JP2019048794A - Detection probe for screening of antibody producing cells, and use thereof - Google Patents

Abstract

To provide detection probes for the screening of antibody producing cells which can obtain cells producing structure recognition antibody with high efficiency, and to provide screening methods of antibody producing cells.SOLUTION: The detection probe for the screening of antibody producing cells comprises an antigen protein and a fluorescent substance bound to the N terminal or C-terminal of the antigen protein and is used in selection by a flow cytometry. The screening method of antibody producing cells comprises a labeling step of bringing into contact with antibody producing cells and detection probes for the screening of the antibody producing cells, and a selection step of selecting the antibody producing cells by a flow cytometry after the labeling step.SELECTED DRAWING: None

Description

  The present invention relates to a detection probe for screening antibody-producing cells and the use thereof. Specifically, the present invention relates to a detection probe for screening antibody-producing cells, a kit for screening antibody-producing cells, a method for screening antibody-producing cells, and a method for producing a monoclonal antibody.

  Monoclonal antibodies are highly sensitive detection probes with high antigen binding specificity and binding strength. In addition to being an essential research tool in the life sciences of the post-genome era, monoclonal antibodies are commercially useful highly functional biological materials because they can mass-produce antibodies of uniform quality. Therefore, the market for disease testing agents using antibodies, diagnostic agents, medicines, etc. continues to expand.

  The most important point in determining the usefulness of a monoclonal antibody is that the monoclonal antibody recognizes and binds to the amino acid sequence of the protein (sequence recognition), or recognizes not only the amino acid sequence but also the three-dimensional structure of the protein. (Structural recognition). In particular, in the case of antibody pharmaceuticals to be administered in vivo, efficacy can not be expected unless a structure-recognizing antibody that recognizes the native structure in vivo. Furthermore, tests and diagnostic agents that use biological material as a subject can not be properly diagnosed without structural recognition antibodies as well. Therefore, it is important to obtain a structural recognition antibody.

The production methods of monoclonal antibodies are roughly divided into the following two methods.
1) A hybridoma method of immunizing an animal with an antigen to produce a hybridoma,
2) In vitro display method for genetically expressing the gene of the antigen binding region of immunoglobulin.

  Although both methods have merits and demerits, the hybridoma method is performed in many laboratories because of easy introduction of the system. However, in the hybridoma method, it took time and effort. In addition, in the hybridoma method, antibody-producing cells are selected by ELISA, and a protein having a modified structure fixed to a solid phase is generally used as an antigen. Therefore, although sequence recognition antibodies can be obtained relatively easily, it has been difficult to obtain structure recognition antibodies.

  As a method for obtaining a structural recognition antibody, Herzenberg et al. Developed a hybridoma method using a flow cytometer in 1979 (see, for example, Non-Patent Document 1). In hybridomas, membrane-type immunoglobulin molecules having the same antigen binding specificity as secretory immunoglobulin are expressed on cell membranes. For this reason, the antigen binds not only to secretory immunoglobulins, ie antibodies, but also to this membrane type immunoglobulin. Therefore, in the method developed by Herzenberg et al., The target hybridoma is fluorescently labeled by screening the antigen fluorescently, and screening and cloning are performed.

  In addition, in 2009, Meagher et al. Developed a hybridoma method using a genetically engineered hybridoma and a flow cytometer (see, for example, Patent Documents 1 to 3 and Non-Patent Document 2). Specifically, first, B cells obtained from mice immunized with an antigen-immunized myeloma cell with an Igα gene, or B cells obtained from a mouse immunized with an antigen-producing myeloma cell and introduced an Igα gene And cell fusion to obtain a hybridoma. In this hybridoma, an Igα molecule is highly expressed on the cell membrane of the hybridoma, which can enhance the expression of membrane-type immunoglobulin. Then, the hybridoma, a biotinylated labeled antigen peptide, and Streptavidin-PE are brought into contact, and cloning is carried out using a flow cytometer.

U.S. Patent No. 7629171 U.S. Pat. No. 7,148,040 U.S. Pat. No. 8,912,385

Parks D R et al., “Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter”, Proc. Natl. Acad. Sci. USA, vol. 76, no. 4, p 1962-1966, 1979. Price P W et al., “Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas”, J. Immunol. Methods, vol. 343, no. 1, p28-41, 2009.

  In the methods described in Patent Literatures 1 to 3 and Non Patent Literatures 1 and 2, a structure recognition antibody can be obtained. However, short-chain peptide antigens that can be easily labeled with fluorescence are used, and a structure-recognizing antibody can be selected only when the short-chain peptide antigens reproduce the structure of a natural protein. In addition, the antibody acquisition efficiency was low, and there was room for improvement.

  The present invention has been made in view of the above circumstances, and is a detection probe for screening antibody-producing cells which can obtain cells producing the structure-recognizing antibody against the target antigen protein with high efficiency, a kit for screening antibody-producing cells, Provided are methods of screening antibody-producing cells. In addition, the present invention provides a method for producing a monoclonal antibody capable of obtaining a structural recognition antibody against a target antigen protein with high efficiency.

That is, the present invention includes the following aspects.
The detection probe for screening antibody-producing cells according to the first aspect of the present invention comprises an antigen protein and a fluorescent substance bound to the N-terminus or C-terminus of the antigen protein, and is used for selection by flow cytometry Be

  In the detection probe for screening antibody-producing cells according to the first aspect, the fluorescent substance may be a fluorescent protein.

  In the detection probe for screening antibody-producing cells according to the first aspect, the antigen protein may be ammonium sulfate precipitate.

  A kit for screening antibody-producing cells according to a second aspect of the present invention comprises a cloning site for inserting a first nucleic acid encoding an antigen protein, and a fluorescent protein linked upstream or downstream of the cloning site. And a second vector comprising: a second vector, and the first vector is a fusion comprising the antigen protein and the fluorescent protein linked to the N-terminus or C-terminus of the antigen protein It is a vector for expressing a protein, and is used for selection by flow cytometry.

  The kit for screening antibody-producing cells according to the second aspect further comprises a second vector comprising a cloning site for inserting a third nucleic acid encoding an antigen protein, wherein the second vector is non- It may be a vector for expressing an antigen protein for human animal immunity.

  The kit for screening antibody-producing cells according to the second aspect may further include ammonium sulfate, and the ammonium sulfate may be for purifying the fusion protein by ammonium sulfate precipitation.

  In the method of screening antibody-producing cells according to the third aspect of the present invention, a labeling step in which antibody-producing cells are brought into contact with the detection probe for screening antibody-producing cells according to the first aspect; And e. A selection step of selecting production cells by flow cytometry.

  Detection for screening of antibody-producing cells containing two or more types of antibody-producing cells producing antibodies against different antigenic proteins in the labeling step, and two or more types of the antigen proteins having different fluorescent substances bound to each other It may be in contact with the probe.

  The method for producing a monoclonal antibody according to the fourth aspect of the present invention is a method comprising a seeding step of seeding the antibody-producing cells obtained by using the method for screening antibody-producing cells according to the third aspect above one by one.

  In the method for producing a monoclonal antibody according to the fourth aspect, the isotype of the monoclonal antibody may be IgG.

  According to the detection probe for screening antibody-producing cells, the kit for screening antibody-producing cells, and the screening method for antibody-producing cells of the above-described embodiment, cells producing a structure-recognizing antibody against the target antigen protein can be obtained with high efficiency. Moreover, according to the method for producing a monoclonal antibody of the above aspect, a structure recognition antibody against a target antigen protein can be obtained with high efficiency.

It is a figure which shows the detection probe for screening of the antibody producing cell which concerns on one Embodiment of this invention. FIG. 1 is a schematic process view showing a method of producing a monoclonal antibody according to an embodiment of the present invention. It is a CBB stained image of fusion protein 1 (EGFP + α-Tubulin partial protein) and fluorescent protein (EGFP) purified using ammonium sulfate of each saturation in Preparation Example 1. It is a graph which shows the result of having detected the hybridoma (control group) to which the fluorescent protein in Example 1 was added, and the hybridoma to which the fusion protein 1 was added by the flow cytometer. It is a fluorescence-microscope image of the hybridoma which added the fluorescence protein (EGFP) in Example 1, or fusion protein 1 (EGFP + (alpha) -Tubulin partial protein). It is an image which shows the result of having performed the western blotting using the antibody obtained in Example 1 in Experiment 1. FIG. 7 is a graph in which the degree of sequence recognition (fluorescence intensity in ELISA) is plotted on the vertical axis and the degree of structural recognition (fluorescence intensity on the flow cytometer) is plotted on the horizontal axis for the antibodies obtained in Example 1 in Test Example 1. . It is a graph which shows the result of having detected the spleen cell which added the fluorescent protein in Example 2 (control group), and the spleen cell which added the fusion protein 1 with the flow cytometer.

<< Detection probe for screening antibody-producing cells >>
In one embodiment of the present invention, the detection probe for screening antibody-producing cells comprises an antigen protein and a fluorescent substance bound to the N-terminus or C-terminus of the antigen protein, and is used for selection by flow cytometry .

  Each component constituting the detection probe for screening antibody-producing cells of the present embodiment will be described in detail below.

<Antigen protein>
The antigen protein contained in the detection probe for screening antibody-producing cells of the present embodiment may be any special one as long as it has a three-dimensional structure in the natural state (hereinafter sometimes referred to as "natural type three-dimensional structure"). There is no limitation. Also, the antigen protein may be a full-length protein or a partial protein including a target site that can be an epitope.

<Fluorescent substance>
The fluorescent substance contained in the detection probe for screening antibody-producing cells of the present embodiment is not particularly limited as long as it is a publicly known one generally used in a flow cytometer. Specific examples of the fluorescent substance include, for example, polycyclic aromatic compounds shown in Table 1 below, and fluorescent proteins shown in Table 2 and Table 3 below.

  Among them, the fluorescent substance is preferably a fluorescent protein. By using a fluorescent protein as a fluorescent substance, as described later, a detection probe for screening antibody-producing cells can be easily produced as a fusion protein of a fluorescent protein and an antigen protein. Further, by using a fluorescent protein as a fluorescent substance, it is not necessary to chemically modify the fluorescent substance to an amino group or a thiol group of an antigen protein at the time of producing a detection probe for screening antibody-producing cells. Therefore, it is possible to prevent the three-dimensional structure of the antigen protein from being changed due to changes in pH, temperature and the like to be in a denatured state.

FIG. 1 is a view showing a detection probe for screening antibody-producing cells according to an embodiment of the present invention.
As shown in FIG. 1, the detection probe 10 for screening antibody-producing cells contains an antigen protein 1 and a fluorescent substance 2. The fluorescent substance 2 may be bound to the antigen protein 1 directly or indirectly.

  When the antigen protein 1 and the fluorescent substance 2 are directly bound, the binding mode of the two is, for example, coordination, covalent bond (eg, disulfide bond, amide bond, ester bond, etc.), hydrogen bond, It may be ionic bonding, hydrophobic interaction, physical adsorption and the like. When the fluorescent substance 2 is a fluorescent protein, it is preferable that the antigen protein 1 and the fluorescent substance 2 be directly bound by an amide bond.

  When the antigen protein 1 and the fluorescent substance 2 are indirectly linked, both may be linked via a linker. Examples of the linker include a peptide linker consisting of 1 to several (for example, about 1 to 5 etc.) amino acids, a linker consisting of a synthetic polymer compound such as polyethylene glycol, and the like, and the invention is not limited thereto.

  Alternatively, when the antigen protein 1 and the fluorescent substance 2 are indirectly bound, for example, there is a method of utilizing proteins that bind to each other such as biotin-avidin. Specifically, a biotin-labeled antigen protein is prepared, and antibody-producing cells are brought into contact with the biotin-labeled antigen protein. Furthermore, by contacting avidin to which a fluorescent substance is bound (for example, streptavidin), a complex consisting of avidin to which an antibody-producing cell-biotin labeled antigen protein-fluorescent substance is bound is formed. Thereby, antibody-producing cells are indirectly labeled with a fluorescent substance via biotin and avidin.

  Further, the binding position of the fluorescent substance 2 in the antigen protein 1 may be a position at which the three-dimensional structure of the antigen protein is maintained, and may be the N-terminus or C-terminus of the antigen protein. The binding position of the fluorescent substance 2 in the antigen protein 1 is preferably opposite to the target site that can be an antibody binding site (epitope) in the antigen protein. For example, when the target site that can be an epitope is present in the N-terminal region side, the binding position of the fluorescent substance 2 is preferably at the C-terminus. On the other hand, when the target site capable of becoming an epitope is present on the C-terminal region side, the binding position of the fluorescent substance 2 is preferably the N-terminus. In addition, when the target site which can be an epitope is present at the central portion of the amino acid sequence of the protein, the binding position of the fluorescent substance 2 may be the N-terminus or the C-terminus.

  In the detection probe 10 for screening antibody-producing cells, the fluorescent substance 2 is linked so as to maintain the native conformation of the antigen protein 1. Therefore, the detection probe 10 for screening antibody-producing cells is bound to the membrane type immunoglobulin molecule 3 present on the cell membrane of the antibody-producing cells producing the structure recognition antibody against the target antigen protein 1 among the antibody-producing cells. It can be labeled. Therefore, by using the detection probe 10 for screening antibody-producing cells, it is possible to obtain cells that produce a structure-recognizing antibody against the target antigen protein with high efficiency.

  In the present specification, the “structure-recognizing antibody” means an antibody that recognizes an epitope that is a natural conformation in an antigen protein. On the other hand, the "sequence recognition antibody" means an antibody that recognizes an epitope having a steric structure (hereinafter, may be referred to as a "denatured steric structure") in a denatured state in an antigen protein. Mainly, the modified steric structure is linear. In particular, as shown in Examples described later, it can be determined that the higher the fluorescence intensity is detected at the time of detection by the flow cytometer, the more antibody-producing cells producing an antibody with high structure recognition ability. In addition, as shown in Examples described later, it can be determined that the higher the fluorescence intensity is detected at the time of detection by ELISA, the more the antibody-producing cells produce an antibody with high sequence recognition ability.

  Conventionally, ELISA is mainly used to evaluate and screen monoclonal antibodies produced by antibody-producing cells. The antigen protein used in the ELISA is in a denatured state, and a sequence recognition antibody is obtained. However, the sequence recognition antibody can not recognize the antigen protein if the native conformation of the epitope in the antigen protein and the denatured conformation differ, and the utility is not very high.

  On the other hand, in the present embodiment, a membrane type immunoglobulin expressed on the cell membrane of antibody-producing cells using the detection probe for screening antibody-producing cells and a flow cytometer, and an antigen retaining the structure Screening is performed by binding of Thus, structure-recognizing antibodies can be screened.

  An antibody that recognizes a structure is essential to bind to a protein that retains physiological activity in vivo. Therefore, the monoclonal antibody produced by the antibody-producing cells obtained using the detection probe for screening antibody-producing cells of the present embodiment is a useful antibody that can be used for test diagnostic agents and medicines. In addition, the present invention is highly useful because it can reliably obtain a structural recognition antibody that has conventionally been missed in screening.

  In the present specification, the "antibody-producing cells" may be any cells that produce antibodies against a target antigen protein, and there is no particular limitation. Specific examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like. Among them, spleen cells or regional lymph node cells are preferable as antibody-producing cells. Alternatively, hybridomas may be used as antibody-producing cells.

  In the present specification, a "hybridoma" is obtained by cell fusion of antibody-producing cells derived from a non-human animal immunized with a target antigen protein and myeloma cells. Alternatively, it can be obtained by cell fusion of a human-derived antibody-producing cell producing an antibody against a target antigen protein and a myeloma cell.

  The non-human animal is preferably a non-human mammal (non-human mammal) or avian animal. Examples of the non-human mammals include, but are not limited to, rodents such as mice, rats and hamsters, rabbits, goats and monkeys. Moreover, as said avian animal, a chicken etc. are mentioned, for example, It is not limited to these.

  For example, antibody-producing cells such as peripheral blood mononuclear cells isolated from human blood may be used as human-derived antibody-producing cells. Alternatively, human antibody-producing cells obtained by causing an antigen protein to act in a medium on cells such as lymphocytes of human origin that have not been immunized may be used. In addition, human-derived antibody-producing cells may be immortalized using an EB virus or the like.

<Method of producing detection probe for screening antibody-producing cells>
When the fluorescent substance is a polycyclic aromatic compound and the antigen protein and the fluorescent substance are directly bound, the detection probe for screening antibody-producing cells of the present embodiment can be produced by the method described below. it can.

  First, a host is transformed using an expression vector containing a nucleic acid encoding an antigen protein. The host is then cultured to express the antigen protein. The conditions such as the composition of the medium, the temperature and time of culture, and the addition of an inducer can be determined by those skilled in the art according to known methods so that the transformant grows and the antigen protein is efficiently produced. Also, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, transformants can be selected by adding an antibiotic to the medium. Then, the antigen protein expressed by the host is purified by an appropriate method to obtain an antigen protein. Then, the fluorescent substance can be chemically modified to the amino group or thiol group of the obtained antigen protein or the like to obtain a detection probe for screening antibody-producing cells.

Alternatively, when the fluorescent substance is a polycyclic aromatic compound and the antigen protein and the fluorescent substance are indirectly bound, the detection probe for screening antibody-producing cells of this embodiment is produced by the method described below. be able to.
First, a host is transformed using an expression vector containing a first nucleic acid encoding an antigen protein and a second nucleic acid encoding AviTag (registered trademark) upstream or downstream of the first nucleic acid. . Then, the host is cultured to express an AviTag (registered trademark) -added antigen protein. Conditions such as the composition of the medium, the temperature and time of culture, and the addition of an inducer are determined according to known methods so that the transformant grows and the antigenic protein to which AviTag (registered trademark) is added is efficiently produced. The vendor can decide. Also, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, transformants can be selected by adding an antibiotic to the medium. Then, the antigen protein to which AviTag (registered trademark) expressed by the host has been added is purified by an appropriate method to obtain the antigen protein to which AviTag (registered trademark) is added. Subsequently, biotin is introduced into the obtained antigenic protein to which AviTag (registered trademark) has been added using biotin ligase. Furthermore, a detection probe for screening antibody-producing cells can be obtained by bringing a biotin-labeled antigen protein into contact with streptavidin chemically bound with a fluorescent substance.

When the fluorescent substance is a fluorescent protein, the detection probe for screening antibody-producing cells of this embodiment can be produced by the method described below.
First, a host is transformed using an expression vector containing a first nucleic acid encoding an antigen protein and a second nucleic acid encoding a fluorescent protein upstream or downstream of the first nucleic acid. At this time, in the case where the antigen protein and the fluorescent protein are indirectly linked via a peptide linker, a fourth encoding of a peptide linker is performed between the first nucleic acid and the second nucleic acid. A nucleic acid may be inserted. Then, the host is cultured to express a fusion protein in which the antigen protein and the fluorescent protein are directly or indirectly linked. Conditions such as the composition of the medium, the temperature and time of culture, and the addition of an inducer can be determined by those skilled in the art according to known methods so that the transformant grows and the fusion protein is efficiently produced. Also, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, transformants can be selected by adding an antibiotic to the medium. Then, the fusion protein expressed by the host is purified to obtain a detection probe for screening antibody-producing cells.

  The method for purifying the fusion protein may be any method as long as it can be performed while maintaining the three-dimensional structure of the antigen protein in the fusion protein, and can be appropriately selected from known protein purification methods. Among them, purification by ammonium sulfate precipitation is preferable. By using ammonium sulfate precipitation, endotoxin (eg, Lipopolysaccharide (LPS) etc.) derived from a host such as E. coli can be removed with high efficiency while maintaining the three-dimensional structure of the antigen protein in the fusion protein. . The degree of saturation of ammonium sulfate used in the ammonium sulfate precipitation method may be appropriately adjusted according to the type and charge state of the antigen protein. For example, as shown in Examples described later, when purifying a fusion protein of α-Tubulin and EGFP, the saturation degree of ammonium sulfate in the sample can be 40% or more and 60% or less, and is about 50%. Is preferred. The obtained ammonium sulfate precipitate (crude product) can be used as an antigen protein contained in a detection probe for screening antibody-producing cells. The ammonium sulfate precipitate (crude product) contains host-derived proteins other than the fusion protein. Therefore, in the screening method to be described later, by using ammonium sulfate precipitate (crude product) as the antigen protein of the detection probe for screening antibody-producing cells, blocking of host-derived proteins other than the fusion protein covering the surface of antibody-producing cells Act as an agent. This prevents nonspecific adsorption of the fluorescent protein on the cell surface of the antibody-producing cells without passing through the antigen protein, and fluorescently labels cells producing a structural recognition antibody against the target antigen protein with higher efficiency. be able to.

  Alternatively, low-molecular impurities such as ammonium sulfate may be removed by further passing the crude product (precipitate of ammonium sulfate) obtained by the ammonium sulfate precipitation method through a gel filtration column. Examples of the carrier used for the gel filtration column include, but not limited to, Sephadex G-50, Sephadex G-75, etc. manufactured by GE Healthcare.

  In addition, the purified product after gel filtration may be filter-sterilized by further passing it through a membrane filter. The pore size of the membrane filter can be, for example, 0.1 μm or more and 0.5 μm or less, and can be 0.2 μm or more and 0.4 μm or less.

<Use>
The detection probe for screening antibody-producing cells of the present embodiment is suitably used to screen and label cells producing a structure-recognizing antibody. In addition, as described later, by selecting cells that are in contact with the detection probe for screening antibody-producing cells of the present embodiment by flow cytometry, cells that produce a fluorescently labeled structure-recognizing antibody are selected one by one. It can be selected and isolated.

«Kit for screening antibody-producing cells»
A kit for screening antibody-producing cells according to one embodiment of the present invention encodes a cloning site for inserting a first nucleic acid encoding an antigen protein, and a fluorescent protein linked upstream or downstream of the cloning site. And a second vector comprising: a second vector, and the first vector is a fusion comprising the antigen protein and the fluorescent protein linked to the N-terminus or C-terminus of the antigen protein It is a vector for expressing a protein, and is used for selection by flow cytometry.

  According to the kit of the present embodiment, it is possible to easily produce a fusion protein of a fluorescent protein and any antigen protein whose conformation is maintained. In addition, by using the fusion protein, it is possible to obtain cells which produce a structural recognition antibody against any antigen protein with high efficiency.

<First vector>
The first vector contained in the kit of the present embodiment is a vector for expressing a fusion protein. The first vector comprises a cloning site for inserting a first nucleic acid encoding an antigenic protein, and a second nucleic acid encoding a fluorescent protein linked upstream or downstream of the cloning site.

  In addition, when a fusion protein in which an antigen protein and a fluorescent protein are indirectly linked via a peptide linker is expressed using a first vector, the first vector may be a first nucleic acid encoding the antigen protein and It is preferred to include a fourth nucleic acid encoding a peptide linker between the second nucleic acid encoding a fluorescent protein.

  The antigen protein and the fluorescent protein are not particularly limited, and include the same ones as exemplified in the above-mentioned “detection probe for screening antibody-producing cells”.

  Moreover, as a kind of 1st vector, what is necessary is just an expression vector, and there is no special limitation. Examples of expression vectors include plasmids derived from E. coli such as pBR322, pBR325, pCold (registered trademark), pUC12 and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5 and pC194; yeast-derived plasmids such as pSH19 and pSH15; Bacteriophages such as phages; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, etc .; and vectors modified from these can be used.

  In the above-mentioned expression vector, the promoter for expressing the fusion protein is not particularly limited. For example, animal cells such as EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter etc. Promoters for expression, 35S promoter for cauliflower mosaic virus (CaMV), promoters for expression using plant cells such as REF (rubber elongation factor) promoter, etc. Hosts for insect cells such as polyhedrin promoter and p10 promoter The promoter for expression can be used. These promoters can be appropriately selected depending on the host expressing the fusion protein.

  The above expression vector may further have an enhancer, a splicing signal, a polyA addition signal, a selection marker, an origin of replication and the like.

<Second vector>
The kit of this embodiment is further. A second vector may be provided. The second vector is a vector for expressing an antigen protein for non-human animal immunity. The second vector also contains a cloning site for inserting a third nucleic acid encoding an antigenic protein. By immunizing a non-human animal with the obtained antigen protein for immunizing a non-human animal, an antibody-producing cell for calculating an antibody against the target antigen protein can be obtained from the animal.

  The third nucleic acid contained in the second vector may be a nucleic acid encoding an antigen protein of the same type as the first nucleic acid contained in the first vector. In addition, the antigen protein obtained by expressing the second vector is identical to the amino acid sequence of the target site that can be an epitope in the antigen protein obtained by expressing the first vector, and consists of different amino acid sequences as a whole It may be a partial protein, or it may be a partial protein or a full-length protein consisting of the same amino acid sequence as the antigen protein obtained by expressing the first vector. Among them, the antigen protein obtained by expressing the second vector is preferably a partial protein or a full-length protein consisting of the same amino acid sequence as the antigen protein obtained by expressing the first vector. Thereby, cells producing a structural recognition antibody against a target antigen protein can be obtained with high efficiency.

  Moreover, as a kind of 1st vector, what is necessary is just an expression vector, and the thing similar to what was illustrated by the above-mentioned "1st vector" is mentioned.

<Ammonium sulfate>
The kit of the present embodiment may further include ammonium sulfate. Ammonium sulfate is for purifying the fusion protein expressed using the above first vector by ammonium sulfate precipitation.

  In the screening method described later, by using a crudely purified product (ammonium sulfate precipitate) obtained by purifying the fusion protein by ammonium sulfate precipitation, a host derived from a host other than the fusion protein contained in the crude purified product (ammonium sulfate precipitate) Protein acts as a blocking agent covering the surface of antibody-producing cells. This prevents nonspecific adsorption of the fluorescent protein on the cell surface of the antibody-producing cells without passing through the antigen protein, and fluorescently labels cells producing a structural recognition antibody against the target antigen protein with higher efficiency. be able to.

<Others>
The kit of the present embodiment may further include a gel filtration column for purifying the fusion protein, a sterilizing filter and the like. Examples of the gel filtration column and the sterilization filter include the same ones as exemplified in the above-mentioned "detection probe for screening antibody-producing cells". In addition, the kit of the present embodiment may further include a fusion protein and a buffer for dissolving and storing an antigen protein for immunizing a non-human animal.

<Use>
Using the first vector contained in the kit of the present embodiment and, if necessary, purification means such as ammonium sulfate, gel filtration column, sterilization filter, etc., to manufacture the above-described detection probe for screening antibody-producing cells it can. Moreover, the antigen protein for non-human animal immunity can be manufactured by using the 2nd vector contained in the kit of this embodiment. Further, by immunizing a non-human animal with the antigen protein for immunizing the non-human animal, antibody-producing cells against the immunized antigen protein can be obtained. The obtained detection probe for screening antibody-producing cells and antibody-producing cells are suitably used for fluorescently labeling and screening cells that produce a structure-recognizing antibody in the antibody-producing cells. In addition, as described later, by selecting cells that are in contact with the detection probe for screening antibody-producing cells of the present embodiment by flow cytometry, cells that produce a fluorescently labeled structure-recognizing antibody are selected one by one. It can be selected and isolated.

«Method for screening antibody producing cells»
In the method of screening antibody-producing cells according to one embodiment of the present invention, a labeling step in which antibody-producing cells are brought into contact with the detection probe for screening antibody-producing cells according to the first aspect, and the antibody step after the labeling step. And e. A selection step of selecting production cells by flow cytometry.

  According to the screening method of the present embodiment, it is possible to obtain cells producing the structural recognition antibody against the target antigen protein with high efficiency.

  FIG. 2 is a schematic flow diagram of a method for producing a monoclonal antibody according to an embodiment of the present invention described later. The screening method of the present embodiment will be described in detail below with reference to FIG.

<Labeling process>
First, in the labeling unit 11, the antibody-producing cells 4 and the detection probe 10 for screening antibody-producing cells are brought into contact in the culture vessel 9a containing the culture medium. In the detection probe 10 for screening antibody-producing cells, a fluorescent substance 2 is bound to the N-terminus or C-terminus of the antigen protein 1. Moreover, the culture container 9a can select and use the well-known container generally used for a cell culture suitably.

  Among them, the fluorescent substance 2 bound to the N-terminus or C-terminus of the antigen protein 1 is preferably a fluorescent protein. By using a fluorescent protein as a fluorescent substance, as described above, a detection probe for screening antibody-producing cells can be easily produced as a fusion protein of a fluorescent protein and an antigen protein. Further, by using a fluorescent protein as a fluorescent substance, it is not necessary to chemically modify the fluorescent substance to the amino group or thiol group of the antigen protein as described above. Therefore, it is possible to prevent the three-dimensional structure of the antigen protein from being changed due to changes in pH, temperature and the like to be in a denatured state.

  When the detection probe 10 for screening antibody-producing cells used in the screening method of the present embodiment is a fusion protein containing an antigen protein and a fluorescent protein, it is a crudely purified product purified by ammonium sulfate precipitation. preferable. By using this crudely purified product, host-derived proteins other than the fusion protein contained in the crudely purified product act as a blocking agent covering the surface of antibody-producing cells. This prevents nonspecific adsorption of the fluorescent protein on the cell surface of the antibody-producing cells without passing through the antigen protein, and fluorescently labels cells producing a structural recognition antibody against the target antigen protein with higher efficiency. be able to.

  In the labeling step, among antibody producing cells 4, antibody producing cells (target antibody producing cells) 4a producing a structural recognition antibody against the target antigen protein 1 are a membrane type immunoglobulin 3 present on the cell membrane, an antigen protein 1 and Specifically bind. Thereby, the target antibody producing cell 4 a is labeled with the fluorescent substance 2 via the antigen protein 1. On the other hand, in the other antibody-producing cells 4b, the membrane-type immunoglobulin present on the cell membrane does not bind to the antigen protein 1. Therefore, the target antibody-producing cells 4 a are not labeled with the fluorescent substance 2 via the antigen protein 1.

  In addition, in the labeling step, two or more types of antibody-producing cells producing antibodies against different antigenic proteins may be brought into contact with a detection probe for screening antibody-producing cells. At this time, the detection probe for screening antibody-producing cells to be used contains two or more types of antigen proteins to which different fluorescent substances are bound.

  The antibody-producing cells to be used may be, for example, antibody-producing cells obtained from one non-human animal immunized with two or more different target antigen proteins. Alternatively, for example, it may be antibody-producing cells obtained by immunizing different target antigen proteins in two or more non-human animals of the same species. Or, for example, in the case of antibody-producing cells of human origin, as described above, human-derived antibody-producing cells such as peripheral blood mononuclear cells isolated from one or two or more individuals of human blood Good. Alternatively, for example, a human-derived antibody obtained by causing two or more different target antigen proteins to act on cells such as non-immunized one or two or more human-derived lymphocytes. It may be a producer cell. In any case, multiple types of antibody-producing cells can be obtained in combination.

  Therefore, in the labeling step in the present embodiment, by using detection probes for screening antibody-producing cells which are bound to different fluorescent substances and which contain an antigen protein of a type corresponding to the immune antigen protein, a plurality of purposes can be obtained. Antibody-producing cells that produce a structure-recognizing antibody against the antigen protein can be screened simultaneously.

In addition, the time for which the antibody-producing cells 4 and the fluorescently labeled antigen 10 are in contact with each other, ie, the labeling time may be, for example, 30 minutes or more and 24 hours or less.
When the labeling time is equal to or more than the above lower limit value, the membrane type immunoglobulin 3 on the cell membrane of the antibody producing cell 4 and the antigen protein 1 can be uniformly contacted to perform an antigen-antibody reaction. In addition, when the labeling time is equal to or less than the upper limit, the antibody-producing cells 4 can be efficiently labeled in a short time.

In particular, when obtaining antibody-producing cells in which the membrane type immunoglobulin is isotype IgG, the labeling time is preferably less than 12 hours and 10 hours or less, more preferably 30 minutes or more and 6 hours or less, and 1 hour or more and 5 hours The following is particularly preferable, and about 2 hours is the most preferable.
When the labeling time is not more than the above upper limit value, among antibody producing cells 4, antibody producing cells of which the isotype of membrane type immunoglobulin on the cell membrane is IgG can be preferentially labeled. It is presumed that this is because IgG is structurally easier to contact with antigen than IgM and easier to label.
This is not known in the method using the conventional flow cytometer such as the method developed by Herzenberg et al. Described above and the method developed by Meagher et al., And was found for the first time by the present inventors.
On the other hand, in the screening method of the present embodiment, the labeling time is equal to or less than the upper limit value, and among the antibody-producing cells 4, antibody-producing cells of which the isotype of membrane type immunoglobulin on the cell membrane is IgG are prioritized. Can be labeled.

  In addition, dead cells may be stained in advance among the antibody-producing cells 4 using 7-Amino-Actinomycin D (7-AAD) or the like in parallel with the labeling step or after the labeling step. At this time, as the fluorescent substance 2 bound to the antigen protein 1 and the fluorescent substance for discriminating dead cells, a combination in which the maximum fluorescence wavelengths of the two are different may be appropriately selected.

[Selection process]
The selection step is a step of selecting target antibody-producing cells 4a among the antibody-producing cells 4 after the labeling step by flow cytometry. As a method of evaluating the selection result, for example, an antibody producing cell showing a fluorescence intensity at least 2 times, preferably 2.5 times or more, more preferably 5 times or more from the mode of the signal in unlabeled antibody producing cells 4 4 may be a positive cell.

  Selection by flow cytometry is performed using a flow cytometer 12. The flow cytometer 12 sorts the antibody-producing cells 4 one by one, detects the fluorescence, and can select the target antibody-producing cells 4a from which the fluorescence is detected, as long as the target antibody-producing cells 4a can be selected. The device may be used as it is. Specific examples thereof include MoFlo Astroios EQ / EQs (manufactured by Beckman Counter Co., Ltd.), MoFlo XDP (manufactured by Beckman Counter Co., Ltd.), and the like, but the invention is not limited thereto.

As shown in FIG. 2, the flow cytometer 12 may include, for example, a first detector 5, a second detector 6, an excitation light source 7, an air sorter 8, and the like.
The first detector 5 is, for example, for detecting fluorescence, and is not particularly limited. Further, the second detector 6 is for measuring, for example, the size of antibody-producing cells and the like, and there is no particular limitation. Also, the excitation light source 7 is for exciting the fluorescent substance bound to the antibody-producing cells, and there is no particular limitation. In addition, the sorter 8 is for discharging other antibody-producing cells 4b out of the system by, for example, a propellant substance 8a such as air; a liquid such as water, etc.

  In the screening method of the present embodiment, screening for antibody-producing cells can be performed in a short time of about one day because selection is performed by flow cytometry. Further, in the screening method of the present embodiment, in the selection by flow cytometry, the screening is performed by binding of the membrane type immunoglobulin expressed on the cell membrane of the antibody-producing cells and the antigen retaining the structure. Thus, it is possible to screen for structural recognition antibodies against the target antigen protein.

Moreover, in order to enhance the sorting efficiency of cells, a washing step may be provided to wash the flow channel of the flow cytometer before the selection step or when using a plurality of samples.
In the washing step, washing may be performed using a known washing solution. Specifically, for example, a commercially available cleaning solution having high detergency and an appropriate viscosity (eg, hypochlorite, about 2% sodium hydroxide, and a surfactant (eg, alkylamine oxide etc.) By using a pipe cleaning agent or the like, proteins and the like remaining in the flow path can be sufficiently cleaned.

<< Method for Producing Monoclonal Antibody >>
The method for producing a monoclonal antibody according to one embodiment of the present invention is a method comprising a seeding step of seeding antibody-producing cells obtained by using the method for screening antibody-producing cells according to the above embodiment one by one.

  According to the manufacturing method of the present embodiment, it is possible to obtain a structure-recognizing antibody against a target antigen protein with high efficiency. An antibody that recognizes a structure is essential to bind to a protein that retains physiological activity in vivo. Therefore, the antibody obtained by the production method of the present embodiment is a useful antibody that can be used as a test diagnostic agent or a medicine. In addition, as shown in Examples described later, according to the conventional production method, since the structure-recognizing antibody which has been missed in the screening can be surely obtained, the utility is high.

  As used herein, "monoclonal antibody" means an antibody with substantially uniform specificity.

  Moreover, the isotype of the monoclonal antibody obtained by the production method of the present embodiment is not particularly limited, and, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2a, IgG2b, IgG3, IgG4), IgM Etc. Among them, the isotype of the monoclonal antibody obtained by the production method of the present embodiment is preferably IgG, since it is most commonly used and is involved in many of the immune responses that occur against pathogen invasion.

Up to the labeling step and the selecting step, antibody-producing cells producing a structure-recognizing antibody against the target antigen protein are screened using the same method as the above-mentioned "Method for screening antibody-producing cells".
The manufacturing method of this embodiment will be described in detail below with reference to FIG.

[Seeding process]
The seeding step is a step of seeding the antibody-producing cells selected in the selection step, that is, antibody-producing cells evaluated as positive one by one. In the manufacturing method of the present embodiment, by using the flow cytometer 12, the antibody-producing cells 4a evaluated as positive can be automatically seeded one by one into the culture container 9b, so that it does not take time for cloning.

  The culture vessel 9b may be any known one used for culturing antibody-producing cells, and the material and the shape thereof are not particularly limited. For example, as the culture vessel 9b, a multiwell plate in which any number of wells are arranged, a petri dish and the like can be mentioned. The number of wells is, for example, 6, 12, 24, 96, 384, 1536, etc. per plate. In addition, the bottom of culture vessel 9b has a small impact when seeding with antibody-producing cells evaluated as positive, and can keep the cell survival rate higher, so that the bottom of the cross-section has a U-shape It may be.

  In addition, the production method of the present embodiment may include an immunization step, a cell fusion step, a proliferation step and the like before the labeling step. In addition, after the seeding step, a culture step, an evaluation step and the like may be provided.

[Immunization process]
The immunization step is a step of immunizing a non-human animal with a target antigen protein.
Examples of non-human animals that are immunized with the target antigen protein include the same ones as exemplified in the above-mentioned “detection probe for screening antibody-producing cells”.

  Examples of immunizing proteins include those similar to those exemplified in the above-mentioned “kit for screening antibody-producing cells”.

  For example, when immunizing a mouse, the immunization is carried out by injecting the antigen protein subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like. Inoculation intervals, inoculum size, etc. vary depending on the inoculation method. For example, when immunizing antigen protein in the peritoneal cavity of a mouse and immunizing, for example, after immunizing once with antigen protein, immunizing after 2 weeks, a total of 2 times Immunization should be performed to some extent. Furthermore, antibody-producing cells such as spleen cells, lymph node cells (eg, B cells), peripheral blood leukocytes, etc. may be removed from the immunized animals, for example, about 3 days after the last immunization.

[Cell fusion process]
The cell fusion step is a step required when using a hybridoma. Specifically, this is a step of cell fusion of antibody-producing cells and myeloma cells to obtain hybridomas.

  Examples of antibody-producing cells used for cell fusion include the same as those exemplified in the above-mentioned "detection probe for screening antibody-producing cells".

  As a myeloma cell used for cell fusion, a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient strain required for the regeneration pathway (salvage pathway) is preferably used. Preferably, the myeloma cells are those into which an Igα gene or the like has not been introduced. That is, in the obtained hybridoma, it is preferable that the expression of the membrane type immunoglobulin is not enhanced, and the membrane type immunoglobulin derived from the antibody producing cell is naturally expressed on the cell membrane. In addition, known cell lines can be used as myeloma cells. Known cell lines include, for example, P3X63-Ag.8 strain (ATCC TIB9), P3X63-Ag.8.U1 strain (JCRB9085), P3 / NSI / 1-, which are HGPRT-deficient cell lines derived from BALB / c mice. Ag4-1 strain (JCRB0009), P3x63Ag8.653 strain (JCRB0028), Sp2 / 0-Ag14 strain (JCRB0029) and the like can be mentioned.

  Myeloma cells are prepared as a preparation procedure prior to cell fusion in a basic medium such as Eagle's minimal basic medium (MEM), Dulbecco's modified MEM, PRMI 1640, etc., 10% CS (calf serum), 10% FCS (fetal calf serum) What is necessary is just to use what was culture | cultivated previously by the culture medium which added etc.).

  The antibody-producing cells and the myeloma cells may be from different animal species as long as they can be fused, but are preferably from the same animal species.

  Moreover, as a method of cell fusion, for example, the well-known method of Koehler and Milstein (see, for example, Kohler & Milstein, Nature, 256: 495, 1975.) and the like can be mentioned. Specifically, as a method of cell fusion, for example, antibody-producing cells and myeloma cells in a ratio of 1: 1 to 20: 1 in a culture medium for animal cell culture such as DMEM, RPMI-1640 medium and the like without serum It may be mixed and fusion reaction may be performed. In addition, polyethylene glycol with a molecular weight of about 10 to 80% and an average molecular weight of 1,500 to 4,000 Dalton, or Sendai virus can be used as a cell fusion promoter. Furthermore, in order to enhance the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide may be used in combination. Furthermore, antibody-producing cells can be fused with myeloma cells using a commercially available cell fusion device utilizing electrical stimulation (eg, electroporation).

[Proliferation process]
The growth step is a step of growing antibody-producing cells (including hybridomas) before the labeling step. In the case of a hybridoma, by culturing in HAT selection medium, only hybridomas fused with antibody-producing cells can be grown. Moreover, when it is antibody production cells other than a hybridoma, it can culture | cultivate using the culture medium normally used for cell culture.

The HAT selection medium is a medium containing hypoxanthin, aminopterin, thymidin. There are de novo pathways and regenerating pathways (salvage pathways) in cells in order to make nucleotides that are materials for DNA synthesis. Because aminopterin inhibits the nascent pathway, when it is included in the culture medium, cells must supply nucleotides only by the regenerative pathway. Thus, cells with defects in the salvage pathway can not proliferate and die. By utilizing this phenomenon, only myeloma cells in which the salvage pathway is deficient or hybridomas in which the myelomas are cell-fused can not proliferate and die. Moreover, in a hybridoma in which only antibody-producing cells or antibody-producing cells are fused, they can be proliferated but are not immortalized, and therefore they die after culture for a certain period of time. On the other hand, a hybridoma in which myeloma cells and antibody-producing cells are fused is immortalized and can continue to grow without limitation, and nucleotides can be produced by the salvage pathway. Therefore, by culturing in a HAT selective medium, only hybridomas in which myeloma cells and antibody-producing cells are fused can be selectively grown.
The HAT medium may contain cell growth factors such as, for example, interleukin-6 (IL-6).

The growth period may be, for example, 3 days or more and 10 days or less, for example, about 5 days.
The growth environment may be a known environment for growing antibody-producing cells (including hybridomas). Specifically, the temperature may be, for example, 20 ° C. or more and 40 ° C. or less, and may be, for example, about 5% CO ₂ conditions.

[Culture process]
The culture step is a step of producing a monoclonal antibody by culturing antibody-producing cells (including hybridomas) evaluated as positive after the seeding step using a HAT selection medium or the like. The culture environment may be a known environment for growing antibody-producing cells (including hybridomas). Specifically, the temperature may be, for example, 20 ° C. or more and 40 ° C. or less, and may be, for example, about 5% CO ₂ conditions.
The monoclonal antibody is contained in a culture solution, and the culture solution may be used as it is, or may be purified or used as needed.

[Evaluation process]
The evaluation step is a step of evaluating the antigen specificity of the monoclonal antibody obtained after the culture step using a known evaluation method.

As the evaluation method, for example, ELISA (Enzyme-Linked Immunosorbent Assay) method, CLEIA (Chemiluminescent Enzyme Immuno Assay) method (chemiluminescent enzyme immunoassay), western blotting, immunoprecipitation, immunostaining, flow cytometry Etc.
In any of the above-described methods, monoclonal antibodies are reacted by performing an antibody-antigen reaction with the obtained monoclonal antibody and an antigen or a carrier on which the antigen is immobilized, and detecting using a labeled secondary antibody or the like. Antigen specificity of the antibody can be assessed.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

Production Example 1 Production of Fusion Protein 1 Construction of Vector 1 First, a vector expressing a fusion protein of a fluorescent protein and an antigen protein was prepared.

  Specifically, a cassette vector (hereinafter sometimes referred to as "cassette vector") in which a nucleic acid (SEQ ID NO: 1) encoding EGFP is inserted upstream of the multicloning site of pCold II vector (Takara Bio Inc.) Was produced. Then, a nucleic acid (SEQ ID NO: 2) encoding a partial protein of α-Tubulin as an antigen protein is inserted into the multi-cloning site of the cassette vector, and a fusion protein of a fluorescent protein and an antigen protein (hereinafter “fusion protein 1” A vector (hereinafter sometimes referred to as "vector 1") into which a nucleic acid encoding the same may be inserted was constructed. The partial protein of α-Tubulin is a partial protein (SEQ ID NO: 3) consisting of the amino acid sequence from the N-terminus to the 165th to 451st positions in the amino acid sequence.

2. Expression of Fusion Protein 1 Next, the cassette vector obtained in “1.” and Vector 1 were transformed into E. coli respectively. Then, after obtaining a clone, liquid culture was performed. Then, when the absorbance (OD) reaches 0.4 to 0.6, 0.1 to 1 mM of isopropyl-β-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside; IPTG) is added, C. overnight at 15.degree. C. to induce protein expression. The E. coli was then recovered and suspended in PBS. The suspension was then sonicated to sonicate the E. coli. Next, the disrupted cell suspension was centrifuged (conditions: 4 ° C., 20 minutes, 8000 rpm), and the soluble fraction of the supernatant (containing fluorescent protein or fusion protein 1) was recovered.

3. Purification of Fusion Protein 1 The soluble fraction containing the collected fluorescent protein or fusion protein 1 also includes endotoxin such as Lipopolysaccharide (LPS) derived from E. coli. Therefore, in order to remove the endotoxins, soluble fractions containing fluorescent protein or fusion protein 1 were purified by ammonium sulfate precipitation, respectively. Specifically, first, the recovery solution was diluted with PBS so that the protein concentration was 1.0 mg / mL. Next, an ammonium sulfate solution was added to the diluted solution until the degree of saturation was 0, 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and stirred at 4 ° C. for 1 hour. Then, the supernatant was removed by centrifugation (conditions: 4 ° C., 20 minutes, 12000 rpm). Next, to the precipitate, an equal amount and an equal concentration of ammonium sulfate solution were added, and operations of stirring and centrifuging were repeated twice to wash the precipitate. The precipitate was then suspended in PBS. Further, as a control, a fusion protein 1 not subjected to the ammonium sulfate precipitation method (hereinafter sometimes referred to as “a fusion protein 1 without ammonium sulfate”) was also prepared.

  The resulting suspension containing fluorescent protein or fusion protein 1 purified with each saturated concentration of ammonium sulfate was electrophoresed, and the result of Coomassie brilliant blue (CBB) staining is shown in FIG.

  From FIG. 3, the fusion protein 1 (EGFP + α-Tubulin partial protein) could be purified with high efficiency by using ammonium sulfate having a saturation degree of about 50%. In addition, fluorescent protein (EGFP) could be purified with high efficiency by using ammonium sulfate having a saturation degree of about 70%.

  The suspension containing the fluorescent protein or fusion protein 1 was then purified using gel filtration columns, respectively, to remove low molecular weight contaminants such as ammonium sulfate. Specifically, a suspension containing a fluorescent protein or fusion protein 1 is applied to a G75 gel filtration column or a G50 gel filtration column (both manufactured by GE Healthcare Bio-Sciences), respectively, and the fluorescence-colored fraction is obtained. Was collected. The resulting fraction was then sterilized by passage through a 0.22 μm filter to obtain fluorescent protein or fusion protein 1.

Production Example 2 Production of Fusion Protein 2 Construction of Vector 2 First, a vector expressing a fusion protein of a fluorescent protein and an antigenic peptide was prepared.

  Specifically, instead of a nucleic acid encoding a partial protein of α-Tubulin, a nucleic acid (SEQ ID NO: 4) encoding a short peptide of α-Tubulin as an antigenic peptide is inserted into the multicloning site of the cassette vector, A vector (hereinafter sometimes referred to as “vector 2”) into which a nucleic acid encoding a fusion protein of a fluorescent protein and an antigenic peptide (hereinafter sometimes referred to as “fusion protein 2”) was inserted was prepared. The inserted short-chain peptide of α-Tubulin is a peptide consisting of an amino acid sequence in which a cysteine for peptide coupling is added to the N-terminus of the amino acid sequence from the N-terminus to the 427th to 441th amino acid sequence (SEQ ID NO: 5 : CALEKDYEEVGVDSVE).

2. Expression of Fusion Protein 2 Next, a fusion protein was prepared using the same method as in “2.” of Production Example 1 except that vector 2 obtained in “1.” was used instead of the cassette vector and vector 1. 2 was expressed to obtain a soluble fraction containing fusion protein 2.

3. Purification of fusion protein 2 Instead of the soluble fraction containing fusion protein 1, purification was carried out using the same method as "3." of Production Example 1 except that the soluble fraction containing fusion protein 2 was used. Fusion protein 2 was obtained.

Example 1
1. Preparation of Immunization Antigen (1) Construction of Vector First, a vector for expressing an immunity antigen protein to a mouse was prepared.

  Specifically, a nucleic acid (SEQ ID NO: 2) encoding a partial protein of α-Tubulin as an antigen protein is inserted into the multi-cloning site of pCold II vector (Takara Bio Inc.), and a vector for antigen protein expression is obtained. It was constructed.

(2) Expression of Immunizing Antigen Subsequently, an immunizing antigen protein was obtained using the same method as in Production Example 1 as in “2.” to “3.”.

2. Screening of Antibody-Producing Cells (1) Immunization of Mice First, a mixed solution of an antigen, a carrier protein, and an autologous preparation adjuvant is injected from the abdominal sole of the BALB / cA mouse, and six days later, the antigen solution and PBS The mixture (antigen solution: PBS = 1: 1) was immunized, and a total of 2 immunizations were performed. For the antigen, the immunizing protein obtained in "1." was immunized.

(2) Recovery of spleen cells Then, 3 days after the last immunization (9 days after the first immunization), mouse spleen was removed and spleen cells were collected.

(3) Cell fusion and proliferation of hybridomas Next, spleen cells and mouse myeloma cells (SP2 / 0-Ag14) (Riken BRC RCB 0209, ATCC CRL-1581) were electrically connected using a cell fusion apparatus (ECFG21 manufactured by Nepagene). The cells were fused by the fusion method to obtain hybridomas. Then, the obtained hybridomas are treated with HAT selection medium (10% calf calf serum (NCS), 1 ng / mL interleukin 6 (IL-6) and RPMI 1640 containing HAT) to obtain 5% CO _2. The cells were cultured at 37 ° C. for 5 days.

(4) Labeling of hybridomas The cultured hybridomas were then collected and suspended in 20 mL of 13% OtiPrep diluted in culture solution. Then, the culture solution was overlaid on the upper layer of the suspension and centrifuged (conditions: 25 ° C., 20 minutes, 2000 rpm). The surviving hybridomas were then recovered from the middle layer, and the hybridomas were seeded in a 24-well plate at 2.5 × 10 ⁵ cells / well. Then, the fusion protein 1 (with ammonium sulfate) obtained in Production Example 1 was added to a concentration of 0.5 nmol per well, and the mixture was incubated for 2 hours under conditions of 5% CO ₂ and 37 ° C. The hybridomas were thus fluorescently labeled. Also, as a control, PBS, 0.5 nmol / well of fluorescent protein, or 0.5 nmol / well of ammonium sulfate-free fusion protein 1 was added and incubated.

(5) Screening and Cloning by Flow Cytometer Next, the fluorescently labeled hybridomas were washed 3 times with PBS, and then the washed hybridomas were suspended in 500 μL of RPMI 1640 containing 2% NCS. Next, 3 μL of 7-AAD Viability Dye (Beckman Coulter, A07704) was added to stain dead cells and viable cells.

  Then, the stained cells were introduced into a high-speed flow cytometer (Becton Dickinson Co., MoFlow Astroios) and screened. The fractionation conditions were analyzed by detecting signals with an optical filter of 512 nm and 644 nm using a 488 nm laser, and ejecting cells with a 60 μm nozzle. The analysis results are shown in FIGS. 4A and 4B. In FIG. 4A, “GFP” indicates the result of a hybridoma to which a fluorescent protein (EGFP) is added, and “GT” indicates a result of a hybridoma to which fusion protein 1 (EGFP + α-Tubulin partial protein) is added. . Also, in FIG. 4B, “GFP recombinant protein” indicates a fluorescence microscopic image of a hybridoma to which a fluorescent protein (EGFP) has been added. "GFP-Tubulin" shows a fluorescence microscopic image (using a fluorescence microscope manufactured by Carle Zeiss, magnification: 400 times) of a hybridoma to which fusion protein 1 (EGFP + α-Tubulin partial protein) has been added.

  Also, the percentage of dead cells in cells to which fusion protein 1 (with ammonium sulfate), PBS or fusion protein 1 without ammonium sulfate was added is shown in Table 4 below.

  From Table 4, purification by ammonium sulfate precipitation revealed that E. coli-derived endotoxin contained in the purified protein was removed, and the toxicity to hybridomas was reduced.

  Further, from FIGS. 4A and 4B, in comparison with the hybridoma to which the fluorescent protein was added, strong fluorescence was detected in the hybridoma to which the fusion protein 1 was added, and the fluorescence intensity was about 14 times.

In addition, cells having a fluorescence intensity of 5 times or more that of unstained cells were sorted one by one in 96-well microplates containing 200 μL of HAT selective medium. The sorted cells were then cultured for 10 days under conditions of 5% CO ₂ and 37 ° C. The resulting cell colonies were used as monoclonal hybridomas that produce the target antibody. Then, using the antibody contained in the culture supernatant of the positive hybridoma, evaluation was performed by ELISA, Western blotting, etc. in Test Example 1 described later.

Comparative Example 1
1. Preparation of Immunizing Antigen An immunizing peptide was prepared as an immunizing antigen to mice. Specifically, an α-Tubulin short-chain peptide (SEQ ID NO: 5) is chemically synthesized to prepare an immune antigen peptide in which keyhole limpet hemocyanin (KLH) is coupled to the α-Tubulin short-chain peptide by the maleimide method. did.

2. Screening of antibody-producing cells (1) Immunization of mice Other than using the immunizing antigen peptide (short chain peptide of α-Tubulin) obtained in “1.” instead of immunizing antigen protein (partial protein of α-Tubulin) The mice were immunized using the same method as (1) in "2." of Example 1.

(2) Recovery of Spleen Cells Subsequently, spleen cells were recovered using the same method as (2) of “2.” in Example 1.

(3) Cell fusion and proliferation of hybridomas Next, hybridomas were prepared and cultured using the same method as (3) in "2." of Example 1.

(4) Labeling of Hybridoma Then, the fusion protein 2 obtained in Production Example 2 was used in place of the fusion protein 1 obtained in Production Example 1; The hybridomas were fluorescently labeled using the same method as in.

(5) Screening and Cloning by Flow Cytometer Next, hybridomas were screened and cloned using the same method as (5) of “2.” in Example 1. Subsequently, evaluation by ELISA, western blotting, etc. was performed in Test Example 1 described later using the antibody contained in the culture supernatant of the hybridoma that is positive.

Comparative Example 2
Monoclonal antibodies were produced by screening of hybridomas by conventional ELISA and single cloning by limiting dilution. Details are as follows.

1. Production of Monoclonal Antibody (1) Immunization of Mice Using the same method as (1) in “2.” of Comparative Example 1, mice were immunized with an immunizing antigen peptide (short-chain peptide of α-Tubulin) as an antigen.

(2) Recovery of spleen cells Then, 17 days after the first immunization, spleen cells were recovered using the same method as (2) of "2." in Example 1.

(3) Cell fusion and growth of hybridomas Then, cell fusion is performed using the same method as (3) of "2." in Example 1, and hybridomas are seeded on a 96-well plate, using HAT selective medium. Culture for 12 days.

(4) Screening by ELISA Next, ELISA was performed using the antibody secreted in the culture solution of hybridoma. Specifically, first, hybridoma culture supernatant was added to a plate on which the short-chain peptide of α-Tubulin was immobilized, and the plate was incubated for 60 minutes. Then, a solution (10,000 fold dilution) containing HRP-conjugated anti-mouse antibody (MBL) was added dropwise and incubated for 60 minutes. Next, Powerscan HT (manufactured by DS-Pharma Biomedical) was used for detection, and hybridomas that were positive were selected.

(5) Cloning by Limiting Dilution Then, hybridomas that were positive by ELISA were diluted by limit dilution so that only one cell could theoretically be contained in one well, and then seeded in a 96 well plate and cultured for 12 days. Evaluation by ELISA, western blotting or the like was performed in Test Example 1 described later using the antibody obtained from each positive cell.

[Test Example 1]
1. Evaluation of monoclonal antibody (1) Evaluation by ELISA (Enzyme-Linked Immunosorbent Assay) A plate obtained by immobilizing each antigen on a solid phase for the antibodies obtained from each positive cell of Example 1, Comparative Example 1 and Comparative Example 2 Performed the ELISA. First, a solution containing each antibody (the culture supernatant of each positive cell) was added to a plate on which the short-chain peptide of α-Tubulin was immobilized, and incubated for 60 minutes. Then, a solution (10,000 fold dilution) containing HRP-conjugated anti-mouse antibody (MBL) was added dropwise and incubated for 60 minutes. Then, it detected using Powerscan HT (made by DS-Pharma Biomedical). The number of samples that were also positive in ELISA was counted, and the positive rate ({(number of positive samples / total number of samples) × 100}) was calculated. The results are shown in Table 5.

(2) Evaluation by Western Blotting The antibodies obtained from the positive cells of Example 1 and Comparative Example 1 and Comparative Example 2 were subjected to electrophoresis and transcription of each antigen (a partial protein or short chain peptide of α-Tubulin). Western blotting was performed using a membrane. First, the membrane to which each antigen was blotted was blocked using skimmed milk solution. Then, a solution containing each antibody (a 2-fold dilution of culture supernatant of each positive cell) was added and incubated for 120 minutes. Then, a solution (diluted 50,000 times) containing HRP-conjugated anti-mouse antibody (MBL) was added dropwise and incubated for 60 minutes. Then, it detected using LAS-4000 mini (made by Fujifilm). The detection results are shown in FIG. Further, from FIG. 5, the number of samples that were also positive in Western blotting was counted, and the positive rate ({(number of positive samples / total number of samples) × 100}) was calculated. In addition, the number of samples showing no cross reactivity was counted, and the percentage (%) of samples showing no cross reactivity to the total number of samples was calculated. The results are shown in Table 5. In Table 5, "WB" is the evaluation result by Western blotting.

  As compared with the positive cells obtained in Comparative Example 1 and Comparative Example 2, the positive cells obtained in Example 1 had a significantly improved positive rate in ELISA and Western blotting. Above all, the ratio in Example 1 was about 150 times the ratio in Comparative Example 2 with respect to the ratio of an antibody having no cross reactivity, ie, an antibody that specifically recognizes an antigen.

  In addition, for the antibody produced by the positive cells obtained in Example 1, the degree of sequence recognition (fluorescence intensity in ELISA) is plotted on the vertical axis, and the degree of structural recognition (fluorescence intensity on the flow cytometer) is plotted on the horizontal axis. The graph is shown in FIG. In FIG. 6, "sequence recognition" indicates fluorescence intensity in ELISA, and "structure recognition degree" indicates fluorescence intensity in a flow cytometer.

  From FIG. 6, many antibodies showed correlations between structure recognition and sequence recognition, and recognized structure recognition and sequence recognition to the same extent. On the other hand, at the lower right of FIG. 6, there was an antibody that recognized the structure more dominantly than the sequence. With this antibody, it did not react with the antigen in the ELISA and did not show positive but in the western blot it showed positive with the antigen. An antibody that recognizes a structure can bind to a protein that retains physiological activity in vivo.

  From the above, it became clear that the method of Example 1 can obtain an antibody that recognizes a structure with high efficiency.

Example 2
1. Screening of Antibody-Producing Cells (1) Immunization of Mice Mice were immunized using a method similar to (1) of “2.” in Example 1.

(2) Recovery of Spleen Cells Subsequently, spleen cells were recovered using the same method as (2) of “2.” in Example 1.

(3) Labeling of spleen cells Then, using the same method as (4) of "2." of Example 1 except that the spleen cells obtained in (2) were used instead of hybridomas. The cells were fluorescently labeled.

(4) Screening and Cloning by Flow Cytometer Subsequently, spleen cells were screened using the same method as (5) of “2.” in Example 1. The analysis result by the flow cytometer is shown in FIG. In FIG. 7, "GFP" shows the result in spleen cells to which a fluorescent protein (EGFP) is added, and "GT" shows the result in spleen cells to which fusion protein 1 (EGFP + α-Tubulin partial protein) is added. .

  It was confirmed from FIG. 7 that, also for spleen cells, as in the case of using hybridomas, positive cells producing a structure-recognizing antibody can be fluorescently labeled and screened.

  According to the detection probe for screening antibody-producing cells of the present embodiment, the kit for screening antibody-producing cells, and the method for screening antibody-producing cells, cells producing a structure-recognizing antibody can be obtained with high efficiency. Further, according to the method for producing a monoclonal antibody of the present embodiment, a structure recognition antibody can be obtained with high efficiency. Moreover, the obtained monoclonal antibody is suitably used for antibody medicine, a test, a diagnostic agent, etc.

  DESCRIPTION OF SYMBOLS 1 ... Antigen protein, 2 ... fluorescent substance, 3 ... membrane type | mold immunoglobulin, 4 ... antibody production cell, 4a ... objective antibody production cell, 4b ... other antibody production cell, 5 ... 1st detector, 6 ... 2nd Detectors 7: Excitation light source 8: Sorter 8: Injection substance 9a, 9b: Culture vessel 10: Detection probe for screening antibody-producing cells 11: Labeling part 12: Flow cytometer 13: Cell Seeding station.

Claims (10)

An antigen protein, and a fluorescent substance linked to the N terminus or C terminus of the antigen protein,
A detection probe for screening antibody-producing cells, which is used for selection by flow cytometry.   The detection probe for screening antibody-producing cells according to claim 1, wherein the fluorescent substance is a fluorescent protein.   The detection probe for screening antibody-producing cells according to claim 1 or 2, wherein the antigen protein is an ammonium sulfate precipitate. A cloning site for inserting a first nucleic acid encoding an antigenic protein,
A second nucleic acid encoding a fluorescent protein linked upstream or downstream of the cloning site;
The first vector is a vector for expressing a fusion protein comprising the antigen protein and the fluorescent protein linked to the N-terminus or C-terminus of the antigen protein,
A kit for screening antibody-producing cells, which is used for selection by flow cytometry. Furthermore, a second vector comprising a cloning site for inserting a third nucleic acid encoding an antigenic protein,
The screening kit for antibody-producing cells according to claim 4, wherein the second vector is a vector for expressing an antigen protein for non-human animal immunity. In addition, ammonium sulfate is provided,
The kit for screening antibody-producing cells according to claim 4 or 5, wherein the ammonium sulfate is for purifying the fusion protein by ammonium sulfate precipitation. A labeling step in which antibody-producing cells are brought into contact with the detection probe for screening antibody-producing cells according to any one of claims 1 to 3;
A screening step of screening said antibody-producing cells by flow cytometry after said labeling step.   Detection for screening of antibody-producing cells containing two or more types of antibody-producing cells producing antibodies against different antigenic proteins in the labeling step, and two or more types of the antigen proteins having different fluorescent substances bound to each other The method for screening antibody-producing cells according to claim 7, which is brought into contact with a probe.   A method for producing a monoclonal antibody, comprising a seeding step of seeding the antibody-producing cells obtained by using the method of screening antibody-producing cells according to claim 7 one by one.   The method for producing a monoclonal antibody according to claim 11, wherein the isotype of the monoclonal antibody is IgG.