JPS62223195A - Monoclonal antibody of anti-rat langerhans' inlet cell - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody for recognizing a tumor cell membrane antigen of rat Langerhans' inlet produced by a hybridoma obtained by cell fusion of a splenocyte of a mouse to which streptozotocin is previously applied and which is immunized against Langerhans' inlet tumor cell of insulin-forming rat and a myeloma cell. USE:A reagent for elucidating crisis mechanism of human insulin-dependent diabetes (IDDM) and a diagnosticum. PREPARATION:For example, a rat Langerhans' inlet tumor cell is administered to cavum abdominis of Balb/c mouse, the mouse is immunized, the splenocyte is collected and subjected to cell fusion with a myeloma cell to give a hybridoma. Then, the hybridoma is cloned by limiting dilution and a monocloned hybridoma is cultivated to give a monoclonal antibody.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention provides a novel monoclonal antidepressant pancreatic Langerhans that can contribute to the elucidation of the onset mechanism of human insulin-dependent diabetes mellitus (IDDM) and the development of diagnostic methods. Regarding islet tumor cell antibodies.

[Background of the invention]

Elucidation of the onset mechanism of diabetes is an area in which research has been particularly advanced recently, but a clear answer has not yet been obtained.

For example, research is progressing on genetic factors such as the relationship with HLA and causal relationships with viral infections, etc.
In the field of insulin-dependent diabetes mellitus (IDDM), in which metabolic abnormalities such as blood sugar can be controlled only by insulin administration, research from the perspective of an autoimmune endocrine disease has been attracting attention in recent years. In other words, there are reports that almost no β (beta) cells are observed in the pancreatic islets of insulin-dependent diabetes mellitus (IDDM) patients, and that significant mononuclear cell infiltration is observed in IDDM patients who have just developed the disease. There have been reports suggesting that there is a certain process that destroys β cells in the pancreatic islets of Langerhans, particularly in the islets of Langerhans. And Lernmark et al. (NewFi:ng
l, J, Med, 299, 375-380.19
78) hypothesized that if diabetes develops due to immunological destruction of β-cells, antibodies that react with the cell membrane of Randall I. As a result of the search, plasma membrane of rat pancreatic islet cells was found in the blood of IDDM patients.
We found an antibody that reacts with the membrane antigen on Lslet C.
e1lSurface Antibody (IC8A
) was reported. This antibody is said to have high organ specificity and extremely low race specificity, and its detection is usually performed using indirect immunofluorescence (IF) using rat or mouse pancreatic islet cells (New Engl. J. Med. , ,
299, 375-380.1978) and 125T-
Labeled protetnA or anti-human Ig
Radioimmunoassay (RIA) method using G (Di
abetologia19.445-451, 1980
: J, Immunol Methods, 31°201-209.19795 Diabetes, 30
．． 226-230° (1981), etc. are used.

Furthermore, recently, a measurement method using an automatic cell analysis and separation device using laser flow cytometry has been studied (Diabetes, 28.1041-1048.1985).

However, since such a method uses cells, it is difficult to standardize it as a measurement method, and it cannot be said to be usable in all clinical settings.

On the other hand, as part of basic research on IC3A, there is research attempting to explore the essence of IC3A0 in diabetic patients by experimentally producing antibodies against Langehans' islets and examining the properties of the antibodies. Lernmark et al. produced antibodies in rabbits against rat islet cells (Diabetrologia, 19, 445-
451 +1980). Also, Eissnbarth
reported the production of monoclonal antibodies in mice using the RIN cell line (Diabetes, 30, 2
26-230.1981).

IC5A present in the blood of diabetic patients is thought to consist of various antibodies directed against the surface of pancreatic islet cell membranes, rather than one type of self-antibody, the body. I'm also interested in To date, it has been reported that autoantibodies that recognize human pancreatic islet cell membrane soluble proteins with a molecular weight of 64 Kd (kilodaltons) or 38 Kd exist in the serum of diabetic patients (Natural + 298 +167～
169.1982:5science, 224.134
8-1350°1984).

[Purpose of the invention]

The present invention was made in view of the above-mentioned situation, and is based on insulin-producing rat pancreatic islet tumor cells (RI).
Provided are a monoclonal antibody against Nr), an islet of Langerhans tumor cell membrane IC8A antigen that can be identified using the antibody, and a method for analyzing IC3A in the serum of a human diabetes mellitus (IDDM) patient using the IC8A antigen. Its purpose is to

[Summary of the invention]

A monoclonal antibody that recognizes rat pancreatic islet of Langerhans tumor cell membrane antigen produced by a hybridoma obtained by fusion of splenocytes and bone marrow tumor cells of a mouse immunized with insulin-producing rat pancreatic islet of Langerhans tumor cells after prior administration of streptozotocin. and a hybridoma producing the antibody, a solubilized membrane protein antigen of rat pancreatic Zinkel 9 Hans islet tumor cell RINr cell whose molecular weight is recognized by the antibody in the range of 15 to 100 Kd, and a human serum using these membrane antigens. IC inside
This is the invention of the analytical method of 8A.

In recent years, many studies have been conducted using cell fusion methods to produce monoclonal antibodies against specific cells, and use this to detect and analyze specific antigens on the cell membrane surface. There have been reports on antigen analysis in human cancer cells and analysis of link cell subsent. Detection and analysis of the antigen on pancreatic islet cell membranes of IC3A, which has been reported as an autoantibody in the serum of insulin-dependent diabetic patients, is difficult due to the difficulty in obtaining human pancreatic islet cells.
Because of its low race specificity, it is mainly carried out using pancreatic islet cells from animals such as mice and rats. Antibodies that can serve as models for IC8A are produced by immunizing isolated rat pancreatic islet cells and established tumor cell lines.

On the other hand, in research investigating the cause of IC8A development, it is known that diabetes can be induced in mice by administering streptozotocin.

The present inventors used insulin-producing rat pancreatic islet of Langerhans tumor cells established as pancreatic islet cells to produce monoclonal antibodies against these pancreatic tumor cells, and the antibodies were found to be good model antibodies for IC5A. Therefore, we thought that it would be possible to detect and analyze cell membrane antigens using this antibody, and through intensive research, we cloned a hybridoma that produced the desired antibody and obtained a monoclonal antibody that recognizes pancreatic islet cells. We have arrived at the present invention.

The present invention uses a new immunization technique in which mice are immunized with tumor cells after primary administration of streptozotocin when producing monoclonal antibodies, instead of immunizing mice with pancreatic tumor cells alone. A major feature is that it increases the response of monoclonal antibody production.

The monoclonal antibody of the present invention can be reacted with solubilized lung tumor cell membrane protein separated by Ir electrophoresis and transferred onto a nitrocellulose membrane, and detected and analyzed by immunostaining to become the membrane antigen of IC8A. The protein can be identified, and further, human IC8A can be analyzed by examining the reactivity of human (II) DM patient serum to this antigen.

In the present invention, hybridomas are prepared by methods known per se, for example, as described in Nature by Milstein et al.
256 +495-497.1975.

Pancreatic Langerhans islet cells serving as antigens may be those directly isolated from animals, but various established cultured cells may also be used. The immunization method involved pre-administering streptozotocin, a drug known to cause diabetes in animals, to Balb/e mice to induce a metabolic immune system that is expected to produce Ic5Ai, and then injecting the pancreatic Langerhans islet cells with physiological saline, etc. The solution is diluted to an appropriate concentration and administered to mice by intraperitoneal injection, etc. Administration is carried out several times every 2 to 3 weeks, and the dose per administration is approximately 1 x 10 mice/mouse. As the immune cells, it is preferable to use pancreatic cells extracted 3 days after the final immunization.

The obtained immune splenocytes and myeloma cells are then fused.

As myeloma cells, various known cells such as N
5-1, P3-Ul, etc. are used. The fusion reaction is carried out according to a known cell fusion method, for example, by incubation in a medium in the presence of a fusion promoter. As the fusion promoter, polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and generally a 40-60% solution of PEG with an average molecular weight of 1000-8000 is used because it is easy to handle.

Adjuvants such as dimethyl sulfoxide can be added to increase fusion efficiency. The ratio of immune cells to myeloma cells used is the same as in general cell fusion methods, for example, about 5 to 20 times as many immune cells as myeloma cells. As a medium for fusion, for example, MEM medium, RPMI
Any medium commonly used in cell fusion can be used, such as -1640 medium and the like.

Fusion is carried out by thoroughly mixing a predetermined amount of the above immune cells and myeloma cells in the above medium, centrifuging, removing the supernatant, and pre-incubating at 37°C.
A PEG solution heated to, for example, an average molecular weight of 1000 to 8
This is carried out by dissolving about 1,000 ml of acetate in a normal medium to a concentration of 40 to 60% and stirring for about 1 to 10 minutes. Thereafter, a suitable medium is sequentially added, centrifuged, and the supernatant is removed to form hybridomas.

Isolation of the target hybridoma is performed by culturing the cells after the cell fusion described above in a normal hybridoma selection medium. The myeloma cell line described above has hypoxanthine-guanine phosphoribosyltransferase (H
GPRT) and therefore cannot grow in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine).

Therefore, cells that grow in HAT medium may be selected. Cells may be cultured in the HAT medium for a period of time sufficient to kill cells other than the target hybridoma, usually from several days to several weeks. Hybridomas are grown in HT medium at 1
After culturing for about 2 weeks, the cells may be cultured in a normal medium. The obtained hybridoma was subjected to the usual limiting dilution method.
A search for a strain producing the antibody of interest and single cloning are performed.

The thus obtained hybridoma producing the monoclonal antibody of the present invention can be subcultured in a conventional medium and can be easily stored for a long period of time in liquid nitrogen. To find out whether the obtained hybridoma produces the desired monoclonal antibody, general antibody detection methods such as ELISA, RIA, ophthalmology, and agglutination reactions are used. When searching for antibodies against
Preferably, a fluorescent antibody method is used.

To obtain the anti-pancreatic islet of Langerhans tumor cell antibody of the present invention from the specific hybridoma obtained, the hybridoma can be cultured according to a conventional method and separated from the culture supernatant, or the entire hybridoma can be administered to compatible animals. There are methods such as allowing the cells to proliferate and isolating them from ascites. The former method is good for obtaining high-purity products, and the latter method is excellent for mass production. Which method to use is arbitrary and may be selected as appropriate depending on the purpose.

To detect and identify a membrane protein antigen using the monoclonal antibody of the present invention, first, a cell membrane fraction obtained from rat pancreatic islet of Langerhans tumor cells (RIN) is treated with, for example, Triton
-100, the resulting solubilized membrane proteins were reduced with mercaptoethanol, separated by sodium dodecyl sulfate (SDS)/acrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. Detection and identification may be performed by immunostaining using a monoclonal antibody. The membrane protein antigens thus detected and identified include those that can be detected and identified in the same manner as IC3A in the serum of human IDDM patients, such as those with a molecular weight of 58 Kd and those with a molecular weight of 38 Kd. Therefore, using this method, it is possible to analyze the specificity of human IC8A.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited in any way by these Examples.

〔Example〕

Example 1 Preparation of monoclonal anti-rat pancreatic islet of Langerhans tumor cell antibody and hybridoma producing the same (1) Immunization Preparation of cells to be used as immunogen was carried out in a long-term in vitro manner.
.

Passage-cultured rat pancreatic islets of Langerhans tumor cells RINr were subcultured with 10% tallow serum and the antibiotic streptomycin (50%
The cells were cultured at 37°C in a humid atmosphere containing 5% carbon dioxide in RPMI-1640 medium supplemented with 0Ag/rnl), henicillin (5oUAnl), and 0.3% glucose. After culturing, the supernatant was removed by centrifugation, and after centrifugal washing with phosphate buffered saline (PBS), the cells were suspended in PBS.

Strezotozotocin (20 μ/ml) dissolved in 0.2 rnl of saline was intraperitoneally administered to Ba1b/c mice.
After a week, immunization was performed by injecting one RINr cell into one cavity.

(2) Cell fusion Three days after the final immunization, the mouse pancreas was removed.

108 splenocytes and 107 MS-1 cells were treated with polyethylene glycol: y-ol. 5g, MEM medium 0. 5 mA'
, DMSO 0. Fusion was achieved by incubating in 15 rnl of solution for 1 minute at room temperature.

After the cells were suspended in RPMI-1640 medium supplemented with 15% FC8, they were dispensed into five 96-well plates, and culture was started in a CO2 incubator. 2 drops of H per hole the next day
AT medium was added. Then, every 3 days, add half of the medium to HA.
Replaced with T medium. Culture supernatants two weeks after fusion were used for detection of antibody production.

(3) Selection of hybridomas In order to select hybridomas producing anti-RINr cell antibodies, each of the above 96-well cell culture supernatants was analyzed by indirect fluorescent antibody method and ELISA method.

First, RIN r cells (106@/rnl) were
%B SA/'P BS and 200 μl of it was added to 9
After dispensing into a 6-well plate, it was centrifuged. 50 μl of the above culture supernatant was added to the cells in each well and reacted at 4° C. for 1 hour. After centrifugation, the supernatant was removed, washed with 4% BSA/PBS, and centrifuged again.

After centrifugal washing three times in this manner, the mixture was reacted with 50 μl of FITC-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO, 10-fold diluted solution) at 4° C. for 1 hour. After centrifugal washing three times, the pellet was transferred onto a slide glass with glycerin buffer and examined under a fluorescence microscope.

Detection by the 1iLISA method was performed using RINr cells cultured in advance in a 96-well plate. The cells were treated with s% glutaraldehyde/PBS for 20 minutes and fixed. After washing, mix this with 50 μl of the above hybridoma culture supernatant at room temperature.
After reacting for an hour and washing again three times with PBS, 1000
It was reacted with peroxidase-conjugated anti-mouse immunoglobulin antibody (manufactured by DAKO) 50# diluted twice at room temperature for 1 hour, and then treated with PBS-Tween 200,0.
It was thoroughly washed with 5%. To this, add 4~/rnl of 0-phenylenediamine and 20tt17k of 1.7% hydrogen peroxide solution.
1-containing citrate buffer (0.1M, pH 4.8)
501 tl was added, a color reaction was carried out for 30 minutes, and 6N
After stopping the reaction with H2sO4 solution, 490 nm
The absorbance was measured.

(4) Monocloning Cloning was performed by the limiting dilution method. 0.0 hybridomas per well of a 96-well plate. The mixture was divided into portions for 5 individuals. This operation was repeated twice to complete monocloning. Nine clones were selected by indirect fluorescence antibody method and gLIsA method and used for the next antibody production.

(5) Preparation of monoclonal antibodies 5 x 10 cells of the above nine hybridomas were intraperitoneally administered to Ba1b/c mice (male, 8 weeks old) that had been treated with pristane 10 days earlier, and about 2 weeks later, the ascites was removed. Recovered.

The ascites was subjected to ammonium sulfate fractionation at 50% saturation, and further column chromatography using DgAg cellulose was performed to obtain nine types of monoclonal antibodies of the present invention.

Example 2 Characteristics of anti-RINr cell antibodies = Identification of immunoglobulin classes and subclasses The immunoglobulin classes of the nine monoclonal antibodies obtained in Example 1 were identified as immunoglobulin oxidase-conjugated anti-mouse immunoglobulin antibodies. immunoglobulin r
gG4, IgG2a, igc,..., IgG3.

The measurement was performed by enzyme-linked solid-phase immunoassay using anti-IgA and anti-IgM (both manufactured by DAKO).

The results are shown in Table 1.

Table 1 Example 3 Rat pancreatic islet of Langerhans tumor cells RINr
Two-layer method (D, M, Brunette and J, E. Ti
1l, J. Membrane Biol, 5.
215-224.1971), and this membrane fraction was solubilized with 1% Triton X-100. Add 5% SDS to this.

Equal volume of glycine Tris buffer W containing 10% 2-mercaptoethanol and 20% glycerin (pH 7,0)
-i was added, and after heating in a water bath for 2 minutes, it was applied to a 10% polyacrylamide gel and subjected to electrophoresis. After electrophoresis, membrane proteins were transferred to a nitrocellulose membrane, and after washing, 4% BSA/P
It was treated with BS at 37°C for 1 hour.

After washing again, monoclonal antibody 300 obtained in Example 1
The mixture was reacted with lJ&/ml at 37°C for 1 hour. After thorough washing, it was reacted with a peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO) diluted 500 times at 37°C for 1 hour, and after thorough washing with PBS-Tween 20, a substrate solution (3,3'-diaminobenzidine 10 (2) The nitrocellulose membrane was soaked in 40 ml of 0.05 M 1 liter hydrochloric acid buffer, pH 7.2, containing 10 μl of 30% hydrogen peroxide solution to develop color.

Similarly, instead of the above monoclonal antibody, human
DDM patient serum (serum diluted 5 times) was reacted with the solubilized membrane protein antigen on the nitrocellulose membrane, and detection was performed using a oxidase-labeled anti-human immunoglobulin antibody (manufactured by DAKO). Molecular weight 15Kd~100Kd
A large number of membrane proteins in the range of monoclonal antibodies and humans)
It was recognized by antibodies in the serum of IDDM patients. Especially monoclonal antibodies 1-8.4-2.8-10.23-2.2
The five antibodies in 4-1 recognized proteins with molecular weights of 38 Kd and 58 Kd, which are characteristic membrane proteins common to antibodies in human IDDM patient serum.

〔Effect of the invention〕

As described above, the present invention provides a novel anti-porous anti-porous pancreatic islet of Langerhans tumor cell antibody, and isolates an islet of Langerhans tumor cell membrane I C3A antigen that can be identified using the antibody. If this is used, I in the serum of human insulin-dependent diabetes mellitus (IDDM) patients
C8A analysis can be carried out on a regular basis and in a standardized manner, which is extremely effective in contributing greatly to the elucidation of the onset mechanism of IDDM and the development of diagnostic methods.

Claims (8)

[Claims] (1) Recognizes rat pancreatic islet of Langerhans tumor cell membrane antigen produced by a hybridoma obtained by fusion of splenocytes and bone marrow tumor cells of a mouse immunized with insulin-producing rat pancreatic islet of Langerhans tumor cells after prior administration of streptozotocin Monoclonal antibodies. (2) Claim 1, wherein the insulin-producing rat pancreatic islet of Langerhans tumor cells are RINr cells established from serially transplanted pancreatic islet of Langerhans tumors created by X-ray irradiation of NEDH rats. The monoclonal antibodies described. (3) The monoclonal antibody according to claim 1 or 2, wherein the bone marrow tumor cells are cells belonging to P3-X63. (4) RIN, rat pancreatic islet of Langerhans tumor cells
The monoclonal antibody according to any one of claims 1 to 3, which recognizes a membrane protein obtained by solubilizing the cell membrane of r cells. (5) obtained by fusion of splenocytes of a mouse immunized with insulin-producing rat pancreatic islet of Langerhans tumor cells and bone marrow tumor cells after pre-administration of streptozotocin;
A hybridoma cell line that produces monoclonal antibodies that recognize rat pancreatic islet of Langerhans tumor cell membrane antigens. (6) Recognizes rat pancreatic islet of Langerhans tumor cell membrane antigen produced by a hybridoma obtained by fusion of splenocytes and bone marrow tumor cells of a mouse immunized with insulin-producing rat pancreatic islet of Langerhans tumor cells after prior administration of streptozotocin An antigen recognized by a monoclonal antibody with a molecular weight of 15 to 100 Kd (
Solubilized membrane protein antigen of rat pancreatic islet of Langerhans tumor cells RINr cells in the kilodalton range. (7) Antibody ICSA (Islet Cell Surf
7. The membrane protein antigen according to claim 6, which is also recognized by Ace Antibody. (8) Recognizes rat pancreatic islet of Langerhans tumor cell membrane antigen produced by a hybridoma obtained by fusion of splenocytes and bone marrow tumor cells of a mouse immunized with insulin-producing rat pancreatic islet of Langerhans tumor cells after prior administration of streptozotocin ICSA (Isl
et Cell Surface Antibody)
analysis method.