JPH054074B2 - - Google Patents

Description

[Detailed description of the invention]

In recent years, the human major histocompatibility system (abbreviated as HLA)
The importance and demand for HLA analysis increases as research on HLA is introduced into the areas of organ transplantation, disease associations, immune response mechanisms, anthropology, genetics, transfusion science, and forensic medicine, with concomitant increases in selectivity and activity. strong
Obtaining HLA antibodies has become important. Traditionally, HLA analysis required collecting blood samples from pregnant women.
HLA antibodies have been used, but there are limits to their quantity, and there is a desire for antibodies with even higher levels of activity. Recently, methods using hybridomas made by fusion of lymphocytes and myeloma cells are entering the practical stage in the field of antibody production, and methods using mouse hybridomas for HLA antibodies are also being researched and put into practical use. However, when producing antibodies that recognize HLA antigens, which are human alloantigens, using mouse lymphocytes, it is difficult to obtain highly specific antibodies. Under such circumstances, the inventor of this invention
By immunizing mice with human B lymphocytes containing human HLA-DR antigen, extracting B lymphocytes from the immunized mice, fusing them with mouse myeloma cells, and cloning them, they can be extracted from the blood of pregnant women. Replace with collected HLA antibody
We succeeded in obtaining monoclonal antibodies that can be used for HLA analysis. These monoclonal antibodies have novel type specificity for HLA-DR antigens. This invention provides five monoclonal antibodies with novel type specificity for HLA-DR antigens, produced by mouse hybridomas,
HLA-DR2, HLA-DRw6.2 and HLA-
It reacts with cultured human lymphoblastoid cells that have DRw9, but does not have the above antigen and has HLA-DR1,
HLA-DRw6, HLA-DR7 and HLA-
An immunoglobulin _G2a class antibody that does not react with cultured human lymphoblastoid cells with 8wDRw6Y, produced by a mouse hybridoma,
HLA−DR1, HLA−DR2, HLA−
Reacts with cultured human lymphoblastoid cells that have 8wDRw6Y and HLA-DRw9, but does not react with cultured human lymphoblastoid cells that do not have the above antigen and have HLA-DRw6.2, HLA-DRw6, and HLA-DR7. Immunoglobulin G ₂ a class antibodies, produced by mouse hybridomas,
HLA−DR1, HLA−DR2, HLA−
Reacts with cultured human lymphoblastoid cells that have 8wDRw6Y and HLA-DRw9, but does not react with cultured human lymphoblastoid cells that do not have the above antigen and have HLA-DRw6.2, HLA-DRw6, and HLA-DR7. Immunoglobulin M class antibodies, produced by mouse hybridomas,
HLA−DR1, HLA−DR2, HLA−
8wDRw6Y, HLA−DRw6.2 and HLA−
An antibody of the immunoglobulin G ₂ a class that reacts with cultured human lymphoblastoid cells having DRw9 but not with cultured human lymphoblastoid cells having HLA-DRw6 and HLA-DR7 without the antigen, mouse・Produced by hybridoma,
HLA−8wDRw6Y, HLA−DRw6.2, HLA−
It reacts with cultured human lymphoblastoid cells that have DR7 and HLA-DRw9, but does not have the above antigens and reacts with HLA-DR1, HLA-DR2 and HLA-
The present invention relates to a monoclonal antibody that is an immunoglobulin G ₃ class antibody that does not react with cultured human lymphoblastoid cells having DRw6. Next, methods for producing the monoclonal antibodies of the present invention and mouse hybridomas that produce these monoclonal antibodies will be explained. Human B lymphocytes Sh (or HO or
Infect HOR or Ky) with EB virus and in
Lymphoblastoid cells that can proliferate in vitro. On the other hand, LG-10, a human cultured lymphoblastoid cell that has homozygous HLA-D and HLA-DR antigens,
is directly grown in vitro and used. After sensitizing mice with each of the lymphoblastoid cells obtained by the above method, the spleen is removed from each mouse and a splenocyte suspension is prepared. Each was then mixed with myeloma cells from a mouse (the same strain of mouse that sensitized the lymphoblastoid cells).
Fuse using polyethylene glycol. After culturing each hybridoma obtained by the above method in a conventional manner, those that are positive for cytotoxicity against the corresponding human B lymphocyte Sh (or HO or HOR or Ky or LG-10) are selected. Each of the things obtained in this way is
For example, cloning is performed by a conventional method such as a method using a fibrin gel plate, and the specificity of the produced monoclonal antibody is examined to obtain the desired monoclonal antibody. Further, the mouse hybridoma of interest can be obtained by collecting a mouse hybridoma producing the monoclonal antibody of interest by a conventional method. The above operating method will be explained in more detail below. As an embodiment of sensitizing a mouse with human B lymphocytes, for example, about 2 to 4 x 10 ⁷ cultured human B lymphocytes are first administered into the abdominal cavity of the mouse to sensitize it, and then as needed. For example, after 1 to 3 weeks, approximately 2 to 4 x 10 ⁷ cultured human B lymphocytes may be injected intravenously or/and intraperitoneally, but the key is to inject the human B lymphocytes into the mouse. It is only necessary to obtain the required antibody-producing cells by sensitization, and therefore, it is needless to say that the method is not limited to the embodiments described above. The mouse used to obtain the monoclonal antibody of this invention is not particularly limited, but in order to obtain hybridomas relatively easily, use a mouse of the same strain as the mouse from which the myeloma cells to be fused are derived. It is desirable to use Table 1 shows the properties of the human B lymphocytes used first as antigens and later in the cytotoxicity test against cultured lymphoblastoid cells.

[Table] Table 2 shows the properties of the obtained monoclonal antibodies.
It is as follows.

[Table] Various ¹²⁵ I monoclonal antibodies obtained
Table 3 shows the binding properties with -labeled membrane antigens and the immunoglobulin classes determined by the Ouchterloni method.

[Table] Next, the monoclonal antibodies obtained by this invention
The reaction against T lymphocytes and B lymphocytes isolated from human peripheral blood of known HLD-DR type was investigated using a conventional HLA typing cytotoxicity test, and the results showed the following specificity. Number of SH-2-60-1 antibody samples: 54

【table】

[Table] Unknown This antibody did not react with the T lymphocytes of each sample. Number of HO-3-43-4 antibody samples: 54

【table】

[Table] Unknown This sample did not react with the T lymphocytes of each sample. Number of HOR-2-16-2 antibody samples: 54

[Table] Unknown This antibody did not react with the T lymphocytes of each sample. *1x intraperitoneally into mice (Balb/c, female) that had been administered pristane one week beforehand.
10 ^Seven hybridomas were transplanted, and 14
A day later, the abdomen was opened and ascites was obtained. Number of Ky-6-16-23 antibody samples: 54

[Table] Unknown This antibody did not react with the T lymphocytes of each sample. Number of Dw7-3-5-10 antibody samples: 53

[Table] Unknown This antibody did not react with the T lymphocytes of each sample. *1×10 ⁷ hybridomas were transplanted into mice (Balb/c, female) to which pristane had been administered one week beforehand, and 14 days after transplantation, the abdomen was opened to obtain ascites. The above data revealed that all the monoclonal antibodies of the present invention have a high correlation with HLA antigens, which are human alloantigens, and can be used in place of multiparous HLA antibodies for HLA analysis. Next, the present invention will be explained in more detail based on examples. Example 1 Lymphoblastoid cells obtained by infecting cultured human lymphocytes Sh with EB virus were incubated in vitro.
and the cells were added with 10% fetal bovine serum.
The cells were cultured and grown in RPMI1640 medium at 37°C in a stream of 5% carbon dioxide gas, and 4 x 10 ⁷ cells were suspended in physiological saline and administered into the abdominal cavity of Balb/c mice (female). 24 days after administration, an additional 1.5 x 10 ⁷ of the above cells
1.5×10 ⁷ cells were administered intraperitoneally.
Three days later, the mouse's abdomen was opened, the spleen was removed, and a splenocyte suspension was prepared. The splenocyte suspension and mouse myeloma P3/×63Ag8U1 cell suspension (myeloma derived from Ba1b/c mice) were mixed at a cell ratio of 10:1 and centrifuged, followed by 45% polyethylene glycol (Sigma, molecular weight 4000). ) was gradually added dropwise and allowed to stand at room temperature for 6 minutes to fuse the cells. The fused cells were grown in Dulbecco's modified Eagle's minimal nutrient medium supplemented with 15% fetal bovine serum (2mM glutamine, 5x
10 ^-5 M2-mercaptoethanol, 80 μg/ml gentamicin sulfate, 100 units/ml penicillin G,
Contains 100μg/ml streptomycin sulfate)
Suspended in
Only the fused cells were allowed to grow by replacing half the volume with HAT medium (hypoxanthine, amethopretene, and thymidine supplemented medium) every ~3 days. The presence or absence of antibody production in the culture medium after 11 days of culture was examined by a cytotoxicity test against Sh cells. For the cytotoxicity test, 2 μ of culture supernatant and 2 μ of cell suspension (1×10 ⁶ cells/ml) were added to each well of the HLA typing plate.
Add 5μ of rabbit complement and keep at 37℃ for 2 hours.
The method used was to observe whether the animal was alive or dead using an inverted microscope. Table 1
Among the cells listed above, cultured B lymphocytes and cultured T lymphocytes other than Sh cells (CCRF-
We investigated the effect on CEM cells and HSB-2 cells, and selected a culture solution that acts on some of the former but not the latter. The cultured cells sorted in this way were placed on a fibrin gel plate (a 6 cm diameter plate containing 1 ml of 0.25% Sigma fibrinogen solution, containing 0.02 units/ml of thrombin).
(prepared by adding 4 ml of RPMI medium) to 10, 200,
After inoculating 500 and 1000 cells and culturing for 10 days, a colony considered to be derived from a single cell was selected and used as a clone. The DR type specificity of the antibody was examined by examining the presence or absence of an effect on the cells listed in Table 1 using the culture supernatant of the cloned cells on the third day of culture under the conditions of the cytotoxicity test described above. To confirm that the antibody is an HLA-DR antibody, see Table 3.
The binding with the membrane-solubilized HLA-DR antigen labeled with ¹²⁵ I was examined by acrylamide gel electrophoresis, and it was found that the antigen reacted with 35,000 Dalton chain (α chain) and 27,000 Dalton chain (β chain). . The immunoglobulin class of the antibody was determined by the Ouchterlony method using an anti-mouse immunoglobulin heavy chain rabbit antibody, and it was determined to be immunoglobulin G _2a class based on the formation of a sedimentation line in the 20-fold concentrated culture supernatant. From the above results, mouse hybridoma SH-2-60-1 antibody is
HLA-DR2, HLA-DRw6.2 and HLA-
It was found that it specifically reacts with DRw9. Example 2 Human lymphocyte H0 with homozygous HLA-DH0
Cultured cells were infected with EB virus to produce lymphoblastoid cells that can proliferate in vitro. this cell
37°C using RPMI1640 medium supplemented with 10% fetal bovine serum.
Cultured and grown in a 5% carbon dioxide gas stream, 1.4
×10 ⁷ mice were suspended in physiological saline and administered into the abdominal cavity of Balb/c mice (♀). Seventeen days after administration, 2×10 ⁷ of the above cells were further administered intravenously and 2×10 ⁷ intraperitoneally. Three days later, the mouse's abdomen was opened, the spleen was removed, and a splenocyte suspension was prepared. The splenocyte suspension and mouse myeloma P3/×
63Ag8U1 cell suspension (myeloma from Balb/c mouse) was mixed at a cell ratio of 10:1 and centrifuged.
45% polyethylene glycol (Sigma, molecular weight
4000) was gradually added dropwise and allowed to stand at room temperature for 6 minutes to fuse the cells. The fused cells were grown in Dulbecco's modified Eagle's minimal nutrient medium (2 m
Mglutamine, 5 x 10 ^-5 M2-mercaptoethanol, 80 μg/ml gentamicin sulfate, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate) and placed in a 24-well plate from Linbro. After implantation, only the fused cells were grown by replacing half of the medium with HAT medium (hypoxanthine, amethoprythene, and thymidine-supplemented medium) the next day, the day after, and every 2 to 3 days thereafter. The presence or absence of antibody production in the culture medium after 11 days of culture was examined by a cytotoxicity test against HO cells. For the cytotoxicity test, 2μ of culture supernatant, 2μ of cell suspension (1×10 ⁶ cells/ml), and 5μ of rabbit complement were added to each well of the HLA typing plate and kept at 37°C for 2 hours. The method used was to observe whether the animal was alive or dead using an inverted microscope. Among the cells listed in Table 1, cultured B lymphocytes and cultured T lymphocytes (CCRF-CEM cells and HSB-2 cells)
We investigated the effect on the latter and selected a culture solution that acts on some of the former but not on the latter. The cultured cells thus sorted were placed on a fibrin gel plate (prepared by adding 1 ml of 0.25% Sigma fibrinogen solution to a 6 cm diameter dish and adding 4 ml of RPMI medium containing 0.002 units/ml of thrombin) for 10 minutes. , 200, 500, and 1000 cells were inoculated, and after culturing for 10 days, a colony considered to be derived from a single cell was selected and used as a clone. The DR type specificity of the antibody was examined by examining the presence or absence of an effect on the cells listed in Table 1 using the culture supernatant of the cloned cells on the third day under the conditions of the cytotoxicity test described above. To confirm that the antibody is an HLA-DR antibody, see Table 3.
The binding with the membrane-solubilized HLA-DR antigen labeled with ¹²⁵ I was examined by acrylamide gel electrophoresis, and it was found that the antigen was reacted with 35,000 Dalton chain (α chain) and 27,000 Dalton chain (β chain). . The immunoglobulin class of the antibody was determined by the Ouchterlony method using an anti-mouse immunoglobulin heavy chain rabbit antibody, and it was determined to be immunoglobulin G _2a class based on the formation of a sedimentation line in the 20-fold concentrated culture supernatant. From the above results, mouse hybridoma HO-3-43-4 antibody is
HLA−DR1, HLA−DR2, HLA−8wDRw6Y
and HLA-DRw9. Example 3 Human lymphocytes with homozygous HLA-DEn
Infect HOR cultured cells with EB virus in
The cells were made into lymphoblastoid cells that can proliferate in vitro. These cells were cultured and grown in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in a stream of 5% carbon dioxide gas.
3.5×10 ⁷ cells thereof were suspended in physiological saline and administered into the abdominal cavity of Balb/c mice (female). Ten days after the administration, 4×10 ⁷ of the above cells were further intraperitoneally administered. Four days later, the mouse's abdomen was opened, the spleen was removed, and a splenocyte suspension was prepared. The splenocyte suspension and mouse myeloma P3/×63Ag8U1 cell suspension (myeloma derived from Banb/c mouse) were mixed at 10:
After mixing at a cell ratio of 1:1 and centrifuging, 45% polyethylene glycol (Sigma, molecular weight 4000) was gradually added dropwise and allowed to stand at room temperature for 6 minutes to fuse the cells.
The fused cells were grown in Dulbecco's modified Eagle's minimal nutrient medium supplemented with 15% fetal bovine serum (2mM glutamine.
×10 ^-5 M2-mercaptoethanol, 80 μg/ml gentamicin sulfate, 100 units/ml penicillin G,
Contains 100μg/ml streptomycin sulfate)
Suspended in
Only the fused cells were allowed to grow by replacing half of the HAT medium (medium supplemented with hypoxanthine, amethopretene, and thymidine) every ~3 days. The presence or absence of antibody production in the culture medium after 11 days of culture was examined by a cytotoxicity test against HOR cells. For the cytotoxicity test, add 2 μ of culture supernatant and 2 μ of cell suspension (1×10 ⁶ cells/ml) to each well of the HLA typing plate.
Add 5μ of rabbit complement and keep at 37℃ for 2 hours.
The method used was to observe whether the animal was alive or dead using an inverted microscope. Table 1
Among the cells listed above, cultured B lymphocytes and cultured T lymphocytes other than HOR cells (CCRF-
We investigated the effect on CEM cells and HSB-2 cells, and selected a culture solution that acts on some of the former but not the latter. The cultured cells thus selected were placed on a fibrin gel plate (prepared by adding 1 ml of 0.25% Sigma fibrinogen solution to a 6 cm diameter dish and adding 4 ml of RPMI medium containing 0.002 units/ml of thrombin) for 10 minutes. ,200,
After 500 and 1000 cells were inoculated and cultured for 10 days, a colony considered to be derived from a single cell was selected and used as a clone. The DR type specificity of the antibody was examined by examining the presence or absence of an effect on the cells listed in Table 1 using the culture supernatant of the cloned cells on the third day of culture under the conditions of the cytotoxicity test described above. To confirm that the antibody is an HLA-DR antibody, see Table 3.
The binding with the membrane-solubilized HLA-DR antigen labeled with ¹²⁵ I was examined by acrylamide gel electrophoresis, and it was found that the antigen was reacted with 35,000 Dalton chain (α chain) and 27,000 Dalton chain (β chain). . The immunoglobulin class of the antibody was determined by the Ouchterlony method using an anti-mouse immunoglobulin heavy chain rabbit antibody, and it was determined to be immunoglobulin M class based on the formation of a sedimentation line in the 20-fold concentrated culture supernatant. From the above results, mouse hybridoma HOR-2-16-2 antibody is
It was found that it specifically reacts with HLA-DR1, HLA-DR2, HLA-8wDRwY and HLA-DRw9. Example 4 Human lymphocyte Ky with homozygous HLA-DKy
Cultured cells were infected with EB virus to produce lymphoblastoid cells that can proliferate in vitro. this cell
37°C using RPMI1640 medium supplemented with 10% fetal bovine serum.
Cultured and grown in a stream of 5% carbon dioxide gas, and
10 ⁷ mice were suspended in physiological saline and administered into the abdominal cavity of Balb/c mice (♀). Ten days after the administration, 4×10 ⁷ of the above cells were further intraperitoneally administered. Three days later, the mouse's abdomen was opened, the spleen was removed, and a splenocyte suspension was prepared. The splenocyte suspension and mouse myeloma P3/×63Ag8U1 cell suspension (Balb/c
Mouse-derived myeloma) were mixed at a cell ratio of 10:1, and after centrifugation, 45% polyethylene glycol (Sigma, molecular weight 4000) was gradually added dropwise and allowed to stand at room temperature for 6 minutes to fuse the cells. This fused cell
Dulbecco's modified Eagle's minimal nutrient medium supplemented with 15% fetal bovine serum (containing 2 mM glutamine, 5 x 10 ^-5 M2-mercaptoethanol, 80 μg/ml gentamicin sulfate, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate). Suspend and inoculate into Linbro 24-well plates, the next day, the day after, and every 2-3 days thereafter.
HAT medium (hypoxanthine, amethopritene,
Only the fused cells were grown by replacing half the volume with thymidine-supplemented medium). The presence or absence of antibody production in the culture medium after 11 days of culture was examined by a cytotoxicity test against Ky cells. For the cytotoxicity test, add 2 μl of culture supernatant and 2 μl to each well of the HLA typing plate.
A cell suspension (1×10 ⁶ cells/ml) and 5 μ of rabbit complement were added, kept at 37° C. for 2 hours, and then observed for viability using an inverted microscope. Among the cells listed in Table 1, cultured B lymphocytes and cultured T lymphocytes other than Ky cells (CCRF-CEM cells and
HSB-2 cells), and selected a culture solution that acts on some of the former but not on the latter.
The cultured cells sorted in this way were placed on a fibrin gel plate (1 ml of 0.25 cm in a 6 cm diameter plate).
Add % Sigma fibrinogen solution,
10, 200, 500 and
After 1000 cells were inoculated and cultured for 10 days, a colony considered to be derived from a single cell was selected and used as a clone. The DR type specificity of the antibody was examined by examining the presence or absence of an effect on the cells listed in Table 1 using the culture supernatant of the cloned cells on the third day under the conditions of the cytotoxicity test described above. To confirm that the antibody is an HLA-DR antibody, see Table 3.
The binding with the membrane-solubilized HLA-DR antigen labeled with ¹²⁵ I was examined by acrylamide gel electrophoresis, and it was found that the antigen was reacted with 35,000 Dalton chain (α chain) and 27,000 Dalton chain (β chain). . The immunoglobulin class of the antibody was determined by the Ouchterlony method using an anti-mouse immunoglobulin heavy chain rabbit antibody, and it was determined to be immunoglobulin G _2a class based on the formation of a sedimentation line in the 20-fold concentrated culture supernatant. From the above results, mouse hybridoma Ky6-16-23 antibody is
HLA-DR1, HLA-DR2, HLA-8wDRw6Y,
It was found that it specifically reacts with HLA-DRw6.2 and HLA-DRw9. Example 5 Human lymphoblastoid cells LG-10 homozygous for HLA-Dw7 were added to RPMI1640 with 10% fetal bovine serum.
The medium was cultured and grown at 37°C in a stream of 5% carbon dioxide gas, and 4 x 10 ⁷ mice were grown as Balb/c mice (♀).
The drug was suspended in physiological saline and administered into the abdominal cavity of the patient. administration
After 14 days, 1.5×10 ⁷ of the above cells were further administered intravenously and 1.5×10 ⁷ intraperitoneally. Three days later, the mouse's abdomen was opened, the spleen was removed, and a splenocyte suspension was prepared. The splenocyte suspension and mouse myeloma P3/×63Ag8U1 cell suspension (myeloma derived from Ba1b/c mice) were mixed at a cell ratio of 10:1 and centrifuged, followed by 45% polyethylene glycol (Sigma, molecular weight 4000). ) was gradually added dropwise and allowed to stand at room temperature for 6 minutes to fuse the cells. 15% of this fused cells
Suspend in Dulbecco's modified Eagle's minimal nutrient medium supplemented with fetal bovine serum (containing 2mM glutamine, 5 x 10 ^-5 M2-mercaptoethanol, 80μg/ml gentamicin sulfate, 100 units/ml penicillin G, and 100μg/ml streptomycin sulfate). and implanted in Linbro 24-well plate.
The next day, the next day, and every 2 to 3 days thereafter, only the fused cells were grown by replacing half of the medium with HAT medium (hypoxanthine, amethopterin, and thymidine supplemented medium). The presence or absence of antibody production in the culture solution after 11 days of culture was examined by a cytotoxicity test against LG-10 cells.
For the cytotoxicity test, 2μ of culture supernatant, 2μ of cell suspension (1×10 ⁶ cells/ml), and 5μ of rabbit complement were added to each well of the HLA typing plate and kept at 37°C for 2 hours. The method used was to observe whether the animal was alive or dead using an inverted microscope. Of the cells listed in Table 1
Cultured B lymphocytes other than LG-10 cells and T
Lymphocyte culture cells (CCRF-CEM cells and
HSB-2 cells), and selected a culture solution that acts on some of the former but not on the latter.
The cultured cells sorted in this way were placed on a fibrin gel plate (1 ml of 0.25 cm in a 6 cm diameter plate).
Add % Sigma fibrinogen solution,
10, 200, 500 and
After 1000 cells were inoculated and cultured for 10 days, a colony considered to be derived from a single cell was selected and used as a clone. The DR type specificity of the antibody was examined by examining the presence or absence of an effect on the cells listed in Table 1 using the culture supernatant of the cloned cells on the third day of culture under the conditions of the cytotoxicity test described above. To confirm that the antibody is an HLA-DR antibody, see Table 3.
The binding with the membrane-solubilized HLA-DR antigen labeled with ¹²⁵ I was examined by acrylamide gel electrophoresis, and it was found that the antigen reacted with 35,000 Dalton chain (α chain) and 27,000 Dalton chain (β chain). . The immunoglobulin class of the antibody was determined by the Ouchterlony method using an anti-mouse immunoglobulin heavy chain rabbit antibody, and it was determined to be immunoglobulin G ₃ class based on the formation of a sedimentation line in the 20-fold concentrated culture supernatant. From the above results, mouse hybridoma Dw7-3-5-10 antibody is
HLA−8wDRw6Y, HLA−DRw6.2, HLA−
It was found that it specifically reacts with DR7 and HLA-DRw9.

Claims (1)

[Claims] 1. Produced by mouse hybridoma,
HLA-DR2, HLA-DRw6.2 and HLA-
It reacts with cultured human lymphoblastoid cells that have DRw9, but does not have the above antigen and has HLA-DR1, HLA-
Antibodies of the immunoglobulin G ₂ a class that do not react with cultured human lymphoblastoid cells having DRw6, HLA-DR7 and HLA-8wDRw6Y, or produced by mouse hybridomas,
HLA−DR1, HLA−DR2, HLA−8wDRw6Y
and human cultured lymphoblastoid cells that have HLA-DRw9, but do not have the above antigen and react with HLA-DRw9.
- an antibody of the immunoglobulin G ₂ a class that does not react with cultured human lymphoblastoid cells having DRw6.2, HLA-DRw6 and HLA-DR7, or produced by a mouse hybridoma;
HLA−DR1, HLA−DR2, HLA−8wDRw6Y
and human cultured lymphoblastoid cells that have HLA-DRw9, but do not have the above antigen and react with HLA-DRw9.
- an immunoglobulin M class antibody that does not react with cultured human lymphoblastoid cells having DRw6.2, HLA-DRw6 and HLA-DR7, or produced by a mouse hybridoma;
HLA-DR1, HLA-DR2, HLA-8wDRw6Y,
Immunoglobulin that reacts with cultured human lymphoblastoid cells that have HLA-DRw6.2 and HLA-DRw9, but does not react with cultured human lymphoblastoid cells that do not have the above antigen and have HLA-DRw6 and HLA-DR7. Produced by G ₂ a class antibodies or mouse hybridomas,
HLA−8wDRw6Y, HLA−DRw6.2, HLA−
Reacts with cultured human lymphoblastoid cells with DR7 and HLA-DRw9, but not with the above antigens
HLA-DR1, HLA-DR2 and HLA-DRw6
A monoclonal antibody that is an immunoglobulin G ₃ class antibody that does not react with cultured human lymphoblastoid cells. 2 Human B lymphocytes Sh or HO or HOR
Alternatively, Ky or LG-10 are used as lymphoblastoid cells, respectively, and sensitized to a mouse, and then the splenocytes of the mouse are obtained, and then the splenocytes are fused with mouse myeloma cells, cloned, and then each of them is used for the desired purpose. Claim 1: Obtaining a hybridoma that produces a monoclonal antibody that specifically reacts with cultured human lymphoblastoid cells having an HLA-DR antigen, and then producing the hybridoma using each of the hybridomas. Monoclonal antibodies as described.