JPWO2013172347A1 - Method for detecting IgA aggregate and method for examining IgA nephropathy - Google Patents

Abstract

Provided is a novel method for detecting IgA nephropathy using a lectin having binding specificity for the characteristic structure of IgA aggregates found in IgA nephropathy patients.

Description

  The present invention provides a simple method for detecting an IgA aggregate in the serum or urine of a patient with IgA nephropathy, and enables early diagnosis of IgA nephropathy.

IgA nephropathy (also referred to as IgAN) is a chronic glomerulonephritis characterized by the formation of immune complexes and IgA, a type of immunoglobulin, that deposits in the glomerular mesangial region. About 40% are extremely poor prognosis diseases that shift to renal failure within 20 years.
This disease is increasing in the number of patients all over the world, but particularly in Asia Pacific and Europe. For example, in Japan, the total number of patients receiving dialysis therapy due to end stage renal failure exceeds 260,000. A large amount of money is spent on medical expenses of 1.4 trillion billion (3% of total medical expenses) annually. In addition, the number of new dialysis patients who are caused by IgA nephropathy is very large at 4 to 5,000 per year, but there are many unclear points about the onset mechanism of this nephropathy, and a method for appropriately treating it has not been established. Therefore, it is necessary to detect this disease at an early stage and to apply dietary therapy and drug therapy.
By the way, at present, a highly invasive renal biopsy is used as the only clinical method for the definitive diagnosis of IgA nephropathy. However, despite the heavy burden on patients, renal biopsy does not provide accurate prognosis, and multiple tests cannot be performed in a short period of time. It is not possible to determine whether the treatment is properly performed. Thus, renal biopsy lacks consistency as a clinical diagnostic marker, and it is difficult to appropriately treat a patient based on the result of renal biopsy.
Based on the above, there is an urgent need for early diagnosis of IgA nephropathy patients and development of new serum markers that can easily and quantitatively determine symptoms in patients with IgA nephropathy before dialysis.

  By the way, IgA nephropathy is characterized by the deposition of an immune complex containing IgA1 in the glomerular mesangial region, but it has been reported that the sugar chain structure on IgA1 in the patient's serum has changed. (Non-patent documents 1 to 3). In particular, there have been many reports on the O-glycan structure on IgA1, such as the presence of an abnormal O-glycan structure lacking galactose on O-glycan in the serum IgA1 of IgAN patients. It is shown by using an antibody detection method (Non-patent Documents 4 to 6, Patent Documents 1 to 5). However, there has been no report that such abnormal IgA having an abnormal O-glycan structure was actually separated, purified or concentrated from serum.

Recently, we compared IgA in patients with monoclonal IgA deposition (mIgA-MIDD), disease control (mIgA), and healthy subjects. As a result, IgA in mIgA-MIDD patients showed that Core-fucose and sialic acid on N-glycan of IgA It has been reported that the structure of is changed (Non-patent Document 7). However, it is not clear how such changes in sugar chain structure are involved in glomerular deposition.
Thus, although it has been reported that changes in glycan structure occur in IgA in patients with nephropathy, how these glycan structure changes are related to the development of IgA nephropathy. It is not clear whether this is the case, and no method for easily detecting this change in sugar chain structure has been found.

J Am Soc Nephrol. 1999 Apr; 10 (4): 760-9. Kidney Int. 2001 Sep; 60 (3): 969-73. Clin Exp Nephrol. 2008 Oct; 12 (5): 332-8. J Clin Invest. 2009 Jun; 119 (6): 1668-77 Nephrol Dial Transplant. 2008 Jun; 23 (6): 1931-9. Nephrol Dial Transplant. 2002 Jan; 17 (1): 50-6. Clin Exp Nephrol. 2010 Aug; 14 (4): 389-95

JP 2010-285419 A JP 2009-001512 A JP 2007-024661 JP 2007-163151 A Japanese Patent Laid-Open No. 10-111291

  An object of the present invention is to provide a method for easily diagnosing or detecting IgA nephropathy using a sample such as serum or urine collected from a patient with IgA nephropathy, which is not invasive and has a low burden on the patient. .

The inventors focused on an abnormal IgA molecule deposited in the glomeruli that causes IgA nephropathy, and it is considered that this abnormal molecule is present in the blood in a large amount. Monoclonal immunoglobulin disease (MGUS) and IgA kidney We used a patient with mIgA-MIDD with comorbid nephropathy as a model, and purified and identified an abnormal IgA molecule in the serum of this patient.
As a result, it was found that IgA1 in the serum of this patient has a molecular structure in which N-glycan is abnormally added and the number of sugar chains per molecule is large. Further, the present inventors have found that the IgA1 forms a multimer that is not found in normal IgA and exists as an aggregate molecule that is highly coated with a sugar chain. Thus, until now, regarding IgA of patients with nephropathy, attention has been paid only to the difference in sugar chain structure from normal (non-disease) IgA. It was clarified that there were differences in the three-dimensional structures such as the number of added chain structures and the formation of IgA aggregates.
Furthermore, when the inventors measured the lectin array for this IgA aggregate (abnormal IgA), they found various lectins that react specifically with the IgA aggregate. And the method of diagnosing / detecting IgA nephropathy simply using the lectin which reacts specifically with this IgA aggregate was discovered.
That is, this specification provides the following inventions.

(1) A method for detecting an IgA aggregate, which comprises contacting a sample containing IgA with a lectin that specifically reacts with the IgA aggregate.
(2) The method according to (1), wherein the binding ability (binding amount) of the lectin to the IgA aggregate is measured.
(3) The method according to (1) or (2), wherein the measurement of the binding ability (binding amount) is based on the recognition property of the lectin for non-reducing terminal β14 galactose on N-glycan.
(4) The lectin is at least one lectin selected from the group consisting of HHL, GNA, NPA, WFA, SBA, VVA, BPL, TJA-II, PHA-L, and AOL lectin (1) The method in any one of-(3).
(5) The method according to any one of (1) to (3), wherein the lectin is a WFA lectin.
(6) The method according to any one of (1) to (5), wherein the sample containing IgA is serum or urine of a subject.
(7) An IgA nephropathy test method, wherein IgA aggregates are detected by the method according to any one of (1) to (6), and IgA nephropathy is tested based on the detection result.
(8) The method according to (7), wherein the IgA nephropathy is mIgA-MIDD.
(9) A method of purifying or concentrating molecules of the IgA aggregate by binding a lectin that specifically reacts with the IgA aggregate.
(10) Examination method of IgA nephropathy including the following procedures:
1) Contact IgA in serum or urine collected from a subject with WFA lectin;
2) The IgA1 monoclonal antibody is analyzed by the antibody overlay method, the amount of IgA bound to the WFA lectin is measured, and IgA nephropathy is determined by comparing the amount of IgA bound to the WFA lectin with that of a healthy person.
(11) The method according to (10), wherein the amount of IgA bound to the WFA lectin is measured by analyzing by an antibody overlay method using an IgA1 monoclonal antibody.

  According to the present invention, IgA nephropathy can be easily diagnosed or detected without invasiveness and less burden on the patient.

The N-glycosylation site (Asn263, Asn459) in the FC region in the α chain in which the structure of IgA1 is conserved and the O-glycosylation site of the hinge part are shown. The results of SDS-PAGE (left: silver staining) of purified IgA1 from various sera and Western blotting analysis (right) using an anti-IgA1 monologous antibody are shown (photos). Lectin microarray analysis using 1,40 ng of IgA purified from each serum. The results for all 43 lectins subjected to analysis are shown. Each lectin signal was corrected by dividing by the highest signal lectin TJA-1. Lectin microarray analysis using 1,100 ng of IgA purified from each serum. Here are 12 excerpts of lectins that were significantly different from patients with mIgA-MIDD. The signal for each lectin was corrected by dividing by the highest signal lectin TJA-1. The lectin microarray analysis result using the trypsin digest. Reactivity of WFA lectin to various glycosidase digested IgA1. The change of the reactivity of WFA lectin with IgA1 derived from mIgA-MIDD by PNGase digestion treatment. Measurement of lectin microarray of heat-treated IgA1. Reactivity of WFA to mIgA-MIDD-derived purified IgA1 under non-reducing conditions (photo). Detection of N-glycan addition number using PNGase F treatment (photo). Measurement of molecular weight of each specimen-derived purified IgA1 (photo). Comparative analysis with Sandwich lectin EILSA using WFA lectin. Separation and purification 1 (photo) of mIgA-MIDD using WFA lectin. Separation and purification of mIgA-MIDD using WFA lectin 2 (photo). Examination of mIgA-MIDD purification conditions using WFA lectin. Lectin microarray measurement on serum using anti-IgA antibody overlay method (photo).

  The method of the present invention is a method for detecting an IgA aggregate containing IgA (abnormal IgA) in which N-glycan is abnormally added and aggregates to form a multimer, and a method for examining IgA nephropathy by this method.

  IgA may include IgA1 and IgA2, but only about IgA1 is detected because IgA1 is predominantly about 90% in monomer or dimer IgA (serotype IgA) present in serum or urine. The method of the present invention may be carried out.

  Here, IgA nephropathy is chronic glomerulonephritis characterized by IgA deposition in the glomerular mesangial region, and includes, for example, monoclonal IgA deposition (mIgA-MIDD).

  For example, a case where the number of N-glycan added per molecule of IgA is 4 or more as compared with healthy individuals is exemplified. Abnormal addition of N-glycan can be confirmed by ordinary sugar chain analysis, but the band pattern of IgA heavy and light chains may be examined by electrophoresis or the like.

  If IgA aggregates are formed by the abnormal addition of N-glycan, and this promotes IgA deposition in the glomeruli, IgA nephropathy is ultimately caused. Therefore, when abnormal addition of N-glycan is observed and / or when the amount thereof is higher than that of healthy subjects, the subject can be determined to have IgA nephropathy.

  In the method of the present invention, the lectin is preferably contacted with β1,4 galactose on the N-glycan of the IgA aggregate.

  The lectins that specifically react on the above IgA aggregates (abnormal IgA) include HHL (Hippeastrum Hybrid-derived lectin), GNA (Galanthus nivalis-derived lectin), NPA (Narcissus pseudonarcissus-derived lectin), WFA (Wisteria floribunda-derived lectin) , SBA (Glycine max-derived lectin), VVA (Vicia villosa-derived lectin), BPL (Bauhinia purpurea-derived lectin), TJA-II (Trichosanthes japonica-derived lectin), PHA-L (Phaseolus vulgaris-derived lectin), AOL (derived from Aspergillus oryzae) Preferably, the lectin is at least one lectin selected from the group consisting of lectins, and more preferably WFA. These lectins are anal biochem.1991 Dec; 199 (2): 286-92, Biochim Biophys Acta.1998 Apr 10; 1380 (2): 268-74, Int J Cancer, 2003, Jul 1; 105 (4): 533-41, Glycobiolog. Sep; 16 (9): 777-85, etc. it can.

The detection of IgA using lectin is performed by, for example, mixing a sample such as serum or urine with a carrier to which lectin is bound, binding IgA in these samples to lectin, and then detecting with a labeled anti-IgA antibody or the like. be able to.
For example, IgA in serum or urine collected from a subject is contacted with WFA lectin immobilized on a plate; IgA1 monoclonal antibody (eg, 7303B) is analyzed by antibody overlay method and IgA bound to WFA lectin The amount can be measured.
Then, the amount of IgA bound to WFA lectin is compared with the amount of IgA bound to WFA lectin when contacted with serum or urine of a healthy person, and can be determined as IgA nephropathy when the amount is higher than that of a healthy person .

In addition, after lectin is biotinylated and IgA in serum or urine is bound to biotinylated lectin, it can also be detected by avidin labeled with an enzyme or fluorescence.
When IgA is detected by WFA, sialidase treatment is more preferable because detection sensitivity is improved.

  IgA nephropathy can also be determined by measuring the binding ability of WFA lectin to IgA in a sample such as serum or urine isolated from a subject. The binding ability can be determined by quantifying the amount (binding amount) of IgA that interacts with the lectin or by calculating the dissociation constant by a commonly used interaction analysis technique.

  In addition, IgA aggregates derived from IgA nephropathy patients can be purified or concentrated using a lectin such as WFA.

  Examples of the present invention will be described below. However, the examples given here are merely specific illustrations, and do not limit the technical scope of the present invention.

<Example 1> Analysis of lectin reactivity of IgA1
(1) Separation and purification method of IgA1 from serum Serum samples used were mIgA-MIDD, benign disease (MGUS patient) (mIgA), and healthy subjects. 500 μL of serum was diluted to 5 mL with 0.01 M Tris-HCl buffer and applied to a Cibacron Blue A agarose column (1 mL Column volume; Sigma Chemical Co). After the sample was provided, it was washed with 0.01 M Tris-HCl buffer, 6 mL, and a flow-through fraction and a washed fraction were collected. Subsequently, the collected sample was applied to a column (500 μL column volume) prepared by binding anti-IgA1 monoclonal antibody (7393B) to Sepharose 4B. After washing with 20 mL of PBS, elution was carried out using 3 mL of 0.1 M Glycine / HCl (pH 2.5) buffer, and immediately neutralized by adding 400 μL of 1 M Tris-HCl (pH 8.0). Furthermore, 100 μL of protein G sepharose (Pierce) was added to the eluted fraction and reacted overnight at 4 ° C. to remove mixed IgG. The supernatant was collected, dialyzed against 0.01M ammonium bicarbonate buffer, and lyophilized. The sample after lyophilization was dissolved in PBS buffer, the concentration was measured using an absorptiometer, and the concentration was adjusted to 1 mg / mL. The purity of the sample was confirmed by the following SDS-PAGE and Western blot.

(2) SDS-PAGE and Western blotting purification IgA1 analysis Purified IgA1 samples were reduced in the presence of 2-mercaptoethanol, then subjected to SDS-PAGE using a 10% polyacrylamide gel, and purified by silver staining. Was confirmed. In addition to the silver staining, the SDS-PAGE gel was transferred to a PVDF membrane and subjected to Western blotting. The transferred PVDF membrane was blocked with 3% BSA / 0.1% Tween / PBS buffer at room temperature for 1 h, and then diluted with a 1000-fold diluted solution of 1 μg / μL anti-IgA1 monoclonal antibody (7303B, Special Immunology Laboratories) The reaction was allowed to proceed overnight at 4 ° C. After washing with 0.1% Tween / PBS buffer for 5 min three times, a solution obtained by diluting anti-IgG-HRP antibody (GE healthcare) 5000 times was used and reacted at room temperature for 1 h. After washing with 0.1% Tween / PBS buffer for 5 min three times, using Western lightning CHemiluminescence Plus (Perkin-Elmer) as a substrate, developing to high-perfomance chemiluminescence film (GE Healthcare), and developing the target IgA1 band Detection was performed. FIG. 2 shows the above results.

(3) Antibody-overlay lectin microarray comparative analysis for purified IgA1
10 ng, 50 ng and 100 ng of each purified IgA1 was diluted to 80 μL with 0.1% Triton x / PBS solution (PBS-Tx), and lectin microarray slide glass (Kuno et al. Nature Methods 2005
Prepared by the method described in Uchiyama et al Methods in Enzymology 2006, Uchiyama et al Proteomics 2008) and reacted at 20 ° C. for 18 hours. Use 1 μg of human serum polyclonal IgG (SIGMA), react at 20 ° C. for 30 min, wash 3 times with 80 μL of PBS-Tx, biotinylated 6.25 μg / mL concentration of biotinylated anti-IgA1 antibody ( 7303B) was applied at 80 μL and reacted at 20 ° C. for 1 h. After further washing 3 times with 80 μL PBS-Tx, 80 μL of 2.5 μg / mL concentration of streptavidin-Cy3 was applied and reacted at 20 ° C. for 1 h. After washing 3 times with 80 μL PBS-Tx, 80 μL PBS-Tx was applied, and measurement was performed using Glycostation (Moritex). The images measured at Gain 125 to 100 were converted into numerical values from various lectin signals using Array Pro analyzer version 4.5, and then subjected to comparative analysis between patients and healthy subjects.

  FIG. 3 shows the results of lectin microarray analysis using IgA1 purified from each serum. Analysis was performed using 1,40 ng of IgA purified from each serum, and various values were obtained for the lectins in which signals were observed. FIG. 3 shows 43 lectins placed on a slide glass, and the signal of each lectin was corrected by dividing by the value of TJA-1 lectin having the highest signal.

In FIG. 4, 100 ng of IgA1 purified from each serum was used, and various numerical values were obtained for the lectins in which signals were observed. Similarly to FIG. 3, the signal of each lectin was corrected by dividing by the value of TJA-1 lectin having the highest signal. In FIG. 4, 12 types of lectins that were remarkably different in mIgA-MIDD patients are extracted and shown.
From the above study, (1) mIgA-MIDD> MGUS and mIgA-MIDD showing healthy lectin binding to IgA patients with high binding to IgA, (2) MGUS and normal> mIgA-MIDD binding These were classified into lectins having high binding properties to mIgA and healthy individuals, and are listed below. Moreover, the recognition site | part of the sugar chain of each lectin reported so far was shown by the arrow.

  Therefore, in order to investigate the reactivity between lectin and IgA1, an experiment was conducted using a tryptic digest of IgA1.

<Example 2> Analysis of structural features of IgA1
(1) Trypsin digestion of purified IgA1 500 μg of purified IgA1 was subjected to reduction treatment in 0.1 M Tris-HCl (pH 7.4) buffer in the presence of 10 mM dithiothreitol at 37 ° C. for 45 min, followed by 50 mM Iodoacetoamide at room temperature. Shading and 90 min alkylation were performed. The reduced alkylated sample was dialyzed against 2.5 L of 50 mM ammonium bicarbonate buffer at 4 ° C. for 48 h. Of the dialyzed sample, 100 μg of IgA1 was reacted with 1 μg trypsin at 37 ° C. for 1 h. The tryptic digest after the reaction was desalted using a reverse phase column Oasis HLB catridge (10 mg / mL; Waters). The operating procedure of the reverse phase column was that the trypsin digest solution was applied to the column which had been equilibrated or operated in the order of 3 mL of MeOH and 3 mL of pure water, and washed with 3 mL of pure water. After washing, the sample eluted with 3 mL of 80% acetonitrile solution was lyophilized.

(2) Lectin microarray analysis of IgA1 trypsin digest The freeze-dried trypsin digest was adjusted to a concentration of 1 mg / ml with a PBS solution. Of the prepared sample, a trypsin digest solution corresponding to 1 μg was labeled with 10 μg of Cy3-succimidyl ester (GE Healthcare) at room temperature for 1 h under light-shielding conditions. In order to invalidate the unreacted Cy3 reagent, 500 mM glycine / 1% Triton X-100 / TBS (probing buffer) was added, and blocking treatment was performed at room temperature for 2 h under light-shielding conditions. Finally, 80 μL of the sample diluted to 10 μg / mL with probing buffer was applied to a lectin microarray and reacted at 20 ° C. for 12 h. After the reaction, the membrane was washed three times with a probing buffer, 80 μL of probing buffer was added, and measurement was performed using Glycostation. Signal detection, data processing methods, and the like are the same as those in the lectin overlay section described above.

As shown in FIG. 5, the lectin PNA recognizing O-glycan was increased in the digested trypsin compared to the whole sample data, suggesting that the trypsin digestion treatment enhanced the reaction with O-glycan. Is done. On the other hand, the WFA signal specifically observed in mIgA-MIDD was significantly reduced by trypsin digestion.
From the above results, it was shown that the specific binding force of WFA mIgA-MIDD to IgA1 molecule recognizes N-glycan, not O-glycan.
Therefore, in order to investigate what structural sites of WFA lectin recognize N-glycan, HPA, VVA, various WFA lectins, and Core1 ( Sandwich lectin ELISA was performed using PNA lectin that recognizes Gal-GalNAc).

(3) Preparation of sugar chain-deficient IgA1 using various glycosidases
Purified IgA derived from mIgA-MIDD (1,10 μg) was diluted to 25 μL with 200 mM sodium acetate buffer and reacted overnight at 37 ° C. using 20 mU sialidase (Nacalai Tesque). Add 5 μL of the reacted sample to the sample, and add 1.1 mU β-galactosidase, 1 mU β1,3 galactosidase, 1.5 mU β1,4 galactosidase to the solution, and continue at 37 ° C overnight. Reacted. Finally, a total of 8 types of untreated, sialidase (neuraminidase), β galactosidase, β1,3 galactosidase, β1,4 galactosidase, sialidase + β galactosidase, sialidase + β1,3 galactosidase, sialidase + β1,4 galactosidase were prepared. Next, each sample was compared by Sandwich lectin ELISA using HPA, PNA, VVA, and WFA lectin.
As shown in FIG. 6, HPA and VVA lectin showed the strongest reaction when treated with sialidase + galactosidase (white). As expected, PNA strongly reacted to IgA treated with sialidase, and IgA that appeared to expose core1 (Gal-GalNAc) on O-glycan. On the other hand, WFA lectin reacted more strongly when sialic acid was digested. On the other hand, when treated with β1,4 galactosidase, which digests β1,4-linked galactose on N-glycan, the WFA signal disappeared significantly (black).
Since β1,4 galactosidase is an enzyme that cleaves Gal bound to glcNAc on N-glycan by β1,4, WFA lectin is not a known substrate specificity for Tn and LacdiNAc reported so far, It has a novel substrate specificity that binds to β1,4-linked Gal, and it is clear that it shows a different reactivity from GalNac-recognizing lectins, HPA and VVA, which were thought to have similar substrate specificity. It was.
From the above results, it was found that the WFA lectin strongly binds to the IgA1 molecule derived from mIgA-MIDD because the WFA lectin recognizes N-glycan on the IgA1 molecule, particularly β1,4 galactose structure.
In addition, WFA lectin binds to IgA1 derived from mIgA-MIDD patients more strongly than IgA1 derived from healthy individuals, so that IgA1 derived from mIgA-MIDD patients may have structural characteristics related to N-glycan. It became clear.

(4) Enzyme-linked immunoassay (ELISA) using various lectins
50 μL of a polyclonal anti-IgA antibody (manufactured by Jackson) at a concentration of 5 μg / mL was subjected to Maxisorp 96-well immunoplate (Thermo Fisher Scientific) and allowed to stand at room temperature for 2 h. After discarding the supernatant, blocking was performed with 100 μL of 1% BSA / PBS solution at room temperature for 1 h. After discarding the supernatant, the various glycosidase digested IgA1 prepared above was diluted to 5 μg / mL with a 1% BSA / PBS solution, 50 μL was applied to a plate, and reacted at 4 ° C. overnight. The reacted plate was washed 3 times with 200 μL of PBS-T and then various biotinylated lectins HPA (5 μg / mL), PNA (2 μg / mL), VVA (5 μg / mL), WFA (5 μg / mL). 50 mL of each) was allowed to react at room temperature for 1 h. After further washing three times with 200 μL of PBS-T, 50 μL of 1 μg / μL of streptavidin-HRP was applied to each sample and allowed to react at room temperature for 1 h. After washing 3 times with 200 μL of PBS-T, 50 μL of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific) was used as a substrate and reacted at room temperature for 1 to 15 min. After sufficient signal was obtained, 50 μL of 2 MH ₂ SO ₄ was added to terminate the reaction. After completion of the reaction, detection was performed at a wavelength of 450 nm using a microplate reader (Wallac 1420ARVO sx Multilabel Counter, Perkin Elmer).

(5) De-N-glycosylation of purified IgA1 using PNGase F
To completely excise N-glycan from IgA1 protein, 10 μg of purified IgA1 was heated at 98 ° C for 5 min in 20 μL of 0.5% SDS / 0.2 M 2-mercaptoethanol / PBS (pH 8.0), Furthermore, 20 μL of 5% Nonidet P-40, 60 μL of PBS (8.0) and 1 mU of Peptide N-glycosidase F (PNGase F; takara) were added and reacted at 37 ° C. overnight. For partial cleavage, heat 2.5 μg of purified IgA1 in 5 μL of 0.5% SDS / 0.2 M 2-mercaptoethanol / PBS (pH 8.0) at 98 ° C. for 5 min, and then add 5 μL of 5% Nonidet P-40, 15 μL of PBS (8.0) and 0.1 mU of PNGase F were added and reacted at 37 ° C. with 15, 30, 60, 120 min over time. Each enzyme reaction was stopped by heat treatment at 98 ° C. for 5 min. The prepared de-N-glycosylated IgA1 was subjected to SDS-PAGE (silver staining) and lectin array measurement.

(6) SDS-PAGE analysis using a PNGase F-treated sample (silver staining)
For purified IgA1 derived from mIgA-MIDD and mIgA, 400 ng of each prepared PNGase F-treated and untreated sample was separated by 10% polyacrylamide gel SDS-PAGE, and bands were detected by silver staining method. .

(7) Lectin microarray analysis using PNGase-treated samples
The mIgA-MIDD-derived purified IgA1 was completely digested with PNGase F and undigested by the above method. The prepared PNGase digested product corresponding to 1 μg and the undigested product IgA1 were labeled with 10 μg of Cy3-succimidyl ester (GE Healthcare) at room temperature for 1 h under light-shielding conditions. In order to invalidate the unreacted Cy3 reagent, 500 mM glycine / 1% Triton X-100 / TBS (probing buffer) was added, and blocking treatment was performed at room temperature for 2 h under light-shielding conditions. Finally, IgA1 equivalent to 100 ng of the sample diluted to 10 μg / mL with probing buffer was applied to a lectin microarray and reacted at 20 ° C. overnight. After the reaction, the membrane was washed three times with a probing buffer, 80 μL of probing buffer was added, and measurement was performed using Glycostation. Signal detection, data processing, and the like were performed using the same method as that used for the above-mentioned measurement of tryptic digest.

As shown in FIG. 7, signals of many lectins (lower than UDA lectin) that recognize N-glycan are lost by treatment with the enzyme PNGaseF that cleaves N-glycan. On the other hand, the signal of the lectin that recognizes O-glycan (above the BPL lectin) is unchanged or increased by the PNGase treatment. Therefore, the WFA lectin was confirmed to have recognized N-glycan since the signal was reduced by the treatment with PNGaseF.
In addition, unexpectedly, the WFA signal was reduced even in samples that were not digested with PNGase. In this experiment, pretreatment with 2-mercaptoethanol that destroys the three-dimensional structure is performed. Similarly, trypsin digestion also eliminates the three-dimensional structure, suggesting that WFA may recognize changes taking into account the three-dimensional structure of IgA. In this way, WFA may recognize the three-dimensional structure of mIgA-MIDD-derived IgA1, so what kind of behavior the WFA lectin signal shows by pre-processing to eliminate the three-dimensional structure I decided to investigate. Therefore, lectin microarray measurement was performed on IgA1 that had been heat-treated in the presence of SDS.

(8) Heat treatment method of purified IgA1 and lectin microarray measurement in heat treated sample
In order to examine how the heat treatment in the presence of a surfactant of mIgA-MIDD-derived purified IgA1 affects the interaction with lectins on lectin microarrays, the following experiment was performed. SDS was added to mIgA-MIDD-derived purified IgA1 to a final concentration of 0.2% and heated at 95 ° C. for 5 minutes. As a target sample, a 0.2% SDS-containing mIgA-MIDD-derived purified IgA1 solution was prepared. 100 ng, 50 ng, and 25 ng of these were added to each lectin array reaction vessel and incubated at 20 ° C. for about 16 hours. The interaction after the reaction was detected by the above-described antibody overlay lectin array method.
As shown in FIG. 8, mIgA-MIDD-derived IgA1 had different sugar chain profiles in the lectin array by heat treatment in the presence of SDS. In particular, WFA lectin signal was significantly reduced by heat treatment. In contrast, other O-glycan-recognizing lectins did not show significant fluctuations due to heat treatment.

(9) WFA reactivity to mIgA-MIDD-derived purified IgA1 under non-reducing conditions
In order to further confirm that the WFA lectin recognizes the conformation change of IgA, SDS-PAGE was performed under reducing or non-reducing conditions, and a WFA lectin blot was performed.
After SDS-PAGE of purified IgA1 derived from mIgA-MIDD under reducing and non-reducing conditions, Western blotting was performed using WFA lectin, anti-IgA1 antibody, and ConA lectin. As shown in FIG. 9, when anti-IgA antibody and ConA lectin were used, the reaction was carried out regardless of non-reducing and reducing conditions (B, C). On the other hand, WFA lectin did not react at all with IgA under reducing conditions, but reacted only with IgA under non-reducing conditions. The sample treated with sialidase + galactosidase (the structure of O-glycan has a Tn structure) reacts even under reducing conditions (A).
From this result, it was found that WFA reacts with non-reduced native IgA. Furthermore, among native IgA, it was found to react only with high molecular weight IgA molecules.
In addition, WFA lectin binds to IgA1 derived from mIgA-MIDD patients more strongly than IgA1 derived from healthy subjects, so that IgA1 derived from mIgA-MIDD patients is a higher polymer than IgA1 derived from healthy subjects. Conceivable.

(10) Detection of N-glycan addition number using PNGase F treatment The difference in the number of added sugar chains was examined using PNGFase F. PNGase F was allowed to react with IgA1 over time, and IgA1-derived bands at each time were detected by SDS-PAGE (silver staining). When PNGaseF was reacted over time, multiple bands depending on the number of N-glycan added were observed (FIG. 10).
In addition, as shown in FIG. 10C, in the benign disease (mIgA), when the PNGaseF reaction was stopped halfway, three heavy chain-derived and one light chain-derived bands were detected (B, lanes 8 to 10). On the other hand, when the PNGase F reaction was stopped midway with mIgA-MIDD, bands derived from 4 heavy chains and 2 light chains were detected (A, lanes 2 to 5).
As a result, three bands were found in healthy subjects, indicating that two N-glycans were present on the heavy chain. On the other hand, in mIgA-MIDD, a total of 4 bands appeared, indicating that 3 N-glycans were present on the heavy chain. It was also found that one N-glycan was added to the light chain. In mIgA-MIDD, it was found that the number of N-glycan was increased, and an abnormal addition was observed in which a total of 4 sugar chains increased per molecule of IgA compared to healthy subjects.

(11) Measurement of molecular weight of each specimen-derived purified IgA1 After treating each specimen-derived purified IgA1 under non-reducing conditions or reducing conditions, the specimens were separated by 10% polyacrylamide gel SDS-PAGE (silver staining) and compared for molecular weight. However, we could not find any significant differences (A). However, when the molecular weight was separated by 2% agarose gel SDS-PAGE (Western blotting with anti-IgA1 antibody) that can detect higher molecular weight, the molecular weight of mIgA-MIDD was higher than 500 kDa. It was found that IgA was formed (B, lane 3). (Fig. 11)
In general, IgA is known to form monomers, dimers, and polymers, and most of the IgA in the serum of healthy subjects is a monomer. Such a high molecular weight substance is considered to have a very strong multivalent sugar chain.
Until now, attention was focused only on the difference in the sugar chain structure between nephropathy IgA and healthy subject IgA. However, in this disease, not the difference in sugar chain structure, but the three-dimensional structure such as the number of added sugar chain structures and the formation of aggregated IgA We found abnormalities that took into account the abnormalities.

<Example 3> Measurement of WFA lectin binding ability
(1) Comparative analysis by Sandwich lectin EILSA using WFA lectin Since the WFA lectin signal was significantly higher than IgA1 of mIgA-MIDD in the lectin microarray measurement, benign diseases, healthy subjects and purified IgA1 of mIgA-MIDD The difference in reactivity to WFA was examined by Sandwich lectin ELISA using WFA lectin. mIgA-MIDD, mIgA and 1,10 μg of purified IgA derived from healthy subjects were diluted to 50 μL with 200 mM sodium acetate buffer and reacted overnight at 37 ° C. with 20 mU sialidase. After the reaction, the solution was reacted overnight at 37 ° C. with 1.1 mU β-galactosidase or 1.5 mU β1,4 galactosidase. Next, samples prepared by enzymatic digestion were compared by Sandwich lectin ELISA using WFA lectin. First, 50 μL of a polyclonal anti-IgA antibody (manufactured by Jackson) at a concentration of 5 μg / mL was subjected to Maxisorp 96-well immunoplate (Thermo Fisher Scientific) and allowed to stand at room temperature for 2 h. After discarding the supernatant, 100 μL of a 1% BSA-containing PBS solution was used and allowed to stand at room temperature for 1 h. After discarding the supernatant, the enzyme-digested IgA1 prepared above was diluted 200-fold with PBS containing 1% BSA, and 50 μL was applied to the plate and reacted at 4 ° C. overnight. The reacted plate was washed 3 times with 200 μL of PBS-T, and then WFA (5 μg / ml; Vector), VVA A (5 μg / ml; Vector), PNA (2 μg / μl) 50 μL of various biotinylated lectins of Vector) and HPA (5 μg / ml; EY laboratories) were reacted at room temperature for 1 h. After further washing with 200 μL of PBS-T three times, 50 μL of 1 μg / μL of streptavidin-HRP was applied and reacted at room temperature for 1 h. After washing 3 times with 200 μL of PBS-T, 50 μL of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific) was used as a substrate and reacted at room temperature for 1 to 15 min. After sufficient signal was obtained, 50 μL of 2 MH ₂ SO ₄ was added to terminate the reaction. After completion of the reaction, detection was performed at a wavelength of 450 nm using a microplate reader (Wallac 1420ARVO sx Multilabel Counter, Perkin Elmer). The detection results are shown in FIG. 12, and it was found that WFA reacted strongly with sialidase-digested IgA1 in all specimens, but particularly reacted with mIgA-MIDD IgA1. Therefore, it was found that IgA1 treated with sialidase had a significant difference in reactivity between benign diseases and healthy subjects and mIgA-MIDD.

<Example 4> Separation and purification (enrichment) of mIgA-MIDD using WFA lectin
(1) Separation and purification method 1
Since WFA lectin strongly reacts with mIgA-MIDD, mIgA-MIDD-derived IgA was separated and purified using WFA lectin. 4 μg of biotinylated WFA lectin was added to 20 μL of mIgA-MIDD and 1 μg / μL of purified IgA1 in PBS buffer of healthy subjects, and reacted overnight at 20 ° C. and 14000 rpm. Dynabeads MyOne streptavidin T1 (Invitrogen) 20 μL (v / v) previously washed with 200 μL PBS-Tx buffer was added and incubated at 20 ° C. for 1 h. The magnetic beads were washed three times with 1 mL of PBS-Tx, and then eluted with 20 μL of 100 mM GalNAc or 100 mM Glycine-HCl (pH 2.5) solution at 20 ° C., 14000 rpm, and 15 min. When it was eluted with the latter acid, it was neutralized with 3 μL 1M Tris-HCl (pH 8.0). For the flow-through fraction and the eluted fraction, bands were detected using SDS-PAGE (silver staining) (FIG. 13).
mIgA-MIDD IgA1 was successfully purified.

(2) Separation and purification method 2
MIgA-MIDD-derived IgA was purified from normal healthy IgA using WFA lectin. As a sample before purification, a sample in which mIgA-MIDD-derived IgA1 was mixed at a constant ratio with healthy-derived IgA1 was prepared. The amount of mIgA-MIDD-derived IgA1 was mixed at a ratio of 1 μg (10%), 100 ng (1%), and 10 ng (0.1%) to 10 μg of healthy subject IgA1 to prepare a sample. Subsequently, Dynabeads MyOne streptavidin T1 (Invitrogen) previously washed with 200 μL PBS-Tx buffer was diluted with 50 μL (v / v) of 8 μg of biotinylated WFA lectin in 50 μL of PBS, 20 ° C., 14000 rpm, 1 h Incubated. Each of the above samples was diluted to 40 μL with PBS, added to WFA-conjugated magnetic beads washed 3 times with 1 mL of PBS-Tx, and incubated overnight at 20 ° C. and 14000 rpm. After washing three times with 1 mL of PBS-Tx, 40 μl of 100 mM GalNAc / PBS solution was added, and elution was performed at 20 ° C., 14000 rpm, and 15 min. Bands were detected using SDS-PAGE (silver staining) for the flow-through and elution fractions.
As shown in FIG. 14, mIgA-MIDD IgA1 (1 μg, 100 ng 10 ng, respectively) at a ratio of 10%, 1% and 0.1% was mixed with 10 μg of purified IgA1 derived from healthy subjects, and WFA lectin was mixed. Was used to purify mIgA-MIDD IgA1. By using WFA lectin, 1% of mIgA-MIDD IgA1 could be sufficiently separated (green frame). Moreover, 0.1% of mIgA-MIDD IgA1 was separable.

(3) Examination of mIgA-MIDD purification conditions using WFA lectin
In order to verify the binding ability of WFA lectin to mIgA-MIDD-derived IgA1, WFA agarose column chromatography was performed. mIgA-MIDD-derived purified IgA1 (600 ng) was added to a minicolumn packed with WFA agarose (130 μL) previously equilibrated with PBSTx buffer, and allowed to stand at 4 ° C. for 2 hours. After the reaction, 120 μL of PBSTx buffer was added to the column, and unbound molecules were dropped and collected. This was defined as the unbound fraction. Prior to the lectin array, SDS-polyacrylamide gel electrophoresis was performed to estimate the amount of IgA in the unbound fraction. As a result, it was found that about 70% of IgA1 was bound to the WFA column. Next, the unbound fraction was converted to the amount of IgA before addition, 50 ng, 25 ng, and 12.5 ng were added to the lectin array, and reacted at 20 ° C. for 16 hours. The antibody overlay lectin array method described above was used to detect the binding reaction. As a control experiment, mIgA-MIDD-derived purified IgA1 was subjected to the same amount (12.5 ng) of lectin array analysis. The result is shown in FIG.
The lectin signal intensity of the unbound fraction (Through) is clearly reduced compared to that of purified IgA1. In particular, in the WFA signal, the signal decreased significantly in the non-binding fraction, indicating that mIgA-MIDD-derived purified IgA1 was adsorbed to WFA agarose. Therefore, it was found that IgA1 derived from mIgA-MIDD can be enriched by adsorption with WFA agarose.

<Example 5> Measurement of lectin microarray for serum using anti-IgA antibody overlay method
Since IgA1 was highly reactive with WFA lectin in patients with mIgA-MIDD, lectin microarray measurements were performed on serum to determine whether mIgA-MIDD can be distinguished from normal subjects directly from serum without purification. It was. As a measuring method, it was measured by an antibody overlay method using an anti-IgA1 monoclonal antibody (7303B). The experimental method is shown below.
mIgA-MIDD and 70 nL of healthy human serum were diluted in 80 μL of PBS-Tx, and all were applied to a lectin microarray slide glass and reacted at 20 ° C. for 18 h. After the reaction, the plate was washed 3 times with 80 μL PBS-Tx, and then 80 μL of 6.25 μg / mL concentration of biotinylated anti-IgA1 antibody (7303B) was used and reacted at 20 ° C. for 1 h. After further washing 3 times with 80 μL PBS-Tx, 80 μL of 2.5 μg / mL concentration of streptavidin-Cy3 was applied and reacted at 20 ° C. for 1 h. After washing 3 times with 80 μL PBS-Tx, 80 μL of PBS-Tx was used, and measurement was performed with a lectin microarray. Measurements were taken at Gain 125-110. The result shows that of Gain125.
As shown in FIG. 16, even when serum was used directly, significantly different sugar chain profiles were obtained between mIgA-MIDD and healthy subjects. In direct serum measurement, it was found that WFA lectin does not react with healthy subjects, but strongly reacts with IgA1 of mIgA-MIDD.
As a result, even when serum was used directly, significantly different sugar chain profiles were obtained between mIgA-MIDD and healthy subjects. In direct serum measurement, it was found that WFA lectin does not react with healthy subjects, but strongly reacts with IgA1 of mIgA-MIDD.

Claims (11)

A method for detecting an IgA aggregate, comprising contacting a sample containing IgA with a lectin that specifically reacts with the IgA aggregate. The method according to claim 1, wherein the binding ability of the lectin to the IgA aggregate is measured. The method according to claim 1 or 2, wherein the measurement of the binding ability is based on the recognition property of the lectin for β 14 galactose on N-glycan. The lectin is at least one lectin selected from the group consisting of HHL, GNA, NPA, WFA, SBA, VVA, BPL, TJA-II, PHA-L, and AOL lectin. The method according to any one. The method according to any one of claims 1 to 3, wherein the lectin is a WFA lectin. The method according to claim 1, wherein the sample containing IgA is serum or urine of a subject. An IgA nephropathy test method, comprising detecting IgA aggregates by the method according to claim 1 and testing IgA nephropathy based on the detection result. The method according to claim 7, wherein the IgA nephropathy is mIgA-MIDD. A method for purifying an IgA aggregate molecule, comprising purifying the IgA aggregate molecule by binding a lectin that specifically reacts with the IgA aggregate molecule. Methods for testing IgA nephropathy, including the following procedures:
1) Contact IgA in serum or urine collected from a subject with WFA lectin;
2) IgA nephropathy is determined by comparing the amount of IgA bound to the WFA lectin with that of a healthy person. The method according to claim 10, wherein the amount of IgA bound to the WFA lectin is measured by performing analysis by an antibody overlay method using an IgA1 monoclonal antibody.