JP5177479B2 - Glycated albumin measuring reagent - Google Patents

Description

  The present invention relates to a method and a reagent for measuring the amount of glycated albumin in a sample.

Glycated albumin is a protein in which albumin is non-enzymatically glycosylated, and the aldehyde group on the sugar, that is, the aldose (monosaccharide and derivatives thereof having an aldehyde group) and the amino group on the protein side are non-enzymatic. Generated as a result of a covalent bond.
Glycated albumin is contained in biological samples such as blood in the living body. The concentration of glycated albumin present in blood strongly depends on the concentration of saccharides such as glucose dissolved in serum. In a diabetic state, the production of glycated albumin is enhanced, and the concentration of glycated albumin in serum reflects the average blood glucose level during a certain period in the past. Measuring glycated albumin is important for diabetes diagnosis and disease state management.

Conventionally, as a method for measuring glycated albumin, a method using high performance liquid chromatography (for example, see Non-Patent Document 1) and a method using an antigen-antibody reaction (for example, see Non-Patent Document 2) are known.
At present, an enzymatic measurement method has been proposed as a method for measuring glycated albumin that is simpler and less expensive than the above-described method and is accurately performed in a short time (see, for example, Patent Documents 1 to 3).
This enzymatic measurement method is a method in which glycated albumin is decomposed with a protease, glycated amino acid oxidase is allowed to act on the released glycated amino acid, and the generated hydrogen peroxide is measured.

However, this enzymatic measurement method has a drawback in that the concentration of protease used is 1,000 U / mL or more, which is a very high concentration.
That is, in the current clinical diagnostic measurement test, it is usual to measure a plurality of clinical diagnostic markers simultaneously with one analytical instrument. Once a high-concentration protease of 1,000 U / mL or more is used once, in normal washing Protease cannot be completely removed from the measurement system of the analytical instrument, and the remaining protease that cannot be removed by normal washing affects the enzyme reaction of other clinical diagnostic measurement tests. The amount of protease used that does not affect the enzymatic reaction of other clinical diagnostic measurement tests should be reduced as much as possible to less than 1,000 U / mL.
At present, in order to avoid the influence of this remaining protease, the number of washing of the analytical instrument after glycated albumin measurement is increased, or simultaneous measurement with other measurement test items is avoided, and only glycated albumin measurement is performed separately. . Therefore, in clinical diagnostic measurement, the implementation of rapid measurement is affected.

  There is a need for a reagent for measuring glycated albumin in which the amount of protease used is reduced to less than 1,000 U / mL as much as possible, the protease activity itself is enhanced, and the effect of residual protease is small. However, in order to suppress the amount of protease used, it is necessary to reduce the amount of sample occupying in the reaction solution or to add a substance that promotes the protease reaction. In the former, the sensitivity of the reaction is reduced, and the accuracy of the measurement is lost. In the latter case, there are problems such as the reaction solution becoming turbid depending on the substance to be added, causing problems affecting measurement.

Conventionally, in the measurement of glycated protein, when a quaternary ammonium salt such as benzyltriethylammonium chloride, benzyl-n-butylammonium chloride, lauryltrimethylammonium chloride (carbon number of alkyl group 12) or lauryldimethylamine oxide is added, globulin It is known that the influence of components and ascorbic acid can be avoided and protease can be stabilized (for example, see Patent Document 4).
However, even when these quaternary ammonium salts are used, the turbidity of the reaction solution as described above cannot be sufficiently avoided, and the amount of protease used cannot be reduced to less than 1,000 U / mL. .

Japanese Patent Publication No. 05-33997 JP-A-11-127895 WO 97/013872 pamphlet International Publication No. 02/061119 Pamphlet Chromatogr. sci. 1979, 10 volumes, p659 Journal of the Japan Society for Clinical Laboratory Automation, 1993, 18 volumes, p620

  The subject of this invention is providing the measuring method and measuring reagent of glycated albumin which reduce the usage-amount of protease and do not generate turbidity in a reaction solution.

  In the measurement of glycated albumin, trimethylammonium salt and / or dimethylammonium salt, which is a quaternary ammonium salt, coexists to suppress the occurrence of turbidity in the reaction solution and to reduce the amount of protease used. .

That is, the present invention relates to the following glycated albumin measuring method and a glycated albumin measuring reagent using the measuring method.
(1) A reagent for measuring glycated albumin that decomposes glycated albumin with a protease, causes glycated amino acid oxidase to act on the released glycated amino acid, and measures the generated hydrogen peroxide, and contains a trimethylammonium salt and / or a dimethylammonium salt. A reagent for measuring glycated albumin characterized by
(2) The trimethylammonium salt is one or more selected from hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide or tetradecyltrimethylammonium bromide, as described in (1) above Glycated albumin measurement reagent.
(3) The reagent for measuring glycated albumin according to (1) above, wherein the dimethylammonium salt is hexadecyldimethylethylammonium bromide.
(4) The reagent for measuring glycated albumin according to any one of (1) to (3) above, wherein the use concentration of trimethylammonium salt and / or dimethylammonium salt is in the range of 1 to 5 g / L.
(5) The reagent for measuring glycated albumin according to any one of the above (1) to (4), wherein the glycated amino acid oxidase is an enzyme derived from a filamentous fungus, an enzyme derived from the genus Aspergillus, or an enzyme derived from the genus Corynebacterium.

1. Glycated albumin measuring reagent The glycated albumin measuring reagent comprises a glycated amino acid oxidase, a protease, and a quaternary ammonium salt such as trimethylammonium salt or dimethylammonium salt. If necessary, ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, Triton-X100; enzymes such as peroxidase, catalase, ascorbate oxidase; 4-aminoantipyrine, phenol, TOOS, 10- (carboxymethylaminocarbonyl) -3,7 -Hydrogen peroxide detection reagents such as bis (dimethylamino) phenothiazine sodium salt (DA-67), N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) diphenylamine sodium salt (DA-64); 1 -Redox coloring reagents such as Methoxy PMS and WST-8 (formazan reagent); Tris, phosphoric acid, acetic acid, citric acid, oxalic acid, MES, BES, HEPES, TES, bicine, tricine, glycine, boric acid, Bis -T Buffers such as ris propane and imidazole; Coenzymes such as FAD, NAD, NADP, NADH and NADPH; Various inorganic salts such as sodium chloride, potassium chloride and sodium azide; Polysaccharides such as dextran; Bovine serum albumin (BSA) Glycerol; amino acid; surfactant; any one or more of chemotherapeutic agents such as antibiotics and sulfa drugs may be contained.
The reagent may be liquid, or may be freeze-dried or spray-dried if necessary, and dissolved at the time of use.
According to the method of the present invention, by using trimethylammonium salt and / or dimethylammonium salt which is a quaternary ammonium salt, turbidity generation due to mixing of the reagent and the sample is suppressed, and the amount of protease used is 1,000 U / A reagent for measuring glycated albumin reduced to less than mL can be prepared.

2. Saccharified Amino Acid Oxidase “Saccharified amino acid oxidase” used in the present invention is an enzyme that catalyzes a reaction of oxidizing glycated amino acid in the presence of oxygen and generating hydrogen peroxide, and can be used from any source. . Any of the saccharified amino acid oxidases produced by microorganisms, plants, animals or the like may be used and is not particularly limited. For example, the genus Gibberella, Aspergillus, Candida, Penicillium, Fusarium, Acremonium, or Debaryomes Preferably, an enzyme derived from a filamentous fungus, an enzyme derived from the genus Aspergillus or an enzyme derived from the genus Corynebacterium is used. More specifically, commercially available glycated amino acid oxidase FAOD-E (manufactured by Kikkoman Corporation) is preferably used.
The concentration of glycated amino acid oxidase is, for example, 0.05 to 1,000 U / ml, preferably 0.2 to 100 U / ml in the case of a solution. If the concentration of glycated amino acid oxidase is less than 0.05 U / ml, the enzymatic reaction becomes insufficient.

3. Proteases Examples of proteases that can coexist include microorganism-derived proteases such as Bacillus, Aspergillus, and Streptomyces. In addition, serine protease is also preferable. It is desirable that the amount of protease used is reduced to less than 1,000 U / mL, which does not affect the enzyme reaction of other clinical diagnostic measurement tests.

4). Quaternary ammonium salt A quaternary ammonium salt is a compound obtained by substituting four hydrocarbon groups for NH in NH ₄ ⁺ . The trimethylammonium salt is an ammonium salt having an ammonium structure having one alkyl group as a main chain of a lipophilic group and three methyl groups (CH ₃ ) and an anion such as chlorine or bromine as a counter ion. The dimethylammonium salt is an ammonium salt having two main chains serving as lipophilic groups and two methyl groups.

  The glycated amino acid oxidase and the trimethylammonium salt and / or dimethylammonium salt can be mixed in advance in the same reagent and used together. Further, since the trimethylammonium salt and / or dimethylammonium salt only needs to be present when the reaction with glycated amino acid oxidase proceeds, the reagent containing glycated amino acid oxidase (reagent A) and the trimethylammonium salt and / or A reagent containing dimethylammonium salt (reagent B) is separately prepared, and reagent A and reagent B are mixed as appropriate to allow the glycated amino acid oxidase reaction to proceed in the presence of the trimethylammonium salt and / or dimethylammonium salt. it can.

  In the present invention, the trimethylammonium salt and dimethylammonium salt which are coexisted in order to eliminate the turbidity of the reaction solution are specifically hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, bromide Examples include tetradecyltrimethylammonium and hexadecyldimethylethylammonium bromide. In the present invention, the trimethylammonium salt and dimethylammonium salt can be used either individually or in combination.

The concentration of the trimethylammonium salt and dimethylammonium salt in the solution coexisting with the glycated amino acid oxidase is within the range where the effect of eliminating the turbidity of the reaction solution is exhibited and there is no inconvenience in handling the reagent containing the enzyme. If it is not specifically limited, it can add at a suitable density | concentration about each compound. For example, the concentration of trimethylammonium salt or dimethylammonium salt is preferably 1 to 5 g / L.
When trimethylammonium salt and / or dimethylammonium salt are allowed to coexist with 1 to 5 g / L, the occurrence of turbidity is suppressed, and the amount of protease used can be reduced to less than 1,000 U / mL. Become.

5. Reaction pH and Buffering Agent Generally, since the stability of an enzyme is greatly affected by the pH during storage, it is preferable to use various buffers in a stable pH range. The buffer used in eliminating turbidity of the reaction system of the present invention preferably has a buffer capacity between pH 6.0 and 8.0. Examples of such a buffer include general-purpose Tris buffer and phosphate buffer, organic acid buffers such as acetic acid, citric acid, and oxalic acid, 2-Morpholinoethane acid (MES), BES, Good buffers such as HEPES, TES, bicine, and tricine, amino acid buffers such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, imidazole buffer, and the like can also be used.

  About the density | concentration of the buffer solution at the time of preserve | saving an enzyme, Preferably it is 5-500 mM, More preferably, it is 20-200 mM. When the trimethylammonium salt and / or dimethylammonium salt of the present invention is added to the buffer, it is added directly or pH 5.0-10.0, preferably pH 6.0-8.0, particularly preferably pH 6. What is necessary is just to add those aqueous solutions adjusted to 0-7.5. When the pH is outside the target range by adding trimethylammonium salt and / or dimethylammonium salt, the pH is within the target range by adding acetic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, aqueous ammonia, etc. It can be adjusted to be inside.

6). Method for measuring glycated albumin Examples of the sample for measuring glycated albumin include urine, serum, and plasma. Regarding the concentration of glycated albumin contained in the measurement sample, it is preferable that the sample is appropriately diluted and is at a concentration suitable for measurement, for example, about 2.5 g / dL.
The pH to be applied is pH 5.0 to 10.0, preferably pH 6.0 to 9.0, particularly preferably pH 6.0 to 7.5, and a normal pH range used for the enzymatic reaction of glycated amino acid oxidase is determined. It can be selected appropriately.
The pH adjustment method can be adjusted using a general-purpose Tris buffer or phosphate buffer. Organic acid buffers such as acetic acid, citric acid, and oxalic acid, MES, BES, HEPES, TES, bicine Good buffer such as tricine, amino acid buffer such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, imidazole buffer and the like can also be used. In this case, a trimethylammonium salt and / or dimethylammonium salt that is a stabilizer for glycated amino acid oxidase, a chelating reagent, and a surfactant that is a substrate specificity improver may be appropriately present.

The concentration of glycated amino acid oxidase may be added so that the final concentration is 0.1 to 50 U / ml, preferably 1 to 10 U / ml.
The action time of glycated amino acid oxidase is 30 seconds to 120 minutes, preferably 1 to 30 minutes. The working temperature is, for example, 20 to 45 ° C., and a temperature used for a normal enzyme reaction can be appropriately selected.

  In the glycated amino acid oxidase solution added to the sample, various inorganic salts such as sodium chloride, potassium chloride and sodium azide, polysaccharides such as dextran, BSA, glycerol and amino acids may coexist if necessary. This liquid preparation may be freeze-dried or spray-dried.

The product resulting from the reaction of glycated amino acid oxidase may be measured by any method. For example, the generated hydrogen peroxide can be measured by an enzymatic method using peroxidase and an appropriate color reagent, a luminescent reagent, or an electrical method using an enzyme electrode.
For example, detection reagents for reaction products using enzymes include tris, phosphoric acid, acetic acid, citric acid, oxalic acid, MES, BES, HEPES, TES, bicine, tricine, glycine, boric acid, Bis-Tris propane, imidazole Buffering agents such as FAD, NAD, NADP, NADH, NADPH, etc., hydrogen peroxide detection reagents such as peroxidase, 4-aminoantipyrine, phenol, TOOS, DA-67, 1-METHOXY PMS and WST-8 ( Any one or more of a redox coloring reagent such as formazan reagent) and an enzyme such as catalase can be contained.
If necessary, various inorganic salts such as sodium chloride, potassium chloride and sodium azide, polysaccharides such as dextran, BSA, glycerol and amino acids may coexist.

7). Turbidity Evaluation Method Turbidity evaluation can be confirmed by measuring a wavelength of 600 nm or more, which is generally less influenced by reagents. Specifically, a wavelength of 600 nm or more is selected from the maximum absorption wavelength of the color former to be used. For example, when the hydrogen peroxide detection reagent DA-67 is used, since the maximum absorption wavelength of DA-67 is 666 nm, a wavelength of 694 nm is used for evaluation of turbidity.
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, an Example does not limit this invention at all.

1. Measurement of glycated albumin using quaternary ammonium salt In order to measure glycated albumin using the quaternary ammonium salt of the present invention, the following reagents were prepared.
<Reagent>
Reagent 1 (R1):
50 mM MES buffer (pH 7.0)
15 mM KCl
0.06% Triton X100
0.05% EGTA
5 U / mL peroxidase (Kikkoman)
5 U / mL saccharified amino acid oxidase (FAOD-E manufactured by Kikkoman Corporation)
Reagent 2 (R2):
150 mM MES buffer (pH 6.5)
0.09% sodium azide 0.05mM DA-67
3.0 g / L hexadecyltrimethylammonium chloride (CH ₃ (CH ₂ ) ₁₅ N (CH ₃ ) ₃ ) Cl or phenyltrimethylammonium iodide (manufactured by Nacalai)
375 U / mL protease (manufactured by Sigma)
<Sample>
Saline and human serum were used as experimental samples. As a control reagent for measuring glycated albumin, Lucika GA-L calibrator (manufactured by Asahi Kasei Pharma) was used.
<Measurement equipment used>
Biochemical automatic analyzer (trade name JCA-BM1650: manufactured by JEOL Ltd.)

<Reaction procedure>
To 78 μL of R1 incubated at 37 ° C., 3 μL of sample (standard diluted specimen in biochemical automatic analyzer (trade name JCA-BM1650: manufactured by JEOL Ltd.)) was added, and the reaction was started at 37 ° C. After 5 minutes of reaction, 26 μL of R2 was added. Absorbance at a main wavelength of 658 nm and a subwavelength of 805 nm was measured before addition of R2 and 5 minutes after the addition.
Lucica GA-L calibrator was used as a control reagent for measuring glycated albumin.
<Measurement results>
As a result of measuring glycated albumin in human serum using hexadecyltrimethylammonium chloride as a quaternary ammonium salt, the added protease was reduced to 375 U / mL and less than 1,000 U / ml. Good reactivity (FIG. 1) was observed. In addition, a good correlation with the control reagent Lucika GA-L was also observed (FIG. 2). This confirmed that hexadecyltrimethylammonium chloride and phenyltrimethylammonium iodide, which are trimethylammonium salts, can be used, and in particular, hexadecyltrimethylammonium chloride was confirmed to be useful.

2. Measurement of glycated albumin using hexadecyldimethylethylammonium bromide as a quaternary ammonium salt <Reagent>
Reagent 1 (R1):
Same as Example 1.
Reagent 2 (R3):
150 mM MES buffer (pH 6.5)
0.09% sodium azide 0.05mM DA-67
1.0 g / L Hexadecyldimethylethylammonium bromide (C ₂₀ H ₄₄ BrN)
375 U / mL protease (manufactured by Sigma)

<Reaction procedure>
Example 3 was the same as Example 1 except that R3 was used instead of R2.
<Measurement results>
By using hexadecyldimethylethylammonium bromide as the quaternary ammonium salt, the reactivity equivalent to that in Example 1 (FIG. 3) and a good correlation with the reference reagent Lucika GA-L (FIG. 4) can be obtained. Obtained. As a result, it was confirmed that hexadecyldimethylethylammonium bromide, which is a dimethylammonium salt, can also be used.

3. Confirmation of turbidity due to quaternary ammonium salt differences <Reagent>
Reagent 1 (R1):
Same as Example 1.
Reagent 2 (R4)
The following quaternary ammonium salt (1.0 g / L) was used as the quaternary ammonium salt, and the reactivity was compared and the presence or absence of turbidity was confirmed.
Phenyltrimethylammonium bromide Lauryltrimethylammonium bromide Tetradecyltrimethylammonium bromide Hexadecyltrimethylammonium bromide Octadecyltrimethylammonium bromide Phenyltrimethylammonium iodide

<Reaction procedure>
The same procedure as in Example 1 was conducted except that the trimethylammonium salt described in R4 was used instead of the quaternary ammonium salt used in R2. In order to confirm the presence or absence of turbidity, the absorbance at 694 nm was confirmed.

<Measurement results>
Rather than containing phenyltrimethylammonium bromide, lauryltrimethylammonium bromide or phenyltrimethylammonium iodide as the quaternary ammonium salt (FIG. 5), tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide or bromide It was confirmed that the reactivity was better when octadecyltrimethylammonium was contained (FIG. 6).
Tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide, which were quaternary ammonium salts with good reactivity, were further checked for the occurrence of turbidity (FIG. 7). ).
These results confirm that glycated albumin can be measured under conditions that are less susceptible to turbidity by containing tetradecyltrimethylammonium bromide or hexadecyltrimethylammonium bromide than by containing octadecyltrimethylammonium bromide. did it.

It is a graph which shows the reactivity with respect to the Lucica GA-L calibrator when not containing a trimethylammonium salt, when hexadecyltrimethylammonium chloride which is a trimethylammonium salt or phenyltrimethylammonium iodide is contained. It is a graph which shows the correlation with Lucika GA-L when containing hexadecyl trimethyl ammonium chloride. It is a graph which shows the reactivity with respect to the Lucika GA-L calibrator when containing hexadecyl dimethyl ethyl ammonium bromide which is a dimethyl ammonium salt. It is a graph which shows the correlation with Lucika GA-L when containing hexadecyl dimethyl ethyl ammonium bromide. It is a graph which shows the reactivity with respect to a Lucika GA-L calibrator when containing phenyltrimethylammonium bromide, lauryltrimethylammonium bromide, or phenyltrimethylammonium iodide. It is a graph which shows the reactivity with respect to a Lucica GA-L calibrator when containing tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, or octadecyl trimethyl ammonium bromide. It is the graph which showed the condition of the turbidity in the reaction solution when containing tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, or octadecyl trimethyl ammonium bromide by the light absorbency in wavelength 694nm.

Claims (3)

Use of tetradecyltrimethylammonium bromide in a reagent for measuring glycated albumin that decomposes glycated albumin at a protease concentration of less than 1,000 U / mL, causes glycated amino acid oxidase to act on the released glycated amino acid, and measures hydrogen peroxide produced A glycated albumin measuring reagent containing a concentration in the range of 1 to 5 g / L.   The 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium salt is used as a hydrogen peroxide detection reagent in a saccharified albumin measurement reagent for measuring the hydrogen peroxide produced. Glycated albumin measurement reagent.   The glycated albumin measuring reagent according to claim 1 or 2, wherein the glycated amino acid oxidase is an enzyme derived from a filamentous fungus, an enzyme derived from the genus Aspergillus, or an enzyme derived from the genus Corynebacterium.

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