JPH0542269B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is used for functional testing of immunocompetent cells and for creating human monoclonal antibody-producing strains.
This invention relates to a method for producing antibodies by stimulating normal human lymphocytes with mitogens. Conventional technical background The production of antibodies by stimulating normal human lymphocytes with mitogen in vitro is an important method for testing the function of immunocompetent cells. 42 , special issue, 1344 (1984)], and also has important significance as a presensitization for the creation of human monoclonal antibody-producing strains, which have been significantly developed in recent years. Traditionally, the preferred mitogens for in vitro stimulation of lymphocytes include phytohemagglutinin (PHA) and pokeweed mitogen (PHA).
(abbreviated as PWM). Incidentally, PHA is a lectin derived from red kidney beans and activates T cells, whereas PWM is a lectin derived from pokeweed and has the property of activating B cells in a T cell-dependent manner and inducing antibody production. In addition, PWM is used as a prestimulus for the production of human monoclonal antibodies, using the Epstein-Barrvirus transformation method [Takada, "Tissue Culture" 10 , 174 (1984)] or cell fusion with malignant tumor lines. [Strihe et al., Journal of Immunology (J.Immunol.) 132 ,
1798 (1984)] with good results. However, in the conventional method of producing antibodies by stimulating normal human lymphocytes with mitogens, there is a problem in that it is actually difficult to increase the amount of antibodies produced. Problems to be Solved by the Invention In view of the importance of antibody production by stimulating normal human lymphocytes with mitogens as described above, the present invention provides a method for greatly increasing the amount of antibodies produced. The purpose is to The present inventors investigated the improvement of antibody production by stimulation of normal human lymphocytes with mitogens, especially pokeweed midogen (PWM), and found that in the presence of specific extracted components from cancer cell lines. By performing the above stimulation, we were able to successfully increase antibody production in the lymphocytes several times, leading to the present invention. The present invention will be explained in detail below. Structure of the Invention The present invention is characterized in that antibodies are produced by culturing normal human lymphocytes in a medium containing a mitogen and an extracted component of a cancer cell line. Means for Solving the Problems The normal human lymphocytes used in the present invention can be prepared from peripheral blood, colostrum, lymph nodes, tonsils, spleen, etc. The lymphocytes prepared from these can be frozen appropriately. It can be frozen and stored at -80°C or lower in a medium. In the present invention, the cancer cell line extract component to be used in combination with mitogen can be prepared by extracting cancer cells with 3M-KCl, but can be prepared by extracting the culture supernatant of the cancer cell line and by freezing and thawing the cells. is not used because it has no effect on antibody production. The components extracted from the above cancer cells using 3M-KCl are:
After dialysis against a balanced salt solution or medium, it is used by adding it to a medium for antibody production. Furthermore, in the present invention, a fraction obtained by further precipitating the above 3M-KCl extract component with ammonium sulfate at 50% saturation can be used as an extract component for cancer cell lines. When preparing extract components from cancer cell lines,
Cancer cell lines, such as lung adenocarcinoma cell line PC-8 (a cancer cell line established from lung adenocarcinoma tissue removed from lung cancer patients, which has adhesive properties, Naito,
"Cancer and Chemotherapy" 5 , 89, (1978) as a medium, e.g.
Cultured cells were cultured in RPMI1640 medium containing FCS, washed twice with PBS, collected by centrifugation, and further washed with PBS, using 0.02M potassium phosphate buffer containing 3M-KCl. , at 4℃
Extract for about 24 hours. The obtained extract is centrifuged to remove impurities, and then dialyzed against PBS to obtain extract components. In addition, the extracted components (crude extract) obtained in this way were mixed with 50% ammonium sulfate.
Fractions can be obtained by precipitation by saturation. In the present invention, normal human lymphocytes are cultured in a medium containing extracted components of cancer cell lines obtained as described above and mitogen, preferably pokeweed mitogen (PWM). Components should be added to the medium at a concentration of about 10 to 50 μg/ml, and mitogens (PWM) should be added to a concentration of about 0.01 to 1%. Cultivation should be carried out in an atmosphere containing 5% carbon dioxide and a humidity of 90% or more. It is best to do this at 37℃. In addition, the culture medium used can be those commonly used for cell culture, such as
RPMI1640 medium or Dulbecco's modified minimum essential medium to which approximately 10% fetal calf serum (FCS) or other serum is added is used. To carry out the above culture, lymphocytes are suspended in a medium containing extract components of the above cancer cell line and mitogen, and cultured in an incubator under the above conditions for several days. Incidentally, a glass or plastic shear dish, T-flask, microtiter plate, spinner bottle, etc. can be used as the culture vessel. The amount of antibodies produced by culturing the lymphocytes can be measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). EXAMPLES The present invention and its effects will be specifically explained below with reference to Examples. Example 1 Preparation of lymphocytes: Heparinized blood collected from a healthy adult was diluted with phosphate buffered saline (PBS), layered on Ficoll Pack (manufactured by Pharmacia), and weighed at 400 x g.
After centrifugation for 30 minutes, the lymphocyte layer at the interface was collected and used as peripheral blood lymphocytes (PBL). In addition, lymphocytes (LNL) were prepared from lymph nodes removed from breast cancer patients. Preparation of cancer cell extract components: Lung adenocarcinoma cell line PC-8 containing 10% FCS
PC-8 cells were cultured in RPMI1640 medium and grown to a confluent state. After removing the medium by suction, the PC-8 cells were washed twice with PBS, and then peeled off using a rubber scraper. The obtained cells were collected by centrifugation and washed with PBS. In addition, when culturing the above cells, the cells are detached from the culture medium with trypsin during subculture, and 1/2 to 1/2
Split to 1/4. Next, the cells obtained as described above were treated with 3M-
Extraction was performed at 4°C for about 24 hours using a 0.02M potassium phosphate buffer containing KCl, and the resulting extract was centrifuged at 15,000 x g for 20 minutes to remove impurities.
Dialysis was performed against PBS. Insoluble matter generated during dialysis was further removed by centrifugation, and the resulting solution was used as a crude extract. Solid ammonium sulfate was added to this crude extract to give 50% saturation, and after standing at 4°C overnight, the mixture was centrifuged to collect a precipitate, which was dissolved and dialyzed against PBS for partial purification. From 2×10 ⁸ lung adenocarcinoma cells, 24 mg of crude protein extract and 8 mg of 50% saturated ammonium sulfate were obtained as a precipitate fraction. Culture for antibody production: Use RPMI1640 medium containing 10% FCS as a medium, add pork weed mitogen to this medium to a final concentration of 0.5%, and use the above crude extract as the cancer cell extract component as shown below. It was added to each amount shown in Table 1. Next, each lymphocyte prepared above was seeded in the above medium in a 96-well microtiter plate (Falcon) at 2 x 10 ⁵ lymphocytes per hole so that the final volume was 200μ, and 5% carbon dioxide gas was added. Culture was carried out for 6 days at 37° C. and 100% humidity in air containing After completion of the culture, a certain amount of the culture supernatant of each culture solution was collected, and the amount of antibodies and IgG produced was measured by ELISA. The results are shown in Table 1.

【table】

[Table] As shown in Table 1, according to the present invention, when lymphocytes were cultured in a medium to which cancer cell extracts and mitogen were added, compared to when the extracts were not added, The amount of antibody produced by mitogen-stimulated lymphocytes increases significantly, reaching more than three times as much at maximum. Furthermore, it can be seen that the amount of antibody produced is higher when LNL is used as the lymphocyte. Example 2 Using lymphocytes LNL prepared according to Example 1,
In addition, 17 μg/g of the above 50% saturated ammonium sulfate precipitate fraction was added as a cancer cell extract component instead of the crude extract.
ml (equivalent to 50μg/ml in terms of crude extract)
Culture was carried out in the same manner as described in Example 1 except that a medium supplemented with was used, and the produced antibodies were measured. The results are shown in Table 2.

[Table] As shown in Table 2, when a medium to which the above precipitate fraction is added as a cancer cell extract component is used, the amount of antibody produced by mitogen-stimulated lymphocytes is increased by more than three times.

Claims (1)

[Scope of Claims] 1. A method for producing antibodies, which comprises culturing normal human lymphocytes in a medium containing a mitogen and a component extracted from a cancer cell line. 2. The method for producing antibodies according to claim 1, wherein the mitogen is porkweed mitogen. 3 Extracted components of cancer cell line are lung adenocarcinoma cell line PC
2. The method for producing an antibody according to claim 1, which is a component obtained by extracting -8 with 3M-KCl. 4 Extracted components of cancer cell lines are extracted from lung adenocarcinoma cell lines.
50% of the components obtained by extracting PC-8 with 3M-KCl
% saturated ammonium sulfate precipitated fraction. 2. The method for producing an antibody according to claim 1, wherein the fraction is precipitated with ammonium sulfate. 5. Production of the antibody according to claim 1, wherein the normal human lymphocytes are selected from the group consisting of peripheral blood lymphocytes, colostrum lymphocytes, tonsillar lymphocytes, spleen lymphocytes, and lymph node lymphocytes. Method.