JPH05304983A - Substrate for determining protease-inhibiting protein in human urine and determination method - Google Patents

Abstract

PURPOSE:To provide the monoclonal antibody recognizing human urine protease- inhibiting protein (UTI) which relates to cancer diseases and which is contained in the urine, and further to provide the method for determining the UTI with the antibody. CONSTITUTION:A mouse is subjected to an immunological treatment using an antigen containing the UTI. The splenic cell of the immunized mouse is fused with a myeloma cell originated from a mouse, and an UTI-resistant monoclonal antibody-producing hybridoma cell is screened from the fused cells. The hybridoma cell is administered into the abdominal cavity of a mouse, and the abdominal dropsy of the mouse is collected. The collected fraction is purified into the monoclonal antibody recognizing the UTI. The monoclonal antibody is reacted with a specimen containing urinary proteins, and an antibody bonded to a label molecule for color generation is bonded to the above- mentioned monoclonal antibody to develop the color of the label molecule for the colorimetry of the UTI. The method is clinically useful for the diagnosis of the disease state of a cancer patient and as an indicator for the process of the treatments of the patient.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention recognizes a human urinary proteinase-inhibiting protein (hereinafter referred to as "UTI"), and a human urinary proteinase-inhibiting protein-like antigen in human cancer tissue. The present invention relates to a mouse monoclonal antibody, a hybridoma cell producing the mouse monoclonal antibody, and a method for immunologically quantifying UTI using the mouse monoclonal antibody.

[0002]

BACKGROUND ART Animals and plants possess proteinaceous inhibitors that inhibit the activity of proteolytic enzymes, and it is believed that these inhibitors control the physiological actions of proteolytic enzymes. There are still many unclear points about the physiological role of.

UTI was also discovered by Bauer and Reich in 1909 as one of these proteolytic enzyme inhibitors [Med. Klin., Vol.46, pp.1744-1747 (1909)].

Since most of urinary proteins are derived from serum components including metabolites of each organ, they are closely related to diseases and are important diagnostic materials in the field of clinical medicine.

It has also been reported that the urinary UTI amount significantly increases in stress conditions such as pregnancy and surgery, infectious diseases, renal and urinary tract diseases, and cancer patients [Cancer Res., Vo.
l.36, pp.1837-1846 (1976); J. Immunol., vol.121, p.
p.1636-1639 (1978); Western Japan Urology, vol.42, pp.35-43
(1980); Acta Haemat., Vol.67, pp.109-113 (1982); Sc
and. J. Clin. Lab. Invest., vol.43, pp.151-155 (19
83); Clinical Pathology, vol.13, pp.445-449 (1985); Hiroshima University Medical Journal, vol.34, pp.857-864 (1986)].

[0006] An increase in urinary UTI level in cancer patients has been observed in many carcinomas such as urinary system tumors, hematological system tumors, head and neck tumors, digestive system tumors, lung-mediastinal tumors, and breast cancers [Clinical Pathology, vol.13, pp.445-449 (1985)], and the presence of UTI-like antigens in the cancer tissues of lung cancer patients [Cancer Re
s., vol.44, pp.2011-2015 (1984); Hiroshima University Medical Journal,
vol.33, pp.1-16 (1985)] is also reported.

Although the biosynthetic mechanism of UTI has not yet been elucidated, urinary UTI has been reported based on the reports of various documents mentioned above.
There is a close relationship between increased dose and cancer.

Therefore, the quantitative value of the amount of UTI in urine or blood of a cancer patient is clinically useful not only as an index of a tumor but also as an index of diagnosis of a medical condition or a course of treatment. Further, as described above, it is considered that the detection of human urinary protease-inhibiting protein-like antigen present in cancer tissue is useful in the pathological diagnosis of cancer.

As a method for detecting UTI, an enzymatic assay method or an immunoassay method using a polyclonal antibody obtained by immunizing a rabbit or the like with purified UTI [Western urine, vol. 42, p.
p.35-43 (1980); Western Japan Urology, vol.74, pp.1641-1652 (1
983); Clinical Pharmacology, vol.19, pp.259-260 (1988)] is often used.

[0010]

However, since the tryptic enzyme used in the assay system is an enzyme that is unstable and the assay method itself uses the enzyme inhibitory activity as an index, the enzyme assay method uses a quantitative value. Since it shows total trypsin inhibitory activity in the sample, it is difficult to quantify only UTI. Furthermore, the immunoassay using the polyclonal antibody described above
It was pointed out that it was not suitable for accurate quantification of UTI from the viewpoint of specificity to I and homogeneity of antibody.

[0011]

In view of the above-mentioned problems, the present invention has been completed by focusing on a monoclonal antibody having superior properties in terms of UTI specificity and antibody homogeneity over a polyclonal antibody. The gist of the present invention is a monoclonal antibody that specifically recognizes UTI and UTI-like antigens in cancer tissues, a hybridoma cell that produces the monoclonal antibody, and a monoclonal antibody that specifically recognizes the UTI. This is the immunological quantification method of UTI used.

The human UTI-specific monoclonal antibody of the present invention is prepared according to the following procedure.

(1) Immunization of animals and production of hybridomas
A mouse is used as the immunized animal.

As the antigen, purified human UTI, a fragment derived from human UTI, and a synthetic peptide bound to an appropriate carrier molecule are administered together with an adjuvant intraperitoneally or subcutaneously to an immunized animal. The antigenic substance alone may be administered intravenously.

At intervals of one to two weeks, a further 3-5
After performing immunization once, blood is collected from the fundus vein using a Pasteur pipette. Human UT in the obtained serum
The antibody activity against I was measured by enzyme-linked immunosorbent assay (hereinafter referred to as “ELISA
"), And if sufficient antibody activity is observed, final immunization is performed and mouse spleen cells are used for cell fusion.

(2) Preparation of myeloma cells As the myeloma cells used for cell fusion, a hypoxanthine guanine phosphoribosyl transferase (HGPRT) -deficient cell line established from BALB / c mice is used. For example, myeloma cells such as P3-X63-Ag8-U1, X63-Ag8.653, SP2 / o-Ag14 were cultured in 10% FCS-supplemented RPMI 1640 medium containing 8-azaguanine or 6-thioguanine, and HGPRT
Defective strains are maintained and passaged.

One week before the experiment Normal RPM with 10% FCS
Culture myeloma cells in I 1640 medium and use the cultured cells for cell fusion.

(3) Cell fusion 3 to 4 days after the final immunization, the spleen is aseptically removed from the immunized mouse, and a spleen cell suspension is prepared in RPMI1640 medium.

RPM for both myeloma cells and immune spleen cells
The cells are thoroughly washed in I 1640 medium, and cell fusion is performed using a conventional method such as the polyethylene glycol method or the electric cell fusion method.

Cell fusion was carried out in HAT medium (hypoxanthine 10
^-4 M, Aminopritene 4 × 10 ^-7 M, Thymidine 1.6 × 10 ^-5
Resuspend in M, 10% FCS-containing RPMI 1640 medium) and dispense 0.2 ml each into a 96-well plate.

The cells were cultured at 37 ° C. in the presence of 5% CO ₂ , and after 1 day and 3 days, half of the medium was freshly HAT.
Replace with medium.

(4) Production of anti-human UTI monoclonal antibody
Selection of ibridoma cells From the wells in which cells grown in a hybridoma cell selection medium (HAT medium) have grown, the culture supernatant is aseptically collected, and the antibody activity is measured by ELISA using a 96-well plate.

That is, UTI protein or a fragment of UTI protein was adsorbed on a plate, the nonspecific binding site of the antibody to the plate was blocked with a 1% BSA-PBS (-) solution, and then the primary antibody of hybridoma cells was used. Add the culture supernatant.

After the reaction, the antibody solution was removed, the primary antibody remaining in the wells was washed with PBS (-) solution, and the solution was washed with 1% BSA-PBS.
The (-) solution is used to block the nonspecific binding site of the antibody to the plate again.

The enzyme-labeled anti-mouse immunoglobulin antibody prepared with a 1% BSA-PBS (-) solution is used as a secondary antibody to screen for an antibody that specifically reacts with the antigen.

Then, an antigen-specific primary antibody is detected by an enzymatic reaction, and the antibody activity is quantified using an ELISA microplate reader. When positive wells are confirmed, the hybridoma cells are cloned according to the limiting dilution method using BALB / c mouse thymocytes as supporting cells to establish the target hybridoma clone.

(5) Preparation of Monoclonal Antibody The monoclonal antibody is purified using the culture supernatant and mouse ascites.

That is, in the case of the culture supernatant, the culture supernatant obtained by large-scale culture of hybridoma cells using a serum-free medium or a medium supplemented with fetal bovine serum, and in the case of mouse ascites, to pristane-treated BALB / c mice, The crude fractions obtained by salting out the ascites obtained by intraperitoneally administering 5-10 × 10 ⁶ antibody-producing hybridoma cells with ammonium sulfate are used as starting materials for the purification of monoclonal antibodies.

The purification operation is performed by high performance liquid chromatography (H
PLC) and anion exchange chromatography using an open column, gel filtration / affinity chromatography, etc.

The purified monoclonal antibody was added to the enzyme (β-galactosidase, alkaline phosphatase,
Peroxidase, glucosidase, etc.) or radioisotope can be labeled to detect the antigen by a direct method without using a secondary antibody.

[0031]

EXAMPLES Examples of the present invention will be described in detail below.

Example 1: Confirmation of anti-UTI monoclonal antibody
Standing (1) Immunization A 6-week-old female BALB / c mouse (supplier: Charles River Japan) was bred for several days in a sterile room and then used in the experiment.

Human UTI (manufactured by Nippon Chemical Research Co., Ltd.) was used to mix human UTI-PBS (-) solution (1 mg / ml) and Freund's complete adjuvant (volume ratio 1: 1) to prepare an emulsion. 500 μl was administered intraperitoneally.

After 3 weeks, an immunogen was prepared by mixing human UTI-PBS (-) solution (1 mg / ml) with Freund's incomplete adjuvant (volume ratio 1: 1), and 250 μl of this was intraperitoneally administered.
Further, 4 weeks after this, the final immunization was similarly performed with the antigen similarly prepared using Freund's incomplete adjuvant, and the spleen cells of the immunized mouse were subjected to cell fusion.

(2) Preparation of mouse myeloma cells 6-thioguanine-resistant Sp2 / o-Ag14 derived from BALB / c mice
The myeloma cells were cultured in RPMI 1640 medium containing 10% FCS (manufactured by Nissui Pharmaceutical Co., Ltd.), and about 6 × 10 ⁷ myeloma cells were used for cell fusion.

(3) Preparation of hybridoma cells and cloni
Spleen cells prepared by ring (1) and (2) and Sp2 / o-Ag
14 Myeloma cells were washed with RPMI 1640 medium and 50% polyethylene glycol (Molecular weight: 4000, Merck) -RPM
Cell fusion was performed using the I 1640 solution.

After fusion, RPMI 1640 medium was poured, the cells were collected by low speed centrifugation (800 rpm), and 100 ml of HAT was collected.
96-well plate (Nunclon, Nunc) after suspending in medium
Dispensed into 5 sheets. Then, the cells were cultured at 37 ° C in the presence of 5% CO ₂ . One day and three days after the start of culture, half of the medium was replaced with fresh HAT medium.

From the wells in which the proliferation of hybridoma cells was high, 96-well plates (Immunoplate II Maxisorp, manufactured by Nunc) were used, and peroxidase-labeled goat anti-mouse immunoglobulin G & M antibody (Jacson Immunores) was used.
Hybridoma cells were screened by ELISA using earch Laboratories) as a secondary antibody.

Cloning of hybridoma cells proliferating in wells in which a positive reaction was observed with human UTI used as an antigen, according to the limiting dilution method using a 96-well plate containing BALB / c mouse thymocytes. Were carried out to establish hybridoma cells producing monoclonal antibody 4G12 (hereinafter referred to as "MAb4G12").

The hybridoma cell established in this Example was deposited as hybridoma cell 4G12 (FERMP-12930).

The obtained hybridoma clone cells are
The cells were cultured in RPMI 1640 medium containing 10% FCS, and the cultured cells were used for the subsequent experiment. The class of MAb4G12 is anti-mouse immunoglobulin antibody (Jacson Immunoresearch Labo
IgG1 was determined by sandwich ELISA using an enzyme-labeled anti-mouse immunoglobulin class / subclass specific antibody (SEROTEC).

(4) Preparation of MAb4G12 Hybridoma cells 4G12 were cultured to a cell concentration limit on a 10 cm dish (manufactured by Corning), and this cell suspension was
The mixture was transferred to a 50 ml centrifuge tube and centrifuged at 2800 rpm to collect the culture supernatant. The obtained culture supernatant contains Na at 0.1%.
N ₃ was added, and it was stored frozen at a temperature of −20 ° C. until use.

Next, the hybridoma cells producing MAb4G12 were intraperitoneally administered to pristane-treated BALB / c mice to obtain the ascites. The obtained ascites was salted out with 33% ammonium sulfate and dialyzed, and the sample was placed on a protein A column (Af
fi-Gel Protein A MAPS II Kit, manufactured by BioRad) to remove excess protein, and a fraction eluted with PBS (-) solution (pH 7.2) was collected.

The dialyzed product of this eluate fraction against a PBS (-) solution was used as a protein A column-purified MAb 4G12 sample, and this purified M
Ab4G12 was stored frozen at −80 ° C. until the time of experiment.

The protein was quantified by using an absorptiometer (absorption wavelength 280 nm) using bovine serum albumin as a standard sample.

MAb4G12 contained in the culture supernatant and purified MAb4G12 derived from mouse ascites show the same antigen specificity,
Since the culture supernatant samples contained bovine serum proteins and protein components derived from hybridoma cells in addition to MAb4G12, these samples were used according to the purpose of the experiment.

Example 2: Reactivity to UTI 50 μl of UTI antigen solution diluted to the optimum concentration with PBS (-) solution
A 96-well plate (ImmunoPlate II MaxiSoap, Nu
(manufactured by NC Inc.) and left overnight at 4 ° C. All the subsequent operations were performed at room temperature.

After discarding the antigen solution, 100 μl of PBS (-) solution was poured into the well to wash the excess antigen solution, and then 1% BSA-PB was added.
100 μl of S (-) solution (blocking solution) was poured into each well, and nonspecific adsorption site of the antibody to the plate was blocked for 15 minutes.

Then, the blocking solution was removed, and 50 μl of the culture supernatant (primary antibody) of the hybridoma cell 4G12 was poured into each well to carry out an antibody reaction.

After 30 minutes from the initiation of the antibody reaction, each well was washed 3 times with PBS (-) solution, further blocked with the above blocking solution, and then washed with 1% BSA-PBS (-) solution. 50 μl of peroxidase-labeled goat anti-mouse immunoglobulin G & M antibody (secondary antibody) diluted to 400 ng / ml was poured into each well and reacted for 30 minutes in the same manner as the primary antibody.

After completion of the reaction, each well was diluted with PBS (-) solution to 3 times.
After washing twice, 100 μl of substrate solution (orthophenylenediamine 0.4 mg / ml, 0.01% H ₂ O ₂ -citrate phosphate buffer, pH
5.0) was poured into each well to carry out an enzymatic reaction. Two minutes after starting the reaction, 30 μl of 2M sulfuric acid was added to each well to stop the enzyme reaction, and the microplate reader (CORONA MTP-1
Colorimetric determination (absorption wavelength: 492 nm) using 00, manufactured by Corona) to quantify the antibody activity, and the results are summarized in Table 1 below. As is clear from Table 1, detection was possible even with UTI at a concentration of 1 ng / ml or less.

[0052]

[Table 1]

Example 3: Specificity to protein in human urine Fresh urine collected from 8 healthy men and SDS sample buffer (8M Urea, 0.5M Tris-HCl, pH 6.8) were mixed (volume ratio 1: 1). After SDS conversion of the sample, 20 μl of it was subjected to SDS polyacrylamide gel (16%) electrophoresis.

The electrophoresed protein was electrically transferred onto a Millipore filter (Immobilon, Millipore) and 5
It was immersed in a% skim milk-PBS (-) solution for blocking.

Using the culture supernatant of hybridoma cells 4G12 (primary antibody), an antibody reaction was carried out at room temperature for 1 hour to obtain PBS (-).
The unreacted MAb4G12 was washed away with the solution, and immersed in a 5% skim milk-PBS (-) solution to perform blocking again.

Then, 500 ng / ml in 1% BSA-PBS (-) solution
The peroxidase-labeled goat anti-mouse immunoglobulin G & M antibody diluted to 1% as a secondary antibody at room temperature for 1 hour,
The reaction was carried out.

After washing the secondary antibody with PBS (-) solution,
The enzymatic reaction was performed using the Konica Immunostain HRP kit. As a result, as shown in FIG. 1, which shows the result of Western blotting of MAb4G12 using urinary protein as an antigen, although there are individual differences in the intensity of the stained band,
A specific staining band was observed at the same migration position as UTI,
It was confirmed that MAb4G12 specifically recognizes UTI in urinary protein.

Example 4 Quantification of UTI in dog serum UTI was quantified using rabbit anti-UTI polyclonal antibody and MAb4.
It was performed by a secondary antibody sandwich ELISA using G12.

With physiological saline (Otsuka raw food, manufactured by Otsuka Pharmaceutical Co., Ltd.)
A UTI solution dissolved at 10000 units / ml (3.5 mg / ml) was administered through the forelimb vein of a male beagle dog to 2000 units per 1 kg of beagle dog body weight.

Immediately before administration (0 minutes), 5 minutes, 10 minutes, 20 minutes after administration
Minutes, 30 minutes, 60 minutes, 120 minutes and 180 minutes later, 2 ml of blood was collected from the opposite forelimb administered with UTI, and serum was separated using a Separabit tube (Sekisui Chemical Co., Ltd.) until use. It was stored frozen under a temperature condition of -20 ° C.

In preparation for the quantification, the serum was quickly thawed under a temperature condition of 37 ° C. and further diluted 100-fold with a PBS (-) solution. Serum collected from 5 to 30 minutes after the start of UTI administration was further diluted 10-fold with a normal beagle dog serum solution diluted 100-fold with PBS (-) solution and used for quantification.

The calibration curve is 0.1, 0.3, 1, for normal beagle dog serum solution diluted 100-fold with PBS (-) solution.
Prepared at concentrations of 3, 10, 30, 100, 300, 1000 ng / ml
It was created using a UTI (manufactured by Japan Chemical Research Co.) solution.

20μ purified using protein A column
50 μl of a g / ml rabbit anti-UTI antibody IgG-PBS (-) solution was poured into the wells of a 96-well plate (Immunoplate II Maxisorp, Nunc) and left overnight at 4 ° C. All subsequent operations were performed at room temperature.

The excess antibody was washed with PBS (-) solution, 100 μl of 1% BSA-PBS (-) solution was poured into each well, and blocking was performed for 1 hour. Remove the blocking solution and dilute the serum sample to the optimum concentration and UTI standard sample for calibration curve preparation, 50 each
μl was poured into the well and the reaction was carried out for 2 hours. PBS (-)
After washing 3 times with the solution, 1 μg / with 1% BSA-PBS (-) solution
Purified MAb 4G12 (50 μl) prepared in 1 ml was poured into each well.
The reaction was run for a minute.

After washing excess antibody with PBS (-) solution, 100 μl of 1% BSA-PBS (-) solution was poured into each well and blocking was performed for 30 minutes. And 400ng / with 1% BSA-PBS (-) solution
50 μl of peroxidase-labeled goat anti-mouse immunoglobulin G & M antibody diluted to ml was poured into each well and reacted for 30 minutes.

After washing the unreacted secondary antibody with a PBS (-) solution,
Substrate solution 100 μl (ortho-phenylenediamine 0.4 mg / m
1, 0.01% H ₂ O ₂ -citrate phosphate buffer, pH 5.0) was poured into each well and the enzyme reaction was carried out for 2 minutes. 2M sulfuric acid 30 μl
Was added to each well, the enzymatic reaction was stopped, and then colorimetric quantification (absorption wavelength 492 nm) was performed using a microplate reader (CORONA MTP-100, manufactured by Corona), and quantification results (Table 2).
A calibration curve (Fig. 2) was prepared based on the above.

[0067]

[Table 2]

From the calibration curve, the UTI detection sensitivity in the two-antibody sandwich ELISA using the monoclonal antibody was 1 ng / ml.
And the UTI concentration between 300 pg / ml and 100 ng / ml
It was possible to measure quantitatively.

As shown in Table 3 below, the UTI concentration in beagle dog serum was 12.6 μg / ml 5 minutes after administration, but was 1.26 μg / ml after 1 hour and 2 hours after administration. 140ng / m
l After 3 hours, 30 ng /, which is close to the detection limit of 100-fold diluted sample
It has dropped to ml.

[0070]

[Table 3]

Example 5: UTI-like antigen in human lung cancer tissue
Detection (1) Immunohistological staining A tissue piece containing a cancer tissue from a paraffin block of human poorly differentiated lung squamous cell carcinoma was sliced with a microtome and placed on a slide glass coated with egg albumin. After the tissue was sufficiently dried, the paraffin was removed using xylene, and the ethanol and the PBS (-) solution were washed in this order.

In order to inactivate the endogenous peroxidase in the tissue, it was immersed in a 0.6% H ₂ O ₂ · 0.2% NaN ₃ -methanol solution for 1 hour, and the tissue piece was washed with a PBS (-) solution.
Blocking was performed with a 0.2% NaN ₃ -5% goat albumin-1% BSA-PBS (-) solution.

Next, purified MAb 4G12 (46 μm) which is the primary antibody
(g / ml) solution was reacted at room temperature for 1 hour.
Thoroughly wash the primary antibody with PBS (-) solution, block with 5% goat albumin-1% BSA-PBS (-) solution, and
The reaction was carried out at room temperature for 1 hour using peroxidase-labeled goat anti-mouse immunoglobulin G & M antibody diluted to g / ml as a secondary antibody.

After the reaction, the tissue pieces were thoroughly washed with PBS (-) solution, and then the substrate solution (diaminobenzidine 0.2 mg / ml,
The staining reaction was stopped by immersing the slide glass in 0.003% H ₂ O ₂ Tris-HCl buffer, pH 7.6) to perform a staining reaction, and washing the tissue with a PBS (-) solution. Subsequently, the nuclei of the cells were stained with hematoxylin and the mounting agent was dropped to seal the stained tissue with a cover glass.

When the stained tissue was observed using an optical microscope, a stained image specific to MAb4G12 was observed. That is, it was confirmed that the stained image was mostly found in the interstitium of lung tissue in which the cancer cells proliferated and invaded, and the antigen recognized by MAb4G12 was present in the interstitium of lung cancer tissue.

(2) Extraction of Antigen A thin piece excised from a paraffin block of human poorly differentiated lung squamous cell carcinoma was treated with xylene and ethanol to remove paraffin, and 0.35 g of dried cancer tissue was obtained. The dried tissue was shredded with scissors, homogenized in a 0.1% SDS-PBS (-) solution using a Teflon homogenizer, and then centrifuged to obtain 8.7 ml of a supernatant.

A part of the supernatant was concentrated to about 10 times using a mol cut (MW 5000 cut, manufactured by Millipore) and Example 3
Electrophoresis was performed in the same manner as above, and Western blotting was performed using the culture supernatant of the hybridoma cell 4G12 as an antibody solution.

As a result, MA using the lung cancer tissue extract as an antigen
As is clear from FIG. 3 showing the electrophoretic state of b4G12 by Western blotting, an antigen showing the same electrophoretic position as UTI recognized by MAb4G12 was confirmed in the lung cancer tissue extract (lane 2 in FIG. 3).

[0079]

According to the present invention, UT among various urinary proteins
MAb4G12, a monoclonal antibody that specifically recognizes I, and hybridoma cell 4G12, which produces the monoclonal antibody MAb4G12, are provided, and as a result, microquantification of UTI using a monoclonal antibody with uniform antigen specificity is possible and clinically possible. It is expected that useful diagnostic means for cancer diseases will be realized.

[Brief description of drawings]

FIG. 1 shows the results of Western blotting of MAb4G12 using urine protein as an antigen.

FIG. 2 is a diagram showing a calibration curve of a standard UTI sample by a two-antibody sandwich ELISA.

FIG. 3 is a diagram showing the results of Western blotting of MAb4G12 using a lung cancer tissue extract as an antigen.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location G01N 33/53 D 8310-2J 33/573 Z 9015-2J 33/577 B 9015-2J // ( C12P 21/08 C12R 1:91)

Claims (5)

[Claims] 1. A monoclonal antibody that recognizes a human urinary proteinase inhibitor protein. 2. The monoclonal antibody according to claim 1, which recognizes a proteinaceous antigen in cancer tissue. 3. A hybridoma cell that produces a monoclonal antibody that recognizes a human urinary protease inhibitor protein. 4. The hybridoma cell according to claim 3, wherein the hybridoma cell is hybridoma cell 4G12 (FERMP-12930). 5. A method for quantifying human urinary proteinase inhibitor protein, which comprises the following steps. That is, (a) a sample containing urine protein is immobilized, (b) the sample is bound with a monoclonal antibody that recognizes a human urinary protease inhibitor protein, (c) the monoclonal antibody, an enzyme,
An antibody to which a labeling molecule such as a dye is bound is bound, and (d) the labeling molecule is quantified.