JP4798600B2 - Method for stabilizing fructosyl peptide oxidase - Google Patents

Description

  The present invention relates to a method for stabilizing fructosyl peptide oxidase (hereinafter referred to as “FPOX”) used in clinical diagnosis in a liquid state.

  A glycated protein is a protein in which the protein is non-enzymatically glycated, and the aldehyde group on the sugar, that is, aldose (monosaccharide and derivate having a potential aldehyde group) side, and the amino group on the protein side are non-enzymatic. It is generated as a result of covalent bonding to. Examples of the amino group of the protein include the α-amino group of the N-terminal amino acid and the ε-amino group of the internal lysine residue side chain. These glycated proteins are also called so-called Amadori compounds because the Schiff base generated as a reaction intermediate undergoes Amadori transfer.

  Glycated proteins are contained in body fluids such as blood in living bodies and biological samples such as hair. The concentration of glycated protein present in blood strongly depends on the concentration of saccharides such as glucose dissolved in serum. In diabetic state, the production of glycated proteins is enhanced, and the concentration of glycated hemoglobin in serum and glycated albumin in serum reflects the average blood glucose level over a certain period of time. Measuring is important for diagnosis and management of symptoms of diabetes.

Conventional methods for quantifying glycated proteins include, for example, a method using high performance liquid chromatography (see, for example, Non-Patent Document 1), and a method using a column packed with a solid to which boric acid is bound (see, for example, Non-Patent Document 2). , A method using electrophoresis (for example, see Non-patent Document 3), a method using an antigen-antibody reaction (for example, refer to Non-Patent Document 4), and a method for colorimetric determination of reducing ability using a tetrazolium salt (for example, non-Patent Document 4) Patent Document 5), a method for colorimetric determination after oxidation using thiobarbituric acid (for example, see Non-Patent Document 6), a method using an enzyme such as glycated amino acid oxidase (for example, see Patent Documents 1 to 16), etc. It has been known. Recently, a new method for measuring glycated protein has been disclosed as a method for measuring glycated protein more accurately than the above method (see, for example, Patent Document 17). In this measurement method, a sample containing a glycated protein is treated with a protease, the fructosyl peptide is released from the glycated protein, FPOX is allowed to act on the released fructosyl peptide, and the product is measured to measure the glycated protein. Therefore, it is attracting attention as a highly accurate measurement method with a short time and simple operation.

  Although the measurement method using FPOX is a very excellent method as described above, there is a problem that the FPOX used is unstable. Regarding the stabilization of FPOX, a method for stabilizing a fructosyl peptide oxidase lyophilized preparation characterized in that one or more reagents selected from chelating reagents, sugar alcohols, amino acids, and metal salts are allowed to coexist with FPOX ( Japanese Patent Application No. 2004-060645).

  Common clinical laboratory reagents are often manufactured and distributed in a lyophilized state or in a solution state. Recently, however, the demand for a solution state has been increasing in order to prevent easy operation and adjustment errors during dissolution. ing. However, a method for stabilizing FPOX in a solution state has not been known so far.

Japanese Patent Publication No. 05-33997 JP-A-11-127895 WO 97/13872 pamphlet Japanese Patent Publication No. 06-65300 Japanese Patent Laid-Open No. 02-195900 Japanese Patent Laid-Open No. 03-155780 Japanese Patent Laid-Open No. 04-4874 JP 05-192193 A Japanese Patent Laid-Open No. 06-46846 JP-A-11-155596 JP 10-313893 A Japanese Patent Laid-Open No. 11-504808 JP 2000-333696 A JP 2001-54398 A JP 2001-204495 A JP 2001-204494 A JP 2001-95598 A Cole RA, 3 others, “A rapid method for the determination of glycosylated hemoglobins using high pressure liquid chromatography”, Metabolism, 1978 5 Moon, Vol. 27, No. 3, p.289-301 Klenk DC, 9 others, “Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric: Quantification of glycosylated hemoglobin by affinity chromatography: Comparison of colorimetric and ion exchange methods, influence of common inhibitors and ion-exchange methods, and effects of common interferences) ", Clin Chem, October 1982, Vol. 28, No. 10, p. 2088-2094. Menard L, 5 others, “Quantitative determination of glycosylated hemoglobin A1 by agar gel electrophoresis”, Clinical Chemistry (Clin Chem), October 1980, Vol. 28, No. 11, pp. 1598-1602 Journal of the Japan Society for Clinical Laboratory Automation, 1993, Vol. 18, p.620 Johnson (RN), two others, “Fructosamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control”, Clinica Clin Chim Acta, January 7, 1983, Vol. 127, No. 1, p. 87-95 Dolhofer R, 1 other, “Improvement of the thiobarbituric acid assay for serum glycosylprotein determination”, Clin Chim Acta May 5, 1981, Vol. 112, No. 2, p. 197-204

  The present invention relates to a method for stabilizing FPOX used in clinical diagnosis in a liquid reagent.

  As a result of intensive studies to solve the above problems, the present inventors have found that ammonium salts, oxycarboxylic acid-based chelating reagents, sugar alcohols, amino acids, which cannot be confirmed alone, are polyaminocarboxylic acids. By coexisting with a chelating reagent of the system, it has been found that a synergistic effect is exhibited and the stability of FPOX in a liquid state is extremely improved, and the present invention has been completed based on these findings.

That is, the present invention
1) A polyaminocarboxylic acid chelating reagent and one or more reagents selected from ammonium salts, oxycarboxylic acid chelating reagents, sugar alcohols, and amino acids are allowed to coexist in a fructosyl peptide oxidase-containing solution. A method for stabilizing fructosyl peptide oxidase.
2) The method for stabilizing fructosyl peptide oxidase according to 1) above, wherein the polyaminocarboxylic acid-based chelating reagent is ethylenediaminetetraacetic acid.
3) The method for stabilizing fructosyl peptide oxidase according to 1) or 2) above, wherein the concentration of fructosyl peptide oxidase is 100 U / ml or less.
About.

  According to the present invention, a stabilized FPOX liquid reagent can be produced by a simple method and at low cost.

  Hereinafter, the present invention will be described in detail. FPOX used in the present invention is an oxidase that acts on fructosyl peptide in the presence of oxygen to produce α-ketoaldehyde, peptide, and hydrogen peroxide, and any oxidase is included as long as it has this action. . The origin of FPOX used in the present invention may be any, for example, filamentous fungi, yeast, actinomycetes, bacteria, archaea, etc., but is not particularly limited, but preferably, for example, Coniochaeta, Eupenicillium ( FPOX originating from Eupenicillium), Achaetomiella, etc. These FPOXs can also be those produced by gene recombinants.

  In the present invention, the polyaminocarboxylic acid-based chelating reagent coexisting to improve the stability of FPOX is a compound that coordinates to a metal ion to give a chelate compound. The polyaminocarboxylic acid-based chelating reagent that can be used in the present invention may be any one that improves the stability of FPOX, such as ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”), trans-1,2- diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (hereinafter referred to as “CyDTA”), diethylenamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA), glycol ether diaminetetraacetic acid (DTPA) EGDTA), 1,6-hexaneethylenediamine-N, N, N ′, N′-tetraacetic acid (HDTA), triethylenetetramine hexaacetic acid (TTHA) and the like. In the present invention, the polyaminocarboxylic acid-based chelating reagent is not limited in any way, but for example, EDTA is preferably used. The EDTA used may be any salt, and examples of the EDTA salt include sodium salt, potassium salt, lithium salt, and ammonium salt.

  In the present invention, any ammonium salt can be used as the ammonium salt to be present in order to improve the stability of FPOX. Examples thereof include ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium tartrate. In the present invention, the ammonium salts are not limited at all, but, for example, ammonium sulfate is preferably used. The ammonium salts can be used alone or in combination.

  In the present invention, any oxycarboxylic acid-based chelating reagent can be used as the oxycarboxylic acid-based chelating reagent that coexists in order to improve the stability of FPOX. Examples thereof include citric acid, malic acid, isocitric acid, and salts thereof. In the present invention, the oxycarboxylic acid-based chelating reagent is not limited at all, but, for example, trisodium citrate is preferably used. The oxycarboxylic acid chelating reagents can be used alone or in combination.

  In the present invention, any sugar alcohol can be used as the sugar alcohol to be coexisted in order to improve the stability of FPOX. Examples include trehalose, xylitol, sorbitol, inositol, mannitol, arabitol, dulcitol, erythritol and the like. In the present invention, the sugar alcohols are not limited at all, but, for example, xylitol is preferably used. The sugar alcohols can be used alone or in combination.

  In the present invention, any amino acid can be used as the amino acid to coexist in order to improve the stability of FPOX. For example, sodium glutamate, glycine, lysine hydrochloride, asparagine, alanine, methionine, threonine and the like can be mentioned. In the present invention, the amino acids are not limited at all, but for example, glycine is preferably used. The amino acids can be used alone or in combination.

  In the stabilization method of the present invention, the FPOX and the polyaminocarboxylic acid chelating reagent and one or more reagents selected from ammonium salts, oxycarboxylic acid chelating reagents, sugar alcohols, and amino acid salts coexist. Let At this time, if necessary, other reagents may coexist. Usually, solid powders such as FPOX and reagents, or a solution thereof can be mixed together as appropriate.

  The concentration of FPOX used in the stabilization method of the present invention is not particularly limited, but is preferably 0.05 to 1000 U / ml, more preferably 0.10 to 100 U / ml. The enzyme titer here is defined as 1 U for the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using fructosyl valyl histidine as a substrate.

  The concentration of polyaminocarboxylic acid-based chelating reagent, ammonium salt, oxycarboxylic acid-based chelating reagent, sugar alcohols and amino acids is effective in stabilizing FPOX and is not inconvenient when handling reagents containing enzymes. Any concentration within the range can be prepared. For example, the preferred concentration of the polyaminocarboxylic acid-based chelating reagent and the oxycarboxylic acid-based chelating reagent is 0.1 mM or more, more preferably 1 to 100 mM. A preferable concentration of the ammonium salt is 0.1 mM or more, more preferably 1 to 500 mM. A preferable concentration of the sugar alcohol is 1% or more, more preferably 2 to 20%. A preferred concentration of amino acids is 1% or more, more preferably 2 to 20%.

  In general, since the stability of an enzyme is greatly affected by the pH during storage, it is preferable to simultaneously use various buffers in a stable pH range as other reagents. The type of buffer solution used in the present invention, its concentration, and pH are not particularly limited. For example, the buffer solution has a buffer capacity between pH 6 and 10, and is set to a concentration that maintains a necessary and sufficient buffer capacity. It is desirable that Examples of such buffers include general-purpose tris buffers and phosphate buffers, good buffers such as BES, HEPES, TES, bicine, and tricine, and amino acid buffers such as glycine-NaOH. A borate buffer solution, a Bis-Tris propane buffer solution, an imidazole buffer solution, or the like can also be used. About the density | concentration of a buffer solution, Preferably it is 1-1000 mM, More preferably, it is 10-100 mM. In the present invention, Tris buffer or phosphate buffer is preferably used. When adding the chelating reagent, ammonium salt, sugar alcohol, and amino acids of the present invention to the buffer, add them directly or add their aqueous solutions adjusted to pH 5-9, preferably 6-8, for example. do it. When the pH deviates from the target range by adding the above-mentioned reagents, the pH is adjusted to fall within the target range by adding, for example, sodium hydroxide, potassium hydroxide, or aqueous ammonia. Is preferred.

  Further, as other reagents, for example, various inorganic salts such as sodium chloride, potassium chloride and sodium azide, polysaccharides such as dextran, bovine serum albumin (BSA), glycerol, surfactants Further, chemotherapeutic agents such as antibiotics and sulfa drugs may coexist. These reagents may be added to the buffer in advance.

  Evaluation of stability is to store the FPOX liquid reagent under various conditions according to the storage conditions, transport conditions and measurement conditions of the enzyme actually used, and measure the change in its activity over time. In general, an abuse test is usually used to perform evaluation in a short time. For example, there may be mentioned a method in which the FPOX liquid reagent is kept at a constant high temperature and the residual activity is measured over time.

  In this way, FPOX is in a liquid state by coexisting a polyaminocarboxylic acid-based chelating reagent and one or more reagents selected from ammonium salts, oxycarboxylic acid-based chelating reagents, sugar alcohols, and amino acids. Can be stabilized. This stabilization method is used to stabilize a FPOX-containing polyaminocarboxylic acid chelating reagent and one or more reagents selected from ammonium salts, oxycarboxylic acid chelating reagents, sugar alcohols, and amino acids. FPOX liquid reagent can be produced. For example, a buffer solution having a pH of 6 to 10 containing EDTA at a concentration of 1 mM or more is prepared, and FPOX (manufactured by Kikkoman) is added to this buffer solution so that the concentration is 0.1 to 100 U. Further, one or more reagents selected from ammonium salts, oxycarboxylic acid chelating reagents, sugar alcohols, and amino acids are added. By stirring this mixed solution and completely dissolving it, a FPOX liquid reagent can be produced. The FPOX liquid reagent of the present invention thus obtained is significantly stabilized as compared with the liquid reagent produced by the conventional method, and is stabilized by a simple method and at a low cost by the method of the present invention. The prepared FPOX liquid reagent can be manufactured.

Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples. However, the technical scope of the present invention is not limited by these examples.
<Experimental example 1>

(FPOX activity measurement method and stability test)
A method for measuring the amount of hydrogen peroxide will be described. Hereinafter, fructosyl valyl histidine is used as a substrate for measurement of the activity of the oxidase of the present invention unless otherwise specified. The enzyme titer was defined as 1 U for the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using fructosyl valyl histidine as a substrate.
A. Preparation of Reagent (1) Reagent 1: POD-4-aminoantipyrine solution 1.0 kU peroxidase (manufactured by Kikkoman), 100 mg of 4-aminoantipyrine (manufactured by Tokyo Chemical Industry), 0.1 M potassium phosphate buffer Dissolve in (pH 8.0) and make up to 1 L.
(2) Reagent 2: 2,4-dichlorophenol sulfate solution 25 ml of a commercially available 2% solution (manufactured by Nacalai) is dissolved in ion-exchanged water, and the volume is adjusted to 100 ml.
(3) Reagent 3: Substrate solution (150 mM; final concentration 5 mM)
Dissolve 624 mg of fructosyl valyl histidine in ion exchange water to a constant volume of 10 ml. Fructosylvaline histidine was prepared by the method described in JP-A No. 2001-95598.
B. Measurement method 2.7 ml of reagent 1, 100 μl of reagent 2, and 100 μl of FPOX enzyme solution (manufactured by Kikkoman) are mixed and preheated at 30 ° C. for 5 minutes. Thereafter, 100 μl of reagent 3 is added and mixed well, and then the absorbance at 510 nm is measured with a spectrophotometer (U-2000A, manufactured by Hitachi, Ltd.). The measured value is the change in absorbance per minute from 1 minute to 3 minutes after 510 nm. The control solution was the same as described above except that 100 μl of ion exchange water was added instead of 100 μl of reagent 3. A hydrogen peroxide standard solution prepared in advance was used in place of the reagent 3 and ion-exchanged water in place of the enzyme solution, and a graph was prepared in which the relationship with the amount of generated dye was examined. Using this graph, the micromol of hydrogen peroxide produced per minute at 30 ° C. was calculated, and this value was used as the activity unit in the enzyme solution. For dilution of the FPOX enzyme solution for activity measurement, a 10 mM potassium phosphate buffer (pH 8.0) containing 0.15% (w / v) BSA was used. The stability test of the FPOX liquid reagent was performed by comparing the residual activity after the abuse test. The residual activity of the enzyme solution after the abuse test was expressed as a relative amount (%) when the change in absorbance at the time of preparing the enzyme solution was taken as 100%.

(Confirmation of effect when reagent is added alone)
First, as a test for adding no stabilizer (control), FPOX-CE (manufactured by Kikkoman) was added to 1 ml of 20 mM Tris-HCl buffer (pH 8.0) so as to be 20 U / ml. This solution was stored at 30 ° C. for 7 days, and then the enzyme activity was measured. As a result, as shown in Table 1, entry 1, it was found that the activity decreased to 1% as compared with the enzyme activity before storage.

  Next, various reagents were added at final concentrations shown in Table 1, and the same test as described above was performed. As shown in Table 1, it was confirmed that the stability was remarkably improved by adding EDTA, a polyaminocarboxylic acid chelating reagent. On the other hand, a reagent other than EDTA alone could not confirm a remarkable effect.

Table 1

(Confirmation of coexistence effect with EDTA)
Reagents for which a remarkable effect was not confirmed in Table 1 were allowed to coexist with EDTA, and the stabilizing effect was confirmed.
Various reagents were added to 20 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA at the final concentrations shown in Table 2, and the same tests as in Example 1 were performed.
In Table 2, entry 1 shows the result of EDTA alone, and entries 2 to 7 show the result of additional addition. Surprisingly, trisodium citrate, xylitol, glycine, and ammonium sulfate, which alone had only 1 to 4% additive stabilizing effect compared to no addition, were allowed to coexist with EDTA. It was confirmed that the stabilization effect is close to 20%.

Table 2

Next, the test of Example 2 was performed by setting the concentration of FPOX-CE to 40 U / ml and extending the storage period.
Various reagents were added to 20 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA at the final concentrations shown in Table 3, and FPOX-CE was further added to a concentration of 40 U / ml. This solution was stored at 30 ° C. for 18 days, and then the enzyme activity was measured.
In Table 3, entry 1 shows the results of EDTA alone, and entries 2 to 7 show the results of additional addition. As in Table 2, it was confirmed that trisodium citrate, xylitol, glycine, and ammonium sulfate coexist with EDTA and show a remarkable stabilizing effect as compared with EDTA alone.

Table 3

According to the present invention, FPOX can be stably maintained in a solution state, and can be easily used in a measurement kit as a diagnostic enzyme for diabetes. Furthermore, it becomes possible to keep the enzyme stable for a long period of time during storage and transportation, and the development of a clinical diagnostic kit excellent in storage stability together with the present invention excellent in stability.

Claims (1)

It is characterized in that ethylenediaminetetraacetic acid is added to a fructosyl peptide oxidase-containing solution having a fructosyl peptide oxidase concentration of 100 U / ml or less, and at least one reagent selected from ammonium sulfate, xylitol, and glycine coexists. Method for stabilizing fructosyl peptide oxidase.