JPH0972906A - Differential diagnostic kit of kidney/urinary tract disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform the differential diagnosis of a kidney/urinary tract disease by setting a granulocyte component of leucocytes in urine and Carcinoembryonic- antigen(CEA) to measuring objects. SOLUTION: Lactoferrin among granulocyte components of leucocytes is easily excreted into urine but, since myeloperoxidase has a large mol.wt. of about 140K and is bonded to hyaluronic acid of a glomerular basement membrane, it is not excreted to urine. If both components are measured at the same time, the differential diagnosis of a kidney/urinary tract disease becomes possible. When myeloperoxidase is present in urine in large quantities, it is shown that leucocytes are present in a urinary tract and CEA is measured to discriminate wheter leucocytes are increased by a urinary tract infection disease. CEA is produced by epithelial cells of the mucous membrane of the urinary tract and, since it is a component preventing the penetration of pathogenic bacteria into a living body, CEA is increased at a time of a urinary tract infection disease. By measuring lactoferrin, myeloperoxidase and CEA, a kidney/ urinary tract disease can be discriminated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a kit for differential diagnosis of renal / urinary tract diseases, and in particular, by performing a combined measurement of lactoferrin, myeloperoxidase and CEA in urine as a measurement target, renal And a kit for reliably differentially diagnosing lower urinary tract diseases.

[0002]

2. Description of the Related Art Urine sediment is an important test method for diagnosing renal and urinary tract diseases. By obtaining sufficient urinary sediment findings, histological changes in the kidney can be estimated. For example, if the occult blood test is positive in a general urinalysis, by observing the presence or absence of erythrocytes and their morphology in the urine sediment, the cause is hematuria, other pigmented urine, or glomerular origin. Can distinguish between hematuria and hematuria caused by urological diseases. In other words, this method attempts to distinguish renal / urinary tract diseases by observing red blood cells in urine. On the other hand, a method of detecting a minute amount of protein in urine is widely used as a marker for early detection of renal disease. Albumin, transferrin, and prealbumin are targeted for measurement, and it is particularly evaluated that transferrin reflects the early lesion of the kidney. In addition, urinary tract infections are mainly caused by ascending bacterial infections, and white blood cells appear in the urine together with the bacteria. Therefore, microscopic examination of urinary sediment and bacteriological examination of urine are performed for the diagnosis. Urinary tract infection is suspected if more than 5 white blood cells / HPF are detected by microscopic examination of urinary sediment (× 400), and diagnosis is confirmed by demonstrating bacteria in urine obtained directly from the bladder or renal pelvis. However, it is rather rare to directly puncture the bladder or renal pelvis to collect urine, and intermediate urine is often collected.

[0003]

However, in the method of distinguishing renal / urinary tract diseases by observing urinary red blood cells in the urine sediment, it is difficult to distinguish between deformed red blood cells and non-deformed red blood cells according to the morphology. Requires skill. It cannot be said that the early diagnosis of renal lesions, which detects trace proteins in urine, is sensitive enough. Regarding the method of demonstrating bacteria in urine by the method of diagnosing urinary tract infection, intermediate urine as a commonly used sample may be contaminated with indigenous bacteria of the urethra and vulva. It is general that ⁵ CFU / ml or more is significant bacteriuria and 10 ³ CFU / ml or less is contaminated urine. Therefore, in order to diagnose a urinary tract infection, it is necessary to perform quantitative culture of urinary bacteria and measure the number of bacteria in 1 ml of urine, which is a complicated and generally untestable test. In addition, leukocytes in urine also undergo severe disintegration, so the reliability of results by sediment microscopy is low. The present invention has been made in view of these problems, and an object thereof is to provide a kit for differential diagnosis of renal / urinary tract diseases by a new test method replacing the above-mentioned conventional method.

[0004]

In order to achieve the above object, in the present invention, CEA produced by intragranular components of leukocytes in urine and urothelial mucosal epithelial cells is measured.
Examples of the intragranular component include lactoferrin, granulocyte elastase, myeloperoxidase, lysozyme, cathepsin G, pronase 3 and the like, and lactoferrin and myeloperoxidase are particularly preferably measured. CEA produced by urothelial mucosal epithelial cells
Besides, secretory IgA can be considered as a measurement target, but CEA is preferably a measurement target as a component for preventing invasion of pathogenic bacteria. To detect lactoferrin, myeloperoxidase, and CEA, enzyme immunoassay (ELISA method), radioimmunoassay (RIA method), luminescent enzyme immunoassay, latex agglutination reaction, colloidal gold method, etc.
A widely used immunological assay method can be used.

In nephritis, a large number of wet cells (white blood cells)
Are collected in the glomerulus, and among the intragranular components of leukocytes, there are components that are easily excreted in urine (at this time, leukocytes are not excreted in urine because they disintegrate in the glomerulus) and components that are difficult to excrete. If so, we considered that renal lesions could be detected early by measuring the components that are easily excreted. However, in the case of urinary tract infection, a large number of leukocytes are infiltrated in the urinary tract, which means that all of the leukocyte granular components are present in urine. Among the intragranular components, those that are not excreted in urine are appropriate). It was theoretically considered possible to differentially diagnose renal / urinary tract lesions by simultaneously measuring these two types of intragranular components. Among the components of leukocyte granules, lactoferrin is suitable as the former (prone to be excreted in urine), and myeloperoxidase is the latter (not excreted in urine, myeloperoxidase has a large molecular weight of about 140 K, and glomerular basement membrane It was found to be suitable as (not excreted in urine because it adheres to hyaluronic acid).

School urinalysis is carried out as an early method for diagnosing renal disease in young people. It is known that the onset age of nephritis found by this screening is 11 to 15 years in 80% of cases. Urinary lactoferrin and myeloperoxidase were measured for each age group from nursery school children to high school students to working adults.
In 0), it was found that urinary lactoferrin and myeloperoxidase were present in good correlation (meaning the presence of white blood cells), and the group of 5th grade and above (n = 72).
Although myeloperoxidase was low in 0),
There were cases in which lactoferrin had abnormally high levels.
(Fig. 1) In addition, urinary lactoferrin and myeloperoxidase were both normal and a group in which myeloperoxidase was low and lactoferrin was high only, but urinary transferrin (highest sensitivity among protein components as an early diagnostic marker for nephritis) It is said that as a result of comparing the concentrations,
Only lactoferrin showed significantly high transferrin in the high value group. (FIG. 2) The transferrin concentrations in this case were all within the normal range, suggesting the usefulness of early diagnosis of nephritis using the urinary lactoferrin concentration as an index. Furthermore, a high level of urinary myeloperoxidase means that leukocytes are present in the urinary system (below the kidneys), but CEA measurement should be used to distinguish whether leukocyte increases due to urinary tract infection. Good. CEA is produced in urinary tract mucosal epithelial cells, and its role is a component that protects pathogenic microorganisms from invading the living body. Therefore, CEA is considered to increase during urinary tract infection. When the CEA concentrations in urine of the urine leukocyte negative group and the urine positive group were compared, the CEA concentration was significantly high in the leukocyte positive group. (FIG. 3) As described above, renal / urinary tract diseases can be distinguished by measuring lactoferrin and myeloperoxidase, and myeloperoxidase and CEA in urine.

[0007]

【Example】

(A) Preparation of urine sample Urine diluted 2-fold with physiological saline containing 0.1% Triton X-100 was used as a sample. (B) Measurement of lactoferrin by ELISA [Immobilization of antibody on microplate] Each well of a microplate (SUMILON, Japan) contains anti-human lactoferrin antibody (DAKOPATTS, Denmark) 5 μg / ml at 0.1 M
100 μl of Tris buffer is dispensed and left overnight at 4 ° C. for physical adsorption to immobilize on the surface. [Preparation of Enzyme-Labeled Antibody] Separately, an alkaline phosphatase (Beehringer-Mannheim, FRG) is enzymatically labeled with an anti-human lactoferrin antibody by the periodate method to prepare. [Measurement of lactoferrin in urine] 100 μl of 1% B in each well
Tris buffer containing SA (Beehringer-Mannheim, FRG) (0.1
(mol / l pH 8.0) is dispensed, then 50 μl of urine sample is added, mixed and allowed to react at 37 ° C. for 1 hour. Then Twee
Wash three times with deionized water containing 0.05% n20. Then, an alkaline phosphatase-labeled anti-human lactoferrin antibody solution (Tris buffer containing 1% BSA) was added to each well.
After adding 100 μl of each well and mixing, react at 37 ° C. for 1 hour and wash 3 times as above. Further King-Kin
Add 100 μl of substrate buffer of method g to each well and incubate at 37 ° C for 3
Incubate for 0 minutes. Here, the substrate buffer is Sisodium Phe
Nyl-phosphate (WAKO Junyaku Japan) 0.215g and 4-ami
0.09 g of noantipyrine (manufactured by WAKO Junyaku Japan) was dissolved in 100 ml of a carbonate buffer solution (0.05 mol / l pH 10.15).
Next, 100 μl of coloring solution is added to each well to develop a color. Here, the coloring liquid was 0.38 g after dissolving 2.6 g boric acid (WAKO Junyaku Japan) in 200 ml deionized water.
Potassium ferricyande (manufactured by WAKO Junyaku Japan). Finally, the color of each well was measured using a colorimeter for microplates (manufactured by Sanko Junyaku Japan).
Colorimetrically with light at a wavelength of / 680 nm, and calculate the lactoferrin concentration in urine from the calibration curve.

(C) Measurement of myeloperoxidase [immobilization of antibody to microplate] Each well of a microplate (SUMILON, Japan) contains 5 μg / ml of anti-human myeloperoxidase antibody (DAKOPATTS, Denmark) at 0.1 M
Dispense 100 μl of Tris buffer solution and leave it at 4 ° C overnight to allow physical adsorption to immobilize on the surface. [Preparation of Enzyme-Labeled Antibody] Separately, alkaline phosphatase (Beehringer-Mannheim, FRG) is enzymatically labeled with an anti-human myeloperoxidase antibody by the periodate method to prepare. [Measurement of myeloperoxidase in urine] 100 μl in each well
Tris buffer (0.1mol / l pH8.0) containing 1% BSA (Beehringer-Mannheim, FRG) was dispensed, and then 50 μl of urine sample was added, mixed and allowed to react at 37 ° C. for 1 hour. .
It is then washed 3 times with deionized water containing 0.05% Tween 20. Then, add 100 μl of an alkaline phosphatase-labeled anti-human myeloperoxidase antibody solution (Tris buffer containing 1% BSA) to each well and mix, and then mix at 37 ° C.
And react for 1 hour, and wash 3 times as before. Further K
100 μl of substrate buffer for the ing-King method is added to each well, and the mixture is reacted at 37 ° C. for 30 minutes. Next, 100 μl of coloring solution is added to each well to develop a color. Finally, the color of each well was measured by a colorimeter for microplates (Sanko Junyaku Japan
(Manufactured by Mitsui Chemicals Co., Ltd.) is used for colorimetry with 510/680 nm wavelength light, and the concentration of myeloperoxidase in urine is calculated from the calibration curve.

(D) Measurement of CEA in urine [immobilization of antibody on microplate] Anti-human CEA monoclonal antibody (special immunity, does not show cross-reactivity with NCA) in each well of microplate (SUMILON, Japan) )
100 μl of 0.1 M Tris buffer containing 5 μg / ml is dispensed, and left overnight at 4 ° C. to immobilize on the surface. [Preparation of Enzyme-Labeled Antibody] Separately, an alkaline phosphatase (Beehringer-Mannheim, FRG) is enzymatically labeled with anti-human CEA antibody (DAKOPATTS, Denmark) by the periodate method. [Measurement of CEA in urine] 100 μl of 1% BSA (B
eehringer-Mannheim, FRG) containing Tris buffer (0.1 mol / l
(pH 8.0) is dispensed, 50 μl of a urine sample is added, mixed and allowed to react at 37 ° C. for 1 hour. Then Tween 20
Wash 3 times with deionized water containing 0.05%. Then, alkaline phosphatase-labeled anti-human CEA antibody solution (1% B
100 μl each of AE-containing Tris buffer) was added to each well, mixed, reacted at 37 ° C. for 1 hour, and washed 3 times in the same manner as above. Furthermore, the King-King substrate buffer 100
μl is added to each well and reacted at 37 ° C. for 30 minutes. Next, 100 μl of coloring solution is added to each well to develop a color.
Finally, the color of each well was measured by a colorimeter (San
(Ko Junyaku Japan) is used for colorimetry with 510/680 nm wavelength light, and the CEA concentration in urine is calculated from the calibration curve.

(E) Myeloperoxidase in urine, lactoferrin, CEA cutoff value Myeloperoxidase concentration in urine of a healthy person is 27.0 ± 34.1
ng / ml, and the cutoff value was set to 95 ng / ml. The urinary lactoferrin concentration was 31.1 ± 36.2 ng / ml, and the cutoff value was set to 105 ng / ml. The CEA concentration in urine is 3.
8 ± 2.1 ng / ml, and the cutoff value was set to 8.0 ng / ml. (F) Determination Based on the measurement results of the urinary lactoferrin concentration and the urine myeloperoxidase concentration, they are sorted into the following (a) to (c). (a) When the urinary lactoferrin concentration (Lf) is 105 ng / ml or less and the urinary myeloperoxidase concentration (MPO) is 95 ng / ml or less, it is determined to be normal. (b) If the urinary lactoferrin concentration (Lf) is 105 ng / ml or higher and the urinary myeloperoxidase concentration (MPO) is 95 ng / ml or lower, and MPO / Lf is less than 0.2, then there is a possibility of nephritis. Is determined to be large. (c) If the urinary lactoferrin concentration (Lf) is 105 ng / ml or more and the urinary myeloperoxidase concentration (MPO) is 95 ng / ml or less, urinary tract infection is suspected. In the case of (c) above, the CEA concentration in urine is further measured, and if it is 8.0 ng / ml or more, it is determined that the possibility of urinary tract infection is high. If you have a diagnosis of urinary tract infection,
Only PO and CEA are measured and observed.

[0011]

Industrial Applicability As described above, the differential diagnosis of renal / urinary tract diseases can be made by combining and measuring the intragranular components of leukocytes in urine, particularly lactoferrin, myeloperoxidase and CEA. .

[Brief description of drawings]

FIG. 1 is a correlation diagram between urinary lactoferrin and myeloperoxidase.

FIG. 2 is a graph showing the relationship between urinary lactoferrin concentration and urinary transferrin concentration.

FIG. 3 shows urinary C in the urine leukocyte negative group and the positive group.
9 is a diagram showing the results of comparing EA concentrations.

Claims (6)

[Claims] 1. An intragranular component of white blood cells in urine and Carcin
A kit for differential diagnosis of renal / urinary tract diseases, which comprises measuring oembryonic antigen (CEA). 2. The kit for differential diagnosis of renal / urinary tract diseases according to claim 1, wherein urinary lactoferrin, myeloperoxidase and CEA are used as measurement targets. 3. The kit for differential diagnosis of renal / urinary tract diseases according to claim 1 or 2, which uses an immunological measurement method such as an enzyme immunoassay, a latex agglutination reaction, or an immunochromatography method. . 4. Urine lactoferrin by an immunological assay method, comprising at least an anti-lactoferrin antibody, an enzyme-labeled anti-lactoferrin antibody, an anti-human myeloperoxidase antibody, an enzyme-labeled anti-human myeloperoxidase antibody, and the enzyme-measuring reagent. And a kit for differential diagnosis of renal and urinary tract diseases, which comprises measuring myeloperoxidase concentration to diagnose urinary tract diseases. 5. At least an anti-human myeloperoxidase antibody, an enzyme-labeled anti-human myeloperoxidase antibody, an anti-human CEA antibody, an enzyme-labeled CEA antibody, and the enzyme-measuring reagent are contained in urine by an immunological assay method. A kit for differential diagnosis of renal and urinary tract diseases, which comprises measuring myeloperoxidase and CEA concentrations to diagnose urinary tract diseases. 6. The cutoff values for urinary myeloperoxidase concentration, urinary lactoferrin concentration, and urinary CEA concentration are about 95 ng / ml, 105 ng / ml, and 8.0 ng / ml, respectively. Item 5. The kit for differential diagnosis of renal / urinary tract diseases according to Item 4 or 5.