JPS6081130A - Anti-hla-a2 antibody and hybridoma producing the same - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody FLA02 having high antibody value, and produced by the fused cell obtained from the spleen cell of mouse immunized with human B lymphoblastoid cell having human HLA-A2 antigen. CONSTITUTION:An Sh cell which is human B lymphocyte cell having HLA-A2 antigen and HLA-A9 antigen in a heterogeneous form is infected with EB virus. The obtained lymphoblastoid cell is proliferated and immunized to a mouse as an immunogen. The spleen cell collected from the mouse is fused with mouse myeloma cell, and the resultant fused cell is cultured, from which hybridoma SH-2-119-6 having the highest cytotoxicity to Sh cell is separated. The objective monoclonal antibody FLA-2 having complement-dependent cytotoxicity and reactive specifically with HLA-A2 antigen is obtained from the supernatant liquid prepared by the cultivation of the hybridoma in a proper medium, or from the ascites obtained by the peritoneal infusion of the hybridoma to a mouse.

Description

[Detailed Description of the Invention] In recent years, research on the human major histocompatibility system (hereinafter abbreviated as HLA) has progressed rapidly.For example, in the field of organ transplantation,
It has already been implemented as a powerful means of predictively determining compatibility between II device providers and recipients.

Furthermore, research results are being gradually introduced in the fields of disease association, immune response mechanisms, anthropology, JD biology, blood transfusion science, and forensic medicine, and as a result, the importance and demand for HLA analysis has increased. Along with this, tL has strong IR selectivity and activity.
Kokichi, who has obtained the A antibody, is getting a nail shape.

Conventionally, for HLA analysis, H-L collected from pregnant women's blood was used.
Antibody A is used, but IIJ is also used! There is a desire for something even higher in terms of activity.

Recently, methods using hybridomas, which are a fusion of lymphocytes and myeloma cells, are entering the practical stage in the field of antibody production, and HLA antibodies are also being developed using mice and hybridomas.
Methods using hybridomas are being researched and put into practical use. However, the human alloantigen IILA
When mouse lymphocytes produce antibodies that recognize antigens, it is difficult to obtain highly specific antibodies.

Under such circumstances, the inventor of this invention
A mouse was immunized with human B lymphoblastoid cells containing the LA-A-2 antigen, and 3927 cells were taken from the immunized macus, fused with mouse myeloma cells, and cloned. We succeeded in producing a seven-clonal antibody FLAO2 (hereinafter referred to as FLAO2 antibody) that can be used for HLA analysis in place of the collected HLA antibody.

This FLAO2 antibody specifically reacts with the HLA-A2 antigen and has a much higher antibody titer than that obtained from conventional blood of pregnant women.

This FLAO2 antibody is hybridoma 5H-2-119
It can be obtained in large quantities by culturing -6 in an appropriate medium and using -i1, or by separating it from the culture supernatant, or by transplanting it into the abdominal cavity of a mouse and separating it from its ascites. .

Specific examples thereof are described in Examples 2 and 3.

-1, hybridoma 5H-2-119-6 producing the FLA 02 antibody of the present invention could be isolated by the method of Example 1.

The physicochemical and biological properties of the FLAO2 antibody of this invention are as follows.

1) Molecule (;1 This FLAO2 antibody obtained in Example 3 was treated with sodium dodecyl sulfate and examined by polyacrylamide gel electrophoresis, and the molecular weight was 54.000 and 2.
It was found that it consists of 7,000 subunits. These subunits correspond to the heavy and light chains of immunoglobulins, and it was confirmed that the E'LAO2 antibody is an immunoglobulin.

11) Immunoglobulin classes and inactive F'LA
The immunoglobulin class and probe of the O2 antibody were investigated by the off-chrony method.

Culture of 300)11 obtained by the method of Example 2
Mix the saturated ammonium sulfate solution of 3C) Ogll with anti-maximum immunoglobulin M + G, + 02a
, G2b.

The formation of sedimentation lines with each of G3+A+ λ and two-chain serum (manufactured by Miles) was examined.

As a result, the FLAO2 antibody was found to be an antibody consisting of a G2b heavy chain and a light chain.

if) Identification of antigen The surface of 1 x 108 SH cells washed with phosphate H-gain saline was labeled with lmCi Na125I using a lactoperoxidase/hydrogen peroxide system, and then labeled with 1% Nonidet P40. (manufactured by Bethesg Research Laboratories) for 15 minutes under water cooling to solubilize the membrane. The purified F obtained by the method of Example 3 was treated with Sepharose 4B (manufactured by Pharmacia) bound to mouse immunoglobulin at 4°C for 30 minutes twice to remove non-specific adsorbates.
The mixture was reacted with Sepharose 4B (manufactured by Pharmacia) bound to LAO2 antibody overnight at 4°C with continuous shaking.

After centrifugation, the precipitate was solubilized with sodium dodecyl sulfate, examined by polyacrylamide electrophoresis, and -FLA
O2 antibodies and molecules xi), 45,000 and 12;000
HLA-A, HLA-B of sh cells consisting of HL
The A-C bond was recognized as (1(r).

iv) Cytotoxicity test using various cultured cells Tests were conducted on cultured cells for which the HLA, -A antigen or HLA-B antigen possessed by each lymphocyte was known.

In Example 2 of 2 pl, f! 1 supernatant and 2 notl of cell suspension solution (6 IXIO/
Add a 2-fold diluted complement solution (manufactured by Belfries) of me) and 5) tl, and incubate for 1 and 2 hours at 37°C in a stream of carbon dioxide gas, then observe with an inverted microscope to determine whether the cells are viable or dead. I judged it. The results are shown in Table 1.

In the table, "1-1" indicates that each cell did not react with the HLA-A antibody within the range used for quipping but with the HLA-B antibody. This indicates that there is a high possibility that neither antigen is retained.

The results of testing the cytotoxicity of sh cells to cultured cells using the purified antibody prepared by the method of Example 3 showed that the cytotoxicity increased in correlation with the concentration, with 50% cytotoxicity. The concentration indicating this is 0.54 no! ! //rue.

■) The cell-if properties of antibodies against human peripheral blood lymphocytes were determined by the National Institutes of Health (NNH) in the United States.
te of Heartb1nri, S, A,)'s HL
Purified 'Il obtained in Example 3 according to the A typing method
'i: Lymphocytes of Japanese dog blood donors were investigated using the body or the culture supernatant IQed in Example 2.

As a result, 331 peripheral blood lymphocytes (including both BIJ lymphocytes and TIJ lymphocytes) and peripheral blood TIJ members of Japanese dogs were cultured using the method of Example 2. The results of the cytotoxicity test are shown in Table 2.
It showed a high correlation with the presence or absence of HLA-A2.

Table 2 a: HLA-A2 anti-B: Presence or absence of retention (11 individuals): A dead cell rate of 60% or more of cells 71j was considered positive.

In addition, in Example 3 for the patient's peripheral blood phosphorus (t)
IJ) - Antibody specificity 11 (, toxicity is HLA
-A2 was added to the lymphocytes of 9 people with Q10/l//m
e1: indicates the iiJ property of TeloOchi, +11. A
- 0.4 to 1 for lymphocytes in 8 people without A2
(It showed no toxicity at a concentration of 10 p/me.

Vi) T='AC8 (own qJJ cell analysis unit;
Detection of antigen-retaining cells by cultured human peripheral blood cells 100 μl of the culture solution of hybridoma S H-'2-119-6 was added to the tube, and the mixture was incubated at 4°C for 60 minutes. Before centrifugation, centrifuge
Anti-maximunoglobulin G FITC-labeled goat immunoglobulin F (ab
') 30 μl of the 2nd fraction (manufactured by Kappel) was added and reacted at 4°C for 30 minutes.

After centrifugation and washing three times, fluorescence-positive cells were measured by applying FAcsll'K and excitation with laser light.

As a result, the number of 50% antibody-bound cells was 0.08) I! A
It was recognized in le.

■1) Antibody binding ability measurement RP by radioimmunoassay
M I medium '/&'+ Saseta 8 x 106
100 p7 of cells/TIIe cultured cells? 0.5%
[Small plastic sample coated with bovine serum albumin solution] Place in a tube (Eiken 1-li), collect cells by lifting before centrifugation, and add culture solution 100/Z of No. hybridoma SH2119G.
j! was added and reacted at 4°C for 90 minutes. After washing twice,
F(ab')2 fraction of anti-maximmunoglobulin GI25I-labeled rabbit immunoglobulin (manufactured by Miles) was
00) 11! In addition, the cells were allowed to stand overnight at 4°C, washed three times before centrifugation, and the radioactivity bound to the cells was measured using a gamma scintillation probe.

As a result, the 50% antibody binding capacity was 0.13 pV/me
It was recognized in

Example 1 (I) of hybridoma 5H-2-119-6)
i) HLA-A2-immunized hepatocytes, '1 HLA
-A2 antigen II JL
BV-Sh cells) are grown and used as an immunogen. That is, EBV-8h was cultured in MI-1640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) containing 10% tallow serum (1AF) at 37'C in a 5% carbon dioxide gas flow, and the grown cells were incubated at 1.500 rpm. Centrifuge for 5 minutes at li3
! tosi, then f'k irt with phosphate buffered saline.
After that, three BALB/C mice (
M' was injected into the peritoneal cavity of 4 x 10' each. 2
After 5 days, EBV-8h cells, which had been similarly cultured and washed, were injected into the abdomen and intravenously of the mouse.
35 x 10' cells were immunized.

After 40 days, the abdomen of each mouse was opened, the liver was loosened with pins, and the cells were suspended in Dulbecco's modified Eagle's minimal nutrient medium to prepare II' (L cell suspension).

II) Harvesting and cloning of hybridomas Obtained hepatocyte suspension and mouse myeloma P3/x63Ag
8U1 (hereinafter referred to as P3U1) cell suspension (BALB
/C macus mountain rice myeloma) were mixed at a cell ratio of 5:1, and after centrifugation, 45% polyethylene glycol (Sigma, molecular Tjs: 4.000) was gradually added dropwise and allowed to stand at room temperature for 7 minutes. :Fused x. The fused cells were cultured in Grubetzko's modified Eagle's minimal nutrient medium supplemented with 15% tallow blood Wt (2mML-glutamine, 2X10 M2-
Mercaptoethanol, 80 μy/me gentamicin sulfate, 100 units/me penicillin G, 100/me
lfj/me streptomycin sulfate 0.25 pV
/me fungizone) (hereinafter referred to as CM medium) and implanted in a 24-well plate (manufactured by Nunc), and the next day, the suspension was transferred to a well plated well with HA T medium (in addition to the above medium, 1X10M hyboxanthin, 4X10M A3). Pterin and 1.6 x 10" (medium supplemented with 5 M thymidine) were added to each well, and the next day after that, half of the culture medium in the well was added to a new well at every 2.30 °C.
Continue culturing from 1-1 with T medium to grow only the fused cells, then replace half of the medium in each well with HT medium (HAT medium minus aminopterin), and continue culturing 1-1. A part of the sample was taken and subjected to a cell test on Sh cells.
Examine the presence or absence of anti-11-sided production against tu, and further culture wells producing antibodies against 8]co cells.''-61
A portion of HO with HLA-A24 antigen was collected.
Cells, ILA-fi, 11, Ky cells with HLA-A26 antigen, and HOR cells with HT, A-A33 antigen were performed, and antibodies against these cells were produced. The hybridoma growing in this well was selected as 5H-2-119.
This hybridoma was named jl'Ii and was recloned using a conventional method. Cloning was carried out using CM medium, BA
LB/C mouse T cells were transferred to a feeder layer (f'e
eder ], ayer ) as 1 (μ-world dilution method).

A hybridoma derived from a single cell that was most cytotoxic to Sh cells was selected and named 5H-2-119-6.

Example 2 Preparation of FLAO2 antibody Jili hybridoma SH2] 19-6 in CM medium
Cultured for 4 tTI in M11 with 5% carbon dioxide gas at °C.
] and centrifuged at 0 (10 rT) m for 10 minutes at 4°C to remove the culture medium Yt containing FL A02 antibody.

Example 3 Production of 1r LA O2 antibody 1 week before, pristane (2,6,10,14-tetramethylbentatecan) was intraperitoneally administered to 5 animals.
B/C (#1t) 1x per Maxx for 1 back cavity of Maxx
107 hyglioma SH-'2-119-6 cells were transplanted. Ascites meter 18 me 1-2 weeks after transplantation
was collected.

Centrifuge the ascitic fluid at 10,000 rpm for 10 minutes, collect the supernatant, and add 50% Acimonicum sulfate to 1 ml.
Add the Japanese solution and stir on ice 1-3 for 30 minutes, and then stir for 30 minutes.
(The immunoglobulin fraction is precipitated,
The precipitate was collected by centrifugation at 15.00 l rpm for 10 minutes, dissolved in a small amount of phosphate buffered saline, and dialyzed overnight against Lis-HCl buffer pH 7.5 to 5 mM). ) DE5 equilibrated with hydrochloric acid buffer
D 1a A using cellulose 2 (manufactured by Sottomasi)
Subjected to E cell IJ-nucleum chromatography, 5
Elution was carried out using a continuous 0 degree gradient method in which 100 mM NaCl was added to the 100 mM NaCl buffer, followed by elution while replacing the solution with 5 mM NaCl. The fraction was separated into 7 fractions each containing Sme, and the fractions showing absorption at 280 mμ were collected, and purified FLAO2 of 52.4 my was isolated.

Claims (1)

[Scope of Claims] [Produced by Max hybridoma 5H-2-119-6, has complement-dependent cell IJL property,
- Monoclonal antibody F that specifically reacts with A2 antigen
LA 02. (2) Hybridoma 5H-2-119-6°, which has complement-dependent cellularity and produces monoclonal antibody FLA02 that specifically reacts with HLA-A2 antigen.