.75% or more.
The upper limit is preferably 10% or less from the viewpoint of solubility and the like. More preferably, the upper limit is 5% or less, and further preferably the upper limit is 3% or less.
In the method of the present invention, since the protease is arranged only in R2, the concentration of the compound represented by the above formula (I) in R1 is appropriately set based on the specifications of the instrument to be used and each reagent. Is defined by the amount of added liquid (liquid volume ratio).

  Moreover, if the pH in a 1st reaction is the range of 5.0-9.5, it will not specifically limit. In addition, this treatment is usually performed in a buffer solution, and the buffer solution is not particularly limited. For example, Tris-HCl buffer solution, phosphate buffer solution, EPPS buffer solution, MES buffer solution, PIPES buffer solution, etc. Can be given.

  Moreover, the density | concentration of FAOD in a 1st reaction will not be specifically limited if it is 0.1-100 U / mL. Preferably it is the range of 0.5-50 U / mL, More preferably, it is 0.5-20 U / mL, More preferably, it is 1-10 U / mL.

  In the present invention, the concentration and pH of each reagent in the first reaction or the second reaction may vary depending on the liquid volume ratio between the sample and the reagent, but in a normal measurement, R1 is higher than the sample liquid volume. In most cases, the liquid volume or the total liquid volume of R1 and R2 is set to dozens to dozens of times. In such a case, the influence of the sample is ignored and the concentration in the reagent is substantially increased. It can be defined by pH.

  Next, in the second reaction, the step of cleaving FVH from HbA1c by protease by R2 input (“fragmentation reaction”) is started, and immediately thereafter (almost simultaneously), FAOD acts on the cleaved FVH. Thus, a process of generating hydrogen peroxide by an oxidation-reduction reaction is started.

  In the second reaction, there is no need to newly add FAOD (no need to arrange FAOD in R2), and the one arranged in R1 can be used as it is, but if necessary, FAOD is also added to R2. It can be added and added in the second reaction.

  In the first reaction, the free glycated amino acid and / or glycated peptide originally present in the object to be measured is eliminated, so even if HbA1c is decomposed by protease in the second reaction, it is no longer FAOD and non-measurable object. There is no reaction with the product, and only the product cleaved from HbA1c by the protease can be reacted with FAOD.

  The pH in the second reaction is not particularly limited as long as it is in the range of 5.0 to 9.5. In addition, this treatment is usually performed in a buffer solution, and the buffer solution is not particularly limited. For example, Tris-HCl buffer solution, phosphate buffer solution, EPPS buffer solution, MES buffer solution, PIPES buffer solution, etc. Can be given.

  Moreover, the density | concentration of the protease in a 2nd reaction will not be specifically limited if it is 500-500000 U / mL. Preferably it is the range of 500-50000 U / mL, More preferably, it is 800-20000 U / mL, More preferably, it is 1000-10000 U / mL.

In the second reaction, a catalase inhibitor such as sodium azide may be arranged in R2 in order to eliminate the influence of catalase added in R1.
Alternatively, by setting the addition ratio of catalase and peroxidase appropriately using the difference in affinity of catalase and peroxidase to the substrate, it is possible to set no catalase inhibitor in R2. For example, in the second reaction, an excessive amount of peroxidase and a chromogenic substrate may be added. In this case, it is preferable that peroxidase is added in an amount of activity (U) that is, for example, 5 to 100 times the amount of catalase added (U).

The peroxidase used in the above quantitative system (coloring system) is not particularly limited, and for example, commercially available products can be used. Suitable examples include those derived from horseradish, microorganisms and the like. Of these, horseradish-derived peroxidase is preferable. The peroxidase is commercially available with high purity and low cost.
The concentration of peroxidase in the present invention is, for example, in the range of 0.01 KU / L to 4 MU / L, preferably 0.1 KU / L to 200 KU / L, more preferably 5 KU / L to 100 KU / L.
(KU indicates kilo units, MU indicates mega units.)

  Although it does not specifically limit as a chromogenic substrate used for this invention, A hydrogen donor, a leuco body, a tetrazolium salt, etc. are mentioned.

The hydrogen donor can be used in combination with a coupler. For example, a combination of a Trinder reagent as a hydrogen donor and 4-aminoantipyrine as a coupler can be mentioned.
Examples of the Trinder reagent include, but are not limited to, phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives, and the like.
For example, these undergo an oxidative condensation reaction with a coupler (such as 4-aminoantipyrine (4-AA)) in the presence of peroxidase to produce a dye.

  Examples of hydrogen donors include N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl. 3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N- (2-hydroxy-3 -Sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy Aniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3 Sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-sulfopropylaniline, N- (2-hydroxy-3-sulfopropyl) ) -2,5-dimethylaniline, N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine, N-ethyl-N- (3-methylphenyl) -N′-acetylethylenediamine and the like. .

  As the coupler, in addition to the 4-aminoantipyrine, aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH) (more specifically, 3-methyl-2-benzothiazolinone hydrazone) Sulfonated methylbenzthiazolinone hydrazone (SMBTH) (more specifically, sulfonated 3-methyl-2-benzothiazolinone hydrazone) and the like can also be used.

Although it does not specifically limit as a leuco body, For example, a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative etc. can be used.
They are directly oxidatively colored in the presence of peroxidase.

  For example, 4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] methane, 1- (Ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, 4,4′-bis (dimethylamino) diphenylamine, N- ( Carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt (for example, sodium salt), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt, and the like.

  Particularly preferred is 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt.

  Examples of the tetrazolium salt include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium salt, 3,3 ′-[3,3′-dimethoxy- (1 , 1′-biphenyl) -4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium] salt, 3,3 ′-[3,3′-dimethoxy- ( 1,1′-biphenyl) -4,4′-diyl] -bis (2,5-diphenyl-2H-tetrazolium) salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5 (2,4-disulfophenyl) -2H-tetrazolium salt, 3,3 ′-(1,1′-biphenyl-4,4′-diyl) -bis (2,5-diphenyl-2H-tetrazolium) salt, 3- (4,5-dimethyl-2 Thiazolyl) -2,5-diphenyl -2H- tetrazolium salts.

  The amount of the chromogenic substrate used is preferably 0.001 to 10 mmol / L as the final reaction concentration in consideration of solubility. More preferably, it is 0.005-0.5 mmol / L.

  When blood cells are included in the measurement target, the action of catalase, glutathione peroxidase or the like originally present in the blood cells may be used to erase the non-measurement target.

In addition, as a method for removing hydrogen peroxide generated from non-measured glycated amino acids and / or glycated peptides, the following methods can also be employed.
For example, there is a method in which FAOD, peroxidase and an electron donor coexist before adding protease. According to this, before adding a protease, hydrogen peroxide generated from a non-measured glycated amino acid and / or glycated peptide is dehydrogenated by a peroxidase reaction, and electrons are donated to an electron donor. Hydrogen peroxide is removed.
It is also possible to add FAOD, peroxidase, and a coloring agent that develops color by oxidation before adding the protease, measure the color development amount in advance, and correct the color development amount.

Which of R1 and R2 the HbA1c and the protease are arranged on can be determined as appropriate according to the purpose of measurement and the sample to be measured.
In addition, specific conditions such as concentration setting when HbA1c and protease are reacted are not particularly limited, and appropriate conditions are examined according to the concentration and type of the sample and the type and concentration of the protease. In addition, it may be adopted.
In addition, when FAOD is also used to eliminate non-measured glycated amino acids and / or glycated peptides, the conditions should be set appropriately so that both reactions can be performed in consideration of the liquid volume ratio of R1 and R2. That's fine.
For example, the protease is in the range of 0.1 to 30 MU / L, preferably 2 to 15 MU / L, more preferably 3 to 10 MU / L.
For example, FAOD is in the range of 0.1 to 45 U / L, preferably 2 to 15 U / L, and more preferably 3 to 10 U / L.
The reaction temperature is, for example, 2 to 60 ° C, preferably 4 to 40 ° C.
The reaction time is not particularly limited. For example, in the case of all steps, the reaction time is 0.5 to 30 minutes, preferably 10 minutes or less, more preferably 5 minutes or less, more preferably 3 minutes or less, More preferably, it is 2 minutes or less, More preferably, it is 1 minute or less.
In the first reaction, although not particularly limited, for example, in the case of all steps, 0.25 to 15 minutes can be exemplified, preferably 5 minutes or less, more preferably 2.5 minutes or less, Preferably it is 1.5 minutes or less, More preferably, it is 1 minute or less, More preferably, it is 0.5 minute or less.
In the second reaction, although not particularly limited, for example, in the case of all steps, 0.25 to 15 minutes can be exemplified, preferably 5 minutes or less, more preferably 2.5 minutes or less, Preferably it is 1.5 minutes or less, More preferably, it is 1 minute or less, More preferably, it is 0.5 minute or less.
Preferred first reaction time-second reaction time combinations are 10 minutes-10 minutes, more preferably 5 minutes-5 minutes, more preferably 2.5 minutes-2.5 minutes, more preferably 1.5 minutes-1. .5 minutes, more preferably 1 minute-1 minutes, and even more preferably 0.5 minutes-0.5 minutes.

In the measurement of HbA1c using the enzymatic method, the protease reaction becomes the rate-determining factor of the measurement time.
The present inventors have found that the protease reaction is significantly promoted by treating the protein with protease in the presence of a compound represented by the following formula (I) of HLB12 or higher, and have reached the present invention.
R-O-X (I)
(Here, R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue. The alkyl group of R has 9 or more carbon atoms, preferably 9 to 18 carbon atoms. More preferably, it is 12 to 16. Most preferably, the alkyl group of R has 12 carbon atoms.)
That is, by using this method, the reaction of the protease is promoted, and accordingly, the time for all the measurement steps can be shortened.
The pH is, for example, in the range of 5-9. In addition, this treatment is usually performed in a buffer solution, and the buffer solution is not particularly limited. For example, Tris-HCl buffer solution, phosphate buffer solution, EPPS buffer solution, MES buffer solution, PIPES buffer solution, etc. Can be given.

In the present invention, the enzyme activity is not described in the specification. As a general rule, for commercial products, the measurement method and unit are described in containers, attached instructions, pamphlets, etc. . Moreover, if it is described in literature, it follows the activity measuring method described in the literature.
In cases where the definition of activity (measurement method) is not known or there are restrictions on the availability of reagents necessary for measurement, etc., the substrate should be reasonably selected based on common knowledge of those skilled in the art, and the measurement conditions determined. Measure with

[Measurement of hemoglobin]
In the above aspect, hemoglobin (hereinafter also referred to as Hb) can be measured. Further, glycated hemoglobin and hemoglobin (hereinafter also referred to as Hb) can be measured simultaneously, and the ratio of glycated hemoglobin to hemoglobin can be determined based on the measurement results.
The measurement of hemoglobin (hereinafter also referred to as Hb) measures the absorbance of red (near 550 nm) inherent to Hb at any point during the execution of the first step in the measurement method of the present invention. Thus, measurement can be performed.
For example, at any point during the measurement of HbA1c, preferably at any point during the execution of the first step in the measurement method of the present invention, Hb and HbA1c can be measured simultaneously by measuring the absorbance at around 550 nm.
Here, in the color development reaction in the measurement of HbA1c, it is preferable to set the measurement wavelength so as not to match between the Hb measurement and the HbA1c measurement by appropriately selecting a substrate that develops color by oxidation. Thereby, the influence which it has on a mutual measurement can be made small.

In addition, Hb is oxidized in the reaction solution to change to methemoglobin, and the absorbance gradually changes, which may affect the measured values of Hb and HbA1c.
In order to prevent this, Hb is changed to methemoglobin in advance by adding an oxidizing agent such as ferrocyanide, nitrite, potassium nitrate, or azide as a pretreatment to the sample (hereinafter referred to as “methoate”). Description) is preferred. Alternatively, as a pretreatment similarly to R1, an oxidizing agent such as ferrocyanide, nitrite, potassium nitrate, azide, etc. may be provided and the first reaction may be subjected to methation.

In the present invention, as shown in the above embodiment, FAOD is formulated in the first reagent and protease is formulated in the second reagent.
With this configuration, by disposing protease in the second reagent, endogenous glycated amino acids or peptides can be eliminated in the first reagent, and by measuring the endogenous glycated amino acids (or peptides), False highs can be eliminated.

In the present invention, in the step 3) described in [0015], the protease is preferably allowed to act in the presence of a compound represented by the above formula (I) of HLB12 or higher and a metal ion.
Examples of metal ions include sodium chloride and potassium chloride.

  In the present invention, it is preferable that the protease is allowed to act in the presence of potassium ferrocyanide and / or nitrite in the step 3) described in [0015].

In the present invention, in addition to the above-described configuration, a buffering agent (for example, a borate buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution, and a GOOD buffer solution) may be included.
Furthermore, the reagent composition includes chelating reagents (such as EDTA and O-dianisidine) that capture ions that interfere with the enzyme reaction, ascorbate oxidase that eliminates ascorbic acid, which is a substance that interferes with the determination of hydrogen peroxide, Surfactants (such as Triton X-100 and NP-40) and various antibacterial and preservatives (such as streptomycin and sodium azide) may be included.
Furthermore, salts, enzyme stabilizers, color source stabilizers and the like can be added as necessary.
These reagents may be a single reagent or a combination of two or more reagents.

  Examples of preservatives include various types of procrine (registered trademark, Superco), azides, chelating agents, antibiotics, antibacterial agents, and the like. Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt. Antibiotics include gentamicin, kanamycin, chloramphenicol and the like. Examples of the antibacterial agent include imidazolidinyl urea.

  Examples of the salts include sodium chloride, potassium chloride, aluminum chloride, calcium chloride and the like.

Enzyme stabilizers include sucrose, trehalose, cyclodextrin, glycerol, gluconate, amino acids and the like.
Moreover, inactive proteins, such as serum albumins, globulins, or fibrous proteins, can be added as enzyme stabilizers. A preferred protein is bovine serum albumin. Preferred inactive proteins are those that do not contain protease impurities that cause enzymatic degradation.

  Examples of the dye stabilizer include calixarene sulfate (6) and calixarene sulfate (8).

  Further, for the purpose of avoiding the influence of reducing substances, ferrocyanides such as potassium ferrocyanide and nitrites such as sodium nitrite may be added. These additives are effective not only in avoiding the influence of reducing substances, but also in Hb methation and protease reaction promotion.

The concentration and conditions of each of the above components (proteases present in the reagent, enzymes such as FAOD, and other components) consider the reactivity and / or stability depending on the type of reagent and enzyme used. And can be determined as appropriate.
In that case, it is preferable to consider the possibility that the components present in the reagent may interfere with each other, thereby affecting the measurement of HbA1c and the stability of the reagent. For example, when protease and FAOD coexist in the second reaction, the conditions can be determined as appropriate so that the characteristics of both enzymes can be utilized as much as possible.
The above determination does not necessarily need to be an optimal solution, and may be performed as long as practically sufficient performance is ensured for the measurement of HbA1c according to the purpose and situation.

Another characteristic configuration of the present invention is a reagent for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin in the same reaction tank, A reagent characterized by a) and (b).
(A) It is composed of two parts, a first reagent and a second reagent.
(B) The first reagent is a compound represented by the following formula (I) of HLB 12 or more, and a buffer solution having a buffer capacity in the range of pH 5.0 to 9.5, 0.1 to 100 U / mL fruct Tosyl amino acid oxidase is contained, and the second reagent contains 500 to 500,000 U / mL protease.
R-O-X (I)
(R is an alkyl group having 9 to 18 carbon atoms or a substituted alkyl group, and X is a polyoxyethylene residue.)
The alkyl group of R has 9 or more carbon atoms, preferably 9 to 18, more preferably 12 to 16. Most preferably, the alkyl group of R has 12 carbon atoms.

  In the reagent of the present invention, the first reagent preferably contains a glycated hemoglobin quantitative dye.

In the reagent of the present invention, it is preferable to contain potassium ferrocyanide and / or nitrite in either the first reagent or the second reagent.

The glycated hemoglobin measurement reagent may further contain an enzyme that acts on a glycated amino acid and / or glycated peptide in any part.
The glycated hemoglobin measurement reagent is preferably composed of two parts, and the first reagent contains an enzyme that acts on a glycated amino acid and / or glycated peptide, and the second reagent contains a protease.
In such a form of the reagent, the first reagent may further contain any one or more of catalase, peroxidase, and dye.

  Examples of the glycated hemoglobin measuring reagent of the present invention include fructosyl valyl histidine oxidase, buffer solution, protease, POD, coloring reagent, chelating reagent that captures ions that interfere with the enzyme reaction, and substances that interfere with the determination of hydrogen peroxide. Ascorbate oxidase to erase certain ascorbic acid, surfactant, stabilizer, excipient, antibacterial agent, preservative, well plate, fluorescence scanner, automatic analyzer, and measurement method according to the present invention are recorded on paper, etc. An instruction manual written on the medium may be included.

  The reagent for measuring glycated hemoglobin of the present invention can be used in the method for measuring glycated hemoglobin of the present invention. Therefore, the kit of this invention may be comprised with the articles | goods used for implementation of the measuring method of this invention. For the description of the article, the description of the buffering agent and the like in the section of the measurement method of the present invention can be used.

  The glycated hemoglobin measurement reagent of the present invention substantially includes not only a set of all the components necessary for the measurement but also each divided component. For example, in the case of a liquid set of R1 and R2, the kit of the present invention is substantially applied to R1 that is assumed to be used in combination with R2 or R2 that is assumed to be used in combination with R2. Is included.

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and technical means disclosed in different embodiments and examples are appropriately used. Embodiments and examples obtained in combination are also included in the technical scope of the present invention. Moreover, all the literatures described in this specification are used as reference.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by a following example.

[Example 1] Screening of protease reaction promoter <Reagent>
R1
PIPES 50 mmol / L (pH 6.5)
NaCl 50mmol / L
NaNO ₂ 7 mmol / L
FPOX-CE (Kikkoman) 2KU / L
Peroxidase (PEO-302 Toyobo) 6KU / L
Catalase 100KU / L (Toyobo)
Various compounds 0.2% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Thermolysin (Amano Enzyme) 5000KU / L
10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt (DA-67) (Wako Pure Chemical Industries) 50 μmol / L
<Operation method>
R1 incubated at 37 ° C; 10 µl of 120 µl of HbA1c sample collected with EDTA-2Na blood collection tube diluted 1/20 times with purified water was added, and the reaction was started at 37 ° C. 40 μl was added. The absorbance at 660 nm before R2 addition and 1.5 minutes after the addition was measured, and calculation was performed by subtracting the absorbance after 1.5 minutes from the absorbance before R2 addition. The absorbance obtained from Sample 1 (the ratio of glycated hemoglobin to hemoglobin (HbA1c%) was 10.6%) was designated as absorbance AH, and the absorbance obtained from Sample 2 (HbA1c% was 5.1%) was designated as absorbance AL.

Tables 1 to 3 show the evaluation results of the protease reaction promoting effect of the test samples. AH-AL shown in Tables 1 to 3 is a difference obtained by subtracting the absorbance AL from the absorbance AH. In addition, HbA1c% of specimens 1 and 2 was measured according to a conventional method using HA-8180 (HPLC method, Arkray).
Here, if AH-AL is large, it can be determined that there is a protease reaction promoting effect. In particular, when AH-AL is 40 or more, there is an excellent promoting effect.
From the results of Tables 1 to 3, the effect was observed for polyoxyethylene lauryl ether having HLB 12 or more. Particularly, a better reaction promoting effect was obtained with HLB 16 or more.

[Example 2] Effect on stability of each enzyme <Reagent>
R1
PIPES 50 mmol / L (pH 6.5)
NaCl 50mmol / L
NaNO ₂ 7 mmol / L
FAOD (FPOX-CE (Kikkoman)) 2KU / L
DA-67 (Wako Pure Chemical Industries) 25μmol / L
Catalase 100KU / L (Toyobo)
Various compounds 0.5% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Thermolysin (Amano Enzyme) 5000KU / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Various compounds 0.5% (wt%)
<Operation method>
The stability of FAOD, catalase, and peroxidase in the above composition was examined by the following methods using the reagents stored at 35 ° C. for 2 weeks. The stability was evaluated by the activity value (%) after storage at 35 ° C. for 2 weeks with respect to the activity value measured in the same manner before storage.

1) FAOD
The enzyme activity was determined by measuring the increase in the absorbance of the dye produced by the peroxidase reaction using hydrogen peroxide produced by the FAOD enzyme reaction using 1.0 mM fructosyl valylhistidine as a substrate.
First, 3 mL of the activity measurement reagent was heated at 37 ° C. for 5 minutes, and then the activity measurement reagent was preliminarily added to the enzyme diluent (50 mM potassium phosphate buffer (pH 6.5) containing 0.1% Triton X100). The reaction was started by adding 0.1 mL of the diluted enzyme solution.
After reacting at 37 ° C. for 5 minutes, the change in absorbance at 500 nm per unit time was measured (ΔOD _test / min). As a blind test, 0.1 mL of the enzyme diluent was used instead of the enzyme solution, and the change in absorbance was measured by performing the same operation (ΔOD _blank / min).
Based on the following calculation formula, the enzyme activity was calculated from the change in absorbance obtained. The amount of enzyme that oxidizes 1 micromole of substrate per minute under the above conditions was defined as 1 unit (U).
(a formula)
Activity value (U / mL) = {((ΔOD _test / min) − (ΔOD _blank /min))×3.1 mL × dilution factor} / (13 × 1.0 cm × 0.1 mL)
In the above formula, “3.1 mL” indicates the total liquid volume, “13” indicates the millimolar extinction coefficient, “1.0 cm” indicates the optical path length of the cell, and “0.1 mL” indicates the enzyme sample. Indicates the liquid volume.

2) Peroxidase Principle 2Pyrogallol + 3H ₂ O ₂ Purpurogallin + 5H ₂ O + CO ₂
The produced Purpurogallin is extracted with ether and measured by the change in absorbance at 420 nm.
2. Definition The amount of enzyme that produces 1.0 mg of Purpurogallin in 20 seconds under the following conditions is defined as 1 Purpurogallin unit (U).
3. Reagent A. 5% (W / V) pyrogallol aqueous solution (prepared when used)
B. 0.147 MH ₂ O ₂ aqueous solution [Dilute 1.67 mL of 30% (W / V) H ₂ O ₂ solution with distilled water to 100 mL]
(Preparation before use)
C. 0.1M phosphate buffer, pH 6.0 (for reaction mixture and enzyme dilution)
D. 2.0 NH ₂ SO ₄ solution Enzyme solution: The enzyme preparation was dissolved in 0.1 M phosphate buffer, pH 6.0, which had been ice-cooled in advance, and 3.0 to 6.0 Purpurogallin U / mL with the same buffer. Dilute and store on ice. 4). Procedure (1) Prepare the following reaction mixture in a test tube (32φ × 200 mm) and preheat at 20 ° C. for about 5 minutes.
14.0 mL distilled water 2.0 mL pyrogallol aqueous solution (A)
1.0 mL H ₂ O ₂ aqueous solution (B)
2.0mL phosphate buffer (C)
(2) Add 1.0 mL of enzyme solution to start the reaction.
(3) After reacting accurately at 20 ° C. for 20 seconds, 1.0 mL of H ₂ SO ₄ solution (D) is added to stop the reaction. Purpurogallin produced from the mixed solution after stopping the reaction is extracted with 15 mL of ether. This operation is repeated 5 times, the extracts are combined, and ether is added to make a total volume of 100 mL. The absorbance at 420 nm is measured for this solution (ODtest).
(4) The blind mixture is prepared by allowing the reaction mixture (1) to stand at 20 ° C. for 20 seconds, adding 1.0 mL of H ₂ SO ₄ solution (D), mixing, and then adding 1.0 mL of the enzyme solution. The liquid is subjected to ether extraction in the same manner as described above, and the absorbance is measured (OD _blank ).
5. Formula U / mL = {ΔOD (OD _test −OD _blank ) × dilution ratio} / {0.117 × 1 (mL)}
= ΔOD x 8.547 x dilution factor U / mg = U / mL x 1 / C
Absorbance at 420 nm of 0.117: 1 mg% Purpurogallin ether solution C: Enzyme concentration at dissolution (c mg / mL)
(Note) One Purpurogallin unit corresponds to 13.5 international units (reaction conditions at 25 ° C. using o-dianisidine as a substrate).

3) Catalase Principle The amount of hydrogen peroxide decrease is measured by the titanium color method.
2. Definition The amount of enzyme capable of decomposing 1 micromole of hydrogen peroxide per minute under the following conditions is defined as 1 unit (U).
3. Reagent A. 10 mM phosphate buffer, pH 7.0 (25 ° C.)
B. H ₂ O ₂ solution: 16 mM (0.182 mL of 30% (W / V) H ₂ O ₂ is dissolved in 100 mL of buffer A (prepared when used)
C. Titanium reagent (Nacalai Tesque)
Enzyme solution: The enzyme equipment is diluted to 0.35-1.35 U / mL with buffer A, which has been ice-cooled in advance. 4). Procedure (1) Take 0.25 mL of the substrate solution (B) in a test tube (32φ × 200 mm) and preheat at 25 ° C. for about 5 minutes.
(2) Add 0.25 mL of enzyme solution and mix gently.
(3) After reacting accurately at 25 ° C. for 5 minutes, 2.5 mL of titanium reagent (C) is added to stop the reaction, and the absorbance at 410 nm is measured using water as a control (OD _test ).
(4) In the blind test, after heating for 5 minutes, 0.25 mL of the substrate solution (B) is first added and mixed with 2.5 mL of the titanium reagent (C), and then the enzyme solution is added (OD _blank ).
5. Formula U / mL = {ΔOD (OD _blank −OD _test ) × 3.0 mL × dilution factor} / {F × 5 (min) × 1.0 × 0.25 (mL)}
= ΔOD × 3.43 × 1 / F × dilution ratio U / mg = U / mL × 1 / C
F: Absorbance coefficient of titanium-colored product with 0.1 mM hydrogen peroxide (F is determined for each lot using hydrogen peroxide with known concentration, usually around 0.7)
1.0: Optical path length (cm)
C: Enzyme concentration at the time of dissolution (c mg / mL)

  Table 4 shows the results. In general, since the reaction rate decreases as the enzyme activity value decreases, in order to maintain the reaction promoting effect, it is preferable that the reaction accelerator does not affect the stability of the enzyme. A stabilizing effect on FAOD, catalase, and peroxidase was observed in polyoxyethylene lauryl ether of HLB 12 or higher.

  As a comparative example, N-acylamino acid, sugar ester, and polyoxyethylene alkyl ether sulfate were subjected to the same test. As a result, a certain degree of reaction promotion effect was observed, but the stability of FAOD, catalase and POD was The result was inferior to the example.

[Example 3] Examination of addition amount of reaction accelerator <Reagent>
R1
MES 50 mmol / L (pH 6.5)
NaNO ₂ 7 mmol / L
FAOD (FPOX-CE (Kikkoman)) 2KU / L
DA-67 (Wako Pure Chemical Industries) 25μmol / L
Catalase 100KU / L (Toyobo)
Various compound concentrations (wt%)
R2
MES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Thermolysin (Amano Enzyme) 5000KU / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
<Operation method>
R1 incubated at 37 ° C; 10 µl of 120 µl of HbA1c sample collected with EDTA-2Na blood collection tube diluted 1/20 times with purified water was added, and the reaction was started at 37 ° C. 40 μl was added. The absorbance at 660 nm before R2 addition and 1.5 minutes after the addition was measured, and calculation was performed by subtracting the absorbance after 1.5 minutes from the absorbance before R2 addition. The absorbance obtained from Sample 3 (the ratio of glycated hemoglobin to hemoglobin (HbA1c%) was 9.3%) was defined as absorbance AH, and the absorbance obtained from Sample 4 (HbA1c% was 4.5%) was defined as absorbance AL.

Table 5 shows the evaluation results of the protease reaction promoting effect of the test sample. AH-AL shown in Table 5 is a difference obtained by subtracting the absorbance AL from the absorbance AH. In addition, HbA1c% of the specimens 3 and 4 was measured according to a conventional method using HA-8180 (HPLC method, Arkray).
Here, if AH-AL is large, it can be determined that there is a protease reaction promoting effect. In particular, when AH-AL is 35 or more, there is an excellent promoting effect.
Table 5 shows the results. From Table 5, it was confirmed that a particularly good reaction was exhibited at 0.2% (in the reaction solution, 0.14%) or more.

[Example 4] Confirmation of elimination ability of endogenous glycated amino acids and glycated peptides <Reagent> Example R1
PIPES 50 mmol / L (pH 7.0)
NaCl 50mmol / L
NaNO ₂ 7 mmol / L
FAOD (FPOX-CE (Kikkoman)) 2KU / L
DA-67 (Wako Pure Chemical Industries) 100 μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 7.0)
CaCl ₂ 10 mmol / L
Thermolysin (Amano Enzyme) 5000kU / L
Peroxidase (PEO-302 Toyobo) 20KU / L
<Operation method>
A Hitachi 7180 automatic analyzer was used.
(Measurement of Hb concentration)
10 μl of the HbA1c sample was added to 120 μl of R1 incubated at 37 ° C. from the following sample diluted 1/20 times with purified water, the reaction was started at 37 ° C., and the absorbance at 505 nm was measured after 5 minutes.
As a result, calibrator 1 (Hb 101.0 μmol / l, HbA1c 3.54 μmol / l, HbA1c% is 5.0%) and calibrator 2 (Hb 153.8 μmol / l, HbA1c 13.70 μmol / l, HbA1c% is 10%). .2%) to obtain a Hb concentration in comparison with a calibration curve prepared using
(Measurement of HbA1c concentration)
Moreover, R2; 40 microliters was added 5 minutes after R1 addition. Absorbance at 660 nm was measured before R2 addition and 5 minutes after addition. The results were compared with a calibration curve created using the same calibrator 1 and calibrator 2 as described above to determine the HbA1c concentration.
(Measurement of HbA1c%)
From the Hb concentration (μmol / l) and HbA1c concentration (μmol / l) of each specimen obtained above, HbA1c (%) was calculated by the following formula.
HbA1c (%) =
{96.3 × HbA1c concentration (μmol / l) / Hb concentration (μmol / l)} + 1.62
Specimens used were 5.79% HbA1c whole blood specimens collected with EDTA-Na blood collection tubes with Unicaric N (Terumo; infusion formulation for nutritional tonic containing amino acids, sugars and electrolytes) added at various concentrations did.
In addition, HbA1c% of the sample was measured according to a conventional method using HA-8180 (HPLC method, Arkray).

  In infusion preparations containing amino acids and sugars, glycated amino acids in which amino acids and sugars are combined during storage are produced, and FAOD reacts with these glycated amino acids, which causes a problem of false highs. As shown in Table 6 and FIG. 1, in the present invention, it was confirmed that endogenous glycated amino acids can be eliminated.

[Example 5] Measurement of standard product <Reagent> Example 5
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPOX-CE (Kikkoman)) 5KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Thermolysin (Amano Enzyme) 5000KU / L
Peroxidase (PEO-302 Toyobo) 20KU / L
<Operation method>
Same as Example 4.
The specimens used were JCCRM 411 (Common reference standard for IFCC method HbA1c measurement), General Standards for Laboratory Medicine (ReCCS) product, 5 levels (from LEBEL1 to LEBEL5), each centrifuged at 800 g × 5 minutes, A blood cell component diluted 1/40 with the following pretreatment solution was used.
-Pretreatment solution; aqueous solution containing MES 10 mM (pH 6.5) and NaNO ₂ 0.5 g / l

  Table 7 shows the results. From the results, it was confirmed that the measured value of the standard substance was consistent with the displayed value.

Furthermore, as Examples 5-1 to 5-15 and Comparative Example 1, the same tests as in Example 5 were performed by changing the pH, the amount of FAOD added, and the amount of protease added.
In Examples 5-1 to 5-6, the pH was changed, in Examples 5-7 to 5-10, the addition amount of FAOD was changed, and in Examples 5-11 to 5-15, the addition amount of protease was changed. went. Moreover, the test was done using polyoxyethylene oleyl ether as a comparative example.
The detailed reagent composition of each Example and Comparative Example is shown below.

<Reagent> Example 5-1
R1
R1
MES 50 mmol / L (pH 6.0)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
MES 50 mmol / L (pH 6.0)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-2
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-3
R1
R1
PIPES 50 mmol / L (pH 7.0)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 7.0)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-4
R1
R1
TES 50 mmol / L (pH 7.5)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
TES 50 mmol / L (pH 7.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-5
R1
R1
HEPPSO 50 mmol / L (pH 8.0)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
HEPPSO 50 mmol / L (pH 8.0)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-6
R1
R1
TAPS 50 mmol / L (pH 8.5)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
TAPS 50 mmol / L (pH 8.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-7
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 0.2 KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-8
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 1.0 KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-9
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 4.0 KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-10
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 10.0 KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Reagent> Example 5-11
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 500KU / L

<Reagent> Example 5-12
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 2000KU / L

<Reagent> Example 5-13
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 8000KU / L

<Reagent> Example 5-14
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 12000KU / L

<Reagent> Example 5-15
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 20000KU / L

<Reagent> Comparative Example 1
R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2.0KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene oleyl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

  Table 8 shows the results. From the results, it was confirmed that the measured value of the standard substance matched the displayed value in the examples.

[Example 6] Confirmation of correlation in actual sample measurement <Reagent> Example Three types of reagents were prepared using the following three types of proteases.
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPOX-CE (Kikkoman)) 5KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Protease (below)
Lot. A; Metalloprotease (Toyobo) 5000KU / L
Lot. B: Protease (derived from Streptomyces grieseus, Fluka) 5000 KU / L
Lot. C: Thermolysin (Amano Enzyme) 5000KU / L
<Operation method>
As the specimen (n = 60), a specimen collected with an EDTA-Na blood collection tube and diluted 1/20 with the following pretreatment solution was used. The operations other than sample dilution are the same as in Example 4.
-Pretreatment solution: Aqueous solution containing 10 mM PIPES (pH 6.5) and 0.5 g / l NaNO2 The control method was HPLC, and measurement was carried out using HA-8180 (ARKRAY) according to a conventional method.

  The results are shown in FIGS. The results confirmed that there was a good correlation with existing methods.

[Example 7] (Measurement in a short time (60 seconds to 60 seconds))
[Enzyme method]
1. Reagent R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPOX-CE (Kikkoman)) 5KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Thermolysin (Amano Enzyme) 5000KU / L

<Operation method>
(Measurement of Hb concentration)
10 μl of the sample diluted by the following method was added to 120 μl of R1 incubated at 37 ° C., the reaction was started at 37 ° C., and the absorbance at 505 nm was measured after 1 minute.
As a result, calibrator 1 (Hb 101.0 μmol / l, HbA1c 3.54 μmol / l, HbA1c% is 5.0%) and calibrator 2 (Hb 153.8 μmol / l, HbA1c 13.70 μmol / l, HbA1c% is 10%). .2%) to obtain a Hb concentration in comparison with a calibration curve prepared using
(Measurement of HbA1c concentration)
Moreover, R2; 40 microliters was added 1 minute after R1 addition. Absorbance at 660 nm was measured before the addition of R2 and 1 minute after the addition. The results were compared with a calibration curve created using the same calibrator 1 and calibrator 2 as described above to determine the HbA1c concentration.
(Measurement of HbA1c%)
From the Hb concentration (μmol / l) and HbA1c concentration (μmol / l) of each specimen obtained above, HbA1c (%) was calculated by the following formula.
HbA1c (%) =
{96.3 × HbA1c concentration (μmol / l) / Hb concentration (μmol / l)} + 1.6

<Sample dilution method>
A specimen (n = 100) collected with an EDTA-Na blood collection tube was diluted 1/20 with the following pretreatment solution.
-Pretreatment solution: aqueous solution containing PIPES 10 mM (pH 6.5) and NaNO2 0.5 g / l

The control method was an HPLC method, and measurement was performed according to a conventional method using HA-8180 (Arkray).
The correlation data is shown in FIG.
From these results, by using the present invention, it is possible to measure for a short time, and it is possible to quickly report the measurement result.

[Example 8] (Measurement in a short time (60 to 60 seconds))
[Enzyme method]
1. Reagent R1
R1
PIPES 50 mmol / L (pH 6.5)
FAOD (FPO-301 (Toyobo) 2KU / L
DA-67 (Wako Pure Chemical Industries) 50μmol / L
Catalase 100KU / L (Toyobo)
Polyoxyethylene lauryl ether (Kao) 1.0% (wt%)
R2
PIPES 50 mmol / L (pH 6.5)
CaCl ₂ 10 mmol / L
Potassium ferrocyanide 0.01g / L
Peroxidase (PEO-302 Toyobo) 20KU / L
Metalloprotease (Toyobo) 4000KU / L

<Operation method>
(Measurement of Hb concentration)
Same as Example 7.
(Measurement of HbA1c concentration)
Same as Example 7.
(Measurement of HbA1c%)
Same as Example 7.

<Sample dilution method>
Same as Example 7.

The control method was an HPLC method, and measurement was performed according to a conventional method using HA-8180 (Arkray).
The correlation data is shown in FIG. FIG. 7 shows the time course for 3 samples out of 64 samples.
Also in this result, the time course and the correlation were good, and a short time measurement was possible by using the present invention. By shortening the measurement time from this result, measurement can be performed quickly and in a short time.

  The present invention is useful as a reagent for quantifying HbA1c.

Claims (6)

A method for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin, the method comprising the following (a) and (b):
(A) The following steps 1) to 3) are performed in the same reaction vessel.
(B) Steps 1) and 2) are performed in the same step, and step 3) is started in the next step.
1) quantifying hemoglobin in the test solution;
2) 0.1-100 U / mL fructosyl amino acid oxidase in the range of pH 5.0 to 9.5 in the presence of sucrose monolaurate or n-Dodesyl-β-maltoside is added to the test solution, A step of previously eliminating sex glycated amino acids and glycated peptides,
3) A step of quantifying glycated hemoglobin by allowing a protease of 500 to 500,000 U / mL to act on the reaction solution of 2) above in the range of pH 5.0 to 9.5 in the presence of potassium ferrocyanide. The method of claim 1, wherein
A method of allowing a protease to act in the presence of a compound represented by the above formula (I) of HLB12 or more and a metal ion in the step 3) The method according to claim 1 or 2, wherein
Method in which protease is allowed to act in the presence of nitrite in step 3) A reagent for measuring at least one of hemoglobin, glycated hemoglobin, and a ratio of glycated hemoglobin to hemoglobin in the same reaction tank, characterized by the following (a) to (c).
(A) It is composed of two parts, a first reagent and a second reagent.
(B) Sucrose monolaurate or n-Dodesyl-β-maltide as a first reagent, and a buffer having a buffer capacity in the range of pH 5.0 to 9.5, 0.1-100 U / mL fructosyl amino acid oxidase And the second reagent contains 500 to 500,000 U / mL protease.
(C) One of the first reagent and the second reagent contains potassium ferrocyanide. The reagent according to claim 4, wherein
A reagent containing a glycated hemoglobin quantitative dye in the first reagent. The reagent according to claim 4 or 5,
A reagent containing nitrite in either the first reagent or the second reagent.