JP3292766B2 - Glycoprotein measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a glycated protein useful as a diagnostic marker for diabetes, for example, in the field of clinical testing, and more particularly, to a method using latex as an insoluble carrier. In addition, a monoclonal antibody to the measurement target and a substance having affinity for sugar are respectively supported, and the measurement target substance is measured using the degree of aggregation by the reaction between the monoclonal antibody-supported latex and the sugar-affinity substance-supported latex and the sample. The present invention relates to a method for measuring glycated proteins.

[0002]

2. Description of the Related Art It is known that albumin, which is a protein component in blood, and hemoglobin in erythrocytes react non-enzymatically with glucose to be saccharified into glycated proteins. Since the amount of protein that undergoes glycation is proportional to the amount of glucose in the body during the time the protein was present in the living body, measuring the amount of glycated protein in the protein in the body can be used for diagnosis of diabetes, etc. Is useful in the field of clinical testing.

As a marker for measuring the amount of glycated protein, for example, hemoglobin Alc (HbAlc)
Alternatively, fructosamine and the like can be mentioned.

[0004] HbAlc is obtained by binding glucose to the N-terminal amino acid of the β subunit of hemoglobin.
It is one of the markers used in clinical tests. H
The measurement of the amount of glycated protein using bAlc is performed by a dedicated high-performance liquid chromatograph (HPLC) using an ion exchange resin as a filler (see, for example, JP-A-63-163).
298063). Since the HbAlc value by this measurement method is indicated by a relative percentage of the HbAlc amount to the total hemoglobin amount, although the measured value is not affected by the amount of hemoglobin contained in the test sample, abnormal hemoglobin (for example, HbS) There is a problem that may be affected by.

[0005] A measurement method using fructosamine as a marker is a colorimetric method utilizing the reducibility of glycated protein, and the glycated protein contained in plasma or serum is measured using an automatic measuring device for biochemical tests. It is a method of measuring the amount.
In this measurement method, since the measured value of fructosamine is indicated as the absolute amount of the glycated protein, there is a problem that the measured value is affected by the amount of protein contained in the sample.

As another measuring method, there is known a method for measuring a glycated protein using HPLC using a boronic acid affinity method. The boronic acid affinity method is a method that utilizes the fact that boronic acid forms a reversible conjugate with a substance having a cis-diol group under weak alkaline conditions.
For example, there has been reported an example in which aminophenylboronic acid is bonded to cellulose or polyacrylamide resin as a packing material for chromatography. Boronic acid becomes a boronate anion under a weak alkaline condition and forms a stable conjugate with a substance having a cis-diol group. This conjugate is reversible and dissociates by bringing the pH to the acidic side. By utilizing boronic acid as a filler utilizing this property, the adsorption / desorption of a substance having a cis-diol group to / from the filler can be controlled only by a change in pH.

[0007] Using boronic acid affinity method, H
Regarding the measurement of glycated albumin using PLC, although a dedicated measuring instrument has not yet been widespread in the art, Japanese Patent Application Laid-Open No. 2-96657 discloses that the glycated albumin value by this measuring method is contained in a plasma or serum sample. It is described that, among the albumins used, the amount of glycated albumin is indicated by a relative percentage to the total amount of albumin. Therefore, since there is no effect on measured values due to fluctuations in the amount of protein in a sample, which is a problem in the measurement method using fructosamine as a marker, it is considered to be very useful in the field of clinical tests such as diagnosis of diabetes. .

That is, as a method of measuring glycated albumin by HPLC using the boronic acid affinity method, the same publication discloses that albumin in a sample is separated from other proteins by a first column, and then only albumin is used. A method is described in which the glycated albumin is separated into glycated albumin and non-glycated albumin by leading to two columns (boronic acid affinity column).

As another method for measuring glycated albumin by HPLC, Clinical Chemistry, Vol. 20, Supplement No. 2 (1.
On page 34b and page 35b in (991), the sample is separated into a saccharified component and a non-saccharified component using a boronic acid affinity column, and then a post-column using a dye (bromcresol purple) that specifically binds only to albumin. A method for detecting albumin by the method is described.

As another measurement method, a measurement method for detecting glycated protein using a specific antibody against glycated protein has been developed. A kit for measuring glycated albumin by ELISA is available from EXOCELL, INC. Of the United States. G from the company
It is sold under the trade name LYCABEN. The ELISA method here is a sandwich method between an immobilized 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 separately measured by the ELISA method, and the total amount of albumin is separately measured by the BCG (bromcresol green) method, and the measured value is expressed as a ratio (percent) of the amount of glycated albumin to the total amount of albumin. It has become.

However, the measurement by the above-mentioned HPLC method has a problem in that it is impossible to measure a large number of samples at the same time because the measurement time per sample is long. In addition, the ELI
In the measurement using a measurement kit by the SA method, it is necessary to measure the glycated albumin amount and the total albumin amount separately in order to measure the ratio of the glycated albumin amount to the total albumin amount, which is very troublesome. Things.

In the measurement using the above-mentioned ELISA kit, a monoclonal antibody recognizing the glycation site of albumin is used as the immobilized anti-human glycated albumin antibody. There is a drawback that the amount of glycated albumin cannot be accurately measured with only one kind of monoclonal antibody due to the presence of a receiving site. As described above, the measurement of glycated albumin using a monoclonal antibody against glycated albumin uses an antigen-antibody reaction and thus has excellent specificity, but it is difficult to prepare an antibody capable of recognizing all glycated albumin. There's a problem.

As another immunological measurement method, Japanese Unexamined Patent Publication (Kokai) 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 protein by a method using a solid phase and a labeled antibody having specific affinity only for a specific protein is described. This method is an excellent method in that the amount of a specific glycated protein can be specifically measured.However, since various glycated proteins bind to phenylboronic acid, the specific protein is clinically significant. There is a problem that it is essential to separately measure the total amount of a specific protein in order to measure the saccharification ratio of the protein.

As another immunological measurement method, JP-A-64-16964 discloses that a test solution is reacted with an immobilized antibody in which an antibody to a specific protein is immobilized on a carrier. Thereafter, 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 during or after the reduction reaction. ,
Separate the solid and liquid phases, measure the amount of labeling in either phase,
A method for measuring the specific glycated protein content from the measured value is described. This method is to use a test solution containing a specific protein at a concentration higher than the specific protein can be bound to all of the predetermined amount of antibody immobilized on a solid phase,
This is an excellent method in that the amount of a specific protein bound to the same immobilized antibody is always constant, thereby eliminating the need to measure the total amount of the specific protein. However, there are problems such as the complexity of the method, and the fact that an anti-reduced glycated protein antibody is required but it is not easy to prepare an antibody having no difference between lots.

Other immunoassays are described in
130274 describes glycated protein molecules as lectin-lectin sandwiches. This lectin-lectin combination can capture glycated proteins, but is not suitable for measuring specific proteins.

JP-A-5-87809 describes an EIA method using an anti-albumin antibody and a peroxidase-labeled boronic acid derivative. However, the EIA method is complicated and requires a procedure performed by an individual. The effect on the system is large.

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

[0018]

Means for Solving the Problems The method for measuring glycated protein according to the present invention has been devised to achieve the above object, and a latex as an insoluble carrier has a monoclonal antibody against a measurement object and an affinity for sugar. Respectively, and reacting the analyte with the monoclonal antibody-supported latex and the sugar-affinity-substance-supported latex, and then optically measuring the degree of aggregation of the latex. is there.

The reaction between the monoclonal antibody-carrying latex and the sample and the reaction between the sugar-affinity-substance-carrying latex and the sample may be carried out with the former first followed by the latter, or both may be carried out simultaneously.

Most of the aggregation of the latex is caused by the reaction between the measurement target and the sugar-affinity substance, and thus can be measured as the saccharification ratio of the measurement target.

Next, the measurement system will be described in more detail.

1) A monoclonal antibody is used as the antibody.

2) The sugar-affinity substance may be directly bound to the latex, or the sugar-affinity substance bound to a certain carrier protein may be bound to the latex.

3) The amount of the monoclonal antibody in the system can be adjusted at the stage of fixing to the latex.
It can be almost constant.

4) A substance that promotes an antigen-antibody reaction (eg, polyethylene glycol) can be used in combination.

5) Aggregation other than the reaction between the object to be measured and the carbophilic substance may be considered as a carbophilic substance-protein-carbophilic substance when a protein has many glycated parts. This can be ignored as noise if the quantity is small. It is also possible to control the reaction time so that this aggregation does not occur much.

6) Examples of the sugar-affinity substance include dihydroxyboryl compounds, lectins and the like.

7) As the latex polymer that can be used, those having a particle size of about 0.05 to 1.0 μm are preferably used. In addition, polystyrene or the like is suitably used as the resin material of the latex.

8) The substance to be measured is not particularly limited as long as it is a glycoprotein, and can be measured as clinically necessary.
Currently, clinically useful analytes include glycated albumin and glycated hemoglobin.

9) As an optical measuring instrument, a general-purpose spectrophotometer, an automatic analyzer for biochemistry using a spectrophotometric measurement principle (Hitachi, Hitachi 7150, 7070, etc.)
Toshiba Corp., Toshiba TBA-80R, etc.), a device using near-infrared as a measurement wavelength (Mitsubishi Kasei Corp., LPIA, etc.), and a device based on integrating sphere turbidity (Kyowa Hakko Co., Ltd., EL system, etc.) , A device for measuring the intensity of scattered light (Boehringer,
BNA system etc.) can be used.

[0031]

EXAMPLES The method for measuring a glycated protein of the present invention will be described below with reference to the case where the protein to be measured is glycated albumin.
This will be described in more detail. However, the glycated protein to be subjected to the measurement method of the present invention is not limited to this. For example, glycated hemoglobin and the like can be subjected to the measurement method of the present invention similarly to glycated albumin. The sample containing the 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.

In this example, the following reagents and test samples were used. Unless otherwise indicated, the same reagents of the same name were used in the following Examples.

PES (phosphate buffer): Monosodium phosphate (dihydrate), disodium phosphate (dihydrate) and sodium chloride were used, and the final concentrations of phosphoric acid and sodium chloride were 0.02M, respectively. 0.15M, pH 7.
It was prepared by adding to purified water so as to be 2.

1% sodium caseinate-PBS: PBS
1% sodium caseinate (Wako Pure Chemical, chemical)
(W / V).

Example 1 (Measurement of glycated albumin) 1) Binding of anti-human albumin antibody (monoclonal antibody) to latex Anti-human albumin monoclonal antibody IgG fraction (mouse-derived, subclass IgG1, manufactured by Cosmo Bio) Is dispersed in PBS so as to be 1 mg / ml,
900 μl of this dispersion and 100 μl of latex having a particle size of 0.2 μl (manufactured by Sekisui Chemical Co., Ltd.) were mixed, and the mixture was allowed to stand at 4 ° C. overnight. After centrifugation, the supernatant was discarded, and then 1 ml of 1% sodium caseinate-PBS was added, followed by blocking at 37 ° C for 2 hours. After that, it is washed three times with physiological saline,
4 ml of 1% sodium caseinate-PBS was added to prepare an anti-human albumin monoclonal antibody-bound latex. 2) Binding of dihydroxyboryl compound to casein (binding of sugar-affinity substance to carrier protein) aminophenylboronic acid 19 mg (140 μmol),
And N-succinimidyl-6-maleimidohexanoate 42 mg (140 mg) as a maleimide group introduction reagent.
μmol) was added to a phosphate buffer adjusted to pH 7.0.
Incubated for 1 hour at 30 ° C. in 200 ml of 1 mol / l. Thereafter, the precipitate was removed by centrifugation, and a product into which a maleimide group was introduced was obtained by gel filtration.

To 1.0 mg (3 μmol) of this product, 32 mg (1100 nmol) of sodium caseinate was added, and the pH of the phosphate buffer adjusted to 6.0 was adjusted to 0.1 mol / mol.
Incubation was performed at 120C for 1 hour in 120 ml. Thereafter, by-products such as aminophenylboronic acid and casein polymer into which the maleimide group was introduced were removed by gel filtration.

Further, by removing unreacted casein by boronic acid affinity chromatography using dextran gel as a filler, 0.1 mol / l Tris-HCl buffer containing 1 mg / ml of casein-bound dihydroxyboryl compound is contained. Liquid (pH 8.0)
Was obtained in an amount of 20 ml.

3) Binding of casein-bound dihydroxyboryl compound to latex (binding of sugar-affinity substance to latex via carrier protein) Casein-bound dihydroxyboryl compound was added to PBS such that its concentration was 0.4 mg / ml. The dispersion was mixed with 900 μl of this dispersion and 100 μl of latex (manufactured by Sekisui Chemical Co., Ltd.) having a particle size of 0.2 μl, and the mixture was allowed to stand at 4 ° C. overnight. After centrifugation, the supernatant was discarded, and then 1 ml of 1% sodium caseinate-PBS was added, followed by blocking at 37 ° C for 2 hours. Then, it was washed three times with physiological saline, and 50 ml of 1% sodium caseinate-PBS was added to prepare a casein-bound dihydroxyboryl compound-bound latex.

4) Measurement Measurement method (1) When two types of latex are allowed to react with the sample one by one Anti-human albumin monoclonal antibody-bound latex 3
To 0 μl, 30 μl of a standard sample was added and incubated at 37 ° C. for 5 minutes. Thereafter, 500 μl of a casein-bonded dihydroxyboryl compound-bound latex was added, and the mixture was stirred.
The change in absorbance for 5 minutes was measured in a cell having an optical path length of 1 cm at a wavelength of nm. 30 μl measurement sample instead of standard sample
Was added, and the change in absorbance was measured in the same manner as described above.
The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample.

<Measurement results>

[0041]

2) Measurement sample No. Absorbance change Saccharified albumin value (%) 1 0.064 12.5 2 0.080 15.5 3 0.113 20.0 4 0.094 17.0 5 0.184 38.5 6 0.182 37.5 7 0.162 33.0 8 0.174 35.5 1-4: healthy person, 5-8: diabetic patient

Measurement method (2) When two types of latex are simultaneously reacted with a sample: Binding of casein-bound dihydroxyboryl compound to latex (binding of sugar-affinity substance to latex via carrier protein) 1% after blocking A casein-bound dihydroboryl compound-bound latex was prepared in the same manner as in step 3), except that the addition amount of sodium caseinate-PBS was 4 ml (50 ml in step 3).

20 μl of the standard sample and 350 μl of PBS were added and incubated at 37 ° C. for 5 minutes. Then, anti-human albumin monoclonal antibody-bound latex 25
μl and 25 μl of a casein-bonded dihydroxyboryl compound-bound latex were added, and after stirring, the light path length was 1 cm at a wavelength of 570 nm using a Hitachi U-3200 type spectrophotometer.
The change in absorbance for 5 minutes was measured in the cell. 20 μl of a measurement sample was added instead of the standard sample, and the change in absorbance was measured in the same manner as described above. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample.

<Measurement results>

[0046]

2) Measurement sample No. Absorbance change Saccharified albumin value (%) 1 0.068 19.0 2 0.062 17.5 3 0.056 16.0 4 0.164 39.0 5 0.160 37.0 6 0.162 37.5 7 0.144 34.0 8 0.158 36.5 1-3: Healthy person, 4-8: Diabetic patient

Example 2 (Measurement of Glycated Albumin) 1) An anti-human albumin monoclonal antibody-bound latex was prepared in the same manner as in Step 1) of Example 1.

2) Binding of Concanavalin A to Latex Concanavalin A (manufactured by Wako Pure Chemical Industries, Inc., for biochemistry) was dispersed in PBS to a concentration of 1.0 mg / ml. 100 μl of 2 μl of latex (manufactured by Sekisui Chemical Co., Ltd.) was mixed, and the mixture was allowed to stand at 4 ° C. overnight. After centrifugation, the supernatant was discarded, and then 1 ml of 1% sodium caseinate-PBS was added, followed by blocking at 37 ° C for 2 hours. Then, it was washed three times with physiological saline, and 50 ml of 1% sodium caseinate-PBS was added to prepare a concanavalin A-bound latex.

4) Measurement Anti-human albumin monoclonal antibody-bound latex 3
To 0 μl, 30 μl of a standard sample was added and incubated at 37 ° C. for 5 minutes. Thereafter, 500 μl of concanavalin A-bound latex was added, and after stirring, Hitachi U-320 was added.
Using a 0-type spectrophotometer, an optical path length of 1 at a wavelength of 570 nm
The change in absorbance for 5 minutes was measured in a cm cell. 30 μl of a measurement sample was added instead of the standard sample, and the change in absorbance was measured in the same manner as described above. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample.

<Measurement results>

[0052]

2) Measurement sample No. Absorbance change Saccharified albumin value (%) 1 0.088 14.0 2 0.099 15.5 3 0.118 19.0 4 0.124 19.0 5 0.292 34.5 6 0.352 39.5 7 0.306 36.0 8 0.272 33.0 1-4: healthy person, 5-8: diabetic patient

Example 3 (Measurement of Glycated Albumin) 1) An anti-human albumin monoclonal antibody-bound latex was prepared in the same manner as in Step 1) of Example 1.

2) Binding of peanut lectin to latex Peanut lectin (manufactured by Wako Pure Chemical Industries, for biochemistry) was dispersed in PBS at a concentration of 0.6 mg / ml. 100 μl of 2 μl of latex (manufactured by Sekisui Chemical Co., Ltd.) was mixed, and the mixture was allowed to stand at 4 ° C. overnight. After centrifugation, the supernatant was discarded, and then 1 ml of 1% sodium caseinate-PBS was added, followed by blocking at 37 ° C for 2 hours. Thereafter, the mixture was washed three times with a physiological saline, and 50 ml of 1% sodium caseinate-PBS was added to prepare a peanut lectin-bound latex.

4) Measurement Anti-human albumin monoclonal antibody-bound latex 3
To 0 μl, 30 μl of a standard sample was added and incubated at 37 ° C. for 5 minutes. Thereafter, 500 μl of peanut lectin-bound latex was added, and after stirring, Hitachi U-320 was added.
Using a 0-type spectrophotometer, an optical path length of 1 at a wavelength of 570 nm
The change in absorbance for 5 minutes was measured in a cm cell. 30 μl of a measurement sample was added instead of the standard sample, and the change in absorbance was measured in the same manner as described above. The glycated albumin value in the sample was determined from the absorbance of the standard sample and the measurement sample.

<Measurement results>

[0058]

2) Measurement sample No. Absorbance change Saccharified albumin value (%) 1 0.044 14.0 2 0.044 14.0 3 0.058 18.0 4 0.054 17.0 5 0.098 35.0 6 0.100 36.5 7 0.098 36.0 8 0.096 33.5 1-4: healthy person, 5-8: diabetic patient

[0060]

As described above, the present invention can easily measure the abundance ratio of glycated protein useful as a diagnostic marker for diabetes, for example.

Claims (1)

(57) [Claims] 1. A latex as an insoluble carrier, on which a monoclonal antibody to a measurement object and a substance having an affinity for sugar are respectively supported, and the monoclonal antibody-supported latex and the sugar-affinity substance-supported latex are respectively loaded on the latex. A method for measuring glycated protein, comprising reacting a sample and optically measuring the degree of aggregation of latex.