JP2005134144A - Urinary protein analysis pathologic analysis method using electrophoresis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the most important problem for diagnosis and remedy not by performing type classification on electrophoretic images but by predicting the existence of a specific disease from an electrophoretic image, which is different from that of normal health and peculiar to a disorder, and to determine which organ and which portion thereof the disease is substantially caused by, in order to perform urinary protein analysis pathologic analysis characterized by classifying electrophoretic images of electrophoresed and silver-stained urinary protein into a group composed mainly of glomerulus damages, a group composed mainly of renal tube damages, a group where Bence-Jones protein comes into being, a group of normal healthy persons, etc. <P>SOLUTION: Pathologic analysis is performed by using peculiar algorithm classified by disorder based not only on a conventional densitogram but also on differences in individual concentration, on differences from the group of normal healthy persons, on whether there are peculiar protein bands, etc. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Among the biochemical analyzes of urine from humans and animals, the field in which urine protein is electrophoresed and the type of disease is identified from the features of the analysis results

Tests using urine are widely used for screening at the time of medical examination and resident screening as test substances that can be collected without causing pain to the human body. Regarding the analysis of urine protein among the measurement items used in these, the so-called negative in which protein does not appear is generally regarded as normal in the test method using a test paper. However, it is known that a very small amount of protein is excreted even in a healthy person using a highly sensitive silver staining method as in Patent Document 1. This method for analyzing trace amounts of proteins by electrophoresis is described in detail in Non-Patent Document 1 and Non-Patent Document 2 by Shiba et al. According to this analysis, the analysis of electrophoretic images of urinary proteins using the silver staining method states that diabetes can be roughly divided into 5 groups according to the shape of the electrophoretic image.
JP 2002-236127 A Biophysical chemistry vol.41, 1997, pp. 25-27 Laboratory test vol.42, 1998, pp. 1106-1109

The present invention uses a highly sensitive urinary protein analysis method using a silver staining method, and does not classify an electrophoretic image that has been conventionally performed. We sought to solve the problem that predicting the presence of the disease and identifying which parts of which organ it was roughly attributed to is most important for diagnosis and treatment.

In order to solve the problem, the characteristics of each electrophoretic image are clarified based on clinical diagnosis by many renal biopsies, and even if you are not a specialist, you can grasp the pathology of renal disease from the characteristics of the electrophoretic image without performing a renal biopsy We aimed to develop a method that can be used.

In general, the appearance of urine protein itself predicts impaired renal function and kidney disease because of the renal function that urine is produced in the kidney and necessary nutrients are reabsorbed and reused. Among the renal diseases, IgA nephropathy, membranous nephropathy, focal glomerulosclerosis and the like are known as renal glomerular disorders. Examples of renal tubular disorders include Fancony syndrome and interstitial nephritis.

Renal biopsy is widely performed to make a definitive diagnosis of the kidney disease. Renal biopsy is a method of puncturing the needle toward the kidney, taking out the kidney tissue and performing pathological diagnosis by a specialist after tissue staining, not only causing considerable pain to the patient, but also collecting tissue and preparing pathological tissue specimens, It took considerable cost, time and skill to judge the specimen.

Furthermore, from the patient's side, complications related to bleeding such as gross hematuria and hematoma formation occur after renal biopsy, and if the degree of bleeding is high, blood pressure may decrease and shock may develop.

These examination methods other than renal biopsy include diagnostic imaging such as CT, quantification of urinary protein, and urine protein fractionation. Among them, quantitative tests of urinary protein include measurement of glomerular proteins such as albumin and transferrin, measurement of tubular proteins such as retinol binding protein, β2 microglobulin, NAG, immunoglobulin light chain (κ, λ) and There are various methods such as measurement of proteins caused by diseases of other organs, but there are many disadvantages such as using too many kinds of proteins that need to be measured or using expensive antibodies such as immunoassays, which are not practical. It was not implemented.

Conventional urine protein fractionation tests are performed by concentrating urine several tens of times because the urinary protein concentration is quite low, and it takes about 10 hours, and the separated electrophoretic image is also concentrated. In many cases, it is difficult to make an accurate diagnosis due to the unclearness of the fractions due to the influence of the above.

As a result of detailed examination of clinical examples of electrophoretic images of urinary proteins of many kidney diseases using the silver staining method of Patent Document 1, the present invention has been able to identify a disease-specific algorithm.

First, as shown in the Examples, the electrophoretic image obtained by silver staining of urine protein gives various and various impressions depending on differences in individual kidney functions and disease conditions. Even if this various miscellaneous image is directly converted into a conventional density diagram (densitogram) and forwarded to a specialist, it is too difficult to determine which disease group it corresponds to.

In the present invention, not only the densitogram but also the differences in individual concentrations and the group of healthy subjects and the presence or absence of specific protein bands are classified into four groups according to the disease, and the pathological analysis of the algorithm specific to that group is performed by a computer. Did and came to the conclusion that it was very useful.

FIG. 1 shows actual electrophoretic images of 4 groups of urine proteins by silver staining. (1) (2) is a glomerular disorder group, (3) (4) is a renal tubular disorder group, (5) (6) is a Bence Jones protein appearance group, (7) (8) is a healthy group. is there.

Since the concentration of excreted urinary protein varies depending on the patient's pathology, the concentration is measured in advance by the quantitative method such as Patent Document 1 described above before electrophoresis, and the patient's urine is transferred to the electrophoresis support according to the concentration. Since the coating amount is constant, the change in color tone in FIG. 1 can be recognized as a difference in density.

The disease names related to the specified algorithm of pathological analysis are as follows.

1. In the silver-stained electrophoretic image, if the fraction values of albumin fraction and β fraction are significantly higher than those of other disease groups, respectively, it is the renal glomerular disorder group. Names of diseases include IgA nephropathy, membranous nephropathy, focal glomerulosclerosis, diabetic nephropathy, membranoproliferative glomerulonephritis, lupus nephritis and the like.

2. The fraction value of the albumin fraction is significantly lower than the fraction value of the albumin fraction of the group considered to have glomerular disorders, and another protein fraction is sandwiched between them instead of the β fraction. If it appears significantly deeper, it is the renal tubule disorder group, and the main disease names are Fancony syndrome, cystinuria, interstitial nephritis, gout kidney, drug tubular necrosis, tubule acidosis, cadmium poisoning, etc. included.

3. A group of Bence Jones proteins that appears when a single, distinct protein staining band is detected in the globulin region. Major disease names include multiple myeloma, oligoclonal and primary amyloidosis. The Bence Jones protein is a so-called monocloned plasma cell that is overproduced by the immunoglobulin light chain, and its blood concentration increases, so the protein that could not be reabsorbed is excreted in the urine. The

4. The silver-stained albumin fraction is significantly lighter in color than the albumin fractions of other diseases and has a single staining band, and the entire globulin region is thinly banded, so-called large dumpling staining band When it becomes, it is a group of healthy people. In healthy individuals, the permeability of albumin through the kidney glomerulus is low, and most of the low molecular weight protein is reabsorbed by the tubules.

Further, the above algorithm is not described at all in patent documents and non-patent documents.

1. A definitive diagnosis of a renal disease that must be examined by a renal biopsy requires the patient to suffer pain such as extra renal puncture, and requires time-consuming preparation of a pathological specimen and diagnosis of a specialist by a microscope of the specimen. A large effect can be expected, such as conducting a screening test using this measurement method for a large number of patients who have been left without a renal biopsy, but who have to do a renal biopsy early.

2. Applying the urine protein analysis pathology analysis method to detect kidney disease and Bence Jones protein dramatically simplifies diagnosis, shortens measurement time and lowers test cost compared to renal biopsy .

3. Early treatment by early diagnosis of kidney disease can delay the start of artificial dialysis and prevent deterioration of the disease state, resulting in a significant reduction in medical costs.

4). Since the electrophoretic image of urine shows various patterns, it is possible to determine the disease group even if it is not a renal specialist by the present urine protein analysis pathological analysis, which is difficult for the specialist to judge.

Electrophoresis of urinary protein, silver staining, and electrophoretic image of the electrophoretic image for groups with mainly glomerular glomerular disorders, groups with mainly renal tubular disorders, groups with Bence Jones protein appearing, groups of healthy subjects, etc. Classify.

FIG. 2 is a schematic diagram (9) of electrophoretic images of healthy serum proteins, and (10) to (15) are six schematic diagrams of electrophoretic images of urine from a patient diagnosed as having a glomerular disorder. (16) is the albumin fraction and (17) is the β fraction. All the patient groups of (10) to (15) are clearly denser than the concentrations of the albumin fraction and the β fraction in FIGS.

Since the α1 and α2 fractions appear in various ways, the patient groups (10) to (15) are classified.

FIG. 3 is a schematic diagram (9) of electrophoretic images of healthy serum proteins and four schematic diagrams of urine electrophoretic images of patients diagnosed as renal tubular disorder patients (18) to (21). (16) is the albumin fraction and (17) is the position of the β fraction, but it is not visible here. (22) and (23) appear in a form sandwiching the β fraction (17), respectively.

Since the α1 and α2 fractions appear in various ways, the patient groups (18) to (20) are classified. Regarding (21), it is acidic urine, which is a degradation of the protein (β2 microglobulin) in the staining zone of (23).

FIG. 4 shows schematic diagrams (9) of electrophoretic images of healthy serum proteins and six schematic diagrams of electropherograms of urine of patients diagnosed as having Bence Jones protein in (24) to (29). (30) is the gamma globulin region. In (31) to (36), distinct protein staining bands were observed at different positions.

FIG. 5 shows a schematic diagram (9) of an electrophoretic image of healthy serum protein and a schematic diagram (37) of an electrophoretic image of urine of a healthy person. (16) is the albumin fraction, and (38) is the α1 globulin to γ globulin region. (16) appears in a single staining band, but the globulin region of (38) is entirely thin and dumpled.

Representative electrophoretic images of urine protein group 4 Schematic diagram of electrophoretic image of healthy serum protein and urine of glomerular disorder patient Schematic diagram of electrophoretic image of healthy serum protein and urine of renal tubule disorder patient Schematic diagram of electrophoretic images of urine from patients with healthy serum protein and Bence Jones protein Schematic diagram of electrophoretic images of healthy serum protein and urine of healthy subjects

Explanation of symbols

(1) (2) Representative electrophoretic images of the glomerular disorder group
(3) (4) Representative electrophoretic images of renal tubule injury group
(5) (6) Representative electrophoresis image of Bence Jones protein appearance group
(7) (8) Representative electrophoretic images of healthy subjects
(9) Schematic diagram of electrophoresis image of normal serum protein
(10)-(15) Schematic diagram of electrophoretic image of urine from glomerular disorder patient
(16) Albumin fraction
(17) β fraction
(18)-(21) Schematic diagram of electrophoretic image of urine from renal tubule disorder patient
(22) Albumin side protein staining band (retinol binding protein) appearing between (17)
(23) Gamma globulin protein staining zone (β2 microglobulin) that appears across (17)
(24)-(29) Schematic diagram of electrophoretic image of urine from patient with Bence Jones protein
(30) Gamma globulin region
(31)-(36) Bence Jones protein with distinct protein staining bands at different positions
(37) Schematic diagram of urine electrophoresis image of healthy subject
(38) α1 globulin to γ globulin region, which is thinly dyed as a whole

Claims (5)

Electrophoretic images of urinary protein electrophoresed and silver-stained are classified into groups mainly composed of glomerular disorders, groups mainly composed of renal tubular disorders, groups where Bence Jones protein appears, and groups of healthy subjects. Urine protein analysis pathological condition analysis method characterized by
2. The urinary protein according to claim 1, wherein the group characterized in that the fractional values of albumin fraction and β fraction appear significantly higher than the fractional values of other disease groups, respectively, is a glomerular disorder group Analyze pathological analysis method. The fraction value of the albumin fraction is significantly lower than the fraction value of the albumin fraction of the group mainly composed of glomerular disorders, and another protein fraction is significant in the form of sandwiching it instead of the β fraction. 2. The urinary protein analysis pathological condition analysis method according to claim 1, wherein the group characterized by appearing deeply in the kidney is a renal tubular disorder group. 2. The urinary protein analysis pathological condition analysis method according to claim 1, wherein the group capable of detecting a clear protein staining band suggesting the presence of myeloma or the like in the globulin region is a group in which Bence Jones protein appears. A group characterized in that the color tone of the silver-stained albumin fraction is significantly lighter than the albumin fraction of other disease groups and has a single staining zone, and the globulin region is thin and becomes a large dumpling-like staining zone. 2. The urinary protein analysis pathological condition analysis method according to claim 1, wherein the group is a group of healthy subjects.

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