JP2529836B2 - Method for diagnosing renal disorder by measuring renal urinary antigen using monoclonal antibody - Google Patents

Description

TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically reacts with an antigen derived from normal human kidney tissue, and a method for diagnosing renal damage using the monoclonal antibody.

In particular, the present invention relates to a method for identifying a lesion site in the kidney and diagnosing the extent of the lesion by measuring the amount of the antigen excreted in urine using the monoclonal antibody.

(Prior art and its problems) Currently, there are about 70,000 patients in Japan who are undergoing dialysis treatment due to renal failure.
An enormous treatment cost of 5 million yen is required, which is one of the major causes of pressure on the government and local governments' finances. Moreover, about 5,000 new renal failure patients are occurring each year.

If you have renal failure, you have to receive dialysis treatment for 4 to 6 hours a day for 2 to 3 days every week, which not only significantly impairs the social life of the patient, but also has a great social impact as described above. This imposes a burden, and if the situation continues as it is, there is a risk that the social medical system will eventually collapse.

Kidney disease is a condition that eventually leads to patients with kidney disease, but it is caused by diseases other than the kidney, such as diabetes, high blood pressure, and gout, and eventually causes kidney failure and renal failure, or damage due to regular use of sleeping pills. In the case of receiving renal failure due to side effects such as antibiotics and anti-cancer agents,
When the course of renal failure is followed, various nephritis such as chronic nephritis, acute nephritis, or rapidly progressive nephritis, or pyelonephritis, renal tuberculosis, renal stones, cystic kidney disease, and other renal diseases may affect the kidney. There are many cases. In particular, due to recent changes in social conditions, the number of patients suffering from diabetes, hypertension, gout, etc. has increased remarkably, and many drugs such as sleeping pills, antibiotics and anti-cancer agents have been used, and the incidence of renal disorders caused by these is also increasing. I'm doing it.

By the way, in renal diseases and renal disorders that accompany with these other diseases, there are no noticeable subjective symptoms until the end stage, that is, near renal failure, and their occurrence is often overlooked. Therefore, except for a small number of cases such as edema and oliguria, renal diseases and disorders are often discovered only by urinalysis during a physical examination.

By the way, in Japan, the School Insurance Law, the Occupational Safety and Health Act, and the Elderly Insurance Act legally require urinalysis at the time of physical examination. Alternatively, a urine test is performed by a local government such as a medical examination for an adult disease.

Thus, in the urinalysis currently performed, urine protein, urine sedimentation, etc. are tested, but the sensitivity of the test strips used for these tests varies depending on the manufacturer, and they are commercially available. The quality, gloss, and color tone of the comparison color paper of the inspection test paper vary, and the reliability of the inspection result is sufficient depending on the lighting of the inspection place, the eyesight / manipulation of the person inspecting, or the reaction time. Is not satisfactory. Urine protein is a protein leaked from the blood into the urine in the kidney or urinary tract, and is excreted daily in normal people as well as in patients with renal disease.
Posture, post-exercise, febrile proteinuria, or proteinuria simply due to semen contamination are also found in normal individuals. Urinary tract disease,
Proteinuria is also seen in bladder disorders and female genital disorders. Therefore, the urine protein test is suitable for screening purposes such as physical examination, but it contains many false positives and is not sufficient for further diagnostic purposes. It is impossible.

Urine sediment is obtained by centrifuging urine and examining the sediment with a microscope. Similar to urinary proteins, erythrocytes in the sediment are found in healthy people, and some are derived from urinary tract-related organs other than the kidney, and are also found in appendicitis, colon diverticulitis, and inflammation of pelvic tumors. The diagnostic significance is almost the same as that of urinary protein.

Of the casts, the glass-like casts may be found in normal people. The appearance of granular casts is considered pathological but does not indicate a particular disorder.

Other epithelial cell erythrocytes may also have pathological significance.

By the way, if there is any abnormality or suspicion of abnormality in the above urinalysis, a more detailed examination should be conducted in a medical facility. Serum creatinine, urea nitrogen, filament overdose,
Tubular function, β ₂ microglobulin measurement, urine concentrating ability,
Even if a renal function test such as urinary diluting ability, renal blood flow and urinary acidification ability is performed, it is often difficult to diagnose a specific disease. Also, N-Acetyl-β-D-glucosamidase
Although there are tests that try to detect tubular damage by measuring the enzyme activity of (NAG), the pH of urine changes from weak alkaline to strongly acidic, and the salt concentration also fluctuates significantly, such as osmotic pressure. Is not effective except for some diseases, acute renal failure, etc. In addition, it may be possible to spend a lot of time and money to perform an image examination such as transvenous renal pelvis, renal scintigraphy, echography, X-ray examination, CT scan, plain abdominal X-ray or renal angiography. That have a morphological change depending on the obtained image and have a certain size or more, that is,
It is effective for renal tumors, cysts, calculi, and malformations, but is almost useless for the majority of medical renal diseases such as various types of nephritis, nephrosclerosis, nephrosis, diabetic nephropathy, and gout. In addition, there are many disadvantages such as X-ray exposure from the outside, radioactive exposure from the inside of the body, and complications associated with surgical procedures.

Therefore, it is necessary to perform a renal biopsy for the final determination of the disease. However, in order to carry out a renal biopsy, it is necessary to be admitted to a hospital having a nephrologist, a fluorescence microscope, an electron microscope, etc., and the risk is high because the patient's kidney is punctured by a needle or resected after laparotomy. Due to its high cost, it can only be carried out in a very limited number of patients who are in great need.

As described above, the urine test by urinalysis can be performed daily or several times a day, and since it is an in vitro test, it is an excellent test method that does not cause complications due to the test. , The urine tests currently conducted there contain a lot of false positives due to the uncertainty involved in the test itself,
It cannot be said to be satisfactory from the viewpoint of diagnosis of renal disorders and diseases. On the other hand, the usefulness of renal biopsy is extremely limited for the above reasons. Therefore, there has been a strong demand for a test method that enables simple and highly sensitive measurement using a urinalysis system and enables early diagnosis of renal disorders and diseases.

(Means for Solving Problems) The present invention has been made to solve the above problems, and is based on the following findings.

In the glomerulus of the kidney, blood is passed through to produce raw urine, which flows through the renal tubules and collects in the collecting duct. The urine passes through the ureter to the bladder and is excreted. We suspected that if lesions or lesions occur in the kidney, some of the renal structure may be destroyed and spilled into the urine and excreted as urine via the urine flow route. Furthermore, if the degree of destruction of a specific site in the kidney or the metabolic abnormality of a specific site is known, the nature of renal lesions can be estimated to some extent and diagnosed. In addition, it was thought that a urine test would lighten the burden on the patient, and if such information is available, it could be a test method that bridges the gap between urinalysis and renal biopsy.

Therefore, using human normal kidney tissue or HeLa cells as an antigen, a monoclonal antibody that specifically reacts with an antigen derived from kidney tissue was prepared, and it was observed in both groups when reacted with normal human urine and renal disease patient urine. It turned out that there were significant differences. That is, in the urine, the renal tissue patients were able to detect significantly more disintegration products of normal tissues localized in the kidney than in normal cases. Also, normal metabolism is suppressed in patients with renal disease,
As a result, it was confirmed that the amount of the antigen, which is a metabolite, was significantly reduced in the urine of the patient. Since the degree of urine concentration varies from person to person from time to time, when the ratio of the amount of the two antigens was determined, the patient group was significantly different from that of a normal person.

It was also found that some so-called unhealthy people showed significant abnormalities as compared with normal people, although not as markedly as those with renal disease.

Therefore, by the test method using the monoclonal antibody of the present invention, it becomes possible to safely and simply determine the site of micro-lesion in the kidney and its severity without any side effect, and also to infer the nature of the lesion. However, the nature of the lesion can be better evaluated when combined with clinical symptoms.

In addition, urine protein test, urine occult blood test, etc. in urinalysis are not abnormal unless glomerular damage progresses to a certain extent.
Since the diagnostic method using the monoclonal antibody of the present invention can detect fine lesions before that, it can be applied to general people who may potentially have renal disorders such as hypertension, diabetes, gout, obesity, and leanness, that is, unhealthy people. In this, renal damage can be detected at the time of urine protein negative, that is, at an early stage.

Further, in renal biopsy, lesions are observed in the form of accumulated history of diseases from the past to the present, whereas in the diagnostic method of the present invention, the current state at the time of excretion in urine is known. be able to.

Also, because you can repeat the inspection every day,
It can provide important information for treatment decision making.

As described above, the diagnostic method of the present invention does not require expensive equipment and can be performed very easily, and is extremely useful as a secondary screening method for urine protein positive or (±) group in conventional health examination. It is useful.

Furthermore, according to the present invention, a rejection reaction associated with renal transplantation can be detected at an early stage.

The present invention provides a monoclonal antibody which is localized in the human kidney and specifically recognizes an adult normal kidney tissue antigen.

The monoclonal antibody of the present invention reacts significantly more strongly with urine of patients with renal disease than that of normal people, and with urine of people with renal impairment more strongly than that of normal people. Such strength of reactivity with urine indicates the severity of renal lesions or the degree of renal damage. As a result, it is possible to know the degree of collapse of the normal structure in the lesioned area in the kidney and the metabolic disorder such as an organelle.

In the present invention, the term "renal disorder" may include all of the above-mentioned lesions of renal disease patients, metabolic disorders, and histological abnormalities of the kidney.

As the monoclonal antibody of the present invention, as shown in Examples described later, human renal tubular lumen wall, glomerular basement membrane,
There are monoclonal antibodies that react specifically with the glomerular and tubular basement membranes, or the tubular cytoplasm, which are IgG ₁
Class or IgG _2a class.

Further, the present invention provides a method for diagnosing the site and degree of renal damage by measuring the antigen in urine using a monoclonal antibody that specifically recognizes the human normal renal tissue antigen described above. A method for diagnosing a renal disorder, which comprises diagnosing a site and a degree thereof. In the diagnostic method of the present invention, diagnosis is performed by comparing the measured value of the antigen in the urine of the sample with the antigen value in the urine of normal humans. In particular, in the method of the present invention, the presence or absence of renal disorder can be diagnosed by comparing the measured value of the antigen in the urine of the sample with the antigen value in the urine of normal humans.

Further, according to the present invention, antibody-producing cells of an animal immunized with normal human kidney tissue or HeLa cells are fused with myeloma cells, and the resulting hybridoma is cultured and specifically reacted with the produced normal human kidney tissue. There is provided a method for producing a monoclonal antibody, which comprises recovering the monoclonal antibody.

Further, the present invention is obtained from antibody-producing cells and myeloma cells of an animal immunized with normal human kidney tissue or HeLa cells,
A hybridoma producing a monoclonal antibody that specifically reacts with the normal human kidney tissue is provided.

Finally, the present invention provides a reagent for diagnosing renal damage, which comprises a monoclonal antibody that specifically reacts with human normal kidney tissue antigen.

 Next, the present invention will be described in more detail by way of examples.

Example 1 Method for Producing Monoclonal Antibody 1. Immunity 1) As a immunogen, a normal cortex of the kidney extracted from an adult male was used. The renal cortex is sequentially washed with sterilized physiological saline containing aminobenzylpenicillin (10 mg / ml) and streptomycin sulfate (10 mg / ml), and then with sterilized physiological saline, and then cut into 1 mm square pieces.

2) Crush the thin section (3.9g) together with physiological saline in a glass homogenizer, then add physiological saline,
Use homogenized solution (300 mg / ml renal cortex).

3) The above homogenized solution is diluted with physiological saline and administered as it is or together with an adjuvant (eg, Freund's complete adjuvant) as an immunogen to BALB / c mice or other immunizable animals. The administration method may be any of intraperitoneal injection, subcutaneous injection, intradermal injection, intravenous injection, etc.,
Subcutaneous or intraperitoneal injection is preferred. For example, in mice, 47 mg of cortex was subcutaneously or intraperitoneally injected.

4) The booster immunization is performed once to several times at appropriate intervals, for example, 3 to 4 weeks. The antigen solution is administered as it is or with an auxiliary agent intraperitoneally or subcutaneously. For example, in mice, 30 mg of cortical portion was intraperitoneally injected as it was.

2. Cell fusion 1) Three to four days after the final immunization, immune splenocytes and mouse myeloma cells such as P3-NS1-1-Ag4-1 cells (NS1)
Cell) fusion.

At the ratio of 5 × 10 ⁷ immune splenocytes and 1 × 10 ⁷ NS1 cells,
Alternatively, the mixture is mixed at another appropriate ratio and centrifuged at 400 × g for 5 minutes to remove the supernatant. Next, 1 ml of polyethylene glycol (PEG) solution, for example, 50% PEG4000 solution is added dropwise with stirring for 1 minute, and after further stirring for 1 minute, 9 ml of Dulbecco's MEM medium (DMEM medium) is slowly added with stirring.
Centrifuge at 00 xg for 5 minutes and remove the supernatant.

2) 10 ml of culture medium (nutrient medium such as DMEM medium + serum such as 15% horse serum + antibiotics such as aminobenzylpenicillin 0.1 mg / ml and streptomycin sulfate 0.1 mg / ml)
Suspend cells in a well and place 10 wells in a 96-well tissue culture plate well.
Dispense 0μ each and incubate at 37 ℃, 5% CO ₂ , humid atmosphere.

3) The day after fusion, hypoxane (100μ
M), aminopterin (0.4 μM) and thymidine (16
100 μl of culture medium (HAT medium) containing 100 μM) is added. Two
Replace half of the medium with HAT medium every ~ 3 days. Replace with HT medium after 10-14 days. HT medium is HAT medium from which aminoplythenes have been removed.

Screening of antibody-producing cells Antibody-producing cells were examined by enzyme immunoassay (EIA method) 10 to 21 days after cell fusion. The screening can also be performed by radioimmunoassay, fluorescent antibody method, or the like.

 The EIA method was carried out as follows.

1) Add human renal cortex antigen solution to a 96-well EIA plate and immobilize it under appropriate conditions. For example, 100 μl of antigen solution is added and left overnight at 4 ° C., then the antigen solution is removed and air dried at room temperature. The antigen adsorption plate is blocked with a solution such as bovine serum albumin (BSA).

2) Add the culture supernatant to each plate well and react under appropriate conditions. For example, 100 μ of the supernatant is reacted at room temperature for 1 hour.

3) Tween 20 containing 1% phosphate buffer (wash buffer)
Wash with. Washing is performed by vibrating the plate filled with the washing buffer with a plate mixer for 3 minutes, and usually this operation is repeated 4 times. Horseradish peroxidase (HRPO) -conjugated anti-mouse IgG + IgM on this plate
The antibody solution was added at 100 μ / well and reacted at room temperature.

4) After washing, the substrate solution is added and the color reaction is performed. A solution (substrate solution) of 0.4 mg / ml of ortho-phenylenediamine and 0.2 μ / ml of 31% hydrogen peroxide in a citric acid-phosphate buffer (pH 5.0) was added at 100 μ / well, and the mixture was kept at room temperature for 30 minutes. After the reaction, 4N sulfuric acid is added at 50 μ / well to stop the reaction. Those that develop color are used as antibody-producing positive cells (hybridomas).

Cloning of antibody-producing positive cells Hybridomas are cloned by the limiting dilution method, soft agar method, fibrin gel method or the like. An example of the limiting dilution method is shown below.

1) The hybridoma was suspended in HT medium at 10 cells / ml, and the mouse or rat thymus or spleen cells to be feeder cells were suspended in HT medium at about 1.7 × 10 ⁷ cells / ml, and 100 μ / in a 96-well tissue culture plate. Dispense wells and culture. On the next day, add 100 μl / well of HT medium and replace by half every 2-3 days.

2) After 2-3 weeks, the same operation as the above-mentioned screening operation is repeated to select antibody-producing positive cells. Cloning can be performed by repeating the limiting dilution method once or several times.

3) As a result, a total of 13 hybridomas were obtained, including the 4 strains shown in Table 1 below.

Example 2 Using HeLa cells as an immunogen Basically, the same procedure as in the case of using a renal tissue antigen as an immunogen was performed.

1) Immunization HeLa cells cultured in a culture medium (eg, Dulbecco's MEM medium + 10% fetal bovine serum + antibiotics) are administered together with Freund's complete adjuvant to higher animals such as BALB / C mice by intraperitoneal injection or intravenous injection. And immunized. HeLa cells were administered as described above with or without an appropriate period of time with or without an adjuvant to perform a booster immunization.

2) Cell fusion 3 days after the final immunization, immune splenocytes and myeloma cells, for example,
P3-NS1-1-Ag4-1 cells were mixed at an appropriate ratio, and polyethylene glycol (30 to 50%) was used, followed by a conventional method.
The fused cells were cultured in HAT medium using a 96-well tissue culture plate.

3) Screening and cloning of antibody-producing hybridomas The culture supernatant of HeLa cells cultured on a 96-well tissue culture plate was removed, blocked with 1% BSA, and the fusion cell culture supernatant was added, followed by 2 hours at room temperature. It was made to react. After washing with a washing buffer, a HRPO-conjugated anti-mouse IgG + IgM antibody dilution was added, and the mixture was reacted at room temperature for 2 hours. After washing with a washing buffer, a substrate solution, for example, an ABTS solution is added and reacted at room temperature for 30 minutes, and then 2 mM NaN ₃ is added to stop the reaction.

The cells in the antibody production positive well are cloned by repeating the limiting dilution method.

Six strains including TCP were obtained as antibody intra-body hybridomas.

Example 3 Preparation of Ascites Fluid Containing Monoclonal Antibody A hybridoma can be transplanted into a living body for growth and the body fluid can be collected from the living body to produce an antibody secreted by a high-lyoma. When hybridomas are transplanted into the abdominal cavity and proliferated, pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered before the transplantation, for example, for 3 to 8 weeks to increase the yield of ascites. You can The living organism used as the host is preferably an animal of the same species and syngeneic as the parent cell of the hybridoma to be transplanted. When animals of other species or other strains are used, immunosuppressive treatment such as X-ray irradiation or administration of immunosuppressive agents to the host is carried out.
When nude mice are used, transplantation is possible without treatment.

1) Using mature BALB / C mice Hybridomas cultured in HT medium on pristane-treated mice were added to DMEM medium at 1 × 10 ⁷ to 2
Float at × 10 ⁷ cells / ml and 5 × 10 ⁶ 〜 1 per mouse
The × 10 ⁷ hybridomas are transplanted one to several times into the abdominal cavity.

2) Collect ascites when the abdomen is enlarged. Ascites is centrifuged (for example, at 4 ° C. and 2500 rpm for 10 minutes), and the supernatant is collected.

Example 4 Typing of Monoclonal Antibody Subclasses of antibodies were identified by the Octaloni method or EIA method.

1) A mouse monoclonal typing kit (Serotec) was used for the Octaloni method. Add 75μ of antibody-containing culture supernatant or ascites to a large rosette well on the agar plate. 10 μ of 6 kinds of anti-mouse immunoglobulin antibodies such as anti-IgG and anti-IgG _2a are put in the surrounding small holes. Allow to react for 24-48 hours at room temperature and observe the formation of sedimentation lines.

2) A Mono Ab-ID EIA kit (Zymed) was used for the EIA method. 50 μl of a culture supernatant containing a monoclonal antibody is added to a solid phase on which an antigen is adsorbed, for example, an EIA plate, reacted at room temperature for 1 hour or more, and then washed with a washing buffer. Next, 50 μl of subclass-specific rabbit anti-mouse immunoglobulin antibody is added, and the mixture is reacted at room temperature for 1 hour and then washed. Then add 50μ of HRPO-conjugated anti-rabbit IgG antibody,
The mixture is reacted at room temperature for 1 hour and then washed. ABTS as substrate
(2,2-Azino-di [3-ethylbenzthiazoline sulfonic acid]) was added to 100 μm of a solution containing 1 mM, and at room temperature, 20-
Let react for 30 minutes and judge. The results are shown in Table 1 below.

Example 5 Purification of Monoclonal Antibody Monoclonal antibody in the culture supernatant or ascites fluid was subjected to gel chromatography, ion exchange chromatography,
It can be purified using a suitable amount of affinity chromatography or the like.

For example, purification by affinity chromatography with protein A is performed as follows.

1) For purification, an Affigel Protein A MAPS-II kit (BIO RAD) was used. Ascites fluid or concentrated culture supernatant is mixed 1: 1 with binding buffer and applied to a column packed with Affigel Protein A. After washing with binding buffer, elution buffer is added to elute the antibody. The obtained fraction is subjected to operations such as concentration and desalting and used as a purified monoclonal antibody.

Example 6 Fluorescent Antibody Method Normal human tissues were stained by the indirect fluorescent antibody method using the antibodies secreted by the four hybridomas TWL, GMB, GTBM and TCP. The kidney tissue section is snap frozen with dry ice-acetone, sliced to about 4 μm using a cryostat, attached to a slide glass, and air-dried at room temperature. Antibody-containing culture supernatant or mouse ascites fluid or the like is added as it is or appropriately diluted and added to this section, and reacted at 37 ° C. for 1 hour in a humid atmosphere. Then, using phosphate buffer (PBS) several times (3 ~
It is preferably washed 4 times), and a fluorescein-conjugated anti-mouse immunoglobulin antibody (Kappel) solution diluted with PBS is added, and the mixture is reacted at room temperature for 2 hours in a moist chamber. Then, wash with PBS and add P containing 10% glycerin.
Enclose with BS and inspect under a fluorescence microscope. The results are shown in Table 1.

Example 7 Measurement of renal disease-related urinary antigens using monoclonal antibody 1) Interviews, physical findings, and weight (Matsugi standard weight) chest X-ray of 986 company employees who normally work for normal urine , Blood pressure, urinalysis (protein, sugar, occult blood), blood biochemistry (seroprecipitation,
White blood cell count, hemoglobin, hematocrit, ZTT, GOT, GP
T, LDH, ALP, γ-GTP, uric acid, BUN, neutral fat, total cholesterol, blood glucose, creatinine) were tested. Of the 400 patients who received all items, 84 were all of the above items were within the standard values, and the above items had a deep body weight associated with renal disease,
Blood pressure, urinalysis (protein, sugar, occult blood), uric acid, BUN, blood glucose, and creatinine were within standard values, and other items were collected from 125 people, 41 people who were recognized as normal although they were slightly off the standard values. Urine was regarded as normal human urine.

2) Patient urine Patients with renal diseases collected from 3 facilities (eg various nephritis, SLE,
Urine collected from 45 patients with nephrotic syndrome, acute renal failure, chronic renal failure, and diabetic nephropathy was used as patient urine.

3) Method for measuring urinary antigen Add 50 μl of urine to a 96-well EIA plate, leave it at 4 ° C overnight, and remove the urine. Add about 400 μl of PBS containing 1% BSA, leave at room temperature for 1 hour, remove PBS containing BSA, and wash twice with a washing buffer.

50 μl of purified antibodies TWL, UAL, GTBM, TCP diluted appropriately with PBS
After reaction at room temperature for 2 hours, the antibody is removed. Then, the plate is washed with a washing buffer (the washing is performed by shaking the plate filled with the washing buffer with the plate mixer for 3 minutes and then changing the washing solution four times).

HRPO-conjugated anti-mouse IgG + IgM antibody (human plasma component non-crossover, Pelle Freeze) was added at 50μ and reacted at room temperature for 2 hours.
Wash with wash buffer.

Add 50μ of substrate solution and react at room temperature for 30 minutes.
Add μ to stop the reaction.

Color development using SJeia Auto Reader (manufactured by Sanko Junyaku) 490
The absorbance at nm and 630 nm was measured, and the difference was used as the measured value.

4) Setting of cutoff value The following cutoff value was set based on the test results of the normal group.

TWL 0.20 TCP 0.60 GBM 0.40 TCP / UAL 1.10 TCP / GTBM 0.80 Here, the normal range is TWL, and for GBM is the cutoff value or more T
For CP, TCP / UAL, and TCP / GTBM, the cutoff values were set.

The frequency distribution of the absorbance, that is, the amount of antigen in the normal group and the patient group is shown in FIG. 1, and the frequency distribution of the absorbance is shown in FIGS.
As is clear from the figure, the distributions in the normal group and the patient group differ greatly in all cases.

Example 8 Using the monoclonal antibody of the present invention, the reactivity to urine of the normal group, the renal disease patient group, and the unhealthy group was compared and examined.
The results are shown in Table 3.1 and Table 3.2.

An unhealthy person is a person who is in a healthy state in which he or she can normally work as a company employee, but is mainly mild to moderate with respect to the inspection items shown in Table 3.1 and Table 3.2 in the medical examination of the contents described in Example 7, 1). The degree of abnormality was recognized.

As shown in Table 2, in the case of TWL, all are in the normal range in the normal group, and if abnormalities are caused by the antibody, all are recognized as renal diseases. With TCP, the normal group has a normal rate of 96% and a false positive rate of 4%.
Only. On the other hand, the abnormal rate was 53% in the patient group. In the renal disease patient group, 98% of TCP / UAL and 93% of TCP / GTBM abnormalities were detected, whereas in the normal group, 0% and 1% were observed, respectively, and false positives were hardly observed, and renal disease was detected. It is a very effective inspection method. 4 out of 12 cases of nephrotic syndrome
An example was GBM abnormality.

From Table 3, the abnormal rate of TWL was highest in the renal disease patient group, followed by the group in which abnormal renal disease was suggested, and no abnormality was observed in the normal group. The abnormal rates of TCP, TCP / UAL and TCP / GTBM were highest in the renal disease group, followed by the unhealthy group, and significantly lower in the normal group. In the group with abnormal antibody test values, many in the group with abnormal liver function tests were far outside the standard range. Renal disorders may be observed when the liver is abnormal such as with hepatorenal syndrome, but it can be interpreted that it is possible that renal disorders have already started when there is moderate or higher hepatic dysfunction.

Thus, the possibility of discovering an early renal disorder that could not be detected by conventional renal function tests was suggested.

[Brief description of drawings]

FIGS. 1 to 3 attached herewith are diagrams showing a comparison of the reactivity of the normal group and the patient group to TWL, TCP / UAL, and TCP / GTBM, respectively.

Front Page Continuation (72) Inventor Akira Matsukawa 532-1-809 Saiki, Sayama-cho, Minamikawachi-gun, Osaka (56) Reference JP-A-59-219299 (JP, A)

Claims (7)

(57) [Claims] 1. A monoclonal antibody which reacts with human renal tubular lumen wall or tubular cytoplasm. 2. The monoclonal antibody according to claim _1, which belongs to the IgG ₁ class. 3. The monoclonal antibody according to claim 1, which belongs to the IgG _2a class. 4. A human normal renal tissue antigen in urine to which the antibody reacts is measured using a monoclonal antibody that reacts with the inner wall of the renal tubule of the kidney or the cytoplasm of the tubule, and the site from which the antigen is derived and the antigen. How to determine the amount. 5. The method according to claim 4, wherein the measured value of the antigen in the urine of the sample is compared with the antigen value in the urine of normal human. 6. A fusion of antibody-producing cells of an animal immunized with normal human kidney tissue or HeLa cells with myeloma cells, culturing the resulting hybridoma, and producing a monoclonal antibody reactive with the normal human kidney tissue. A method for producing a monoclonal antibody, which comprises reacting with the lumen wall of human renal tubules or the cytoplasm of the tubules, which comprises recovering. 7. A reagent for diagnosing renal disorders, which comprises a monoclonal antibody that reacts with the lumen wall of human renal tubules or the cytoplasm of the tubules.