JP2009284771A - Method for preparing antibody-producing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably preparing a monoclonal antibody by establishing a means capable of reproducibly obtaining B cells which has acquired antibody-producing ability. <P>SOLUTION: Provided is a method for preparing an antibody-producing cell which produces a mouse monoclonal antibody wherein the method is featured by collecting a tumid lymph node obtained from a mouse immunized by administering an antigen to the neck or back and by using cells obtained from the lymph node for cell fusion. Since the lymph nodes of each part of the mouse are tumefied by the immunization method, these lymph nodes are available as a source of B cell. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a mouse monoclonal antibody useful in fields such as research, diagnosis, and medical care.

  A monoclonal antibody is an antibody derived from a single antibody-producing cell. Monoclonal antibodies are a collection of antibody molecules that recognize a single antigenic determinant, making them excellent tools for detecting various substances with high specificity and sensitivity, and are essential tools for research, diagnosis, and medical fields. It has become.

  Antibody-producing cells that produce monoclonal antibodies are obtained by fusing B cells or a group of cells containing B cells collected from animals immunized with a desired antigen with myeloma cells, and then obtaining the antigen from the resulting fused cell (hybridoma) population. It is produced by isolating a cell line that produces an antibody that recognizes. Conventionally, the spleen has been used as the origin of B cells, but in recent years, methods using lymph node-derived lymphocytes for hybridoma production have become widespread. Among them, the method using the iliac lymph node is said to have excellent characteristics such as a single administration (immunization) of the antigen and a cell fusion operation in about 2 weeks after the immunization.

  According to this method, for example, in rats, by administering an antigen to the hind footpad, the iliac lymph nodes can be swollen in a short period of time, and this can be used as a material for hybridoma production (Patent Document 1, Non-patent Document 1). Patent Document 1). In the case of mice, there is a problem with the amount of antigen administered in the same manner as in rats, ie, antigen administration to the hind footpad, and the iliac lymph node does not swell, so the root of the tail (ridge part) as the antigen administration site It is said that it is necessary to select the intramuscular muscle (mouse iliac lymph node method: Patent Document 2, Non-Patent Document 2). Further, Non-Patent Document 3 describes that for an antigen administration site to a mouse, an emulsion is injected with a needle tip of about 1 cm in the ridge muscle.

JP-A-6-209769 JP 2007-20547 A Cell Structure and Function, Vol. 20, pp. 151-156, 1995 Acta Histochemistry and Cytochemistry, 39, 89-94, 2006 Biochemistry, 78, 1092-1094, 2006

  An animal's immune response is greatly influenced by the nature (species, sex, age, health status) of the individual used. Some antigens have a low effect of causing an immune response in an individual, and in many cases, sufficient lymph node swelling is not observed in the immunized individual. For this reason, in order to stably produce a monoclonal antibody, it is necessary to establish a technique capable of obtaining B cells having acquired antibody-producing ability with high reproducibility.

  As a result of intensive studies, the present inventors have found that when an antigen is administered to the neck or back of a mouse, enlargement is seen in lymph nodes at each site of the mouse. On the other hand, when the needle is inserted from the ridge as described above and the emulsion is injected into the muscle 1 cm ahead (hereinafter referred to as tail administration), almost no enlargement of lymph nodes other than the iliac lymph nodes is observed. I couldn't. Further, the present invention was completed by confirming that a hybridoma producing a monoclonal antibody that recognizes the antigen can be prepared using the enlarged lymph node obtained by antigen administration to the neck or back of the mouse as a B cell source. I let you.

That is, the present invention
[1] Producing a mouse monoclonal antibody characterized by collecting a swollen lymph node from a mouse immunized by administering an antigen to the neck or back and subjecting the cell obtained from the lymph node to cell fusion A method for producing antibody-producing cells,
[2] The method for producing antibody-producing cells according to [1], wherein an antigen administration site is subcutaneous or intramuscular,
[3] The method for producing an antibody-producing cell according to [1], wherein cells obtained from an enlarged lymph node selected from an inferior lymph node, an inguinal lymph node, or an iliac lymph node are subjected to cell fusion,
[4] A method for producing a monoclonal antibody, comprising the steps of producing antibody-producing cells by any one of the methods [1] to [3] and collecting a monoclonal antibody produced by the antibody-producing cells; [5] The method for producing a monoclonal antibody according to [4], wherein the antibody-producing cells are transplanted into a mouse abdominal cavity and the monoclonal antibody is collected from the mouse ascites.
About.

  According to the immunization method of the present invention, swollen lymph nodes at each site of the mouse can be obtained and used for cell fusion. Therefore, even when there is a problem with the state of the mouse used and the immunogenicity of the antigen, the risk that sensitized B cells necessary for production of monoclonal antibodies cannot be obtained is greatly reduced. The method of the present invention can greatly contribute to more reliable production of mouse monoclonal antibodies.

  The antigen used in the present invention is not particularly limited, and may be any antigen for which acquisition of a monoclonal antibody that recognizes the antigen is desired. Antigens can be used alone or as a mixture of a plurality of antigens. The antigen is preferably mixed with an appropriate adjuvant to form an antigen solution (emulsion) to be administered to mice and used for immunization of mice.

  Mice are immunized by injecting an antigen solution into the neck or back of the mouse. In a preferred embodiment when the antigen is administered to the back in the present invention, the antigen solution is administered to the back of the mouse behind the ear and in front of the torso. The antigen is preferably administered at two positions on the left and right sides of the spine, and may be administered on the spine in addition to the above two positions. The administration site may be selected from subcutaneous and intramuscular, but subcutaneous administration is simple and may be intradermal.

  The antigen solution is administered in an amount usually used for immunization of mice. The dose may be set in consideration of the concentration of the antigen, the body weight of the mouse, etc. For example, 50 to 300 μL, preferably 100 to 200 μL is administered per mouse.

  In the method of the present invention, usually, only one immunization is required, but the booster immunization is not prevented.

  From the time when 10 days have passed since immunization (from the first immunization in the case of booster immunization), enlargement of the lymph nodes such as the armpit, groin, and iliac of the mouse occurs. In the present invention, enlarged lymph nodes are selected and collected 10 to 25 days, preferably 12 to 18 days after immunization. Thus, the present invention is not limited to the use of specific lymph nodes. In general, according to the method of the present invention, the swallowing lymph node is larger than the other lymph nodes, and therefore the swallowing lymph node is preferably used. Furthermore, cells derived from enlarged lymph nodes at different sites may be combined.

  The cells are dispersed from the collected lymph nodes by an appropriate method, and then the obtained cells are washed. Cell dispersion can be carried out using a known method such as a stainless mesh. These cells contain B cells (sensitized B cells) that have acquired antibody-producing ability, and are used for the following cell fusion operations.

  For fusion, myeloma cells derived from mouse, rat, human and the like can be used. Cell fusion techniques are known to those skilled in the art. The method described in G. Kehler “Nature, Vol. 256, pp. 495 (1975)” or a method similar thereto may be used. For example, both cells can be fused by reacting lymph node-derived cells with myeloma cells for about 1 to 3 minutes in 30-50% polyethylene glycol (PEG, molecular weight 1000-6000). Thus, a hybridoma that is an immortalized antibody-producing cell can be produced.

  The hybridoma obtained by cell fusion is subjected to screening. The antibody that recognizes the antigen or an epitope thereof can be detected by an ELISA method using an antigen used for immunization or a substance having the same epitope as the antigen. Thus, an isolated hybridoma strain can be established by selecting a cell producing a desired antibody and further performing cell cloning.

  Preferably, the antigen recognition specificity of antibodies produced for a plurality of hybridoma strains, the affinity for the antigen, the amount of antibody produced, etc. are examined, and a hybridoma strain that meets the purpose, that is, a monoclonal antibody-producing cell, is selected.

  The monoclonal antibody-producing cells obtained by the method of the present invention shown above can be cultured, and the antibody produced in the culture can be collected to produce a desired monoclonal antibody. Examples of a method for obtaining monoclonal cells from the above cells include a method for culturing the cells in vitro, a method for transplanting the cells into the abdominal cavity of an animal, for example, a mouse, and producing a monoclonal antibody in ascites. However, it is not limited to these. Furthermore, a monoclonal antibody purified using a known method such as ammonium sulfate salting out or various chromatographies can be obtained from the monoclonal antibody-containing sample (culture fluid supernatant, ascites, etc.) thus obtained.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited only to the scope of the following examples.

Example 1 Enlargement of Lymph Node at the Antigen Administration Site Using Freund's complete adjuvant, an emulsion containing keyhole limpet hemocyanin at a concentration of 1 mg / mL was prepared. Three groups of BALBc mice, each consisting of two mice, were prepared, and this emulsion was subcutaneously administered to the neck, intracervical intramuscularly, and tailed to each group under the conditions of 150 μL / shot / body. Twelve days after administration, the mice were laparotomized to confirm the enlarged state of the lymph nodes. Table 1 shows the status of swollen lymph nodes, inguinal lymph nodes, and iliac lymph nodes. In the table,-indicates that no swelling was observed, ± indicates that some swelling was observed, + indicates that swelling was observed, and ++ indicates that swelling was large. In addition, since one mouse died in the cervical intramuscular administration group, the result of this group is an observation result of only one mouse.

  In this study, it was confirmed that antigen administration to the cervix was useful for the enlargement of the inguinal lymph nodes and inguinal lymph nodes. In addition, the antigen administration to the back above the tail shows the same result as the cervical administration.

Example 2 Production of Hybridoma (1) Antigen Immunity Thermococcus litoralis-derived ribonuclease H (RNase H) was used as an antigen. RNase H purified by the method described in WO 02/22831 International Publication Pamphlet was mixed with Freund's complete adjuvant to prepare an emulsion containing RNase H at a concentration of 1 mg / mL. Two groups of BALBc mice, each consisting of 3 mice, were prepared. One group was subjected to the tail administration of the emulsion, and the other group was subcutaneously administered to the neck. The dose was 100 μL / shot / body.

  Fourteen days after immunization, the mice were opened, and swollen lymph nodes were collected. There was no clear difference between the two groups regarding iliac lymph nodes. In the tail-administered group, no swollen lymph nodes other than the iliac lymph nodes were observed, except that one mouse had swallowed lymph nodes and one had swollen inguinal lymph nodes. On the other hand, in all mice in the cervical administration group, the swallowing lymph node was larger than the iliac lymph node. In one animal, in addition to the two lymph nodes, the inguinal lymph nodes were also enlarged greatly.

(2) Cell fusion The armpit lymph node of the cervical administration group and the iliac lymph node of the tail administration group were dispersed in a serum-free medium (RPMI1640 medium) and washed, and then 5: 1 with cell fusion myeloma (P3U1). Mixed at a ratio of (lymph node cell: myeloma). The cell mixture was centrifuged to remove the supernatant and a cell pellet was prepared. To this cell pellet, a 50% PEG solution prepared by dissolving in RPMI 1640 medium was added and mixed at a constant speed with light shaking, and then 20 mL of RPMI 1640 medium was similarly added at a constant speed and filled up to 40 mL. In this operation, cell fusion was performed.

(3) HAT selection The fused cells obtained in (2) were suspended in 100 mL of RPMI 1640 containing 10% fetal bovine serum, and dispensed at 100 μL / well into 10 96-well plates. From the next day, the medium was changed to HAT (H: hypoxanthine, A: aminopterin, T: thymidine) added to S-cron cloning medium (manufactured by Sanko Junyaku Co., Ltd.) and the culture was continued for 10 days. went. During this period, the medium was changed three times with the HAT medium including the day after the fusion date. Cells that have grown in this medium are fusion cells that have a de novo synthesis system and are immortalized.

(4) Screening 5 μg / mL PBS (phosphate buffered saline) solution of RNaseH as an immunogen is added at 50 μL / well to an immunoplate (manufactured by Nargenunk) and left overnight at 4 ° C. to give RNaseH. Physically adsorbed. On the next day, the antigen solution was discarded and 50% Blocker Casein (Pierce) was added to 200 μL / well and allowed to stand at room temperature (20-30 ° C.) for 1 hour for blocking operation. Thereafter, the blocking solution was discarded and used as an antigen plate for the following operations.

  Of the 960-well fusion cell cultures obtained in (3) above, the 959-well culture supernatant (using the stock solution) was numbered and placed on the antigen plate, and the primary reaction was performed at room temperature (20-30 ° C). ) For 1 hour. Each well after completion of the reaction was washed 3 times with PBS, and then thoroughly drained with a paper towel. For detection, an anti-mouse IgG rat monoclonal antibody cocktail-peroxidase labeled antibody was used. A 1 μg / mL solution of the labeled antibody was added at 50 μL / well and reacted at room temperature (20-30 ° C.) for 1 hour. Thereafter, the labeled antibody solution was discarded, and the wells were washed 4 times with PBS. The wash solution was thoroughly removed by applying to a paper towel, and then the peroxidase substrate ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] solution (Pierce) was added at 50 μL / well. Color was allowed to develop for 15-30 minutes at room temperature. After completion of the reaction, an equal amount of 150 mM oxalic acid was added to stop the reaction, and positive strains were confirmed with the naked eye and a plate reader. From the reaction of 10 96-well plates, a well having an absorbance at 405 nm exceeding 0.1 in a plate reader was selected as a positive well. 317 wells were positive in the tail administration group and 291 wells in the cervical administration group, and both groups showed substantially equivalent results.

  50 hybridomas in which production of RNase H-binding antibody was observed in the above test were selected from both groups, and tested for inhibition of RNase H activity by the culture supernatant of each cell line. As a result, several strains that significantly inhibited RNase H activity were confirmed in both groups. This indicates that there is no significant difference in the variation of antibodies produced by both groups of hybridomas.

  The present invention provides a method for more reliably obtaining enlarged lymph nodes containing B cells that have acquired antibody-producing ability. By carrying out cell fusion using the enlarged lymph node obtained by the above method, antibody-producing cells can be obtained more efficiently than before. The method of the present invention is a very useful technique for producing mouse monoclonal antibodies, and can be used in various fields.

Claims (5)

  Antibody-producing cells that produce mouse monoclonal antibodies characterized by collecting swollen lymph nodes from mice immunized by administering an antigen to the neck or back, and subjecting the cells obtained from the lymph nodes to cell fusion Manufacturing method.   The method for producing an antibody-producing cell according to claim 1, wherein the administration site of the antigen is subcutaneous or intramuscular.   The method for producing an antibody-producing cell according to claim 1, wherein cells obtained from an enlarged lymph node selected from the armpit lymph node, the inguinal lymph node, or the iliac lymph node are subjected to cell fusion.   The manufacturing method of a monoclonal antibody including the process of producing an antibody producing cell by the method in any one of Claims 1-3, and the process of extract | collecting the monoclonal antibody which the said antibody producing cell produces.   The method for producing a monoclonal antibody according to claim 4, wherein the antibody-producing cells are transplanted into a mouse abdominal cavity and the monoclonal antibody is collected from the mouse ascites.