Screening method for hybridoma cells

Abstract

The present invention aims to provide a means for screening positive hybridomas in which antigens can be used while maintaining their three-dimensional structure in vivo. [Solution] A method for screening hybridoma cells that produce antibodies that selectively recognize the three-dimensional structure of an antigen, The antigen used for screening is a protein that maintains its three-dimensional structure in vivo. The aforementioned protein is exposed and presented on the surface of a vesicle composed of a biomaterial with a lipid bilayer, The vesicles are characterized by containing a fluorescent protein. A screening method for hybridoma cells.

Description

Technical Field

[0001] The present invention relates to a method for screening hybridoma cells that produce antibodies. In particular, it relates to a method for efficiently screening hybridoma cells that produce antibodies that recognize a three-dimensional structure.

Background Art

[0002] Monoclonal antibodies are highly sensitive detection probes having high antigen-binding specificity and binding strength. Monoclonal antibodies are not only essential research tools in life sciences in the post-genomic era, but also highly functional biological materials useful commercially because antibodies of uniform quality can be mass-produced. Therefore, currently, the markets for disease test drugs, diagnostic agents, pharmaceuticals, etc. using antibodies are continuously expanding.

[0003] There are roughly two methods for producing monoclonal antibodies. 1) The hybridoma method in which an animal is immunized with an antigen to produce hybridomas, 2) The in vitro display method in which a gene for the antigen-binding region of an immunoglobulin is expressed genetically. Although each method has its advantages and disadvantages, the hybridoma method is widely used in many research institutes because it is easy to introduce the system. However, in the hybridoma method, it takes time and effort, and screening by ELISA in which an antigen is directly immobilized hardly retains the three-dimensional structure of the antigen, so it has been difficult to obtain an antibody that recognizes the three-dimensional structure of the antigen.

[0004] In 1979, Herzenberg et al. developed a hybridoma method using a flow cytometer (see, for example, Non-Patent Document 1). Hybridomas express membrane-bound immunoglobulin molecules on their cell membranes that have the same antigen-binding specificity as secretory immunoglobulins. Therefore, antigens bind not only to secretory immunoglobulins, i.e., antibodies, but also to these membrane-bound immunoglobulins. Thus, in the method developed by Herzenberg et al., the target hybridoma is fluorescently labeled by fluorescently labeling the antigen, and then screened and cloned.

[0005] Patent Document 1 discloses a method developed by Herzenberg et al. (Non-Patent Document 1) and Meagher et al. (Non-Patent Document 2) that completes the most time-consuming and laborious screening and cloning processes in a single flow cytometer operation. Patent Document 2 describes a method for screening antibodies secreted by hybridomas using microwells, utilizing vesicles with target substances on their membrane surface. However, a method for directly screening hybridoma cells by contacting hybridomas with vesicles containing target substances on their membrane surface is not disclosed.

[0006] One method for screening for structure-specific antibodies is cell-based ELISA screening, which applies cell-based assay methods using cultured cell lines that present antigens on their cell membranes. Cell-based assays use cells that express or artificially express a specific antigen (antigen-displaying cells) and react them with antibodies secreted by hybridoma cells to detect the antibodies that react with the antigen-displaying cells. This method screens hybridomas that secrete the target antibody. When applying this cell-based assay method to flow cytometry screening, the analysis is performed with the antigen-displaying cells and hybridoma cells in contact (cell aggregate). However, since flow cytometry is a method that dissociates cell aggregates into single cells under mild conditions and analyzes them at the individual cell level, there are limitations in terms of equipment when analyzing cell aggregates, and cell-based Performing a screening method that employs the principles of an assay using a flow cytometer was technically difficult. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2018-148832 [Patent Document 2] WO2024 / 071374 issue [Non-patent literature]

[0008] [Non-Patent Document 1] Parks DR et al.,Proc.Natl.Acad.Sci.USA,Vol76,No.4,p1962-1966,1979 [Non-Patent Document 2] Price PW et al.,J.Immunol.Methods,Vol,343,p28-41,2009 [Overview of the project] [Problems that the invention aims to solve]

[0009] In the screening process for isolating hybridoma cells that produce the target antibody from hybridoma cells created from lymphocytes of animals immunized with an antigen, it was difficult to screen for positive hybridomas in which the antigenic membrane protein retains its three-dimensional structure in vivo.

[0010] Therefore, the object of the present invention is to provide a means for using antigens while maintaining their three-dimensional structure in vivo when screening positive hybridomas. [Means for solving the problem]

[0011] After diligent research, the inventors discovered a method for using vesicles composed of biomaterials with a lipid bilayer on the surface, which retains the three-dimensional structure of proteins within the body, as antigens in the screening process.

[0012] In other words, the present invention encompasses the following embodiments shown in [1] to [9]. [1] A method for screening hybridoma cells that produce antibodies that selectively recognize the three-dimensional structure of an antigen, The antigen used for screening is a protein that maintains its three-dimensional structure in vivo. The aforementioned protein is exposed and presented on the surface of a vesicle composed of a biomaterial with a lipid bilayer, The vesicles are characterized by containing a fluorescent protein. A screening method for hybridoma cells. [2] The method according to [1], wherein the protein that maintains the three-dimensional structure is a membrane protein. [3] The method according to [1] or [2], wherein the fluorescent protein is a GFP protein. [4] The method according to any one of [1] to [3], wherein the vesicle composed of the biomaterial composed of the lipid bilayer is an extracellular vesicle. [5] The method according to any one of [1] to [4], wherein the cells that produce the vesicles are prepared to express a fluorescent protein, thereby encapsulating the fluorescent protein in the vesicles. [6] The method according to [5], wherein cells are prepared to express a fluorescent protein by introducing an exogenous GFP gene. [7] The method according to any one of [2] to [6], wherein the membrane protein is selected from either a single-pass transmembrane protein or a multiple-pass transmembrane protein. [8] The membrane protein is platelet-derived growth factor receptor (PDGFR), The method described in any of [2] to [6]. [9] The method according to any one of the above [1] to [8], wherein the screening method is performed using a flow cytometer. [Effect of the Invention]

[0013] According to the invention described in the present application, in the step of screening for positive hybridomas, it becomes easy to use an antigen that maintains its three-dimensional structure in vivo.

[0014] In addition, antibodies that recognize antigens that maintain their three-dimensional structure in vivo and are obtained by the method described in the present application are expected to be used for antibody pharmaceuticals, antibodies used for in vitro diagnostic pharmaceuticals, etc., and are expected to promote the development of next-generation pharmaceuticals.

[0015] In a preferred embodiment of the invention of the present application, in the step of screening for positive hybridomas using a flow cytometer, it becomes easy to use an antigen that maintains its three-dimensional structure in vivo.

[0016] Furthermore, by performing screening of hybridoma cells by the flow cytometry method, screening evaluation of antibody-producing cells and isolation of cells can be carried out simultaneously, and efficient and large-scale screening of antibody-producing hybridoma cells can be carried out. [Brief Description of the Drawings]

[0017] [Figure 1] It is a diagram showing the binding activity of the ALP recognition antibody obtained in Example 7 (Example 8), and shows the fluorescence intensity for each well. [Modes for Carrying Out the Invention]

[0018] Antibody-producing cells As used herein, the "antibody-producing cells" are hybridoma cells obtained by cell fusion of spleen-derived B cells, lymph node-derived B cells or peripheral blood-derived B cells derived from an animal immunized with the target antigen and any one of the above B cells and myeloma cells.

[0019] The method for producing hybridoma cells is not particularly limited and can be any method commonly chosen by those skilled in the art, but examples include a method using electrofusion.

[0020] antigen In this specification, the term "antigen" may be any protein containing a polypeptide fragment with an antibody recognition site, and is not particularly limited as long as it can screen for antibody-producing cells. Furthermore, the protein used in this embodiment is preferably a transmembrane protein that retains its native structure. In addition, in this embodiment, the protein may be a complex of multiple molecules.

[0021] screening In this specification, "screening" refers to the process of selecting antibodies or hybridoma cells from a group of antibodies with different specificities, or from a group of hybridoma cells that secrete those antibodies, based on specific evaluation criteria. These specific evaluation criteria are not limited to, but may include the recognition epitope of the antibody, the structural selectivity of the antigen, the strength of its binding, and the system performance of the antigen-antibody reaction under specific conditions.

[0022] The aforementioned specific evaluation criteria can be determined based on predetermined thresholds. Such predetermined thresholds can be appropriately set and used by those skilled in the art. For example, if the measured value is above a predetermined threshold, the desired antibody or hybridoma cells can be selected. It is possible.

[0023] Antibodies that selectively recognize the three-dimensional structure of antigens. In this specification, an "antibody that selectively recognizes the three-dimensional structure of an antigen" is an antibody that binds only to the three-dimensional structure that a protein can adopt when it is performing its function. It is known that proteins have linear polypeptide chains (primary structure) that take on higher-order structures (conformations) through interactions with amino acids within the same chain and with amino acid residues of multiple different polypeptide chains. Generally, it is thought that for a protein to perform its function, the polypeptide chain must be in a stable conformation, and that the protein will not perform its original function in any conformation other than a specific one. In other words, if an antibody that selectively recognizes the three-dimensional structure of an antigen is an antibody that recognizes a protein that maintains its three-dimensional structure in vivo, then that antibody can be said to be an antibody that recognizes a protein that has a structure that functions in vivo, and is useful, for example, when investigating how a particular protein that functions in vivo is distributed.

[0024] In this specification, "maintaining the three-dimensional structure in vivo" means that the three-dimensional structure that a protein as a biomolecule can adopt under physiological conditions is maintained, and that the three-dimensional structure that the same protein or protein complex can adopt in different environments within a living organism (e.g., intracellular, extracellular, on the cell membrane) is maintained.

[0025] Furthermore, in antibody drugs used as pharmaceutical raw materials to control the function of specific proteins in the body, antibodies that recognize proteins that maintain their three-dimensional structure in the body are useful. On the other hand, if an antibody that selectively recognizes three-dimensional structure does not recognize proteins that maintain their normal three-dimensional structure in the body, but instead selects proteins that maintain a three-dimensional structure different from the normal one in the body—so-called denatured proteins—it can be used to investigate the distribution of specific proteins that are not functioning in the body, and is therefore also considered to be industrially useful. In particular, it is a well known fact that certain proteins become non-functional depending on the disease, and if proteins that do not have their original function appear in the bloodstream, they can be used as so-called tumor markers.

[0026] membrane proteins In this specification, "membrane protein" refers to a protein that has a hydrophobic transmembrane domain in its amino acid sequence. Typically, proteins are classified as single-pass transmembrane proteins or multi-pass transmembrane proteins depending on the number of transmembrane domains. The presence or absence of protein localization signals in the amino acid sequence, the presence or absence of signal sequences, and the number of transmembrane domains determine the portion of the protein exposed outside the cell.

[0027] Examples of single-pass transmembrane proteins include, but are not limited to, tyrosine kinase receptors (EGFR, FGFR, PDGFR, VEGFR) that recognize factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0028] Examples of multi-pass transmembrane proteins include, but are not limited to, ion channel-embedded receptors such as nicotine, gamma-aminobutyric acid (GABA), and glycine; receptors that break down inositol phosphate, such as alpha-1, histamine-1 (H1), bradykinin (B2), muscarinic-1 (M1), and angiotensin-II (AT1); receptors such as beta-histamine-2 (H2), prostacyclin (IP), and glucagon; and receptors for various peptide hormones such as thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH).

[0029] Furthermore, since "membrane proteins" localize to lipid bilayer membranes derived from cells, they are incorporated not only into the cell membrane on the cell surface, but also into the lipid bilayer membranes that constitute organelles released from cells, such as extracellular vesicles (EVs), exosomes, and secretory granules. For example, membrane proteins to be presented on EVs include ion channel-embedded receptors such as nicotine, GABA, and glycine; receptors that degrade inositol phosphate, such as alpha-1, histamine-1 (H1), bradykinin (B2), muscarinic-1 (M1), and angiotensin-II (AT1); receptors that synthesize cAMP, such as beta-, histamine-2 (H2), prostacyclin (IP), and glucagon; receptors that synthesize cAMP, such as TSH, FSH, and LH, and GTP-binding protein-coupled receptors; and insulin receptors, tyrosine kinase receptors with tyrosine kinase activity, such as EGFR, FGFR, and PDGFR. However, these are not the only examples.

[0030] Vesicles are composed of biomolecules made up of lipid bilayers. In this specification, "vesicles composed of lipid bilayers" include artificially created vesicles called liposomes, as well as cells, extracellular vesicles, exosomes, and secretory granules.

[0031] When using liposomes, a separately prepared protein can be mixed with one or more lipid components capable of forming a lipid bilayer, and then liposomes can be formed using known methods. Among the purified proteins, it is known that some molecular species can reproduce the in vivo three-dimensional structure on liposomes.

[0032] When using cells or cell-derived vesicles (extracellular vesicles, exosomes, secretory granules), the intracellular localization of a protein is determined by the presence or absence of a localization signal sequence within the cell, based on the amino acid sequence. In the case of membrane proteins with localization signals expressed on the cell membrane, they are presented on the biological membrane while maintaining their correct three-dimensional structure. Therefore, the purification process of the antigen protein is unnecessary, and it is expected that the protein will be presented on the lipid bilayer surface while maintaining its in vivo three-dimensional structure on the biological membrane. Furthermore, it is well known to those skilled in the art that membrane proteins with localization signals expressed on the cell membrane can be incorporated into extracellular vesicles, exosomes, secretory granules, etc.

[0033] Extracellular vesicles, exosomes, secretory granules In this specification, "extracellular vesicles, exosomes, and secretory granules" are a general term for vesicles secreted from cells with a diameter of approximately 50 to 1000 nm, and there is no strict distinction between them. They are extremely small compared to the size of cells, and it is possible to separate cells from extracellular vesicles, exosomes, and secretory granules by centrifugation or other methods. It is known that cell-derived vesicles take up cell-derived biomolecules such as proteins and microRNAs expressed in the cells from which they originate, and similarly, membrane proteins expressed on the cell membrane of cells are thought to be stably expressed on the surface of vesicles while maintaining their three-dimensional structure in vivo. Therefore, in the culture supernatant of cultured cells in which a specific membrane protein is expressed on the cell membrane, there are vesicles in which that specific membrane protein is presented on the cell membrane, and these extracellular vesicles can be recovered by an appropriate method. In particular, although not limited, the invention of this application prefers to use extracellular vesicles, exosomes, and secretory granules, and extracellular vesicles are particularly preferred.

[0034] Exposed and presented on the surface In this specification, "exposed and presented on the surface" means that a portion of a membrane protein is exposed on the outer surface of a cell or cell-derived vesicle (extracellular vesicle, exosome, secretory granule), allowing antibodies or other substances to come into contact with it. During screening, antibodies or antibody-expressing cells can capture cells or cell-derived vesicles (extracellular vesicles, exosomes, secretory granules) by coming into contact with the portion of the membrane protein exposed on the surface.

[0035] Fluorescent label In this specification, "fluorescent label" refers to a substance that emits fluorescence when excited by exposure to light of a specific wavelength, and includes chemically modified fluorescent groups or fluorescent proteins.

[0036] Fluorescent groups for chemical modification In this specification, "fluorescent groups for chemical modification" refers to reagents that have reactive groups with various functional groups of biomolecules, and examples include, but are not limited to, fluorescein derivatives, rhodamine derivatives, Cy3 derivatives, Cy5 derivatives, etc.

[0037] fluorescent protein In this specification, "fluorescent protein" includes, but is not limited to, green fluorescent protein GFP, yellow fluorescent protein YFP, cyan fluorescent protein CFP, red fluorescent protein RFP, and blue fluorescent protein BFP.

[0038] fluorescently labeled vesicles In this specification, "fluorescently labeled vesicles" refers to vesicles containing a fluorescent protein, which are isolated from the culture supernatant of cells that produce vesicles and are designed to express a fluorescent protein. The method for preparing cells that produce vesicles to express a fluorescent protein is not particularly limited as long as it is a method usually chosen by those skilled in the art, but examples include a method of gene transfer using an expression plasmid designed to express an exogenous green fluorescent protein (GFP) in the cells. Examples of exogenous green fluorescent protein (GFP) include GFP derived from the jellyfish Aequorea victoria.

[0039] Screening method for hybridoma cells In embodiments of the present invention, screening for positive hybridoma cells is performed by detecting hybridoma cells that recognize antigens presented by fluorescently labeled vesicles. Detection of fluorescently labeled vesicles is not particularly limited, but may be performed using methods commonly used by those skilled in the art, such as a fluorescence microscope, optical detector, or flow cytometer, and is preferably performed using a flow cytometer. [Examples]

[0040] Example 1: Immunization of experimental animals to isolate ALP-recognizing antibodies Ten female mice (ICR, 4 weeks old) were prepared as immunization animals. 50 μg of ALP-480 (SEQ ID NO: 1), described in Japanese Patent Application Publication No. 2017-192381, was used as the immune antigen per mouse. The 50 μg immune antigen was mixed with an equal volume of Freund's Complete Adjuvant (Sigma-Aldrich), and the resulting emulsion was injected entirely into the subcutaneous tissue of the mouse tail to immunize the mice (primary immunization).

[0041] The antigen solution was prepared in the same manner as for the first immunization, except that the adjuvant was changed to Freund's Incomplete Adjuvant (Sigma-Aldrich). The second immunization was performed one week after the first, and subsequent immunizations were performed every week until the ninth immunization.

[0042] Example 2: Production of ALP-recognizing antibody-producing hybridoma cells by electrocellular fusion. After the ninth immunization, an immune antigen not mixed with an adjuvant was administered subcutaneously to the tail of mice. Two days later, spleen tissue was removed from 10 mice, and B cells were isolated. The B cells isolated from the 10 mice were mixed with myeloma cells (cell name: sp2 / 0-Ag14), and hybridoma cells were created by cell fusion using electrofusion.

[0043] The hybridoma cells created were placed in a fusion cell culture medium at a rate of 4 × 10 5 The cells were suspended to a concentration of cells / mL. For the fusion cell culture medium, E-RDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) was used, supplemented with 10% fetal bovine serum, 5% BM Condified (manufactured by Merck), and HAT additive (manufactured by Merck).

[0044] The hybridoma cell suspension was seeded into 15 cm dish cell culture vessels and cultured for 5 days in a mammalian cell culture incubator (37°C, 5 vol% CO2 atmosphere), after which the entire culture medium was collected.

[0045] Example 3: Recovery of hybridoma cells using density gradient centrifugation medium From the culture medium containing the recovered hybridoma cells, density gradient centrifugation medium (Abbott Hybridoma cells were isolated using OptiPrep (Diagnostics Technologies) according to the attached protocol.

[0046] In detail, the culture medium containing hybridoma cells was centrifuged (180 × g, 5 minutes), and the precipitated cells containing hybridoma cells were collected. The precipitated cells were suspended in 10 mL of E-RDF medium, and the cells were again centrifuged (180 × g, 5 minutes) to precipitate them, and the cells were washed. Next, the washed and precipitated cells containing hybridoma cells were suspended in 6 mL of E-RDF medium containing 13% OptiPrep. 6 mL of E-RDF medium was gently added to this suspension, and centrifugation (740 × g, 20 minutes) was performed. The centrifuged solution separated into two layers, and the intermediate layer containing hybridoma cells was collected and suspended in 40 mL of E-RDF medium. The cells in this suspension were precipitated by centrifugation (400 × g, 10 minutes), and the cells were washed. The cell washing procedure was repeated two more times, and the collected cells were fermented in fusion cell medium at 1.25 × 10⁶ 5 The cells were suspended to a concentration of cells / mL.

[0047] Example 4: Preparation of an alkaline phosphatase expression vector A gene sequence encoding a protein in which the transmembrane domain of PDGFR is fused to the C-terminus of ALP was designed (Sequence Number 5). This sequence was created by concatenating the nucleotide sequence described in Sequence Number 2, which encodes the extracellular region containing the active domain of bovine alkaline phosphatase (hereinafter referred to as "ALP"), with the nucleotide sequence (Sequence Number 4), which encodes amino acids 512 to 561 of the amino acid sequence of human platelet-derived growth factor receptor (hereinafter referred to as "PDGFR"), described in Sequence Number 3, along with their translation frames. This gene sequence was inserted downstream of the CMV promoter in an expression vector for mammalian cell expression to create an ALP-PDGFR fusion protein expression vector (hereinafter referred to as "ALP-PDGFR / CMV").

[0048] Example 5: Preparation of a green fluorescent protein (GFP) expression vector derived from the jellyfish Aequorea victoria. The intracellular GFP expression vector (hereinafter referred to as "GFP / CMV") was constructed by inserting the gene sequence described in Sequence ID No. 6, which was designed based on the GFP gene sequence described in Sravanthi Shastri, (2017) 89:49-56, downstream of the CMV promoter in an expression vector for mammalian cell expression.

[0049] Example 6: Preparation of extracellular vesicles containing ALP-PDGFR fusion protein and GFP 293 cultured human kidney tissue cells were simultaneously transfected with ALP-PDGFR / CMV prepared in Example 4 and GFP / CMV prepared in Example 5 using Lipofectamine 2000 (Thermo Fisher Scientific) to create a stable expression cell line that stably expresses ALP-PDGFR fusion protein on the cell membrane and GFP in the cytoplasm, according to a standard method. Extracellular vesicles containing ALP-PDGFR fusion protein and GFP were prepared from the culture supernatant of this stable cell line cultured for 5 days, according to a standard method.

[0050] In detail, the culture supernatant containing extracellular vesicles was centrifuged (2,300 × g, 30 minutes) to remove insoluble sediment, and then centrifuged again (10,000 × g, 30 minutes) to remove the insoluble sediment and prepare a clarified supernatant. Next, this clarified supernatant was ultracentrifuged (100,000 × g, 2 hours) and the precipitate containing extracellular vesicles was collected. An equal volume of physiological saline (8.1 mmol / L disodium hydrogen phosphate, 1.5 mmol / L potassium hydrogen phosphate, 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, pH 7.5; hereafter referred to as "PBS") was added to a centrifuge tube and ultracentrifuged (100,000 × g, 2 hours), and the resulting precipitate was collected as extracellular vesicles containing ALP-PDGFR fusion protein and GFP.

[0051] The recovered extracellular vesicles were quantified using a protein quantification kit (Pierce).

[0052] Example 7 Selection of hybridoma cells bound to extracellular vesicles containing ALP-PDGFR fusion protein using a flow cytometer. The hybridoma cell suspension prepared in Example 3 was seeded at a dose of 1 mL into each well of a 24-well cell culture plate and cultured for 24 hours. After 24 hours, only the culture supernatant was aspirated and removed, and 300 μL of fusion cell medium (0.25 mg / mL) containing extracellular vesicles prepared in Example 6 was added to each well. Culture was continued for another 24 hours to promote contact between hybridoma cells and extracellular vesicles. After 24 hours, the mixed solution of extracellular vesicles and hybridoma cells was collected in a new tube, washed three times with PBS, and the mixture of extracellular vesicles and hybridoma cells was recovered. The sample containing these hybridoma cells was analyzed using a flow cytometer (On-chip® Sort, manufactured by On-chip Biotechnologies, Inc.), and cells showing strong fluorescence were isolated (excitation wavelength 488 nm / fluorescence wavelength 543 nm). The separated cells were diluted 100-fold in fusion cell medium, suspended, and then seeded in 50 μL portions into each well of a 384-well cell culture plate. The cells were then cultured for 7 days in a mammalian cell culture incubator (37°C, 5 vol% CO2 atmosphere).

[0053] Example 8: Evaluation of ALP-recognizing antibody-producing hybridomas In Example 7, ALP-recognizing antibodies secreted into the culture supernatant of hybridoma cells cultured in a 384-well cell culture plate were detected by an ELISA method using inactive ALP-480 as the antigen, as described in SEQ ID NO: 7, to evaluate the acquisition rate of hybridomas that secrete anti-ALP-recognizing antibodies. The ELISA method was performed according to standard procedures, using a 384-well ELISA plate immobilized with streptavidin (12.5 μg / well) and an ELISA plate in which biotinylated ALP antigen was indirectly immobilized (12.5 μg / well).

[0054] 50 μL of 1% BSA-PBST solution (PBS containing 1% BSA and 0.05% Tween-20) was dispensed into each well of the aforementioned plate. 2 μL of culture supernatant from each well of a 384-well cell culture plate in which hybridoma cells were cultured was added, and an antigen-antibody reaction was carried out at room temperature for 1 hour. After washing each well, ALP-labeled anti-mouse IgG antibody diluted in 1% BSA-PBST solution was added to each well, and the reaction was carried out at room temperature for 1 hour.

[0055] To detect ALP-labeled anti-mouse IgG antibodies bound to ELISA plates, 50 μL of ALP detection substrate solution (10 mmol / L 4-methylumbelliferyl phosphate, 1 mol / L diethanolamine, 0.5 mmol / L magnesium chloride, pH 9.8) was added to each well. After reacting at room temperature for 30 minutes, the fluorescence intensity of each well was measured (excitation wavelength 360 nm / fluorescence wavelength 465 nm) to detect wells containing hybridoma cells secreting ALP-recognizing antibodies into the culture medium. The acquisition rate of hybridomas secreting anti-ALP-recognizing antibodies was evaluated.

[0056] Acquisition rate of hybridomas that secrete anti-ALP-recognizing antibodies

[0057] Figure 1 shows the ELISA results. Upon observation of the 384-well cell culture plate, single colonies were observed in 95 wells. Of these 95 wells, a reaction to biotinylated ALP antigen was detected in 22 wells (23.2%). [Industrial applicability]

[0058] By using the method of this invention to acquire antibodies, antibodies against membrane proteins with complex structures, which were difficult to obtain with conventional techniques, can be efficiently acquired. Furthermore, it facilitates the acquisition of structure-recognizing antibodies, which play an important role in the fields of antibody drugs and diagnostic agents.

Claims

1. A method for screening hybridoma cells that produce antibodies that selectively recognize the three-dimensional structure of an antigen, The antigen used for screening is a protein that maintains its three-dimensional structure in vivo. The aforementioned protein is exposed and presented on the surface of a vesicle composed of a biomaterial with a lipid bilayer, The vesicles are characterized by containing a fluorescent protein. A screening method for hybridoma cells.

2. The method according to claim 1, wherein the protein that maintains the three-dimensional structure is a membrane protein.

3. The method according to claim 1 or 2, wherein the fluorescent protein is a GFP protein.

4. The method according to claim 3, wherein the vesicle composed of the biomaterial composed of the lipid bilayer is an extracellular vesicle.

5. The method according to claim 4, wherein the vesicles contain a fluorescent protein by preparing the cells that produce the vesicles to express a fluorescent protein.

6. The method according to claim 5, wherein the cells are prepared to express a fluorescent protein by introducing an exogenous GFP gene.

7. The method according to claim 2, wherein the membrane protein is selected from either a single-pass transmembrane protein or a multiple-pass transmembrane protein.

8. The method according to claim 2, wherein the membrane protein is platelet-derived growth factor receptor (PDGFR).

9. The method according to claim 1, wherein the screening method is performed using a flow cytometer.

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