JPH08134099A - Monoclonal antibody against inositol-1,4,5-triphosphate receptor - Google Patents

Abstract

PURPOSE: To obtain an anti-inositol 1,4,5-triphosphate (IP3 ) receptor type 2 monoclonal antibody and an anti-inositol-1,4,5-triphosphate receptor type 3 monoclonal antibody each of which is useful for judging the subtypes of expressed inositol 1,4,5-triphosphate and controlling their functions on cell diagnosis or tissue diagnosis. CONSTITUTION: A monoclonal antibody recognizing the epitope from 2687 amino acid to 2701 amino acid from the N-terminus of the IP3 receptor type 2, but not IP3 receptors types 1 and 3, a monoclonal antibody recognizing the epitope from 2657 amino acid to 2671 amino acid of the IP3 receptor type 3, but not IP3 receptors types 1 and 2, and a monoclonal antibody recognizing the epitope from 2410 amino acid to 2424 amino acid of the IP3 receptor type 3, but not the IP3 receptors types 1 and 2, and the hybridomas producing these antibodies are provided.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention is useful for determining the subtype of inositol 1,4,5-trisphosphate receptor which is expressed and controlling its function in cell diagnosis and tissue diagnosis. Anti-inositol 1,4,5-trisphosphate receptor type 2 monoclonal antibody and anti-inositol 1,
Provided is a 4,5-triphosphate receptor type 3 monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION By binding hormones, cytokines or neurotransmitters to their respective receptors on target cells, intracellular inositol 1,4,5-trisphosphate (insi
tol 1,4,5-trisphosphate, hereinafter abbreviated as IP ₃ . )
Production is induced, and the produced IP ₃ is present on the endoplasmic reticulum inositol 1,4,5-trisphosphate receptor (hereinafter, IP ₃
Abbreviated as receptor. ). The binding of IP ₃ to the IP ₃ receptor induces the release of calcium present in the endoplasmic reticulum to the cytoplasm, resulting in phagocytosis, granule secretion, motility and nerve cell excitement. Thus, IP
₃ receptors are important proteins involved in the transmission of cellular responses.

There are multiple subtypes of the IP ₃ receptor, and there are at least three subtypes in mammals:
The presence of IP ₃ receptors type 1, type 2 and type 3 was revealed from full length cDNA cloning, and partial cDNA cloning also suggested the presence of type 4. The amino acid homology between types is high. Also, there is not much difference due to animal species differences (Nature, 342, 32 (1989), EMBO J., 10 , 3199 (199).
1), J. Biol. Chem., 268 , 11356 (1993), Recept. Ch.
annel., 2 , 9 (1994), J. Biol. Chem., 269 , 1222 (1
994)].

The IP ₃ receptor is presumed to be a tetrameric membrane protein [J. Biol. Chem., 266 , 1109 (199).
1), Proc. Natl. Acad. Sci. USA, 88 , 4911 (1991)
], Accurate knowledge about the amino acid region exposed outside the membrane, the amino acid region existing inside the membrane, and the amino acid sequence suggesting the specificity of the subtype have not been specified.
Northern blotting and immunohistochemical and immunocytochemical techniques using antibodies have been used to analyze the subtypes of IP ₃ receptors present in tissues and various hematopoietic cells.

In Northern blotting, transcription from DNA
IP₃Confirm the messenger RNA of the receptor
, But the messenger RNA is translated
Or translated IP₃The receptor protein is working
No information on whether or not it is available. However, using antibodies
Present in tissues and various hematopoietic cells
IP ₃It is possible to detect the receptor and analyze its function. IP₃Acceptance
For body type 1, IP ₃Receptor type 1 specifically
Since a monoclonal antibody that recognizes it has been obtained [J.
 Neurochem,51, 1724 (1988)], on tissues and cells.
IP₃Receptor type 1 analysis is possible, but IP₃Acceptance
No antibody has been obtained for body type 2
IP using the method₃Analysis of receptor type 2 is not possible
Yes. IP₃For receptor type 3
Although the body has been acquired [J. Biol. Chem.,268, 11356
(1993), Recept. Channel.,2, 9 (1994), J. Biol.
Chem.,269, 1222 (1994)], the polyclonal antibody
In the method used, IP in tissues and cells₃Receptor
The type-specific expression of is difficult to identify.

[0006]

[Problems to be Solved by the Invention] In order to obtain a monoclonal antibody that recognizes an IP ₃ receptor in a type-specific manner, it is necessary to determine a region of an amino acid sequence that differs between the types of IP ₃ receptors or a type-specific three-dimensional structure. It is necessary to pay attention to obtain an anti-IP ₃ receptor type-specific monoclonal antibody using that region as an epitope, but accurate knowledge of the amino acid sequence suggesting the three-dimensional structure and subtype of the IP ₃ receptor is not available. Absent. Focusing on a different amino acid sequence among the type of IP ₃ receptor, even when producing antibodies, if the region of the amino acid sequence of the IP ₃ receptor is present in the film, the recognition site of the produced antibody to present in the film, not only able to identify the type of IP ₃ receptors and may not recognize the IP ₃ receptor itself. As described above, there is no knowledge as to which region of the IP ₃ receptor should be used as an epitope to obtain a monoclonal antibody that recognizes the IP ₃ receptor in a type-specific manner.

An object of the present invention, IP ₃ receptor type 1 and monoclonal antibodies reactive with IP ₃ receptor type 2 without reacting to type 3, and IP ₃ receptor type 1 and IP ₃ does not react to the Type 2 The object is to provide a monoclonal antibody that reacts with receptor type 3. Anti IP ₃ receptor type specific monoclonal antibodies to bind to a certain epitope of IP ₃ receptor, which is useful for the diagnosis of tissues and cells expressing IP ₃ receptor. Moreover, the monoclonal antibody that specifically control the functions of IP ₃ receptor type 2 or type 3, IP ₃ receptor type 2 or type 3
Since the function of cells expressing the is modified, the role of the IP ₃ receptor in these cells can be elucidated.
It is also possible to modify the cell function using such a monoclonal antibody and apply it to therapy.

[0008]

The present invention recognizes the epitopes present at positions 2687-2701 from the N-terminal amino acid of IP ₃ receptor type 2, and recognizes IP ₃ receptor types 1 and I.
IP, a monoclonal antibody that does not recognize P ₃ receptor type 3
_{3 From} the N-terminal amino acid of receptor type 3 to 2657-267
It recognizes an epitope present in the first monoclonal antibody that does not recognize the IP ₃ receptor type 1 and IP ₃ receptor type 2, the N-terminal amino acids of IP ₃ receptor type 3 24
Recognizes the 10th to 2424th epitopes and recognizes IP
_{The present invention} relates to a monoclonal antibody that does not recognize ₃ receptor type 1 and IP ₃ receptor type 2 and a hybridoma that produces the antibody.

The outline of the method for producing a monoclonal antibody against the IP ₃ receptor of the present invention is as follows. IP ₃ and part of the amino acid sequence of the receptor type 2 or type 3 and animal spleen cells immunized with a synthetic peptide having the same sequence, by fusing the mouse myeloma cells, to produce hybridomas, IP ₃ receptor A hybridoma that produces a monoclonal antibody that does not react to type 1 and 3 in response to body type 2 and a monoclonal antibody that does not react to type 1 and 2 in response to type 3 is established, and the hybridoma is cultured in a medium or an animal. To produce ascites tumor, and the culture supernatant or ascites fluid is collected to obtain a monoclonal antibody. Western blotting is carried out using this, and it is confirmed that the monoclonal antibody is specific to type 2 and type 3 of IP ₃ receptor, respectively. The thus-obtained monoclonal antibodies can be utilized for the control of IP ₃ detection of receptor type 2 and type 3 and IP ₃ receptor type 2 and type 3 functions in cytology and histology.

The method for producing a monoclonal antibody against the IP ₃ receptor of the present invention will be described in detail below. (1) Synthesis of Antigen Peptide The peptide serving as an antigen can be synthesized by the solid phase method using an automatic peptide synthesizer. The peptide-bonded solid-phase carrier obtained by the solid-phase method is treated with a mixed solution of trifluoroacetic acid (hereinafter abbreviated as TFA) / thioanisole / ethanedithiol (90: 5: 5) to solid-phase the peptide. At the same time as it is released from the carrier, the protecting group of the amino acid side chain is removed. The crude peptide product thus obtained is purified by high performance liquid chromatography (hereinafter abbreviated as HPLC) using a reverse phase column and used as an antigen peptide.

(2) Immunization of animals and preparation of antibody-producing cells In mice, rats or hamsters of 3 to 20 weeks of age,
The antigenic peptide obtained in (1), Keyhole Limpet Hemocyanin (hereinafter, abbreviated as KLH), bovine serum albumin (hereinafter, B)
Abbreviated as SA. ), Thyroglobulin (hereinafter abbreviated as THY), carrier proteins such as ovalbumin or human serum albumin by known methods [Cell, 28 , 477 (1982).
Etc.] in an amount of 50 to 100 μg / mouse, which is subcutaneously, intravenously, or intraperitoneally administered to an animal, and an appropriate adjuvant [eg, Freund's complete adjuvant (Co
mplete Freund's Adjuvant) or aluminum hydroxide gel and B. pertussis vaccine].

The immunogen is administered 1-2 times after the first administration.
Repeat every 5-10 times every week. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is confirmed by enzyme-linked immunosorbent assay (ELIS
Act A): Published by Ikushosho, 1976, Antibodies-A Lab
oratory Manual, Cold Spring Harbor Laboratory, 198
8) and so on. Mice, rats, or hamsters whose sera exhibit a sufficient antibody titer against the antigen used for immunization are provided as a source of antibody-producing cells.

In providing the fusion of splenocytes and myeloma cells, the spleen is excised from the immunized mouse or rat 3 to 7 days after the final administration of the antigen, and splenocytes are collected. The spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged (1200 rpm, 5 minutes), the supernatant is discarded, and the Tris-ammonium chloride buffer solution (p.
H7.65) for 1-2 minutes to remove red blood cells,
The cells are washed three times with EM medium and provided as antibody-producing cells.

(3) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (Ba
lb / c derived) myeloma cell line P3-X63Ag8-U1
(P3-U1) [Current Topics in Microbiology and
Immunology, 18 , 1-7 (1978), European J. Immunolog
y, 6 , 511-519 (1976)], SP2 / O-Ag14 (SP
-2) [Nature, 276 , 269-270 (1978)], P3-X6
3-Ag8653 (653) [J. Immunology, 123 , 15
48-1550 (1979)], P3-X63-Ag8 (X63)
[Nature, 256 , 495-497 (1975)] and the like are used.
These cell lines were prepared from 8-azaguanine medium [RPMI-
Glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 ⁻⁵ M), gentamicin (10 μg / ml) and fetal calf serum (CSL, 1) in 1640 medium.
0%) added medium (hereinafter referred to as normal medium) to which 8-azaguanine (15 μg / ml) was added], but subcultured to normal medium 3 to 4 days before cell fusion. Then, on the day of fusion, secure a cell number of 2 × 10 ⁷ or more.

(4) Preparation of hybridoma The antibody-producing cells immunized in (2) and the myeloma cells obtained in (3) were treated with MEM medium or PBS buffer (1.83 g disodium hydrogen phosphate, 0.21 g phosphorus). Acid dihydrogen dihydrogen, and 7.65 g sodium chloride were dissolved in distilled water to make 1 liter, and washed well with a buffer solution, pH 7.2),
Mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, and centrifuge (1,200 rpm, 5 minutes)
After that, the supernatant is discarded, and the precipitated cell group is thoroughly loosened, and then polyethylene glycol-1,000 (hereinafter abbreviated as PEG-1,000) 2 at 37 ° C. with stirring.
g, MEM medium 2 ml and dimethyl sulfoxide 0.
7 ml of the mixed solution 0.2 to 1 ml / 10 ⁸ antibody-producing cells are added, and 1 to 2 ml of MEM medium is added several times every 1 to 2 minutes, and then MEM medium is added to make the total volume 50 ml. After centrifugation (900 rpm, 5 minutes), discard the supernatant,
After gently loosening the precipitated cells, the cells are gently sucked in by a measuring pipette and gently blown to remove the cells from HAT medium [normal medium containing hypoxanthine (10 ⁻⁴ M) and thymidine (1.
5 × 10 ^-5 M) and aminopterin (4 × 10 ^-7 M)
Suspended in 100 ml. This suspension was dispensed into a 96-well culture plate at 100 μl / well, and 5
Incubate at 37 ° C in a CO ₂ incubator for 7 to 14 days.

After culturing, a part of the culture supernatant is taken and a hybridoma strain producing an antibody that specifically reacts with the antigen peptide used for immunization is selected by an enzyme immunoassay or the like. 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. Are selected as hybridoma strains producing anti-IP ₃ receptor monoclonal antibody.

Specific examples of the hybridoma cell line producing the anti-IP ₃ receptor type 1 monoclonal antibody include 2681 to the N-terminal amino acid of IP ₃ receptor type 1.
The hybridoma cell line KM1112, etc., which produces an antibody that recognizes the epitope present at the 2695th position, is the anti-IP ₃
Specific examples of the hybridoma cell line that produces the receptor type 2 monoclonal antibody include hybridoma cell lines KM1083 and KM1085 that produce an antibody that recognizes an epitope present at positions 2687-2701 from the N-terminal amino acid of IP ₃ receptor type 2. As a specific example of the hybridoma cell line producing an anti-IP ₃ receptor type 3 monoclonal antibody, an antibody recognizing an epitope existing at positions 2657 to 2671 from the N-terminal amino acid of IP ₃ receptor type 3 is produced. Hybridoma cell line KM10
81, KM1082, and KM1109, which produces an antibody that recognizes an epitope located at the 2410 to 2424th position from the N-terminal amino acid of IP ₃ receptor type 3. Hybridoma cell lines KM1082, KM108
3 and KM1109 were assigned to FERM BP- at the Institute of Biotechnology, National Institute of Biotechnology, on November 01, 1994.
4860, FERM BP-4861, FERM BP
Deposited as -4862.

(5) Purification of Monoclonal Antibody Pristane-treated mice of 8 to 10 weeks old treated with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane, intraperitoneally and raised for 2 weeks) or In the nude mouse, the anti-IP ₃ receptor monoclonal antibody-producing hybridoma cells obtained in (4) 2 × 10 ^{6 to} 5
Inject 10 ⁷ cells / mouse intraperitoneally. The hybridoma develops ascites tumor in 10 to 21 days. Ascites fluid was collected from this mouse, centrifuged (3,000 rpm, 5 minutes) to remove solids, salted out with 40 to 50% saturated ammonium sulfate, caprylic acid precipitation method, DEAE-Sepharose column, protein A- After passing through a column or a gel filtration column, the IgG or IgM fractions are collected and used as a purified anti-IP ₃ receptor monoclonal antibody.

The subclass of the antibody is determined by enzyme immunoassay using a subclass typing kit. The amount of protein is quantified by the Lowry method and the absorbance at 280 nm.

(6) Anti-IP ₃ receptor type 2 monoclonal antibody and anti-I using Western blotting
Confirmation of specificity of P ₃ receptor type 3 monoclonal antibody Anti-IP ₃ receptor type 2 selected in (4) or (5)
The reaction specificity of the monoclonal antibody and the anti-IP ₃ receptor type 3 monoclonal antibody is confirmed by Western blotting.

Proc. Natl. Acad. Sci. USA., 89 , 4265
(1992), Natur from the FEBS Letters, 349, 191 (1994 ) was obtained by the method shown in the like, IP ₃ receptor expressing cell cultures
The microsome fraction is obtained according to the method described in e, 342 , 32 (1989). 1 to 10 μg of protein contained in the microsome fraction was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE), blotted on a nitrocellulose membrane, and further blocked with a skim milk solution. Prepare the membrane.

Separately, in accordance with the method described in the above section (5) Purification of monoclonal antibody, a hybridoma producing an anti-IP ₃ receptor type 2 monoclonal antibody or an anti-IP ₃ receptor type 3 monoclonal antibody is cultured. A clear antibody solution or an antibody solution comprising an antibody purified from the culture supernatant is prepared. Further, 10 μg / ml of the corresponding antigenic peptide is added to the antibody solution to prepare a treated antibody solution in which the antibody in the antibody solution is bound. The antibody solution or the treated antibody solution was added to a PBS buffer solution in an amount of 0.
A solution containing 1% Tween-20 (hereinafter referred to as PBS-0.
Abbreviated as 1Tween. ) Or the like to dilute 0.1 to 1 μg / m
Adjust to l.

The antibody solution or the antibody solution treated with the antigen peptide prepared at 0.1 to 1 μg / ml is added to the nitrocellulose membrane prepared above and reacted at room temperature for 2 hours or at 4 ° C. overnight. PBS buffer or PBS-0.1Twee
After thoroughly washing the nitrocellulose membrane with n etc., anti-IP ₃
An antibody against immunoglobulin of the same animal species as the animal species from which the receptor monoclonal antibody-producing cells were obtained is labeled with biotin, an enzyme, a chemiluminescent substance or a radiation compound, and the prepared second antibody 1 to 50 μg / ml is added, Allow to react for 1-2 hours at room temperature. After washing well with PBS-0.1 Tween, the reaction corresponding to the labeling substance of the second antibody was performed, and the anti-IP ₃ receptor type 2 monoclonal antibody was treated with IP ₃
Anti-IP ₃ receptor type 3 monoclonal antibody reacts with receptor type 2 only to IP ₃ receptor type 3 and IP ₃
Make sure it no longer responds to the receptor.

[0024] When the anti-IP ₃ receptor monoclonal antibody does not inhibit the binding of IP ₃ to the IP ₃ receptor, using the binding to IP ₃ receptor IP _3, the following method, the anti-IP ₃ It can be confirmed that the receptor monoclonal antibody binds to the IP ₃ receptor. A lysate containing Triton-X100 etc. is added to the microsome fraction of IP ₃ receptor-expressing cells containing 2 mg of protein, and the mixture is reacted at 4 ° C. for 1 hour. After the reaction, the insoluble fraction was removed by centrifugation (14,000 rpm, 10 minutes),
1/20 volume of normal mouse serum is added to the obtained supernatant containing the solubilized protein, and the mixture is reacted at 4 ° C. for 1 hour. Next, protein G agarose was added to the reaction solution, and further 4
React at 1 ° C for 1 hour. The reaction solution was centrifuged (10,0
00 rpm, 30 seconds), and 6 mg / ml in the resulting supernatant
The addition of an anti-IP ₃ receptor monoclonal antibody. After reacting at 4 ° C for 1 hour, protein G agarose is added and further reacted at 4 ° C for 1 hour. The reaction solution is centrifuged (10,
000 rpm, 30 seconds), the precipitate obtained was thoroughly washed with a solubilizing solution, and then 15 mM IP ₃ and 10 nM [ ³ H] -IP ₃
Or Tris-hydrochloric acid buffer of pH 8 containing 10 nM [ ³ H] -IP ₃ is added and reacted for 10 minutes on ice. The reaction solution was centrifuged (14,500 rpm, 5 minutes), and the obtained precipitate was SOLVABLE (DuPon / NEN Reseach Products).
After dissolution in company), etc., it was added scintillator to measure the radiation dose by confirming that the IP ₃ are bound by a liquid scintillation counter, anti IP ₃ receptor monoclonal antibody binds to the IP ₃ receptor You can confirm that you are doing.

(7) Confirmation of reaction specificity of anti-IP ₃ receptor monoclonal antibody using immunoprecipitation and Western blotting The solubilization buffer [50 mM Tris was added to the microsome fraction derived from Jurkat cells, which is a T cell line. Hydrochloric acid, 1% Triton-X100, 5 mM ethylenediaminetetraacetic acid (EDT
A), 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 10 μM pepstatin A, 10 μM leupeptin, 1 mM 2-mercaptoethanol, pH 7.
5] was added to 3 mg protein / ml at 4 ° C.
React for 30 minutes. Solubilized solution II [6M urea, 20 mM dithiothreitol (DT
T), 1% sodium dodecyl sulfate, 1% Triton-X
100] is added and boiled at 100 ° C. for 5 minutes. This solution is subjected to dialysis buffer (10 mM sodium phosphate, 150 mM sodium chloride, 5 mM EDTA, 0.1 mM PMS) using microdialysis (manufactured by Bio-Tech) or the like.
F, 10 μM pepstatin A, 10 μM leupeptin,
In 1 mM 2-mercaptoethanol, pH 7.2),
Dialyze at 4 ° C. for 3 hours.

To 100 μl of this dialysate, 6 μg / ml of
An antibody against immunoglobulin of the same animal species as the IP ₃ receptor monoclonal antibody-producing cells was added, reacted at 4 ° C for 30 minutes, and then pansorbin (Calb
50 μl (manufactured by iochem) is added and reacted at 4 ° C. for 30 minutes. This reaction solution was centrifuged (12,000 rpm, 2
Then, 6 μg / ml of anti-IP ₃ receptor monoclonal antibody is added to the obtained supernatant, and the mixture is reacted at 4 ° C. for 1 hour. After the reaction, 6 μg / ml anti-IP ₃ receptor monoclonal antibody-producing cells are added with an antibody against immunoglobulin of the same animal species as the obtained animal species and reacted at 4 ° C. for 1 hour. Add 50 μl of pansorbin to this and mix at 4 ℃ for 30
React for minutes. Centrifuge the reaction mixture (12,000
rpm, 2 minutes), and the resulting immunoprecipitated precipitate was washed with a buffer (10% Nonidet P-40, 10% Triton-X).
After washing 3 times with 100, 0.1% BSA, PBS buffer solution), the proteins contained in the precipitate were separated by SDS-PAGE, and then blotted on a nitrocellulose membrane using a semi-dry blotting device, etc. Prepare a nitrocellulose membrane blocked with milk solution.

A culture supernatant of a hybridoma strain producing an anti-IP ₃ receptor monoclonal antibody or an antibody purified from the culture supernatant was diluted with PBS-0.1 Tween or the like to give a 0.1.
Adjust to 1 to 1 μg / ml. The antibody solution and the prepared nitrocellulose membrane are reacted overnight at 4 ° C. PBS
After thoroughly washing the nitrocellulose membrane with a buffer solution, PBS-0.1 Tween, or the like, an antibody against immunoglobulin of the same animal species as the animal species from which the anti-IP ₃ receptor monoclonal antibody-producing cells were obtained was treated with biotin, an enzyme, and a chemiluminescent substance. Alternatively, 1 to 50 μg / ml of the second antibody prepared by labeling with a radiation compound or the like is added, and the mixture is reacted at room temperature for 1 to 2 hours. After washing well with PBS-0.1 Tween, the reaction corresponding to the labeling substance of the second antibody is performed.

The protein immunoprecipitated with the anti-IP ₃ receptor type 2 monoclonal antibody was anti-IP ₃ receptor type 2
Other types of anti-IP detected only with monoclonal antibodies
₃ may not be detected in the receptor monoclonal antibody, confirming that the obtained similar results for anti-IP ₃ receptor type 3 monoclonal antibody and an anti-IP ₃ receptor type 1 monoclonal antibody similar results.

[0029] (8) by inhibition with anti-IP ₃ receptor monoclonal antibody is trigger by binding to IP ₃ receptor IP ₃ Ca ²⁺ release IP ₃ binds to IP ₃ receptor, IP ₃ receptor Ca ²⁺ is released from the endoplasmic reticulum of somatic cells, but this release
By binding to IP ₃ receptor monoclonal antibody of IP ₃ receptor, checks whether is inhibited.

The cells expressing the IP ₃ receptor were treated with buffer A [20 mM
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 110 mM potassium chloride, 10 mM sodium chloride, 2 mM magnesium chloride, 5 mM potassium dihydrogen phosphate, 5 mM ethylene glycol bis (2-aminoethyl ether) Tetraacetic acid, 1m
After suspending in MDTT, pH 7.2], the cells are disrupted using a homogenizer. 1,500 rpm for the cell lysate
Centrifuge for 15 minutes to collect the supernatant. The supernatant was centrifuged (35,000 g, 2 minutes), and the resulting precipitate fraction was added to buffer B [20 mM HEPES, 110 mM potassium chloride, 10 mM sodium chloride, 2 mM magnesium chloride, 5 mM potassium dihydrogen phosphate, pH 7.
2]. The suspension is centrifuged (35,000).
g, 20 minutes), and the resulting precipitate fraction is resuspended in buffer B to give a microsome fraction for measuring Ca ²⁺ release.

10 units / ml creatine kinase, 10 m in the microsome fraction (2-10 mg protein / ml)
Several solutions of M creatine phosphate, 10 μg / ml oligomycin and 0 to several 100 μg / ml of anti-IP ₃ receptor monoclonal antibody are prepared. The above operation is performed on ice, and the subsequent operation is performed at 22 ° C. In the solution
The Ca ²⁺ was added Indo-1 Ca ²⁺ was fluorescent reagent or radiolabel for Ca ²⁺ measurements (Dojin Chemical Laboratories, Ltd.) for measuring, further, by adding 2 mM ATP Ca ^{2 +}
Are incorporated into microsomes. Then a proper amount of IP ₃
And to release Ca ²⁺ from microsomes by adding, this Ca ²⁺
The release amount of Ca ²⁺ is measured to confirm whether or not Ca ²⁺ release is inhibited by the anti-IP ₃ receptor monoclonal antibody.

(9) Application of Anti-IP ₃ Receptor Type 2 Monoclonal Antibody and Anti-IP ₃ Receptor Type 3 Monoclonal Antibody to Cell Diagnosis (Immune Cell Staining Method) Floating type was applied to a slide glass coated with poly L-lysine. The suspension of cultured cells is added dropwise, and the cells are incubated at 37 ° C. for 15 minutes in a 5% CO ₂ incubator. After washing with PBS buffer, 4% paraformaldehyde (PF
A) or 2% periodate lysine paraformaldehyde (PLP), and let stand for 30 minutes. PB
After immersing in S buffer and washing, PBS buffer solution containing 0.1% Triton-X100 (PBS-0.1 Trito
Soak in n) for 10 minutes at room temperature. Next, using 2% normal serum of the same animal species that produces the antibody used to prepare the second antibody described below, the slide glass was blocked at room temperature for 30 minutes, and then the slide glass was (5) From a culture supernatant of an anti-IP ₃ receptor type 2 monoclonal antibody or a hybridoma expressing an anti-IP ₃ receptor type 3 monoclonal antibody, or a culture supernatant thereof, which has been cultured by the method described in the section of preparation of monoclonal antibody Add 1 to 10 μg / ml of purified antibody and
React for 1 hour to overnight. Put the slide glass on P
After washing with a BS buffer, an antibody against immunoglobulin of the same animal species as the animal species from which the anti-IP ₃ receptor monoclonal antibody-producing cells were obtained was labeled with an enzyme or a chemiluminescent substance to prepare a second antibody 1 to 10 μg / Add ml and allow to react for 1 hour at room temperature. After thorough washing with a PBS buffer solution, a coloring reaction using the labeling substance of the second antibody is performed.
An encapsulating material such as PBS-80 glycerol (glycerol dissolved in PBS buffer to be 80%) is added to the colored slide glass, and after encapsulation, observation is performed with a microscope.

(10) Application of Anti-IP ₃ Receptor Type 2 Monoclonal Antibody and Anti-IP ₃ Receptor Type 3 Monoclonal Antibody to Histological Diagnosis (Immunohistological Staining Method) Enzyme Antibody Method [Keiichi Watanabe et al., Revised Third Edition, Interdisciplinary] Planning (199
2)] In accordance with the method described above, various animal tissues were directly or PFA-fixed, frozen and embedded in OCT compound (manufactured by MILES), sliced to a thickness of 4 to 10 μm, and coated with poly-L-lysine. Stick it on a slide glass. This is fixed again with PFA or the like and washed with PBS buffer.

When a section of 6 μm or more is prepared, P
10 to 30 at room temperature in a solution (PBS-0.05 Triton) in which 0.05% Triton-X100 was added to BS buffer.
Immerse for a minute, and after soaking, wash the slide glass with PBS buffer. When peroxidase is used as a labeling substance during the preparation of the second antibody described below, 0.3
Immerse in methanol containing% hydrogen peroxide for 30 minutes and wash again with PBS buffer.

After washing, the slide glass is blocked at room temperature for 30 minutes using 2% normal serum of the same animal species as the antibody-producing animal used for preparing the secondary antibody described below. After blocking, 1 to 10 μg / ml of a purified antibody of anti-IP ₃ receptor type 2 monoclonal antibody or anti-IP ₃ receptor type 3 monoclonal antibody is added, and the mixture is reacted at 4 ° C. overnight. After washing the slide glass, an antibody against immunoglobulin of an anti-IP ₃ receptor monoclonal antibody-producing cells obtained species and the same animals were labeled with an enzyme or a chemiluminescent substance such as a second antibody 1~10μ prepared
Add g / ml and react at room temperature for 1-3 hours. After washing well with a PBS buffer solution, a coloring reaction using a labeling substance of the second antibody is performed. After encapsulating by adding an encapsulating material such as PBS-80 glycerol to the colored slide glass,
Observe with a microscope.

[0036]

【Example】

Example 1 Synthesis of Antigen Peptide Antigen peptides IP3RC1, IP3RC2, IP3RC3, IP having the amino acid sequences shown in SEQ ID NOs: 1 to 6
The synthesis of 3RL1, IP3RL2 and IP3RL3 is
The peptide synthesizer PSSM8 manufactured by Shimadzu Corporation was used and the reagents and solvents manufactured by Shimadzu Corporation were used according to the synthesis program of the company. The condensation reaction of amino acids is performed by the Fmoc method [basics and experiments of peptide synthesis, Nobuo Izumiya et al., Maruzen (198
5)] under standard conditions. In addition, mass spectrometry of the antigen peptide was performed using JEOL JMS-HX110A.

(1) Synthesis of IP3RC1 Cys is further added to the N-terminus of the peptide shown in SEQ ID NO: 1 and having the same sequence as the amino acid sequence present at positions 2681 to 2695 from the N-terminus amino acid of IP ₃ receptor type 1. The synthesis of bound antigenic peptide IP3RC1 was performed by the following method. Nα-9-fluorenylmethyloxycarbonyl-L-alanine (Fmoc-Ala-OH) 3
30 mg (p-benzyloxybenzyl alcohol type) of carrier resin having 0 μmol bound thereto was placed in a reactor of an automatic synthesizer and subjected to the following treatment and washing according to a synthesis program of Shimadzu Corporation to obtain a carrier resin bound with Ala. It was

(A) N, N-dimethylformamide (D
Wash with MF) for 3 minutes. (B) DMF solution containing 30% piperidine for 4 minutes, 2
Process once. (C) Treat with DMF 5 times for 1 minute. The Ala-bonded carrier resin thus obtained was further treated and washed as follows.

(D) Nα-9-fluorenylmethyloxycarbonyl-L-proline (Fmoc-Pro-O)
H) 300 μmol, benzotriazol-1-yl-
Oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) 300 μmo
1, N-hydroxybenzotriazole (HOBt) 30
0 μmol, N-methylmorpholine (NMM) 450 μ
Add 1.05 ml DMF containing mol and stir for 3 minutes, and treat with the resulting solution for 30 minutes.

(E) DMF is treated 5 times for 1 minute. In this way, Fmoc-Pro-Ala is synthesized on the carrier resin. Next, after performing the treatments (a) to (c) and washing, the step (d) is performed by Fmoc-Pro-
Instead of OH, Nα-9-fluorenylmethyloxycarbonyl-Nδ-trityl-L-glutamine [Fmoc
-Gln (Trt) -OH], and then the washing step (e) above is carried out to perform Fmoc-Gln (Trt)-.
Pro-Ala is synthesized on a carrier resin.

Thereafter, steps (a) to (e) were sequentially repeated to obtain a carrier resin having a protected peptide bound thereto. In the step (d), Fmoc-Gln (Trt) -OH,
Fmoc-Pro-OH, Nα-9-fluorenylmethyloxycarbonyl-Nγ-trityl-L-asparagine [Fmoc-Asn (Trt) -OH], Fmoc-
Val-OH, Fmoc-Asn (Trt) -OH, F
moc-Met-OH, Nα-9-fluorenylmethyloxycarbonyl-Nim-trityl-L-histidine [Fmoc-His (Trt) -OH], Fmoc-P
ro-OH, Fmoc-Pro-OH, Fmoc-Hi
s (Trt) -OH, Fmoc-Gly-OH, Fmo
c-Leu-OH and Nα-9-fluorenylmethyloxycarbonyl-L-Nim-trityl-L-cysteine [Fmoc-Cys (Trt) -OH] were used.

After completion of the synthesis reaction, the obtained carrier resin was washed with methanol and butyl ether, and dried under reduced pressure for 3 hours. To this, 200 μl of a mixed solution of TFA / thioanisole / ethanedithiol (90: 5: 5) was added, and the mixture was allowed to stand at room temperature for 2 hours to cut out the peptide from the resin. The carrier resin was removed by filtration, and the obtained filtrate was added with about 10 ml of e-
The crude peptide was precipitated by adding tellurium, and 34.4 mg of the crude peptide was obtained by filtration. This crude peptide was
By LC method, reverse phase column (CAPCELL PAK C18SG120 S-5
μm 30 × 250m, manufactured by Shiseido Co., Ltd.
Fractionation containing IP3RC1 was obtained by performing linear concentration gradient elution with acetonitrile containing FA and detecting at 220 nm. This fraction was freeze-dried to obtain 9.8 mg of IP3RC1.

The physicochemical properties of IP3RC1 are as follows. Mass spectrometry; M + H = 1741 Amino acid analysis Measured value (calculated value): Pro3.8 (4), Val1.0
(1), Met1.0 (1), Leu1.0 (1), Cys1.2 (1), Gly1.1 (1), H
is2.2 (2), Asx2.0 (2), Glx2.1 (2), Ala1.0 (1) (2) Synthesis of IP3RC2 N-terminal of IP ₃ receptor type 2 shown in SEQ ID NO: 2 The antigen peptide IP3RC2 was synthesized by the following method in which Cys was further bound to the N-terminus of a peptide having the same amino acid sequence as the 2687-2701th position from the amino acid.

60 mg of a carrier resin having Nα-trityl-Nim-trityl-L-histidine [Trt-His (Trt) -OH] bound thereto was used, and methylene chloride containing 0.2M hydrochloric acid / methanol / dioxane (90: 5: 5) in 5
After washing for a minute, a carrier resin having His bound thereto was obtained. After that,
The antigen peptide was synthesized according to Example 1- (1).

In step (d), Fmoc-Pro-
OH, Fmoc-Pro-OH, Fmoc-Met-O
H, Fmoc-His (Trt) -OH, Fmoc-H
is (Trt) -OH, Fmoc-Asn (Trt)-
OH, Fmoc-Val-OH, Fmoc-His (T
rt) -OH, Fmoc-Pro-OH, Nα-9-fluorenylmethyloxycarbonyl-Ot-butyl-
L-threonine [Fmoc-Thr (tBu) -O
H], Fmoc-Asn (Trt) -OH, Fmoc-
Ser-OH, Fmoc-Gly-OH, Fmoc-L
eu-OH and Fmoc-Cys (Trt) -OH were used. Further, by the same method as in Example 1- (1), 19.3 mg of the crude peptide and 6.5 mg of IP3RC2 obtained by purifying the crude peptide by HPLC were obtained.

The physicochemical properties of IP3RC2 are as follows. Mass spectrometry; M + H = 1778 Amino acid analysis Measured value (calculated value): Pro3.0 (3), Val0.9
(1), Met1.1 (1), Leu1.0 (1), Cys1.2 (1), Gly1.1 (1), H
is4.0 (4), Asx2.1 (2), Ser1.0 (1), Thr0.9 (1)

(3) Synthesis of IP3RC3 A peptide having the same sequence as the amino acid sequence present at positions 2657 to 2671 from the N-terminal amino acid of IP ₃ receptor type 3 shown in SEQ ID NO: 3 is the fourth from the C-terminal. Cy
The antigen peptide IP3RC3 in which s was substituted with Ser and Cys was bound to the N-terminus was synthesized by the following method.

Nα-9-fluorenylmethyloxycarbonyl-N-δ-2,2,5,7,8, -pentamethylchroman-6-sulfonyl-L-arginine [Fmoc
-Arg (Pmc) -OH] -bonded carrier resin 30 m
Using g, the antigen peptide was synthesized according to Example 1- (1).

In step (d), Fmoc-Ser-
OH, Fmoc-IIe-OH, Fmoc-Ser-O
H, Fmoc-Asn (Trt) -OH, Fmoc-G
ln (Trt) -OH, Fmoc-Val-OH, Nα
-9-Fluorenylmethyloxycarbonyl-L-as
Paraginic acid-β-t-butyl ester [Fmoc-As
p (OtBu) -OH], Fmoc-Val-OH, F
moc-Phe-OH, Fmoc-Gly-OH, Fm
oc-Leu-OH, Fmoc-Arg (Pmc) -O
H, Fmoc-Gln (Trt) -OH, Fmoc-A
rg (Pmc) -OH, Fmoc-Cys (Trt)-
OH was used. After synthesis, TFA / H ₂O / Thioani
Sol / ethanedithiol / ethylmethyl sulfide
/ Of thiophenol (82.5: 5: 2.5: 3: 2)
Add 200 μl of the mixed solution and leave it for 8 hours.
Deprotection and peptide excision were performed. Since then
The crude peptide was treated with 2 by the same method as in Example 1- (1).
6.2 mg, HPLC-purified IP3 of the crude peptide
8.6 mg of RC2 was obtained.

The physicochemical properties of IP3RC2 are as follows. Mass spectrometry; M + H = 1879 Amino acid analysis Measured value (calculated value): Val1.8 (2), Leu1.0
(1), Cys1.2 (1), Gly1.1 (1), Asx2.1 (2), Glx2.0 (2), I
Ie0.9 (1), Ser2.0 (2), Phe1.0 (1), Arg2.8 (3)

(4) Synthesis of IP3RL1 The peptide having the same sequence as the amino acid sequence present at 2483 to 2497 from the N-terminal amino acid of IP ₃ receptor type 1 shown in SEQ ID NO: 4 is the second from the C-terminal. Cy
The antigen peptide IP3RL1 in which s was substituted with Ser and Cys was bound to the N terminus was synthesized by the following method.

The antigen peptide was synthesized according to Example 1- (1) using 30 mg of the carrier resin having Fmoc-Arg (Pmc) -OH bound thereto.

In step (d), Fmoc-Ser-
OH, Fmoc-Val-OH, Fmoc-Asp (O
tBu) -OH, Fmoc-Ser-OH, Nα-9-
Fluorenylmethyloxycarbonyl-Ot-butyl
-L-tyrosine [Fmoc-Tyr (tBu) -O
H], Fmoc-Leu-OH, Fmoc-Phe-
OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Asn (Trt) -OH, Fmoc-Ala-OH,
Fmoc-Leu-OH, Fmoc-Ser-OH, N
α-9-fluorenylmethyloxycarbonyl-L-g
Lutamate-γ-t-butyl ester [Fmoc-Gl
u (OtBu) -OH], Fmoc-Gly-OH, F
moc-Cys (Trt) -OH was used. Completion
Later, TFA / H ₂O / thioanisole / ethanedithio-
/ Ethyl methyl sulfide / thiophenol (8
2.5: 5: 2.5: 3: 2) 200 μl of mixed solution
Add and leave for 8 hours to deprotect and peptize
I cut it out. Thereafter, similar to Example 1- (1)
19.0 mg of crude peptide according to the method of
Was obtained by HPLC to obtain 4.1 mg of IP3RL1.
Was.

The physicochemical properties of IP3RL1 are as follows. Mass spectrometry; M + H = 1777 Amino acid analysis Measured value (calculated value): Val1.0 (1), Leu2.0
(2), Cys1.9 (2), Gly1.0 (1), Asx3.0 (3), Ser1.8 (2), G
lx0.9 (1), Ala0.9 (1), Phe1.0 (1), Tyr1.1 (1), Arg0.9
(1)

(5) Synthesis of IP3RL2 Cys is further added to the N-terminus of the peptide shown in SEQ ID NO: 5 and having the same sequence as the amino acid sequence present at positions 2436 to 2450 from the N-terminus amino acid of IP ₃ receptor type 2. Synthesis of the bound antigenic peptide IP3RL2 was performed by the following method. Using 30 mg of a carrier resin having Fmoc-Glu (OtBu) -OH bound thereto, an antigen peptide was synthesized according to Example 1- (1).

In step (d), Fmoc-Met-
OH, Fmoc-Met-OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Thr (tBu) -OH, F
moc-Leu-OH, Fmoc-Thr (tBu)-
OH, Fmoc-Met-OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Pro-OH, Fmoc-V
al-OH, Fmoc-Gln (Trt) -OH, Fm
oc-His (Trt) OH, Nα-9-fluorenylmethyloxycarbonyl-Ot-butyl-L-serine [Fmoc-Ser (tBu) -OH], Fmoc-G.
ly-OH and Fmoc-Cys (Trt) -OH were used. Further, by the same method as in Example 1- (1), 10.0 mg of the crude peptide and 2.9 mg of IP3RL2 obtained by purifying the crude peptide by HPLC were obtained.

The physicochemical properties of IP3RL1 are as follows. Mass spectrometry; M + H = 1768 Amino acid analysis Measured value (calculated value): Val0.9 (1), Leu1.0
(1), Cys1.1 (1), Gly1.1 (1), Ser1.0 (1), Glx2.0 (2), H
is0.9 (1), Pro1.0 (1), Thr3.6 (4), Met2.8 (3)

(6) Synthesis of IP3RL3 Cys is further added to the N-terminus of the peptide shown in SEQ ID NO: 6 and having the same sequence as the amino acid sequence present at the 2410-2424th position from the N-terminus amino acid of IP ₃ receptor type 3. Synthesis of the bound antigenic peptide IP3RL3 was performed by the following method. Using 30 mg of a carrier resin having Fmoc-Thr (tBu) -OH bound thereto, an antigen peptide was synthesized according to Example 1- (1).

In the step (d), Fmoc-Asp was sequentially added.
(OtBu) -OH, Fmoc-Val-OH, Fmo
c-Phe-OH, Fmoc-Ala-OH, Fmoc
-Ala-OH, Fmoc-Ala-OH, Fmoc-
Gly-OH, Fmoc-His (Trt) -OH, F
moc-Pro-OH, Fmoc-Met-OH, Fm
oc-Gly-OH, Fmoc-Leu-OH, Fmo
c-Pro-OH, Fmoc-Ser (tBu) -O
H, Fmoc-Cys (Trt) -OH was used. Further, in the same manner as in Example 1- (1), 9.8 mg of the crude peptide and IP3 obtained by purifying the crude peptide by HPLC were used.
4.6 mg of RL2 was obtained.

The physicochemical properties of IP3RL1 are as follows. Mass spectrometry; M + H = 1575 Amino acid analysis Measured value (calculated value): Val1.0 (1), Leu0.9
(1), Cys1.0 (1), Gly2.0 (2), Ser1.0 (1), His1.0 (1), P
ro1.8 (2), Thr1.1 (1), Met1.2 (1), Ala3.1 (3), Phe0.9
(1), Asx1.0 (1) Example 2 (1) Immunization of animals and preparation of antibody-producing cells Synthetic peptides IP3RC1 to IP3 prepared in Example 1
RL2 was used to immunize animals and screen for antibody producing cells. Each synthetic peptide was bound to KLH (CALBIOCHEM) using m-maleimido-benzoyl-n-hydroxysuccil (manufactured by Nacalai Tesque, Inc .; hereinafter abbreviated as MBS) as a cross-linking agent for the purpose of enhancing immunogenicity. Used as an immunogen. The N-terminal Cys of each synthetic peptide was used for binding to MBS.

The immunogen preparation method is shown below. KLH Dissolve in 1M sodium phosphate buffer (pH 7.0) to prepare 10 mg / ml, and prepare 1/10 volume of 25 mg.
/ Ml MBS, and stir for 30 minutes at room temperature. After stirring, the mixture is passed through a Sephadex G-25 column that has been equilibrated with the same buffer in advance to remove unreacted MBS. 2.5 mg of the obtained KLH-MBS complex was mixed with 1 mg of each synthetic peptide, and the mixture was stirred at room temperature for 3 hours. After stirring, it was dialyzed against a PBS buffer containing 0.5 M sodium chloride and used as an immunogen.

100 μg of each immunogen was used together with 2 mg of aluminum gel for the first time and 1 × 10 ⁹ cells of pertussis vaccine (manufactured by Chiba Prefectural Serum Research Institute) in a 5-week-old female mouse (Bal.
b / c), and 2 weeks later, 100 μg of each immunogen
Was administered once a week for a total of 4 times. Blood was collected from the fundus venous plexus, and the serum antibody titer was examined by the enzyme immunoassay method shown below.

The results are shown in FIGS. 1 and 2. IP3RC
The antisera of the group immunized with 1 to IP3RC3 and the group immunized with IP3RL3 each showed a binding activity to a positive antigen up to a 1000-fold dilution, showed low reactivity to a negative antigen, and had an IP ₃ receptor type specificity. I was able to obtain a proper clone. However, IP3RL
No increase in antibody titer was observed in the group immunized with 1 or IP3RL2, and an IP ₃ receptor type-specific clone could not be obtained.

Spleens were extracted from the mice showing a sufficient antibody titer 3 days after the final immunization. The spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged (1,200 rpm, 5 minutes), the supernatant is discarded, and the Tris-ammonium chloride buffer solution (pH 7.65). And 1-2
Treatment for minutes to remove red blood cells, wash 3 times with MEM medium,
Used for cell fusion.

Enzyme-linked immunosorbent assay 1 mg of the synthetic peptide prepared in Example 1 and 5 mg of thyroglobulin (THY) were separately dissolved in 0.1 M ammonium acetate buffer (pH 7.0), and after mixing, the same buffer was added to 1 ml. I chose 540 μl of 0.02 M glutaraldehyde was added dropwise with stirring, and the mixture was stirred at room temperature for 5 hours. After stirring, dialyzing against PBS buffer overnight, each THY
-A synthetic peptide complex was made. A THY-synthetic peptide complex corresponding to the antibody to be measured was used as a positive antigen, and another THY-synthetic peptide complex was used as a negative dive antigen in the enzyme immunoassay.

On a 96-well EIA plate (manufactured by Greiner), 10 to 100 μg / ml of TH prepared above was prepared.
Y-synthetic peptide (positive antigen) or another THY
-Synthetic peptide (negative antigen) was dispensed at 50 µl / well and left overnight at 4 ° C. Synthetic peptide IP3RC
Because of the high amino acid sequence homology 1 and IP3RC2, IP3RC1 (having the IP ₃ same sequence as 2681 to 2695 amino acid sequences from the N-terminal amino acid of the receptor type 1) and IP3RC2 (the IP ₃ receptor type 2 N THY-IP3RC1 and THY-IP3RC2 were used as positive and negative antigens to each other in order to select mice producing antibodies that can be strictly distinguished from the last amino acid having the same amino acid sequence as the positions 2687 to 2701). I was there. After washing, 100-200 μl of PBS buffer containing 1% BSA (BSA-PBS)
/ Hole dispensed and left at room temperature for 1-2 hours or at 4 ° C for 1-2 nights. After allowing to stand, the BSA-PBS is discarded, and after thoroughly washing with a PBS buffer solution, the first antibody is BSA-PBS.
20-100 μl / well of mouse serum diluted with
It was left at room temperature for 2-3 hours or at 4 ° C. overnight. PBS
After thorough washing with a buffer solution or PBS-0.05 Tween, a peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO) as a second antibody was added to 50-100.
μl / well was dispensed and left at room temperature for 2 hours.

After thoroughly washing with PBS-0.05 Tween, 550 mg of ABTS substrate solution [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium was added to 0.1 M citrate buffer solution (pH 4.2). ) 1
Immediately before use, add 1μ of hydrogen peroxide to the solution dissolved in 1 liter.
1 / ml was added] to develop color, and the absorbance at OD _{415 nm} was measured.

(2) 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.

(3) Preparation of hybridoma Mouse splenocytes obtained in Example 2 (1) and Example 2
The myeloma cells obtained in (2) were mixed at a ratio of 10: 1, centrifuged (1,200 rpm, 5 minutes), the supernatant was discarded, and the precipitated cells were thoroughly loosened and stirred. However, at 37 ° C., 0.2 to 1 ml / 10 ⁸ mouse splenocytes were added to a mixed solution of 2 g of PEG-1,000, 2 ml of MEM medium and 0.7 ml of dimethyl sulfoxide, and MEM was added every 1 to 2 minutes. After adding 1-2 ml of the medium several times, MEM medium was added to adjust the total amount to 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, the cells were loosely loosened, and then the cells were gently sucked in by a measuring pipette and gently sucked to suspend the cells in 100 ml of HAT medium.

10 times this suspension was added to a 96-well culture plate.
The solution was dispensed in an amount of 0 μl / well and cultured in an incubator at 37 ° C. in 5% CO ₂ for 10 to 14 days. Using the BSA-PBS diluted solution of the obtained hybridoma culture supernatant as the first antibody, enzyme immunoassay was carried out according to the method described in Example 2 (1) to specifically react with the peptide used for immunization. A hybridoma producing an anti-IP ₃ receptor monoclonal antibody was selected, and cloning by the limiting dilution method was repeated twice to establish a hybridoma strain.

As shown in FIGS. 3A and 3B, a hybridoma cell line KM1112 producing an anti-IP ₃ receptor type 1 monoclonal antibody and a hybridoma cell line KM 108 producing an anti-IP ₃ receptor type 2 monoclonal antibody.
3, KM1085 and hybridoma cell line KM108 producing anti-IP ₃ receptor type 3 monoclonal antibody
1, KM1082 and KM1109 were acquired.

(4) Purification of Monoclonal Antibody 5 to 20 hybridoma strains obtained in Example 2 (3) were applied to 8-week-old nude female mice (Balb / c) treated with pristane.
Each of 10 ⁶ cells / mouse was intraperitoneally injected. 10-21
A day later, the hybridoma developed ascites tumor. Ascites was collected (1 to 8 ml / mouse) from the mouse in which ascites was collected, and centrifuged (3,000 rpm, 5 minutes) to remove solids. When the monoclonal antibody is IgM, it is salted out with 50% ammonium sulfate, dialyzed with PBS buffer containing 0.5 M sodium chloride, and then Cellulofine GSL2.
000 (Seikagaku Corporation) (Bet volume 750m
The column of 1) was passed through the column at a flow rate of 15 ml / hour, and the IgM fraction was collected to give a purified monoclonal antibody.

When the monoclonal antibody is IgG, the caprylic acid precipitation method (Antibodies-A Laboratory Manual, Cold
Purified by Spring Harbor Laboratory, 1988) to obtain a purified monoclonal antibody. The antibody subclass was determined by enzyme immunoassay using a mouse monoclonal antibody typing kit. The subclass of each antibody is shown in Table 1.

[0074]

[Table 1]

(5) Anti-IP ₃ receptor type 2 monoclonal antibody and anti-I using Western blotting
P ₃ COS-7 cell lines by introducing the reaction specificity of the confirmation pcDNAI vector DNA receptor type 3 monoclonal antibody, have been transformed with a vector DNA inserted a full-length cDNA of human IP ₃ receptor type 3 in pcDNAI COS-
Jurkat cells, which are 7 cell lines and T cell lines, were used in RPMI1640 medium containing 10% fetal bovine serum.
Each culture was performed at 37 ° C. in a% CO ₂ incubator. The cell lines and mouse cerebellum obtained from each of the culture solutions were washed with PBS buffer, and then 8 volumes of homogenization buffer [5 mM Tris-HCl, 1 mM EDTA, 0.25 M sucrose, 0.1 mM PMSF (Wako Pure Chemical
Industries, Lt), 10 μM Pepstatin A (Peptide
Institute. Inc), 10 μM leupeptin (Peptide In)
Institute. Inc.), pH 7.4], and the cells were disrupted by using a Potter homogenizer at 1,000 rpm for 10 times. The cell lysate is centrifuged (1,500 rpm, 10 minutes), and the resulting supernatant is centrifuged again (70,000 rpm, 20 minutes). The resulting precipitate fraction was resuspended in buffer (50 mM Tris-HCl, 1 m
M EDTA, 0.1 mM PMSF, 10 μM pepstatin A and 10 μM leupeptin, pH 7.4) were suspended and used as a microsome fraction.

10 μg of protein contained in the microsome fraction derived from Jurkat cells and 2 μg of protein contained in each microsome fraction derived from other cells were added to SDS-P.
After separation by AGE, blotting was performed on a nitrocellulose membrane using a semi-dry blotting apparatus (manufactured by Atto), and a nitrocellulose membrane blocked with a skim milk solution was prepared.

Separately, the culture supernatant of the hybridoma strain producing the anti-IP ₃ receptor type 2 monoclonal antibody or the anti-IP ₃ receptor type 3 monoclonal antibody obtained in Example 2 (3) was added to PBS-0.1 Tween. Synthetic peptide 1 prepared in Example 1 in which the antibody solution diluted with 50 times and the antibody contained in the culture supernatant can be recognized in the culture supernatant
Add 0 μg / ml and add 50 with PBS-0.1Tween.
A double-diluted treated antibody solution was prepared.

These antibody solutions or treated antibody solutions were reacted with the above prepared nitrocellulose membrane at 4 ° C. overnight. After thoroughly washing these with PBS-0.1 Tween, peroxidase-labeled anti-mouse immunoglobulin antibody (DA
(Manufactured by KO) was reacted at room temperature for 1 hour. PBS-0.1T
After washing well with ween, autoradiography film was exposed using ECL (Western blotting detection kit manufactured by Amersham).

As shown in FIG. 4, the anti-IP ₃ receptor type 3 monoclonal antibodies KM1082 and KM1109 were
It does not react with mouse cerebellum-derived microsomal fraction proteins (IP ₃ receptor type 1 is more abundant than other types),
Microsome fraction derived from COS-7 cell line into which pcDNAI vector DNA has been introduced. Microsome fraction derived from COS-7 cell line which weakly reacts with a protein of about 250 kDa and expresses full-length cDNA of human IP ₃ receptor type 3. It reacted strongly with a protein of about 250 kDa. The anti-IP ₃ receptor type 2 monoclonal antibody KM1083 was
Vector DNA-introduced COS-7 cell line and human
COS expressing full length cDNA of IP ₃ receptor type 3
The microsome fraction derived from the -7 cell line reacted with the protein of about 250 kDa to the same extent.

As shown in FIG. 5, anti-IP ₃ receptor type 3
Monoclonal antibodies KM1082 and KM1109
, Jurkat cells, and human IP ₃ receptor type 3
Reacts with a protein of about 250 kDa in the microsomal fraction derived from the COS-7 cell line expressing the full-length cDNA of
This anti-IP ₃ receptor type 3 monoclonal antibody was preliminarily added and treated with a synthetic peptide specific for IP ₃ receptor type 3, so that it did not react with a protein of about 250 kDa in the microsome fraction. Similarly IP ₃ receptor type 2 monoclonal antibody, IP ₃ by adding pre-processed in a specific synthetic peptide receptor type 2 monoclonal antibody, reactive with the protein of approximately 250kDa microsomal fraction from Jurkat cells I stopped doing it.

[0081] the Example 2 (3) anti-IP ₃ receptor type 2 monoclonal antibody KM1083 selected in binds to IP ₃ receptor was identified using binding to IP ₃ receptor IP ₃ . 2.8 mg of Jurkat cell microsomal fraction was 1 in solubilized solution (10 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, 1% (w / v) Triton-X100, 0.1 mM PMSF, pH 8.0). After stirring for 5 hours, centrifugation (14,500 rpm,
10 minutes), and 5% (V / V) normal mouse serum was added to the obtained supernatant. After reacting for 1 hour, 2% (V /
V) Protein G Agarose (VIVAX BIOTECHNOLOGY
(Manufactured by K.K.) was added and the reaction was continued for 1 hour. This was centrifuged (10,000 rpm, 30 seconds), and the obtained precipitate was used as a non-specific binding product with normal mouse serum. To the supernatant, 6 μg / ml of purified anti-IP ₃ receptor type 2 monoclonal antibody KM1083 was added, and after reacting for 1 hour, 2% (V / V) of protein G agarose was added, and further reacted for 1 hour. It was This is centrifuged (10,
(000 rpm, 30 seconds), and the resulting precipitate was used as a specific binding product with the anti-IP ₃ receptor type 2 monoclonal antibody. The non-specific binding product and the specific binding product were each washed with a solubilizing solution three times. All the above operations were performed at 4 ° C.

Next, the binding of IP ₃ to non-specific and specific binding substances was examined. Binding buffer (10 mM Tris-HCl, 1 mM EDT) to non-specific and specific binders of IP ₃
A, pH 8.0) is added to make 100 ml. A solution to which 10 nM [ ³ H] IP ₃ is added and reacted for 10 minutes on ice and a solution to which 15 mM IP ₃ and 10 nM [ ³ H] IP ₃ are simultaneously added and reacted for 10 minutes on ice are prepared. These solutions were centrifuged (14,500 rpm, 5 minutes) at 4 ° C, 250 ml of SOLVABLE was added to the precipitate, and the mixture was reacted at 42 ° C for 20 minutes. To this, 25 ml of acetic acid and 6 ml of scintillator were added, and the radiation dose was measured with a scintillation counter.

[0083]

[Table 2]

As shown in Table 2, IP ₃ specific binding (1,0) to the non-specific binding product precipitated by normal mouse serum was performed.
56.9 cpm) was excluded, and the specific binding product with anti-IP ₃ receptor type 2 monoclonal antibody was treated with IP ₃
There was specific binding (2,238 cpm). Therefore anti-IP ₃
The receptor type 2 monoclonal antibody was shown to recognize the IP ₃ receptor.

(6) Confirmation of reaction specificity of anti-IP ₃ receptor type 1 monoclonal antibody, anti-IP ₃ receptor type 2 monoclonal antibody and anti-IP ₃ receptor type 3 monoclonal antibody using immunoprecipitation and Western blotting According to the method described in Example 2 (5), microsome fractions were prepared from Jurkat cells, which are T cell lines. Add 3 mg protein / ml of this microsome fraction to the solubilization buffer.
And added thereto so as to react at 4 ° C. for 30 minutes. To this reaction solution was added 1.5 times the amount of the solubilized solution II, and the mixture was boiled at 100 ° C. for 5 minutes. This solution is used for microdialysis (Bi
(manufactured by o-Tech) was dialyzed in dialysis buffer at 4 ° C. for 3 hours. To 100 μl of this dialysate, 6 μg / ml of anti-mouse immunoglobulin antibody (γ-chain specific) was added and reacted at 4 ° C. for 30 minutes, and then 50 μl of pansorbin was added and reacted at 4 ° C. for 30 minutes. This reaction solution was centrifuged (12,000 rpm, 2 minutes), and the resulting supernatant was mixed with 6
μg / ml of anti-IP ₃ receptor type 1 monoclonal antibody KM1112, anti-IP ₃ receptor type 2 monoclonal antibody KM1083 or anti-IP ₃ receptor type 3 monoclonal antibody KM1082 was added and reacted at 4 ° C. for 1 hour. After the reaction, 6 μg / ml of anti-mouse immunoglobulin antibody (γ chain-specific) was added, and the mixture was reacted at 4 ° C. for 1 hour. Add 50 μl of pansorbin to this and mix at 4 ℃ for 30
Let react for minutes. Centrifuge the reaction mixture (12,000
(rpm, 2 minutes), the obtained precipitate was washed 3 times with a washing buffer, and the protein contained in the precipitate was separated by SDS-PAGE and then blotted on a nitrocellulose membrane using a semi-dry blotting apparatus. Furthermore, a nitrocellulose membrane blocked with a skim milk solution was prepared.

The anti-IP ₃ receptor type 1 monoclonal antibody KM11 obtained according to the method described in Example 2 (3).
12, anti-IP ₃ receptor type 2 monoclonal antibody KM1
The culture supernatant of the hybridoma strain producing 083 or the anti-IP ₃ receptor type 3 monoclonal antibody KM1082 was diluted 50 times with PBS-0.1Tween, and these antibody solutions were reacted with the above prepared nitrocellulose membrane overnight at 4 ° C. . After washing them well with PBS-0.1 Tween, they were reacted with a peroxidase-labeled anti-mouse immunoglobulin antibody at room temperature for 1 hour. PBS-0.1Tween
After thorough washing with, the autoradiographic film was exposed to light using ECL (Western blotting detection kit manufactured by Amersham).

As shown in lane 1 of FIG. 6-a, KM1
The 250 kDa protein immunoprecipitated with 112 is KM1.
Although it was detected in 112, it was not detected in KM1083 and KM1082 as shown in lane 1 of FIGS. 6-b and 6c. Similarly, KM1083
The 250-kDa protein immunoprecipitated with Escherichia coli was KM1083.
Was detected by KM1112 and KM1
082 could not be detected. KM1082
Similarly, 250 kDa immunoprecipitated with KM1082.
Was detected with KM1082,
It could not be detected by M1112 and KM1083.

[0088] Thus, each antibody is specific to the type of IP ₃ receptor, anti-IP ₃ receptor type 2 monoclonal antibodies only IP ₃ receptor type 2, anti-IP ₃ receptor type 3 monoclonal antibodies IP ₃ It was confirmed that only receptor type 3 was recognized. Example 3 (1) Inhibition of Ca ²⁺ Release Induced by Binding of IP ₃ to IP ₃ Receptor Type 3 by Anti-IP ₃ Receptor Type 3 Monoclonal Antibody Anti-selection in Example 2 (3) confirmation that IP ₃ receptor type 3 monoclonal antibody KM1082 to inhibit the release of Ca ²⁺ due to IP ₃ from microsomes of IP ₃ receptor type 3 expressing cells was performed with a fluorescent dye for Ca ²⁺ measurement.

The microsome fraction for measuring Ca ^{2+ in} the human cell line HPB-ALL expressing IP ₃ receptor type 3 was set to 4 m.
It was adjusted to g protein / ml. This is 490μ
Dispense l by 10 units / ml creatine kinase, 1
0 mM creatine phosphate, 10 μg / ml oligomycin was added. Final concentration of 0, 10, 20 and 50μ
Anti-IP ₃ Receptor Type 3 Monoclonal Antibody KM1082 Purified to g / ml and 4 Kinds of Solution and 50 μg / ml KM231 [Antibodies Not Recognizing IP ₃ Receptor, Anticancer Research, 11 , 2003 (1991) ))
Was added to prepare a solution, and the mixture was reacted on ice for 2 hours. 6 mM Indo-1 (Dojindo Laboratories Inc.) was added to these solutions. The subsequent operation was performed at 22 ° C. Changes in intracellular Ca ²⁺ concentration are measured by an intracellular ion measuring device [CAF-
110 (manufactured by JASCO Corporation)], the fluorescence values of 405 nm and 500 nm by excitation light of 340 nm are measured, and the ratio of the fluorescence value of 405 nm and the fluorescence value of 500 nm (fluorescence value of 405 nm / fluorescence value of 500 nm) is used. Tracked. Ca ²⁺ is incorporated into microsomes by adding 2 mM ATP, Ca ²⁺ is released by adding 1 mM IP ₃ , and finally 2 mM ionomycin (Calbio Chem) is added. All Ca ^{2+ in} microsomes was released.

FIG. 7 shows the ratio of fluorescence values immediately before and immediately after the addition of IP ₃ with the ratio of fluorescence values at the time of maximum Ca ²⁺ release in microsomes by ionomycin being taken as 1. Anti IP ₃
There is no difference in the ratio of the initial fluorescence value immediately before the addition of IP ₃ regardless of whether or not the receptor type 3 monoclonal antibody is added, and thus the amount of Ca ²⁺ incorporated into the microsomes after the addition of ATP is the same. I understand. Therefore, anti
Ca by adding IP ₃ receptor type 3 monoclonal antibody
Uptake of ²⁺ into microsomes is not inhibited. However, Ca ²⁺ release from IP ₃ after addition microsomes anti IP ₃
It was suppressed depending on the concentration of the receptor type 3 monoclonal antibody added.

[IP]₃KM231, which does not recognize the receptor, is anti-I
P₃Receptor type 3 monoclonal antibody reveals Ca
²⁺Ca, even at concentrations where release is observed²⁺Emission suppression
The system was not recognized. From the above, anti-IP₃Receptor
Type 3 monoclonal antibody is IP₃Connected to receptor type 3
IP by combining₃IP₃For binding to receptor type 3
More triggered Ca ²⁺To specifically suppress the release of
won.

Example 4 (1) Immune Cell Staining Using Anti-IP ₃ Receptor Type 2 Monoclonal Antibody and Anti-IP ₃ Receptor Type 3 Monoclonal Antibody Jurkat cells were suspended on a slide glass coated with poly-L-lysine. The suspension was added dropwise, and the mixture was incubated at 37 ° C for 15 minutes in a 5% CO ₂ incubator. After washing with PBS buffer, the cells were fixed by immersing them in 4% PFA prepared at the time of use and leaving them on ice for 30 minutes. After washing with PBS buffer, P
It was treated with BS-0.1 Triton at room temperature for 10 minutes. Then, using 2% normal horse serum and blocking at room temperature for 30 minutes, 2 μg / ml of anti-IP ₃ receptor type 2 monoclonal antibody or purified antibody of anti-IP ₃ receptor type 3 monoclonal antibody was added, and the mixture was incubated at 4 ° C. Allowed to react overnight. PBS
After washing with a buffer solution, 2 μg / ml of biotinylated anti-mouse immunoglobulin antibody (manufactured by vector) was added and the mixture was allowed to stand at room temperature for 1
Allowed to react for hours. After washing well with PBS buffer, 2μ
Fluorescein avidin DCS (manufactured by vector) of g / ml was added and reacted at room temperature for 30 minutes. After thoroughly washing with PBS buffer, PBS-80 glycerol encapsulating material was added, and then observed with a fluorescence microscope.

As shown in Table 3, both the anti-IP ₃ receptor type 2 monoclonal antibody and the anti-IP ₃ receptor type 3 monoclonal antibody strongly react with Jurkat cells, and these antibodies can be applied to cytodiagnosis. Was shown.

[0094]

[Table 3]

(2) Immunohistological staining using anti-IP ₃ receptor type 2 monoclonal antibody and anti-IP ₃ receptor type 3 monoclonal antibody Unfixed rat submandibular gland and brain tissue were frozen and embedded in OCT compound, It was sliced to a thickness of 4 mm and attached to a slide glass coated with poly L-lysine. Fix this on ice for 30 minutes with 4% PFA prepared just before use, and
Washed with S buffer. Then add 2% normal horse serum,
After blocking at room temperature for 30 minutes, purified anti-IP ₃ receptor type 2 monoclonal antibody or anti-IP ₃ receptor type 3 monoclonal antibody 2 μg / ml was added, and the mixture was added at 4 ° C.
And reacted overnight at. 2 μg / m after washing with PBS buffer
l biotinylated anti-mouse immunoglobulin antibody (vector
(Manufactured by the company) was added and the reaction was carried out at room temperature for 1 hour. After thorough washing with a PBS buffer, p-dimethylaminoazobenzene (DAB) was used to develop color by the VECTASTAIN ABC method (manufactured by vector). Next, Methyl Green (Muto Chemical Co., Ltd.)
After nuclear staining with Permount (Fisher Scientifi
(manufactured by Company c) and then observed with an optical microscope.

As shown in Table 4, the anti-IP ₃ receptor type 2 monoclonal antibody reacted with the striatal duct of the submandibular gland. On the other hand, the anti-IP ₃ receptor type 3 monoclonal antibody reacted well with cerebellar astrocytes. Thus these monoclonal antibodies are useful to diagnose tissue-specific expression of IP ₃ receptor type 2, IP ₃ receptor type 3 is shown.

[0097]

[Table 4]

Immunohistochemical staining was performed on mouse submandibular duct epithelial cells, duodenal goblet cells, epididymal spermatic epithelial cells, bronchial epithelial secretory cells and brain glial cells using the same procedure. The results are shown in Table 5.

[0099]

[Table 5]

Mouse submandibular duct epithelial cells, duodenal goblet cells, epididymal spermatic duct epithelial cells and bronchial epithelial secretory cells are anti-IP ₃ receptor type 1 monoclonal antibody KM11.
12 and anti-IP ₃ receptor type 3 monoclonal antibody K
It did not stain with M1082, and it was possible to stain only with the anti-IP ₃ receptor type 2 monoclonal antibody KM1083. For brain glial cells, it was possible to stain only with anti-IP ₃ receptor type 3 monoclonal antibody KM1082. Thus, in order to analyze the IP ₃ receptor in each tissue, not only anti-IP ₃ receptor type 1 monoclonal antibody but also anti-IP ₃ receptor type 2 monoclonal antibody and anti-IP ₃ receptor type 3 monoclonal antibody Has been shown to be essential.

[0101]

INDUSTRIAL APPLICABILITY According to the present invention, IP ₃ receptor type 1,
It is possible to provide a monoclonal antibody which does not react to type 3 and reacts to type 2 and a monoclonal antibody which does not react to types 1 and 2 of IP ₃ receptors but reacts to type 3. Monoclonal antibodies having such specificity is useful for diagnosis histology and cell, by controlling the function of the IP ₃ receptor can reveal a role in cell of IP ₃ receptor.

[0102]

[Sequence listing] SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: Human sequence Cys Leu Gly His Pro Pro His Met Asn Val Asn Pro Gln Gln Pro Ala 1 5 10 15

SEQ ID NO: 2 Sequence Length: 16 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: Human Sequence Cys Leu Gly Ser Asn Thr Pro His Val Asn His His Met Pro Pro His 1 5 10 15

SEQ ID NO: 3 Sequence Length: 16 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: Human Sequence Cys Arg Gln Arg Leu Gly Phe Val Asp Val Gln Asn Ser IIe Ser Arg 1 5 10 15

SEQ ID NO: 4 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: Human Sequence Cys Gly Glu Ser Leu Ala Asn Asp Phe Leu Tyr Ser Asp Val Ser Arg 1 5 10 15

SEQ ID NO: 5 Sequence Length: 16 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: Human Sequence Cys Gly Ser His Gln Val Pro Thr Met Thr Leu Thr Thr Met Met Glu 1 5 10 15

SEQ ID NO: 6 Sequence Length: 16 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Origin: Human Sequence Cys Ser Pro Leu Gly Met Pro His Gly Ala Ala Ala Phe Val Asp Thr 1 5 10 15

[0108]

[Brief description of drawings]

1 shows IP3RC1, IP3RC2 and IP3R
The measurement result of the antibody titer of each antiserum with respect to C3 peptide is shown.

FIG. 2 shows IP3RL1, IP3RL2 and IP3R
The measurement result of the antibody titer of each antiserum with respect to L3 peptide is shown.

FIG. 3 shows the binding reactivity of anti-IP ₃ receptor monoclonal antibody to a synthetic peptide-THY complex.

FIG. 4 shows the reaction specificity of an anti-IP ₃ receptor monoclonal antibody for the IP ₃ receptor.

FIG. 5 shows the effect of synthetic peptides on the reaction specificity of an anti-IP ₃ receptor monoclonal antibody for the IP ₃ receptor.

FIG. 6 shows the reaction specificity of anti-IP ₃ receptor monoclonal antibody.

Figure 7 shows a functional inhibition by anti-IP ₃ receptor monoclonal antibodies to IP ₃ receptor.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 1, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

1 shows IP3RC1, IP3RC2 and IP3R
The measurement result of the antibody titer of each antiserum with respect to C3 peptide is shown.

FIG. 2 shows IP3RL1, IP3RL2 and IP3R
The measurement result of the antibody titer of each antiserum with respect to L3 peptide is shown.

FIG. 3 shows the binding reactivity of anti-IP ₃ receptor monoclonal antibody to a synthetic peptide-THY complex.

FIG. 4 shows the reaction specificity of an anti-IP ₃ receptor monoclonal antibody for the IP ₃ receptor. (It is a photograph of electrophoresis.)

FIG. 5 shows the effect of synthetic peptides on the reaction specificity of an anti-IP ₃ receptor monoclonal antibody for the IP ₃ receptor. (It is a photograph of electrophoresis.)

FIG. 6 shows the reaction specificity of anti-IP ₃ receptor monoclonal antibody. (It is a photograph of electrophoresis.)

Figure 7 shows a functional inhibition by anti-IP ₃ receptor monoclonal antibodies to IP ₃ receptor.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 21/08 9358-4B G01N 33/53 DY 33/536 DE 33/577 B // ( (12) C12P 21/08 C12R 1:91) (72) Inventor Yama ▲ Saki ▼ Motoki 3-9-13 Nakamachi, Machida-shi, Tokyo (72) Inventor Mamoru Hasegawa 1-9-26 Katahira, Aso-ku, Kawasaki-shi, Kanagawa

Claims (12)

[Claims] 1. A monoclonal antibody which specifically recognizes inositol 1,4,5-trisphosphate receptor type 2 or inositol 1,4,5-trisphosphate receptor type 3. 2. Inositol 1,4,5-trisphosphate receptor type 2 recognizes the epitopes present at positions 2687-2701 from the N-terminal amino acid of inositol 1,4,5-inositol
-Triphosphate acceptor type 1 and inositol 1,4,5
-The monoclonal antibody according to claim 1, which does not recognize triphosphate receptor type 3. 3. Inositol 1,4,5-trisphosphate receptor type 3 recognizes an epitope existing at positions 2657 to 2671 from the N-terminal amino acid, and inositol 1,4,5
-Triphosphate acceptor type 1 and inositol 1,4,5
-The monoclonal antibody according to claim 1, which does not recognize triphosphate receptor type 2. 4. Inositol 1,4,5-trisphosphate receptor type 3 recognizes an epitope existing at 2410 to 2424th position from the N-terminal amino acid of inositol 1,4,5.
-Triphosphate acceptor type 1 and inositol 1,4,5
-The monoclonal antibody according to claim 1, which does not recognize triphosphate receptor type 2. 5. The monoclonal antibody according to claim 2, which is capable of detecting inositol 1,4,5-trisphosphate receptor type 2 by Western blotting, immunohistochemical staining, or immunocyte staining. 6. Inositol 1,4,5-trisphosphate receptor type 3 can be detected by Western blotting, immunohistological staining, and immunocyte staining, and inositol 1,
The monoclonal antibody according to claim 3, which inhibits the function of 4,5-trisphosphate receptor type 3. 7. The monoclonal antibody according to claim 4, which is capable of detecting inositol 1,4,5-trisphosphate receptor type 3 by Western blotting, immunohistological staining, or immunocyte staining. 8. The monoclonal antibody according to claim 5, which is selected from KM1083 and KM1085. 9. The monoclonal antibody according to claim 6, which is selected from KM1081 and KM1082. 10. The monoclonal antibody according to claim 7, which is KM1109. 11. A method for detecting an inositol 1,4,5-trisphosphate receptor subtype by an immunocyte staining method using the monoclonal antibody according to claim 1, 2, 3, 4, 5, 6 or 7. 12. A method for detecting an inositol 1,4,5-trisphosphate receptor subtype by an immunohistochemical staining method using the monoclonal antibody according to claim 1, 2, 3, 4, 5, 6 or 7.