JP2013040771A - METHOD FOR DETERMINING DISORDER ON THE BASIS OF IgA-HINGE O-COUPLING TYPE SUGAR CHAIN STRUCTURE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a low-invasive method for determining disorder by which a doctor can objectively determine the disorder.SOLUTION: The method for determining the disorder is provided for determining whether or not a subject is affected with the disorder on the basis of the number of coupled N-acetyl-galactosamines in the IgA-hinge O-coupling type sugar chain isolated from body fluid of the subject. The inflammatory bowel disease and the immunoglobulin A nephropathy exemplify disorders to be determined by the method.

Description

    The present invention provides a method for determining a less invasive disease. Specifically, the present invention relates to a method for determining a disease based on a change in the structure of an IgAO-linked sugar chain.

  Inflammatory bowel diseases including IgA nephropathy and Crohn's disease and ulcerative colitis, all of which are of unknown cause diseases specified in the specific disease (intractable) by the Ministry of Health, Labor and Welfare.

  IgA nephropathy is the most frequent disease of chronic glomerulonephritis, and the number of patients currently being treated in Japan is estimated to be about 20,000 to 30,000. IgA nephropathy is often found by proteinuria or hematuria at the time of urinalysis, but renal biopsy is necessary for its definitive diagnosis. In recent years, it has been reported that galactose deficiency is observed in the sugar chains of IgA and serum IgA deposited on the glomeruli of IgA nephropathy patients. Based on such findings, diagnosis of IgA nephropathy by examining IgA sugar chains in serum has been proposed (Non-patent Document 1, Patent Document 1).

  Here, the structure of IgA is shown in FIG. It is known that an IgA molecule is composed of two heavy chains and two light chains, and has an O-linked sugar chain at the hinge shown in FIG. The general structure of the O-linked sugar chain of the IgA hinge part is shown in FIG. IgA is known to act as a first-line defense mechanism in the mucous membrane including the digestive tract, and IgA contained in mucus is associated with microorganisms that infest or infect living organisms, and due to immunological interactions. It is thought to be a cause of various diseases. In many cases, IgA deposited in the serum or glomeruli of IgA nephropathy patients lacks galactose in the O-linked sugar chain at the hinge as shown in FIG.

  On the other hand, inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's disease is known as an intractable disease that currently has about 120,000 patients nationwide. At present, examination by an endoscope is indispensable for diagnosing an IBD patient, and the burden on the patient is very large. There is a need for a diagnostic method that is non-invasive and capable of objective judgment.

  Some of the inventors of the present application examined blood IgG sugar chains of patients with inflammatory bowel disease, and patients with inflammatory bowel disease have a high rate of galactose deficiency of IgG fucosylated sugar chains, which is related to disease activity. (Non-Patent Document 2). Further, the differential diagnosis of patients Crohn's disease patients and ulcerative colitis among inflammatory bowel disease patients, found that it is possible to perform on the basis of the difference in blood IgG oligosaccharide (Patent Document 2). However, the relationship between inflammatory bowel disease and IgA sugar chains is not known. In addition, there has been no report analyzing immunoglobulin sugar chains obtained from body fluids other than blood.

Japanese Patent Laid-Open No. 10-111291 WO2007 / 136001

Moldoveanu Z, Wyatt RJ, Lee JY, Tomana M, Julian BA, Mestecky J, Huang WQ, Anreddy SR, Hall S, Hastings MC, Lau KK, Cook WJ, Novak J. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int 2007; 71: 1148-54. Shinzaki S, Iijima H, Nakagawa T, Egawa S, Nakajima S, Ishii S, Irie T, Kakiuchi Y, Nishida T, Yasumaru M, Kanto T, Tsujii M, Tsuji S, Mizushima T, Yoshihara H, Kondo A, Miyoshi E , Hayashi N. IgG oligosaccharide alterations are a novel diagnostic marker for disease activity and the clinical course of inflammatory bowel disease. Am J Gastroenterol 2008; 103: 1173-81.

  An object of the present invention is to provide a disease determination method that is low in invasiveness and can be objectively determined.

  The present inventors have it hinges O-linked sugar chain structure of IgA in blood and saliva is approximately the same in the same human, and the present invention found that the structure corresponding to the presence and severity of the disease Was completed.

The present invention relates to:
(1) In the determination of whether or not the subject is afflicted with the disease based on the number of N-acetylgalactosamine bonds in the hinge part O-linked sugar chain of IgA isolated from the subject's body fluid Method.
(2) The values of IgA (5N) having a sugar chain structure in which 5 N-acetylgalactosamines are bonded per IgA hinge part and IgA (4N) having a sugar chain structure in which 4 bonds are bonded are measured. The disease determination method according to (1), wherein the disease is determined to be affected when the 4N ratio (5N / 4N) is lower than a predetermined value.
(3) The method according to (1) or (2), wherein the body fluid is saliva.
(4) The method according to (1) or (2), wherein the body fluid is blood.
(5) The method according to any one of (1) to (4), wherein the disease is inflammatory bowel disease.
(6) The method according to any one of (1) to (4), wherein the disease is IgA nephropathy.
(7) A method for determining a disease, wherein IgA is isolated from the saliva of a subject, the hinge O-linked sugar chain structure of IgA is measured, and the presence or severity of the disease is determined based on the result.
(8) The method according to (7), wherein the disease is inflammatory bowel disease or IgA nephropathy.
(9) The method according to (8), wherein the disease is inflammatory bowel disease (10) Galactose (Gal) and N-acetylgalactosamine (GalNAc) contained in the hinge part O-linked sugar chain of IgA in the saliva of the subject The method according to (7), wherein the presence number of each is examined, and when the Gal / GalNAc ratio is lower than a predetermined value, it is determined that the subject suffers from or is likely to have IgA nephropathy.

  According to the method of the present invention, a patient's disease can be determined by means of extremely low invasiveness, which is considered to contribute to early detection of the disease. For diseases that require invasive examinations such as renal biopsy and endoscopy for definitive diagnosis, such as IgA nephropathy and inflammatory bowel disease, invasive examinations can be performed by using the method of the present invention. It is possible to determine in advance whether or not it is necessary, and unnecessary tests can be eliminated. It may be possible to eliminate invasive tests. In particular, both diseases have a low onset age, and it can be avoided that the method of the present invention imposes pain on pediatric patients, which is considered to contribute to an improvement in the disease detection rate. By introducing early treatment based on early diagnosis, it is possible to improve the patient's QOL and return to society, and it is expected to contribute to the reduction of patients requiring expensive medical care such as artificial dialysis, kidney transplantation, intestinal resection, etc. The social effect is great.

The relationship between IgA and a sugar chain is shown. The structure of an IgAO-linked sugar chain is shown. An example of galactose and GalNAc deficiency in the hinge part O-linked sugar chain of IgA is shown. The schematic diagram of the analysis method of the hinge part O-linked sugar chain structure of IgA is shown. An example of the MALDI-TOF MS chart of the hinge part O-linked sugar chain of IgA derived from healthy human serum is shown. An example of the MALDI-TOF MS chart of the hinge part O-linked sugar chain of IgA isolated from each serum of a healthy person and a Crohn disease patient is shown. The number of GalNAc additions per hinge part (HP) of IgA and the abundance ratio of IgA having the addition number (HV), disease control patients (DC), ulcerative colitis patients (UC), Crohn's disease patients ( CD), mean values in IgA nephropathy patients (IgAN) are shown. A method for calculating the 5N / 4N ratio in the first embodiment will be described. The average value of 5N / 4N ratio of the hinge part O-linked sugar chain of IgA derived from patients with inflammatory bowel disease, healthy subjects, and disease control serum is shown. The ROC curve (Crohn's disease patient vs. healthy person) with a 5N / 4N ratio is shown. The positive rate of each disease and a healthy person group at the time of dividing positive / negative by the cut-off value calculated from the ROC curve of FIG. 10A is shown. The relationship between 5N / 4N and Crohn's disease activity index (CDAI) is shown. The relationship between 5N / 4N and an ulcerative colitis activity index (CAI) is shown. The example of the MALDI-TOF MS analysis chart of the hinge part O-linked sugar chain of serum-derived IgA before and after infliximab treatment of a Crohn disease patient is shown. The change of 5N / 4N ratio before and after use of infliximab of Crohn's disease patient is shown. The example of the MALDI-TOF MS analysis chart of each hinge part O-linked sugar chain of IgA derived from serum, saliva and intestinal fluid of healthy persons is shown. The example of the MALDI-TOF MS analysis chart of each hinge part O-linked sugar chain of IgA derived from serum and saliva of Crohn's disease patients is shown. The example of the MALDI-TOF MS analysis chart of each of the hinge part O-linked sugar chain of IgA derived from the serum of a healthy person, Crohn's disease patient and IgA nephropathy patient and from the saliva of an IgA nephropathy patient is shown. The average of the 5N / 4N ratio of the hinge part O-linked sugar chain of IgA derived from serum of healthy subjects, patients with inflammatory bowel disease and IgA nephropathy is shown. The average of the Gal addition rate of the serum origin of a healthy subject, a patient with inflammatory bowel disease, and a patient with IgA nephropathy and the hinge part O-linked sugar chain of IgA is shown. The average of the Gal addition rate of the hinge part O-linked sugar chain of IgA derived from healthy subject serum, IgA nephropathy patient serum, and IgA patient saliva is shown.

  The present invention relates to a method for determining a disease based on the structure of a hinge part O-linked sugar chain of IgA contained in a body fluid of a subject. In the method of the present invention, the body fluid includes blood, particularly serum, saliva, intestinal fluid, etc., but it is particularly preferable to use blood or saliva. Although the relationship between the specific structure of the blood-derived IgA hinge part O-linked sugar chain and some diseases is already known (Non-patent Document 1), the sugar chain analysis is performed in saliva etc. There has been no report that examined the relationship before this application. For the first time, the inventors of the present invention have the same structure in both the serum and saliva of IgA hinge O-linked sugar chains in the same person. Like blood IgA, the hinge of IgA in saliva. It was found that the partial O-linked sugar chain structure can be used for disease determination.

  The inventors of the present application have also found a new relationship between the hinge part O-linked sugar chain of IgA and the disease contained in the subject body fluid.

  Differences in the O-linked sugar chain structure of the hinge part of IgA can be easily determined by quantifying the difference based on the number of N-acetylgalactosamine, galactose, or N-acetylgalactosamine and galactose added per hinge part. It can be used as a criterion.

  In determining the disease, after determining the measurement protocol and numerical method of the O-linked sugar chain binding structure of the IgA hinge part, the protocol has a disease to be determined according to the conventional determination criteria. Alternatively, data on body fluid of a patient whose severity is diagnosed and body fluid of a healthy person are collected, the data is analyzed by an analysis method such as an ROC curve, and a determination criterion is set in advance. The presence or severity of the target disease may be determined based on the above.

In the method of the present invention, the structure of the hinge part O-linked sugar chain of IgA in body fluid can be easily measured by combining conventionally known methods.
When saliva is employed as the body fluid, IgA is isolated from the saliva of the subject using an anti-IgA affinity gel. The isolated IgA is reduced and alkylated with dithiothreitol and iodoacetamide to release the higher order structure of IgA. The protein is then broken down into peptides with trypsin as well as lysyl endopeptidase. In organic solvents, the linkage between carbohydrate polymer (Sepharose) and sugar chain is emphasized, and this principle is used to concentrate and separate glycopeptides. What is necessary is just to mass-analyze this by MALDI-TOF MS and identify a sugar chain structure. FIG. 4 shows a schematic diagram of the purification procedure.

  When blood is used as a body fluid, serum is obtained from blood collected as usual, IgA is isolated from the obtained serum using an anti-IgA affinity gel, and glycopeptides are then isolated in the same manner as in the case of saliva. This may be separated and mass spectrometry may be performed by MALDI-TOF MS.

  In a preferred embodiment of the present invention, the obtained glycopeptide is heated with acetic acid to remove sialic acid, and mass spectrometry is performed by MALDI-TOF MS to identify the sugar chain structure. The distribution of the number of bindings of galactose and N-acetylgalactosamine per hinge part is examined by MALDI-TOF MS. By using MALDI-TOF MS, the addition pattern of galactose (Gal) and N-acetylgalactosamine (GalNac) per hinge part can be known, and the relative ratio of each can be obtained. FIG. 4 shows a glycopeptide purification scheme, and FIG. 5 shows a measurement example. FIG. 5 shows the results of examining the addition pattern of GalNAC and Gal in the serum of healthy subjects. The relative intensity (peak height) of each MS spectrum correlates with the number of each additional pattern.

  One index for determining the difference in the structure of the hinge part O-linked sugar chain of IgA provided by the present invention is the GalNAc addition rate of the hinge part O-linked sugar chain of IgA. This is determined by using the added number of GalNAc per hinge as an index. As an example of the index of the addition rate of GalNAc, a ratio (5N / 4N ratio) of (5N) IgA to which five GalNAcs are added and (4N) IgA to which four GalNAcs are added can be used.

  In patients with inflammatory bowel disease, the number of GalNAc additions in the hinge part O-linked sugar chain of IgA decreases. This decrease is more pronounced in Crohn's disease patients.

  Therefore, in the present invention, the disease is determined using the GalNAc addition rate as an index. As an index of the addition rate of GalNAc, the ratio (5N / 4N ratio) of the peak corresponding to the hinge part to which five GalNAcs were added and the peak corresponding to the hinge part to which four GalNAcs were added (5N2H + 5N3H + 5N4H + 5N5H) / (4N2H + 4N3H + 4N4H) Calculate using the formula.

  Comparing the ratio of IgA in 5N / 4N of and saliva blood between healthy subjects and inflammatory bowel disease patients, in inflammatory bowel disease patients is lower significantly 5N / 4N ratio. In particular, the decrease in the 5N / 4N ratio is significant in Crohn's disease. The 5N / 4N ratio shows a negative correlation with the disease activity index. That is, the lower the disease activity index is, the higher the 5N / 4N ratio is, which is an index indicating the severity of the disease. Moreover, since the increase of 5N / 4N ratio is recognized by treatment, it can also be used for judgment of the effectiveness of treatment.

  In this embodiment, when the 5N / 4N ratio in the hinge part O-linked sugar chain of IgA obtained from the subject body fluid is lower than a preset value, the patient is suffering from inflammatory bowel disease or the possibility thereof Can be determined to be high. Furthermore, it can be determined that the lower the value, the higher the severity.

  Further, in a patient suffering from inflammatory bowel disease, when the 5N / 4N ratio is lower than a preset value, it can be determined that the inflammatory bowel disease is Crohn's disease.

  Here, "predetermined value", after determining the measurement protocol of O-linked sugar chain binding patterns of IgA hinge at the protocol, diagnosed with inflammatory bowel disease by criteria conventionally patients are either Crohn's disease, if necessary, is a value that is set in terms of the data accumulation of ulcerative colitis in which one of a body fluid of a patient diagnosis is, and for healthy individuals body fluids. The bodily fluid used in this embodiment is exemplified by saliva or serum.

  For example, when the 5N / 4N ratio in the hinge part O-linked sugar chain of IgA in serum, which is the method used in the examples described in the present specification, is used, a negative / positive cutoff value of 5N / 4N is obtained. For example, by setting a value lower than 0.90 as positive, it becomes possible to distinguish inflammatory bowel disease patients with high probability.

  Furthermore, by monitoring the 5N / 4N ratio in O-linked sugar chains of IgA hinge portion of the blood in inflammatory bowel disease patients, it is possible to determine the severity and treatment efficacy of the disease.

  The 5N / 4N ratio of the O-linked sugar chain of the IgA hinge region is also decreased in IgA nephropathy patients. Therefore, the present invention determines that the patient is suffering from or highly likely to suffer from IgA nephropathy when the 5A / 4N ratio of the hinge part O-linked IgA obtained from the subject body fluid is lower than a preset value. Provide a way to Here, blood and saliva are exemplified as the body fluid. Similarly to the above, the preset value is a value set based on the data of the healthy person and the patient after determining the measurement protocol.

  In another aspect of the present invention, the galactose addition rate is calculated from the relative ratio of each addition number of galactose (Gal) and N-acetylgalactosamine (GalNAc) per hinge obtained by MALDI-TOF MS and used. To determine the patient. As the galactose addition rate, for example, a value obtained by adding Gal addition number × peak height for each pattern and dividing the result by adding GalNAC addition number × peak height for each pattern may be used. it can. In this specification, this value is referred to as “Gal addition rate”.

  In the serum of IgA nephropathy patients, the Gal addition rate is significantly lower than that of healthy individuals. This is consistent with the report that the serum IgA sugar chain of a patient with IgA nephropathy known in the prior art is Gal deficient. And since the Gal addition rate of the hinge part O-linked sugar chain of IgA derived from saliva derived from IgA nephropathy also shows the same decrease as the sugar chain derived from serum, the hinge part O-linked sugar of IgA derived from the target saliva Provided is a method for determining IgA nephropathy, in which it is determined that the chain has a Gal addition rate lower than a predetermined value and is suffering from or likely to have IgA nephropathy.

  In even a "predetermined value" this embodiment, after determining the measurement protocol of the hinge portion O-linked sugar chain binding patterns of saliva from IgA, in the protocol, is IgA nephropathy by criteria conventionally May be set based on the data on the saliva of a patient diagnosed with and the saliva of a healthy person.

  In the above description, inflammatory bowel disease and IgA nephropathy have been described as examples. However, the method of the present invention is expected to be applied to other diseases involving IgA. Examples of such diseases include diseases in which IgA contained in mucus contributes to the pathological condition in the oral cavity, dentistry, otolaryngology, digestive organs, respiratory organs, immunity, ophthalmology, dermatology, and the like, including inflammatory bowel disease and IgA kidney Examples include periodontal disease, atopic dermatitis, allergic rhinitis, keratoconjunctivitis and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples. The examples are not intended to limit the present application in any way.

  IgA sugar chains were isolated from blood and saliva of patients with inflammatory bowel disease and healthy subjects, and the binding patterns of galactose (Gal) and N-acetylgalactosamine (GalNAc) in the hinge O-linked sugar chain were examined.

30 healthy individuals (HV), 30 patients with ulcerative colitis (UC), 32 patients with Crohn's disease (CD), and 17 patients with disease control (DC) listed in Table 1, 9 patients with IgA nephropathy (IgAN) Serum was obtained from the peripheral blood of the case by a conventional method.

  Purification of IgA from serum was performed using an anti-IgA affinity gel prepared using anti-human IgA antibody (MBL) and HiTrap ™ NHS-activated HP Columns (GE healthcare) according to the column protocol. 20 μL of serum, 20 μL of gel, and 500 μL of PBS were mixed in a tube and stirred for 2 hours. After the gel was washed, IgA was eluted with 50 μL of 0.1 M glycine (pH 2.0).

  The obtained glycopeptide was analyzed by Voyager DE (trademark) Pro MALDI-TOF-MS (Applied Biosystems). 0.1 to 1 pmol of purified glycopeptide was dissolved in 10 mg / mL 2,5-dihydroxybenzoic acid (DHB), 1 μL was placed on a sample plate, dried and crystallized, and then measured in positive ion mode. did.

  By MALDI-TOF MS analysis, the relative amount for each binding pattern of Gal and GalNAc per hinge part can be obtained. FIG. 5 shows a MALDI-TOF MS chart of GalNAC and Gal binding pattern contained in the hinge part O-linked sugar chain of IgA in a typical healthy human serum. FIG. 6 shows a MALDI-TOF MS chart of the hinge part O-linked sugar chain of IgA between healthy subjects and Crohn's disease patients.

  From the chart obtained from each subject, the number of GalNAc additions per hinge part and the abundance ratio of IgA indicating the number of additions were examined, and average values were obtained for each patient, healthy subject, and disease control group. The results are shown in FIG. From FIG. 7, it was found that the abundance ratio of glycopeptides to which GalNAC is 5-bonded was significantly low in Crohn's disease patients.

The number of GalNAc additions per IgA hinge was adopted as an index of sugar chain change. Regardless of the number of Gal additions, the abundance ratio of the hinge part (5N) to which 5 GalNacs are added and the hinge part (4N) to which 4 GalNAcs are added, that is, the 5N / 4N ratio is (5N2H + 5N3H + 5N4H + 5N5H) / (4N2H + 4N3H + 4N4H) As calculated. A method for calculating the 5N / 4N ratio is illustrated in FIG. Moreover, the average value of each group was calculated. The results are shown in FIG.
As shown in FIG. 9, the 5N / 4N ratio was significantly lower in patients with inflammatory bowel disease than in healthy individuals and disease controls. This decrease was particularly remarkable in patients with Crohn's disease.

  An ROC curve was created between the 5N / 4N ratio and the disease specificity of Crohn's disease patients and healthy individuals. The ROC curve is shown in FIG. The most distinction point was obtained from the obtained ROC curve, and 0.90 was calculated as a cutoff value. When 0.90 and cut-off value of prevalence, but 87.5% of CD patients are included in the positive group, only 6.7% in healthy people not become positive group, both differential is sufficiently possible (FIG. 11).

Furthermore, the relationship between the 5N / 4N ratio of patients with Crohn's disease (CD) and ulcerative colitis (UC) and the clinical parameters of each patient was examined. Disease activity was determined by Crohn's disease activity index (CDAI) for Crohn's disease patients and by Ulcerative colitis activity index (CAI) for patients with ulcerative colitis. FIG. 11 shows a graph in which the disease activity of each patient is plotted on the vertical axis and the 5N / 4N ratio is plotted on the horizontal axis.
As is clear from FIG. 11, the 5N / 4N value showed a negative correlation, and it was shown that the 5N / 4N ratio can also be used to determine the severity of the disease.

For 5 patients with Crohn's disease, blood was collected before and after treatment with infliximab (trade name: Remicade), and the 5N / 4N ratio of the hinge O-linked sugar chain of IgA in blood was measured.
Patients: Age 27 ± 7 years, 3 males, 2 females, CRP 2.0 ± 1.0, CDAI 218 ± 70 (before infliximab administration)
Treatment: Infliximab was administered at a dose of 5 mg / Kg at 0, 2, 6 weeks, and thereafter maintenance was administered every 8 weeks. Serum was collected before infliximab and 6 weeks after the first dose.

  The same technique as in Example 1 was used for isolation of IgA and measurement of the hinge part O-linked sugar chain by MALDI-TOF. FIG. 12 shows a typical MALDI-TOF MS chart of healthy subjects and Crohn's disease patients before and after treatment, and FIG. 13 shows changes in the 5N / 4N ratio before and after infliximab treatment.

  As is clear from FIG. 13, the 5N / 4N ratio showed an upward trend with infliximab treatment. Therefore, the 5N / 4N ratio is useful as an indicator of changes in the degree of disease due to treatment.

Comparison of the hinge part O-linked sugar chain structure of IgA in blood, saliva, and intestinal fluid IgA was isolated from blood, saliva, and intestinal fluid of a healthy person (31 years old, male) and treated in the same manner as in Example 1. A MALDI-TOF MS chart of the hinge part O-linked sugar chain was prepared.
Mix 100 μL of saliva and intestinal fluid with 20 μL of affinity gel and 500 μL of PBS and stir for 2 hours. As in the case of serum, the supernatant is collected after centrifugation and washed. Wash three times with 500 μL of PBS and elute with 0.1 M glycine (pH 2.0) to isolate.
The results are shown in FIG. It is a chart which shows distribution of the sugar chain structure of IgA isolated from serum, saliva, and intestinal fluid. It is clear from the chart that the three have almost the same structure.

  When the 5N / 4N ratio was calculated based on the obtained chart, it was serum: 1.08, saliva: 1.08, and intestinal fluid: 1.04. From the viewpoint of the 5N / 4N ratio, IgA in saliva and intestinal fluid was It can be seen that the sugar chain structure is almost the same as IgA in blood.

  IgA was isolated from the blood and saliva of a Crohn's disease patient (41-year-old female, small intestine large intestine type), and a MALDI-TOF MS chart of the hinge O-linked sugar chain was obtained in the same manner as in Example 1. The obtained chart is shown in FIG. It is clear from the chart that both the serum-derived and saliva-derived structures have the same structure.

  When a 5N / 4N ratio based on the obtained chart was calculated, serum: 0.76 and saliva: 0.85, the IgA in saliva has substantially the same sugar chain structure as IgA in blood I understand. Therefore, even if the same test performed using blood in Examples 1 and 2 is performed using saliva, it is presumed that similar results can be obtained.

The relationship between IgA nephropathy and IgA hinge O-linked sugar chain was examined.
Specimens of IgA nephropathy patients (4 males, 5 females, age 33.2 ± 6.8 years, creatinine 0.82 ± 0.18) that were confirmed by renal biopsy were used.

  Blood and saliva were collected from patients with IgA nephropathy, and IgA was isolated by the same method as in Example 1 and Example 3. A pattern by MALDI-TOF MS of the obtained IgA hinge O-linked sugar chain was obtained in the same manner as in Example 1. FIG. 16 shows a MALDI-TOF MS chart of a typical IgA nephropathy patient's serum and saliva-derived IgA hinge O-linked sugar chain. For comparison, a chart of the hinge part O-linked sugar chain of serum-derived IgA from healthy subjects and Crohn's disease patients obtained in Example 1 is also shown.

  As is clear from FIG. 16, the patterns of IgA nephropathy patients' serum and saliva-derived IgA hinge O-linked sugar chains were almost the same. This pattern behaved differently from the charts of healthy individuals and Crohn's disease patients.

The 5N / 4N ratio was obtained from the MALD-TOF MS chart of the O-linked sugar chain of each IgA nephropathy patient serum-derived IgA hinge part, and the average value was obtained. The results are shown in FIG. For comparison, the data are shown together with HV, DC, UC and CD data obtained in Example 1.
As is clear from FIG. 17, the 5N / 4N ratio of serum-derived IgA of IgA nephropathy patients was significantly lower than that of healthy subjects and disease control groups.

  As another index, the galactose addition rate was calculated. The galactose addition rate is obtained by adding Gal addition number × peak height for each pattern, and dividing this by adding GalNAC addition number × peak height for each pattern. The galactose addition rate was similarly calculated based on the MALDI-TOF MS chart obtained from healthy subjects obtained in Example 1, patients with disease control, Crohn's disease, and ulcerative colitis. The results are shown in FIG.

  In IgA obtained from the sera of patients with Crohn's disease (CD) and ulcerative colitis (UC), the galactose addition rate was not different from that of healthy individuals (HV) and disease control (DC) groups. In the serum IgA hinge O-linked sugar chain, a clear decrease in the galactose addition rate was observed.

  Subsequently, the galactose addition rate was calculated from the MALDI-TOF MS chart of the hinge part O-linked sugar chain of IgA derived from saliva of IgA nephropathy patients. Although the number of samples was small and no significant difference was found, almost the same value as blood-derived IgA was obtained. The results are shown in FIG.

  The present invention provides a method for determining a less invasive disease used for the determination of a wide range of diseases including refractory diseases such as inflammatory bowel disease and IgA nephropathy. The present invention, simple and provides a determination method of sharing is very little disease to a patient, the method alternative to kidney biopsy and endoscopy, or avoid unnecessary renal biopsy and endoscopy Can be used effectively.

Claims (10)

A method for determining a disease, comprising determining whether or not the subject suffers from a disease based on the number of N-acetylgalactosamine bonds in a hinge O-linked sugar chain of IgA isolated from the body fluid of the subject. The respective values of IgA (5N) having a sugar chain structure in which 5 N-acetylgalactosamine is bonded per IgA hinge part and IgA (4N) having a sugar chain structure in which 4 is bonded are measured, and the ratio of 5N to 4N is measured. The disease determination method according to claim 1, wherein the disease is determined to be affected when (5N / 4N) is lower than a predetermined value. The method according to claim 1 or 2, wherein the body fluid is saliva. The method according to claim 1 or 2, wherein the body fluid is blood. The method according to any one of claims 1 to 4, wherein the disease is inflammatory bowel disease. The method according to any one of claims 1 to 4, wherein the disease is IgA nephropathy. A method for determining a disease, wherein IgA is isolated from the saliva of a subject, the hinge O-linked sugar chain structure of IgA is measured, and the presence or severity of the disease is determined based on the result. 8. The method of claim 7, wherein the disease is inflammatory bowel disease or IgA nephropathy. 9. The method of claim 8, wherein the disease is inflammatory bowel disease. When the number of galactose (Gal) and N-acetylgalactosamine (GalNAc) contained in the hinge part O-linked sugar chain of IgA in the subject's saliva is examined, and the Gal / GalNAc ratio is lower than a predetermined value 9. The method of claim 8, wherein the subject is determined to have or likely have IgA nephropathy.