JP4825828B2 - Method for determining pathological conditions such as renal diseases by electrophoresis using silver staining - Google Patents

Description

Fields in which proteins in human and animal urine are electrophoresed to identify the type of disease based on the characteristics of the analysis results

Tests using urine are widely used in screening at screening and resident screening as test substances that can be collected without causing pain to the human body. Among the measurement items used in these, the analysis of urine protein is generally considered to be negative so-called negative in which no protein appears in the test method using a test paper. The measurement sensitivity of this test paper is 20-30 mg / dL.
However, it is known that when a highly sensitive silver staining method such as Patent Document 1, Patent Document 2, and Patent Document 3 is used, a very small amount of protein is excreted even in a healthy person. The method for analyzing this minute amount of protein by electrophoresis is described in detail in Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3 by Shiba et al. According to this, from the analysis of electrophoretic images of urinary proteins using silver staining, diabetes is roughly divided into 5 groups according to the shape of the electrophoretic image.
JP 2002-236127 A JP 2003-215130 A JP 2005-134144 A Biophysical Chemistry Vol. 41, 1997, pp. 25-27 Clinical Laboratory Vol. 42, 1998, pp. 1106-1109 Clinical pathology, extra edition, extra edition 107, 1998, pp. 49-55

The present invention uses a highly sensitive urinary protein analysis method using a silver staining method, so that not only analysis of electrophoretic images conventionally performed, but also glomerular disorder group, renal tubular disorder group, A specific algorithm is extracted from a disease-specific electrophoretic image, such as a group in which Bence Jones protein appears, or a mixed type renal disorder group, to determine the type of kidney disease that is difficult for a specialist to determine.

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

One of the important functions of the kidney is that urine is produced in the kidney and necessary nutrients such as proteins are reabsorbed and reused. Therefore, when protein appears in urine, impaired renal function or renal disease is predicted. 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.

Test methods other than renal biopsy include image diagnosis such as CT, urinary protein quantification, and urine protein fractionation. Among them, quantitative tests for urinary proteins 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 the concentration of protein to be measured is low or there are many types of items to be measured, and expensive antibodies such as immunoassay are used. There are many drawbacks and it is not practical and is not implemented very often.

In the conventional urine protein fraction test, there was a staining method with Ponceau 3R or Acid Bio Red 17, but the protein staining ability was low and the protein concentration in urine was quite low. The concentration time was as long as 10 hours, and the separated electrophoretic image was often difficult to accurately diagnose due to the distortion and unclearness of the fraction due to the effect of concentration.

The present invention uses the silver staining method of Patent Document 1 described above, and it is known that the detection sensitivity can be detected in a trace amount of several mg / dL. The inventors measured the concentration of urine protein in a large number of healthy subjects and kidney diseases, and as a result, it was also found that patients with less than 10 mg / dL do not have severe kidney disease. As a result of electrophoretic analysis of silver-stained electrophoretic images of urinary proteins from patients with known renal pathology, we were able to identify disease-specific algorithms.

First, as shown in the Examples, electrophoretic images obtained by silver staining of urinary proteins exhibit a variety of variations depending on differences in individual renal functions and disease conditions. Even if measurement results obtained by directly converting these various electrophoretic images into conventional concentration diagrams (zymograms) were sent to the attending physician, there were too many conditions to determine which disease group they belonged to, and it was extremely difficult.

In the invention of the present application, as a result of classifying into four groups according to diseases based on not only the densitogram but also the concentration difference and presence / absence of a specific protein band, etc. It was concluded that it is 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 condition of the patient, in order to obtain a beautiful electrophoretic image, the concentration is measured in advance by the quantitative method described in Patent Document 1 before electrophoresis, The application amount of patient urine to the electrophoresis support is made constant according to the concentration. In order to apply a certain amount with a sample applicator (applicator), when using a device to which 0.8 μL is applied once, the sample is applied approximately 3.2 μL about 4 times. When the concentration is low, it can be applied up to 8 times. Conversely, if the concentration is high, the specimen is diluted and, as a result, an almost constant amount is applied.
Accordingly, it is impossible to judge the quantitative ratio of urine protein by looking at the changes in color tone in FIG. Of course, it is very difficult to judge the pathological condition such as kidney disease by this alone.

Therefore, in order to determine the pathology of kidney disease, etc., not only the concentration difference in the electrophoretic image, but also the location of the protein that identifies the disease, the presence or absence of the peak of the staining band, the size, etc. are also determined. You must determine the pathology of the disease. The inventors of the present application determined the so-called algorithms for the first time in the world from a large number of actual measurement examples, and actually applied them.

An electrophoretic image obtained by electrophoresis of serum protein with a cellulose acetate membrane without electroosmosis, such as Separax SP (manufactured by Fujifilm), can detect five normal peaks in a healthy person, and albumin, α1, α2, β, It is called γ. The protein appearing in urine is considered to be essentially the same as the protein in this serum, and its mobility is almost the same.

In the electrophoresis method, it is well known that there is a slight difference in protein mobility depending on the cellulose acetate membrane and buffer used, the size of the electrophoresis tank, the humidity, temperature and voltage of the electrophoresis environment. Therefore, the concept of relative mobility (Relative Mobility = Rm) may be used as a method for fixing the position of the appearing protein regardless of the electrophoresis conditions.

The relative mobility will be described with reference to FIG. FIG. 2 is a zymograph of electrophoresis image of serum protein. The vertex B of the albumin fraction, the position B (11) where the perpendicular from the peak (9) intersects the reference line (10) is Rm = 1.00, and the specimen application position A point (12) is Rm = 0.00. The points (13), (14), (15), and (16) in which the peak positions of the peaks of the α1, α2, β, and γ fractions are represented by Rm are {(α1, α2, β, and γ fractional peaks). (Position) -A} / (BA) (1).
The Rm values of the α1, α2, β, and γ fractions are almost the same for the electrophoretic image of the urine protein that was electrophoresed simultaneously with the serum protein and was stained with silver.

[Neuroglomerular disorder group]
The protein concentration of many patient urine determined to be renal glomerular injury by renal biopsy is 10 mg / dL or more, and the results of examination of those expressed by concentration diagram by cellulose acetate membrane electrophoresis using silver staining The β-fraction position has a clear staining band with a peak-to-valley difference of 3% or more, and the peak position Rm is 0.49 to 0.59 and the albumin fraction Rm = 1.00. The image% was in the range of 30-85%. Therefore, the group corresponding to these conditions could be a glomerular disorder group. This disease group included IgA nephropathy, membranous nephropathy, focal glomerulosclerosis, diabetic nephropathy, membranoproliferative glomerulonephritis, lupus nephritis and the like.

[Renal tubular disorder group]
The protein concentration of many patient urine determined to be renal tubule injury by renal biopsy is 10 mg / dL or more, and as a result of examining the cellulose acetate membrane electrophoretic image using silver staining in a density diagram, The difference between the peak and the valley is 3% or more, and the peak Rm is 0.59 to 0.69 and the albumin fraction Rm is 1.00. Fraction% was in the range of 5-49%.
In addition to the above algorithm, in this disease group, a unique staining band may appear around Rm 0.38 to 0.51 at the position on the cathode side of the β fraction, and it may be an important judgment material when confusing. it can. Therefore, the group corresponding to these conditions could be a renal tubule disorder group. However, this Rm may vary depending on the therapeutic agent during treatment. This group included Fancony's syndrome, cystinuria, interstitial nephritis, gout kidney, drug-induced tubular necrosis, tubular acidosis, cadmium poisoning, and others.

[Group of Bence Jones proteins appearing]
The protein concentration of many patient urines in which the appearance of Bence Jones protein was confirmed by the immunofixation method was 10 mg / dL or more, and the patient's urine was converted into a cellulose acetate membrane electrophoretic image using silver staining, which was represented by a density diagram. As a result of close examination of the thing, in the wide part of the globulin region (α to γ fraction region) with Rm ranging from 0.15 to 0.80, a clear staining band where the difference between the peaks and valleys is 10% or more in fraction% When there is a dyeing band of a large height so as to overlap the dyeing band, and the width at half h / 2 of the entire height h including the dyeing band is half-value width = θ, albumin When the distance between the apex and the coating position is assigned as 1.00, and each band is expressed by relative mobility, when the width of θ is expressed by Rm, θ takes a value of 0.2 or less, and the albumin fraction Rm = The fraction of 1.00 is less than 20% It took the value.
As described above, those satisfying these conditions could be made a group in which the Bence Jones protein appears.
This disease group included multiple myeloma, oligoclonal bands, primary amyloidosis and the like. Bence Jones protein is a so-called monocloned plasma cell that is overproduced by the immunoglobulin light chain, and because its blood concentration increases, the protein that could not be reabsorbed is excreted in the urine. Is a protein that has emerged as a result.

The protein concentration of a large number of healthy examinees who were diagnosed as healthy in a health examination using blood tests and urinalysis was 10 mg / dL or less. As a result of examining the urine of a cellulose acetate membrane electrophoretic image using silver staining in a density diagram, the fraction% of the albumin fraction Rm = 1.00 was in the range of 20 to 70%. In addition, α1, α2, β, and γ fractions appeared uniformly.
Regarding the quantitative value of urine protein, the sensitivity of the urine test paper used in normal medical examinations is generally 20-30 mg / dL, and it is a reasonable result considering that nephropathy was also present in the urine protein negative group. It was.
In healthy individuals, the permeability of albumin in the kidney glomerulus is low, and most low molecular weight proteins are reabsorbed by the tubules, so a protein staining band appears in addition to albumin. Indicated. In cellulose acetate membrane electrophoresis using silver staining, the concentration of urine protein is measured in advance, and if the concentration is low, the amount of sample to be applied is increased, so only the fraction% value and the waveform size are observed. It should be noted that it is not possible to determine whether the amount is large or small as described above.

Silver staining of urine from two or more patients affected by renal biopsy and those who have been diagnosed with renal glomerular disorder and renal tubule disorder and patients with Bence Jones protein confirmed by immunofixation As a result of careful examination of the concentration diagram of the cellulose acetate membrane electrophoresis image used, it has the characteristics of [renal glomerular disorder group], [renal tubule disorder group], and [group in which Bence Jones protein appears]. It was confirmed. Since these disease groups are affected by each disease, they are referred to as a mixed kidney disorder group.

In the early stages of renal glomerular injury and renal tubular injury, it may be difficult to differentiate from a healthy group. In this case, the position of the β fraction is first determined by accurately overlaying the waveform of albumin and γ globulin with the waveform of the serum protein of the healthy subject on the waveform of the patient, and separately from the fraction% of this β fraction. By multiplying the concentrations determined by quantification, the concentrations of β fraction and other fractions can be calculated. For example, even in a sample that appears to be a healthy person, even if a stained band is observed at the position of the β fraction, when the β concentration is calculated as described above, the β protein amount is 5 mg / dL or less. When it is difficult to distinguish a glomerular disorder, renal tubular disorder, and a group of healthy subjects, the above algorithm can be added to the determination.

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. Therefore, since there are too many patients who actually need a renal biopsy, early screening of mild kidney disease has been avoided. If the present invention is screened for a large number of patients, it can be expected to have a great effect such as early diagnosis of kidney disease and early detection of patients who have to undergo a renal biopsy.

By applying the urine protein analysis pathophysiology analysis method for detecting renal diseases and Bence Jones protein, the renal biopsy itself can be reduced, which leads to not only a reduction in medical costs but also an emergency medical accident.

Since the present invention is a urine test, it not only causes pain to the patient but also avoids an invasive test such as a doctor's renal tissue sampling and simplifies the diagnosis.

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.

Since electrophoretic images of urine show a wide variety of forms, even specialists required considerable skill and experience for judgment, but by implementing the present invention, it becomes possible to judge disease groups even if not a renal specialist.
For example, detection of Bence Jones protein is well known as a marker for cancers such as myeloma, but it was determined by an immunofixation method using a specific antibody after first hitting with a visual inspection by a skilled laboratory technician. This first visual inspection by a skilled laboratory technician could not be judged at a facility without a skilled laboratory technician and was overlooked. In this invention, since it can detect by adoption of the prescribed algorithm rather than individual subjectivity, if this inspection method is applied even if it is not a specific facility, its presence can be detected by screening. Therefore, it can be utilized for early detection of this cancer type and confirmation of therapeutic effect.

Specific examples of desirable forms are shown below.

A healthy human serum specimen is electrophoresed by cellulose acetate membrane electrophoresis, silver-stained, and the appearance position of each band in the electrophoretic image is represented by relative mobility (Relative Mobility = Rm). .
As the cellulose acetate membrane, Separax SP manufactured by Fuji Photo Film Co., Ltd. without electroosmosis was used. The buffer used was Barbital buffer pH 8.6, the ionic strength was 0.06, the electrophoresis tank and the direct current stabilized power supply were manufactured by Joko Co., Ltd.
The electrophoresis conditions were in accordance with the standard operation method of the Electrophoresis Society. The reagent of patent document 1 was used for the silver dyeing | staining liquid. A finger printer manufactured by Joko was used to create the density map.

In FIG. 2, the baseline without the dyeing zone is called the baseline (10). (9) is the albumin fraction, and the point (11) where the perpendicular of the peak position of this apex intersects the base line is Rm = 1.00. (13) is the α1 fraction, (14) is the α2 fraction, (15) is the β fraction, and (16) is the Rm position of the γ fraction.
In (12), the Rm at this point was set to 0.00 when it hits the specimen application position.

An example of calculation of Rm. In FIG. 2, when the distance between the application point (12) and the Rm position (11) of albumin is 5.0 cm, the distance to the application point of each fraction is as follows: a. Application point to albumin peak Distance to position 5.0cm
b. Distance from application point to peak position of α1 4.4 cm
c. Distance from application point to peak position of α2 3.6 cm
d. Distance from application point to peak position of β 2.6 cm

e. Distance from application point to peak position of γ 1.5cm

f. Application point 0.0cm

From these, the Rm value is calculated as follows using Equation (1).
Alb. Rm 5.0 / 5.0 = 1.00
Rm of α1 4.4 / 5.0 = 0.88
Rm of α2 3.6 / 5.0 = 0.72
Rm of β 2.6 / 5.0 = 0.52
Rm of γ 1.5 / 5.0 = 0.3
Rm of application point 0.0 / 5.0 = 0.00
Rm is expressed in this way, but in the case of a gentle peak, this value varies somewhat depending on how the peak position is selected.

FIG. 3 is a concentration diagram when the urine (265 mg / dL) of a glomerular disease patient is electrophoresed and the protein is silver stained. This patient had an albumin fraction of 44.1%, a β of 49.7% and an Rm of 0.55. When judged using the algorithm of the present invention, it was judged as a renal glomerular disorder group.

FIG. 4 is a concentration diagram when the urine (173 mg / dL) of a patient diagnosed as a renal tubular disorder patient is electrophoresed and silver stained. The albumin fraction of this patient was 22.3%, and a large staining band of 9.7% was observed at a position where the Rm corresponding to the anode side from the β fraction was 0.60. When judged using the algorithm of the present invention, it was judged as a renal tubular disorder group.
A staining band of Rm0.48 is also found at the position on the cathode side of the β fraction that appears relatively frequently in this disease group, strongly suggesting renal tubular damage.

FIG. 5 is a concentration diagram when the patient urine containing Bence Jones protein 80 mg / dL is electrophoresed and silver-stained. The albumin fraction is 89.7%, and there is a peak with a tip at the position of Rm 0.53, the height h is 29 mm, 1/2 h is 14.5 mm, the half width is 6.3, Rm 1.0. Since the half-width Rm was calculated as 6.3 / 53 = 0.119, it was determined that the Bence Jones protein appeared.

FIG. 6 is a diagram in which the concentration diagram (17) of the electrophoretic image obtained by using the silver staining method of the healthy serum protein is represented by a solid line and the concentration diagram (18) of the Bence Jones protein appearing person is superimposed.
When the concentration diagrams of serum proteins albumin (19), α1 (20), α2 (21), β (22), and γ (23) are superimposed on the concentration diagram of the patient's urine, the position and amount of the special fraction It becomes easy to identify.

Electrophoretic image of urinary protein by silver staining method Concentration map and relative mobility of healthy serum proteins Density diagram of glomerular disorder Density diagram of renal tubule disorder Concentration map of Bence Jones protein appearances A figure showing the concentration diagram of healthy serum protein superimposed on the concentration diagram of Bence Jones protein appearances

Explanation of symbols

(1) (2) Representative electrophoretic image of renal glomerular disorder group (3) (4) Representative electrophoretic image of renal tubular disorder group (5) (6) Representative of Bence Jones protein appearance group Electrophoretic image (7) (8) Representative electrophoretic image of healthy subjects (9) Albumin (10) Reference line (baseline)
(11) Rm = 1.00 position (12) Application position (Rm = 0.00)
(13) α1
(14) α2
(15) β
(16) γ
(17) Serum protein concentration diagram in healthy subjects (18) Concentration diagram of Bence Jones protein appearances

Claims (3)

Electrophoretic images of serum proteins electrophoresed on a cellulose acetate membrane and silver-stained electrophoretic images represented by density diagrams, relative mobility of albumin fraction peak position and sample application position are Rm = 1.00 and Rm, respectively. Electrophoretic electrophoresis of patient urine having a urine protein of 10 mg / dL or more , using the analysis method of setting = 0.00 and the analysis method of electrophoresis image, the fraction%, and the method of defining the urine protein concentration value , For the electrophoretic image of silver-stained electrophoretic images represented by a density map, there is a clear staining band with a difference between peaks and valleys of 3% or more in the β fraction position, and the peak position of the β fraction is Rm is judged to glomerular disorders group with that fraction percent and a 0.49 to 0.59 albumin fraction Rm = 1.00 is in the range of 30% to 85%, likewise urinary protein 10 mg / dL or higher Electrophoresis of human urine The electrophoretic image of silver-stained electrophoretic images expressed as a density map has a clear staining band with a difference between peaks and valleys of 3% or more at the position close to the anode of the β fraction. kidney Rm at the peak of the image of the anode nearer is and fractionation% albumin fraction Rm = 1.00 in 0.59 to 0.69 is a renal tubular disorder group have to be in the range of 5 to 49% Disease state diagnosis method Electrophoretic images of serum proteins electrophoresed on a cellulose acetate membrane and silver-stained electrophoretic images represented by density diagrams, relative mobility of albumin fraction peak position and sample application position are Rm = 1.00 and Rm, respectively. Electrophoresis of human urine having a urinary protein of 10 mg / dL or more using an analysis method for setting = 0.00 and an analysis method for electrophoretic images, a method for defining fraction%, urine protein concentration and half-value width In the case of a silver-stained electrophoretic image expressed as a density diagram, the difference between the peaks and valleys is 10% or more in the fractional% from α to γ fractional region where Rm is 0.15 to 0.80. There is a clear dyeing band, and there is a dyeing band with a large height so as to overlap the dyeing band, and the width at the half h / 2 of the entire height h including the dyeing band is defined as the half-value width θ. The distance between the albumin and the application position is 1.0. Assignment, theta and a value of theta is 0.2 or less when expressed by the value of Rm, and fractionation% albumin fraction Rm = 1.00 is with taking the value of 20% or less Bence Jones proteins as Method for determining pathological conditions such as kidney disease that is determined as an appearing group Electrophoresis images obtained by electrophoresis of urine of a human having urine protein of 10 mg / dL or more and silver staining are represented by concentration diagrams, and two or more types of kidney disease-specific proteins are simultaneously expressed in claim 1 and claim 2 For determining pathological conditions such as kidney disease when the disease is recognized as a mixed renal disorder group