JPH08277300A - Monoclonal antibody against anti-ribonucleotide reductase r2 subunit - Google Patents

Abstract

PURPOSE: To obtain a new antibody specifically reacting with a ribonucleotide reductase(RNR) R2 subunit, having a RNR activity-inhibiting action, and capable of detecting the RNR for cell cycle markers on the diagnosis of tissues, etc. CONSTITUTION: The C-terminal peptide of a human ribonucleotide reductase(RNR) R2 subunit having an amino acid sequence of the formula is synthesized, and bound to keyhole limpet hemocyanin(KLH). The produced conjugate is administered as an immunogen into a rat to immunize the rat. After the immunization is finished, splenic cells are collected from the immunized rat, fused with myeloma cells, and subsequently selectively cultured in a HAT culture medium. The obtained hybridoma cells are screened to select an antibody- producing strain. The stain is cloned and subsequently cultured to obtain the subject new antibody specifically reacting with the human RNR R2 subunit, having an action for inhibiting the activity of the RNR and useful as a cell cycle marker, etc., on the diagnosis of tissues, the diagnosis of cells, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a ribonucleotide reductase useful as a cell cycle marker or a proliferation marker in tissue diagnosis and cytodiagnosis, and a monoclonal antibody that specifically reacts with the R2 subunit, and a ribonucleotide reductase using the antibody. It relates to a method for immunological detection of R2 subunit.

[0002]

2. Description of the Related Art Ribonucleotide reductase (hereinafter referred to as
Abbreviated as RNR. ) Is a deoxyribonucleoside 2 that reduces ribonucleoside 2 phosphate and becomes a material for DNA.
It is an enzyme that produces phosphoric acid. RNR activity is strongly correlated with cell growth rate, and the intracellular pool of deoxyribonucleotides is small.
Is responsible for the rate-determining step of DNA synthesis and is believed to be involved in its control (Annu. Rev. Biochem., 48 , 133-15).
8,1979).

Therefore, it is considered that RNR can be used as a cell cycle marker or a proliferation marker. In fact, it is known that the RNR activity increases / decreases depending on the cell cycle and is low in the G0 / G1 period and highest in the S period. RNR is R1 which has no activity by itself.
It is composed of two subunits, R and R2.
The expression level of 1 subunit is always constant and sufficient.
It has been reported that R activity is controlled by the expression level of R2 subunit that increases and decreases in a cell cycle-dependent manner [JBC, 256 ( 18), 9436-9440, 1981]. Also,
RNR activity has been reported to be higher in human tumor tissue than in normal tissue (Life Science , 28, 1007-1014 , 198).
1), RNR can be used as a tumor marker or a tumor growth marker.

However, the conventional method for measuring RNR activity (Analytical Biochemistry, 34 , 123-130, 1970) is complicated and unsuitable for the purpose of examining the localization in tissues, so that it is specific and simple. Various detection methods are desired. R1 is a monoclonal antibody against RNR.
Antibodies that react with subunits and inhibit RNR activity are known [Acta Chem. Scand. , B36 (5), 343, 198].
2]. Monoclonal antibodies that react with the R2 subunit are known [The EMBO Journal, 7 (6), 161.
5,1988], but the monoclonal antibody does not have an action of inhibiting RNR activity.

[0005]

The object of the present invention is a monoclonal antibody which reacts specifically with the R2 subunit of RNR, which is useful as a cell cycle marker or a proliferation marker, and which has the property of inhibiting RNR activity. To provide an antibody. The monoclonal antibody of the present invention has a neutralizing activity against RNR,
It is possible to inhibit RNR activity in a concentration-dependent manner. Therefore, the monoclonal antibody of the present invention is effective not only for immunologically detecting the R2 subunit of RNR in cells and tissues but also for studying the intracellular role of RNR and biological function, and You can obtain new knowledge about basic research.

[0006]

The present inventors have found that human RN
A C-terminal peptide of R2 subunit of R is used as an immunogen to prepare a hybridoma, and a monoclonal antibody-producing hybridoma strain that specifically reacts with the peptide is established.
The hybridoma was cultured in a medium or administered to an animal to cause ascites tumor, and a culture supernatant or ascites was collected to obtain a monoclonal antibody. When Western blotting and immunoprecipitation reaction were carried out using this, the monoclonal antibody reacts with a protein of 45 K daltons corresponding to the molecular weight of the R2 subunit of RNR, and the monoclonal antibody has an action of inhibiting RNR activity. I found that. Furthermore, immunological staining of cultured cells and various human tissues was performed using the monoclonal antibody, and it was found that the antibody can specifically detect RNR in cells and human tissues, thus completing the present invention. .

The present invention relates to a monoclonal antibody which reacts specifically with the R2 subunit of RNR and has an action of inhibiting RNR activity. As a specific example of the monoclonal antibody which reacts specifically with the ribonucleotide reductase R2 subunit of the present invention (hereinafter referred to as anti-RNR.R2 subunit monoclonal antibody), the monoclonal antibody KM1054 produced by the hybridoma cell line KM1054 can be mentioned. You can

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below. (1) Immunization of animals and preparation of antibody-producing cells Peptides used as antigens have the purpose of enhancing immunogenicity,
Carrier proteins such as keyhole limpet hemocyanin (hereinafter abbreviated as KLH) and bovine serum albumin (hereinafter abbreviated as BSA), glutaraldehyde, and N- (m-maleimidobenzoyloxy) succinimide (hereinafter MBS) It is abbreviated as ").

The R of human RNR having the amino acid sequence shown in SEQ ID NO: 1 in 3 to 20-week-old mice or rats
By immunizing a conjugate of the 2 subunit C-terminal peptide with the above-mentioned carrier protein, spleen, lymph node,
Antibody-producing cells are collected from peripheral blood or the like. Immunization is performed by subcutaneously or intravenously or intraperitoneally administering an appropriate adjuvant [eg Freund's complete adjuvant (Com
plete Freund's Adjuvant) or aluminum hydroxide gel and B. pertussis vaccine]
Together with the antigen. The administration of the antigen is performed 5 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with an antigen is examined by enzyme immunoassay [enzyme immunoassay (ELISA): published by Ikushosho 1976].

For the peptide used for immunization, mouse or rat whose serum shows a sufficient antibody titer is used as a source of antibody-producing cells. In using the fusion of antibody-producing cells and myeloma cells, for example, when spleen cells are used as antibody-producing cells, the spleen is extracted from the immunized mouse 3 to 7 days after the final administration of the antigen, and spleen cells are extracted. To collect. The spleen is cut into small pieces in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1,200 rpm, 5).
After 10 minutes), the supernatant is discarded, treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, and washed with MEM medium 3 times to obtain splenocytes for fusion.

(2) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mouse (BAL
B / c derived) myeloma cell line P3-X63Ag8-U1
(P3-U1) [Current Topics in Microbiology and Immunology (Current Topics
in Microbiology and Immunology) 81, 1-7 (1978)],
European Journal of Immunology (Euro
pean J. Immunology) 6 , 511-519 (1976)], SP2 /
0-Ag14 (SP-2) [Nature 276 ,
269-270 (1978)], P3-X63-Ag8653 (65
3) [Journal of Immunology (J. Immunolog
y) 123, 1548-1550 (1979)], P3-X63-Ag8
(X63) [Nature 256 , 495-497 (1975)
] Is used. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 x 10
^-5 M), gentamicin (10 μg / ml) and fetal calf serum (FCS) (CSL, 10%) added to a medium (hereinafter referred to as normal medium), and 8-azaguanine (15 μg / ml). Medium added),
3-4 days before cell fusion, subculture to normal medium, and
Ensure a cell number of × 10 ⁷ or more.

(3) Cell fusion The antibody-producing cells obtained in (1) and the myeloma cells obtained in (2) were mixed with MEM medium or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g). , Salt 7.6
Wash thoroughly with 5 g, distilled water 1 liter, pH 7.2),
After mixing so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuging (1,200 rpm, 5 minutes), discarding the supernatant, and thoroughly loosening the precipitated cell group , With stirring, at 37 ° C., 2 g of polyethylene glycol-1,000 (PEG-1,000), MEM2
mixed solution of 0.7 ml and dimethyl sulfoxide 0.7 ml.
2-1 ml / 10 ⁸ antibody-producing cells are added, and 1 to 2 ml of MEM medium is added every 1 to 2 minutes several times, and then MEM medium is added to bring the total volume to 50 ml. Centrifuge (9
(00 rpm, 5 minutes), after discarding the supernatant and gently loosening the cells, the cells are gently sucked by a measuring pipette and gently blown to remove the cells from HAT medium [hypoxanthine (10 ⁻⁴ M) in normal medium, thymidine (1. Medium ^{supplemented with} 5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M)] 100
Suspend in ml. 100 μl / well of this suspension is dispensed to a 96-well culture plate, and cultured at 37 ° C. in a 5% CO ₂ incubator for 7 to 14 days.

After culturing, a part of the culture supernatant is taken, and a hole that specifically reacts with the C-terminal peptide of the R2 subunit of human RNR is selected by the enzyme immunoassay described below. Then, cloning was repeated twice by the limiting dilution method [the first time was HT medium (medium in which aminopterin was removed from HAT medium), the second time was normal medium], and a stable and strong antibody titer was observed. Anti-RNR
-Select as a hybridoma strain producing R2 subunit monoclonal antibody. Specific examples of the hybridoma strain include hybridoma strains KM1054 and KM105.
6 and KM1060. The hybridoma strain KM1054 was transferred to FERM BP-4875 at the Institute of Biotechnology, National Institute of Biotechnology, on November 8, 1994.
Has been deposited as.

Enzyme-linked immunosorbent assay The C-terminal peptide of the R2 subunit of RNR used as an antigen uses a protein different from that used for immunization as a carrier protein, and is bound using a cross-linking agent such as glutaraldehyde or MBS. . Conjugate of C-terminal peptide of R2 subunit of human RNR and carrier protein 1 to 50 μg / ml, 10 to 100 μl / well 9
Dispense into a 6-well plate and let stand at 4 ° C. overnight to adsorb to the plate. After blocking with a BSA solution or the like, the hybridoma culture supernatant is used as the first antibody in an amount of 50 to 100 μm.
Dispense 1 / well, and react at room temperature for 2 hours or at 4 ° C. overnight. 0.05% Tween-in PBS or PBS
After washing with a solution containing 20 (hereinafter abbreviated as Tween-PBS), an anti-mouse immunoglobulin antibody or an anti-rat immunoglobulin antibody 1 labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like as a second antibody -50 μg / ml is dispensed at 50-100 μl / well, and the reaction is carried out at room temperature for 1-2 hours. After washing well,
The reaction depending on the labeling substance of the second antibody is carried out, and human RNR
The hole that specifically reacts with the C-terminal peptide of the R2 subunit of is selected as a hybridoma that produces an anti-RNR.R2 subunit monoclonal antibody.

(4) Preparation of Monoclonal Antibody Pristane-treated mice of 8 to 10 weeks of age treated with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane, intraperitoneally and raised for 2 weeks) or Nude mice are intraperitoneally injected with 2 × 10 ^{7 to} 5 × 10 ⁶ cells / mouse of hybridoma cells producing the anti-RNR / R2 subunit monoclonal antibody obtained in (3). 1
The hybridoma becomes ascites tumor in 0 to 21 days. Ascites fluid was collected from this mouse and centrifuged (3,000 rpm,
(5 minutes) to remove the solid content, and then salting out with 40 to 50% saturated ammonium sulfate to obtain a purified monoclonal antibody by the caprylic acid precipitation method, or a DEAE-Sepharose column, protein A column or Cellulofine GSL20.
It is passed through a column of 00 (manufactured by Seikagaku Corporation), and IgG or IgM fractions are collected to obtain a purified monoclonal antibody.

The subclass of the antibody is determined using a mouse monoclonal antibody typing kit (Zymet) or a rat monoclonal antibody typing kit (Nordic Immunology). The amount of protein is quantified by the Lowry method and the absorbance at 280 nm.

(5) Examination of Specificity of Monoclonal Antibody Using Western Blotting The reaction specificity of the anti-RNR.R2 subunit monoclonal antibody obtained in (4) was examined by the following Western blotting. First, a roughly purified RNR fraction is prepared from a human tumor cell line such as HelaS ₃ as follows.

A homogenate was prepared from human tumor cells,
Centrifuge and collect the supernatant. Final concentration is 0.65%
Streptomycin sulfate was added to give (w / v),
After stirring at 4 ° C for 30 minutes, centrifugation is performed to collect the supernatant.
Then add ammonium sulfate to a final concentration of 50% saturation,
After stirring at 4 ° C for 45 minutes, the precipitate is collected by centrifugation.
Tris-HCl buffer [50 mM Tris-HCl (Tris-HCl)
(pH 7.6), 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 2 mM dithiothreitol (DTT)], and dialyzed against the above buffer overnight, which is used as a crude RNR fraction.

The RNR crude purified fraction prepared as described above was fractionated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then a polyvinylidene difluoride membrane (hereinafter referred to as P
(Abbreviated as VDF membrane). PBS containing 1% bovine serum albumin (BSA) (hereinafter referred to as BSA
Abbreviated as a solution), and the anti-RNR / R2 subunit monoclonal antibodies 1 to 1 obtained in (4)
React with 0 μg / ml for 2 hours at room temperature or overnight at 4 ° C. After thoroughly washing with PBS or PBS-Tween, 1 to 50 μg / ml of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with biotin, enzyme, chemiluminescent substance, radioactive compound, etc. as a secondary antibody
Is dispensed at 50 to 100 μl / well and reacted at room temperature for 1 to 2 hours. After thorough washing, a reaction depending on the labeling substance of the second antibody is carried out, and it is confirmed that the anti-RNR.R2 subunit monoclonal antibody reacts with a protein of 45 K dalton which corresponds to the molecular weight of the R2 subunit of RNR.

(6) Examination of Specificity of Monoclonal Antibody Using Immunoprecipitation Reaction The reaction specificity of the anti-RNR.R2 subunit monoclonal antibody obtained in (4) was examined by the following immunoprecipitation reaction. Anti-RN obtained in (4) on EIA plate
Purify the R / R2 subunit monoclonal antibody and the control antibody from 50 to 200 μg with 5 to 50 μg / ml of the purified antibody.
l / well so that the plate is left to stand overnight at 4 ° C. for adsorption. After washing 3 times with PBS, add 300 mL of BSA solution.
Add μl / well and block. After adding C-terminal peptide (100 μg / ml) of RNR subunit of RNR or PBS at 50 μl / well and reacting at room temperature for 2 hours, He
A crudely purified fraction of RNR prepared from a human tumor cell line such as laS _{3 is} added at 100 μl / well and mixed, and the mixture is reacted at 4 ° C. overnight. After washing with Tween-PBS, 50 μl / well of SDS-PAGE sample buffer (5-fold concentration solution) is added, and the mixture is shaken at room temperature for 2 hours. After diluting 5 times with PBS,
After fractionation using SDS-PAGE at 20 μl / lane, blotting is performed on a PVDF membrane by a conventional method. After blocking with BSA solution, etc., the purified antibody of anti-RNR / R2 subunit monoclonal antibody 1 to 10 μg / ml is used at room temperature for 2
React for hours. After washing with Tween-PBS, 1 to 50 μg / ml of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with biotin, enzyme, chemiluminescent substance, radioactive compound, etc. as a second antibody is added, and the mixture is kept at room temperature for 1 to 2 hours. React. After washing well with PBS containing 0.02% Tween, a reaction depending on the labeling substance of the second antibody was performed, and the anti-RNR / R2 subunit monoclonal antibody was a 45 K dalton protein whose molecular weight was the same as that of the R2 subunit of RNR. Make sure to settle.

(7) Study on inhibition of RNR activity of monoclonal antibody The conversion amount of CDP to deoxyCDP by the R388 crudely purified fraction derived from P388 cells was used as an index of RNR activity, and the anti-RNR / R2 subunit monoclonal antibody was RNR.
Whether or not the activity can be inhibited is examined as follows.
The purified antibody of the anti-RNR / R2 subunit monoclonal antibody obtained in (4) is serially diluted and dispensed, and the P388 cell-derived RNR crude purified fraction prepared in the same manner as in (5) is mixed to obtain 4 React at 1-2C for 1-2 hours. A solution containing tritium-labeled CDP is added thereto, reacted at 37 ° C. for 30 minutes, and then boiled at 95 ° C. for 2 minutes to stop the reaction. Furthermore, a snake venom derived from Crotalus adamanteus was added, and the mixture was incubated at 37 ° C for 2 hours to convert nucleotides into nucleosides, which was then centrifuged to separate the supernatant from polyethyleneimine-borate-cellulose [polyethylenei
mine (PEI) -borate-cellulose] Spot on the sheet. Development solution (20 mM ammonium formate: ethanol =
6: 4), and after drying, the portions corresponding to nucleosides and deoxynucleosides are cut out and the radioactivity is measured using a liquid scintillation counter.

(8) Immune cell staining using monoclonal antibody As for floating cells, trypsin EDTA (0.1% trypsin and 0.02% E) was used for adherent cells.
After peeling the cells with PBS containing DTA), 1 × 10 ⁶
Dispense one by one. After washing with PBS, dispense 100% -500 μl of 4% paraformaldehyde prepared at the time of use,
Fix at room temperature for 30 minutes. After washing with PBS, add methanol to 100-5 to increase the antibody permeability of the cell membrane.
Dispense 00 μl each and treat at −20 ° C. for 2 minutes. PB
After washing with S, 100-500 μl of 10% normal human serum
Aliquot and block for 30 minutes at room temperature, then (4)
Purified anti-RNR / R2 subunit monoclonal antibody obtained in 1.
React for minutes. After washing with PBS, 1 to 50 μg / ml of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with a fluorescent dye such as FITC
Dispense each in an amount of 00 to 500 μl and allow the reaction to occur at 4 ° C. for 30 minutes while protected from light. After the reaction, the cells are thoroughly washed with PBS and then analyzed by a cell sorter.

In the case of carrying out double staining of antibody staining and DNA staining, after performing antibody staining as described above, 0.25
mg / ml RNase (Sigma) and 0.1% N
0.9 ml of PBS containing P40 per 1 × 10 ⁶ cells
In addition, the mixture is reacted at 37 ° C for 30 minutes. 500 μg
/ Ml PI (propium iodide) and 1.0% N
PBS containing P40 0.1 ml per 1 × 10 ⁶ cells
In addition, they are mixed, reacted for 20 minutes or more in ice-cooling, and analyzed by a cell sorter.

(9) Immunohistological Staining Using Monoclonal Antibody 1
Slice to ~ 5 micron thickness and immobilize on ovalbumin-coated slides. After deparaffinization with xylene, hydrophilicity is gradually made hydrophilic with alcohol-water. For frozen sections, fix in cold acetone for 20 minutes. Then, it is treated with methanol containing 0.3% hydrogen peroxide for 30 minutes to block the endogenous peroxidase. The section was washed with PBS, blocked in diluted horse normal serum for 20 minutes, and purified from the anti-RNR / R2 subunit monoclonal antibody obtained in (4). Antibody 1-10 μg /
The ml is reacted at 4 ° C. for 12 hours. After thoroughly washing, add 1 to 50 μg / ml of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with biotin, enzyme, chemiluminescent substance, radiation compound, etc. as the second antibody, and let react at room temperature for 30 minutes, then wash After that, the reaction according to the labeling substance of the second antibody is performed. After stopping the reaction in ice-cold, in the case of a tissue containing paraffin-fixed paraffin, nuclear staining is performed with hematoxylene, dehydration with alcohol-water and xylene, fixation with Canadian balsam, and observation under a microscope. In the case of a frozen section, it is fixed with glycerin-PBS and observed under a microscope.

Examples of the present invention will be shown below.

[0026]

【Example】

Example 1 (1) Preparation of Immunogen After synthesizing the C-terminal peptide of the R2 subunit of human RNR having the amino acid sequence shown in SEQ ID NO: 1, KLH (Calbiochem) was prepared as follows for the purpose of enhancing immunogenicity. (Manufactured by the company) was prepared as an immunogen.

10 mg / ml by dissolving KLH in PBS
1/10 volume of 25 mg / ml N-
(M-Maleimidobenzoyloxy) succinimide (MBS; manufactured by Nacalai Tesque, Inc.) was added dropwise and reacted for 30 minutes. Sephadex G- pre-equilibrated with PBS
R of human RNR obtained by dissolving 2.5 mg of KLH-MBS obtained by removing free MBS on a 25 column (Pharmacia) in 0.1 M sodium phosphate buffer (pH 7.0).
It was mixed with 1 mg of the C-terminal peptide of 2 subunits, and reacted by stirring at room temperature for 3 hours. After the reaction, what was dialyzed with PBS containing 0.5 M sodium chloride was used as an immunogen.

(2) Immunization of Animal and Preparation of Antibody-Producing Cells 100 μg of C-terminal peptide-KLH conjugate of R2 subunit of human RNR obtained in (1), 2 mg of aluminum gel and pertussis vaccine (Chiba Prefectural Serum Research Institute) 1 × 10 ⁹ cells were administered to 5 week-old female SD rats, and 2 weeks later, 100 μg of the above conjugate was administered once a week for a total of 4 times. Blood was collected from the fundus venous plexus, and the serum antibody titer thereof was examined by the enzyme immunoassay described below, and the spleen was extracted from the rat showing a sufficient antibody titer 3 days after the final immunization.

The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged (1,200 rp).
m 5 minutes), the supernatant was discarded, treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, washed 3 times with MEM medium, and used for cell fusion. . Enzyme Immunoassay Using the glutaraldehyde method, a C-terminal peptide cycloglobulin (THY) conjugate of human RNR / R2 subunit was prepared as an antigen for enzyme immunoassay as follows.

1 mg of the C-terminal peptide of human RNR / R2 subunit was dissolved in 0.1 M ammonium acetate buffer, and 5 mg of THY dissolved in the same buffer was added to make the total volume 1 ml. Under stirring, 540 μl of 0.02 M glutaraldehyde was added dropwise, and the mixture was reacted at room temperature for 5 hours with stirring. After the reaction, dialyzed overnight with PBS was used as an antigen.

A THY-human RNR.R prepared as described above was added to a 96-well EIA plate (manufactured by Greiner).
THY-SOX47-3 as a C-terminal peptide conjugate of 2 subunits or a control antigen (conjugate of SOX47-3 peptide and THY; SOX in SEQ ID NO: 2)
The amino acid sequence of the 47-3 peptide is shown. ) 10 μg
/ Ml was dispensed at 50 μl / well and left at 4 ° C. overnight to be adsorbed on the plate. After washing with PBS, 1% BSA-
PBS (100 μl / well) was added, and the reaction was performed at room temperature for 1 hour to block the remaining active groups. 1% BSA-PBS
The cells were discarded and the hybridoma culture supernatant or the immunized rat antiserum was dispensed at 50 μl / well and reacted for 2 hours. After washing with Tween-PBS, 50 peroxidase-labeled rabbit anti-rat immunoglobulin (manufactured by Dako) was used.
Add μl / well each and react at room temperature for 1 hour.
-After washing with PBS, color is developed using ABTS substrate solution [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] and the absorbance at OD415nm is measured by an absorptiometer (NJ2001; manufactured by Nippon Intermed). It was measured.

(3) Preparation of mouse myeloma cells The 8-azaguanine-resistant mouse myeloma cell line P3-U1 was cultured in a normal medium to secure 2 × 10 ⁷ or more cells at the time of cell fusion, and used as a parent strain for cell fusion. I served. (4) Preparation of hybridoma The mouse splenocytes obtained in (2) and the myeloma cells obtained in (3) were mixed at a ratio of 10: 1 and centrifuged (1,
(200 rpm, 5 minutes), the supernatant was discarded, and the precipitated cell group was thoroughly loosened, and then polyethylene glycol-1,000 (PEG-100) was added at 37 ° C with stirring.
0) A mixed solution of 2 g, 2 ml of MEM medium and 0.7 ml of dimethyl sulfoxide was added so as to be 0.2 to 1 ml per 1 × 10 ⁸ mouse splenocytes, and ME was added every 1 to 2 minutes.
After adding 1-2 ml of M medium several times, MEM medium was added so that the total amount became 50 ml. Centrifuge (900r
(pm, 5 minutes), the supernatant was discarded, the cells were loosely loosened, and then the cells were gently suspended in a 100 ml HAT medium by suctioning with a measuring pipette and sucking out.

This suspension was added to a 96-well culture plate at 10
Each 0 μl / well was dispensed, and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 10 to 14 days under CO ₂ 5%.
This culture supernatant was examined by the enzyme immunoassay described above, and the RNR R
A hole that specifically reacts with the 2-subunit C-terminal peptide was selected, the HT medium was replaced with a normal medium, and cloning was repeated twice to establish a hybridoma strain producing an anti-RNR.R2 subunit monoclonal antibody. As a result, as shown in FIG. 1, hybridoma strain KM10
54, KM1056 and KM1060 were selected. The antibody class of the monoclonal antibody produced by each hybridoma was determined by the enzyme immunoassay using a subcluster typing kit. The results are shown in Table 1.

[0034]

[Table 1]

(5) Purification of Monoclonal Antibody The hybridoma obtained in (4) was intraperitoneally injected into pristane-treated 8-week-old nude female mice (Balb / c) by 5 to 20 × 10 ⁶ cells / mouse. After 10 to 21 days, the hybridoma became ascites tumor. Ascites was collected from mice with accumulated ascites (1 to 8 ml / mouse), and centrifuged (3, 3).
After removing solids at 000 rpm for 5 minutes, the caprylic acid precipitation method (Antibodies-A Laboratory Manual, Cold)
Purified by Spring Harbor Laboratory, 1988) to obtain a purified monoclonal antibody.

(6) Examination of Specificity of Monoclonal Antibody by Western Blotting A crude RNR purified fraction was prepared from HelaS ₃ cells as follows. 1 × 10 ⁹ HelaS ₃ cells were added to a cell solubilization buffer [20 mM Hepes (pH 7.
6), 10 mM MgCl ₂ , 2 mM DTT, 1 mM
PMSF] was suspended in 30 ml and allowed to stand for 30 minutes under ice cooling to prepare a homogenate. 30 at 35,000 × g
Centrifuge for minutes and collect the supernatant to a final concentration of 0.65%
Streptomycin sulfate was added to give (w / v). After stirring at 4 ° C. for 30 minutes, centrifugation was performed at 13,000 × g for 20 minutes to collect the supernatant. Ammonium sulfate was added to the obtained supernatant so that the final concentration became 50%,
After stirring at 4 ° C for 45 minutes, centrifugation was performed at 13,000 xg for 20 minutes to obtain a precipitate. 4 ml of Tris-hydrochloric acid buffer solution [50 mM Tris-hydrochloric acid (pH 7.6), 0.1 m
M PMSF, 2 mM DTT] and further dialyzed overnight against the above buffer were used as the RNR crude purified fraction.

The RNR crude purified fraction was fractionated by SDS-PAGE at 40 μg / lane and 8 μg / lane, and then blotted on a PVDF membrane by a conventional method. 1% BSA-
Anti-human RN obtained in (5) after blocking with PBS
R / R2 subunit monoclonal antibody KM105
4, 10 μg / each of KM1056 and KM1060
ml and normal rat serum (1% BSA-
(Diluted 500 times with PBS) was reacted at room temperature for 2 hours or at 4 ° C. overnight. In addition, for each antibody, the C-terminal peptide of RNR (final concentration 10 μg /
ml) was reacted at room temperature for 1 hour, and the same reaction was performed. After thoroughly washing with Tween-PBS, peroxidase-labeled anti-rat immunoglobulin antibody (manufactured by Dako)
Was reacted at room temperature for 1 hour. After thoroughly washing with Tween-PBS, the liquid was thoroughly removed, and ECL reagent (Amersham)
Was added and reacted for 1 minute. After the reaction, the excess reagent was removed, and the film was exposed for 10 seconds to 2 minutes for detection. As a result, as shown in FIG. 2, KM1054, KM1
Both 056 and KM1060 reacted with a protein of 45 K daltons corresponding to the molecular weight of the RNR · R2 subunit, but the antibody previously reacted with the C-terminal peptide of the R2 subunit did not react.

(7) Examination of specificity of monoclonal antibody using immunoprecipitation reaction Anti-RNR / R2 subunit monoclonal antibodies KM1054, KM1056, KM106 were plated on EIA plates.
0 and rat IgG antibody KM1024 as control antibody
(50 μg / ml) was added at 200 μl / well, and the plate was coated overnight at 4 ° C. [In addition, although there is no description or deposit of KM1024, KM987, which is an identical antibody, can be used instead of KM1024. For KM987, refer to (JBC 268 (34) 2584
6, 1993). ] After washing 3 times with PBS,
300 μl / well of 1% BSA-PBS was added, and blocking was performed at room temperature for 1 hour. To this, C-terminal peptide (100 μg / ml) of RNR / R2 subunit or PBS was added.
After adding 0 μl / well and reacting at room temperature for 2 hours, a crudely purified fraction of RNR (7.67 mg / ml) was added at 100 μl / well and mixed, and reacted at 4 ° C. overnight. Tween-PB
After washing with S, 50 μl / well of SDS-PAGE sample buffer (25% 2-mercaptoethanol added, 5-fold concentration solution) was added and shaken at room temperature for 2 hours. P
After diluting 5 times with BS, SD at 20 μl / lane each
After fractionation by S-PAGE, it was blotted on a PVDF membrane by a conventional method. After blocking with PBS containing 1% BSA, antibody mixture (monoclonal antibody KM1054, K
M1056, KM1060 and KM1024 each 10
1% BSA-PBS solution containing μg / ml) at room temperature for 2
Allowed to react for hours. After washing with Tween-PBS, peroxidase-labeled rabbit anti-rat immunoglobulin (manufactured by Dako) was added, and the mixture was reacted at room temperature for 1 hour. Then, a reaction using an ECL reagent was performed in the same manner as in (6) for detection. It was As a result, as shown in FIG.
Only M1054 and KM1056 precipitated a protein of 45 K daltons corresponding to the molecular weight of the RNR / R2 subunit, but the antibody previously reacted with the R2 subunit C-terminal peptide did not react.

(8) Inhibitory Effect of RNR Activity of Monoclonal Antibody The conversion amount of CDP to deoxyCDP by the RNR crude purified fraction derived from P388 cells was used as an index of RNR activity, and the anti-RNR / R2 subunit monoclonal antibody was used as RNR.
Whether or not the activity could be inhibited was examined as follows.
The purified antibodies of the anti-RNR / R2 subunit monoclonal antibodies KM1054 and KM1056 obtained in (5) were serially diluted to 0.1 to 100 μg / ml, and then 50 μl
Was collected and the RNR crude purified fraction 0.75 derived from P388 cells was collected.
The mixture was mixed with mg / 50 μl and reacted at 4 ° C. for 1 to 2 hours. A substrate solution containing CDP labeled with tritium [5 mM ATP, 5 mM MgCl ₂ , 50 mM H
epes (pH 7.4), 5 mM DTT, 10 mM
NaF, 0.5 mM CDP, ³ H-CDP 1.25
μCi] (25 μl) was added, and the mixture was reacted at 37 ° C. for 30 minutes and then boiled at 95 ° C. for 2 minutes to stop the reaction. 20 mg / ml of Crotalus adamanteus snake venom 25
After adding μl and incubating at 37 ° C. for 2 hours to convert nucleotides into nucleosides, the reaction was stopped by boiling at 95 ° C. for 2 minutes. Centrifuge (5000 rpm,
10 minutes of the supernatant obtained after 2 minutes)
Borate-cellulose (polyethyleneimine (PEI) -borat
e-cellulose] sheet, develop in a developing buffer (20 mM ammonium formate: ethanol = 6: 4), dry, cut out the portions corresponding to nucleosides and deoxynucleosides, and measure the radioactivity with a liquid scintillation counter. did. As a result, as shown in FIG. 4, monoclonal antibodies KM1054 and KM10
All 56 inhibited RNR activity in a concentration-dependent manner.

Example 2 (1) Investigation of Reactivity of Monoclonal Antibody with Cancer Cells The reactivity of the monoclonal antibodies KM1054 and KM1056 obtained in Example 1 with various cancer cells was examined as follows. As a cancer cell, a human gastric cancer cell line NUG
C-4, human thyroid cancer cell line TPC-1, human lung cancer Ca
lu-1 (ATCC HTB54) and human leukemia cell line U937 (ATCC CRL1593) were used. Floating cells remain intact, trypsin ED for adherent cells
Peel off the cells with TA and dispense 1 x 10 ⁶ cells each
After washing with BS, 500 μl of 4% paraformaldehyde prepared at the time of use was dispensed and left standing at room temperature for 30 minutes for fixation. After washing with PBS, in order to increase the antibody permeability of the cell membrane, 500 μl of methanol was dispensed at 2 ° C at -20 ° C.
Processed for a minute. After washing with PBS, 500 μl of 10% normal human serum was dispensed and blocking was performed at room temperature for 30 minutes, followed by monoclonal antibodies KM1054 and KM1056.
Alternatively, 200 μl each of KM1024 (10 μg / ml) was added and reacted at room temperature for 30 minutes. After washing with PBS, F
ITC-labeled anti-rat immunoglobulin antibody (manufactured by Dako)
Was dispensed in 200 μl aliquots and reacted at 4 ° C. for 30 minutes while protected from light. After the reaction, the plate was washed well with PBS and then analyzed using a cell sorter [Epics Elite (manufactured by Coulter)]. As a result, as shown in FIG. 5, it was shown that both of the monoclonal antibodies KM1054 and KM1056 react with all cancer cell lines and can be used for detection of RNR widely existing in cancer cells.

(2) Examination of Reactivity of Monoclonal Antibody and Cell Cycle For the purpose of investigating the relationship between the reactivity of monoclonal antibodies KM1054 and KM1056 and the cell cycle, double staining of antibody staining and DNA staining was performed as follows. It was (A) Human T-cell lymphoma cell line CCRF-CEM
(ATCC CCL119), (B) Po from human peripheral blood
Lymphocytes collected by centrifugation using lymorphprep (Daiichi Kagaku) and OKT-3 [hybridoma OK
The culture supernatant of T-3 (ATCC CRL8001) was purified using a protein A column] and IL-2 (manufactured by Shionogi Pharmaceutical Co., Ltd.) were used to stimulate lymphocytes.

Monoclonal antibodies KM1054 and KM
After performing antibody staining in the same manner as (1) using 1024,
0.25 mg / ml RNase (manufactured by Sigma) and 0.
0 per 1 × 10 ⁶ cells in PBS containing 1% NP40.
9 ml was added and reacted at 37 ° C. for 30 minutes. 50 more
0 μg / ml propium iodide (PI) and 1.
0 1 × 10 ⁶ per cell of PBS containing 0% NP40.
Add 1 ml and mix, react for 20 minutes or more in ice-cooled,
It analyzed using the cell sorter [Epics elite (made by Coulter)]. The results are shown in FIGS. 6 and 7. As shown in FIG. 6, human T-cell lymphoma CCRF
-In CEM, the monoclonal antibody KM1054 is G1
Some cells in the phase became positive for cells in S phase, G2 phase, and M phase. In addition, as shown in FIG. 7, the monoclonal antibody KM1054 did not react at all with unstimulated peripheral blood lymphocytes, which is a cell population in the G0 phase, like the control antibody KM1024. In lymphocyte blasts in which lymphocytes started to proliferate synchronously using OKT-3 and IL-2, in addition to the G0 / G1 phase population, S phase, G phase
Appearance of populations in the 2nd and Mth stages was observed, but the control antibody KM1024 did not react with any of the populations, but the monoclonal antibody KM
1054 responded selectively to the S phase, G2 phase, and M phase populations with high DNA levels.

As described above, the monoclonal antibody KM1
The reactivity of 054 is low in the G0 / G1 phase,
2. Consistent with cell cycle-dependent expression of high RNR / R2 subunits in M phase, monoclonal antibody KM10
It was suggested that 54 can be used for detection of RNR useful as a cell cycle marker or a proliferation marker.

(3) Examination of cytodiagnosis using monoclonal antibody For the purpose of examining the application of the anti-RNR / R2 subunit monoclonal antibody to cytodiagnosis, ascites cells or pleural effusion cells were selected from 3 cases of cancerous pneumonia. 5 ml aliquots were taken and fixed with 95% alcohol. After the fixation, immunocyte staining with the monoclonal antibody KM1054 was performed in the same manner as in (1) except that alcohol was used instead of paraformaldehyde. As a result, as shown in Table 2, all the samples were positive. Further, similar to the result of (1) immunostaining using a cancer cell line, the reactivity of various cells was different.

[0045]

[Table 2]

(4) Immunohistochemical staining using monoclonal antibody 1
It was sliced to a thickness of ˜5 μm and fixed on a slide glass coated with ovalbumin. After deparaffinization with xylene, the mixture was hydrophilized stepwise with alcohol-water. In the case of frozen sections, they were fixed in cold acetone for 20 minutes. Then, the cells were treated with methanol containing 0.3% hydrogen peroxide for 30 minutes to block the endogenous peroxidase. The sections were washed with PBS, blocked in diluted horse normal serum for 20 minutes, and purified antibody of anti-RNR / R2 subunit monoclonal antibody KM1054 was added at 4 μg / ml.
The reaction was performed at 12 ° C for 12 hours. After washing well with PBS, 1 μg / ml biotin-labeled anti-rat immunoglobulin antibody (manufactured by Jackson Immunoresearch Lab) as a second antibody
100 μl of the reaction mixture was reacted at room temperature for 30 minutes, washed with PBS, added with avidin-labeled peroxidase (Nichirei), reacted at room temperature for 30 minutes, washed in water for 10 minutes, and then 0.01% hydrogen peroxide was added. Including 0.05%
It was reacted with diaminobenzidine for 1 minute. After stopping the reaction in cold water, nuclear staining was performed using hematoxylin, and in the case of formalin-fixed paraffin-embedded tissue, dehydrated with alcohol-water and xylene and then fixed with Canadian balsam,
I looked through a microscope. In the case of a frozen section, it was embedded in glycerin-PBS and observed under a microscope. As a result, as shown in Table 3, the monoclonal antibody KM1054 reacted with the cancer cells in all the samples in the 8 cases of breast cancer. However, it did not respond to the surrounding normal part and 4 cases of benign disease.

[0047]

[Table 3]

[0048]

INDUSTRIAL APPLICABILITY The present invention provides a monoclonal antibody having a function of specifically reacting with RNR useful as a cell cycle marker or a proliferation marker and having an action of inhibiting RNR activity. INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is useful for immunological detection of RNR in tissue diagnosis and cytodiagnosis, and analysis of biological function.

[0049]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: CysAsnSerPheThrLeuAspAlaAspPhe

SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: CysLeuSerProGlyGlnLysGlnLysGluGluProLysGluVa
lLys

[Brief description of drawings]

FIG. 1 shows the binding reactivity of anti-RNR / R2 subunit monoclonal antibodies KM1054, KM1056, and KM1060 to the C-terminal peptide of R2 subunit in the enzyme immunoassay.

FIG. 2 shows the reactivity of anti-RNR · R2 subunit monoclonal antibodies KM1054, KM1056, and KM1060 with a roughly purified RNR fraction derived from HelaS ₃ cells in Western blotting. In the figure, + indicates the reactivity when the antibody was added after the reaction with the C-terminal peptide of the RNR / R2 subunit in advance, and-represents the reactivity when the antibody was added as it was.

FIG. 3 shows the reactivity of anti-RNR · R2 subunit monoclonal antibodies KM1054, KM1056 and KM1060 with HelaS ₃ cell-derived RNR crude purified fraction in the immunoprecipitation reaction. In the figure, + means RNR / R in advance
After reacting with the C-terminal peptide of 2 subunits, reactivity is shown when an antibody is added, and-represents the reactivity when an antibody is added as it is.

FIG. 4 shows RNR inhibitory activities of anti-RNR / R2 subunit monoclonal antibodies KM1054 and KM1056.

FIG. 5 shows the results of cell reactivity analysis of the reactivity of anti-RNR / R2 subunit monoclonal antibodies KM1054 and KM1056 with various cancer cell lines by immunocyte staining.

FIG. 6: Human T-cell lymphoma cell line CCRF-CEM
The results obtained by analyzing the relationship between the reactivity of the anti-RNR / R2 subunit monoclonal antibody KM1054 and the cell cycle using a cell sorter are shown below.

FIG. 7 shows the results of a cell sorter analysis of the relationship between the reactivity of anti-RNR / R2 subunit monoclonal antibody KM1054 and the cell cycle using human peripheral blood lymphocytes and human peripheral blood lymphocyte blasts.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/577 9281-4B C12N 5/00 B // A61K 39/395 9162-4B 15/00 ZNAC (C12P 21/08 C12R 1:91) (72) Inventor Masami Okabe 30-5 Kamo, Mishima City, Shizuoka Prefecture (72) Inventor Yama ▲ ▼ Motoki 3-9-13 Nakamachi, Machida City, Tokyo

Claims (8)

[Claims] 1. A monoclonal antibody which reacts specifically with a ribonucleotide reductase R2 subunit and has an action of inhibiting the activity of ribonucleotide reductase. 2. The monoclonal antibody KM1054 according to claim 1, which is a rat monoclonal antibody belonging to the IgG2a subclass. 3. A hybridoma which produces the monoclonal antibody according to claim 1. 4. A hybridoma KM1054 (FERM BP-, which produces the monoclonal antibody according to claim 2.
4875). 5. A method for immunologically detecting a ribonucleotide reductase R2 subunit using the monoclonal antibody according to claim 1. 6. A method for detecting a ribonucleotide reductase R2 subunit by immunocytostaining using the monoclonal antibody according to claim 1. 7. A method for detecting a ribonucleotide reductase R2 subunit by immunohistostaining using the monoclonal antibody according to claim 1. 8. A method for diagnosing cancer of various human tissues using the monoclonal antibody according to claim 1.