JPH0231691A - Anti-human glucocorticoid receptor antibody and use thereof - Google Patents

Abstract

PURPOSE:To accomplish simple quantification of human glococorticoid receptor in high accuracy through the enzyme immunoassay by using anti-human glucocorticoid receptor monoclonal antibody. CONSTITUTION:Using a monoclonal antibody to human glucocortiocid receptor, the human glucocorticoid receptor is quantified through the enzyme immunoassay. Also, using the above monoclonal antibody, human glucocorticoid receptor-positive cells are detected through a immunohistochemical technique. To prepare the human glucocorticoid receptor, human leukemia cell strain containing glucocorticoid receptor is put to mass culture and the resultant cells collected are used as a raw material.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a monoclonal antibody against human glucocorticoid receptor and its use.

The present invention also relates to a method for quantifying and detecting glucocorticoid receptors present in human body fluids, tissues, and cells.

It is well known that accurate measurement of glucocorticoid receptors is an important factor when performing steroid therapy.

Since the monoclonal antibody against human glucocorticoid receptor of the present invention can quantify glucocorticoid receptor, it will greatly contribute to the medical field.

(Prior Art) It is generally well known that glucocorticoid receptors exist in small amounts in many normal tissues and regulate cell proliferation.

However, it is also known that in certain hematopoietic tumor cases, glucocorticoid receptors in tumor cells are significantly increased.

When the amount of glucocorticoid receptors in the body increases in this way, the sensitivity to steroid drugs increases, making steroid therapy effective.

In general, steroid therapy is widely applied to diseases of organs such as the lungs, liver, and kidneys, but it often causes strong side effects, so it is currently performed with extreme caution.

Furthermore, in steroid therapy, glucocorticoid receptors are quantified and appropriate drug dosages are determined accordingly to enhance therapeutic effects and reduce side effects.

Traditional glucocorticoid receptor measurements are performed using radioisotope-labeled steroids (glucocorticoids).
It was a complicated method using

(Problems to be Solved by the Invention) Measurement of glucocorticoid receptors using radioisotope-labeled steroids is not common because there are significant limitations in equipment, measurement sites, etc. due to the radioisotope.

Generally, the content of glucocorticoid receptors in human tissues or cells is extremely low, and measuring this requires antibodies with high specificity and reactivity; however, glucocorticoid receptors are unstable and cannot be purified. Because of the difficulty in producing anti-human glucocorticoid receptor monoclonal antibodies, a sufficient amount of human glucocorticoid receptor as an antigen has not been obtained, and therefore, anti-human glucocorticoid receptor antibodies are also unknown. It wasn't.

(Means for Solving the Problems) In order to solve these problems, the present inventors have conducted extensive research and have made human glucocorticoid receptors purified from human leukemia cells into a single cell strain into antigens. A hybridoma cell line obtained by immunizing an animal and fusing that animal's lymphocytes with myeloma cells.

The aim is to produce an anti-human glucocorticoid receptor monoclonal antibody, to be able to easily and accurately quantify human glucocorticoid receptor using an enzyme immunoassay using this antibody, and to identify glucocorticoid receptor-positive cells using an immunohistochemical method. The present invention was completed by discovering that detection can be performed with high sensitivity.

The present invention relates to monoclonal antibodies directed against human glucocorticoid receptors.

Further, the present invention is a method for quantifying human glucocorticoid receptor, which is characterized by quantifying human glucocorticoid receptor by enzyme immunoassay using a monoclonal antibody against human glucocorticoid receptor.

Further, the present invention is a method for detecting human glucocorticoid receptor, which comprises detecting human glucocorticoid receptor-positive cells by an immunohistochemical method using a monoclonal antibody against human glucocorticoid receptor.

In producing the monoclonal antibody of the present invention, first, a human glucocorticoid receptor serving as an antigen is prepared.

To prepare human glucofluticoid receptor, a human leukemia cell line containing glucocorticoid receptor is cultured in large quantities, and the collected cells are used as a material. Conventionally, purification of glucocorticoid receptors has been carried out using, for example, Journal of Biological Chemistry, Vol. 254, 92.
There is a method described on page 84, but perhaps because this method uses rat liver as the material, glucocorticoid receptors of sufficient purity to be used as an antigen cannot be obtained, and blood antibody titers are low even after immunizing animals. It became clear that there was almost no increase in

In the present invention, in order to obtain a highly pure human glucocorticoid receptor, a gel is prepared in which a glucocorticoid, for example, deoxycorticosterone, is bound to Sepharose, and an extract made from the human leukemia cell line is applied to the gel. Mix and shake to adsorb glucocorticoid receptors. Furthermore, the conjugate of glucocorticoid receptor and glucocorticoid is converted into a DNA-bound form by heating. After washing, an excess amount of glucocorticoid, such as triamcinolone acetonide, is added to selectively elute glucocorticoid receptors, and this eluate is applied to a DNA-cellulose column to obtain highly pure human glucocorticoid receptors.

It became possible to sufficiently immunize animals for the first time by using human glucocorticoids purified in this way as antigens.

A mammal, preferably a mouse or a rat, is immunized with the antigen obtained here; for example, when using a mouse, 5 to 1
O week old is preferable, human glucocorticoid receptor is 0.001-1 mg per mouse,
Preferably, 1 to 10 μg is administered mixed well with an adjuvant, such as Freund's complete adjuvant.

Administration is carried out by subcutaneous injection, intravenous injection, intraperitoneal injection, etc. Thereafter, every 1 to 3 weeks, the mixture is thoroughly mixed with an adjuvant, such as Freund's incomplete adjuvant, and administered subcutaneously, intravenously, intraperitoneally, etc. 3 to 5 times to sufficiently immunize.

From animals thus immunized, 2 to 4
After a day, lymphocytes are collected. Lung for lymphocyte preparation;
Lymph nodes, peripheral blood, etc. are used.

These lymphocytes are fused with myeloma cells.

Myeloma cells are cells that lack specific enzymes.

For example mouse mini o-7P3-X63-Ag8-11
(P3-Ul).

P3-X63-Ag8-653 (X63-653), P
3-NSI-1-Ag4-1 (MS-1), rat myeloma 210-RCY3-Ag123, and the like are preferred.

Cell fusion can be performed by the method of Keller and Milstein (Nature 256.495-497.1975).

After completion of cell fusion, hybridomas are selected using a selective medium, such as HAT (hypoxanthine-aminopterin-thymidine) medium.

Hybridomas producing antibodies against human glucocorticoid receptors are searched for by the enzyme immunoassay described below and reactivity with tritium-labeled glucocorticoid-human glucocorticoid receptor complexes. By repeating cloning by limiting dilution 2 to 4 times using this hybridoma, a large number of hybridoma cell lines that produce monoclonal antibodies against human glucocorticoid receptors can be obtained.

The monoclonal antibody-producing hybridoma cell line thus obtained can be intraperitoneally inoculated into a syngeneic animal, allowed to proliferate, and then the ascites fluid collected or cultured in an incubator to obtain the desired monoclonal antibody. .

The antibody thus obtained can be purified and used if necessary. That is, it can be purified using methods commonly applied to proteins, such as ammonium sulfate fractionation, ion exchanger, gel filtration, and affinity chromatography.

Since the monoclonal antibody of the present invention specifically reacts with human glucocorticoid receptors, it can be used for quantifying glucocorticoid receptors contained in human tissues or cells by enzyme immunoassay and for detection by immunohistochemical methods. be able to.

[Enzyme immunoassay]

Pour 50 μQ/well of purified human glucocorticoid receptor (0.1-1 μg/Otori Q) into a 96-well plate for ELISA (manufactured by Nunc), leave it at 4°C for about 5 hours, and apply it to the bottom of the plate hole. Let it absorb. O, 10mM phosphate buffer containing 15M sodium chloride (P
After washing three times with BS, ρn 7 , 6 ), each formula was filled with PBS containing 1% bovine serum albumin (BSA) and allowed to stand at room temperature for 1 hour, and the remaining portion was completely coated with BSA. After discarding the solution for each formula, wash with PBS, add 50μρ/well of hybridoma culture supernatant, and
Leave overnight at °C. After washing with PBS, biotin-conjugated anti-mouse IgG (Cubbell 11) was used as a secondary antibody.
Dispense 50 μQ/6 aliquots of a 300-fold dilution of the solution and leave at room temperature for 3 hours. After washing with PBS, 50 μQ/6 portions of a 500-fold diluted solution of streptavidin-conjugated peroxidase (Zymet Co., Ltd. 111) are dispensed and left at room temperature for 1 hour.

After washing with PBS, 2,2-azinodi(3-ethylbenzthiozolinesulfonic acid) (ABTS) solution (manufactured by Boehringer) (contains 1μQ/■Q hydrogen peroxide solution)
Dispense 50μQ/6 portions and leave at room temperature for 10 to 30 minutes.
Measured at 414n■. When searching for hybridomas, it is sufficient to select strains whose supernatant fluid shows high measured values.

The present invention will be specifically explained below using examples.

(1) Purification of human glucocorticoid receptor 1010 cells of human leukemia cell line Na1m-18 cultured in RPMI-1640 medium containing 5% tallow serum (Fe2)
After collecting and washing with PBS, 5% glycerol, 1
■Add 50mQ of 10mM Tris buffer (TGNP buffer, pH 8,0) containing M2-mercaptoethanol and 0.01mM phenylmethylsulfonyl fluoride, suspend, sonicate, and then TGMP containing 1% Triton X-1oo. Buffer solution: 50 fl ml of buffer solution, left at 0°C for 1 hour, and centrifuged at 1105000X to obtain a crude extract.

50 Ilf of the crude extract was dialyzed against TGMP II fi solution at 4°C overnight.

Mix with deoxycorticosterone-Sepharose gel 10a+12 prepared in advance and equilibrated with the same buffer solution,
Shake gently overnight at ℃. After being left at 25°C for 1 hour, the gel was thoroughly washed with TGNP buffer.

The gel was packed into a column, and the column was thoroughly washed with the same buffer containing 1M sodium chloride.

Triamcinolone acetonide (Sigma) at 1 x 10-
Elution was performed with TGNP buffer containing 'M. 4 eluate
The mixture was dialyzed against TGNP buffer overnight at °C and applied to a DNA-cellulose column (Pharmacia). Wash the column thoroughly with TGMP buffer, elute with the same buffer containing 1M sodium chloride, and dialyze against TGMP buffer.
A purified product was obtained.

[Method for producing deoxycorticosterone-Sepharose]

150 mg of AH-Sepharose 4B (Pharmacia) was added to Gel 10+aQ, which was suspended in 70% dioxane.
20ml of a solution of deoxyfluticosterone-21-hemisuccinate (manufactured by Sigma) dissolved in 70% dioxane
Q, as well as 1-ethyl-3-( at a concentration of 0.2 g/+d
3-(dimethylamino)propyl]carbodiimide (Sigma) aqueous solution 0.8a+Q was mixed and reacted by shaking slowly at room temperature overnight. 100% dioxane, 8
The gel was thoroughly washed with 0% ethanol and then distilled water to prepare a deoxycorticosterone-Sepharose gel.

(2) Preparation of immunized mouse tile cells The human glucocol obtained in (1) above was applied to 5-week-old Ba1b/c female mice (obtained from Charles River Japan).

The animals were immunized by subcutaneously administering ticoid receptor together with Freund's complete adjuvant at a rate of 1 μg per animal.

Human glucocorticoid receptor 1 every 2 weeks thereafter
A booster immunization was performed by subcutaneously administering μg/mouse. At the 6th time, 0.5 μg/mouse of human glucocorticoid receptor was intravenously injected for boosting.

Four days later, the lungs were removed, loosened in RPMI-1640 medium, suspended, and washed to prepare lung cells for fusion.

(3) Preparation of mouse myeloma cells (myeloma) Mouse myeloma cells N5-1 were cultured in RPMI-1640 medium,
Cells in logarithmic growth phase were collected and used as the parent strain for fusion.

(4) Mix the lung cells (2 x 10' cells) obtained in cell fusion (2) and (3) with the mouse myeloma cells (2 x 10' cells), centrifuge, discard the supernatant, and loosen the pellet well. , RPMI-1640 medium containing 45% polyethylene glycol 6000 i
n+R was gradually added over 1 minute with stirring. After allowing the mixture to stand for 7 minutes, RPMI-1640 medium 20o+R was gradually added dropwise while stirring. Discard the supernatant polyethylene glycol solution by centrifugation, suspend the pellet by adding RPMI-1640 medium containing 20% FC5, dispense it into a 96-well culture plate at 0.1 mR/well, and add 5% CO, 3
The cells were cultured at 7°C. After 24 hours, each culture plate was supplemented with IIAT medium (100 μN hypoxanthine, 0.
4μ aminopterin, 16μ athymidine, 20% FC
Add 0.1 mQ of RPMI-1640 medium containing 3). Thereafter, half of the culture medium was discarded every 24 hours for 3 days, fresh HAT medium was added, and the culture was continued for 10 days.

(5) Preparation and cloning of hybridomas For wells in which growth of hybridomas was observed, half of the culture solution was replaced with IT medium (HAT medium with aminopterin removed) and culture was continued.

A portion of the culture supernatant from the well where hybridoma growth was observed was taken, and the amount of anti-human glucocorticoid receptor antibody produced was measured by the enzyme immunoassay method described above. For hybridomas in wells in which production of anti-human glucocorticoid receptor antibodies was observed, the culture solution volume was increased to 2 mQ, and culture was continued. When the cells have grown to approximately 1X10' cells/company, the culture supernatant is collected, and the reaction with human glucocorticoid receptors is confirmed by Western blotting, and
We investigated the response to complexes of glucocorticoids and human glucocorticoid receptors. Regarding the latter, 2
Antibodies purified from the culture supernatant of mQ using protein A-Sepharose (Biorat) or lentil lectin-Sepharose (Pharmacia) were bound to activated Sepharose 4B (Pharmacia) to form an antibody column. The pre-prepared tritium-labeled triamcinolone acetonide (Amajam Co. 1)-human glucocorticoid receptor complex was passed through this column and washed, followed by O, IN.
The radioactivity of the solution eluted with sodium hydroxide was measured.

Hybridomas in the wells that were positive by the above measurement method were selected and cloned using the limiting dilution method to produce clone 1, which produced anti-human glucocorticoid receptor antibodies.
Two strains (h-1 to h-12) were selected.

(6) Purification of monoclonal antibodies The 12 hybridoma strains obtained in (5) above were cultured in serum-free medium A.
Cultured in SF103 (manufactured by Ajinomoto Co., Ltd.) at 50 mR each.
The culture at a cell concentration of 1X10'/IIQ was centrifuged, and the supernatant was applied to protein A-agarose (Bio-Rad) or lentil lectin-Sepharose (Pharmacia). After elution with 0.0 citrate buffer and PBS containing 10% methyl-α-D-mannopyranoside in the latter case, each purified monoclonal antibody (hLG-1 to hLG-12) was obtained by dialysis against PBS.

(7) Specificity of monoclonal antibody The specificity of the monoclonal antibody obtained in (6) above was confirmed using the Western blot method.

[Confirmation of specificity by Western blot method] 0.1%
A crude extract obtained from cells of the human glucocorticoid receptor-positive human leukemia cell line Na1m-18 was electrophoresed in a 10% polyacrylamide gel containing SDS and then containing 20% methanol. 25
Proteins were electrotransferred from polyacrylamide gels to nitrocellulose paper in 11 IM trishydroxyaminomethane-192 mM glycine buffer (Pi 48.3). Nitrocellulose paper with 1% BSA
- Soaked in PBS at room temperature for 1 hour to coat. Next, each monoclonal antibody was reacted with each at 4°C overnight, washed,
peroxidase-labeled anti-mouse digcO++L) (
A 100-fold diluted solution (manufactured by Kappel) was reacted at room temperature for about 2 hours. After washing, 4-chloro-1-naphthol (manufactured by Seikagaku Corporation) 0.5+*g/mQ.

A peroxidase substrate solution containing 0.02% hydrogen peroxide solution was reacted at room temperature for 30 minutes, and the colored protein band was examined.

All of the 12 monoclonal antibodies obtained in (6) above are human glucocorticoid receptor molecules ji920.
It specifically reacted only with the protein matching 00.

Example 2 Amount of human glucocorticoid receptor by enzyme-linked immunosorbent assay Each monoclonal antibody obtained in (6) above was labeled with biotin to serve as a secondary antibody, and an unlabeled monoclonal antibody was adsorbed to the same phase as the primary antibody. Human glucocorticoid receptor was quantified using an enzyme-linked immunosorbent assay using the sandwich method.

The biotin labeling method for monoclonal antibodies is explained below. A monoclonal antibody-PBS solution prepared to a concentration of 60.1B/mfl was prepared using IIIQtr0. After dialysis against IM sodium bicarbonate aqueous solution IQ overnight at 4°C,
After adding 0 μQ of biotinyl-N-hydroxysufushiimide solution (mg/aQ dimethyl sulfoxide) and allowing it to stand at room temperature for about 4 hours, it was dialyzed against PBS containing 0.1% sodium azide overnight at 4°C. , a biotin-labeled monoclonal antibody was obtained.

A method for preparing a sample containing human glucocorticoid receptors will be described below.

Cells of 6 types of human leukemia cell lines and 4 types of human liver cancer cell lines were cultured in 10% FC5-RPM11640 medium, centrifuged to collect cells, and washed to a concentration of 10 cells/cell.
The pellet was suspended in PBS so that it became IIIQ, and the cytoplasmic components obtained by ultrasonic disruption were used as a sample. The amount of glucocorticoid receptor contained in each sample was determined by radioreceptor assay. The measurement method will be explained below.

After washing the cultured cells, l? Cells are suspended in PMI-1640 solution at a concentration of 2X]O' cells/IIIQ, tritium-labeled triamcinolone acetonide is added to a concentration of 20++M, and the cells are incubated at 25°C for 2 hours. On the other hand, in order to determine non-specific binding, tritium-labeled triamcinolone acetonide and 10-'H unlabeled triamcinolone acetonide are added and incubated in parallel. After incubation, PBS at 4°C.
Cell components are collected by repeated washing and centrifugation, and the incorporated radioactivity is measured.

The added tritium-labeled triamcinolone acetonite binds to almost all of the lyabuter. The difference between the binding at this time and the non-specific binding measured by adding a large amount of unlabeled triamcinolone acetonide is the specific binding and becomes the receptor amount. The amount of glucocorticoid receptors in each sample obtained by this method ranged from 1,000 to 100,000/cell.

The method and results of measuring glucocorticoid receptors in each sample using the monoclonal antibody will be described below.

Purified monoclonal antibody hLG-1 at 5μg/mQ
Adjust the concentration to 50μ in a 96-well ELISA plate.
The antibody was dispensed into Q/hole and left at 4°C for 3 hours to coat the bottom of the hole.

Next, each formula was filled with 1% BSA-PBS and left to stand at room temperature for 1 hour to coat the unadsorbed portions. After thorough washing with PBS, each sample was added at 50 μQ/well, left standing at room temperature for 3 hours, and then washed with PBS. continue.

Biotin-labeled monoclonal antibody hLG-2 adjusted to a concentration of 5 μg/rnQ was dispensed at 50 μQ/well and left at room temperature for 2 hours. After washing with PBS, a 500-fold diluted solution of peroxidase-labeled streptavidin (Zymet) was dispensed at 50 μQ/well and allowed to stand for 1 hour.

After washing with PBS, ABTS solution (400 μg/aQ,
50μQ/containing 1μQ/IIIfl of hydrogen peroxide solution
The absorbance was measured at 414 nm after color development for 30 minutes at room temperature. The results are shown in FIG.

A similar test was conducted using purified monoclonal antibody hLG-3 and biotin-labeled monoclonal antibody hLG-4. The results are shown in Figure 2.

From Figures 1 and 2, a correlation was observed between the values measured by the measurement method using the monoclonal antibody and the number of receptors measured by the conventional method5. It was confirmed that it can be used for quantitative determination.

A method for preparing a cell sample containing human glucocorticoids will be described below.

Human leukemia-derived cell line 5 used in Example 1 (1) above
10% FC5-
After culturing in 1'l PMII640 medium, collecting cells by centrifugation, and washing with PBS, the cell concentration was approximately 1 x 10.
The pellet was suspended in PBS at r' cells/IIIQ and used as a cell sample.

Each cell sample was subjected to a cell collection centrifuge (Cytospin ■ type, manufactured by Shanton), and the cells were smeared onto a glass slide. The smear was treated with Bouin's fixative (saturated picric acid 1
5: formalin 5: acetic acid 1) overnight at 4°C. P
After washing with BS, each monoclonal antibody obtained in Example 1 (6) was adjusted to 10 μg/mQ and reacted overnight at 4°C in a humid chamber. After washing with PBS, anti-mouse IgG goat Antibodies (Cubbell, 20-fold dilution) were allowed to react in a humid box for 2 hours at room temperature. Next, wash with PBS,
A peroxidase-anti-peroxidase mouse antibody conjugate (Cubbell, diluted 50 times) was allowed to react in a humid chamber at room temperature for 30 minutes. After washing with PBS, a 50mMh lysate saline buffer containing 0.3a+g/ml diaminobenzidine hydrochloride (manufactured by Sigma) and 0.03% hydrogen peroxide was reacted in a humid chamber for 30 minutes at room temperature. . After washing with distilled water, glycerin was placed on the surface, a cover glass was placed on the surface, and the surface was examined using a microscope. In the same manner, normal mouse immunoglobulin (manufactured by Kappel) was used as a control instead of the monoclonal antibody and examined under a microscope. The difference in color development between the control and the sample was defined as the color development specific to human glucocorticoid receptors. All 12 monoclonal antibodies exhibited specific color development in cell samples containing 4,000 or more receptors/cell, confirming that these monoclonal antibodies can be used to detect human glucocorticoid receptor-positive cells by immunohistochemical methods. . The number of glucocorticoid receptors in human normal lymphocytes is reported to be around 3000 per cell (Journal.
of Immunology, vol. 118, p. 1977, 197
7 years).

The results are shown in Table 1.

[Brief explanation of the drawing]

Figure 1 shows the number of glucocorticoid receptors in 1000
Using a large number of samples adjusted to ~100,000 cells/cell,
Figure 2 shows the same number of receptors measured using monoclonal antibody hLG-1 as the primary antibody and biotin-labeled monoclonal antibody hLG-2 as the secondary antibody. year,
It is a figure in which the number of same receptors was measured using monoclonal antibody hLG-4 as a secondary antibody. Agent Patent attorney 1) Parent Male

Claims (3)

[Claims] (1) Monoclonal antibody against human glucocorticoid receptor. (2) A method for quantifying human glucocorticoid receptor, which comprises quantifying human glucocorticoid receptor by enzyme immunoassay using a monoclonal antibody against human glucocorticoid receptor. (3) A method for detecting human glucocorticoid receptor, which comprises detecting human glucocorticoid receptor-positive cells by an immunohistochemical method using a monoclonal antibody against human glucocorticoid receptor.