JPH0658936A - Method for measuring saccharifying protein - Google Patents

Abstract

PURPOSE:To make it possible to determine simply the relative ratio of the amount of saccharifying albumin to the amount of total albumin without measuring the amount of the saccharifying albumin and the amount of the total albumin discretely, for instance, and thus to measure saccharifying protein easily. CONSTITUTION:This is a method for measuring specific saccharifying protein by a sandwich method using a solidified antibody having a specific power of combination with the part of protein of saccharifying protein to be measured and a tagged compound, and the tagged compound is an enzyme-tagged dihydroxy boryl compound.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for measuring glycated protein. More specifically, for example, it relates to a method for measuring a glycated protein useful as a diagnostic marker for diabetes,
The present invention relates to a measurement method used in the clinical laboratory field and the like.

[0002]

BACKGROUND OF THE INVENTION It is known that albumin, which is a protein component in blood, and hemoglobin in red blood cells, react non-enzymatically with glucose and are saccharified to form a glycated protein. The amount of protein that undergoes this glycation is proportional to the amount of glucose in the body during the period in which the protein was present in the living body, so measuring the amount of glycated protein in the protein in the body is useful for diagnosing diabetes, etc. It is useful in the field of clinical examination.

A marker for measuring the amount of glycated protein is, for example, hemoglobin Alc (HbAlc).
Alternatively, fructosamine and the like can be mentioned. HbAlc
Is glucose bound to the N-terminal amino acid of the β subunit of hemoglobin and is one of the markers used in clinical tests. The amount of glycated protein using HbAlc is measured by a dedicated high performance liquid chromatograph (HPLC) using an ion exchange resin as a packing material (for example, Japanese Patent Laid-Open No. 63-298063). Since the HbAlc value by this measurement method is shown as a relative percentage of the HbAlc amount with respect to the total hemoglobin amount, the measured value is not affected by the hemoglobin amount contained in the test sample, but abnormal hemoglobin (eg, HbS) There is a problem that may be affected by.

Further, the assay method using fructosamine as a marker is a colorimetric method utilizing the reducing property of glycated protein, and the glycated protein contained in plasma or serum using an automatic measuring instrument for biochemical tests. It is a method of measuring quantity.
According to this measuring method, since the measured value of fructosamine is shown as the absolute amount of glycated protein, there is a problem that the measured value is affected by the amount of protein contained in the sample.

[0005] As another measuring method, a method for measuring glycated protein using HPLC utilizing the boronic acid affinity method is known. The boronic acid affinity method is
This is a method that utilizes the formation of a reversible conjugate of a boronic acid with a substance having a cis-diol group under weak alkaline conditions. For example, aminophenylboronic acid is used as a packing material for chromatography in cellulose or polyacrylamide resin. An example in which it is bound to is reported. Boronic acid becomes a boronic acid anion under weak alkaline conditions and forms a stable bond with a substance having a cis-diol group. This conjugate is reversible and dissociates when the pH is adjusted to the acidic side.
By utilizing this property and using boronic acid as a filler, adsorption / desorption of a substance having a cis-diol group to the filler can be controlled only by changing the pH.

H using the boronic acid affinity method
Regarding the measurement of glycated albumin using PLC, although a dedicated measuring instrument thereof has not yet spread in the industry, according to Japanese Patent Laid-Open No. 2-96657, the glycated albumin value according to this measurement method is included in a plasma or serum sample. It is described that among the albumin present, the amount of saccharified albumin is shown as a relative percentage with respect to the total amount of albumin. Therefore, it is considered to be very useful in the clinical laboratory field such as the diagnosis of diabetes since there is no influence on the measured value due to the fluctuation of the amount of protein in the sample, which is a problem in the measurement method using fructosamine as a marker. .

That is, as a method for measuring glycated albumin by the above-mentioned HPLC using the boronic acid affinity method, the publication discloses that albumin in a sample is separated from other proteins in a first column, and then only albumin is added to a second sample. A method for introducing a column (boronic acid affinity column) to separate glycated albumin and non-glycated albumin is described. In addition, as another method for measuring glycated albumin by HPLC, on pages 34b and 35b in Clinical Chemistry, Volume 20, Supplement 2, (1991), the sample is separated into a glycated component and a non-glycated component by a boronic acid affinity column. After that, a method for detecting albumin by a post-column method using a dye (bromcresol purple) that specifically binds to albumin alone is described.

[0008] As another assay method, a method for detecting and assaying a glycosylated protein using a specific antibody against the glycosylated protein has been developed, and a kit for assaying a glycosylated albumin by the ELISA method is available in the US EXOCELL, IN.
C. It is sold by the company under the product name GLYCABEN. The ELISA method here is a sandwich method of a solid-phased anti-human glycated albumin monoclonal antibody and an enzyme-labeled anti-human albumin polyclonal antibody. In this kit, the amount of glycated albumin in the sample is measured separately by the ELISA method and the amount of total albumin is measured by the BCG (Bromcresol Green) method, and the measured value is expressed as the ratio (percent) of the glycated albumin amount to the total albumin amount. It has become.

However, since the measurement by the above-mentioned HPLC method takes a long measurement time per sample, there is a problem that a large number of samples cannot be measured at the same time. In addition, the ELI
In the measurement using the measurement kit by the SA method, it is necessary to separately measure the glycated albumin amount and the total albumin amount in order to measure the abundance ratio of the glycated albumin amount to the total albumin amount, which is very troublesome. It is a thing.
Further, in the measurement using the measurement kit by the ELISA method, a monoclonal antibody that recognizes the glycation site of albumin is used as the immobilized anti-human glycated albumin antibody, but human albumin has four glycation sites. Therefore, it has a drawback that the amount of glycated albumin cannot be accurately measured with only one kind of monoclonal antibody. As described above, the measurement of glycated albumin using a monoclonal antibody against glycated albumin is excellent in specificity because it utilizes an antigen-antibody reaction,
There is a problem that it is difficult to produce an antibody that can recognize all glycated albumin.

Further, as another immunological assay method, Japanese Patent Laid-Open No. 62-100660 discloses that an adsorbent having a substrate specificity / affinity such as phenylboronic acid which specifically adsorbs a glycated protein is bound. A method of specifically measuring only a specific glycated protein by a method using a labeled antibody having a specific affinity only for the solid phase thus prepared and a specific protein is described. This method is an excellent method in that the abundance of a specific glycated protein can be specifically measured, but since various glycated proteins bind to phenylboronic acid,
There is a problem that it is essential to measure the total amount of the specific protein separately in order to measure the glycation ratio of the specific protein which has clinical significance.

As another immunological measuring method, in JP-A-64-16964, a solid-phased antibody having an antibody against a specific protein immobilized on a carrier is reacted with a test solution. Then, the free sugar on the solid phase is removed, the glycated portion of the glycated protein is reduced to a reduced form using a reducing agent, and the labeled anti-reduced glycated protein antibody is reacted at the time of the reduction reaction or after the reduction reaction, A method is described in which the solid phase and the liquid phase are separated, the labeled amount of either phase is measured, and the specific glycated protein content is measured from the measured value. In this method, the same immobilized antibody can be prepared by using a test solution that can contain the specific protein at a concentration higher than the concentration at which the specific protein can bind to all of the predetermined amount of the antibody immobilized on the solid phase. Since the amount of the specific protein bound is always constant, it is an excellent method in that it can save the labor of measuring the total amount of the specific protein, but it requires a reduction step, which complicates the operation and Although a reduced glycosylated protein antibody is required, there is a problem that it is not easy to produce an antibody that does not differ between lots.

Therefore, an object of the present invention is to provide a method for measuring glycated protein, which can easily measure the abundance ratio of glycated protein.

[0013]

The present inventors have arrived at the present invention as a result of extensive studies to solve the above-mentioned problems. That is, the gist of the present invention is a method for measuring a specific glycated protein by a sandwich method using a solid-phased antibody having a specific binding ability to a protein portion of a glycated protein to be measured and a labeled compound. The present invention also relates to a method for measuring a glycated protein, wherein the labeling compound is an enzyme-labeled dihydroxyboryl compound. Specific modes include the following modes.

(1) A method for measuring a glycated protein having the following steps, a solid-phased antibody having a specific binding ability to a protein portion of a glycated protein to be measured is reacted with a glycated protein in a sample, Step of forming a solid-phased antibody-glycated protein complex, reacting an enzyme-labeled dihydroxyboryl compound with the solid-phased antibody-glycated protein complex, and through the solid-phased antibody-glycated protein on a solid phase The step of binding the enzyme-labeled dihydroxyboryl compound, and the step of measuring the enzyme activity on the solid phase after washing, (2) the measurement of glycated protein, which further comprises a washing step between the steps described in (1) above Method, (3) Method for measuring glycated protein having the following steps, enzyme-labeled dihydroxyboryl compound and glycated protein in a sample are reacted to form enzyme-labeled dihydroxyboryl compound-glycated protein complex And the step of reacting the enzyme-labeled dihydroxyboryl compound-glycated protein complex with a solid-phased antibody having a specific binding ability to the protein portion of the glycated protein to be measured, and then solid-phased onto the solid phase. Step of binding the enzyme-labeled dihydroxyboryl compound via antibody-glycated protein, and step of measuring enzyme activity on the solid phase after washing, (4) Specific to protein portion of glycated protein to be measured The immobilized antibody having binding ability, the glycated protein in the sample and the enzyme-labeled dihydroxyboryl compound are reacted substantially simultaneously to form a complex of the immobilized antibody-glycated protein-enzyme-labeled dihydroxyboryl compound, and then A method for measuring glycated protein, which comprises measuring the enzyme activity on a solid phase after washing.

The assay method of the present invention comprises a specific glycated protein by the sandwich method using a solid-phased antibody having a specific binding ability to the protein portion of the glycated protein to be measured and a labeling compound as described above. The sandwich method in the present invention is a sandwich method in which a protein having a specific binding ability to a protein portion of a glycated protein to be measured is sandwiched between an immobilized antibody and a labeled compound. It is a method of measuring the abundance of the protein to be measured using the label of the labeled compound by forming a complex of immobilized antibody-protein to be measured-labeled compound by sandwiching the two.

The method for measuring a glycated protein of the present invention will be described below in more detail with reference to a case where the protein to be measured is glycated albumin, and the method will be described in more detail. However, glycated hemoglobin and the like are also subject to the measurement method of the present invention. Further, the sample having a glycated protein to be measured may be derived from a living body containing the glycated protein as described above, and examples thereof include urine, blood and various organ extracts.

The principle of measurement according to the present invention will be described below. (1) By reacting a sample with an antibody having a specific binding ability to the protein portion of glycated albumin immobilized on a plate or the like, the albumin in the sample and the protein portion of glycated albumin are A solid-phased antibody-albumin complex and a solid-phased antibody-glycated albumin complex bound to the solid-phased antibody are formed. (2) The components in the sample not bound to the immobilized antibody are removed by suction to wash the solid phase. (3) An excess amount of the enzyme-labeled dihydroxyboryl compound with respect to the bound glycated albumin is added to the solid phase obtained by the washing step of (2), and the solid-phased antibody-glycated albumin complex is mixed with a weak alkaline solution. React under conditions. In this reaction, the cis-diol group in glycated albumin bound to the solid phase is bound to the boronic acid anion of the enzyme-labeled dihydroxyboryl compound. On the other hand, the solid-phased antibody-albumin complex does not have a cis-diol group and therefore exists without any reaction with the enzyme-labeled dihydroxyboryl compound. (4) The free enzyme-labeled dihydroxyboryl compound that did not participate in the reaction is removed by suction, the solid phase is washed, and then a substrate solution is added to develop color, and the enzyme activity is measured. (5) A calibration curve is prepared from the relationship between the amount of glycated albumin obtained by the measurement of the standard sample and the amount of color development, and this is used to calculate the amount of glycated albumin in the measurement sample.

Each step of the above measuring method of the present invention will be described in more detail below. The antibody used as the solid-phased antibody in the step (1) has an ability to specifically bind to the protein portion of glycated albumin. Therefore, it may have a specific binding ability to non-glycated albumin itself, but may be a monoclonal antibody or a polyclonal antibody as long as the binding ability is not different between glycated albumin and non-glycated albumin. Among them, a monoclonal antibody is preferable from the viewpoint of ensuring higher reproducibility of measurement results, and in that case, a monoclonal antibody having an antigenic determinant at a position sterically separated from a lysine residue to which glucose binds to albumin. However, it is more preferable to obtain an antibody having no difference in specific binding ability between glycated albumin and non-glycated albumin.

The solid phase on which such an antibody is immobilized does not contain a cis-diol group, and it is sufficient that each step in the present measuring method can be carried out. The material thereof is mainly inorganic substance powder or synthetic polymer. Examples of organic substances include: Examples of the inorganic substance powder include glass, silica or alumina, or metal pieces such as gold, titanium, iron or nickel. In addition, as the shape, flakes, beads,
Either a tubular shape or a plate-like shape obtained by molding a resin can be used. A microplate is particularly preferable from the viewpoint of automating each step in the measurement. In order to bind the antibody to the solid phase, a known method for binding the protein to the solid phase can be appropriately used, and either physical adsorption or chemical binding can be used. Since the antibody used has a sugar chain, the sugar chain is previously removed from the Fc fragment by enzymatic treatment with glycopeptidase (derived from almond) or N-glycanase, or treated with pepsin to form F (ab ') ₂ . The antibody may be bound to the solid phase after removing the influence of the sugar chain derived from the antibody by a known method such as removing the Fc fragment having a sugar chain portion.

In the method of the present invention, when calculating the abundance ratio of glycated albumin and non-glycated albumin, it is necessary to keep the amount of antibody immobilized on the solid phase constant.
That is, by keeping the total sum of albumin and glycated albumin bound to the immobilized antibody constant, the calculated value of glycated albumin by the calibration curve in the step (5) described later is calculated as the relative amount of glycated albumin to the total albumin amount. It can be a ratio. From this point of view, it is preferable to use a blocking agent after immobilizing a predetermined amount of the antibody on the solid phase so that the excess antibody is not immobilized on the solid phase. Examples of such a blocking agent include bovine serum albumin (BSA), casein, and a surfactant. When the blocking agent has a cis-diol group, the background becomes large.
Boronic acid affinity column chromatography method,
It is desirable to remove the glycated component from the components of these blocking agents by a known method such as a method for removing a sugar chain with an enzyme in order to improve the measurement sensitivity. However,
Even if the blocking agent has a cis-diol group, it is possible to measure the amount of glycated albumin as long as the amount of the cis-diol group in the components of the blocking agent is constant in each lot (measurement batch). is there.

In the step (1), the amount of immobilized antibody is kept constant, and a sample containing sufficient amounts of albumin and glycated albumin to react with all antibodies of the immobilized antibody is immobilized on the solid phase. The total amount of bound albumin and glycated albumin can be made to be the same in each measurement batch by reacting with the conjugated antibody. Further, as the solid-phased antibody, as described above, by using an antibody that has no difference in specificity for glycated albumin and specificity for non-glycated albumin as described above, the solid-phased antibody after reaction with the sample The abundance ratio of the albumin complex and the immobilized antibody-glycated albumin complex can be regarded as the abundance ratio of glycated albumin and non-glycated albumin in the sample.

The washing solution used in the washing step (2) is preferably a buffer solution such as physiological saline, phosphoric acid or Tris-hydrochloric acid. In addition, these buffers and the like may contain a surfactant and the like. This step can be omitted because it can be replaced by the washing step (4).

Examples of the enzyme-labeled dihydroxyboryl compound used in step (3) include enzyme-labeled substances such as aminophenylboronic acid, and aminophenylboronic acid enzyme-labeled substances are particularly preferable. For enzymatic labeling of dihydroxyboryl compounds, known labeling methods such as the two-step glutaraldehyde method, maleimide method and pyridyl disulfide method (Eiji Ishikawa et al., Enzyme Immunoassay 3rd Edition, Medical Shoin (1987)) are used. It is possible to easily obtain an enzyme-labeled dihydroxyboryl compound by a cross-linking reaction between an amino group in the dihydroxyboryl compound and a functional group in the enzyme (for example, an amino group or a thiol group) using The maleimide method is preferably selected in order to obtain yield and high reproducibility.

The labeling enzyme for the dihydroxyboryl compound does not contain a cis-diol group, has a functional group such as an amino group or a thiol group used for the binding reaction with the dihydroxyboryl compound, and is subjected to an enzyme-substrate reaction. There is no particular limitation as long as it exhibits color developability. The cis-diol group in glycated albumin and the boronic acid anion of the enzyme-labeled dihydroxyboryl compound are
Since it binds under a weak alkali having a pH of about 7.5 to 9.5, it does not contain a cis-diol group and has a pH of 7.5 to 9.5 for the purpose of reducing the kinds of reaction reagents and simplifying the process.
It is desirable to select an enzyme that allows the enzymatic reaction to proceed under a weak alkali of about 9.5. For example, β-D-galactosidase, alkaline phosphatase and the like can be mentioned, and β-D-galactosidase is particularly preferable from the viewpoint of the optimum pH of the enzyme. When using an enzyme that undergoes an enzymatic reaction under pH conditions other than under weak alkali, bind glycated albumin and the enzyme-labeled dihydroxyboryl compound under weak alkali,
After the excess free enzyme-labeled dihydroxyboryl compound is removed by suction in the step (4) and the solid phase is washed, the pH may be adjusted to a pH condition suitable for the enzyme-substrate reaction of the enzyme used. The reaction between the enzyme-labeled dihydroxyboryl compound and the immobilized antibody-glycated albumin complex is performed at pH 7.5 to
The reaction temperature is 20 to 40 ° C. under 9.5 and the reaction time is 30 minutes to 2 hours. As a result, the enzyme-labeled dihydroxyboryl compound can be bound to the solid phase via the immobilized antibody-glycated protein.

The substrate solution used in the step (4) is (3)
This is a solution containing a substrate for the labeling enzyme of the enzyme-labeled dihydroxyboryl compound used in the step of. As the washing liquid used in this washing step, Tris-hydrochloric acid buffer solution or the like is preferable as in the step (2).

In the step (5), the amount of glycated albumin obtained by calculation based on the calibration curve obtained from the relationship between the amount of glycated albumin obtained by measurement of the standard sample and the amount of color development (absorbance) is an absolute amount. , A sample that reacts with an antibody if the amount of immobilized antibody is fixed in advance and brought into contact with an excess sample (standard sample or measurement sample)
Since the amount of albumin in the sample is constant, the absolute amount of glycated albumin (horizontal axis) is replaced by the relative ratio (percent, horizontal axis) of glycated albumin to total albumin in the process of calculating from the calibration curve. It can be a value (percent). That is, the ratio of the glycated albumin amount to the total albumin amount can be measured at one time.

In the measuring method of the present invention, the step (1) (the step of forming a solid-phased antibody-albumin complex, the solid-phased antibody-glycated albumin complex) and the step (3). The order of the steps of (the step of binding the glycated albumin and the enzyme-labeled dihydroxyboryl compound) may be reversed. That is, first, (a) the glycated albumin in the sample and the enzyme-labeled dihydroxyboryl compound are reacted and bonded to each other to form an enzyme-labeled dihydroxyboryl compound-glycated protein complex. In this case, non-glycated non-glycated albumin is mixed as it is without reacting with the enzyme-labeled dihydroxyboryl compound. In addition,
In this step (a), the reaction for binding the glycated albumin and the enzyme-labeled dihydroxyboryl compound can be carried out under the same reaction conditions as in the case of the step (3). Next, (b) the immobilized antibody having a specific binding ability to the protein portion of the glycated protein to be measured is reacted with the enzyme-labeled dihydroxyboryl compound-glycated protein complex, and The enzyme-labeled dihydroxyboryl compound is bound via the immobilized antibody-glycated protein. As the solid-phased antibody used here, those having no difference in specificity for glycated albumin and specificity for non-glycated albumin as described above are used, and therefore, depending on the abundance ratio of glycated albumin. The amount binds to the immobilized antibody. As a result, the enzyme-labeled dihydroxyboryl compound can be bound to the solid phase via the immobilized antibody-glycated protein. Next, (c) a free enzyme-labeled dihydroxyboryl compound or an enzyme-labeled dihydroxyboryl compound not bound to the solid phase-
The glycated protein complex is removed and the solid phase is washed, and (d) enzyme activity on the solid phase is measured in the same manner as in the above method. According to this method, between the step (a) of binding the glycated albumin and the enzyme-labeled dihydroxyboryl compound and the step (b) of forming the immobilized antibody-albumin complex and the immobilized antibody-glycated albumin complex. Can be said to be a simple method in that a washing step is unnecessary.

Further, in the present invention, it is also possible to carry out the steps (4) and (5) by carrying out the steps (1) and (3) substantially at the same time. That is, the immobilized antibody having specific binding ability to the protein portion of the glycated protein to be measured, the glycated protein in the sample and the enzyme-labeled dihydroxyboryl compound are reacted substantially at the same time to give the immobilized antibody- A glycosylated protein-enzyme-labeled dihydroxyboryl compound complex is formed, and after washing, the enzyme activity on the solid phase is measured. In this case, the reaction for binding the immobilized antibody, glycated albumin and enzyme-labeled dihydroxyboryl compound is carried out at a reaction temperature of 20 to 20 at pH 7.5 to 9.5.
The reaction time is 40 ° C. and the reaction time is 30 minutes to 2 hours.

[0029]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. The following reagents and samples were used in this example. (1) Preparation of PBS (phosphate buffer solution): Monosodium phosphate (dihydrate), disodium phosphate (dihydrate) and sodium chloride were added so that the final concentrations of phosphoric acid and sodium chloride were 0. It was prepared by adding purified water so as to have a concentration of 02M and 0.15M and a pH of 7.2. (2) Preparation of 1% sodium caseinate-PBS: the above P
1 casein sodium (for Wako Pure Chemical) for BS
It melt | dissolved so that it might become% (W / V). (3) Preparation of measurement sample: Blood collected from 5 healthy subjects and 5 diabetic patients was left at room temperature for 60 minutes, and then 3
After centrifugation at 000 rpm for 10 minutes, the supernatant (serum) was used as a measurement sample. In Tables 1 to 4, 1 to 5 are healthy individuals, and 6 to 10 are samples obtained from diabetic patients. (4) Preparation of standard sample: Human serum albumin (HSA, manufactured by Sigma) and glucose in vitro, distilled water 1
L was added so that 40 g of HSA and 18 g of glucose were added, and the mixture was incubated (37 ° C., 7 days). Then, by using boronic acid affinity column chromatography (manufactured by Isolab), glycated albumin and non-glycated albumin are separated and separated, and the glycation ratio is 0.
After mixing them so as to be 0, 10.0, 20.0, 40.0%, physiological saline was used to prepare a total albumin concentration of 4 g / dl, which was used as a standard sample.

Experimental Example 1 Immobilization of anti-human albumin antibody (polyclonal antibody) on a microtiter plate An anti-human albumin antibody IgG fraction was used as an antibody capable of specifically binding to the protein portion of glycated albumin. Immobilization of this antibody on the microtiter plate was performed by physical adsorption. That is, the anti-human albumin polyclonal antibody IgG fraction (goat-derived, manufactured by Cosmo Bio Inc.) was dispersed in PBS at 30 μg / ml, and 100 μl of this solution was added to each of 96 96-well microtiter plates (manufactured by Falcon). Added to wells and left overnight at 4 ° C. In order to examine the amount of adsorption in each well, 50 μl of the supernatant of one plate was left after standing overnight, and the amount of protein (IgG) in the supernatant was measured with a protein quantification kit of Pierce.
Adsorbed IgG amount (added IgG amount-supernatant IgG amount) is 8 on average
2 ng (average value for 96 wells; coefficient of variation is 4.
1%), and it was considered that each well was almost uniformly adsorbed. The other plate was washed with physiological saline three times after removing the IgG solution with an aspirator. Then 200% 1% sodium caseinate-PBS to each well.
μl was added and blocking was performed at 37 ° C. for 2 hours. Then, it was washed with physiological saline three times to prepare an anti-human albumin antibody-immobilized microtiter plate.

Experimental Example 2 Immobilization of anti-human albumin antibody (monoclonal antibody) on a microtiter plate An anti-human albumin antibody IgG fraction was used as an antibody having a specific binding ability to the protein portion of glycated albumin. Immobilization of this antibody on the microtiter plate was performed by physical adsorption. That is, the anti-human albumin monoclonal antibody IgG fraction (mouse-derived, subclass IgG1, manufactured by Cosmo Bio Inc.) was dispersed in PBS at 30 μg / ml, and 100 μl of this solution was added to 96
Two wells of a microtiter plate (manufactured by Falcon) were added to each well and left overnight at 4 ° C. In order to check the amount of adsorption in each well, leave it overnight and then 50 μl of the supernatant of one plate
1 was taken and the amount of protein (IgG) in the supernatant was measured with a protein quantification kit of Pierce. The adsorbed IgG amount (added IgG amount-supernatant IgG amount) was 68 ng on average (average value for 96 wells, coefficient of variation was 3.6%), and it was considered that each well was adsorbed almost uniformly. . The other plate was washed with physiological saline three times after removing the IgG solution with an aspirator. Then 1% sodium caseinate-
200 μl of PBS was added to each well and blocking was performed at 37 ° C. for 2 hours. Then, it was washed three times with physiological saline to prepare an anti-human albumin antibody-immobilized microtiter plate.

Experimental Example 3 Preparation of enzyme-labeled dihydroxyboryl compound 9.5 mg (70 μmol) of aminophenylboronic acid and 21 mg (70 μmo) of N-succinimidyl 6-maleimidohexanoate as a maleimide group-introducing reagent.
l) is a phosphate buffer adjusted to pH 7.0 with 0.1 mo
Incubation was performed at 30 ° C. for 1 hour in 100 ml of 1 / l. Then, the precipitate was removed by centrifugation, and the product introduced with the maleimide group was purified by gel filtration chromatography. Next, 0.5 mg (1.5 μ) of the purified product
mol) to β-galactosidase 300 mg (550 n)
mol) or alkaline phosphatase 60 mg (5
50 nmol) and 0.1 mol / l, 10 ml of phosphate buffer adjusted to pH 6.0 at 30 ° C.
Incubation was carried out for an hour. The obtained reaction product was treated by gel filtration chromatography to remove unreacted maleimide group-introduced aminophenylboronic acid and side reaction products such as β-galactosidase or a polymer of alkaline phosphatase. further,
By removing unreacted β-galactosidase or alkaline phosphatase by boronic acid affinity chromatography using dextran gel as a packing material, β-galactosidase-bound dihydroxyboryl compound was 11 mg / ml (about 20 nmol / ml).
Alternatively, 100 mM containing 100 mM sodium chloride containing 2 mg / ml (about 20 nmol / ml) of an alkaline phosphatase-conjugated dihydroxyboryl compound.
20 ml of Tris-hydrochloric acid buffer solution (pH 8.0) was prepared.

Example 1 Measurement of Glycated Albumin Level in Human Serum Using Anti-Human Albumin Antibody (Polyclonal Antibody) and β-Galactosidase-Binding Dihydroxyboryl Compound Each well of the anti-human albumin antibody-binding microplate obtained in Experimental Example 1 5 standard samples or 5 samples each
0 μl was weighed in and incubated at room temperature for 60 minutes.
After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer containing 100 mM sodium chloride (pH
8.0) Each well was washed 3 times with 300 μl to remove unreacted albumin and glycated albumin in the sample.
Then, 200 μl of the β-galactosidase-bonded dihydroxyboryl compound solution obtained in Experimental Example 3 was weighed in and further incubated at room temperature for 120 minutes. After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride.
Each well was washed 3 times with 300 μl, and the washing solution was removed from each well, and then 100 mM sodium chloride, 1 mM magnesium chloride, for measuring β-galactosidase activity.
100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 2 mM o-nitrophenyl-β-D-galactoside
50 μl was weighed in and incubated for 60 minutes at room temperature. 1N sodium hydroxide solution 2 to stop the reaction
00 μl was mixed and the absorbance was measured at a wavelength of 420 nm. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample. The results are shown in Table 1.

[0034]

[Table 1]

Example 2 Measurement of Glycated Albumin Level in Human Serum Using Anti-Human Albumin Antibody (Monoclonal Antibody) and β-Galactosidase-Binding Dihydroxyboryl Compound Each well of the anti-human albumin antibody-binding microplate obtained in Experimental Example 2 5 standard and 5 measurement samples each
0 μl was weighed in and incubated at room temperature for 60 minutes.
After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer containing 100 mM sodium chloride (pH
8.0) Each well was washed 3 times with 300 μl to remove unreacted albumin and glycated albumin in the sample.
Then, 200 μl of the β-galactosidase-bonded dihydroxyboryl compound solution obtained in Experimental Example 3 was weighed in and further incubated at room temperature for 120 minutes. After removing the reaction solution from each well, 100 mM Tris-HCl buffer (pH 8.0) containing 100 mM sodium chloride
Each well was washed 3 times with 300 μl, and the washing solution was removed from each well, and then 100 mM sodium chloride, 1 mM magnesium chloride, for measuring β-galactosidase activity.
100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 2 mM o-nitrophenyl-β-D-galactoside
50 μl was weighed in and incubated for 60 minutes at room temperature. 1N sodium hydroxide solution 2 to stop the reaction
00 μl was mixed and the absorbance was measured at a wavelength of 420 nm. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample. The results are shown in Table 2.

[0036]

[Table 2]

Example 3 Measurement of Glycated Albumin Level in Human Serum Using Anti-Human Albumin Antibody (Polyclonal Antibody) and Alkaline Phosphatase-Binding Dihydroxyboryl Compound In each well of the anti-human albumin antibody-binding microplate obtained in Experimental Example 1 Standard sample and measurement sample 5 each
0 μl was weighed in and incubated at room temperature for 60 minutes.
After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer containing 100 mM sodium chloride (pH
8.0) Each well was washed 3 times with 300 μl to remove unreacted albumin and glycated albumin in the sample.
Then, 200 μl of the alkaline phosphatase-bonded dihydroxyboryl compound solution obtained in Experimental Example 3 was weighed in and further incubated at room temperature for 120 minutes. After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer solution (pH 8.
0) Each well was washed 3 times with 300 μl, and the washing solution was removed from each well, and then 1 mM containing 100 mM sodium chloride, 1 mM magnesium chloride, 2 mM p-nitrophenyl phosphate for measuring alkaline phosphatase activity.
50 μl of 00 mM Tris-HCl buffer (pH 8.0) was weighed in and incubated at room temperature for 60 minutes. To stop the reaction, 200 μl of 1N sodium hydroxide solution is mixed and the absorbance is measured at a wavelength of 410 nm. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample. The results are shown in Table 3.

[0038]

[Table 3]

Example 4 Measurement of Glycated Albumin Level in Human Serum Using Anti-Human Albumin Antibody (Monoclonal Antibody) and Alkaline Phosphatase-Binding Dihydroxyboryl Compound In each well of the anti-human albumin antibody-binding microplate obtained in Experimental Example 2 Standard sample and measurement sample 5 each
0 μl was weighed in and incubated at room temperature for 60 minutes.
After removing the reaction solution from each well, 100 mM Tris-hydrochloric acid buffer containing 100 mM sodium chloride (pH
8.0) Each well was washed 3 times with 300 μl to remove unreacted albumin and glycated albumin in the sample.
Then, 200 μl of the alkaline phosphatase-bonded dihydroxyboryl compound solution was weighed in, and further 120
Incubated for minutes. After removing the reaction solution from each well, each well was washed three times with 300 μl of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride, and after removing the wash solution from each well, the alkaline phosphatase activity was measured. For this purpose, 50 μl of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride, 1 mM magnesium chloride, 2 mM p-nitrophenyl phosphate was measured and incubated at room temperature for 60 minutes. To stop the reaction, 200 μl of 1N sodium hydroxide solution was mixed, and the absorbance was measured at a wavelength of 410 nm. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample. The results are shown in Table 4.

[0040]

[Table 4]

[0041]

According to the measuring method of the present invention, in the measurement of glycated protein, for example, the relative ratio of the glycated albumin amount to the total albumin amount can be simply determined without separately measuring the glycated albumin amount and the total albumin amount. Therefore, it becomes possible to easily measure glycated proteins.

Claims (3)

[Claims] 1. A method for measuring a specific glycated protein by a sandwich method using a solid-phased antibody having a specific binding ability to a protein portion of a glycated protein to be measured and a labeling compound, the method comprising: A method for measuring a glycated protein, wherein the compound is an enzyme-labeled dihydroxyboryl compound. 2. A method for measuring a glycated protein, which comprises the following steps. A step of reacting a solid-phased antibody having specific binding ability with a protein portion of a glycated protein to be measured with a glycated protein in a sample to form a solid-phased antibody-glycated protein complex, enzyme-labeled dihydroxy A step of reacting the boryl compound with the immobilized antibody-glycated protein complex to bind the enzyme-labeled dihydroxyboryl compound on the solid phase via the immobilized antibody-glycated protein, and after washing, solid phase Measuring the above enzyme activity. 3. The measuring method according to claim 2, further comprising a washing step between the steps according to claim 2.