JP5178070B2 - Method for determining diseases caused by glycated immunoglobulin - Google Patents

Description

  The present invention relates to a method for determining a disease such as malignant IgA type M proteinemia by measuring the level of glycated immunoglobulin in a sample, a kit for measuring glycated immunoglobulin, and kynurenine in an immunoglobulin molecule in the sample. The present invention relates to a method for determining diseases such as malignant IgA-type M proteinemia by measuring the level of residues.

An immunoglobulin has a dimer structure in which a heavy chain and a light chain are S—S bonded. There are five types of heavy chains, γ (IgG), α (IgA), μ (IgM), δ (IgD), and ε (IgE), and the L chain is of κ type and λ type common to each immunoglobulin. Two types are known.
Immunoglobulins usually secrete antibodies with different variable region structures, which are antigen-binding regions, from antibody-producing cells corresponding to the diversity of antigen structures. A heterogeneous broad peak (polyclonal) is formed. However, when antibody-producing cells proliferate in a neoplastic or reactive manner, an immunoglobulin having a highly uniform variable part of one class, type, and structure, that is, a monoclonal protein (M protein) is produced from one clonal cell. Come. In this case, since the charge uniformity is high on electrophoresis, a single sharp peak is shown. M proteinemia (M-proteinemia) in which such M protein is recognized includes malignant M proteinemia and benign M proteinemia (also referred to as MGUS) (Non-patent Document 1).
In the case of malignant M proteinemia, some are associated with neoplastic diseases such as multiple myeloma and primary macroglobulinemia. In the case of benign M proteinemia, it may be observed even in healthy middle-aged and elderly people and is almost unrelated to such diseases. Therefore, when M protein is detected, it must be distinguished whether the M protein is malignant M proteinemia due to neoplastic growth or benign M proteinemia appearing due to reactive proliferation. Benign M proteinemia MGUS accounts for about 3/4 of M proteinemia. However, no means has been found at this time to clearly distinguish between malignant and benign.

On the other hand, in the measurement of fructosamine, a glycated protein, which is a parameter for diabetes diagnosis, it has been reported that the presence of immunoglobulins, particularly IgA type M protein, has an influence (Non-patent Documents 2 and 3). It is not clear whether glycated immunoglobulin can be used to differentiate malignant M proteinemia from benign M proteinemia.
Annual Review Blood, 2005, 207-223 Clinical examination vol. 47 no. 10, October 2003, pp. 1066-1068 J Electrophoresis 2006; 50:19

  An object of the present invention is to provide a method for accurately determining a disease, preferably a disease such as IgA multiple myeloma. Furthermore, the subject of this invention is providing the measuring method of the glycated immunoglobulin in a test substance.

  In this situation, the present inventors examined in detail the IgA / albumin complex collected from patients with malignant IgA type M proteinemia, and found that only the IgA / albumin complex collected from patients with malignant IgA type M proteinemia. In particular, a peptide having a kynurenine residue that is easily glycosylated is observed, and therefore, whether or not a disease such as malignant IgA type M proteinemia can be determined by measuring the level of glycated immunoglobulin in a sample. As a result of repeated research, a new method for measuring glycated immunoglobulin such as glycated IgA in a specimen was developed, and by measuring the level of glycated immunoglobulin using the measurement method, malignant IgA type M protein blood The present invention has been completed by finding that it can be easily determined whether the disease is benign or benign IgA type M proteinemia.

Therefore, the present invention relates to a determination method for measuring the level of glycated immunoglobulin in a specimen and determining the presence and / or extent of a disease from the level of the level.
Furthermore, the present invention relates to the step of binding an immunoglobulin site in a glycated immunoglobulin in a specimen with a substance capable of binding to the immunoglobulin, and then measuring the level of the sugar site of the bound glycated immunoglobulin. The present invention relates to a method for measuring glycated immunoglobulin.
Furthermore, the present invention relates to a determination method for measuring the level of glycated immunoglobulin in a sample by the above-described measurement method and determining the presence and / or extent of the disease from the level of the level.
Furthermore, the present invention is a kit for measuring glycated immunoglobulin in a specimen,
i) a substance capable of binding to the immunoglobulin site in the glycated immunoglobulin; and ii) a reagent for measuring the level of the sugar site of the glycated immunoglobulin bound to the substance;
Relates to a kit comprising
Furthermore, the present invention relates to a determination method for measuring the level of kynurenine residue in immunoglobulin present in a specimen and determining the presence and / or extent of disease from the level of the level.

  According to the present invention, diseases such as IgA multiple myeloma can be accurately determined. Furthermore, according to the present invention, glycated immunoglobulin in a specimen can be accurately measured.

The present invention is a determination method for measuring the level of glycated immunoglobulin in a specimen and determining the presence and / or extent of a disease from the level of the level.
In the present invention, the specimen can be exemplified by biological samples such as serum, plasma, urine, etc., or their dilutions, or model samples thereof.
In the present invention, the glycated immunoglobulin is not particularly limited as long as it has an immunoglobulin moiety and a sugar moiety bonded to a protein such as the immunoglobulin. For example, the glycated immunoglobulin includes those obtained by glycation of a protein (for example, glycated immunoglobulin / albumin complex) in which an immunoglobulin and a protein other than immunoglobulin such as albumin are bound. In the present invention, examples of the immune blobulin in the glycated immunoglobulin include IgA, IgG, IgM, IgD, IgE, and all of these immunoglobulins, but the disease includes malignant IgA type M such as malignant IgA type multiple myeloma. IgA is preferred for determining whether it is proteinemia. In this case, a healthy person or a benign glycated IgA value is low, and a glycated IgA value of a malignant IgA type M proteinemia patient shows a high value. Therefore, this disease can be determined.

In the present invention, in order to measure the level of glycated immunoglobulin in a sample, for example, the immunoglobulin site in the glycated immunoglobulin in the sample is bound to a substance that can bind to the immunoglobulin, and then the bound. This can be achieved by measuring the level of the sugar site of the glycated immunoglobulin.
In the present invention, the substance capable of binding to immunoglobulin is not particularly limited as long as it is a substance that specifically binds to immunoglobulin, but anti-immunoglobulin antibody, protein G, protein A and the like are preferable. In the case where the glycated immunoglobulin is glycated IgA, glycated IgG, glycated IgM, glycated IgD, or glycated IgE as the measurement target, the substances that can bind to the immunoglobulin are anti-IgA antibody, anti-IgG antibody, anti-IgM antibody, anti-IgM antibody, respectively. It is preferable to use an IgD antibody or an anti-IgE antibody. Here, as such an antibody, either a monoclonal antibody or a polyclonal antibody can be used, but a monoclonal antibody is preferable from the viewpoint of specificity.

  In order to bind the immunoglobulin site in the glycated immunoglobulin in the sample to a substance capable of binding to the immunoglobulin, for example, a solid support such as a plate is specifically bound to the immunoglobulin such as an anti-human IgA antibody. Add substance to bind and sensitize. Next, the solid support is washed with a washing solution, and a blocking agent (for example, PBS containing BSA) is added. The obtained solid support such as a plate is stored at a low temperature to prepare an antibody-sensitized solid support such as an antibody-sensitized plate. The solid support is washed with a washing solution before use, and then a sample that may contain glycated immunoglobulin is added to the support, and an antibody or the like is reacted with glycated immunoglobulin. The immunoglobulin site in the glycated immunoglobulin can be bound to a substance capable of binding to the immunoglobulin.

In the present invention, the level of the sugar site of the bound glycated immunoglobulin is measured, for example, by allowing the enzyme to act on the bound glycated immunoglobulin to release glucose and measuring the glucose. be able to. That is, in order to measure the level of the sugar site of the bound glycated immunoglobulin (preferably glycated IgA), for example, the binding site between the sugar site and the immunoglobulin site in the glycated immunoglobulin is cleaved. It can be achieved by measuring the level of sugar such as glucose that is cleaved by the enzyme to be cleaved and produced by cleaving.
In this case, an enzyme that hydrolyzes the binding site, such as glucosidase, is used to cleave the binding site between the sugar site and the immunoglobulin site in the saccharified immunoglobulin bound to the antibody or the like. When a hydrolase is used, glucose is released by the action of the enzyme. For example, the glucose is measured. Glucose can be measured by a known method and is not particularly limited, but i) a method in which glucose oxidase is allowed to act, and a hydrogen is produced by acting a Trinder reagent and peroxidase to produce a dye, ii) a method of measuring the increase in NADH by acting glucose with hexokinase, glucose-6-phosphate dehydrogenase, NAD, iii) inducing glucose to glucose-6-phosphate with hexokinase, A reagent containing two types of coenzymes, glucose-6-phosphate dehydrogenase, oxidized or reduced thio-NAD (P) and reduced or oxidized NAD (P), acts on 6-phosphate The amount of glucose is measured by forming a cycling reaction and measuring the amount of coenzyme changed by the reaction. How to (e.g., JP-A-4-335898, see JP-A-10-225300) can be exemplified. Among these, from the viewpoint of measurement sensitivity, the method of measuring glucose by enzyme cycling of the above iii) is preferable. In this case, for example, the first reagent contains glucosidase, hexokinase, magnesium ion, thio-NAD and glucose-6-phosphate dehydrogenase, the second reagent contains a chelating agent and NADH, The glycated immunoglobulin bound to the solid phase can be measured by adding the first reagent to the phase support and allowing it to react, and then adding the second reagent to cause an enzyme cycling reaction.

  In the present invention, a kit can be used to measure glycated immunoglobulin in a sample. Such a kit includes: i) a substance capable of binding to an immunoglobulin site in a glycated immunoglobulin; and ii) a reagent for measuring the level of the sugar site of the glycated immunoglobulin bound to the substance. There is. Examples of the substance that can be bound to the immunoglobulin site in the saccharified immunoglobulin used in the kit include the same substances as described above, and the reagent for measuring the level of the saccharified immunoglobulin sugar part is also used. As described above, a reagent for measuring glucose and the like can be mentioned. These kits may contain a buffer solution, a surfactant and the like, which are usually used, as necessary.

In the present invention, a disease, for example, malignant IgA, is measured by measuring the level of kynurenine residue in an immunoglobulin (for example, IgA) such as an immunoglobulin / albumin complex (for example, IgA / albumin complex) in a sample. Whether or not there is a disease such as type M proteinemia can be determined. A method for measuring the level of kynurenine residue in the IgA / albumin complex will be described below as an example.
When the IgA / albumin complex purified from a patient sample is digested with trypsin and decomposed, the resulting peptide is analyzed by reversed phase chromatography (HPLC), and the peptide detected only with the IgA / albumin complex is analyzed, kynurenine A peptide with residues is obtained. The level of kynurenine residues can be determined from the amount of the peptide.
In this case, for example, IgA and albumin of a healthy person are first purified separately, mixed at a ratio of 1: 1, and then trypsinized in a test tube. Thereafter, the decomposed peptide is fractionated by reverse phase HPLC to prepare a basic peptide map. The purified IgA / albumin complex is treated with trypsin by exactly the same operation, and compared with a peptide map previously prepared by a healthy person, a peak observed only in the IgA-albumin complex is detected and analyzed. N-terminal amino acid sequence analysis is performed on the peak to detect a peptide containing a kynurenine residue (for example, Gly-Leu-Glu-kynurenine-Val). Thus, malignant IgA-type M proteinemia can be determined by measuring IgA in a specimen, particularly kynurenine residue of IgA-albumin complex.

According to another aspect of the present invention, there is provided a peptide (hereinafter referred to as a kynurenine-containing peptide) that is present in IgA containing kynurenine, for example, having an amino acid sequence of 8 or more and Glu-kynurenine-Val. The level of the kynurenine residue in IgA can be measured using an antibody as an epitope (anti-kynurenine-containing IgA antibody), preferably a monoclonal antibody.
In this case, for example, by using a combination of an anti-IgA antibody and an anti-kynurenine-containing IgA antibody, the level of kynurenine residues in IgA can be measured. For example, first, an anti-IgA antibody is added to a solid support such as a plate and allowed to stand. After binding, blocking with a protein such as BSA is performed based on a conventional method in order to block the non-specific adsorption site of the protein. Next, the anti-IgA antibody thus bound to the solid phase is reacted with the specimen, and the kynurenine-containing IgA in the specimen is reacted with the solid phase via the anti-IgA antibody bound to the solid phase by an antigen-antibody reaction. Combine. Next, after washing, the kynurenine-containing IgA bound to the solid phase is reacted with an anti-kynurenine-containing IgA antibody by an antigen-antibody reaction. The kynurenine-containing IgA antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody in order to improve measurement accuracy.

  Next, after washing, anti-kynurenine-containing IgA antibody bound to the solid phase via kynurenine-containing IgA is measured. This can be performed by various methods conventionally known in the field of immunoassay. For example, it can be performed by labeling an anti-kynurenine-containing IgA antibody with an enzyme, a fluorescent dye or a radioactive substance, and measuring the label. Alternatively, a labeled antibody that specifically reacts with a kynurenine-containing IgA antibody can be further reacted to measure the label.

  Hereinafter, the present invention will be described in more detail with reference examples and examples, but the present invention is not limited to these reference examples and examples.

Reference example 1
Purification of IgA / Albumin Complex from Malignant IgA Type M Proteinemia Patient Purification of IgA / Albumin Complex from Malignant IgA Type M Proteinemia Multiple Myeloma Patient Sera is described in the literature (J. Electrophoresis, 50, pages 19-23, 2006).
Serum of a patient with malignant IgA proteinemia was added to a Jacalin-Agarose (Funakoshi) column (0.9 × 15 cm) equilibrated with pH 7.2, 0.1 mol / L phosphate buffer, After the fraction was eluted, the IgA fraction bound with 0.8 mol / L galactose-pH 7.2, 0.1 mol / L phosphate buffer was eluted. The fraction was dialyzed against pH 8.0, 0.2 mol / L Tris-HCl buffer, concentrated with Minicon B15, and the sample was applied to DEAE-Sephacel (Amersham Pharmacia Biotech) which is an ion exchanger. The IgA-albumin complex was purified by elution with a NaCl linear concentration gradient from 0 to 0.5 mol / L.

Example 1
Determination of malignant IgA-type M proteinemia based on the presence or absence of kynurenine residues in IgA IgA- albumin complex, normal human IgA and human albumin purified from trypsin treatment, decomposed, and the resulting peptide reversed Analysis by phase chromatography (HPLC) and analysis of the peptide detected only with the IgA-albumin complex gave a peptide having a kynurenine residue only in the case of the IgA-albumin complex.

  The process is briefly described below. First, normal human IgA and albumin were purified separately, mixed at a ratio of 1: 1, and then trypsinized in a test tube. Thereafter, the decomposed peptide was fractionated by reverse phase HPLC to prepare a basic peptide map. The purified IgA / albumin complex obtained in Reference Example 1 was treated with trypsin by exactly the same operation, and compared with a peptide map previously prepared by a healthy person, a peak observed only in the IgA / albumin complex was detected and analyzed. did.

  FIG. 1 compares peptide maps of i) purified IgA / albumin complex and ii) purified normal IgA and albumin mixture by reverse phase HPLC. A peak that seems to be specific to the IgA / albumin complex was confirmed. When N-terminal amino acid sequence analysis was performed on the peak, a peptide containing oxidized tryptophan was detected at fraction 66 (arrow). The amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing. Oxidized tryptophan is a substance called kynurenine. As a result, it was found that malignant IgA-type M proteinemia can be determined by measuring IgA in a specimen, particularly kynurenine residue of IgA-albumin complex.

  This kynurenine is a substance that is known to be accumulated in the brain of Alzheimer's disease and to be involved in white turbidity in the aging of the lens of the eye, but for the first time it has been proved from IgA in serum. It is. In addition, kynurenine is known to be a site where sugars are easily added to 3-OH kynurenine, further 3-OH kynurenine glucoside, and O-β-D-glucoside. By the way, it is also known that IgA / albumin complex appears in all cases of IgA type M protein in which glycation was observed (Clinical Laboratory, 47, 1066-1068). Therefore, in malignant IgA type M proteinemia, an IgA-albumin complex containing a kynurenine residue is produced, and IgA is glycated via the kynurenine residue to produce a glycated IgA-albumin complex.

Example 2
Measurement of Glycated IgA As shown in Example 1, since the inventors first demonstrated the involvement of kynurenine in the glycation of IgA / albumin complex, the measurement of the amount of glycated IgA was performed for diseases (IgA multiple myeloma). Therefore, a glycated immunoglobulin measurement system such as the following glycated IgA measurement system was constructed. This measurement system binds the site of an immunoglobulin (eg, IgA) in a glycated immunoglobulin (eg, glycated IgA) in a specimen to a substance that can bind to the immunoglobulin, and then This is a measurement system that measures the level of a sugar moiety.

  For example, in the measurement of glycated IgA, an anti-human IgA antibody-binding plate can be prepared and measured by an enzyme cycling method containing α-glucosidase by the same procedure as the immunoenzyme assay (EIA). Specifically, an anti-human IgA antibody (Dakocytomation) was added at 100 μg / 100 μL to a 96-well plate (Nunc). After sensitization at 4 ° C. for 1 day, the plate was washed three times with 200 μL of a washing solution (PBS containing 0.05% Tween 20), and 300 μL of a blocking agent (PBS containing 1.5% BSA) was added. It was stored again at 4 ° C. for 2 days to obtain an antibody-sensitized plate. The plate is washed once with 200 μL of washing solution before use, and 50 μL of informed consent volunteer and patient sample and 50 μL of 50 mM Tris buffer (pH 7.4) are added and allowed to react at room temperature for 1 hour. It was. After completion of the reaction, wash 3 times with 300 μL of the washing solution, add 100 μl of the following first reagent to the plate and further react at 37 ° C. for 1 hour, then add 50 μl of the following second reagent, and after a certain period of time, develop the color value. Was colorimetrically at 405 nm.

First reagent composition Trishydroxyaminomethane 100 mM pH 6.60
Imidazole 100 mM
EDTA · 2Na 2mM
Magnesium acetate tetrahydrate 10 mM
N-acetylcysteine 25 mM
Thio NAD (Thio-NAD) 1 mM
Hexokinase (HK) 3000U / L
Glucose-6-phosphate dehydrogenase (G6PDH) 50000U / L
α-Glucosidase 500U / L
Second reagent composition Trishydroxyaminomethane 400 mM pH 9.00
EDTA · 2Na 2mM
ATP 8 mM
β-NADH 20 mM

In these reactions, α-glucosidase in the following first reagent hydrolyzes the glycoside bond of glycated IgA bound to the plate, whereby glucose (sugar) bound to IgA is obtained. Next, this glucose is converted into glucose-6-phosphate or the like, and the glucose-6-phosphate is measured by the enzyme cycling method described in JP-A-4-335898. The measured value of the absorbance is based on the principle that the glucose concentration and finally the amount of glycated IgA in the sample are reflected. FIG. 2 shows the measurement principle, and Table 1 shows the relationship between the glucose concentration of the standard solution and the absorbance.

Example 3
Determination of IgA type multiple myeloma using glycated IgA measurement system Patient specimen measurement was performed using the glycated IgA measurement system established in Example 2. Samples include IgA type multiple myeloma (M protein, malignant) 6 cases, IgA type multiple myeloma (M protein, benign) 4 cases, IgG, M type multiple myeloma (M protein, malignant) 2 cases, IgG , M type multiple myeloma (M protein, benign) 2 cases, diabetes 11 cases, healthy people 6 cases. The measurement results are shown in Table 2. From the results shown in Table 2, it was shown that diseases such as multiple myeloma, particularly IgA type multiple myeloma (malignant M proteinemia) can be determined by measuring glycated IgA.

  According to the present invention, it is possible to accurately measure glycated immunoglobulin in a sample. By the method for measuring glycated immunoglobulin of the present invention, the level of glycated immunoglobulin in a sample is measured to obtain IgA multiple myeloma, etc. The disease can be accurately determined. Furthermore, according to the present invention, it is possible to accurately determine diseases such as IgA multiple myeloma by measuring kynurenine residues in immunoglobulins in specimens.

FIG. 1 is a comparison of peptide maps of purified IgA / albumin complex from a patient with malignant IgA type M proteinemia and a mixture of purified normal IgA and albumin by reverse phase HPLC. FIG. 2 shows the measurement principle for measuring glucose by the enzyme cycling method.

Claims (5)

A determination method for measuring the level of glycated immunoglobulin in a sample and determining the presence and / or extent of a disease from the level of the level , wherein glycated immunoglobulin can generate glucose by the action of an enzyme. Determination method that is globulin .   The determination method according to claim 1, wherein the immunoglobulin in the glycated immunoglobulin is IgA.   The method according to claim 1 or 2, wherein the disease is malignant IgA type M proteinemia.   4. The determination method according to claim 1, wherein the disease is IgA multiple myeloma.   The determination method according to claim 1, wherein the glycated immunoglobulin is a glycated immunoglobulin / albumin complex.

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