JPH05302922A - Diagnosing method for kidney disease - Google Patents

Abstract

PURPOSE:To obtain the diagnosing method for kidney disease at an early period by detecting the decomposed material of human albumin in human urine. CONSTITUTION:When the decomposed material of human albumins in the healthy person's urine and the urine of the kidney disease patient are detected, the urines of the persons under test are separated by (1) SDS polyacrylamide gel electrophoresis method, (2) isoelectric gel electrophoresis method, (3) cellulose acetate film electrophoresis method and (4) high-performance liquid chromatograph method. The human albumin and the decomposed material of the human albumin are specifically detected by immunity transcription method, direct immunity coloring method or the detection of ultraviolet-part absorbing spectrum by the combination of affinity chromatography and high-performance liquid chromatography. Thus, the kidney disease is diagnosed at an early period. The diagnosis for the kidney disease at the earlier period than a conventional method can be performed by this method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for diagnosing renal diseases. More specifically, the present invention relates to a method for diagnosing renal diseases by detecting albumin degradation products in human urine.

[0002]

[Prior Art and Problems to be Solved by the Invention] Nephropathy,
In particular, since diabetic nephropathy is difficult to biopsy, clinical diagnosis has been made based on findings such as persistent proteinuria, renal dysfunction, and hypertension. However, when proteinuria appears, treatment is difficult.
It is customary to fall into end-stage renal failure after 6 years. Therefore, from the standpoint of clinical medicine, it is strongly desired to diagnose renal lesions and perform early treatment before urine protein becomes positive by the usual urine test strip method. A micrometric method for urinary albumin was developed for this purpose, and in recent years, it has become an essential test method for medical treatment of nephropathy.

Microalbumin in urine can be accurately quantified by RIA (radioimmunoassay) or immunoprecipitation method, but recently a simple kit is commercially available, and Arbushua (Eisai Co., Ltd.) is one of them. This is based on the immunoagglutination inhibition reaction, the reagents are albumin-sensitized latex and albumin antibody, and if there is almost no albumin in urine, the latex will agglutinate due to the antigen-antibody reaction,
If albumin is present in urine, it interferes with albumin and prevents aggregation.

Although there is a method for diagnosing renal disease by measuring a minute amount of albumin as described above, there is no method for diagnosing nephropathy at an earlier stage. That is, Mogen
Sen et al. ( N. Engl . J. Med ., 310 , 35.
6, 1984), microalbuminuria is a period of early nephropathy 6 to 20 years after the onset of diabetes mellitus, and there is a high risk of transition to the 4th stage (overt nephropathy). It is desired to develop a method for diagnosing the disease.

[0005]

[Means for Solving the Problems] As a result of diligent examination of a test method useful for early diagnosis of nephropathy, the present inventors have focused on albumin degradation products in human urine, which have been analyzed by electrophoresis and immunoblotting. Succeeded in detection and further separation and analysis using liquid chromatography, etc., and found that nephropathy can be discovered much earlier than conventionally known methods for diagnosing early nephropathy, and completed the present invention. It was

The human albumin hydrolyzate in the present invention is substantially all human albumin that has been degraded in vivo by an enzyme or the like and the original molecular weight is reduced to about 69,000. The presence of a degradation product of human albumin can be detected by separating it from human albumin by utilizing the difference in molecular weight or the difference in charge state and carrying out an antigen-antibody reaction.

[0007] A specific antigen-antibody reaction is generally used for the detection of human albumin and human albumin degradation products, and the immunological methods for these are EIA (enzyme immunoassay) method, RIA method and immunological method. Although there are blotting methods, immunoblotting method from the viewpoint of operability and speed, considering the combination with SDS polyacrylamide gel electrophoresis method, cellulose acetate membrane electrophoresis method, and isoelectric focusing gel electrophoresis method. Is the best.

On the other hand, in recent years, the progress of analysis of biological components by liquid chromatography has been remarkable, and particularly in high performance liquid chromatography, the number of theoretical plates of the column has increased,
With improvements in metering pumps and detectors, technologies such as autosampler and autoinjector system to complete the automation have been culminated, and it has become possible to operate day and night. ..

Particularly in the field of clinical examination, higher accuracy and faster analysis are desired. For example, in the analysis of hemoglobin A _IC, which is an index of the transition of blood glucose level in the past 1-2 months in diabetic patients. This high performance liquid chromatography is now becoming an indispensable analytical instrument.

When liquid chromatography including high performance liquid chromatography is applied to the separation and analysis of albumin degradation products in urine, urine contains complex and many kinds of biological components, so that they are separated at once by the same column. However, in order to improve the efficiency of the separation column, only the albumin and albumin degradation products in urine are selectively adsorbed to the post column (pre-column) and then eluted with the eluent to elute this column. It is also possible to separate and analyze each albumin degradation product with a separation column that is. Hereinafter, a method for separating and analyzing a human albumin degradation product will be described in detail, but this method introduces a part of the basic technique, and is not limited to these.

[SDS polyacrylamide electrophoresis method]
(Hereinafter, it is abbreviated as SDS-PAGE method.) For SDS-PAGE method, Shapiro et al. ( Bioch
em . Biophys . Res . Commun . , 2
8 , 815, 1967) and Weber et al. ( J. Bio ).
l . Chem . , 244 , 4406, 1969) and Laemmli ( Natur).
Re , 277 , 680, 1970) and the discontinuous buffer method, and any method can be used, but the method of Laemmli, which has excellent separation, is most preferable.

The concentration of the separating gel is usually 5%.
Although about 12.5% is used, it is not particularly specified as long as human albumin and a human albumin degradation product can be separated. Further, there are a disc migration method and a slab migration method, and either method can be used, but the slab migration method is preferable. In the immunoblotting method, human albumin and a human albumin degradation product are transferred onto a support, and then color is developed by an antigen-antibody reaction.

That is, after separating human albumin and human albumin degradation product by the SDS-PAGE method, each protein in the gel was rapidly transferred to a nitrocellulose membrane,
Adsorb to the film without diffusion. Since the protein on the membrane does not diffuse, the subsequent treatment can be performed over time. Next, a primary antibody (in the present invention, an anti-human albumin antibody is shown) is made to react and after washing,
The enzyme is reacted with a labeled secondary antibody. After further washing,
The presence or absence is confirmed by adding an enzyme detection reagent.

There are various transfer methods, among which the method of Towbin et al. ( Proc . Natl . Aca
d . Sci . , 76 , 4350-4354, 1979).
Is the best. As the support used for transfer, nitrocellulose, aminophenyl thioether paper, aminobenzyloxymethyl paper, diaminoethyl cellulose or the like can be used, but a nitrocellulose membrane is particularly suitable. The nitrocellulose membrane is BA85 (Schle
HAHY (Micher & Schuell)
(commercially available from Llipore). In addition, S
The DS polyacrylamide gel, electrophoretic device, nitrocellulose membrane, and transfer device are all commercially available from Tefco Corporation, so it is convenient to use them.

When an antigen-antibody reaction is carried out on a nitrocellulose membrane, bovine serum albumin (BSA), skim milk or the like is used to suppress non-specific reaction, but preferably cross-reactivity with anti-human albumin antibody is possible. Low skim milk is suitable. The anti-human albumin antibody can be easily obtained by immunizing a rabbit, but a commercially available product can also be used. More preferably, a highly pure antibody is preferably used to prevent non-specific reaction.
To obtain high purity, for example, purification by affinity chromatography is performed.

The secondary antibody differs depending on the animal species that produced the anti-human albumin antibody, but an antibody against IgG of rabbit, goat, mouse, etc. is usually used. More preferably, purification is performed in order to prevent non-specific reaction as in the case of anti-human albumin. Peroxidase, alkaline phosphatase, glucose oxidase, and the like can be used as the enzyme used for the enzyme-labeled product, but a peroxidase for which a pure product is easily available and an activity detection method is established is preferable.

[0017] It should be noted that the method of labeling the enzyme in the secondary antibody glutaraldehyde method (S.Arrameas, Imm
unchemistry , 6 , 43, 1969) and the periodate oxidation method (P.K. Nakane, A. Kawa).
oi, J. Hisrochem , Cytochem . ，
22 , 1084, 1974) and the like, and the labeled substance can be easily produced. The enzyme-labeled secondary antibody is also commercially available from Cappel and the like and can be easily obtained.

As a reagent for detecting an enzyme, hydrogen peroxide solution and diaminobenzidine, chloronaphthol, aminoethylcarbazole, etc. can be used for peroxidase, and a combination of hydrogen peroxide solution and diaminobenzidine is suitable. On the other hand, in the case of alkaline phosphatase, β-naphthyl phosphate, bromochloroindolyl phosphate, umbelliferyl phosphate and the like are used. Furthermore, glucose oxidase uses D-glucose.

The outline of this diagnostic method is shown below. First, urinary protein is solubilized. That is, it deactivates proteolytic enzymes present in urine, and at the same time SDS and β
-Heat treatment in boiling water for a certain time in order to effectively denature the protein with mercaptoethanol. Next, a fixed amount of the solubilized urine is injected into each lane of the SDS-polyacrylamide slab gel, and the glycine-Tris buffer containing SDS is used as a migration buffer to migrate at a constant current for a fixed time.

After the electrophoresis, the gel is equilibrated by immersing the gel in a chilled glycine-Tris buffer solution (transfer buffer solution) containing methanol for a certain period of time. The gel is mounted on the blotting device with the cathode side and the transfer support as the anode side. A transfer buffer solution is added to the transfer tank, and transfer is performed at a constant voltage for a fixed time under ice cooling. After transfer, the transfer support was washed with a phosphate physiological buffer solution (PBS) containing Tween 20,
Masking is performed with PBS containing skim milk for a certain time at a certain temperature.

Next, the anti-human albumin antibody is diluted with PBS containing BSA and reacted at a fixed temperature for a fixed time.
Furthermore, after the reaction, washing with PBS containing Tween 20
An enzyme-labeled secondary antibody previously diluted to a certain concentration with PBS containing BSA is reacted at a certain temperature for a certain period of time to detect a human albumin degradation product.

[Isoelectric Focusing Gel Electrophoresis] In general, electrophoresis utilizes the difference in the charge state of electrolytes at a specific pH, whereas isoelectric focusing gel electrophoresis uses amphoteric electrolytes at a certain value. At this pH, the effective charge becomes 0, and migration is stopped, so that the medium is migrated in a medium having a pH gradient to separate the proteins according to the value of the isoelectric point.

A carrier for forming a pH gradient can be easily formed by an ampholyte (Carrier ampholytes). Amphoteric carriers are described by Vesteberg et al. ( Acta . Chem . Scand ., 20 , 820-
834, 1966),
Recently, it has been marketed by several companies such as the brand name Pharmalyte (manufactured by Pharmacia).

As a support used for isoelectric focusing gel electrophoresis, polyacrylamide gel can be mentioned (provided that there is no electropermeability, but this condition is satisfied). This gel usually has a molecular sieving action, but when used for isoelectric focusing gel electrophoresis, the molecular sieving effect adversely deteriorates the separation, so that it is usually preferable to use a gel of 5% or less.

The preparation of gels containing an amphoteric carrier is described in various experimental books. For example, Yoshiyuki Kuchino, Hisamaru Hirai, Ikunosuke Sakurabayashi (Experimental operation blotting method, 239
-241, Soft Science Co., 1987) and the like can be referred to. As a commercial product, IEF PAGE mi
ni (registered trademark) (Tefco Corporation) can also be used. These commercially available products are slab gels and are suitable for processing a large number of specimens.

The conditions for isoelectric focusing polyacrylamide gel electrophoresis are not particularly limited, but usually 0.1 to 1.0 M sodium hydroxide is used for the catholyte and 0 for the anolyte as the electrode solution. 0.01-1 M phosphoric acid is used. The sample is dissolved in a liquid containing an amphoteric carrier and glycerol and used as a sample liquid. The migration voltage is preferably changed to about 100 to 500V.

When transferring various carriers after migration, for example, a nitrocellulose membrane is used as a support for transfer.
Aminophenyl thioether paper, aminobenzyloxymethyl paper, diaminoethyl cellulose and the like can be used, but a nitrocellulose membrane is particularly suitable. Nitrocellulose membrane is BA85 (Schleich
er & Schuell) HAHY (Milli
(manufactured by Pore Co.).

The transfer from the isoelectric point gel after the electrophoresis is performed by the method of Towtin et al. ( Proc . Natl . Aca .
d . Sci . , 76 , 4350-4354, 1979).
Carry out according to. In this case, 0.5 to 1.0% acetic acid is used as the blotting buffer, the gel and the membrane are reversed, and the membrane is mounted on the cathode side. In order to specifically detect a human albumin degradation product on a support such as a nitrocellulose membrane transferred under electric current, it is possible to detect it by the same operation as the method used in the SDS-PAGE method.

[0029] For example, after masking the transferred film on the nitrocellulose film with BSA, skim milk, etc., anti-human albumin antibody is made to react, and further an enzyme-labeled antibody is allowed to act, and a coloring agent such as hydrogen peroxide solution and diaminobench is added. Human albumin and a human albumin degradation product can be colored and detected using gin or the like. The isoelectric focusing gel electrophoresis, the transfer and the coloring method will be described in more detail below, but this method is an example and is not limited to this method.

That is, for isoelectric focusing, transfer and color development methods, first, an amphoteric carrier and glycerol are added to a fixed amount of urine of a subject to prepare a sample solution. Next, a fixed amount of the sample solution is injected into each lane of a polyacrylamide slab gel containing an amphoteric carrier, a sodium hydroxide solution is added to the cathode side and a phosphoric acid solution is added to the anode side as migration buffers, and electrophoresis is performed for a certain period of time.

After electrophoresis, the gel is immersed in a pre-cooled acetic acid solution for a certain period of time to equilibrate. The transfer support is attached to the blotting device with the cathode side and the gel on the anode side. An acetic acid solution is added to the transfer tank, and transfer is performed at a constant voltage for a fixed time under ice cooling. After transfer, the transfer support was washed with PBS containing Tween 20, and P containing skim milk was added.
Masking is performed with BS at a constant temperature for a constant time. Next, the anti-human albumin antibody is diluted with PBS containing BSA and reacted at a constant temperature for a constant time. After the reaction, Twee
After washing with PBS containing n20, an enzyme-labeled secondary antibody previously diluted with PBS containing BSA to a certain concentration is reacted at a certain temperature for a certain period of time to detect a human albumin degradation product.

[Cellulose Acetate Membrane Electrophoresis Method] In this method, a urine sample of a subject is applied onto a cellulose acetate membrane, electrophoresis is performed, and then the migrated cellulose acetate membrane is transferred to a nitrocellulose membrane. Or, after directly immobilizing the protein on the cellulose acetate membrane,
An antigen-antibody reaction is performed to selectively detect a human albumin degradation product. Generally, a cellulose acetate membrane is a membrane in which a part or all of the hydroxyl groups of cellulose are replaced with acetyl groups and which has uniform pores. This membrane has the following advantages over a support such as a filter paper.

That is, a. A thin, homogeneous porous membrane, b.
Can be performed with a small amount of sample, c. Almost no tailing phenomenon during migration, d. It is characterized by clear separation of each component. In particular, it has the advantage that separation of each component can be achieved sufficiently with a short migration distance, so that the migration time can be shortened and the support can be made narrower. Can be simultaneously processed and automated. Cellulose acetate membrane electrophoresis is widely used as one of screening tests in daily tests such as serum protein fractionation.

The cellulose acetate membrane and the electrophoretic device used in this method may be those commonly used. Although various cellulose acetate membranes are commercially available, Titan
III (manufactured by Helena Laboratories) is suitable in terms of strength because the back surface is laminated with a plastic plate.

The conditions of the cellulose acetate membrane electrophoresis are not particularly limited, but as the migration buffer, a veronal buffer (pH 8.6) generally used in the serum protein fractionation method can be used, but it is more clear. 0.2-0.4M Tris-glycine buffer (p
H 9.0 to 9.2) is preferable. Specimen application amount is 1c wide
It is generally 0.4 to 1.2 μl per m, but it is not particularly specified as long as it is possible to separate human albumin and human albumin degradation product. Regarding the energization condition, there is no problem as long as it is a commonly used method, but 50 to 2
It is desirable to select a current of about 00V.

The transfer method from the cellulose acetate membrane after the migration is usually accomplished by stacking supports such as a nitrocellulose membrane and pressing them at room temperature for several minutes. The support used for transfer may be the same product as that used for SDS polyacrylamide gel or isoelectric point gel. Alternatively, a protein denaturing agent may be added to the cellulose acetate membrane after the migration to directly immobilize it.

When an antigen-antibody reaction is carried out on a nitrocellulose membrane or a cellulose acetate membrane, it is necessary to suppress the non-specific reaction. This method uses SDS-PAGE.
Method and isoelectric focusing gel electrophoresis method. Since the amount of sample added is small in cellulose acetate membrane electrophoresis, it is necessary to further increase the sensitivity of the antigen-antibody reaction.

There are various methods for increasing the detection sensitivity, but the avidin-biotin method is suitable. That is, it is a method in which an anti-human albumin antibody is reacted, a biotinylated secondary antibody in which biotin is bound to the secondary antibody is reacted, and then an enzyme-labeled avidin is further reacted. Biotinylated antibody is Cappel, enzyme-labeled avidin is Zymed
It is commercially available from other companies and can be easily obtained.
In addition, anti-human albumin antibodies other than avidin and biotin,
The enzyme detection reagent may be the one used in the SDS-PAGE method or the isoelectric focusing gel electrophoresis method.

The electrophoresis, transfer and color development methods of the cellulose acetate film will be illustrated below, but this example is only an example and is not limited to this. That is,
Regarding the electrophoresis, transfer and color development method of the cellulose acetate film, first, a certain amount of urine of the subject is applied to the cellulose acetate film with an applicator. The cellulose acetate membrane is equilibrated with a migration buffer solution in advance.

It was mounted on a commercially available electrophoretic apparatus, and tris-glycine buffer was used on the anode side, and barbital sodium was used on the cathode side.
Add borate buffer and run at constant voltage. After the electrophoresis, the cellulose acetate membrane is pressure-bonded to the nitrocellulose membrane equilibrated with a glycine-Tris buffer solution containing methanol in advance for several minutes. An anti-human albumin antibody is reacted with a nitrocellulose membrane on which human albumin and a human albumin degradation product are transferred at a constant temperature for a constant time.

The nitrocellulose membrane is washed with PBS containing a surfactant, and the biotinylated anti-IgG antibody is reacted at a constant temperature for a fixed time. After further washing, the enzyme-labeled avidin is reacted at a constant temperature for a constant time. After washing, color is developed with a coloring agent to confirm the presence or absence of human albumin degradation products. Next, after migration on a cellulose acetate membrane, a method for specifically detecting only human amibmine and a human albumin degradation product by directly immobilizing human albumin and a human albumin degradation product, and then exemplifying the method is described below. This is an example and the present invention is not limited to this.

A certain amount of urine of the subject is applied to the cellulose acetate membrane with an applicator. The cellulose membrane is equilibrated with a buffer for migration in advance. Attached to a commercially available electrophoretic device, Tris-glycine buffer solution on the anode side,
Sodium barbital-borate buffer is added to the cathode side, and electrophoresis is performed at a constant voltage. After the migration, a trichloroacetic acid-sulfosalicylic acid solution is added to the cellulose acetate membrane to fix human albumin and a human albumin degradation product. Then, it is washed with pure water and reacted with an anti-human albumin antibody at a constant temperature for a predetermined time. The cellulose acetate membrane is washed with PBS containing a surfactant, and the biotinylated anti-IgG antibody is reacted at a constant temperature for a constant time.
After further washing, the enzyme-labeled avidin is reacted at a constant temperature for a constant time. After washing, color is developed with a coloring agent to confirm the presence or absence of human albumin degradation products.

[Liquid chromatographic method] Liquid chromatography methods include adsorption chromatography, ion exchange chromatography, partition chromatography, gel filtration chromatography and affinity chromatography. These liquid chromatographic techniques can be used alone or in combination with various liquid chromatographic methods for the separation and analysis of decomposed products. In the present invention, an example of a combination of affinity chromatography and gel filtration chromatography is shown, but the present invention is not limited to this.

First, in order to obtain a mixture of human albumin and a human albumin degradation product from the urine of a subject, affinity chromatography using a carrier to which an anti-human albumin antibody is bound can be advantageously used. For the binding of an antibody to a carrier, a method of covalently binding a polysaccharide such as agarose to activated agarose obtained by treating a polysaccharide with cyanogen halide, particularly cyanogen bromide (BrCN) via an amino group of a protein is generally used. Has been done. (A
xen, R.M. , Porath, J .; & Ernbac
k, S. Nature , 214 , 1302, 1967).

Various polysaccharides and the like have been reported as carriers used for affinity chromatography, and the carrier and antibody can be easily bound. For example, Sepharose 4B
(Made by Pharmacia) under alkaline conditions (pH 11-1)
2) React under BrCN to make BrCN-activated Sepharose 4B. The target carrier for affinity chromatography can be obtained by adding a protein solution, in this case, an anti-human albumin antibody or the like, usually under conditions of pH 8-10 and stirring.

Since BrCN is a poison, CNBr-a
Proteins, in this case anti-human albumin antibody, etc., can be easily coupled by using activated Sepharose 4B (registered trademark) (Pharmacia). Furthermore, in recent years, activated carriers for high-performance liquid chromatography have been marketed and can be used. That is, TSKgel using hydrophilic polymer
There is TSKgel Tresyl-5PW (manufactured by Tosoh Corporation) in which a tresyl group is introduced into G5000PW (manufactured by Tosoh Corporation), which is a carrier that reacts with primary amino groups or thiol groups of protein molecules.

The protein used for the ligand moiety, for example, an anti-human albumin antibody can be a polyclonal antibody or a monoclonal antibody obtained from any animal species, and can be easily obtained by self-preparation. In this case, the antiserum obtained by immunizing a rabbit with human albumin is purified with an affinity column (human albumin-Sepharose 4B) to obtain an anti-human albumin antibody with even higher purity. A mixture of human albumin and human albumin degradation products obtained by affinity chromatography can be separated and analyzed into human albumin and human albumin degradation products by gel filtration chromatography.

On the other hand, although it is possible to use an ordinary carrier such as Sephadex (Pharmacia) for gel filtration chromatography, it requires a large amount of sample, a long operation time and reproducibility. However, it is usually not so preferable. On the other hand, high performance liquid chromatography can perform separation analysis with a small amount of sample in a short time,
Further, it has advantages such as excellent reproducibility.
Usually, as a packing material for high performance liquid chromatography of proteins, TSKgel (manufactured by Tosoh Corporation), CPG-10 (El
Electro-Nucleonics), Prote
in column I-125 (manufactured by Waters)
Among them, TSKgel among them is commercially available, and carriers having various exclusion limits in terms of molecular weight are available, and non-specific adsorption to the carrier is very weak, which is suitable for protein separation and analysis. In particular, TSKgel
G3000SW, TSKgel G3000SWxL and TSKgel G2000SW, TSKgel G
The exclusion limits of 2000SWxL are 500 KDa and 100 KDa in terms of molecular weight, and both columns are suitable for separating human albumin (molecular weight 69,000) and human albumin degradation products.

The outline of the measuring method will be described below, but the present method is only an example and is not limited to this. A fixed amount of a urine sample of a subject is adsorbed on an anti-human albumin binding carrier, washed, and then eluted under acidic conditions to obtain human albumin and a human albumin degradation product. The obtained mixed solution is directly subjected to high performance gel filtration chromatography, and the elution pattern is determined by absorption in the ultraviolet (A280 nm).
If it is desired to further improve the detection sensitivity, a fluorescent substance such as FITC may be bound to the eluate of affinity chromatography to obtain the elution pattern of high performance gel filtration chromatography by the fluorescence intensity.

[0050]

EXAMPLES The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention. [Example 1] By SDS-PAGE method of human albumin degradation product in urine
Detection (SDS-PAGE method) Solubilization of urine: Equivalent amount of urine was added to 20 mM Tris-hydrochloric acid buffer solution (pH 6.8) containing 2% SDS, 2% 2-mercaptoethanol and 40% glycerin, and boiled for 5 minutes. Solubilized in. Electrophoresis: Performed according to the method of Laemmli. A polyacrylamide gel having a concentration gel of 4% and a separation gel of 12% (trade name SDS-PAGEmini, manufactured by Tefco Ltd.) was used. 10 μl of solubilized test urine in each lane
Injected. The running buffer is 380 containing 0.1% SDS.
Electrophoresis was carried out at 20 mA for 1.5 hours using mM glycine-50 mM Tris buffer (pH 8.3).

(Immunoblotting method) Transcription: The polyacrylamide gel after electrophoresis is treated with Towbin.
It was transferred to a nitrocellulose membrane (manufactured by Tefco Ltd.) by the method described above. That is, the gel after migration was pre-cooled with a buffer for transfer (190 mM glycine-25 mM Tris buffer containing 20% methanol pH 8.
Soak in 3) for 30 minutes to equilibrate. The polyacrylamide gel is mounted on the blotting device with the cathode side and the nitrocellulose membrane on the anode side. A pre-cooled transfer buffer is placed in the transfer tank. Transfer is under ice cooling 42V, 2
Done in time.

Detection of human albumin: 0.05 after transcription
The nitrocellulose membrane was washed three times with% Tween20-PBS (pH 7.2) and then masked with 3% skim milk / PBS at 4 ° C. overnight. Next, a rabbit anti-human albumin serum (manufactured by MBL) was 1: 2 with 1% BSA / PBS.
It was diluted to 00 and reacted at room temperature for 1 hour. 0.0 after reaction
Wash 3 times with 5% Tween 20-PBS and 1% BSA
HRPO anti-rabbit I diluted 1: 400 in / PBS
It was reacted with a gG antibody (manufactured by Cappel) at room temperature for 1 hour. After cleaning, 0.007% hydrogen peroxide solution and 0.025%
Color was developed with 0.05M Tris-hydrochloric acid buffer (pH 7.2) containing 3,3'-diaminobenzidine (Kenji Ogata, et al., Clinical Pathology, 31 , 215, 1983). The reaction was stopped with distilled water, and the presence or absence of a human albumin degradation product was confirmed. When it was carried out on a healthy person, a band was detected only in human albumin (molecular weight 69000), and the presence of a human albumin degradation product was not recognized.

[Comparative Example 1] Human albumin degradation products were subjected to the same procedures as in Example 1 for urine of 5 healthy subjects, 9 patients with IgA nephropathy, 4 patients with diabetic nephropathy, and 9 patients with diabetes. Was detected. FIG. 1 shows the results of immunoblot patterns of urine of healthy subjects. In all 5 healthy individuals, only one band derived from human albumin (molecular weight 69,000) was detected. FIG. 2 shows the results of immunoblot patterns of urine of patients with IgA nephropathy. In the patients with IgA nephropathy, in addition to human albumin, all 6 human albumin degradation products were detected in the low molecular weight region. FIG. 3 shows the results of immunoblot patterns of urine from patients with diabetic nephropathy. In the patients with diabetic nephropathy, human albumin degradation products were detected in all four patients in addition to human albumin in the low molecular weight region. FIG. 4 shows the results of immunoblot patterns of diabetic patient urine. In 9 out of 9 diabetic patients, in addition to human albumin, human albumin degradation products were detected in the low molecular weight region in addition to human albumin.

[Comparative Example 2] A trace amount of albumin was detected using the urine sample used in Comparative Example 1 and a comparison was made in sensitivity with this method. Albushua (Eisai Co., Ltd.) was used for detection of trace albumin. The results are shown in Table 1.

[Example 2] By isoelectric focusing of human albumin degradation products in urine
Detected human urine was diluted 5-fold with the sample buffer shown below. [Sample buffer: Survey (Ser
va) pH 3-10, 0.2 ml, glycerol 3.
0 ml to 10 ml with distilled water. ] The urine sample solution prepared above is subjected to IEF PAGEmini
10 μl was injected into a lane of pH3-10 (manufactured by Tefco). The running buffer was 0.05 M NaOH on the cathode side and 0.01 MH ₃ PO ₄ on the anode side.
Electrophoresis was performed by gradually applying a voltage of 200 V for 30 minutes and 500 V for 60 minutes. After the electrophoresis, the gel was immersed in 0.5% acetic acid which had been cooled in advance for 30 minutes for equilibration. A nitrocellulose membrane (manufactured by S & S) was attached to the cathode side of the blotting device, and a polyacrylamide gel was attached to the anode side. Pre-cooled 0.5% acetic acid was placed in the transfer tank, and transfer was performed at 42 V for 2 hours under ice cooling.

After transfer, 0.05% Tween20-PB
After washing the nitrocellulose membrane three times with S (pH 7.2),
Masking was performed with 3% skim milk / PBS at 4 ° C. overnight. Next, rabbit anti-human albumin serum (manufactured by MBL) was diluted 1: 200 with 1% BSA / PBS, and was diluted at room temperature with 1%.
Reacted for hours. After the reaction, 0.05% Tween20-
Wash 3 times with PBS and 1:40 with 1% BSA / PBS
HRPO anti-rabbit IgG antibody diluted to 0 (Cappe
(manufactured by Company I) at room temperature for 1 hour. After washing 0.00
0.05M Tris-hydrochloric acid buffer solution containing 7% hydrogen peroxide and 0.025% 3,3'-diaminobenzidine (pH
7.2) (Kenji Ogata, et al., Clinical Pathology, 31 , 215, 1)
983). After color development, the product was washed with distilled water and the presence or absence of a human albumin degradation product was confirmed. When the urine of a healthy person was tested, it was detected as a single band near the isoelectric point (hereinafter abbreviated as pI) 4.0, which has the same mobility as the control human albumin (molecular weight 69,000).

Comparative Example 3 One healthy subject and two diabetic patients
And 1 patient with diabetic nephropathy was detected by isoelectric focusing electrophoresis. The result is shown in FIG. A band derived from human albumin (molecular weight 69,000) was recognized as a single band around pI 4.0 in healthy subjects and diabetic patients 1, but pI5 to diabetic patients 2 and diabetic nephropathy patients were found. A human albumin degradation product was recognized in the vicinity of 7 as several to more than 10 bands.

[Example 3] In urine using a transfer coloring method from a cellulose acetate film
Detection of human albumin degradation product (electrophoretic operation) Urine sample on cellulose acetate membrane (trade name: Titan III-ZZ, manufactured by Helena Laboratories) equilibrated with 0.34M Tris-glycine buffer (pH 9.1) Was applied using an applicator (trade name: Super Z Applicator, Helena Laboratory Co., Ltd.). The running buffer was 0.34 M Tris-glycine buffer (pH 9.1) on the anode side and 0.05 M sodium barbital-borate buffer (pH 8.9) on the cathode side (trade name: Electra AC buffer, Helena Laboratory). (Manufactured by K.K.), and the electrophoretic conditions were 100 V for 45 minutes.

(Transfer operation) In transferring the migrated cellulose acetate membrane to the nitrocellulose membrane, the migrated cellulose acetate membrane contains 20% methal.
19M glycine-0.025M Tris buffer (pH 8.
The nitrocellulose membrane equilibrated in 3) was pressed and transferred at room temperature for 5 minutes.

(Coloring operation) Rabbit anti-human albumin antibody (manufactured by MBL) was diluted 1: 400 with 1% BSA / PBS and reacted at room temperature for 30 minutes. After reaction, 0.05%
Wash 3 times with Tween 20-PBS, 1% BSA / P
Biotin anti-rabbit Ig diluted 1: 2000 in BS
It was reacted with G antibody (manufactured by Cappel) at room temperature for 30 minutes. Next, after washing with 0.05% Tween 20-PBS,
Streptavidin-HRPO (manufactured by Zymed) 1
Dilute 1: 1000 with% BSA / PBS and mix at room temperature for 3
The reaction was allowed for 0 minutes. After the reaction, 0.05% Tween 20
-Wash with PBS and add 0.007% hydrogen peroxide and 0.025% 3,3'-diaminobenzidine
Color was developed with 05M Tris-hydrochloric acid buffer (pH 7.2). After color development, the product was washed with pure water and the presence or absence of a human albumin degradation product was confirmed. When urine of a healthy person was analyzed, a single band having the same mobility as that of the control human albumin (molecular weight 69,000) was detected, and the presence of a human albumin degradation product was not recognized.

Example 4 Direct Immobilization on Cellulose Acetate Membrane by Coloring Method
Detection of human albumin degradation products in urine (electrophoretic operation) On a cellulose acetate membrane (trade name Titan III-ZZ, manufactured by Helena Laboratories) equilibrated with 0.34 M Tris-glycine buffer (pH 9.1). About 1 μl of the test urine was applied using an applicator (trade name: Super Z applicator, manufactured by Helena Laboratories). The running buffer was 0.34 M Tris-glycine buffer (pH 9.1) on the anode side and 0.05 M sodium barbital-borate buffer (pH 8.9) on the cathode side (trade name: Electra AC buffer, Helena Laboratory). (Manufactured by Mfg. Co., Ltd.), and the electrophoretic conditions were 100 V and 45 minutes.

(Protein immobilization operation) After the electrophoresis,
Immobilize the protein on the cellulose acetate membrane with 5% trichloroacetic acid-5% sulfosalicylic acid solution, and add 3 with distilled water.
Washed twice.

(Coloring operation) A rabbit anti-human albumin antibody (manufactured by MBL) was diluted 1: 400 with 1% BSA / PBS and reacted at room temperature for 30 minutes. After reaction, 0.05%
Wash 3 times with Tween 20-PBS, 1% BSA / P
Biotin anti-rabbit Ig diluted 1: 2000 in BS
It was reacted with G antibody (manufactured by Cappel) at room temperature for 30 minutes. Next, after washing with 0.05% Tween 20-PBS, streptavidin-HRPO (manufactured by Zymed).
Was diluted 1: 1000 with 1% BSA / PBS and reacted at room temperature for 30 minutes. After the reaction, 0.05% Tween2
The cells were washed with 0-PBS and developed with 0.05M Tris-hydrochloric acid buffer solution (pH 7.2) containing 0.007% hydrogen peroxide solution and 0.025% 3,3'-diaminobenzidine. . After color development, the product was washed with distilled water to confirm the presence or absence of a human albumin degradation product. When urine of a healthy person was analyzed, a single band having the same mobility as that of the control human albumin (molecular weight 69,000) was detected, and the presence of a human albumin degradation product was not recognized.

Comparative Example 4 One healthy subject and two diabetic patients
For one patient with diabetic nephropathy, the urine was analyzed by the method described in Examples 3 and 4, that is, the transfer coloring method from the cellulose acetate film and the direct fixing coloring method on the cellulose acetate film. The respective electrophoretic patterns were compared with each other. The results are shown in FIG. 6 which is an immunoblot pattern obtained by the transfer coloring method from a cellulose acetate film. In healthy subjects and diabetic patients 1, only a single band derived from human albumin (molecular weight 69,000) was detected. On the other hand, in diabetic patient 2 and diabetic nephropathy patient, albumin degradation products were detected as many bands. On the other hand, FIG. 7 shows a result of an immunoblot pattern of a direct immobilization coloring method on a cellulose acetate membrane. A migration pattern similar to that of the transfer coloring method from the cellulose acetate membrane was shown, but each band of the human albumin degradation product was detected as a wide band due to the intensity of diffusion or coloring.

Example 5 Human by Gel Filtration Chromatography
Of albumin and human albumin degradation products (BrCN degradation)
Separation and Analysis (Preparation of Specimen) As a specimen of human albumin, a purified product by chromatography (manufactured by Cappel) was used. On the other hand, the albumin degradation product by BrCN is McMenam.
Y. et al. ( J. Biol , Chem ., 246 , 47.
44-4750, 1971). That is, human albumin (fraction V, manufactured by Sigma) 1
g in distilled water 4 ml, then formic acid 16 ml, BrC
N1g was added and it was made to react at 4 degreeC for 24 hours. Next, Sephadex G, which had been previously equilibrated with 1% propionic acid,
-25 (manufactured by Pharmacia) and the fractions showing absorption at 280 nm were collected to obtain 800 mg of albumin degradation product.

(Gel filtration chromatography) TSKgel G3000SW (7.5 mmφ ×
60 cm) to make a 0.55 M glycine-hydrochloric acid buffer solution having a pH of 3.0, to which NaCl is added at 0.15 mol, and SDS is added at 0.1% by weight to prepare a uniform solution. And was used for equilibration. Human albumin and a human albumin degradation product were dissolved in the above buffer at 1 mg / ml. S for human albumin molecule
A sample to which 1% 2-mercaptoethanol was added was prepared separately because the -S bond exists at 17 sites. 100 μl of this sample was injected into the column, and the eluent was flown at a flow rate of 0.
The solution was passed at 6 ml / min and was detected in the ultraviolet (A280n
m). As shown in FIG. 8, the result shows that human albumin was shown as a single peak under non-reducing conditions, but the human albumin degradation product was separated into four peaks.
In addition, under reducing conditions, human albumin has a single peak as in non-reducing conditions, but in the case of human albumin degradation products, the previous 4 peaks move to a lower molecular region, and chromatography under reducing conditions has a higher molecular weight. Separation due to the difference of was clearly shown.

[Example 6] Affinity chromatography using urine of patients with nephropathy
-, A combination of gel filtration chromatography
Detection of Human Albumin Degradation Products by Settling (Part 1) (Preparation Method of Anti-Human Albumin Antibody-Sepharose 4B)
CNBr-activated Sepharose
4B (registered trademark) (Pharmacia) 3 g (about 10 m)
1 gel) is swollen in 1 mM HCl to give 1 mM H
After washing with 500 ml of Cl, 500 ml of a 0.1 M NaHCO ₃ solution (pH 8.3) containing 0.5 M NaCl (hereinafter referred to as coupling buffer) was used. 50 mg of a rabbit anti-human albumin antibody purified by affinity chromatography which had been dissolved in a coupling buffer in advance was added, and the mixture was reacted at room temperature for 2 hours. Then, after washing with 500 ml of coupling buffer, 50 ml of 0.2 M glycine solution at pH 8.0 was used at room temperature for 2 hours to block residual active groups, and to remove excess adsorbed protein, 0.5 ml of coupling buffer was added.
The carrier for affinity chromatography was washed with 5M glycine-hydrochloric acid buffer (pH 3.0) alternately. This carrier had 5 mg of anti-human albumin antibody bound per 1 ml of gel.

(Immunoaffinity chromatography) A urine sample (2 ml) was adsorbed to anti-human albumin antibody-Sepharose 4B (3 ml) which had been equilibrated with 5 mM borate buffer (pH 8.0) in advance. After adsorption 5
Wash with double volume of 5 mM borate buffer (pH 8.0),
Elution was performed with 0.5 M glycine buffer (pH 3.0). Fractions showing ultraviolet absorption (A280 nm) were collected and mixed with human albumin and a human albumin degradation product (2
ml) was obtained.

(Separation analysis of albumin and albumin degradation products using TSKgel G3000SW) TSKge
l G3000SW (7.5 mmφ x 60 cm), p
A 0.55 M glycine-hydrochloric acid buffer solution of H3.0 is prepared, and NaCl is added to this in an amount of 0.15 mol to further add S.
DS was added to 0.1% by weight to form a uniform solution, which was used for equilibration. The final concentration of 0.1 was added to the eluent of immunoaffinity chromatography [mixture of human albumin and human albumin degradation product (1 ml)].
SDS was added so that the concentration became 100%, and 100 μl thereof was injected. The eluent was passed through at a flow rate of 0.6 ml / min, and detection was performed in the ultraviolet (A280 nm).

(Urine sample) 1 healthy subject, 2 diabetic patients
, 1 diabetic nephropathy, 1 IgA nephropathy patient, 1 SLE patient were analyzed. The results are shown in FIG. 9. A single peak of human albumin was found in the urine of healthy subjects and diabetic patient 1, but a peak of human albumin was found in the urine of diabetic patient 2 in addition to the peak of human albumin. Was given. Furthermore, a human albumin degradation product was remarkably observed also in the urine of diabetic nephropathy patients, IgA nephropathy patients, and SLE patients.

[Example 7] Affinity chromatography using urine of patients with nephropathy
-, Combination of gel filtration chromatography
Detection of human albumin degradation product by (2) (Preparation of anti-human albumin antibody-TSKgel 5PW) To 0.5 g of TSKgel Tresyl-5PW, 2 ml of 1.0 M potassium phosphate and 20 mg of rabbit anti-human albumin antibody were added, and the mixture was placed in an Erlenmeyer flask at room temperature. Immobilization was performed while shaking for 16 hours. The amount of immobilized anti-human albumin antibody was 9 mg per 1 ml of gel. This anti-human albumin antibody-TSKgel 5PW (1.0
(ml) was packed in 10 mmφ × 20 mm under reduced pressure by an aspirator to obtain a high-speed immunoaffinity column. The amount of human albumin bound on the column was 2 mg per 1 ml of gel.

(Anti-human albumin antibody-TSKgel
Purification of human albumin and human albumin degradation products using 5PW) Anti-human albumin antibody-TSKgel 5
A PW column (10 mmφ × 20 mm) was attached to a high performance liquid chromatograph, and 0.1 M phosphate buffer (pH
7.4) was washed at a flow rate of 2.0 ml / min. Inject 50 to 100 μl of urine sample and elute with 0.1 M citric acid-
It was carried out with hydrochloric acid (pH 1.6), and the elution time was completed within 10 minutes.

(Separation of human albumin and human albumin degradation product using TSKgel G3000SW) TSK
gel G3000SW (7.5 mmφ x 60 cm)
Was made into a 0.55 M glycine-hydrochloric acid buffer solution having a pH of 3.0, and NaCl was added to the solution to a concentration of 0.15 mol, and SDS was added to a concentration of 0.1% by weight to form a uniform solution. Was used for equilibration. The eluent for immunoaffinity chromatography [mixture of human albumin and human albumin degradation product (2 ml)] was adjusted to pH 3.
The sample was adjusted to 0 and 100 μl of a sample to which SDS was added so that the final concentration was 0.1% was injected. Flow rate of eluent is 0.6 ml
/ Min, and the detection was performed in the ultraviolet (A280 nm).

(Urine sample) 1 healthy subject, 2 diabetic patients
, 1 diabetic nephropathy, 1 IgA nephropathy patient, 1 SLE patient were analyzed, but the same results as in Example 6 were obtained, so the data were omitted.

[0075]

[Table 1]

As a result of analysis by SDS-PAGE, I
All cases were positive in gA nephropathy, but one case was negative in the microalbumin detection method. In diabetes, 6 out of 9 of the present methods were positive, whereas the microalbumin detection method was negative in all cases. Furthermore, in diabetic nephropathy, this method and trace albumin detection were all positive.
On the other hand, in healthy subjects, both cases were negative. Therefore, this method was found to be more sensitive to renal diseases than conventional methods and particularly useful for early detection of diabetic nephropathy.

[Brief description of drawings]

FIG. 1 is a photograph showing a pattern of an immune transcription method of 5 healthy subjects by SDS-PAGE.

FIG. 2 is a photograph showing a pattern of immunotranscription method of 6 IgA nephropathy patients by SDS-PAGE method.

FIG. 3 is a photograph showing a pattern of immunotranscription method of 4 diabetic nephropathy patients by SDS-PAGE method.

FIG. 4 is a photograph of a pattern of the immunotranscription method of 9 diabetes patients by SDS-PAGE method.

FIG. 5 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by isoelectric focusing gel-immunotranscription method.

FIG. 6 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by cellulose acetate membrane electrophoresis-immunotranscription method.

FIG. 7 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by cellulose acetate membrane electrophoresis-immunodirect coloring method.

FIG. 8 is a high performance liquid chromatogram of a human albumin degradation product obtained by decomposing human albumin with BrCN.

FIG. 9 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and patients with each renal disease by high performance liquid chromatography.

[Procedure amendment]

[Submission date] April 23, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 SDS polyacrylamide gel electrophoresis (SDS
-PAGE method) is a photograph showing the pattern of the immunotranscription method of 5 healthy subjects.

FIG. 2 SDS polyacrylamide gel electrophoresis (SDS
-PAGE method) is a photograph showing a pattern of immunotranscription method of 6 IgA nephropathy patients.

FIG. 3 SDS polyacrylamide gel electrophoresis (SDS
-PAGE method) is a photograph showing the pattern of the immunotranscription method of 4 diabetic nephropathy patients.

FIG. 4 SDS polyacrylamide gel electrophoresis (SDS
-PAGE method) is a photograph showing the pattern of the immunotranscription method of 9 people with diabetes.

FIG. 5 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by isoelectric focusing gel-immunotranscription method.

FIG. 6 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by cellulose acetate membrane electrophoresis-immunotranscription method.

FIG. 7 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and diabetic patients by cellulose acetate membrane electrophoresis-immunodirect coloring method.

FIG. 8 is a high performance liquid chromatogram of a human albumin degradation product obtained by decomposing human albumin with BrCN.

FIG. 9 is a comparison of human albumin and human albumin degradation products in urine of healthy subjects and patients with each renal disease by high performance liquid chromatography.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G01N 33/68 7055-2J

Claims (8)

[Claims] 1. A method for diagnosing renal disease by detecting an albumin degradation product in human urine. 2. The method according to claim 1, wherein the detection of the albumin degradation product in human urine is an immunological analysis method using electrophoresis. 3. The detection of albumin hydrolyzate in human urine is carried out by separating human urine into human albumin and human albumin hydrolyzate by SDS polyacrylamide gel electrophoresis or isoelectric focusing gel electrophoresis. After transfer of the albumin degradation product to a transfer support to immobilize the solid phase and reacting the immobilizing support with an anti-human albumin antibody as a primary antibody,
The method according to claim 2, wherein a secondary antibody labeled with an enzyme is reacted, and then a degradation product of human albumin is colored with a detection reagent for the enzyme. 4. The detection of the albumin degradation product in human urine is performed by subjecting human urine to electrophoresis on a cellulose acetate membrane to separate human albumin and human albumin degradation product, and the albumin degradation product on the membrane is detected. Immobilize it directly or on a support for transcription, react the anti-human albumin antibody as a primary antibody with the immobilized support, then react with biotin anti-IgG antibody as a secondary antibody, and further react with enzyme-labeled avidin 3. The method according to claim 2, wherein the digested product of human albumin is colored with an enzyme detection reagent. 5. The method according to claim 1, wherein the detection of the albumin degradation product in human urine is an analysis method using liquid chromatography. 6. The method according to claim 5, wherein the detection of the albumin degradation product in human urine is an analysis method using affinity chromatography and gel filtration. 7. The method according to claim 5, wherein the affinity chromatography ligand is an anti-human albumin antibody. 8. The method according to claim 5, wherein the carrier used for gel filtration is used for separation and analysis of proteins.