JP2002296276A - DIAGNOSTIC METHOD FOR IgA NEPHROPATHY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an IgA nephropathy diagnosing method by a simple method, instead of an invasive inspection means such as nephros biopsy or an immunological means requiring an antibody of high precision. SOLUTION: In this IgA nephropathy diagnosing method, a human urine specimen is fractionated by cellulose membrane electrophoresis to confirm the existence of an albumin fraction extended to a negative electrode side.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for diagnosing IgA nephropathy. More specifically, the present invention relates to a method for diagnosing IgA from a human urine sample by using cellulose acetate membrane electrophoresis.
The present invention relates to a method for diagnosing IgA nephropathy by confirming the presence of a nephropathy-specific protein.

[0002]

2. Description of the Related Art There are various cases of kidney diseases such as chronic nephritis and acute renal failure. Diabetic nephropathy, membranous nephropathy, focal glomerulosclerosis, membranous proliferative glomerulonephritis, lupus Nephritis,
IgA nephropathy and the like. Among them, IgA nephropathy (also known as IgA nephropathy, Berger's disease) accounts for about 30% of patients with chronic nephritis, and about 30% of the patients with IgA nephropathy do not improve their symptoms by medication, and do not improve the condition such as hemodialysis. It is said that the treatment will shift.

[0003] Criteria for diagnosing IgA nephropathy were presented in 1995 by the "Research Group for Investigation of Specific Disease Progressive Kidney Diseases of the Ministry of Health and Welfare and a joint committee of the Japanese Society of Nephrology". The criteria include a urinalysis (continuous microscopic hematuria, continuous or intermittent proteinuria, gross hematuria), a blood test (IgA value of 350 mg
/ dL), and the only method for definitive diagnosis is "observation of glomeruli by renal biopsy". However, renal biopsy not only requires a high level of skill for the medical staff to diagnose, but also has a problem that the physical and mental distress of the patient is great.

[0004] In order to solve such a problem, there have been proposed several methods for immunologically detecting IgA (immunoglobulin A) from a body fluid of a patient or the like to diagnose IgA nephropathy. It is known that patients with IgA nephropathy have IgA that binds to collagen IV produced from the glomerular basement membrane. Since this IgA exists as an immune complex (IgA-fibronectin complex) circulating in the body fluid of the patient, the IgA is bound to fibronectin or IgA from the body fluid of the subject using a substrate or an antibody that binds to IgA.
A method for diagnosing IgA nephropathy by immunologically detecting (Japanese Patent No. 2592121) has been proposed. Furthermore, as a means for immunological measurement of the IgA-fibronectin complex, an IgA-containing solid carrier that binds a human-fibronectin antibody and an IgA-containing human-IgA antibody are used.
A method using a fibronectin complex measurement reagent has been proposed (JP-A-2000-241431).

[0005] However, in these immunological diagnoses, it is essential to obtain a plurality of antibodies with high accuracy, and for that purpose, purified antigens are required, and the cost and preparation are complicated. Had. In addition, since serum is used as a sample, it was necessary to collect blood from a patient.

On the other hand, a method for detecting human albumin or an albumin degradation product in human urine by an electrophoresis method is disclosed in JP-A-5-302922 and JP-A-6-1603.
No. 84 is disclosed. As the electrophoresis method, S
Examples include DS polyacrylamide gel electrophoresis, isoelectric focusing gel electrophoresis, and cellulose acetate membrane electrophoresis. However, even in these detection methods, after fractionation of the protein by electrophoresis, detection is finally performed by an immunological technique, so that it is costly to obtain antibodies and the preparation of the measurement system is complicated. There was a problem.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a method for diagnosing IgA nephropathy from a human urine sample, without using complicated procedures such as immunological detection of IgA. It is intended to provide a method that can be diagnosed.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to provide a method for diagnosing IgA nephropathy by a simpler technique. In the electrophoresed image, a phenomenon in which the albumin fraction elongates to the cathode side, which was not observed in the protein electrophoresis image obtained by electrophoresing another patient with a renal disorder, was found, and the present invention was reached.

[0009] That is, the present invention is a method for diagnosing IgA nephropathy, which comprises confirming the presence of an albumin fraction elongated on the cathode side in an image obtained by subjecting a human urine sample to cellulose acetate membrane electrophoresis.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Cellulose acetate membrane electrophoresis is a method for separating proteins by applying an electric field to a sample containing proteins etc. coated on a cellulose acetate membrane, and utilizing the fact that the transfer speed of each protein differs due to the difference in surface charge. This is the most widely used electrophoresis method as one of the screening tests for plasma protein abnormalities. When the protein standard serum fractionated by cellulose acetate membrane electrophoresis, albumin fraction from the anode side, alpha ₁ fraction, alpha ₂ fraction, beta fraction, divided into five bands of γ fraction. Diagnosis of malignant tumors, myeloma, and the like is performed by the appearance of other abnormal proteins (M protein, α-fetoprotein) and abnormal band shapes (key albumin). However, regarding the urine sample, since the relationship between the electrophoresis image and the disease has not been clarified, the cellulose acetate membrane electrophoresis is not used for general tests as in the serum sample at present.

In the cellulose acetate membrane electrophoresis of the present invention, since a specimen is usually applied to the cathode side, a substance having a negative charge in the specimen flows from the cathode side to the anode side. Proteins present in urine flow to the anode side according to their charges. The present invention aims at diagnosing IgA nephropathy, and fractionates urine samples from patients with renal impairment suspected of having IgA nephropathy by cellulose acetate membrane electrophoresis, and confirms albumin fraction extended to the cathode side. In this case, IgA nephropathy is diagnosed.

The cellulose acetate membrane used in the present invention is a uniform porous membrane made of cellulose acetate obtained by acetylating a hydroxyl group of cellulose, and may be any of those used in ordinary screening for abnormal plasma proteins. Commercially available ones can be used. Examples of the cellulose acetate membrane include Separax SP (manufactured by Fuji Photo Film), Seraphor (manufactured by Shadon),
Seleka V (manufactured by Toyo Roshi Kaisha) is suitable. Examples of the electrophoresis apparatus used in the present invention include a 238 type electrophoresis apparatus (manufactured by Joko), EC-100 (manufactured by Toyo Filter Paper), and AE-6170A.
S type (manufactured by ATTO) is suitable.

The conditions for the cellulose acetate membrane electrophoresis are not particularly limited, but a veronal buffer, a Tris buffer, or the like can be used as a buffer for electrophoresis. For a clearer separation, use Veronal buffer (p
(H8.6, ionic strength = 0.06) is suitable, and the sample application amount is generally 0.8 to 2.4 μL per 1 cm width. The energizing condition is not particularly limited as long as it is a condition usually performed, but it is 0.6 per cm of the film width.
It is desirable to pass a current of about 0.8 mA.

The electrophoretic image is obtained by staining the fractionated protein on the cellulose acetate membrane after electrophoresis. As a method for staining a protein on a cellulose acetate membrane, for example, the following methods are available. 1. Dye (acid violet-17, nigrosine,
1. Dyeing method using Ponceau 3R) Silver stain

In the present invention, the term “albumin fraction extended to the cathode side” refers to the migration direction as compared with the urine sample of a non-IgA nephropathy patient, the urine sample of a healthy person, or a standard serum obtained by electrophoresis on a cellulose acetate membrane. Is an albumin fraction whose length has been confirmed to have extended to the cathode side. The “albumin fraction extended to the cathode side” is a phenomenon observed in urine of five patients with IgA nephropathy before treatment, which was observed in five urines among the protein electrophoresis images of cellulose acetate membrane electrophoresis. This is a phenomenon in which no urine was observed in the urine of eight patients with the disease (renal disorders other than IgA nephropathy). Specifically, the IgA nephropathy-specific phenomenon in the present invention is a phenomenon observed in Reference Example 1 described later.

The protein in the “albumin fraction extended to the cathode side” causes an immune reaction with an anti-albumin antibody, and
_Since it does not react with the ₁ -antitrypsin antibody, it is presumed that it is a protein that appeared due to denaturation of albumin.

In the present invention, a renal disorder patient is a patient having a renal disease, and the renal disease is diabetic nephropathy, membranous nephropathy, focal glomerulosclerosis, and membranous proliferative thread as described above. Includes spherical nephritis, lupus nephritis, IgA nephropathy and the like.
In finding the IgA nephropathy-specific protein in the present invention, the tested IgA nephropathy patients were presented in 1995 by the "Committee of the Ministry of Health and Welfare Specific Disease Progressive Kidney Disease Investigation and Research Group and the Japanese Society of Nephrology" The patient was diagnosed with IgA nephropathy based on diagnostic criteria. IgA nephropathy patients after treatment
The patient is an IgA nephropathy patient whose symptoms such as urinary occult blood and an increase in blood IgA concentration have been stabilized after treatment of the tonsils after the diagnosis and subsequent treatment with steroid drugs. I before treatment
The gA nephropathy patient is an IgA nephropathy patient who has not been treated for tonsillectomy and steroid administration after diagnosis. Non-IgA
Nephropathy patients are patients with renal disorders other than IgA nephropathy, specifically, diabetic nephropathy, membranous nephropathy, focal glomerulosclerosis,
Refers to patients with membranous proliferative glomerulonephritis.

[0018]

The present invention will be specifically described below with reference to Reference Examples and Examples. (Reference Example 1) Cellulose acetate membrane electrophoresis of urine sample 2 cm from the cathode side of a cellulose acetate membrane (7 cm × 12 cm) buffered with Veronal buffer,
A urine sample was directly applied so that the protein amount per sample was about 1.6 μg. The urine samples consisted of seven IgA nephropathy patients before treatment (Sample Nos. 1 to 7, hereinafter, group A), nine IgA kidney patients after treatment (Sample Nos. 8 to 16, hereinafter, group B), non- I
gA nephropathy patient 8 specimens (diabetic nephropathy 2, membranous nephropathy 3, focal glomerulosclerosis 2, membranous proliferative glomerulonephritis 1) (sample N
o. 17 to 24, hereinafter, group C). The membrane was set in a 238 type electrophoresis apparatus (manufactured by Joko) filled with veronal buffer such that the application point on the cellulose acetate membrane was 1 cm away from the cathode. Then, a current was passed from the anode to the cathode at a constant current of 0.7 mA / film width cm for 30 minutes, and the specimen on the membrane was subjected to electrophoresis. The membrane after the electrophoresis was immersed in a fixing solution (3 w / v% trichloroacetic acid) and shaken for 3 minutes (75 strokes / minute). Next, a preliminary staining solution (0.125 w / v% acid violet-17, 5
% Acetic acid) for 2 minutes and then stain solution (0.25w
/ V% acid violet-17, 5% acetic acid)
Shake for minutes. After shaking in a decolorizing solution (5% acetic acid) for 10 minutes, the membrane was placed on a filter paper and dried at room temperature. A protein band was visually observed on the membrane.

As a result, for each of the groups A to C, FIG.
3 were obtained (the explanatory diagram shows the albumin fraction on the cellulose acetate membrane actually observed with the naked eye). In each of the groups A to C, the length of the migration direction of the band that can be determined by the naked eye was visually measured, and the measured value was compared with the standard serum (fractionated trawl, manufactured by Jokomitsu) that had been simultaneously electrophoresed in each group. ) Divided by the length of the albumin fraction in the migration direction (ratio of the length of the albumin fraction in the migration direction)
Are shown in Tables 1 and 2.

[0020]

[Table 1]

[0021]

[Table 2]

In the results shown in Tables 1 and 2, when the ratio of the length of the albumin fraction in the direction of electrophoresis (sample / standard serum) was 1.4 or more, the albumin fraction elongation was positive. In Group A (7 samples of IgA nephropathy patients before treatment), 5 samples were determined to be positive for the extension of albumin fraction. In group C (samples of non-IgA nephropathy patients), all were negative. Therefore, the result for the differential diagnosis of IgA nephropathy before treatment is 71.4% (5/7 cases) with a correct diagnosis rate.
The correct diagnosis rate was 100% (8/8 cases). Also,
For group B (post-treatment IgA nephropathy), a positive rate of 0%
(0 cases / 9 cases). From the above results, it was found that patients determined to be positive for elongation of the albumin fraction by cellulose acetate membrane electrophoresis had a high possibility of having disease-active IgA nephropathy.

Reference Example 2 Cellulose Acetate Membrane Using Anti-Human Albumin Antibody
The specimens showed albumin fraction elongated positive in immunostaining Reference Example 1 (Sample No.3), were similarly carried cellulose acetate membrane electrophoresis as in Example 1. Transfer buffer (20
v / v% methanol, 25 mM trisaminomethane, 192
filter paper (10 cm × 7.5 c) buffered with mM glycine
m), a polyvinylidene fluoride (hereinafter abbreviated as PVDF) membrane, a cellulose acetate membrane after electrophoresis, and a filter paper were set in this order, and energized at a constant current of 5.5 mA / cm ² membrane for 5 minutes. The PVDF membrane was immersed in a blocking solution (trade name, Block Ace, manufactured by Dainippon Pharmaceutical) and shaken at room temperature for 60 minutes. Nonionic surfactant, Tween2
0 for 5 minutes in a phosphate buffer solution (PBS) containing 0.1% 3
The plate was washed by shaking twice. Then, the PVDF membrane was coated with 0.5 μg / m
The cells were placed in PBS containing L anti-human albumin antibody (host goat, manufactured by Betyl) and shaken at room temperature for 60 minutes. After washing, 5 μg / mL alkaline phosphatase-labeled anti-goat Ig
Put in PBS containing G antibody (host rabbit, Sigma)
Shake at room temperature for 60 minutes. After washing, the substrate solution (0.17
mg / mL BCIP, 0.33 mg / mL NBT, 12 mg / mL
Trisaminomethane, 5.8 mg / mL sodium chloride,
(1.0 mg / mL sodium azide) at room temperature for about 5 minutes.

FIG. 4 shows the colored film. It was found that the albumin fraction stained by Acid Violet-17 staining of the cellulose acetate membrane contained albumin, including the elongated portion.

(Reference Example 3) Anti-human α ₁ -antitrypsin on cellulose acetate membrane
Because emissions antibody according to the position of the alpha ₁ fraction in the electrophoretic image of immunostaining standard serum position of "albumin fraction elongated than usual" overlap,
It was examined using the α ₁ -antitrypsin antibody to determine whether the “albumin fraction extended from the normal” contains α ₁ -antitrypsin, which is the main component of the α ₁ fraction.

In Reference Example 1, the sample No. For No. 3, cellulose acetate membrane electrophoresis was performed in the same manner as in Reference Example 1. Transfer buffer (2
0 v / v% methanol, 25 mM trisaminomethane,
Filter paper buffered with 192 mM glycine (10 cm ×
7.5 cm) and a PVDF membrane (9 cm × 7 cm). Semi-dry trans blotter (Bio-Rad)
, A filter paper, a PVDF membrane, a cellulose acetate membrane after electrophoresis, and a filter paper were set in this order, and a constant current of 5.5 mA / cm ² was applied for 5 minutes. Next, the PVDF membrane was immersed in a blocking solution (Block Ace, manufactured by Dainippon Pharmaceutical) and shaken at room temperature for 60 minutes. Nonionic surfactant, Tween
After shaking and washing three times for 5 minutes each in PBS containing 0.1% of n20, the cells were placed in PBS containing 10 μg / mL of anti-human α ₁ -antitrypsin antibody (host goat, manufactured by Betyl) and shaken at room temperature for 60 minutes. did. After washing, the plate was placed in PBS containing 5 μg / mL of an alkaline phosphatase-labeled anti-goat IgG antibody (host rabbit, manufactured by Sigma) and shaken at room temperature for 60 minutes. After washing, the substrate solution (0.17 mg / mL BCIP,
(0.33 mg / mL NBT, 12 mg / mL trisaminomethane, 5.8 mg / mL sodium chloride, 1.0 mg / mL sodium azide) at room temperature for about 5 minutes.

FIG. 5 shows the colored film. It was found that α ₁ -antitrypsin was not present in the elongated portion of the albumin fraction stained by Acid Violet-17 staining of the cellulose acetate membrane. That is,
The “albumin fraction that is longer than usual” is not derived from α ₁ -antitrypsin, but is presumed to be a protein that has appeared due to denaturation of albumin.

Example 1 Urine of a patient suspected of having IgA nephropathy was collected and 2 cm from the upper end of a cellulose acetate membrane (7 cm × 12 cm) buffered with veronal buffer.
The urine sample is directly applied to the sample at a concentration of about 1.6 μg per sample. The membrane is set in a 238 type electrophoresis apparatus (manufactured by Joko) filled with veronal buffer so that the application point on the cellulose acetate membrane is 1 cm away from the cathode. Then, from the anode to the cathode, 0.7 mA / c
m is passed for 30 minutes, and the sample on the membrane is subjected to electrophoresis. The membrane after the electrophoresis is immersed in a fixing solution (3 w / v% trichloroacetic acid) and shaken for 3 minutes (75 strokes / min). Then, after shaking for 2 minutes in a pre-staining solution (0.125 w / v% acid violet-17, 5% acetic acid), the mixture was shaken in a staining solution (0.25 w / v% acid violet-17, 5% acetic acid). Shake for minutes. After shaking for 10 minutes in the decolorizing solution (5% acetic acid), the membrane is placed on a filter paper and dried at room temperature. Visually observe the band on the membrane,
Patients in which the albumin fraction extended to the cathode side was observed
Diagnose gA nephropathy.

[0029]

According to the present invention, IgA nephropathy can be diagnosed non-invasively by measuring the elongation degree of the albumin fraction obtained by subjecting a human urine sample to cellulose acetate membrane electrophoresis.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a photograph and an explanatory diagram instead of a diagram showing cellulose acetate membrane electrophoresis of a urine sample of a renal disorder patient.

FIG. 2 is a photograph and an explanatory diagram which are alternatives to the diagram showing cellulose acetate membrane electrophoresis of a urine specimen of a renal disorder patient.

FIG. 3 is a photograph and an explanatory diagram, which are alternatives to the diagram showing cellulose acetate membrane electrophoresis of a urine sample of a patient with renal impairment.

FIG. 4 is a photograph (right) in place of the immunoblot using an albumin antibody in a urine specimen subjected to cellulose acetate membrane electrophoresis, in which the albumin fraction was elongated, and a photograph stained with Acid Violet-17. (Left).

FIG. 5 is a photograph (right) in place of an immunoblot using α ₁ -antitrypsin antibody for a sample in which elongation of the albumin fraction was observed in a urine sample subjected to cellulose acetate membrane electrophoresis, and acid violet. 17 is a stained photograph (left).

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Claims (5)

[Claims] 1. An Ig which is characterized by confirming the presence of an albumin fraction elongated on the cathode side in a protein electrophoresis image obtained by subjecting a human urine sample to cellulose acetate membrane electrophoresis.
A method for diagnosing nephropathy. 2. The method according to claim 1, wherein the albumin fraction extended to the cathode side is compared with an albumin fraction in a urine specimen of a non-IgA nephropathy patient, a urine specimen of a healthy person, or a standard serum obtained by electrophoresis on a cellulose acetate membrane. The method for diagnosing IgA nephropathy according to claim 1, wherein the length in the direction is an albumin fraction confirmed to have extended to the cathode side. 3. The Ig according to claim 1, wherein the presence of the albumin fraction extended to the cathode side is confirmed by measuring the length, area or density of the albumin fraction.
A method for diagnosing nephropathy. 4. The method for diagnosing IgA nephropathy according to claim 1, wherein a urine sample of a non-IgA nephropathy patient, a urine sample of a healthy person or a standard serum is electrophoresed as a marker simultaneously with the human urine sample. . 5. The method for diagnosing IgA nephropathy according to claim 1, wherein the human urine sample is a urine sample of a patient with renal impairment.