JPH09311132A - Diagnostic method of immunoglobulin-a nephropathy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform a diagnoses in a short time with a simple operation by detecting the binding capacity between IgA1 molecules separated from human serum, and comparing it with a standard value. SOLUTION: IgA1 is separated from serum, and a plastic-made microtitre plate is coated with it. This is washed with a proper buffer solution, and the IgA1 separated from serum which is labeled with biotin is added and reacted. After washing with a proper buffer solution, a composite of avidin of protein and horseradish peroxidase is added thereto. A coloring agent is added and reacted thereto, and the absorbance of 490nm is measured. The stronger the binding capacity between IgA1 molecules is, the higher the absorbance value is, and the binding capacity can be thus shown by the absorbance value. Thus, the binding capacity between IgA1 molecules of a subject is compared with the binding capacity between IgA1 molecules (standard value) of a non-IgA nephropathy patient group. When a statistical significant difference is present between them, it is judged positive, and when no difference is present, it is judged negative.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel diagnostic method for IgA nephropathy. More specifically, the present invention provides
Based on the detection of the difference in the binding ability between human serum IgA1 molecules, prompt IgA nephropathy with less risk of psychological distress to patients, perirenal hemorrhage, etc. and economic burden
The present invention provides a simple and novel diagnostic method.

[0002]

2. Description of the Related Art IgA (immunoglobulin A) nephropathy is
A disease concept proposed by Berger et al. In 1968, which is clinically poor in clinical symptoms in addition to continuous proteinuria and hematuria, and histologically shows deposits mainly composed of IgA in the glomerular mesangial region. Glomerulonephritis. The frequency of IgA nephropathy in Japan is high, accounting for about 30% of chronic nephritis. In recent years, it has been revealed that the long-term prognosis is not always good, and 10 to 15% of patients after 10 years and about 30% of patients after 20 years have end-stage renal failure. For this reason, IgA nephropathy has received particular attention as a causative disease leading to end-stage renal failure.

At present, the only diagnostic method for IgA nephropathy is a method based on renal biopsy. This diagnostic method may cause great mental distress to the examinee and may cause perirenal bleeding or the like after the examination. Furthermore, after a renal biopsy test, absolute rest is required for at least 24 hours, and the patient is forced to be hospitalized for several days, which presents a large financial burden on the patient. Further, this diagnostic method has a drawback that it requires a lot of equipment and takes a long time for diagnosis. Therefore, a method for diagnosing IgA nephropathy that can be performed in a shorter time by a simpler operation is desired.

[0004]

SUMMARY OF THE INVENTION The object of the present invention is to improve
As a diagnostic method for A nephropathy, it is to provide a simpler diagnostic method that does not rely on renal biopsy, which has been essential until now.

[0005]

FIG. 1 is a schematic diagram showing the outline of the structure of an IgA1 molecule. IgA, which is considered to be involved in the pathogenesis of IgA nephropathy, is a type of immunoglobulin. This IgA molecule is composed of two heavy chains and two light chains, and is classified into two subtypes, IgA1 and IgA2, based on the difference in heavy chain structure. As shown in FIG. 1, Baenziger et al. Reported that five O-linked sugar chains (one is N-acetylgalactosamine and the other four are 2-acetamido-) are located in the hinge region of the IgA1 molecule. 2
-Deoxy-3-O-β-galactopyranosyl-galactopyranose) (Journal of Biological Chemistry, Vol. 249, 7).
270-7281, 1974). On the other hand, no O-linked sugar chain is bound to the hinge of the IgA2 molecule.

In IgA nephropathy, IgA deposited in the glomerulus is mainly composed of the IgA1 subtype. Therefore, there is a difference in the properties of serum IgA1 between IgA nephropathy patients and non-IgA nephropathy patients such as healthy subjects. Assuming that there is, the present inventors have made various studies. As a result, serum Ig of IgA nephropathy patients
A1 was found to have a significantly increased binding ability between IgA1 molecules as compared with serum IgA1 of non-IgA nephropathy patients such as healthy subjects, and this finding can be used for diagnosis of IgA nephropathy. Therefore, the present invention has been completed.

That is, IgA1 nephropathy can be rapidly diagnosed by measuring the binding ability between IgA1 molecules in the serum of a subject, preferably IgA1 separated from the serum. That is, the present invention provides: A diagnostic method for IgA nephropathy, which detects a difference in binding ability of human serum IgA1 between IgA1 molecules. Also provided is a diagnostic method for IgA nephropathy, which detects a difference in binding ability between IgA1 molecules of IgA1 separated from human serum. In addition, labeled IgA1 separated and prepared from human serum was used,
Provided is a diagnostic method for IgA nephropathy, which detects a difference in binding ability between IgA1 molecules. It is also characterized in that it detects a difference in binding ability between IgA1 molecules through the hinge portion of the IgA1 molecule.

Hereinafter, the present invention will be described in detail. In the present invention, when examining the binding ability between IgA1 molecules, the serum of the subject can be used as it is, but in order to examine the binding ability with high accuracy, it is desirable to separate IgA1 from the serum. As a method for separating IgA1 from serum, a method using jacalin which is one of lectins, anti-human I
A method using gA1 is known.

In the present invention, the IgA1 hinge portion comprises
The part of the amino acid sequence from where to where in the gA1 heavy chain is not clearly defined. As shown in FIG.
The gA1 hinge region refers to a region between the CH1 domain and the CH2 domain in the heavy chain constituting the IgA1 molecule. This region contains the disulfide bond between the heavy chains and has a high proline content. In addition, as described above, Bae
Nziger et al. reported that an O-linked sugar chain was bound to the IgA1 hinge region. O-linked sugar chains are bound to the serine residues at 5 positions in the IgA1 hinge region, and the sugar chain structure has N-acetylglucosamine at 1 position and 2-acetamido-2- at 4 other positions. It is reported to be deoxy-3-O-β-galactopyranosyl-galactopyranose (Journal of Biological Chemistry, Vol. 249, 7270-728).
1 page, 1974).

In the present invention, labeling the IgA1 molecule is desirable for more highly sensitively examining the binding ability between the IgA1 molecules. For example, using the ELISA method, IgA1
When examining the binding ability between molecules, labeling with biotin, peroxidase, alkaline phosphatase or the like is preferable, and when using the fluorescence polarization method, labeling with fluorescein or the like is generally used.

Further, in the present invention, the difference in the binding ability between IgA1 molecules is based on the binding ability between the serum IgA1 molecules of the non-IgA nephropathy patient and the serum IgA of the IgA nephropathy patient.
The binding ability between one molecule shows a high value. Subject's serum IgA
The binding ability between one molecule was examined, and the serum I of the non-IgA nephropathy patient group was examined.
gA1 molecule binding ability (reference value), and this difference in binding ability [= binding ability between serum IgA1 molecules of subject-value of binding ability between serum IgA1 molecules of non-IgA nephropathy patient (reference value) Is positive (IgA1 nephropathy), and if there is no statistically significant difference (nearly 0), it is judged negative (not IgA1 nephropathy).

Next, as means for measuring the binding ability between IgA1 molecules in the present invention, in addition to the enzyme immunoassay (ELISA) method which is generally widely used, a fluorescence polarization method,
A surface plasmon resonance method or the like can be used. 1) The outline when the ELISA method is used is as follows. Roque-Barreira et al., Using jacalin (Journal of Immunology, No. 134, 1).
740-1743, 1985).
A1 is separated and coated (immobilized) on a plastic 96-well microtiter plate.

After washing with an appropriate buffer solution, serum which has been subjected to biotin labeling treatment which is generally used for highly sensitive detection of binding ability, preferably IgA separated from serum
Biotin-labeled 1 is added to IgA1-coated microtiter plate and reacted. After washing with an appropriate buffer, the bound biotin-labeled IgA1
A complex of avidin, a protein that specifically binds to biotin, and horseradish peroxidase, an enzyme, is added. To this, a coloring agent for producing color was added by utilizing the reaction with western peroxidase, and the mixture was reacted.
Measure absorbance at m. The value of the absorbance becomes higher as the binding ability between IgA1 molecules is stronger.

For example, from the absorbance values of the test subjects, a criterion is used, in which the average value of the absorbance of 20 non-IgA nephropathy patient groups + 2 × standard deviation is positive and the others are negative. It is possible to diagnose whether IgA nephropathy or not. Not only the ELISA method but also IgA1 as described above
Any method capable of measuring the binding ability between molecules can be used for this diagnostic method.

2) The fluorescence polarization method is a method whose measurement principle is that the value of the fluorescence polarization degree changes when another molecule binds to the fluorescently labeled molecule and the size of the molecule changes. According to this, the binding ability between IgA1 molecules can be measured and can be used for this diagnostic method. 3) The surface plasmon resonance method can also measure the binding ability between IgA1 molecules by a method that utilizes a phenomenon of detecting a change in the concentration of a solution in contact with a metal surface as a change in the reflection angle of light. It can be used for diagnostics.

[0016]

As described above, the present inventors have found that the serum IgA1 of a patient with IgA nephropathy is the serum I of patients with non-IgA nephropathy such as healthy subjects.
It was found that the binding ability between IgA1 molecules was significantly increased as compared with gA1. Further experiments were performed to identify at which site in the IgA1 molecule this binding occurred.
As shown in Reference Example 1, this bond between IgA1 molecules is inhibited not only by IgA1 but also by the hinge glycopeptide separated from IgA1, the peptide fragment of 20 amino acid residues constituting the hinge region, and the sugar chain. I understood.

From this result, the binding between IgA1 molecules is I
It could be revealed that the binding was via the hinge part of the gA1 molecule. That is, serum I of IgA nephropathy patients
gA1 is serum IgA1 of non-IgA nephropathy patients such as healthy people.
It was revealed that the binding ability between IgA1 molecules via the IgA1 molecule hinge region was significantly increased.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail, but these do not limit the scope of the present invention. (Example 1) (Separation of IgA1 used for biotin labeling) Serum 5 ml
Was injected into a column (1 × 15 cm) packed with jacalin agarose (Vector) equilibrated with 0.01 M phosphate buffer (pH 7.5, hereinafter abbreviated as PBS) containing 0.15 M NaCl, and 70 ml of PBS and 7
After the components not adsorbed to jacalin were washed with 0 ml of 0.8 M glucose-containing PBS, the jacalin-adsorbed components were collected with 70 ml of 0.1 M melibiose-containing PBS. This solution was applied to a dialysis membrane (Visase seals, si
After dialysis against PBS at 4 ° C. overnight using ze20 / 32), it was freeze-dried to obtain an IgA1 sample.

(IgA used for coating the plate
1) Separation) Ammonium sulfate was added to 45 ml of serum of a non-IgA nephropathy patient so as to have a 50% saturation concentration, and the resulting insoluble matter was collected by centrifugation. Dissolve this in PBS,
Anti-human IgA affinity column (Organon
An anti-human IgA antibody having specificity for α chain purchased from Tecknika was purchased from Pharmacia Sepharose 4
It was injected into a column packed with gel immobilized on B), washed with a sufficient amount of PBS, and then the adsorbed component was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.5). Put this solution in P
After dialysis against BS, similarly to the above-described separation of IgA1 used for biotin labeling, injection into a Jacalin agarose column, washing with PBS, components eluted with PBS containing 0.1 M melibiose, dialysis, and freezing. After drying, the IgA1 used for coating the plate was separated.

(Biotin labeling of serum IgA1) The biotin labeling of IgA1 separated from serum was performed by the following method. Ig of 20 IgA nephropathy patients isolated from serum
A1 (IgA nephropathy patient group) and IgA1 (non-IgA nephropathy) of 20 healthy subjects and non-IgA nephropathy patients of other renal disease patients
Nephropathy patient group) 100 μg for each American Qu
alex Biotin Labeling Reagent Kit (Code No. K)
8000) according to the method attached thereto.
A reaction for biotin labeling gA1 was performed. Then, the dialysis membrane (manufactured by Visase seals, size20 /
32), and dialyzed against PBS overnight at 4 ° C. This solution was freeze-dried to obtain biotin-labeled IgA1.

(Analysis of binding ability between biotin-labeled IgA1-IgA1) The binding ability between biotin-labeled IgA1-IgA1 was measured by the ELISA method, and the results are shown below. Non-Ig
IgA1 isolated from the serum of patients with A nephropathy
M in a carbonate buffer (pH 9.6) to a concentration of 50 μg / ml, and a 96-well microtiter plate (Linbro / Titertek EIA Micr manufactured by A Flow General Company).
Iteration plate, cat no. 7
6-381-04) was added to each well in an amount of 100 μl,
Incubation (rest) at 4 ° C. overnight.

Each well contained 200 μl of PB containing 0.1% bovine serum albumin and 0.05% Tween 20.
After washing three times with S (hereinafter abbreviated as PS / BSA / Tween), 1% bovine serum albumin (f) was added to each well.
fraction V, Sigma) containing 20 PBS
0 μl each was added and incubated at 4 ° C. overnight. The concentration of biotin-labeled IgA1 in the IgA nephropathy patient group and the non-IgA nephropathy patient group was 100 μg / m in each well.
100 μl of the solution prepared to be 1-PBS was added thereto, and the mixture was incubated at 4 ° C. overnight. Each well was washed 12 times with 200 μl of PBS / BSA / Tween, and then Amer diluted 500-fold with PBS.
sham ABC (Streptavidin b
iotinylated horseradishpe
roxidase complex, code no. RP
N1051) Add 100 μl of reagent to each well,
Incubate for 1 hour at room temperature.

200 μl of PBS / BSA / each well
After washing with Tween six times, a color former (orthophenylenediamine 20 mg, disodium hydrogen phosphate 12
1.795 g of water, 0.525 g of citric acid, hydrogen peroxide 1
(5 μl dissolved in 50 ml of water) was added to each well in an amount of 100 μl, and the mixture was incubated at room temperature for 1 hour. BioRad Microplate Reader Model4
Using 50), the absorbance at OD 490 nm was measured.

FIG. 2 shows a group of patients with IgA nephropathy and non-IgA.
It is the graph which measured the light absorbency of OD490nm of each 20 patient group, and plotted the value. In that case, non-IgA
The average (0.28) + 2 × standard deviation = 0.64 or more absorbance of the group of 20 nephropathy patients was regarded as positive. As a result of the experiment, in the non-IgA nephropathy patient group, all 20 cases were negative, while in the IgA nephropathy patient group, 5 cases were positive, and the positive rate was statistically significant between the two groups. A significant difference was confirmed.

Reference Example 1 (Isolation of IgA1 Hinge Glycopeptide) From serum of a healthy individual,
IgA1 separated by the same operation as the above-mentioned separation of IgA1 used for biotin labeling was subjected to S-carboxymethylation by an ordinary method. This S-carboxymethylated IgA130
mg of trypsin (Sigma) at 1 mg / ml
0.1 M ammonium bicarbonate buffer (pH 8.0) 3
and incubated at 37 ° C. overnight.

A column (1) packed with Jacarin agarose (Vector) equilibrated with this trypsin digest solution by 0.175 M Tris-HCl buffer (pH 7.5).
0 ml), and the components that are not adsorbed on jacalin were washed with 60 ml of 0.175 M Tris-HCl buffer (pH 7.5) and 60 ml of the same buffer containing 0.8 M glucose. Jacalin-adsorbed components were recovered with the same buffer containing galactose. After concentrating this liquid, a gel filtration column (Se
(Phadex G-50, 1.5 x 68 cm) and eluted with 1.0 M acetic acid was fractionated into 1.2 ml portions. A part of each fraction was subjected to the phenol sulfate method, and a fraction containing a glycopeptide that developed color (excluding a fraction containing uncleaved IgA1 and a monosaccharide) was collected and freeze-dried to obtain an IgA1 hinge glycopeptide sample. .

(Reference Example 2) (IgA1 intermolecular bond inhibition experiment) I shown in Example 1
In order to investigate which site of the IgA1 molecule is involved in the binding between the gA1 molecules, IgA1
It was investigated whether or not it had an effect of inhibiting the binding between one molecule.
Those whose inhibitory action was investigated were the above-mentioned IgA1 hinge glycopeptide, IgA1 (isolated from the serum of IgA nephropathy patient by the above-mentioned method), and IgA2 (Athens rear
Purchased from ch and technology. Monoclonal), IgG (Athens res)
Purchased from search and technology. Polyclonal), galactose (Gal,
(Purchased from Sigma),

N-acetylgalactosamine (GalNA
c, purchased from Sigma), N-acetylneuraminic acid (NANA, purchased from Sigma), 2-acetamide-
2-deoxy-3-O-β-galactopyranosyl-galactopyranose (Galβ1-3GalNAc, Tro
nto Research Chemicals), synthetic hinge peptide (PVPSTPPTPSP)
STPPTPSPS, purchased from Bio-Synthesis, Inc. and tetrapeptides (PTPS, Front
o Research Chemicals).

Biotinylated IgA1 used in this inhibition experiment
Was used from an IgA nephropathy patient determined to be positive in Example 1. Biotinylated IgA of Example 1
Biotinylated Ig of 200 μg / ml-PBS instead of l
A 150 μl and 0.006 μM, 0.06 μM, 0.6
μM, 6 μM IgA1 hinge glycopeptide, IgA1,
IgA2, IgG solution in PBS and 0.002 mM,
0.02 mM, 0.2 mM, 2 mM Gal, GalN
An ELISA experiment was performed in the same manner as in Example 1 except that 50 μl of a solution of Ac, NANA, Galβ1-3GalNAc, synthetic hinge peptide, and tetrapeptide in PBS was used.

The inhibitory rate of each inhibitor against the IgA1-IgA1 intermolecular bond was calculated using the following formula. Inhibition rate (%) = (OD490 nm value without addition of inhibitor−OD490 nm value with addition of each inhibitor) / (OD490 nm value without addition of inhibitor) × 100. FIG. 3 is a bar graph showing the inhibitory rate of each inhibitor for IgA1 intermolecular binding. As shown in FIG. 3, the respective inhibition rates at an inhibitory substance concentration of 3 μM were Ig
A1 hinge glycopeptide = 66.1%, IgA1 = 60.
5%, IgA2 = 20.3%, and IgG = 1.0%.

According to FIG. 3, the inhibitory effect on the IgA1 intermolecular bond was observed with the IgA1 hinge glycopeptide and IgA1, but the inhibitory effect with IgA2 was weak and that with IgG was not observed. The error bar in FIG. 3 indicates the error range of the average value of the experiments performed three times + 2 × the standard deviation value.

FIG. 4 shows IgA1 of each inhibitor different from FIG.
It is the figure which showed the inhibition rate with respect to intermolecular binding with the bar graph. As shown in FIG. 4, the respective inhibitory rates at an inhibitor concentration of 1 mM were as follows: synthetic hinge peptide = 69.3%,
Galβ1-3GalNAc = 29.4%, Gal = 1
2.9%, GalNAc = 14.6%, NANA = 0
%, Tetrapeptide = 5.9%. According to FIG. 4, it was found that the inhibitory effect on the IgA1 intermolecular bond was observed with the synthetic hinge peptide and Galβ1-3GalNAc, but not with the others. The error bars in the figure show the error range of the average value of the experiments performed three times + 2 × the standard deviation value.

From the above results, the binding between IgA1 molecules is not limited to IgA1, and is related to the hinge portion of the IgA1 molecule, IgA1 hinge glycopeptide, synthetic hinge peptide of 20 amino acid residues, and sugar chain binding to the hinge portion. Gal
Due to inhibition by β1-3GalNAc, IgA
It was found that the connection was through the hinge part of No. 1.

[0034]

According to the diagnostic method of the present invention, compared with the conventional diagnostic method based on renal biopsy, the risk of mental distress and perinephric hemorrhage to the examinee and the economic burden are reduced, and the diagnostic method is simpler. With simple operation, diagnosis can be performed in a short time.
It is suitable as a quick and simple diagnostic method for A nephropathy.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an outline of the structure of an IgA1 molecule.

FIG. 2: IgA nephropathy patients and non-IgA patients 20 each
It is the graph which plotted the value which measured the light absorbency of OD490nm of an example.

FIG. 3 is a bar graph showing the inhibitory rate of each inhibitor for IgA1 intermolecular binding.

FIG. 4 is a bar graph showing the inhibitory rate of each inhibitor other than that of FIG. 3 on IgA1 intermolecular binding.

Claims (4)

[Claims] 1. An Ig characterized by detecting a difference in binding ability between human IgA1 molecules and IgA1 molecules.
A diagnosis of nephropathy. 2. Ig of IgA1 isolated from human serum
A diagnostic method for IgA nephropathy, which comprises detecting a difference in binding ability between A1 molecules. 3. A labeled IgA1 separated and prepared from human serum.
A method for diagnosing IgA nephropathy, which comprises detecting the difference in binding ability between IgA1 molecules using 4. IgA through the hinge part of IgA1 molecule
The method for diagnosing IgA nephropathy according to any one of claims 1 to 3, which comprises detecting a difference in binding ability between one molecule.