JPH03108497A - Antibody against mouse interleukin 5 receptor - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody against mouse interleukin 5 receptor having specific reactivity to mouse interleukin 5 receptor. EXAMPLE:A reagent for simply detecting and measuring cell having mouse interleukin 5 receptor in high sensitivity and precision. USE:For example, mouse interleukin-5 (IL-5) responding first-term B cell T88-M strain is cultured in a culture medium containing IL-5 and then centrifuged to collect the cell and the collected cell is suspended in a buffer and homogenized and then centrifuged to afford a cell membrane fraction rich in mouse IL-5 receptor, which is then administered to wister type rat as an antigen to immunize the rat and after final immune, the spleen cell is collected and fused with a myeloma cell and the fused cell is cultured in HAT culture medium and then subjected to cloning and the resultant hybridoma cell is cultured to provide the monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to antibodies against mouse interleukin-5 (hereinafter abbreviated as IL-5) receptors, specifically antibodies against mouse IL-5 receptors.
This invention relates to a new monoclonal antibody against mouse IL-5 receptor that enables immunological purification, measurement, etc. of the receptor.

Prior Art IL-5 is a 47Kd protein secreted from T cells. IL-5 binds to receptors on the cell surface and promotes B cell proliferation and Ig production [Takatsu,
K,,Proc,Soc,Exp,Biol.

Med,, 188.243. 1988; Taka
Tsu, K.

et at, I mmunol, Rev.
102. 107° (1988)]. IL-5 has already been partially purified using a combination of various column chromatography techniques [Harada, N. et al.
J. Immunol.

134.3944. (1985)]. More recently, IL-
The cDNA of 5 was also isolated [K1nashi, T.
.

et al., Nature, 324.70.
(1986)] and by the generation of monoclonal antibodies against IL-5 [Harada, N., et al.
at, Proc, Natl.

Acad, Sci, USA, 84.4581° (19
87)], genetically engineered IL-5 is
Used in research on L-5 receptors. and! L
It has been revealed that there are two types of receptors for -5-sensitive cells: low affinity and high affinity [Mita
, S., et al., J. Exp.

Med, 168, 836° (1988)]. However, it has recently been discovered that IL-5 acts not only on B cells but also on other cell populations.
There are still many unknowns about its receptor and signal transduction mechanism. Furthermore, IL-5 is involved in hematopoiesis in emergency situations, and is also thought to be involved in biological defense by increasing the number of mast cells and eosinophils during infection with ticks and parasites. Therefore, antibodies against the IL-5 receptor are considered useful as a means for studying biological defense mechanisms.

Problems to be Solved by the Invention An object of the present invention is to provide an antibody against the mouse IL-5 receptor.

Another object of the present invention is to provide a method for detecting cells having mouse IL-5 receptor.

Another object of the present invention is to provide antibodies that can be used in research to elucidate the mechanism of immune abnormalities caused by IL-5 overreaction.

Another object of the present invention is to provide a method for producing the above antibody.

Means for Solving the Problems According to the present invention, a monoclonal antibody against the mouse IL-5 receptor, which is characterized by specifically reacting with the mouse IL-5 receptor, and a method for producing the same are provided.

Utilization of the antibody of the present invention provides an immunoassay method capable of detecting and measuring cells having the mouse IL-5 receptor with high sensitivity, high accuracy, and ease of use.

Furthermore, the antibodies of the present invention can be used in research centered on mice to elucidate the mechanism of immune abnormalities caused by IL-5 overreaction.

In addition, the antibody of the present invention can be used in mice! Since it is specific for the L-5 receptor, its use also provides a means for specific purification of mouse IL-5 receptor, for example by techniques such as affinity chromatography.

The antibody of the present invention can be produced using mouse IL-5 receptor as an immunizing antigen. More specifically, for example, fusion cells (hybr1d
oa+a), select a clone that produces the desired antibody that recognizes the mouse IL-5 receptor, and culture the clone.

Mouse I as an immunizing antigen used in the above method
The L-5 receptor is not particularly limited as long as it is a mouse It, -5 receptor, and may be a mouse cell line having a known IL-5 receptor, such as mouse IL-5 responsive early B cell T88-M.

Cell fractions such as mouse chronic B-cell leukemia cell BCLI-820 and mouse myeloma cell MOPC104E, or preparations of mouse IL-5 receptor purified from these cell fractions, mouse IL-5 produced using genetic recombination technology Any of receptors and peptides having partial amino acid sequences thereof may be used. In addition, the mammal to be immunized with the immunizing antigen in the above method is not particularly limited, but it is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and is generally selected such as rats. is advantageously used.

Immunization can be carried out by a conventional method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous, intradermal, subcutaneous, or intraperitoneal injection. More specifically, the immunogenic antigen,
Administer to test animals several times every 2 to 14 days, if desired in combination with a conventional adjuvant, for a total dose of approximately 100 to 50
It is preferable to adjust the amount to about 0 μg/rat. As the immunizing antigen, it is preferable to use spleen cells extracted about 3 days after the final administration.

Furthermore, as the mammalian plasmacytoma cell as the other parent cell to be fused with the above-mentioned immune cell, there are various known mammalian plasmacytoma cells, such as P3 (P3/X63-Ag8) [Nat
ure, 256, 495-497 (1975)],
p 3-U 1 [CurrentTopics 1
n Microbiology and Immu
no+ogy-81, 1-7 (1978) I N5-
1 [Eur, J.

I ma+uno1. , Coro, 511-519
(1976) Ko.

MP C-11 (Ce11.8.405-415 (19
76)), 5P210 [Nature, 276°26
9-270 (1978)], FO [J.

Iamunol, Meth, 35. 1 to 21 (
1980)], x63.6.5.3. [J.

Immunol, 123. 1548-1550 (1
979)], 8194 [J, Exp, Med,.

148.313-323 (1978)] and R210 in rats [Nature, 277. 131
-133 (1979) 1, etc. can be used.

The above-mentioned fusion reaction between immune cells and plasmacytoma cells can be carried out using known methods such as the method of Milstein et al. [Meth.

Enzymol, Vol. 73. pp3 (1
981) ] etc. More specifically, the above fusion reaction is carried out using conventional fusion promoters such as polyethylene glycol (PEG), Sendai virus (H
VJ), etc., in a conventional medium, and an adjuvant such as dimethyl sulfoxide may be added to the medium as necessary to further increase the fusion efficiency. The ratio of immune cells to plasmacytoma cells used is the same as in the usual method, for example, about 1 to 10% of immune cells to plasmacytoma cells.
It is normal to use about 10 times as much. As the medium for the fusion reaction, various media commonly used for the proliferation of plasmacytoma cells can be exemplified, such as RPM11640 medium, MEM medium, and other media commonly used for culturing this type of cells. It is recommended to remove serum supplements such as fetal bovine serum (Fe2). For fusion, predetermined amounts of the above immune cells and plasmacytoma cells are thoroughly mixed in the above medium, and 37
Heating it to about ℃ and adding a PEG solution, for example, an average molecular
000 to 6,000 to a normal medium of 30 to 60
This is done by adding and mixing at a concentration of %w/V. Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The resulting desired hybridoma is isolated by culturing it in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is subjected to the search for the desired antibody and single cloning by the usual limiting dilution method.

Search for target antibody-producing strains can be performed, for example, by ELISA method [Eng.
vall, E., Meth, Enzymol, 70
．． 419-439 (1980)], plaque method, spot method, agglutination reaction method, offtelony (Ouchter
lony) method, radioimmunoassay (RIA) method, etc., which are generally used for detecting antibodies ["Hybridoma method and monoclonal antibodies", R & Co., Ltd.
Published by D Planning, pp. 30-53, March 5, 1982], and further inhibits biological activity of IL-5, such as inhibition of IL-5-dependent proliferation of 788-M cells. A combination of the test and a binding test to IL-5 reactive cells using FAC8 or the like is performed. The above-mentioned immune antigen can be used for this search.

The thus obtained hybridoma producing the desired monoclonal antibody that recognizes the mouse IL-5 receptor can be subcultured in a conventional medium and can be stored for a long period of time in liquid nitrogen.

The desired antibody can be collected from the above hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a compatible mammal and allowing it to grow, and obtaining it as ascites. etc. will be adopted. The former method is suitable for obtaining highly purified antibodies, and the latter method is suitable for large-scale production of antibodies.

Furthermore, the antibody obtained as described above can be further purified by conventional means such as salting out, gel permeability, and affinity chromatography.

The monoclonal antibody of the present invention thus obtained has specific reactivity with mouse IL-5 receptor.

Furthermore, the antibody of the present invention is a type of antibody that has the activity of binding to cells having a mouse IL-5 receptor, and such antibodies are suitable for detecting cells having a mouse IL-5 receptor.

Effects of the Invention According to the present invention, a monoclonal antibody specific to the mouse IL-5 receptor is provided, and by using the antibody of the present invention, cells having the mouse IL-5 receptor can be detected with high sensitivity and precision. An immunoassay method that can be easily detected and measured is provided. Furthermore, a method for studying immune abnormalities caused by IL-5 overreaction is provided, mainly in mice.

EXAMPLES Examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 1) Preparation of mouse IL-5 receptor antigen IL-5-responsive early B cell T88-M strain was cultured in a medium containing 10 pM IL-5 (RPM11640 medium containing 5% FC8 and 50 μM β-mercaptoethanol). The cells were cultured at 37°C for 120 hours.

After culturing, T88-M cells (9X 109 cells) 4
The cells were collected by centrifugation at 00×g for 10 minutes at 4°C, and the cells were washed four times at 4°C with Hanks' solution.

Then 10mM Tris-HCρ (pH 7°2)/100
It was resuspended in buffer 100 consisting of OKIU/-aprotinin/1 mM phenylmethanesulfonyl fluoride (PMSF)/12 μM leupeptin/10 μM pepstatin A. After floating in ice water for 10 minutes, D ounce
Homogenize with an e-homogenizer (manufactured by Pyrex), add 10mM) Lis-HCΩ (pH 7,2
) 10.5M sucrose/1100III KCJ2/10
mmM MgCΩ2/2mMCaCΩ2 /100OK
IU/mA7 protinin/1mM PMS F/
An equal volume of buffer containing 12JIM leupeptin/10 μM pepstatin A was added. This cell suspension was added for 20 minutes.
The supernatant was centrifuged at 1,100,000X and 4°C for 90 minutes.

The thus obtained mouse IL-5 receptor-enriched cell membrane fraction was added to 20mM Hepes buffer (pH 7.2).
10.15M NaCΩ/10% glycerol/
450KIU/-Aprotinin/1mM PMSF1
5μM leupeptin 15μM pepstatin A10.05
Suspend in 15% of buffer containing NaN3 and incubate at -80 °C.
Saved with. Proteins were measured using the Bradford method [Bradf
ord, M, M,, Anal.

Biochem, 72,248. (1976)].

By each of the above operations, the IL-5 receptor content per protein was doubled compared to that at the beginning of the culture.

ii) Preparation of Antibody The membrane fraction rich in IL-5 receptor obtained above was used as an antigen for mouse IL-5 receptor. That is, 3 to 4 mg of the membrane-rich portion obtained by the above method (T88-M
(corresponding to approximately 1 x 108 cells) to complete F reu
The antigen was mixed with nd''S adjuvant and administered intramuscularly to Wistar rats.Thereafter, the antigen mixed with incomplete Freund's adjuvant was administered in the same manner every week, and 3 to 4 days after the final immunization, the phlegm cells were harvested. , according to the conventional method [Ha
rada, N, , etal, , Pro, Natl,
, Acad, Sci, USA, 84°4581,
(1987)]X63. The cells were fused with Ag8,653 myeloma cells. The fused cells were treated with 10% inactivated beef tallow serum, 100 U/uQ of penicillin, and 1 streptomycin.
00μg/mf2.2-mercaptoethanol 50μM
suspended in RPM11640 medium containing 24-well plate (L 1nbr.

#76-033-05. Flow Laborat
ories.

I nc, , Mclcan, V A ) to I
Add at a ratio of 106 cells /-/vel l, 3
The cells were cultured in a 7°C, 5% CO2 incubator.

After selecting hybridoma cells using HAT medium, the supernatant was screened using two different assay systems.

In the first test, hybridoma culture supernatant was used to biosynthetically label T88-M cells with 353-methionine.
The activity of competitively inhibiting the binding of 5 was measured. That is, 4
% bovine serum albumin (BSA) coated with 0.1% BSA.
Add T88-M cells (containing 5×10 5 cells) suspended in 50 μΩ of RPMI medium containing SA and 20 mM Hepes buffer. Next, 50 μρ of culture supernatant of each hybridoma was added to each well, and after culturing at 37°C for 1 hour, 1 μΩ of 358-IL-5 was added thereto to a final concentration of 200 pH at 37°C. They were washed by warming for 15 minutes. Non-specific binding was determined by simultaneously adding unlabeled IL-5 to a final concentration of 10 mM and measuring the radioactivity bound to the cells.

The second assay method used T88-M cells to determine whether each hybrid supernatant competitively inhibited the BCGFII activity of IL-5. This measurement method uses T88-
M cells were washed three times with RPM11640 medium and 1x10
5 cells/- were suspended in a 96-well microplate (Corning).

#25860), add various concentrations of hybridoma culture supernatant, and pre-culture for 30 minutes at room temperature. Thereafter, 5U/- of purified IL-5 was added to a final concentration of 1
0% and cultured for 24 hours at 37°C in a 5% CO2 incubator. End of culture 12
0.2 hours ago. 3H-methylthymidine (S
pe.

act, 5ci/mmol; Daiichi Rad
iochemicalCo, Ltd.) was added to each well. After culturing, use a multiple automated cell harvester (multiple automated cell harvester).
The cells were transferred onto a glass filter using a ted cell harvester), and the radioactivity bound to the cells was measured by liquid scintillation to detect a strain producing an antibody against the mouse interleukin-5 receptor of interest.

Cloning was repeated using the limiting dilution method to obtain the desired antibody-producing clone.

The clone (hybridoma producing the antibody of the present invention) was sent to the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
onoclonal anti-murlne IL
-5Receptor antibody prod
uclng hybridoma (H7)J, FERM P-110
19).

The antibody of the present invention obtained from this clone (this antibody “H7
) is shown below.

■ Antibody subclass Rat antibody subclass detection kit (Serotic (Sc
The subclass of the above antibody determined using (rotcc) was (IgG2a, ).

■ Antibody production level When the hybridoma reached its maximum cell density, the IgGffl in the culture supernatant was approximately 10 μg/ml.

■ After culturing molecular weight hybridomas intraperitoneally in mice, salting out ammonium sulfate and injecting chain-specific monoclonal antibodies (M
AR18,5) Binding affinity column allows Ig
It was purified to G2a.

After this, when 5DS-PAGE was performed and stained with Coomassie brilliant blue, no bands other than antibody (H7) were observed. The molecular weight of this antibody determined by 5DS-PAGE (the sum of the molecular weights of the heavy chain and the light chain is the molecular weight of the antibody) was approximately 160 Kd.

■ The 50% inhibition concentration measured by the following method is approximately 250p.
It was M.

Titer measurement method 788-M cells were washed three times with RPMI 1640 medium, suspended in IX 105 cells/ml), and placed in a 96-well microplate (Corning #2586).
0), and then add purified H7 antibody at various concentrations and preincubate for 30 minutes at room temperature. After that, I.L.
-5 was added to a final concentration of 20 pM, and 200 μΩ/
Make it a well. This was heated to 37℃ and 5% CO2.
The cells were cultured for 24 hours under these conditions. Then 0.2μCi/well
Add 1μ of 3H-methylthymidine to each well so that the
Ω was added and cultured for an additional 12 hours.

After culturing, the cells were collected using an autoharvester, and the radioactivity incorporated into the DNA of the proliferated cells was measured using a liquid scintillation counter.

When IL-5 was not added, radioactivity of 428 cpm was observed (Figure 1).

■ Dissociation constant Kd IL-5 expressed on the cell surface of T88-M cells
In order to determine the binding and dissociation constant Kd of H7 antibody to the receptor, 12J-labeled H7 antibody was prepared and S catch
As a result of ard analysis, Kd was calculated to be 5 to 10 nM.

■ Cross-reactivity Harada et al.'s method [Proc, Natl, Acad
, Sci.

USA84.4581-4585 (1987), IL-2-responsive strains (IL-2-dependent
t CTLL (MTH)), IL-3 responsive strain (
As a result of examining the cross-reactivity to FDC-Pi), I
There was no cross-reaction with L-2 and IL-3 responsive strains. Furthermore, BSF-1 assay [Howard, M., et.
al, J, Exp, Med, 155°914~
923. (1982)], the cross-reactivity with IL-4-responsive B cells was examined, and as a result, there was no cross-reactivity with IL-4-responsive B cells.

(2) Neutralizing Activity Neutralizing activity was measured by inhibiting the binding of IL-5 to the IL-5 receptor.

(A) 5 x 10" T88-M cells at a final concentration of 11
00p of 35S-IL-5 and various concentrations of purified H7
Add antibody to 200 μΩ.

Then, the cells were heated at 37°C for 10 minutes, and the radioactivity bound to the cells was measured.

As shown in FIG. 2, the concentration of H7 antibody that inhibited the binding of IL-5 to the IL-5 receptor by 50% was approximately 5 nM.

(B) 5 x 10 788-M cells at a final concentration of 1 n.
+25i H7 of M and various concentrations of non-radiolabeled I
Add L-5 to make 200 μg.

Then, the cells were heated at 37° C. for 10 minutes to determine the radioactivity bound to the cells.

As shown in Figure 3, the concentration of IL-5 that inhibits the binding of H7 to the IL-5 receptor by 50% is approximately 80%.
It was 0 pM.

Example 2 Measurement sensitivity of the antibody of the present invention in flow cytometry (FAC8 analysis) and correlation with biological activity (1) FAC8
Measurement sensitivity in analysis T88-MSMOPC10 with IL-5 binding site
Measurements were performed using 4E and BCLI-B20 cells.

According to the results of the IL-5 binding test, the binding site of IL-5 is T.
Approximately 10,000 cells/cell for 88-M cells, MOPC104
It was about 20,000 cells/cell for E cells and about 5,000 cells/cell for BCLI-B20 cells. These cells were reacted with H7 antibody, and FITC-MAR18,
5 and FAC8 analysis revealed that all cells were clearly positive. MTH (IL-2 dependent strain), FDC-Pi (IL
-3 dependent strain) was completely negative even when stained by the above method.

(2) Correlation with biological activity IL-5 responsiveness and H7 binding to the cell surface are completely correlated.

[Brief explanation of drawings]

Figure 1 shows the H7 antibody titration curve. Figure 2 shows 358-IL- to T88-M cell receptor.
Figure 2 shows the neutralizing activity of H7 monoclonal antibody against 5 binding. Figure 3 shows non-radiolabeled I for 1251 labeled H7 monoclonal antibody binding to T88-M cell receptor.
It shows the neutralizing activity of L-5. (That's all) Figure 2 Figure 1 Piece 1 made H7 monoclonal f: Stop i shin J Kan (9 intervals) Ki T
Manufactured H7 monoclonal J7L'tll (pM)
632-

Claims (1)

[Claims] (1) Mouse interleukin 5 characterized by having specific reactivity to mouse interleukin 5 receptor
Monoclonal antibody against the receptor.