JP2005261383A - Calibration method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply, inexpensively and accurately carrying out calibration by using a calibration substance having only one glycated protein concentration when measuring a glycated protein by using an enzyme. <P>SOLUTION: The calibration method enables simpler, inexpensive and accurate calibration by using an enzyme-containing liquid in which residual activity of the enzyme after one week refrigeration is ≥90% and by using the calibration substance having the one glycated protein concentration. The calibration method enables accurate measurement of glycated albumin by using a reagent containing bromocresol purple as a reagent for albumin when the protein is albumin. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for preparing a calibration curve useful for measuring glycated protein and a method for measuring glycated protein using the calibration curve.

The measurement of glycated protein is very important in the diagnosis and management of diabetes, and reflects the average blood glucose level in the past about 1 to 2 months, hemoglobin A1c (HbA1c), and the average blood glucose level in the past about 2 weeks Glycated albumin (GA) and the like are routinely measured. Among them, GA is useful for confirming the therapeutic effect and adjusting the dosage, and the number of examinations has increased in recent years.
Various methods have been devised by various companies as methods for measuring GA using an enzyme (Non-patent Document 1). However, there are few reports on the calibration substance used to prepare the calibration curve, and it is only known that GA purified from healthy subjects is preferable to GA prepared by artificial saccharification reaction. (Patent Document 1).
In addition, Lucika GA (manufactured by Asahi Kasei Pharma) and GlyAlb-OY (manufactured by Oriental Yeast Co., Ltd.) have been released as enzyme measuring methods for GA, but the calibrators used in these kits are those for normal and abnormal areas. A calibration that linearly regresses two calibration materials and three water points is adopted. The same applies to Non-Patent Document 1 and Patent Document 1. When using two calibrators, a calibration curve is drawn at a total of three points with the blank and two calibrators. However, the calibration curve may not be completely straight, and this kind of error will occur. A method using a single calibration substance that is not present is preferred.

Patent Document 2 shows that calibration using one calibration substance and water is also possible for measuring glycated protein. However, when stored for about one week in the refrigerator, the two-point calibration can be calibrated more accurately, and the one-point calibration is not satisfactory. Therefore, considering the storage of reagents, no method has been reported that can accurately calibrate with one calibration substance in measuring GA.
On the other hand, methods for measuring glycated hemoglobin using an enzyme are known (Patent Documents 3 to 6). However, there has been no report on an example in which calibration is performed with one calibration substance in a method for measuring glycated hemoglobin using an enzyme.

WO 01/94618 WO 02/061119 JP 2001-57897 A WO 00/50579 WO 00/61732 JP2000-300294 Kouzuma et.al.An enzymatic method for the measurement of glycated albumin in biological sample.Clinica Chimica Acta 324 (2002) 61-71

  An object of the present invention is to provide a simpler, less expensive, and more accurate calibration method by using only one calibration substance when measuring a glycated protein using an enzyme. . More specifically, an object of the present invention is to provide a calibration method for measuring glycated protein using an enzyme useful in clinical biochemical examination.

In measuring glycated protein, it is preferable to create a calibration with only one calibration material and blank. Therefore, the present inventors diligently studied the reason why calibration using only one calibration substance cannot be applied to the conventional glycated protein measurement methods using enzymes. As a result, when the reagent is stored, the sensitivity of the reagent decreases, and the calibration curve in the range to be measured deviates slightly from the straight line, especially in the high concentration region, so it is better to use two low and high calibration materials. And found that calibration can be performed more accurately. Furthermore, this phenomenon was found to be due to the progressive inactivation of protease and ketoamine oxidase.

Next, since the present inventors have already found a technique for stabilizing enzymes with various stabilizers (Patent Document 1), an ingenious calibration method is used in a stabilized composition using a stabilizer. As a result of the examination, it was found that GA can be measured more accurately with a single calibration material than with a conventional calibration using two calibration materials in a stable composition.
In addition, by simultaneously displaying the protein concentration and glycated protein concentration on this calibration substance, the same glycated protein ratio can always be obtained from the same sample even if the calibration substance is concentrated. When the protein is albumin, it has been found that by using a reagent containing bromocresol purple as the albumin reagent, the displayed value of the calibration substance can be accurately determined without being influenced by globulin or the like, and the present invention has been completed.

That is, the present invention is (1) a method for preparing a calibration curve using only one calibration substance in the measurement of glycated protein with an enzyme, wherein the enzyme-containing solution is refrigerated and calibrated daily for one week. A calibration curve generating method characterized in that the difference between calibrations is 1.5% or less.
(2) A method for preparing a calibration curve with only one calibration substance in the measurement of the proportion of glycated protein with an enzyme, between calibrations when the enzyme-containing solution is stored refrigerated and calibrated daily for one week. A method for preparing a calibration curve, wherein the difference is 1.5% or less.
(3) The method according to (1) or (2), wherein the residual activity of the enzyme after refrigerated storage of the enzyme-containing solution for 1 week is 90% or more.
(4) The method according to any one of (1) to (3), wherein the enzymes are protease and ketoamine oxidase.
(5) The method according to (4), comprising a protease and a stabilizer for ketoamine oxidase.
(6) The method according to any one of (1) to (5), wherein the glycated protein is glycated albumin or glycated hemoglobin.
(7) The method according to any one of (2) to (6), wherein, in the glycated protein ratio measurement method, the protein is albumin, and the method for measuring albumin is a method using bromocresol purple.
(8) A calibration curve preparation kit comprising at least the following a) to e):
a) Protease b) Ketoamine oxidase c) Protease stabilizer d) Ketoamine oxidase stabilizer e) Single point calibrator

The present invention will be specifically described below.
<2-point calibration substance>
The two inspection substances in the present invention are calibration substances having two glycated protein concentrations with different concentrations, and are usually two points of low concentration and high concentration. The calibration curve is a calibration curve obtained by multiple measurements of a substance having a glycated protein concentration of zero, such as water or physiological saline, and a low value and a high value of these substances, and linear regression using the least square method. On the other hand, in the case of using one inspection quantity substance, a substance having a glycated protein concentration of zero such as water or physiological saline and a calibration substance are subjected to multiple measurement, and a straight line connecting the two points is used as a calibration curve.
<Glycated hemoglobin, hemoglobin A1c>
A glycated protein obtained by non-enzymatic reaction of hemoglobin with glucose is called glycated hemoglobin. It is said that glycated hemoglobin is glycated at the α-chain and β-chain N-terminal valine and the ε-position of lysine in the molecule. Among glycated hemoglobins, hemoglobin β-chain N-terminal valine is glycated. Called A1C. The hemoglobin A1c does not include unstable hemoglobin that is the Schiff base before the Amadori transition.
The calibration substance in the present invention may be any substance as long as it contains a glycated protein. However, when the protein is albumin, the glycated protein is glycated albumin, and when the protein is hemoglobin. The glycated protein refers to glycated hemoglobin or hemoglobin A1c.

  Examples of calibrated substances containing glycated albumin include albumin solutions, solutions containing at least albumin, serum, plasma, whole blood, etc. Examples of calibrated substances containing glycated hemoglobin or hemoglobin A1c include hemoglobin solutions, solutions containing at least hemoglobin. , Whole blood, red blood cells, hemolyzed blood and the like. In addition, since these materials do not necessarily show high glycated protein concentration, glycated protein is purified from the blood of healthy subjects, etc. as necessary, the blood of hyperglycemic people is used, glycated protein by artificial glycation reaction of blood A glycated protein having a target concentration can be obtained using synthesis or the like. The glycated protein can be purified by a chromatographic method using boric acid or zinc-sugar chelate. An artificial saccharification reaction can be obtained by incubation with glucose, for example, at 37 degrees. Usually, it may be about 2 weeks, but the reaction may be continued for a longer time.

The calibration substance may be a pure glycated protein or a mixture thereof, or a glycated protein-containing substance containing a blood component, a non-glycated protein-containing substance containing a blood component, or a mixture thereof. Further, for example, it can be provided as a liquid product, a frozen product of the liquid product, or a freeze-dried product.
Furthermore, the composition of the calibration substance described in the present invention may be added with, for example, a substance that deteriorates the storage stability of the calibration substance and a substance that improves the storage stability. For example, substances that deteriorate the storage stability of a calibration substance include reducing sugars that cause a saccharification reaction, such as glucose, galactose, fructose, and the like, and blood coagulation proteins that cause turbidity, such as fibrinogen, which are removed as much as possible. It is preferable. To confirm the degree of removal, a commercially available measurement kit may be used to measure the content of a substance that deteriorates stability. The target for removal is preferably below the measurement limit with a commercially available kit, for example, 10 mg / dl or less for glucose and 40 mg / dl or less for fibrinogen. As a removal method, a reducing sugar typified by glucose may be a method of removing low molecules such as dialysis, low molecular removal using a molecular sieve, separation using chromatography, or the like. In order to remove proteins related to coagulation, such as fibrinogen, blood may be sufficiently coagulated or purified using a column method, a precipitation method, or the like.

Examples of substances that improve storage stability include non-reducing sugars such as glucitol, galactitol, mannitol, trehalose, sucrose, inositol, glycerol, and the like, and the addition of these substances is effective. There is no problem even if a stabilizer is added. For example, preservatives such as caisson and sodium azide are also considered to be substances that improve storage stability.
As the glycated protein and protein concentration of one standard substance that can be used in the present invention, the value measured before and after the most frequently measured value in the clinical field or larger than the most frequently measured value Is preferable, for example, when the protein is albumin, the most frequent albumin concentration is 4 to 5 g / dl, and the most frequent glycoalbumin concentration is 0.4 to 1.0 g / dl. Therefore, an albumin concentration of 3 to 7 g / dl and a glycoalbumin concentration of 0.3 to 2.0 g / dl are preferable.

On the other hand, for example, when the protein is hemoglobin, the most frequent hemoglobin concentration is 13 to 18 g / dl, and the most frequent hemoglobin A1c concentration is 0.65 to 1.0 g / dl. 20 g / dl and hemoglobin A1c concentration of 0.4 to 2.0 g / dl are preferable.
When a standard substance with a known concentration such as a protein (for example, albumin or hemoglobin) and a glycated protein (for example, hemoglobin A1c) is readily available, the standard substance, for example, CRM670, etc. The value may be transferred from the standard substance to the calibration substance using the reagent to be used. On the other hand, for glycated albumin that does not have a standard substance, the saccharification ratio of albumin is determined using a known physical glycated albumin measurement method (HPLC method; glycoalbumin meter; GAA2000, ARKRAY, Inc.). The concentration may be obtained and the glycated albumin concentration may be calculated from the value. Further, as shown in Non-Patent Document 1, the same result can be obtained by dividing the glycated amino acid concentration in albumin by the albumin concentration and further dividing by the average amount of sugar bonds in albumin (about 2). .

As a method for preparing a calibration curve using one calibration substance in the present invention, a blank substance such as one calibration substance and water or physiological saline is used. Multiple measurement is used, and a straight line connecting two points may be used as a calibration curve.
The difference between calibrations in the present invention refers to reproducibility when, for example, calibration is performed every day for a week and a sample is measured, and any method can be used as long as the reproducibility is statistically significantly reduced. May be used. In the present invention, the difference between calibrations is determined under conditions in which the enzyme-containing solution is stored refrigerated. Refrigeration usually means 4 ° C to 10 ° C, preferably 4 ° C. The enzyme-containing solution refers to a solution containing an enzyme used for measurement of glycated protein. For example, if a glycated protein is measured using a protease and ketoamine oxidase, a protease-containing solution or a ketoamine oxidase-containing solution is used. Say. The enzyme-containing solution may contain a stabilizer as exemplified below if necessary.

For example, the difference between calibrations is usually 1.5% or more when two calibrators are used, but a reagent whose sensitivity does not change for one week is used when only one calibrator is used. In the case, it is about 0 to 1.0%. More specifically, for example, the reproducibility when a sample is measured every day for one week is preferably 1.5% or less, more preferably 1.0% or less, and 0.7% or less. Most preferred.
In addition, as a stable reagent composition in the present invention, for example, the residual activity of the enzyme after 1 week of refrigeration is preferably 90% or more, more preferably 95% or more, and the activity is not substantially decreased. preferable.

The enzyme measurement method for glycated protein in the present invention may be any method as long as it uses an enzyme capable of measuring the glycated protein concentration. For example, well-studied protease and ketoamine oxidase The method using is preferable.
As the protease that can be used in the present invention, any protease can be used as long as it effectively acts on a glycated protein contained in a test solution and effectively produces a glycated amino acid and / or glycated peptide derived from the protein. For example, animals, plants, genus Bacillus, Aspergillus, Rhizopus, Penicillium, Streptomyces, Staphylococcus, Clostridium, Lysobacter Examples include proteases derived from microorganisms such as (Lysobacter), Glifila, Yeast, Tritirachium, Thermus, Pseudomonus, and Achromobacter.
In addition, when the glycated protein to be measured is glycoalbumin, microorganism-derived proteases of the genus Bacillus and Streptomyces are more preferable because they have a large effect on human albumin. Further, when the glycated protein to be measured is glycated hemoglobin or hemoglobin A1c, proteases derived from the genus Bacillus, Aspergillus, Streptomyces, or Trityratium are more preferable because they have a large effect on human hemoglobin.

The protease activity was measured using a known casein-forin method.
The enzyme that can be used for the glycated amino acid or glycated peptide produced by the treatment with the protease as described above is any enzyme that acts on the glycated amino acid or glycated peptide fragmented by the protease treatment that can be used in the present invention. Enzymes may be used, but oxidases are best studied and preferred as described above. However, kinases and dehydrogenases may be used and are not limited at all.
Specificity of the enzyme used to measure glycated amino acid or glycated peptide is that it works well on glycated amino acid or glycated peptide in which α-amino group is glycated, and glycated amino acid or glycated peptide in which ε-amino group is glycated An enzyme etc. are mentioned.
Among the oxidases that act on glycated amino acids or glycated peptides that have been fragmented by protease treatment, examples of enzymes that frequently act on glycated amino acids or glycated peptides in which the ε-amino group is glycated include Gibberella genus and Aspergillus (Aspergillus) And ketoamine oxidase derived from the genera Candida, Penicillium, Fusarium, Acremonium, or Debaryomyces.

An example of an enzyme that frequently acts on a glycated amino acid or glycated peptide in which the α-amino group is glycated is an enzyme derived from Corynebacterium.
Furthermore, examples of enzymes that have sufficient activity even in the presence of proteases and can be produced at low cost include genetically modified ketoamine oxidase (KAOD; manufactured by Asahi Kasei Pharma; described in Non-Patent Document 1) and In addition, a mutant FOD (KAOD-V; manufactured by Asahi Kasei Pharma Co., Ltd.) and a transformed microorganism JM109 / pcmFOD5 (FERM BP-7848) containing the FOD gene described in Patent Document 2 is produced that has significantly reduced glycated valine reactivity. KAOD).
The activity of ketoamine oxidase was measured by the following method.

<Composition of reaction solution>
50 mM Tris buffer pH 7.5
0.03% 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.)
0.02% Phenol (Wako Pure Chemical Industries)
4.5U / ml peroxidase (Sigma)
1.0 mM α-carbobenzoxy-ε-D-fructosyl-L-lysine or fructosyl valine (synthesized and purified based on the method of Hashiba et al. Hashiba H, J. Agric. Food Chem. 24:70, 1976 and below ZFL, (Abbreviated as FV)
1 ml of the above reaction solution is put in a small test tube, preheated at 37 ° C. for 5 minutes, 0.02 ml of an appropriately diluted enzyme solution is added and stirred, and the reaction is started. After the reaction for exactly 10 minutes, 2 ml of 0.5% SDS is added to stop the reaction, and the absorbance at a wavelength of 500 nm is measured (As). Further, the absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution as a blank (Ab). The enzyme activity is determined from the absorbance difference (As-Ab) between the absorbance (As) of this enzyme action and the absorbance (Ab) of the blind test. Separately, the relationship between the absorbance and the generated hydrogen peroxide was examined in advance using a standard solution of hydrogen peroxide, and the amount of enzyme that generates 1 μmol of hydrogen peroxide at 37 ° C.-1 minute is defined as 1 U.
In the present invention, refrigerated storage of an enzyme-containing solution for one week means that, if the enzyme is a lyophilized product, it is refrigerated for one week after preparation with a buffer solution, a solution or distilled water, and the enzyme is a liquid product. In the case of, it refers to refrigerated storage for 1 week after opening.

In general, the proteases and ketoamine oxidases are not stable in solution. Therefore, it can be stabilized in a solution by adding a protease, a ketoamine oxidase stabilizer and the like as required.
Examples of the protease stabilizer include dimethyl sulfoxide, alcohol, calcium, sodium chloride, quaternary ammonium salt or quaternary ammonium salt type cationic surfactant. Examples of alcohols include ethanol, propanol, ethylene glycol, glycerin and the like, and examples of quaternary ammonium salt quaternary ammonium salt type cationic surfactants include lauryl sulfate triethanolamine, lauryltrimethylammonium chloride, and the like. It is done.

These protease stabilizers may be used at any concentration as long as it is a substance that suppresses the decrease in protease activity during storage of the reagent. Particularly, the protease activity decreases during storage of the reagent in a liquid state. If it is the density | concentration which suppresses, it is preferable. Examples of preferable concentrations are usually 0.01% to 50%, preferably 0.1% to 40%, and other concentrations may be used.
As a stabilizer for ketoamine oxidase, any substance can be used as long as it suppresses the decrease in the activity of an enzyme that acts on glycated amino acids during storage of the reagent. In particular, at least saccharification is performed during storage of the reagent in a liquid state. Any substance that suppresses a decrease in the activity of an enzyme that acts on amino acids is preferable.

Examples of preferable stabilizers include sugar alcohol, sucrose, magnesium, calcium, ammonium sulfate, amino acid, and sarcosine. Examples of sugar alcohols include sorbitol, mannitol, trehalose, glycerin, etc., and as amino acids, all amino acids have strong stability or effect, but more preferred are proline, glutamic acid, alanine, valine, glycine, lysine, etc. It is done.
These ketoamine oxidase stabilizers may be used at any concentration as long as it is a substance that suppresses at least a decrease in the activity of the enzyme acting on the glycated amino acid during storage of the reagent. Therefore, it is preferable to use a concentration that suppresses at least a decrease in the activity of an enzyme that acts on glycated amino acids during storage. Examples of preferable concentrations include, for example, sugar alcohol, sucrose, amino acid, sarcosine, usually 0.01% to 30%, preferably 0.1% to 20%. Magnesium, calcium, ammonium sulfate is usually 1 mM. It may be used at a concentration of ˜1M, preferably 10 mM to 500 mM, and may be used at other concentrations.

As described above, as a calibration curve preparation kit for glycated proteins that can be used in the present invention, a glycated proteolytic reagent comprising a protease and its stabilizer, ketoamine oxidase and its stabilizer, and a single-point calibration substance Is included. If necessary, add coloring reagents (for example, peroxidase and Trinder type dyes or leuco type dyes), surfactants, metal salts, and enzymes that eliminate coexisting substances (for example, ascorbate oxidase and bilirubin oxidase). You can also .
The composition of the glycated proteolytic reagent is such that the pH, buffer and protease concentration are determined so that the proteolytic reaction proceeds efficiently, and then the protease stabilizer is appropriately prepared and added to the aforementioned effective concentration. That's fine.
For example, when protease type XXIV (manufactured by Sigma) is used, the proteolytic activity is strong at a pH of around 7 to 10, and the pH of the reaction can be selected from 7 to 10. As the buffer solution, for example, a POPSO buffer solution having a buffering action at pH 7.2 to 8.5 can be used, and the concentration of POPSO may be 1 to 100 mM, preferably 10 to 500 mM.
The protease addition concentration is sufficient if it can sufficiently decompose the glycated protein in the test solution during the actual reaction time, and is preferably 100 to 500,000 PU / ml, preferably 500 to 100,000 PU / ml. More preferred is ml. As the stabilizer, dimethyl sulfoxide may be added in an amount of 5 to 60%, preferably about 10 to 50%.

On the other hand, as the composition of the color reagent, the pH, buffer and ketoamine oxidase concentration are determined so that the glycated amino acid or peptide generated by the action of the glycated proteolytic reagent can be detected efficiently, and then the ketoamine oxidase stabilizer is added. What is necessary is just to adjust and add suitably so that it may become the above-mentioned effective density | concentration.
For example, when FOD (manufactured by Asahi Kasei Pharma) is used, the region showing an activity of 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the pH of the reaction can be selected from 6.5 to 10. Moreover, the enzyme addition concentration should just be a density | concentration which can fully detect glycated amino acid in the reaction liquid used, 0.5-200 U / ml is preferable and 1-50 U / ml is more preferable.

As a stabilizer for ketoamine oxidase, for example, glutamic acid can be used, and it may be used at a concentration of 0.01% to 30%, preferably 0.1% to 20%.
As a method for quantifying a glycated protein that can be used in the present invention, 0.001 to 0.5 ml of a test solution is added to 0.01 to 10 ml of the glycated proteolytic reagent, and the mixture is reacted at a temperature of 37 ° C. to perform first photometry (A0). Subsequently, 0.01 to 5 ml of a coloring reagent is added and reacted at a temperature of 37 ° C., and the amount of hydrogen peroxide or other products changed after a certain time from the start of the reaction may be measured. In this case, the amount of glycated protein in the test solution can be determined by comparing with changes in absorbance or the like when measured using a glycated protein at a known concentration.
As a method for measuring the glycated protein ratio in the present invention, it is necessary to separately measure the protein and calculate the glycated protein ratio in order to obtain the glycated protein ratio. The protein measurement method can be performed by, for example, separating the target protein from the sample using chromatography and quantifying the separated protein by a known method, or by directly measuring using an antibody or a coloring dye. Also good.

As a method of measuring albumin using a direct coloring dye, for example, bromocresol green (BCG), bromocresol purple (BCP), bromophenol blue, methyl orange, or 2- (4-hydroxybenzeneazo) benzoic acid ( Although a method using a dye such as HABA) is known, a method using bromocresol purple (BCP) is preferable from the viewpoint of specificity.
Examples of methods for measuring hemoglobin include the methemoglobin method, the cyan methemoglobin method, the azide methemoglobin method, the green chromophore formation method, and the oxyhemoglobin method. The green chromophore formation method is a method in which a green chromophore forming reagent and hemoglobin are reacted to form a stable product (green chromophore). The green chromophore is an alkaline hematin D described in British Patent Publication No. 2052056. It has an absorption spectrum similar to -575.

In order to measure the protein in a sample, when using, for example, HABA in a normal sample, for example, 0.01 μl to 10 ml, pH 3 to 10, preferably pH 4 to 9, 0.001% to 10%, preferably 0 It may be used at a concentration of 0.01% to 5%, and a change in absorbance at 480 to 550 nm may be measured. Similarly, when BCP is used, a surfactant that suppresses coloration at pH 4 to 8, preferably 4.5 to 7.5, such as Brij 35, is used in an amount of 0.01% to 5%, preferably 0.05%. It may be used at 0.0001 to 0.2%, preferably 0.0005 to 0.1% in the presence of ˜3%, and the change in absorbance near 600 nm may be measured.
In the case of measuring hemoglobin and using the oxyhemoglobin method, for example, the sample may be diluted with distilled water and converted to oxyhemoglobin, and then absorption near 540 nm may be measured. In order to prevent hemoglobin denaturation, a surfactant, for example, a surfactant having at least a sulfate group, and / or a nonionic surfactant, and / or an amphoteric surfactant is preferably 0.001 to 10%. It is better to add at a concentration of.

The present invention will be described based on examples.
[Example 1] Effect of 1-check quantity curve and 2-check quantity curve on measurement of glycated albumin Using glycated albumin measuring reagent (stabilizer +/-), the performance of 1-check quantity curve and 2-check quantity curve is determined as a reagent Comparison was made immediately after preparation and after 1 week of refrigerated storage.

<Reaction solution composition>
R-1 Proteolytic reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
2500U / ml protease type XXIV (manufactured by Sigma)
± 30% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd .; stabilizer)
R-2 glycated amino acid quantitative reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
0.12% 4-aminoantipyrine (Wako Pure Chemical Industries, Ltd.)
0.08% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS; manufactured by Doujin Chemical Laboratory)
24U / ml ketoamine oxidase (KAOD; manufactured by Asahi Kasei Pharma)
20 U / ml peroxidase (Sigma)
± 1% sorbitol (manufactured by Wako Pure Chemical Industries, Ltd .; stabilizer)
R-3 Albumin quantification reagent (method using bromocresol purple)
Aqua Auto Kinos ALB reagent (manufactured by Kinos) was used.
In order to compare albumin measuring methods, a method using bromocresol green and a Kinos Auto Series ALB reagent (manufactured by Kinos) were simultaneously measured.

<Sample>
1; Healthy person pool serum; 12.1%, patient pool serum 1; 25.7%, patient pool serum 2; 33.2% (GA% was measured by ARKRAY High Auto GAA2000)
<Calibrator>
Using a calibrator for Lucika GA, lot0202A L (GA concentration = 0.539g / dl, ALB concentration = 4.56g / dl, HGA concentration = 1.95g / dl; manufactured by Asahi Kasei Pharma) When used, a total of three points of water and two calibration substances were measured twice, and a calibration curve was obtained by the least square method. When one calibration substance was used, water and one calibration substance (using H) were measured twice to obtain a calibration curve.

<Reaction procedure>
8 μl of substrate solution (HSA substrate solution, GI substrate solution) was added to 240 μL of R-1 incubated at 37 ° C., the reaction was started at 37 ° C., and R-2; 80 μL was added exactly 5 minutes later. The absorbance at 546 nm before and after the addition of R-2 was measured, and the difference was defined as the change in absorbance. Moreover, the same operation was performed using distilled water instead of the sample to obtain a blank sample.

The glycated albumin concentration was determined by comparing ΔA obtained by subtracting the absorbance change of the blank sample from the absorbance change obtained from the sample with the calibration curve obtained from the calibration. The quantification of albumin using the BCP reagent was measured based on the dosage of the reagent.
The sample and the blank sample were calibrated using a stabilizer ± reagent, n = 3 was measured, and this was performed for one week to confirm the reproducibility of the calibration. Reagents were stored refrigerated (approximately 4 ° C.). The results are shown in Table 1.

表１から分かるように、安定化剤がない場合のキャリブレーション間差は、２点の検量用物質を用いる場合の方が１点の検量物質を用いる場合よりも小さく、健常者プール血清で1.85％であった。一方安定化剤がある場合のキャリブレーション間差は、１点の検量用物質を用いる場合の方が２点の検量物質を用いる場合よりも小さく0.59％であった。
冷蔵１週間保存後の酵素の残存活性は安定化剤がない場合、プロテアーゼ：85%、ケトアミンオキシダーゼ：88%、安定化剤がある場合プロテアーゼ：95%、ケトアミンオキシダーゼ：98%であった。

As can be seen from Table 1, the difference between calibrations without a stabilizer is smaller when using two calibration substances than when using one calibration substance, and 1.85 for pooled healthy subjects. %Met. On the other hand, the difference between calibrations in the presence of the stabilizer was 0.59% in the case of using one calibration substance, which was smaller than in the case of using two calibration substances.
The remaining activity of the enzyme after storage for 1 week in the refrigerator was protease: 85%, ketoamine oxidase: 88%, when stabilizer was present, protease: 95%, ketoamine oxidase: 98%. .

In other words, when the residual activity of the enzyme after 1 week of refrigeration is 90% or more, it is clear that the use of only one calibration substance has the smallest difference between calibrations and that glycoalbumin is accurately measured. Met.
In addition, it is known that a method for measuring albumin using bromocresol green, which is currently widely used, reacts with α-globulin. As a result of measuring the actual sample from the standard substance CRM470 using bromocresol green and bromocresol purple as the three calibrators, bromocresol purple had the same sample value regardless of which calibrator was used. However, when bromocresol green was used, the value was different for each calibrator, and the measured value of the specimen had a maximum difference of 0.2 g / dl. From this, it is apparent that the accuracy of measuring albumin when measuring glycoalbumin is better when bromocresol purple is used.

[Example 2] Effect of 1 inspection curve and 2 inspection curves on measurement of glycated hemoglobin
R-1 Proteolytic reagent
150 mM Tris buffer (Wako Pure Chemical Industries, Ltd.) pH 8.0
2500U / ml protease type XIV (manufactured by Sigma)
± 30% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd .; stabilizer)
2mM 2- (4-lodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, mono sodium salt (manufactured by Dojindo Laboratories)
R-2 glycated amino acid quantitative reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 8.5
24U / ml ketoamine oxidase (KAOD; manufactured by Asahi Kasei Pharma)
0.08mM N, N, N ', N', N '', N ''-Hexa (3-sulfopropyl) -4,4 ', 4''-triaminotriphenyl methane, hexasodium salt. (Dojindo Laboratories)
20 U / ml peroxidase (Sigma)
± 1% sorbitol (manufactured by Wako Pure Chemical Industries, Ltd .; stabilizer)
R-3 Hemoglobin quantitative reagent (Wako Reagent)

<Sample>
1; healthy controls; 4.69%, patient controls; 9.91%
<Calibrator>
JDS HbA1c Lot2 (manufactured by the Welfare and Medical Technology Promotion Association) was used as an actual sample standard substance (primary calibrator) for HbA1c measurement. When using two calibrators (level 2: 5.38% and level 4: 9.88%), measure water and two calibrators for a total of three points, and calibrate using the least squares method. Sought the line. In addition, when one calibration substance (level 3: 7.32%) was used, water and one calibration substance were measured twice to obtain a calibration curve.

<Reaction procedure>
24 μL of sample was added to R-1; 216 μL incubated at 37 degrees, the reaction was started at 37 ° C., and R-2; 54 μL was added exactly 5 minutes later. The absorbance at 546 nm before and after the addition of R-2 was measured, and the difference was defined as the change in absorbance. Moreover, the same operation was performed using distilled water instead of the sample to obtain a blank sample.

The glycated hemoglobin concentration was determined by comparing ΔA obtained by subtracting the absorbance change of the blank sample from the absorbance change obtained from the sample with a calibration curve obtained from the calibration. Hemoglobin quantification was measured based on the dosage of the reagent.
The sample and the blank sample were calibrated using a stabilizer ± reagent, n = 3 was measured, and this was performed for one week to confirm the reproducibility of the calibration. Reagents were stored at 4 ° C. The results are the same as for glycated albumin,
The difference between the calibrations without the stabilizer was smaller when using two calibration materials than when using one calibration material, which was 2.15% at the normal level. On the other hand, the difference between calibrations in the presence of the stabilizer was 0.87%, which was smaller when using one calibration substance than when using two calibration substances. The remaining activity of the enzyme after storage for 1 week in the refrigerator was protease: 88%, ketoamine oxidase: 85%, when stabilizer was present, protease: 97%, ketoamine oxidase: 97% .
That is, when the residual activity of the enzyme after 1 week of refrigeration is 90% or more, the use of one calibration substance has the smallest difference between calibrations, and glycated hemoglobin or HbA1c is accurately measured. It was obvious.

The measuring method of the present invention can be suitably used in the field of clinical examination, particularly in the field of measuring glycated protein using an enzyme.

Claims (8)

In the measurement of glycated protein with an enzyme, a calibration curve is prepared with only one calibration substance, and the difference between calibrations when the enzyme-containing solution is stored refrigerated and calibrated daily for one week is 1. A method for preparing a calibration curve, characterized in that it is 5% or less. In the measurement of glycated protein ratio with an enzyme, a calibration curve is prepared with only one calibration substance, and the difference between calibrations is 1 when the enzyme-containing solution is stored refrigerated and calibrated daily for one week. A method for creating a calibration curve, which is 5% or less. The method according to claim 1 or 2, wherein the residual activity of the enzyme after the enzyme-containing solution is refrigerated for 1 week is 90% or more. The method according to claims 1 to 3, wherein the enzyme is a protease and a ketoamine oxidase. The method according to claim 4, comprising a protease and a stabilizer for ketoamine oxidase. The method according to any one of claims 1 to 5, wherein the glycated protein is glycated albumin or glycated hemoglobin. 7. The method according to claim 2, wherein the protein is albumin and the method for measuring albumin is a method using bromocresol purple. A calibration curve creating kit comprising at least the following a) to e).
a) Protease b) Ketoamine oxidase c) Protease stabilizer d) Ketoamine oxidase stabilizer e) Single point calibrator