JPH07111888A - The hybridoma, the monoclonal antibody produced by this hybridoma, and the mouse lymphocyte activation antigen recognized by this monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a hybridoma originated from a fused cell of e.g. spleen cell of hamster immunized with an immature thymocyte of mouse, capable of finely and functionally fractionating the immature thymocyte and useful for the treatment, etc., of immune system diseases, etc. CONSTITUTION:This hybridoma is FERM P-13893 originated from a fused cell of a mouse myeloma cell and a spleen cell of hamster immunized with an immature thymocyte of mouse. The monoclonal antibody produced by the hybridoma efficiently recognizes a mouse lymphocyte activation antigen which is a GPI anchor protein having a molecular weight of about 12 kDa and expressing in about 80% of mouse immature thymocyte.

Description

Detailed Description of the Invention

[0001]

This invention relates to a hybridoma,
The present invention relates to a monoclonal antibody produced by this hybridoma and a mouse lymphocyte activation antigen recognized by this monoclonal antibody. INDUSTRIAL APPLICABILITY The monoclonal antibody and activating antigen of the present invention are useful, for example, in basic research fields related to the immune system of the living body, medical fields related to immune system diseases, and the like.

[0002]

[Prior art and its problems] Expression of immune response in living body
T cells that play a central role in regulation are known to differentiate from T precursor cells to mature T cells in the thymus. T progenitor cells migrate to the thymus and then rapidly divide and proliferate, and at the same time, T cell receptor (TCR receptor) gene rearrangement occurs, and the gene product is expressed on the cell surface. . The repertoire of antigens recognized by T cells is considered to be largely determined during the differentiation process in the thymus because the TCR gene causes almost no somatic mutation.

The selection of this repertoire of T cells is based on the fact that CD4 ⁺ 8 ⁺ (double positive; hereinafter DP) in immature thymocytes is selected.
It is known to occur at the cell stage.
That is, DP cells recognize the major histocompatibility complex of stromal cells via the TCR molecule and are positively selected or negatively removed. Thus, DP cells are a heterogeneous cell population, but there are few markers that further finely and functionally fractionate this population.

In recent years, it has become clear that an activating antigen expressed when lymphocytes such as CD69 are activated can serve as an important marker for functionally fractionating DP cells. However, the means for identifying, activating and separating / extracting such an activation antigen have not been known so far, and the characteristics of the lymphocyte activation antigen itself have not been clarified at present.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a monoclonal antibody that specifically recognizes a mouse lymphocyte activation antigen, and a hybridoma that produces this antibody. I am trying. It is also an object of the present invention to make industrial use by clarifying the characteristics of the lymphocyte activation antigen recognized by the above monoclonal antibody.

[0006]

As a solution to the above problems, the present invention provides a hybridoma derived from a fused cell of a hamster spleen cell immunized with a mouse immature thymocyte and a mouse myeloma cell. To do. The present invention also provides a monoclonal antibody produced by the above hybridoma.

The present invention further provides a GPI having a molecular weight of about 12 kDa which is expressed in about 80% of immature mouse thymocytes.
It also provides a mouse lymphocyte activation antigen, which is an anchor protein and is recognized by the above-mentioned monoclonal antibody. Hereinafter, the configuration of the present invention will be described in detail. (1) Production of hybridoma The method for producing a hybridoma producing the monoclonal antibody of the present invention is a known method (Nature, 256, p49.
5, 1975) and the like, for example, as follows. 1) Sensitization of animals Using thymocytes of mice as an antigen, admixing it with complete Freund's or incomplete Freund's adjuvant, and injecting hamsters 1 to 4 times at intervals of 1 week to several months, To sensitize. 2) Cell fusion Spleen cells extracted from the immunized animal and mouse myeloma cells are fused by a conventional method to obtain fused cells. As the mouse myeloma cells, SP2 / 0-Ag14 and the like can be used. Although there are several known methods for cell fusion, for example, a method using polyethylene glycol (hereinafter sometimes referred to as PEG) or a method using electrofusion can be used. In the present invention, fused cells can be obtained by any of these methods. 3) Selection of fused cells Thymocytes are stained with the culture supernatant of the fused cells obtained by the above method, and analyzed using a flow cytometer (flow cytometry) to determine the cell surface antigen of the thymocytes. Among the antibodies recognizing., Those showing a staining pattern different from the known antigen are selected. Furthermore, considering that many known activating antigens are not expressed in unstimulated peripheral T cells, antibody-producing strains that stain thymocytes and do not stain peripheral T cells among the above antibodies were selected. . 4) Cloning In order to make the specific antibody-producing cells selected above into a uniform cell population derived from one clone, a cloning operation is performed using a known limiting dilution method to stabilize the target monoclonal antibody. A cell line which produces so-called hybridoma is obtained.

The hybridoma obtained as described above has a functional characteristic of producing a monoclonal antibody that specifically recognizes a previously unknown mouse lymphocyte activation antigen. As a typical example, a hybridoma obtained by fusing hamster spleen cells and mouse myeloma cells was designated as hybridoma PRST1 and deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and FE
The accession number RM P-13893 was obtained.

This hybridoma PRST1 has the following properties. a) Origin: hybridoma, which is a fusion cell of Armenian hamster spleen cells and mouse myeloma cells SP2 / 0-Ag14 b) Form: non-spindle-like, almost spherical form c) Subculture: Possible d) Functional characteristics: Produces a monoclonal antibody that specifically recognizes mouse lymphocyte activation antigen e) Cell proliferation: Good both in attachment and suspension f) Storage conditions: Storage in liquid nitrogen at −80 ° C. or lower g) Serum requirement: Usually cultured in a medium supplemented with 10% fetal calf serum (2) Acquisition of antibody The antibody of the present invention is inoculated with the hybridoma from the culture supernatant of the above hybridoma,
The biological exudate obtained by in-vivo culturing can be purified by a biochemical method commonly used for protein purification.

Among the several monoclonal antibodies thus obtained, the antibody of the present invention is a hamster-derived antibody which has been confirmed to specifically react with the mouse lymphocyte activation antigen described below. It is characterized by being. The antigen recognized by the monoclonal antibody of the present invention obtained as described above has the following functional and physical properties.

The antigen recognized by the above-mentioned monoclonal antibody is expressed in about 80% of mouse DP cells, but more mature CD4 ⁺ 8 ⁻ or CD4 ⁻ 8 ⁺
The expression decreases rapidly in (single positive, SP) cells, and the expression is not observed in peripheral T cells. Even in peripheral T cells, it was
This antigen is an activation antigen of mouse lymphocytes because its expression becomes visible when stimulated with nA or the like.

This antigen is also expressed on mouse B cells, since its expression is enhanced when mouse B cells are stimulated with LPS. The cells are treated with PI-specific phospholipase C (PI-
This antigen is a GPI-anchored protein because its cell surface expression level is significantly reduced by treatment with PLC).

The molecular weight of this antigen, quantified by immunoprecipitation using solubilized hybridomas, is approximately 12 kDa under reducing conditions. The lymphocyte activation antigen of the present invention recognized by the monoclonal antibody of the present invention and confirmed to have the above-mentioned properties can be a good marker for functionally fractionating DP cells. And, it is considered that many findings obtained as a result will increase the understanding of differentiation and activation of immune cells and will greatly contribute to the establishment of a new classification diagnostic method for leukemia and the like in the future.

Next, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0015]

【Example】

Example 1 (Creation of hybridoma) (1) Immunization of animal 5 × mouse thymocytes suspended in 1 ml of phosphate buffer
10 ⁷ cells were intraperitoneally administered to 6-week-old hamsters. Booster immunization was performed every 2-3 weeks until a sufficiently high antibody titer was observed. (2) Cell fusion The spleen of a hamster with high antibody titer was removed. PEG1500 of mouse myeloma cells SP2 / 0-Ag14 and the hamster splenocytes at a cell number ratio of 1: 2.
Was fused using. The fused cells were suspended in medium and
Each well of the well plate was dispensed and cultured at 37 ° C. under 5% CO ₂ for 7 days. (3) Screening and Cloning A portion of the culture supernatant of each well in which the growth of the hybridoma was completed was sampled, thymocytes were stained with each, and these were analyzed by a flow cytometer, and the cell surface of thymocytes was analyzed. Among the antibodies recognizing the antigen, those showing a staining pattern different from that of the known antigen were selected, and the target hybridoma was screened using this as an index. Next, among the above-mentioned antibodies, antibodies that stain thymocytes and not peripheral T cells were selected, and the wells of the antibody-producing strains were screened by the limiting dilution method.

Thus, the hybridoma PRST1 producing the monoclonal antibody of the present invention was produced. Example 2 (acquisition of monoclonal antibody) The hybridoma PRST1 obtained in Example 1 was cultured,
Monoclonal antibody PRST1 was purified from the culture supernatant using a protein A sepharose column. Example 3 (isolation of mouse lymphocyte activation antigen) Using the monoclonal antibody PRST1 obtained in Example 2,
Mouse lymphocyte activation antigen was isolated.

That is, the monoclonal antibody PRST1
The Sepharose-beads to which was bound were prepared and used to immunoprecipitate the antigen from the T cell hybridoma expressing the lymphocyte activation antigen. When analyzed by electrophoresis, this antigen protein showed a molecular weight of 12 kDa under reducing conditions.

[0018]

As described in detail above, the present invention provides a mouse lymphocyte activation antigen, a monoclonal antibody that specifically recognizes this antigen, and a hybridoma that produces this monoclonal antibody. This makes it possible to finely and functionally fractionate immature thymocytes, and open a way to elucidate the mechanism of differentiation and activation of immune cells.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12N 15/02 C12P 21/08 9161-4B // (C12P 21/08 C12R 1:91) (54 ) [Title of Invention] Hybridoma, monoclonal antibody produced by this hybridoma, and mouse lymphocyte activation antigen recognized by this monoclonal antibody

Claims (4)

[Claims] 1. A hybridoma derived from a fusion cell of spleen cells of a hamster and mouse myeloma cells immunized with immature mouse thymocytes. 2. The hybridoma strain is FERM P-13.
The hybridoma of claim 1, which is 893. 3. A monoclonal antibody produced by the hybridoma according to claim 1 or 2. 4. A mouse lymphocyte activating antigen which is a GPI anchor protein having a molecular weight of about 12 kDa which is expressed in about 80% of immature mouse thymocytes, and which is recognized by the monoclonal antibody of claim 3.