JPH07138299A - Monoclonal antibody against hs1 and its production - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody against an HS1 protein for carrying out the functional analysis of the HS1 protein. CONSTITUTION:This monoclonal antibody is against HS1 or a synthetic peptide derived from the HS1. The characteristic of this method for producing the monoclonal antibody against the HS1 comprises immunizing and sensitizing a rodential animal using a synthetic peptide derived from the HS1, fusing a cell of the spleen in the immunized rodential animal to a cell of a myeloma in a mouse according to a conventional method, sorting the fused cell from the unfused cell of the myeloma, confirming the presence of an antibody using the reactivity of a supernatant containing the resultant hybridoma in each culture well toward the immunized synthetic peptide as an index, sorting the hybridoma capable of producing a desired antibody, then providing a monoclone according to a limiting dilution method and recovering the resultant monoclonal antibody from the culture supernatant of the monoclonal hybridoma. Furthermore this hybridoma is capable of producing the monoclonal antibody against the HS1.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody, and more particularly to a monoclonal antibody which specifically reacts with HS1, a method for producing the monoclonal antibody, and a hybridoma producing the monoclonal antibody.

[0002]

2. Description of the Related Art The HS1 protein is a molecule that is specifically expressed in blood-type lymphoid cells and has a molecular weight of 75 kd. When its structure is analyzed, a domain consisting of 37 amino acids is repeated at the N-terminal side three and a half times, and at the C-terminal side, Src-homology3 is well conserved in non-receptor tyrosine kinases and cytoskeletal molecules.
It has a (SH3) area. In recent years, a protein p80 / 85 that is tyrosine phosphorylated by pp60Src was identified in a chicken embryocarcinoma cell line transformed with the Src gene. Its amino acid sequence is HS1 protein and 37 amino acid repeat sequence and S
It has been shown to have high homology in the H3 region.

In addition, it was found that the HS1 protein undergoes tyrosine phosphorylation by the 1yn protein in B cells when stimulated with an anti-μ chain antibody. From these findings, HS1 protein is
It has been suggested to be deeply involved in intracellular signal transduction via a tyrosine kinase from a membrane receptor.

[0004]

However, analysis of the function of the phosphorylated HS1 protein and how the HS1 protein is regulated expression in cells reveals that a large amount of the protein cannot be obtained. Moreover, since there is no antibody etc., detailed analysis has hardly been done. The purpose of the present invention is to analyze H1 protein in order to analyze its function.
To produce a monoclonal antibody against the S1 protein.

[0005]

Therefore, the inventor of the present invention
It was thought that the functional analysis of the HS1 protein could be easily carried out by producing a monoclonal antibody against the S1 protein, and an attempt was made to produce the antibody.

That is, by immunizing with the HS1 peptide, a hybridoma producing a monoclonal antibody that reacts with the HS1 protein itself was successfully established, and the present invention was completed. Thus, according to the invention, a monoclonal antibody against HS1, and H
Monoclonal antibodies to synthetic peptides derived from S1 are provided.

Further, according to the present invention, (1) a rodent is immunized with a synthetic peptide derived from HS1, and (2) a spleen is excised from the immunized rodent to suspend spleen cells. A suspension is formed, and (3) the splenocyte suspension is mixed with mouse myeloma cells in the presence of a fusion promoter to fuse both cells,
(4) The fused cells are diluted and cultured in a medium that does not support unfused myeloma cells to select hybridomas in which antibody-producing cells and myeloma cells are fused, and (5) in each culture well containing the hybridomas. About the supernatant of
The presence of the antibody is confirmed using the reactivity with the immunized synthetic peptide as an index, and (6) a hybridoma that produces the desired antibody is selected, and then a single clone is obtained by the limiting dilution method. There is provided a method for producing a monoclonal antibody against HS1, which comprises recovering the monoclonal antibody from the culture supernatant of a single clone hybridoma.

Further, according to the present invention, a hybridoma producing a monoclonal antibody against HS1 is provided. The hybridoma of the present invention can be obtained, for example, from the Institute of Biotechnology, National Institute of Industrial Science, Contract No. FERM P-13.
Hybridoma 2D2- deposited as 879
20 can be mentioned.

The present invention will be described in detail below. The monoclonal antibody against the HS1 protein according to the present invention and the hybridoma producing the monoclonal antibody can be produced as follows. (1) Rats were immunized with an HS1 synthetic peptide as an antigen, splenocytes were prepared from the immunized rats, and fused with mouse myeloma cells, and then a hybridoma producing a monoclonal antibody that reacts with the HS1 synthetic peptide was prepared. Selected,
(2) The hybridoma obtained in (1) is cultured under appropriate conditions to recover a monoclonal antibody, and it is confirmed that this monoclonal antibody also reacts with the HS1 protein, and finally, the monoclonal antibody against the HS1 protein. Selecting a hybridoma that produces (3)
The monoclonal antibody is recovered by culturing the hybridoma obtained in (2). The monoclonal antibody against HS1 of the present invention has the specificity shown in the following examples.

[0010]

The present invention will be described in more detail with reference to the following examples.

[Example 1] 1. Preparation of Monoclonal Antibody (A) Immunization 500 μg of HS1 peptide (8 mg) bound to KLH (5 mg) was mixed with Freund's complete adjuvant in an equal amount, and the mixture was intraperitoneally inoculated into Wistar rats. Peptide-KLH after 2 weeks and 4 months
Was mixed with Freund's incomplete adjuvant in an equivalent amount, and the mixture was intraperitoneally inoculated into a rat, and immunization was performed three times in total.

(B) Fusion The spleen was taken out from the rat immunized as described above. Next, after cutting the removed spleen, it is filtered with a mesh to remove RP.
Suspension in MI1640 medium gave 2 × 10 ⁸ splenocytes. These splenocytes were derived from a mouse 8-azaguanine resistant strain (hypoxanthine guanine phosphoribosyl transferase deficient strain) X63Ag8.653 (2 × 10 ⁷ cells).
And mixed at a ratio of about 5: 1 and centrifuged (1500 rp
m, 5 minutes). To the obtained cell pellet, 1 ml of a 50% polyethylene glycol 4000 (manufactured by Merck) / MEM solution (37 ° C) was added as a fusion promoter, and the mixture was allowed to stand at room temperature for 2 hours.
The cells were fused by stirring for a minute. After fusion, a large amount (about 4
0 ml) of MEM solution was gradually added, and the mixture was allowed to stand at room temperature for 30 minutes and then centrifuged to remove the supernatant. Then hypoxanthine (100 μM), aminobuterin (0.4 μM
M), and 10% FBS-containing thymidine (10 μM).
The number of splenocytes was 5 in RPMI1640 medium (HAT) medium.
It was adjusted to be 10 ⁵ cells / ml.

(C) Selection of hybridoma The cell suspension prepared in the above B was used for 24 wells (culture holes).
Dispense 1 ml each onto 18 microplates, 37 ° C 5
Cells were cultured in a CO ₂ incubator under% CO ₂ . After 2 weeks, it was confirmed that only the hybridoma formed colonies and grew.

(D) Detection of antibody Since it was confirmed that the hybridoma was sufficiently proliferated, the culture supernatant was used to detect the antibody by the enzyme-linked immunosorbent assay (ELISA) described below. The HS1 peptide bound to BSA was dispensed in 50 μl aliquots at a concentration of 10 μg / ml into an assay plate (Dynatech Imron 2) and antigen-fixed (4 ° C./overnight). Then
After removing the antigen solution, blocking solution (5% dry mil
k.PBS) was added to block (room temperature, 2
h). After that, the blocking solution was removed, and 45 μl of the hybridoma supernatant was dispensed and reacted at room temperature for 2 hours. After washing the plate with PBS / Tween 20, 50 μl of a 500-fold diluted solution of perefoxidase-labeled anti-rat Ig antibody was added and allowed to bind (room temperature, 1 hour). Then, it was washed again with PBS / Tween 20 and then tetramethylbenzidine (0.01
mg / ml) and 50 μl of sodium acetate solution (pH 6.0) containing 0.012% hydrogen peroxide solution are added to these plates, and 10 to 30 minutes later, 50 μl of 1M sulfuric acid is further added to allow the antibody to react by a color reaction. Detected. As a result, H
Wells were selected that reacted with the S1 peptide.

(E) Cloning The hybridoma growing in the well selected in the above D was cloned by the limiting dilution method. 10% FCS / RPMI1 so that the cell concentration after dilution is 1 cell / well
It was diluted with 640 medium and 100 μl was dispensed to a 96-well microplate. Culturing was carried out at 37 ° C. in the presence of 5% CO ₂ , and after the colonies had grown to some extent, the detection of the antibody against the HS1 peptide and the morphology of the colonies were examined. As a result, 2D2-20 clone that reacts with the HS1 peptide could be obtained.

2. Specificity of Monoclonal Antibody (Western Blot) Whether the obtained monoclonal antibody reacts with HS1 protein or not was subjected to Western blot using mouse B cell WEHI231. 1x WEHI231 cells
After culturing 10 ⁷ cells, they were collected and dissolved. This is 10% S
It was electrophoresed by DS-PAGE. Transfer to a nitrocellulose membrane at 10V. Nitrocellulose membrane with 5% dry milk / TBS
Blocked at T for 1 hour at room temperature. After washing with TBS (TBST) containing 0.05% Tween 20 for 5 minutes, 2
1 ml of culture supernatant of D2-20 hybridoma was added,
The reaction was carried out at room temperature for 1 hour. 5 in TBS with Tween 20
After washing for 3 times for 3 minutes, the alkaline phosphatase-labeled anti-rat IgG antibody diluted 500 times was used for 1 m.
1 was added.

After reacting at room temperature for 1 hour, a washing operation was performed. After that, NBT (nitro blue tetrazolium) 1
0 mg and BCIP (bromochloroindolyl phosphate) 5 mg were added to 100 mM Tris-hydrochloric acid pH 9.
5, 100 mM NaCl, 5 mM MgCl ₂ 30 m
Color was developed using 1 liter of liquid. As a result, it was found that it specifically reacts with the protein of 75 Kd. Moreover, this molecular weight was in agreement with the molecular weight of the HS1 protein deduced from the HS1 cDNA, and it was confirmed that the monoclonal antibody of the present invention specifically recognizes the HS1 protein.

The hybridoma was deposited under the contract number FERM P-13 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
Hybridoma 2D2- deposited as 879
Twenty.

[0019]

The monoclonal antibody against the HS1 protein of the present invention reacts with intracellular HS1.
The distribution of HS1 in cells can be analyzed, and the ratio of HS1 to phosphorylated proteins and the function of HS1 in various activation reactions involving phosphorylation can be analyzed. Further, it becomes possible to analyze whether or not this HS1 protein moves into the nucleus with phosphorylation.

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Claims (6)

[Claims] 1. A monoclonal antibody against HS1. 2. A monoclonal antibody against a synthetic peptide derived from HS1. 3. (1) Immunizing a rodent with a synthetic peptide derived from HS1, (2) extracting the spleen from the immunized rodent to form a splenocyte suspension, (3) The splenocyte suspension is mixed with mouse myeloma cells in the presence of a fusion promoter to fuse both cells, and (4) the fused cells are diluted in a medium that does not support unfused myeloma cells. And culturing is performed to select hybridomas in which antibody-producing cells and myeloma cells are fused, and (5) the supernatant liquid in each culture well containing the hybridomas is used as an index for the reactivity with the immunized synthetic peptide. (6) After selecting a hybridoma that produces the desired antibody, the clone was made into a single clone by the limiting dilution method, and (7) the monoclonal antibody was recovered from the culture supernatant of the hybridoma of this single clone. What to do A method for producing a monoclonal antibody against HS1, which comprises: 4. The method according to claim 3, which further confirms that the recovered monoclonal antibody has a feature of identifying the HS1 protein by Western blotting with cells expressing the HS1 protein. Production method. 5. The rodent is Wistar rat,
In addition, mouse myeloma cells are P3X63Ag8.6.
The manufacturing method according to claim 3, which is 53. 6. A hybridoma producing a monoclonal antibody against HS1.