JPH0783922A - Method for measuring saccharification protein - Google Patents

Abstract

PURPOSE:To easily measure the presence of saccharification protein of a sample by measuring a specific saccharification protein by the sandwich method using a solid-phase antibody with a specifical combination capacity for the protein part of saccharification protein and enzyme labeled lectin. CONSTITUTION:For example, an antibody with a specifical combination capacity for the protein part of saccharification albumin is subjected to solid phase to a plate, etc. The solid-phase antibody and a sample are reacted to form a solid-phase antibody - albumin composite and solid-phase antibody saccharification albumin composite where the protein part of albumin and saccharification albumin within the sample are combined with the solid-phase antibody. The constituents within the sample which are not combined to the solid-phase antibody are eliminated by suction and washing. An excessive amount of enzyme labeled lectin is added and reacted with the saccharification albumin which is not combined with the solid-phase antibody. After surplus lectin is sucked and eliminated and the solid phase is washed, substrate liquid is added for allowing saccharification albumin to develop color, and then enzyme activity is measured by a spectrophotometer. Then, the amount of saccharification albumin within the sample is calculated by a calibration curve.

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 field of clinical examination.

[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.

As a marker for measuring the amount of glycated protein, for example, hemoglobin Alc (HbAl
c), fructosamine and the like. HbAlc is
Glucose is 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 examination. 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 amount is affected by the amount of protein contained in the sample.

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 a method that utilizes that boronic acid forms a reversible conjugate with a substance having a cis-diol group under weak alkaline conditions. For example, aminophenylboronic acid is used as a packing material for chromatography. It has been reported that cellulose is bound to cellulose or polyacrylamide resin. 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 of a substance having a cis-diol group to the filler,
Desorption can be controlled only by changing the pH.

HP using the boronic acid affinity method
Regarding the measurement of glycated albumin using LC, a dedicated measuring instrument for the measurement is not yet widespread, but it is disclosed in JP-A-2-966.
According to Japanese Patent Laid-Open No. 57-57, the glycated albumin value by this measurement method is described as a relative percentage of the glycated albumin amount of albumin contained in a plasma or serum sample to the total albumin amount. . 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 GYL CABEN. 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.

As another immunological assay method, Japanese Patent Laid-Open No. 62-100660 discloses a method of binding a substrate specific / affinity adsorbent such as phenylboronic acid which specifically adsorbs glycated proteins. A method of specifically measuring only a specific glycosylated 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 glycosylated protein can be specifically measured. However, since various glycosylated proteins bind to phenylboronic acid, a clinically significant specific protein There is a problem that it is essential to measure the total amount of the specific protein separately in order to measure the saccharification ratio of the.

As another immunological measuring method, in Japanese Patent Laid-Open No. 4-130274, a lectin that specifically binds to a saccharide of a glycoprotein is insolubilized on the surface of a solid phase made of plastic, and After reacting a sample, a labeled lectin that specifically binds to the same saccharide is added to make a glycoprotein molecule into a lectin-lectin sandwich, and a substrate is added to determine the coloration. The lectin-lectin combination can supplement sugars, but has a problem that it is not suitable for a method for measuring a specific protein.

[0012] As another immunological measuring method, in Japanese Patent Laid-Open No. 5-87809, a carrier containing an anti-human serum albumin antibody is added to a sample containing human serum albumin, and the carrier is separated. Then, a technique of bringing a peroxidase-labeled phenylboronic acid derivative solution into contact with the separated carrier is described, but this method is an EIA method using an anti-albumin antibody and a boronic acid derivative labeled with OPD. The method is complicated to operate and has a large influence on the measurement system by an individual procedure.

Further, in the measurement system using boronic acid, it is necessary to adjust the pH to alkali when reacting with the boronic acid derivative, and therefore, depending on the substance to be measured, p on the alkali side may be present.
There is a problem that deactivation etc. may occur when H is set.

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. After that, the free sugar on the solid phase is removed, the glycated part 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 in which a solid phase and a 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 is prepared by using a test solution that can contain the specific protein at a 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 to be bound is always constant, it is an excellent method in that the labor of measuring the total amount of the specific protein can be saved, but it requires a reduction step, which complicates the operation, Although a reduced glycated protein antibody is required, there is a problem that it is not easy to prepare an antibody that does not differ between lots.

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

[0016]

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 specific binding ability to a protein portion of a glycated protein to be measured and a labeled compound. The labeled compound is an enzyme-labeled lectin. 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, A step of forming a solid-phased antibody-glycated protein complex, reacting an enzyme-labeled lectin with the solid-phased antibody-glycated protein complex, and forming the enzyme on the solid phase via the solid-phased antibody-glycated protein A step of binding the labeled lectin, and a step of measuring the enzyme activity on the solid phase after washing, (2) a method for measuring a glycated protein further comprising a washing step between the steps described in (1) above, and (3) ) A method for measuring a glycated protein having the following steps: a step of reacting an enzyme-labeled lectin with a glycated protein in a sample to form an enzyme-labeled lectin-glycated protein complex, for a protein portion of the glycated protein to be measured. Specific binding ability The immobilized antibody having the enzyme-labeled lectin-glycated protein complex is reacted with the immobilized antibody on the solid phase.
A step of binding the enzyme-labeled lectin via a glycated protein, and a step of measuring the enzyme activity on the solid phase after washing, (4) having specific binding ability to the protein portion of the glycated protein to be measured The immobilized antibody, the glycoprotein in the sample and the enzyme-labeled lectin are reacted substantially at the same time to form a complex of the immobilized antibody-glycated protein-enzyme-labeled lectin, and after washing, the enzyme on the solid phase is washed. A method for measuring a glycated protein, which comprises measuring the activity.

The measuring method of the present invention comprises a specific glycated protein obtained by a sandwich method using a labeled antibody and a solid-phased antibody capable of specifically binding to the protein portion of the glycated protein to be measured 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 the case where the protein to be measured is glycated albumin. The glycated protein to be the object of the measuring method of the present invention will be particularly described below. However, glycated hemoglobin and the like are also subject to the measurement method of the present invention. The sample having a glycated protein to be measured may be of biological origin 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 and the protein portion of glycated albumin in the sample are reacted with each other. 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 lectin 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 reacted. (4) The free enzyme-labeled lectin that was not involved 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-described 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.

As the solid phase on which such an antibody is immobilized, it suffices that each step in the present measuring method can be carried out, and the material thereof mainly includes an inorganic substance powder and an organic substance containing a synthetic polymer. Examples of the inorganic substance powder include glass, silica or alumina, and metal pieces such as gold, titanium, iron, and nickel. Also, as its shape,
A flake shape, a bead shape, a tube shape, or a plate 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 removed from the Fc fragment by enzymatic treatment with glycopeptidase (derived from almond) or N-glycanase in advance, or F (ab ') is treated with pepsin.
_The antibody may be bound to the solid phase after removing the effect of the sugar chain derived from the antibody by a known method such as the method of removing the Fc fragment having a saccharide moiety in the form of ₂ .

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 amount of albumin and glycated albumin bound to the immobilized antibody constant (5)
The calculated value of glycated albumin by the calibration curve in the step can be used as the relative ratio of the glycated albumin amount to the total albumin amount. 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. As such a blocking agent, bovine serum albumin (BS
A), casein, surfactants and the like. When the blocking agent has a cis-diol group, the background becomes large, so that the blocking agent can be prepared by a known method such as a boronic acid affinity column chromatography method or an enzyme-based sugar chain removal method in advance. It is desirable to remove saccharified components from the components in order to improve the measurement sensitivity. However, even if the blocking agent has a cis-diol group,
Within each lot (measurement batch), the amount of glycated albumin can be measured as long as the amount of cis-diol group in the component of the blocking agent is constant.

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 saline solution, a buffer solution such as 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 lectin used in the step (3) include an enzyme-labeled lectin found from animals and plants by a conventional method. Such a lectin is present in, for example, Lotus japonicus, eel serum and the like. L-fucous-binding lectin, peanut, soybean, castor bean, mokwanju, kidney bean, etc. D-galactose, N-acetyl-D-galactosamine-binding lectin, jack bean (concanavalin A), lentil, pea, broad bean, etc. -N-acetylchitobiose-binding lectin present in D-mannose-binding lectin, wheat embryo (wheat germ agglutinin), pokeweed (PWM lectin), potato, Datura strechata (DSA lectin), etc. ,as well as,
Examples thereof include sialic acid-binding lectin present in horseshoe crab. To label the lectin with an enzyme, known labeling methods such as the two-step glutaraldehyde method, the maleimide method, and the pyridyl disulfide method (Eiji Ishikawa et al., Enzyme-linked immunosorbent assay 3rd edition, Medical Institute (1987), etc. are described. , The enzyme-labeled lectin can be easily obtained by a cross-linking reaction between the amino group in the lectin and the functional group in the enzyme (eg, amino group, thiol group, etc.), but high yield and high reproducibility In order to obtain the above, it is preferable to select the maleimide method.

The labeling enzyme for lectins 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 above lectins, and exhibits color development by an enzyme-substrate reaction. If something
It is not particularly limited. The cis-diol group and the enzyme-labeled lectin in glycated albumin have a pH of 7.5 to 9.
Since it binds under a weak alkali of about 5, the enzyme does not contain a cis-diol group and has a pH of about 7.5 to 9.5 for the purpose of simplifying the process by reducing the types of reaction reagents. It is desirable to select an enzyme in which the reaction proceeds.
For example, β-D-galactosidase, alkaline phosphatase and the like can be mentioned, and β is particularly preferable from the viewpoint of the optimum pH of the enzyme.
-D-galactosidase is preferred. Also, when using an enzyme that undergoes an enzymatic reaction under pH conditions other than under weak alkaline, glycated albumin and enzyme-labeled lectin are bound under weak alkaline, and excess free enzyme-labeled lectin is removed by suction in step (4). After washing the solid phase, 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 lectin and the immobilized antibody-glycated albumin complex is carried out at pH 7.5 to 9.5 at a reaction temperature of 20 to 40 ° C and a reaction time of 30 minutes to 2 hours. As a result, the enzyme-labeled lectin can be bound to the solid phase via the immobilized antibody-glycoprotein.

The substrate liquid used in the step (4) is (3)
This is a solution containing a substrate for the labeling enzyme of the enzyme-labeled lectin used in the step. 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 the calculation based on the calibration curve obtained from the relationship between the amount of glycated albumin obtained by the measurement of the standard sample and the amount of color development (absorbance) is an absolute amount. , If the amount of immobilized antibody is fixed in advance and brought into contact with an excess sample, the amount of albumin in the antibody-reactive sample (standard sample or measurement sample) will also be constant, so calculate from the calibration curve. The glycated albumin value (percent) can be obtained by replacing the absolute amount of glycated albumin (horizontal axis) with the relative ratio (percent, horizontal axis) of the glycated albumin amount to the total albumin amount in the process. 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 (step of binding glycated albumin and enzyme-labeled lectin) may be reversed. That is,
First, (a) glycated albumin in a sample and an enzyme-labeled lectin are reacted and bound to form an enzyme-labeled lectin-glycated protein complex. In this case, non-glycated albumin that has not been glycated is mixed as it is without reacting with the enzyme-labeled lectin. In this step (a), the reaction for binding the glycated albumin and the enzyme-labeled lectin can be performed under the same reaction conditions as in the case of the step (3). Next, (b) the enzyme-labeled lectin-glycated protein complex is reacted with a solid-phased antibody having a specific binding ability with respect to the protein portion of the glycated protein to be measured, and the solid phase is fixed on the solid phase. The enzyme-labeled lectin is bound via a glycosylated 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. Thereby, the enzyme-labeled lectin can be bound to the solid phase via the immobilized antibody-glycated protein. Next, (c) the free enzyme-labeled lectin not bound to the solid phase, the enzyme-labeled lectin-glycated protein complex, etc. are removed to wash the solid phase, and (d) the solid phase is applied in the same manner as described above. The enzyme activity of is measured. According to this method, washing is performed between the step (a) of binding glycated albumin and the enzyme-labeled lectin and the step (b) of forming the immobilized antibody-albumin complex and the immobilized antibody-glycated albumin complex. It can be said that it is a simple method in that the process is unnecessary.

Further, in the present invention, it is 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 a specific binding ability to the protein portion of the glycated protein to be measured, the glycated protein in the sample and the enzyme-labeled lectin are reacted substantially at the same time to obtain the immobilized antibody-glycated protein. -A complex of enzyme-labeled lectins is formed, then 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 lectin is carried out at a reaction temperature of 20-4 at pH 7.5-9.5.
The reaction is performed at 0 ° C. for 30 minutes to 2 hours.

[0032]

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.

[0033]Preparation of PBS (phosphate buffer) Monosodium phosphate (dihydrate), disodium phosphate
(Dihydrate) and sodium chloride with phosphoric acid and sodium chloride
The final concentrations of helium were 0.02M and 0.15M, respectively.
Water, and add purified water to adjust the pH to 7.2.
I adjusted it. Preparation of 1% sodium caseinate-PBS Sodium casein (manufactured by Wako Pure Chemical Industries, Chemical
Was dissolved to 1% (W / V).Adjustment of measurement sample Blood collected from 4 healthy subjects and 4 diabetic patients
Leave at room temperature for 60 minutes, then 3000 rpm for 10 minutes
After centrifugation, the supernatant (serum) was used as a measurement sample. Tables 1 to 4
1 to 4 are healthy subjects and 5 to 8 are trials obtained from diabetic patients.
Indicated the fee.Preparation of standard sample Human serum albumin in vitro (HSA, Sigma)
HSA 40g in 1L of distilled water,
Add 18 g of glucose and add
(37 ° C., 7 days). And this is boronic acid
Affinity column chromatography (isolab
Glycated albumin and non-glycated albumin
Separation and fractionation, the saccharification ratio is 0.0, 10.0, 20.
After mixing each of them to 0, 40.0%,
Adjust the total albumin concentration to 4 g / dl using saline
Was adjusted to a standard sample.

Anti-human albumin antibody (polyclonal anti-
Body) Immobilization on microtiter plate Immobilization was performed by physical adsorption. 1 mg of IgG fraction of anti-human albumin antibody (derived from goat, manufactured by Cosmo Bio)
100 ml of this solution
μl was added to each well of two 96-well microtiter plates (Falcon) and left overnight at 4 ° C. In order to examine the amount of adsorption in each well, after leaving it overnight, 50 μl of the supernatant of one well was taken and the amount of protein (IgG) in the supernatant was measured with a protein quantification kit (manufactured by Pierce). The adsorbed IgG amount (added IgG amount-supernatant IgG amount) was on average 82 μg (n = 96
C. V. = 4.1%), and it was considered that each well was almost uniformly adsorbed. The other plate is
After removing the IgG solution with an aspirator, add 3 times with physiological saline.
Washed twice. Thereafter, 200 μl of 1% sodium caseinate-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.

Anti-human albumin antibody (monoclonal anti-
Body) Immobilization on microtiter plate Immobilization was performed by physical adsorption. An anti-human albumin monoclonal antibody IgG fraction (mouse-derived, subclass IgG1, manufactured by Cosmo Bio Inc.) was dispersed in PBS at 1 mg / ml, and 100 μl of this solution was added to each well of two 96-well microtiter plates. Left overnight at ° C. In order to examine the amount of adsorption in each well, after leaving it overnight, 50 μl of the supernatant of one well was taken and the amount of protein (IgG) in the supernatant was measured with a protein quantification kit. The average amount of adsorbed IgG (amount of added IgG-amount of supernatant IgG) was 68 μg (n =
96C. V. = 3.6%), and it was considered that they were adsorbed to each well almost uniformly. For the other plate, remove the IgG solution with an aspirator and add 3 times with physiological saline.
Washed twice. Thereafter, 200 μl of 1% sodium caseinate-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 monoclonal antibody-immobilized microtiter plate.

Concanavalin A, β of peanut lectin
-Binding to galactosidase Concanavalin A 714 mg (70 μmol) or peanut lectin 455 mg (70 μmol) was used as a maleimide group-introducing reagent, N-succinimidinle 6
-Maleimidohexanoate 21 mg (70 μmol)
Was adjusted to pH 7.0 to obtain a phosphate buffer solution of 0.1 mol /
Incubation was performed in 100 ml at 30 ° C. for 1 hour. Then, the precipitate was removed by centrifugation, and a product having a maleimide group introduced was obtained by gel filtration. Β-galactosidase 60 was added to 1.0 mg (3 μmol) of this product.
0 mg (1100 (nmol) was added, and the mixture was incubated in 20 ml of 0.1 mol / l phosphate buffer adjusted to pH 6.0 at 30 ° C. for 1 hour. After that, the product in which the maleimide group was introduced by gel filtration was used. By-products of concanavalin A, β-galactosidase polymer of peanut lectin, etc. were removed, and further, casein of the reaction was removed by boronic acid affinity column chromatography using dextran gel as a packing material. 10 ml of 0.1 mol / l Tris-hydrochloric acid buffer solution (pH 8.0) containing 5 mg / ml of concanavalin A-binding β-galactosidase, and 0.1 mol / l of 10 mg / ml containing peanut lectin-binding β-galactosidase. Tris-HCl buffer (pH
10 ml of 8.0) was obtained.

Example 1 Concatenated with anti-human albumin antibody (polyclonal antibody)
Human serum using navalin A-linked β-galactosidase
Measurement of glycated albumin level in the wells 50 μl each of a standard sample and a measurement sample was weighed into each well of an anti-human albumin antibody-bound microtiter plate, and incubated at room temperature for 30 minutes or more. After removing the reaction solution from each well, 300 μm of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride.
Each well was washed 3 times with 1 to remove unreacted albumin. Then, 200 μl of a concanavalin A-binding β-galactosidase solution was weighed in and further incubated at room temperature for 60 minutes or more. After removing the reaction solution from each well, 100 mM Tris-containing 100 mM sodium chloride was added.
Each well was washed 3 times with 300 μl of a hydrochloric acid buffer solution (pH 8.0), and after removing the washing solution from each well, 100 mM sodium chloride for measuring β-galactosidase activity, 1
mM magnesium chloride, 2 mM o-nitrophenyl-
50 μl of 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing β-D-galactoside was measured and incubated at room temperature for 60 minutes. In order to stop the reaction, 200 μl of 1N sodium hydroxide solution was mixed and the wavelength of 4
Absorbance was measured at 20 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.

[0038]

[Table 1]

Example 2 Anti-human albumin antibody (monoclonal antibody) and conca
Human serum using navalin A-linked β-galactosidase
Measurement of glycated albumin level in the wells 50 μl each of a standard sample and a measurement sample was weighed into each well of an anti-human albumin antibody-bound microtiter plate, and incubated at room temperature for 30 minutes or more. After removing the reaction solution from each well, 300 μm of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride.
Each well was washed 3 times with 1 to remove unreacted albumin. Then, 200 μl of a concanavalin A-binding β-galactosidase solution was weighed in and further incubated at room temperature for 60 minutes or more. After removing the reaction solution from each well, 100 mM Tris-containing 100 mM sodium chloride was added.
Each well was washed 3 times with 300 μl of a hydrochloric acid buffer solution (pH 8.0), and after removing the washing solution from each well, 100 mM sodium chloride for measuring β-galactosidase activity, 1
mM magnesium chloride, 2 mM o-nitrophenyl-
50 μl of 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing β-D-galactoside was measured and incubated at room temperature for 60 minutes. In order to stop the reaction, 200 μl of 1N sodium hydroxide solution was mixed and the wavelength of 4
Absorbance was measured at 20 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.

[0040]

[Table 2]

Example 3 Anti-human albumin antibody (polyclonal antibody) and pea
Human serum using threctin-binding β-galactosidase
Measurement of glycated albumin level in the wells 50 μl each of a standard sample and a measurement sample was weighed into each well of an anti-human albumin antibody-bound microtiter plate, and incubated at room temperature for 30 minutes or more. After removing the reaction solution from each well, 300 μm of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride.
Each well was washed 3 times with 1 to remove unreacted albumin. Then, 200 μl of a peanut lectin-binding β-galactosidase solution was weighed in and further incubated at room temperature for 60 minutes or more. After removing the reaction solution from each well, 100 mM Tris-containing 100 mM sodium chloride was added.
Each well was washed 3 times with 300 μl of a hydrochloric acid buffer solution (pH 8.0), and after removing the washing solution from each well, 100 mM sodium chloride for measuring β-galactosidase activity, 1
mM magnesium chloride, 2 mMo-nitrophenyl-β
50 μl of 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing -D-galactoside was measured and incubated at room temperature for 60 minutes. In order to stop the reaction, 200 μl of 1N sodium hydroxide solution was mixed and the wavelength of 42
Absorbance was measured at 0 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.
It was shown to.

[0042]

[Table 3]

Example 4 Anti-human albumin antibody (monoclonal antibody) and pea
Human serum using threctin-binding β-galactosidase
Measurement of glycated albumin level in the wells 50 μl each of a standard sample and a measurement sample was weighed into each well of an anti-human albumin antibody-bound microtiter plate, and incubated at room temperature for 30 minutes or more. After removing the reaction solution from each well, 300 μm of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 100 mM sodium chloride.
Each well was washed 3 times with 1 to remove unreacted albumin. Then, 200 μl of a peanut lectin-binding β-galactosidase solution was weighed in and further incubated at room temperature for 60 minutes or more. After removing the reaction solution from each well, 100 mM Tris-containing 100 mM sodium chloride was added.
Each well was washed 3 times with 300 μl of a hydrochloric acid buffer solution (pH 8.0), and after removing the washing solution from each well, 100 mM sodium chloride for measuring β-galactosidase activity, 1
mM magnesium chloride, 2 mMo-nitrophenyl-β
50 μl of 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing -D-galactoside was measured and incubated at room temperature for 60 minutes. In order to stop the reaction, 200 μl of 1N sodium hydroxide solution was mixed and the wavelength of 42
Absorbance was measured at 0 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.
It was shown to.

[0044]

[Table 4]

[0045]

EFFECTS OF THE INVENTION According to the present invention, for example, a glycated protein useful as a diagnostic marker for diabetes and the like can be accurately and reliably measured, so that a clinical test can be carried out very easily.

Claims (3)

[Claims] 1. A method for measuring a specific glycated protein by a sandwich method using a labeled antibody and a solid-phased antibody capable of specifically binding to a protein portion of a glycated protein to be measured, wherein the label A method for measuring a glycated protein, wherein the compound is an enzyme-labeled lectin. 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 lectin And the step of reacting the immobilized antibody-glycated protein complex with the enzyme-labeled lectin via the immobilized antibody-glycated protein on the solid phase, and the enzyme activity on the solid phase after washing Measuring step. 3. The method for measuring glycated protein according to claim 2, further comprising a washing step between the steps.