JP2561217B2 - Hybridoma producing a monoclonal antibody for use in a method for diagnosing renal damage by measuring renal antigen in urine - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hybridoma producing a monoclonal antibody which specifically reacts with an antigen derived from normal human kidney tissue.

[0002]

[Background Art] [Problems to be Solved by the Invention] Currently, in Japan, there are about 7 patients undergoing dialysis treatment for renal failure.
There are 10,000 people, and those patients require enormous treatment costs of about 5 million yen per person per year, which is one of the major causes of pressure on the government and local governments. Moreover, about 5,000 new cases of renal failure are occurring each year.

If you fall into renal failure, every 2 to 3 days, 4 to 4 days a day
The patient has to receive dialysis treatment for 6 hours, which not only significantly hinders the social life of the patient himself, but also imposes a heavy social burden on the patient as mentioned above. There is a risk that the medical system will collapse.

[0004] Renal failure is a condition that eventually leads to patients with renal disease, but it is caused by diseases other than the original kidney such as diabetes, high blood pressure and gout, and eventually the kidney is damaged and becomes renal failure, or a sleeping pill is commonly used. If renal failure occurs due to side effects such as antibiotics and anti-cancer agents, etc., various nephritis such as chronic nephritis, acute nephritis or rapidly progressive nephritis, or There are many cases where the kidney is damaged by a condition other than the original renal disease such as pyelonephritis, renal tuberculosis, renal stone, and cystic kidney disease. 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.

[0005] By the way, in renal diseases and renal disorders that appear in association with these other diseases, there is no noticeable subjective symptom until the end stage, that is, near renal failure, and its occurrence may be overlooked. Many. 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 Act, the Occupational Safety and Health Act, and the Elderly People Insurance Act legally obligate 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.

As described above, in the urinalysis currently performed, urine protein, urine sediment and the like are inspected, but the sensitivity of the test paper used for these examinations differs depending on the manufacturer, and The quality, gloss, and color tone of the colorimetric contrast paper of the commercial inspection test paper used in 1. The sex is not fully satisfactory. Urine protein is a protein leaked from the blood into the urine of the kidney or urinary tract, and the protein is excreted daily in normal people as well as in patients with renal disease. After exercising, febrile proteinuria, or simply proteinuria due to contamination with semen, is also seen in normal people. In addition, proteinuria is also seen in urinary tract diseases, bladder diseases, female genital diseases, etc. 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.

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

Other epithelial cell leukocytes may also have pathological significance.

If there is an abnormality or a suspicion of abnormality in the above-mentioned urinalysis, a more detailed examination will be conducted in a medical facility. Serum creatinine, urea nitrogen, glomerular filtration rate, renal tubules. Function, measurement of β ₂ microglobulin,
Even if a renal function test such as urine concentrating ability, urine diluting ability, renal blood flow and urinary acidification ability is performed, it is often difficult to diagnose a specific disease from it. Also, N-Acetyl-β-D-glucosami
Some tests try to detect tubular damage by measuring the enzyme activity of dase (NAG), but the pH of urine changes from a weak alkaline side to a strongly acidic side, salt concentration also fluctuates significantly, and osmotic pressure is high. It may not be effective except for some diseases and acute renal failure, etc. In addition, it may be possible to spend a lot of time and money on an image examination such as transvenous renal pelvis, renal scintigraphy, echography, X-ray examination, CT scan, plain abdominal X-ray or renal angiography. Morphologically changed according to the obtained image and more than a certain size, that is, it is effective for renal tumors, cysts, stones and malformations, but various nephritis, nephrosclerosis, nephrosis, diabetes It is of little use in the vast majority of medical renal diseases such as gonadosis 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 carry out 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. To be expensive
At present, it can be performed only for a very limited number of patients who have extremely high needs.

As described above, the urine test by urinalysis can be performed every day 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. However, the urine tests currently performed there include many false positives due to the uncertainty included in the tests themselves, and are not 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, using the urinalysis system, it is possible to measure easily and with high sensitivity,
There has been a strong demand for a test method that enables early diagnosis of renal disease.

[0014]

The present invention has been made to solve the above problems, and is based on the following knowledge.

In the glomerulus of the kidney, blood is filtered to form raw urine, which flows through the renal tubules and collects in the collecting duct to be excreted as urine through the ureter to the bladder for excretion. 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 which reacts specifically with the antigen derived from the kidney tissue was prepared, and reacted with normal human urine and urine of patients with renal disease. It was found that there was a significant difference between the two groups. 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. Moreover, it was confirmed that normal metabolism was suppressed in the renal disease patient, and as a result, the amount of the antigen, which is a metabolite, was significantly decreased 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 has also been found that some so-called unhealthy people show significant abnormalities as compared with normal people, although they are not as significant as those with renal disease.

Therefore, by the test method using the monoclonal antibody produced by the hybridoma of the present invention, it becomes possible to safely and simply determine the site of micro-lesion in the kidney and its severity without side effects. The nature of the lesion can be estimated in part, and the nature of the lesion can be better evaluated when combined with clinical symptoms.

In the urine protein test, urine occult blood test and the like in the urinalysis, no abnormality is observed unless the glomerular damage progresses to a certain extent. However, the diagnostic method using the monoclonal antibody produced by the hybridoma of the present invention was previously subtle. General people who may have potential renal disorders such as hypertension, diabetes, gout, obesity, and thinness, because various lesions can be found.
That is, in an unhealthy person, renal damage can be detected at the time when the proteinuria is negative, that is, at an early stage.

Further, in renal biopsy, lesions are observed in a form in which the history of accumulated diseases from the past to the present is overlapped, whereas in the diagnostic method of the present invention, the current state at the time of excretion in urine is shown. You can know the condition.

Further, since the examination can be repeated every day, it can provide important information for determining a treatment policy.

As described above, the diagnostic method using the monoclonal antibody produced by the hybridoma of the present invention does not require expensive equipment and can be performed very easily. Second of (±) group
It is extremely useful as a next screening method.

Furthermore, a rejection reaction associated with renal transplantation can be detected early by a measuring method using a monoclonal antibody produced by the hybridoma of the present invention.

The present invention is directed to an adult positive human, localized in the human kidney.
Hybridomas that produce a monoclonal antibody that specifically recognizes a renal tissue antigen, in particular the renal tubular lumen wall or the tubular cytoplasm .

The monoclonal antibody produced by the hybridoma of the present invention reacts significantly more strongly with urine of patients with renal disease than that of normal people, and more strongly with urine of people with renal impairment 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 the lesions, metabolic disorders, and renal histological abnormalities of patients with renal diseases as described above.

As the monoclonal antibody produced by the hybridoma of the present invention, as shown in Examples below,
There are monoclonal antibodies that specifically react with human renal tubular lumen wall, glomerular basement membrane, glomerular and tubular basement membrane, or tubular cytoplasm, and these belong to the IgG ₁ class or IgG _2a class.

The present invention also relates to the diagnosis of the site and extent of renal damage by measuring the antigen in urine using a monoclonal antibody that specifically recognizes the human normal kidney tissue antigen described above. Provide a fault diagnosis method.
In this diagnostic method, 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, the resulting hybridoma is cultured, and is specific to the produced normal human kidney tissue. There is provided a method for producing a monoclonal antibody, which comprises recovering a monoclonal antibody that reacts chemically.

The present invention is directed to normal human kidney tissue or HeLa.
Obtained from antibody-producing cells and myeloma cells of an animal immunized with cells, the normal human renal tissue, particularly human renal tubular lumen wall or
Provide a hybridoma producing a monoclonal antibody that specifically reacts with the tubular cytoplasm .

Finally, the present invention relates to human normal kidney tissue, especially human.
( EN) A reagent for diagnosing renal damage, which comprises a monoclonal antibody that specifically reacts with the lumen wall of renal tubule or the cytoplasm of the tubule .

The present invention will be described in more detail by way of examples.

[0033]

EXAMPLES Example 1 Method for preparing hybridoma 1. Immunity 1) was used as a normal cortex of the kidney was removed from adult male as the immunogen. Aminobenzylpenicillin (10
mg / ml) and streptomycin sulfate (10 mg / ml) in sterile physiological saline, and then successively in sterile physiological saline, and then cut into 1 mm square pieces.

2) The sliced pieces (3.9 g) were crushed together with physiological saline in a glass homogenizer, and then physiological saline was added to prepare a homogenizing solution (renal cortex 300 mg / ml).

3) The above homogenizing solution is diluted with physiological saline and administered as it is or together with an adjuvant (such as 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 and the like, but subcutaneous or intraperitoneal injection is preferred.
For example, in mice, 47 mg of cortex was subcutaneously or intraperitoneally injected.

4) Booster immunizations at appropriate intervals, eg 3-4
Do this once or several times a week. 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) Immunized splenocytes and mouse myeloma cells, such as P3-NS1-1-Ag4-1 cells (NS1 cells) 3 to 4 days after the final immunization
Fusion with.

5 × 10 ⁷ immune splenocytes and 1 × 10 NS1 cells
Mix at a ratio of ⁷ or any other suitable ratio to give 400
Xg, centrifuge for 5 minutes and remove the supernatant. Then, 1 ml of polyethylene glycol (PEG) solution such as 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, at 400 × g. Centrifuge for 5 minutes and remove the supernatant.

2) The cells are suspended in 10 ml of a culture medium (nutrient medium such as DMEM medium + serum, for example, 15% horse serum + antibiotics such as aminobenzylpenicillin 0.1 mg / ml streptomycin sulfate 0.1 mg / ml), and the cells are suspended in 96 wells. Dispense 100 μl each into the wells of a tissue culture plate, 37 ℃, 5% CO
_2. Incubate in a humid atmosphere.

3) The day after fusion, hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine were added to each well.
Add 100 μl of culture medium (HAT medium) containing (16 μM). Half of the medium is replaced with HAT medium every 2-3 days.
Replace with HT medium after 10 to 14 days. What is HT medium HA
It is the T medium without aminopterin.

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) A human renal cortical antigen solution is added to a 96-well EIA plate and adsorbed and solidified under appropriate conditions. For example, 100μ
l of the antigen solution is added, and the mixture is 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 of bovine serum albumin (BSA) or the like.

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

3) Wash with a phosphate buffer solution (wash buffer solution) containing 1% of Tween 20. 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. 100 μl / well of a horseradish peroxidase (HRPO) -conjugated anti-mouse IgG + IgM antibody solution was added to this plate and reacted at room temperature.

4) After washing, a substrate solution is added to carry out a color reaction. Add 0.4 mg / ml ortho-phenylenediamine and 0.2% of 31% hydrogen peroxide solution to citric acid-phosphate buffer (pH 5.0).
100 μl / well of a solution (substrate solution) added with 1 / ml was added, and after reacting at room temperature for 30 minutes, 50 μl / well of 4N sulfuric acid was added to stop the reaction. Those that develop color are used as antibody-producing positive cells (hybridomas).

Cloning of antibody-producing positive cells Cloning of hybridomas is carried out by the limiting dilution method, soft agar method,
The fibrin gel method is used. An example of the limiting dilution method is shown below.

1) The hybridomas were suspended in HT medium at 10 cells / ml, and the mouse or rat thymus or spleen cells serving as feeder cells were suspended in HT medium at about 1.7 × 10 ⁷ cells / ml, and a 96-well tissue culture plate was prepared. Dispense 100 μl / well into each well and incubate. The next day, 100 μl / well of HT medium is added, and a half volume is replaced 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.

[0052] 1) immune culture medium (e.g., Dulbecco's in MEM medium + 10% fetal calf serum + antibiotics) at cultured HeLa cells of higher animals with complete Freund's adjuvant, for example, BALB /
The mice were immunized by intraperitoneal injection or intravenous injection. HeLa cells were administered as described above with or without an appropriate period of time, with or without adjuvant, to perform a booster immunization.

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

3) Screening of antibody-producing hybridoma
Cloning and cloning After removing the culture supernatant of HeLa cells cultured on a 96-well tissue culture plate and blocking with 1% BSA, the fusion cell culture supernatant was added and reacted at room temperature for 2 hours. After washing with washing buffer, HRPO-conjugated anti-mouse IgG + IgM
The antibody dilution was added and the reaction was carried out at room temperature for 2 hours. After washing with a washing buffer, a substrate solution such as an ABTS solution is added,
After reacting for 30 minutes at room temperature, 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-producing hybridomas.

Reference Example 1 Preparation of Ascites Fluid Containing Monoclonal Antibody The hybridoma is transplanted into a living body and allowed to proliferate, and body fluid is collected from the living body to produce an antibody secreted by the hybridoma. When hybridomas are transplanted into the abdominal cavity and proliferated, pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered before transplantation, for example, for 3 to 8 weeks to increase the ascites yield. 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) Use of mature BALB / c mice Hybridomas cultured in HT medium in mice treated with pristane in advance were added to DMEM medium in an amount of 1 × 10 5.
Float ^{7 to} 2 x 10 ⁷ cells / ml, 5 x per mouse
10 ⁶ to 1 × 10 ⁷ hybridomas are intraperitoneally transplanted once to several times.

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

Reference Example 2 Typing of monoclonal antibody The subclass of the antibody was identified by the Octaloni method or EIA method.

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

2) Mono-Ab-ID EIA kit (Zy
med) was used. Solid phase with adsorbed antigen, eg EI
50 ml of culture supernatant containing monoclonal antibody on A plate
After adding μl and reacting at room temperature for 1 hour or more,
Wash with wash buffer. Then add 50 μl of subclass-specific rabbit anti-mouse immunoglobulin antibody at room temperature for 1
After reacting for a time, it is washed. Next, 50 μl of HRPO-conjugated anti-rabbit IgG antibody is added, reacted at room temperature for 1 hour, and then washed. 100 μl of a solution containing 1 mM of ABTS (2,2-azinodi [3-ethylbenzthiazoline sulfonic acid]) as a substrate was added, and the mixture was allowed to react at room temperature for 20 to 30 minutes for determination.
The results are shown in Table 1 below.

Reference Example 3 Purification of Monoclonal Antibody Monoclonal antibody in the culture supernatant or ascites can be purified using gel chromatography, ion exchange chromatography, affinity chromatography and the like.

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

1) For purification, Affi-gel protein AMAPS-
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.

Reference Example 4 Fluorescent antibody method hybridomas TWL, GBM, GTBM, TCP 4
Normal human kidney tissue was stained by the indirect fluorescent antibody method using an antibody secreted by the species. 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, it is washed several times (preferably 3 to 4 times) with a phosphate buffer (PBS), and a fluorescein-conjugated anti-mouse immunoglobulin antibody (Kappel) solution diluted with PBS is added, and the mixture is kept in a moist chamber at room temperature for 2 hours. React with. Then wash with PBS 10
The cells are sealed with PBS containing% glycerin and examined under a fluorescence microscope. The results are shown in Table 1.

[0067]

[Table 1]

Reference Example 5 Measurement of Renal Disease-Related Urinary Antigen Using Monoclonal Antibody 1) Interviews with 986 company employees who normally work for normal urine
Physical findings, weight (Matsugi's standard weight), chest X-ray, blood pressure, urinalysis (protein, sugar, occult blood), blood biochemical test (blood sedimentation,
White blood cell count, hemoglobin, hematocrit, ZTT, G
OT, GPT, LDH, ALP, γ-GTP, uric acid, B
UN, triglyceride, total cholesterol, blood sugar, creatinine) were tested. Of 400 patients who received all items, 84 were all of the above items within the standard values, and the above items were deeply related to kidney disease, blood pressure, and urinalysis (protein, sugar,
Occult blood, uric acid, BUN, blood sugar, and creatinine were within the standard values, and other items were slightly deviated from the standard values, but urine collected from a total of 125 persons, 41 persons who were recognized as normal, was regarded as normal human urine.

2) Patient urine Patients with renal disease collected from 3 facilities (eg various nephritis, SL
E, nephrotic syndrome, acute renal failure, chronic renal failure, diabetic nephropathy).

3) Method for measuring urinary antigen 50 μl of urine is added to a 96-well EIA plate, left at 4 ° C. overnight, and then urine is removed. About 400 μl of PBS supplemented with 1% BSA is added, and the mixture is allowed to stand at room temperature for 1 hour, then the PBS supplemented with BSA is removed, and washed twice with a washing buffer.

Purified antibody TWL, U diluted appropriately with PBS
50 μl of AL, GTBM and TCP are added and reacted at room temperature for 2 hours, and then the antibody is removed. Then, the plate is washed with a washing buffer (for washing, the operation in which the plate filled with the washing buffer is shaken on the plate mixer for 3 minutes and then the washing solution is exchanged is repeated 4 times).

50 μl of HRPO-conjugated anti-mouse IgG + IgM antibody (human plasma component non-crossover, Pelle Freeze) was added, and the mixture was reacted at room temperature for 2 hours and then washed with a washing buffer.

50 μl of the substrate solution was added and reacted at room temperature for 30 minutes, and 50 μl of 4N-sulfuric acid was added to stop the reaction.

For color development, SJeia Auto Reader (manufactured by Sanko Junyaku) was used to measure the absorbance at 490 nm and 630 nm, 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 was TWL and the cutoff value for GBM and below the cutoff value for TCP, TCP / UAL, and TCP / GTBM.

[0077]

[Table 2]

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

Reference Example 6 The monoclonal antibody of the present invention was used to compare and examine the reactivity to urine of a normal group, a renal disease patient group and an unhealthy group. The results are shown in Table 3.1 and Table 3.2.

An unhealthy person is a person who is in a healthy condition in which he or she can normally work as a company employee, but mainly in regard to the inspection items shown in Table 3.1 and Table 3.2 in the medical examination of the contents described in Reference Example 5 and 1). It was a mild to moderate abnormality.

[0081]

[Table 3]

[0082]

[Table 4]

As shown in Table 2, in the case of TWL, in the normal group, all are in the normal range, and if abnormalities are caused by the antibody, all are recognized as renal diseases. With TCP, the normal rate of the normal group is 96%,
The false positive rate is only 4%. On the other hand, in the patient group, the abnormal rate was 53%
Reach TCP / UAL 98%, T in renal disease patient group
While 93% of CP / GTBM abnormalities are observed, 0% and 1% in the normal group show almost no false positives, which is a very effective test method for detecting renal disease.
Abnormal GBM was observed in 4 out of 12 cases of nephrotic syndrome.

From Table 3, the abnormal rate of TWL was highest in the renal disease patient group, followed by a group in which abnormal renal disease was suggested, and no abnormality was observed in the normal group. TCP, TCP / UA
The abnormal rates of L and TCP / GTBM are highest in the renal disease patient group, followed by the unhealthy group, and remarkably low 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 recognized when there is an abnormality in the liver such as hepatorenal syndrome, but it can be interpreted that there is a possibility that renal disorders have already started when there is moderate or higher hepatic dysfunction.

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

[Brief description of drawings]

FIG. 1 is a diagram showing a comparison of responsiveness of a normal group and a patient group to TWL.

FIG. 2 is a diagram showing a comparison of reactivity between a normal group and a patient group to TCP / UAL.

FIG. 3 is a diagram showing a comparison of reactivity between TCP / GTBM in a normal group and a patient group.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/53 G01N 33/577 B 9281-4B C12N 5/00 B 33/577 9162-4B 15 / 00 C // (C12P 21/08 C12R 1:91) (72) Inventor Kazuya Kunigami 85-58 Yamanobo-cho, Kashihara-shi, Nara (72) Akira Matsukawa Akira Matsuki, Sayama-cho, Minamikawachi-gun, Osaka 532 -1-809 (56) Reference JP-A-59-219299 (JP, A)

Claims (1)

(57) [Claims] 1. A hybridoma, which is obtained from antibody-producing cells and myeloma cells of an animal immunized with normal human renal tissue or HeLa cells , and produces a monoclonal antibody that reacts with human renal tubular lumen wall or tubular cytoplasm .