Monoclonal antibody against cypress pollen antigen Cha o 2 and its applications

Abstract

To provide a monoclonal antibody against Japanese cypress pollen allergen Cha o 2.SOLUTION: The present invention provides a monoclonal antibody against Japanese cypress pollen allergen Cha o 2. The monoclonal antibody reacts with or does not react with Cha o 1 and Japanese cedar pollen allergen Cry j 2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a monoclonal antibody against Cha o 2, the major allergen of cypress pollen. [Background technology]

[0002] Japanese cypress pollen is considered one of the substances that cause hay fever symptoms, and Cha o 2 is known as the major allergen of Japanese cypress pollen (Patent Document 1, Non-Patent Documents 2, 3). Other Japanese cypress pollen allergens that have been reported include Cha o 1 (Non-Patent Document 1, Patent Document 1), and more recently, Cha o 3 (Patent Document 2, Non-Patent Document 4).

[0003] It is known that approximately one-third of the Japanese population suffers from cedar pollen allergy. Cypress pollen has been reported to share antigenicity with cedar pollen (Non-Patent Literature 5), and a report that 76.4% of spring pollen allergy sufferers test positive for both cedar and cypress pollen (Non-Patent Literature 6) suggests that cypress pollen is one of the causative allergens that cause hay fever in the spring.

[0004] Antigen-specific monoclonal antibodies are useful for analyzing and researching pollen allergens in hay fever patients, enabling highly sensitive detection and quantification of antigens. For example, monoclonal antibodies against cedar pollen antigens Cry j 1 or Cry j 2, cypress pollen antigen Cha o 1 (Patent Documents 3 and 5), and mite antigen Der f 1 (Patent Document 4) are already known, along with their hybridomas and applications. However, there are no reports of sufficiently validated antibodies or their applications for the major cypress pollen allergen Cha o 2. The only commercially available detection system is "Cha o 2 polyclonal antibody" (Agresera), but currently it is only sold as an antibody, without any description of its performance, making it difficult to consider it a reliable detection system (Non-Patent Document 7). [Prior art documents] [Patent Documents]

[0005] [License 1] Special Announcement No. 8-176192 [License 2] International Publication No. WO2012 / 105541 [License 3] Special Announcement No. 2005-289929 [License 4] Special Announcement No. 9-87298 [Patent Document 5] Special Announcement No. 2021-50146 [Non-licensed literature]

[0006] [Non-licensed Document 1] Susuki M. et al., Mol Immunol, 33, 451-460 (1996): Purification, characterization and molecular cloning of Cha o 1, A major allergen of Chamaecyparis obtuse (Japanese cypress) pollen. [Non-licensed Document 2] Mori T. et al., Biochem Biophys Res Commun,263,166-171 (1999): Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen [Non-licensed Document 3] Yasueda H. et al., Clin Exp Allergy,30,546-550 (2000): Identification and characterization of a group 2 conifer pollen allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonica [Non-Patent Document 4] Osada T. et al., J Allergy Clin Immunol, 138, 911-913 (2016): Identification and gene cloning of a new major allergen Cha o 3 from Chamaecyparis obtusa (Japanese cypress) pollen [Non-Patent Document 5] Sone T. et al., Clin Exp Allergy, 35, 664-671 (2005): Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level. [Non-Patent Document 6] Okano et al., Allergy, 43, 1179-1184 (1994): Clinical study of patients positive for cypress-specific IgE antibodies: Using cypress AlaSTAT testing [Non-Patent Document 7] Anti-Cha o 2 | Major pollen allergen Cha o 2 antibodies (agrisera.com) [Overview of the Initiative]

Problems to be Solved by the Invention

[0007] There has been a need to stably produce monoclonal antibodies against Cryptomeria japonica pollen antigen Cha o 2 and to develop an analysis and quantification system for Cryptomeria japonica pollen antigen using the monoclonal antibodies. Polymorphisms in amino acids have been reported for Cha o 2 (Tateno M. et al., PLoS ONE 16, e0261327 (2021)), and when the epitope contains polymorphisms, the affinity between the monoclonal antibody and Cha o 2 may change. The monoclonal antibody to be used needs to stably bind to Cha o 2 regardless of the presence or absence of amino acid polymorphisms.

[0008] As a result of intensive studies to solve the above problems, the present inventors have produced a monoclonal antibody that is specific to Cha o 2, does not contain amino acid polymorphisms in the epitope, and has a specific recognition site, thus completing the present invention.

Means for Solving the Problems

[0009] That is, the present invention is as follows. (1) A monoclonal antibody against Cryptomeria japonica pollen allergen Cha o 2, which does not react with Cha o 1. (2) The monoclonal antibody according to (1), which does not react with Japanese cedar pollen allergen Cry j 2. (3) The monoclonal antibody according to (1), which reacts with Japanese cedar pollen allergen Cry j 2. (4) The monoclonal antibody according to any one of (1) to (3), which recognizes all or part of the amino acid sequence shown in SEQ ID NO: 2 to 6 as an epitope. (5) The monoclonal antibody according to (3), wherein the minimum detectable concentration of Cha o 2 when combined with the monoclonal antibody according to (2) is 5 ng / mL or less. (6) The monoclonal antibody according to (2), wherein the minimum detectable concentration of Cha o 2 when combined with the monoclonal antibody according to (3) is 5 ng / mL or less. A monoclonal antibody according to any one of (1) to (6), having a coefficient of variation of 20% or less. An antibody that binds to an epitope to which the monoclonal antibody according to any one of (1) to (7) binds. (9) A monoclonal antibody according to any one of (1) to (7), or a fragment of the antibody according to (8). (10) A hybridoma that produces the monoclonal antibody according to any one of (1) to (7). (11) A method for detecting Chao 2, comprising reacting a monoclonal antibody according to any one of (1) to (7), or the antibody according to (8), or a fragment thereof with a sample to detect Chao 2. (12) A kit for detecting Chao 2, comprising a monoclonal antibody according to any one of (1) to (7), or the antibody according to (8), or a fragment thereof. [Advantages of the Invention]

[0010] According to the present invention, by using a monoclonal antibody specific to Chao 2 and having a specific epitope, problems in antigen identification and quantification systems are solved regardless of the presence or absence of amino acid polymorphisms, and a test system capable of highly sensitively and simply identifying and quantifying Chao 2 in Cryptomeria pollen extract immunologically has been developed. [Brief Description of the Drawings]

[0011] [Figure 1] It is a diagram showing the detectability of Chao 2 by a combination of a capture antibody and a biotinylated detection antibody. [Figure 2] It is a diagram showing epitope mapping. [Figure 3] It is a diagram showing the epitope sequences of the Chao 2 amino acid polymorphism and the anti-Chao 2 antibodies 9A11-1F8 and 4H5-3C3. [Figure 4] It is a diagram showing the reactivity of the anti-Chao 2 antibodies 9A11-1F8 and 4H5-3C3 to Chao 2 and Cry j 2 in indirect ELISA. [Figure 5-1] This figure shows the specificity of anti-Cha o 2 antibodies 9A11-1F8 and 4H5-3C3 in Western blotting using purified Cha o 2 and purified Cry j 2. [Figure 5-2] This figure shows the specificity of anti-Chao2 antibodies 9A11-1F8 and 4H5-3C3 in Western blotting using cypress pollen extract. [Figure 6] This figure shows the epitope sequences of the anti-Cha o 2 antibody 9A11-1F8 and 4H5-3C3, and the homologous sequence of Cry j 2. [Figure 7] This figure shows the specificity of antigen detection using Sandwich ELISA. [Figure 8] This figure shows the concentration range (0.313 to 50 ng / mL) for which a 4-paralogistic regression curve with R²=1 was created in the Sandwich ELISA. [Figure 9] This figure shows the cross-reactivity with cedar pollen extract. [Modes for carrying out the invention]

[0012] This invention relates to multiple monoclonal antibodies against Cha o 2, the major allergen of cypress pollen. In this invention, multiple monoclonal antibodies against the cypress antigen were newly created, and their epitopes were successfully identified. Furthermore, by using these monoclonal antibodies, a highly sensitive and accurate detection method for Cha o 2 in cypress pollen extract was developed.

[0013] In research on cypress pollen antigens, there is generally no validated specific measurement method. Test systems using polyclonal antibodies or monoclonal antibodies against cedar antigens, utilizing antigen homology, are commonly used. In the case of polyclonal antibodies against cypress antigens, the epitope of the detected antigen cannot be determined, and they are generally inferior to monoclonal antibodies in terms of specificity. When using monoclonal antibodies against cedar antigens that have homology to cypress antigens, the cedar antigen is also recognized, which imposes certain limitations on the range of samples that can be measured and the interpretation of the results. The Cha o 2-specific monoclonal antibody produced by the present invention has the characteristic of recognizing the cypress pollen antigen Cha o 2 in a sample with high sensitivity, and can be widely applied to immunological assays such as Western blotting, ELISA, and Sandwich ELISA, thus offering broad advantages and usefulness, including use in reagents and kits. In this specification, "recognizing" an antigen such as Cha o 2 can be used synonymously with "reacting" with the antigen or "binding" to the antigen.

[0014] 1. Monoclonal antibody of the present invention The method for obtaining the monoclonal antibody (also called "anti-Cha o 2 antibody") of the present invention involves first immunizing a non-human mammal with Cha o 2 antigen protein or a partial peptide of Cha o 2 protein, and then collecting antibody-producing cells (e.g., B cells) from the immunized animal. These antibody-producing cells are fused with myeloma cells to create a hybridoma (fusion cell line). The target monoclonal antibody is then obtained by collecting the antibody produced from this hybridoma.

[0015] (1) Preparation of antigen In this invention, the full-length Cha o 2 protein is used as an immunoantigen. The full-length Cha o 2 protein can be obtained by combining various column chromatography purification methods from a cypress pollen extract. It can also be obtained by expressing it as a recombinant protein in E. coli or other host cells and then purifying the extract using various chromatography methods. Furthermore, in this invention, a partial peptide can also be synthesized and used as an immunoantigen.

[0016] The basic amino acid sequence for synthesizing the antigen peptide is a partial sequence of the full-length amino acid sequence of Cha o 2, and from that amino acid sequence, a sequence of 10 to 50 amino acids, preferably 10 to 30, and more preferably 15 to 20 amino acids of any length is selected. The peptides that can be used as antigens are the full-length sequence of Cha o 2 (SEQ ID NO: 1) or peptides containing a portion of the sequence. In the case of a portion of the sequence, the sequence from position 113 to 314 (SEQ ID NO: 2) of the full-length amino acid sequence of Cha o 2 can be selected. In the present invention, for example, peptides from the region 113 to 132 (SEQ ID NO: 3), peptides from the region 288 to 307 (SEQ ID NO: 4), or peptides from the region 295 to 314 (SEQ ID NO: 5) can also be used. Furthermore, peptides from the above regions that share a common amino acid sequence, specifically from position 295 to 307 (SEQ ID NO: 6), can also be used.

[0017] Partially synthesized peptides are small molecules, making it difficult to obtain antibodies by immunizing mice with them in their original form. Therefore, an immune antigen is created by disulfide bonding the synthetic peptide to a carrier protein. The chemical synthesis of peptides can be carried out by methods known to those skilled in the art, such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Examples of carrier proteins include BSA (Bovin serum albumin), KLH (Keyhole Limpet Hemocyanin), and OVA (Ovalbumin).

[0018] (2) Animal immunity The types of non-human mammals to be immunized are not particularly limited, and include, for example, mice, rats, guinea pigs, rabbits, dogs, goats, etc., but mice are preferred. The total dose of antigen per animal is 10-150 μg. When immunizing with the antigen, it is common to mix the adjuvant with the antigen solution. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is mainly performed by injection intravenously, subcutaneously, intraperitoneally, intramuscularly, or subcutaneously in the soles of the feet. There is no particular limit to the interval between immunizations, but 1 to 10 immunizations, preferably 2 to 5, are performed at intervals of several days to several weeks, preferably every 2 to 3 weeks.

[0019] (3) Preparation of antibody-producing cells Antibody-producing cells are prepared from spleen cells or regional lymph nodes of immunized non-human mammals. Examples of lymph nodes include iliac lymph nodes and inguinal lymph nodes. While it is not necessary to specifically isolate antibody-producing cells from the collected cell population, it is preferable to isolate only antibody-producing cells from the cell population. However, when preparing antibody-producing cells, it is preferable to remove as much tissue debris and red blood cells as possible.

[0020] (4)Cell fusion Cell fusion is a process performed to create a hybridoma—a cell that continuously proliferates semi-permanently while producing antibodies—by fusing antibody-producing cells with myeloma cells. Those skilled in the art can fuse antibody-producing cells with myeloma cells using known cell fusion methods. As the myeloma cells to be fused with antibody-producing cells, commonly available cell lines from animals such as mice can be used. Preferably, the cell line used is one that cannot survive in HAT selective medium (a medium containing hypoxanthine, thymidine, and aminopterin) and can only survive when fused with antibody-producing cells. Examples of myeloma cells include SP 2 / 0, P3U1, NS I, and P3-X63-Ag8.653.

[0021] Cell fusion can be performed in commercially available media such as DMEM or RPMI1640 medium, which do not contain fetal bovine serum (FCS), etc., with a result of 1 × 10⁶ 6 ~1 × 10 7 cells / mL of spleen cells or lymph node cells and 1 × 10⁶ 5 ~1 × 10 6 Mix with myeloma cells at a concentration of cells / mL and perform fusion in the presence of a cell fusion promoter. A cell ratio of spleen cells or lymph node cells to myeloma cells (spleen cells or lymph node cells:myeloma cells) of 5:1 is preferred. Polyethylene glycol with an average molecular weight of 200 to 20,000 daltons can be used as the cell fusion promoter.

[0022] Furthermore, fusion can also be achieved using commercially available cell fusion devices that utilize electrical stimulation (e.g., electroporation), or by using Sendai virus. After fusion, the cells were diluted with HAT medium prepared, for example, RPMI1640 medium containing 10-20% FCS, and then 0.5-3 × 10¹ cells were placed in each well of a 96-well culture plate. 5 The cells are seeded one by one and cultured in a CO2 incubator.

[0023] (5) Establishment of hybridomas Next, hybridomas that produce the target antibody are selected from the cells after cell fusion. Ten to fourteen days after cell fusion, the selected cells form colonies in HAT medium. The culture supernatant is collected from each well of the colony-positive 96-well culture plate, and the antibody titer against Cha o 2 is checked. This can be done using enzyme immunosorbent assay (ELISA) or radioimmunoassay (RIA). When a conjugate of a partial peptide and a carrier protein is used as the immunoantigen, the antibodies produced by the cells also include antibodies against the carrier proteins KLH and BSA. Therefore, by measuring the antibody titer against KLH, etc., it is possible to exclude KLH antibody-positive wells with high antibody titers against KLH, etc.

[0024] After confirming that the wells are positive for antibody production against Cha o 2, transfer the cells to 24-well or 12-well culture plates. It is preferable to replace the culture medium with HT medium (Hydrogen-Trichosteroids) without aminopterin. HT medium is used as a recovery medium for hybridomas, continuing to supply purine and pyrimidine precursors to the salvage pathway while residual aminopterin in the cells remains. After culturing in HT medium for a period, check the antibody titer in the culture supernatant again.

[0025] As described above, in the present invention, it is necessary to obtain hybridomas that have high specificity for Cha o 2 while confirming cross-reactivity with Cry j 2. Therefore, cross-reactivity with Cry j 2 is confirmed at the culture supernatant level using ELISA, RIA, etc.

[0026] The cells in the final selected wells are cloned to form single cells. Cloning is performed by appropriately diluting the cell suspension with, for example, RPMI1640 medium containing 10-20% FCS (preferably 20%), and then seeding the cells so that 0.3-2 cells are placed in each well of a 96-well culture plate. The number of cells placed in each well of the 96-well culture plate is preferably 1 cell per well to increase the probability that there is 1 cell in each well. After seeding the cells, the culture supernatant of the colony-positive wells is collected 7-10 days later. At this time, it is preferable to confirm that there are single colonies after 3-5 days. The antibody titer of the collected culture supernatant is checked. Here, clones with high specificity for Cha o 2 and low cross-reactivity with Cry j 2, as well as clones with high specificity, are selected. Furthermore, the cells in the selected wells are increased to a certain extent to establish hybridoma strains. Cloning may be performed several times as needed. In this invention, 11 clones were obtained as established hybridomas and were named "2D6-2C3", "9A11-1F8", "3A8-2F2", "4H5-3C3", "8G10-1H7", "4G6-1D6", "9E12-2B7", "4F6-4B11", "5A8-4B2", "3E1-1C8", and "6H5-2F2".

[0027] (6) Preparation of monoclonal antibodies From the established hybridoma strains, Cha o 2-specific monoclonal antibodies are purified and collected by the following methods: preparing antibodies from the culture supernatant obtained by culturing in a medium with reduced serum concentration; preparing antibodies from the culture supernatant obtained by culturing in commercially available serum-free medium; and injecting hybridomas into the peritoneal cavity of animals, collecting ascites fluid, and preparing antibodies from that ascites fluid. The culture supernatant contains cells of 0.1 to 4 × 10⁶ 5 Prepare at a concentration of cells / mL and collect from cells cultured for 1-2 weeks. In the case of ascites, hybridomas are collected in the peritoneal cavity of mammalian allogenes derived from myeloma cells, ranging from 0.1 to 1 × 10⁶ cells. 7 The drug is administered individually to induce massive proliferation of hybridomas. Then, ascites fluid is collected 1-2 weeks later.

[0028] Culture methods include using culture flasks, spinner flasks, shaker flasks, and bioreactors. Antibodies can be purified using protein G affinity columns or protein A affinity columns, Chao 2 affinity columns, gel filtration chromatography from ammonium sulfate salting-out fractions, or ion exchange chromatography. These known methods can be appropriately selected or combined to achieve purification.

[0029] 2. Epitope of anti-Chao2 antibody The epitope (antigenic determinant) of the anti-Cha o 2 antibody of the present invention is not limited to any part of the antigen Cha o 2, but for example, the amino acid sequence of the region from position 113 to 314 (SEQ ID NO: 2). In the present invention, it is preferable that the epitope is the region consisting of the amino acid sequence from position 113 to 132: WWAGQCKVVNGRTVCNDRNR (SEQ ID NO: 3) or at least a part of said region, the region consisting of the amino acid sequence from position 288 to 307 (NPILINQFYCTSASACQNQR (SEQ ID NO: 4)) or at least a part of said region, the region consisting of the amino acid sequence from position 295 to 314 (FYCTSASACQNQRSAVQIQG (SEQ ID NO: 5)) or at least a part of said region, or the region consisting of the amino acid sequence from position 295 to 307 (FYCTSASACQNQR (SEQ ID NO: 6)) or at least a part of said region. In particular, the region consisting of amino acid sequences 295 to 307: FYCTSASACQNQR (SEQ ID NO: 6), or at least a portion of said region, is more preferred.

[0030] Anti-Chao2 antibodies that recognize (bind to or react with) these regions are Chao2-specific, highly sensitive, and extremely useful for detecting Chao2. The monoclonal antibodies of the present invention are antibodies that specifically bind to Cha o 2. However, depending on the clone, there are antibodies that react with the cedar pollen allergen Cry j 2 (antibodies that have cross-reactivity with Cry j 2) and antibodies that do not react with Cry j 2 (antibodies that do not have cross-reactivity with Cry j 2). The present invention encompasses both of these embodiments of antibodies.

[0031] Furthermore, antibodies against Cha o 2 are not limited to the monoclonal antibodies against Cha o 2 produced by the hybridomas mentioned above, but are included in the anti-Cha o 2 antibodies of the present invention as long as they bind to the epitopes recognized by the monoclonal antibodies produced by these hybridomas. Here, "epitope" refers to the epitope recognized by the monoclonal antibodies produced by the hybridomas mentioned above. This epitope may be the amino acid residues in the region from position 113 to 314 of the amino acid sequence of Cha o 2, for example, amino acid residues from position 113 to 132, 288 to 307, 295 to 314, or amino acid residues from 295 to 307, or even a part of these regions.

[0032] 3. Recombinant antibodies The monoclonal antibody of the present invention may also be a recombinant antibody or antigen-binding fragment prepared and expressed by a recombinant means. For example, the anti-Chao2 antibody of the present invention may be a chimeric antibody, a humanized antibody, or a fully human antibody. The recombinant antibody of the present invention can be produced by recombinant expression of the heavy chain and light chain. To express a recombinant antibody, for example, a recombinant expression vector containing nucleic acids encoding the heavy and light chains of the antibody is introduced into host cells, and the host cells into which the vector has been introduced are cultured. Then, the target antibody is recovered from the culture of these host cells. To obtain the genes for the heavy and light chains of the antibody, incorporate these nucleic acids into an expression vector, and introduce it into host cells, the standard recombinant DNA method in this field (Green and Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012) can be used.

[0033] For example, a mouse-human chimeric antibody can be prepared by isolating the antibody gene from mouse cells that produce antibodies against the Cha o 2 protein, recombining its heavy chain (H chain) constant region and light chain (L chain) constant region with the human IgG H chain constant region gene and the human IgG L chain constant region gene, respectively, and then introducing these into mouse myeloma cells. Nucleic acids (e.g., DNA) encoding the heavy chain variable region (VH) are constructed into a full-length heavy chain gene by expressively binding the VH-encoding DNA to the heavy chain constant region-encoding DNA. Similarly, nucleic acids (e.g., DNA) encoding the light chain variable region (VL) are constructed into a full-length light chain gene by expressively binding the VL-encoding DNA to the light chain constant region-encoding DNA. If the nucleic acids encoding the full-length light chains VH and VL are, for example, derived from mouse, and the nucleic acids encoding the heavy chain and light chain constant region are derived from human, then a mouse-human chimeric antibody or antigen-binding fragment is formed. The nucleic acid sequences of the heavy chain constant region derived from humans, mice, etc., are well known in this field.

[0034] When producing humanized antibodies, a technique known as CDR grafting (CDR transplantation) can be employed. CDR grafting is a method of creating a reconstituted variable region by transplanting the complementarity-determining region (CDR) from the variable region of a mouse antibody into the human variable region, so that the framework region (FR) is of human origin and the CDR is of mouse origin. Antibody fragments (peptides) containing CDRs are composed of at least one region of VH or VL CDRs (CDR1-3). Antibody fragments containing multiple CDRs can be conjugated directly or via an appropriate peptide linker.

[0035] Next, these humanized reconstituted human variable regions are ligated to the human constant region. Methods for producing such humanized antibodies are well known in this field (Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989)).

[0036] Human antibodies (fully human antibodies) generally have the same structure as human antibodies in the hypervariable region (the antigen-binding site of the variable region), the rest of the V region, and the constant region. Techniques for producing human antibodies are also well-known, and methods for producing antibodies using genetic engineering techniques have been established for gene sequences common to humans. Human antibodies can be obtained, for example, by methods using human antibody-producing mice that possess human chromosome fragments containing the genes for the H and L chains of human antibodies (e.g., Tomizuka, K. et al., Nature Genetics, (1977)16, 133-143; Kuroiwa, Y. et.al., Nuc. Acids Res., (1998)26, 3447-3448), or by methods for obtaining human antibodies derived from phage displays selected from human antibody libraries (e.g., Wormstone, IMet.al, Investigative Ophthalmology & Visual Science., (2002)43 (7), 2301-8; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics, (2002)1 (2), 189-203).

[0037] Furthermore, in the present invention, chimeric antibodies, humanized antibodies, and humanized antibodies can be produced using hybridomas or DNA or RNA extracted from said hybridomas as raw materials, in accordance with the well-known methods described above.

[0038] 4. Preparation of antibody fragments The antibody fragments used in this invention refer to a portion of the antibody of the present invention, such as Fab, Fab', F(ab')2, Fv, diabody (dibodies), dsFv, scFv (single chain Fv), etc. These antibody fragments can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes as needed. For example, Fab can be obtained by treating an antibody molecule with papain, and F(ab')2 can be obtained by treating an antibody molecule with pepsin. Furthermore, Fab' can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0039] In the case of scFv, cDNA encoding the VH and VL of the antibody is obtained, and DNA encoding scFv is constructed. This DNA is inserted into an expression vector, and scFv can be produced by introducing this expression vector into a host organism and expressing it.

[0040] In the case of diabody, cDNA encoding the VH and VL of the antibody is obtained, and DNA encoding scFv is constructed such that the length of the peptide linker amino acid sequence is 8 residues or less. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, diabody can be produced. In the case of dsFv, cDNA encoding the VH and VL of the antibody is obtained, and DNA encoding dsFv is constructed. This DNA is inserted into an expression vector, and dsFv can be produced by introducing this expression vector into a host organism and expressing it.

[0041] 5. Detection method and detection kit Since Cha o 2 is an allergen in cypress pollen allergy, Cha o 2 can be detected by reacting the anti-Cha o 2 antibody of the present invention with a sample and measuring the amount of Cha o 2 in the sample. Accordingly, the present invention provides a method for detecting Cha o 2, characterized by reacting the antibody or a fragment thereof with Cha o 2 to detect Cha o 2. Furthermore, in the present invention, an anti-Chao2 antibody can be used as a reagent or kit for detecting Chao2.

[0042] When using the anti-Cha o 2 antibody of the present invention as a kit, the anti-Cha o 2 antibody can be combined with other solvents or solutes to form a composition. For example, distilled water, pH buffering reagents, salts, proteins, surfactants, etc., can be used in combination. The anti-Cha o 2 antibody can also be used after being enzyme-labeled or biotin-labeled. As labeling enzymes, in addition to HRP (horseradish peroxidase), alkaline phosphatase, malate dehydrogenase, α-glucosidase, β-galactosidase, gold colloid, etc., can be used.

[0043] When the present invention is used in a kit, in addition to the antibody of the present invention, the kit may include the above-mentioned solvent, solute, enzyme-labeled reagent, antigen-immobilized microplate, antibody dilution solution, OPD (orthophenylenediamine) tablets, substrate solution, reaction stop solution, concentrated washing solution, instructions for use, etc. Furthermore, reaction media such as buffers that provide optimal reaction conditions, buffers useful for stabilizing reaction products, and stabilizers for reaction substances may also be included in the kit of the present invention.

[0044] The anti-Cha o 2 antibody and kit of the present invention can measure Cha o 2 specifically and with high sensitivity, while also achieving sufficient measurement accuracy. Therefore, the anti-Cha o 2 antibody and kit of the present invention can be used for quality evaluation of cypress pollen extracts and other materials used in clinical applications such as allergen immunotherapy and measurement of antibody titers against cypress pollen allergens in human serum. Measurement accuracy is an indicator of how much the measured values ​​vary when the same sample is divided into multiple test tubes or wells and an assay is performed once. Statistically, this is expressed as the coefficient of variation (CV), i.e., the ratio (%) of the standard deviation to the mean. In the present invention, this coefficient of variation (CV) is called reproducibility. Reproducibility is 20% or less, but preferably 15% or less, or 10% or less, and more preferably 5% or less.

[0045] The minimum detection concentration of the kit of the present invention is 5 ng / mL or less, for example, 1 to 3 ng / mL. Furthermore, the within-run reproducibility is 10% or less, preferably around 5%, and the daily reproducibility is 20% or less, or 15% or less, preferably about 8 to 10%. All reproducibility levels have a CV of 20% or less. As described above, the antibodies of the present invention are antibodies that specifically bind to (react specifically with) Cha o 2, but they also include antibodies that react with the cedar pollen allergen Cry j 2 and antibodies that do not react with Cry j 2. In this case, the minimum detection concentration of Cha o 2 when combining an antibody that reacts with Cry j 2 and an antibody that does not react with Cry j 2 is, for example, 5 ng / mL or less.

[0046] Examples The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. [Examples]

[0047] 1. Materials and Methods 1.1 Materials 1.2 Reagents Cypress pollen: Yamizo Pollen Research Association or Torii Pharmaceutical Co., Ltd. Cedar pollen: Torii Pharmaceutical HiTrap Octyl FF:Cytiva HiTrap SP HP:Cytiva HiTrap IMAC HP :Cytiva ADJUVANT COMPLETE FREUND:DIFCO Hybridoma-SFM: GIBCO NaHCO3: Fujifilm Wako Pure Chemical Industries HCl: Fujifilm Wako Pure Chemical Industries NaOH: Fujifilm Wako Pure Chemical Industries FBS: Biological Industry Recombinant human IL-6:R&D Systems D-MEM: Fujifilm Wako Pure Chemical Industries Hypoxanthine: SIGMA-ALDRICH Aminopterin: SIGMA-ALDRICH Thymidine: Fujifilm Wako Pure Chemical Industries PEG 1500: Roche Penicillin-Streptomycin-Amphotericin B Suspension: Fujifilm Wako Pure Chemical Industries Buffer Kit: Protenova Ammonium sulfate: Fujifilm Wako Pure Chemical Industries Oriole Fluorescent Gel Stain:BIO-RAD TMB ONE: Kem-en-tec Goat, anti mouse-HRP:R&D systems Avidin, Horseradish Peroxidase Conjugated (Avidin-HRP):Pierce PBS: Fujifilm Wako Pure Chemical Industries Tween20: Pure Chemistry BSA: Sigma-Aldrich Tris-Glycine Sample Buffer (2X): TEFCO DTT (1,4-Dithiothreitol): Pure Chemical Bullet PAGE One Precast Gel, 5-15% (17 wells): Nacalai Tesque Electrophoresis buffer: Fujifilm Wako Pure Chemical Industries Precision plus Protein Dual color Standard:Bio-RAD 10x Tris / Glycine (Transfer Buffer): Bio-RAD Block Ace: KAC ECL Prime RPN2232: GE Healthcare NH4HCO3: Fujifilm Wako Pure Chemical Industries NaCl: Pure Chemical Anti-Cry j 2 antibody (8D6): Torii Pharmaceutical

[0048] 1.3 Equipment Zeba Spin Desalting Columns (7K MWCO) :Thermo Scientific 96-well microplate Maxisorp:NUNC 96-well microplate IMMULON 2HB: Thermo Scientific Immoblon-PSQ membrane 7x8.4:Millipore

[0049] 1.4 Reagent 10% FBS, Hybridoma-SFM medium Hybridoma-SFM was treated with 50 mL of inactivated FBS, 5 mL of penicillin-streptomycin-amphotericin B suspension, and 50 μL of 10 μg / mL IL-6, bringing the total volume to 500 mL.

[0050] serum-free culture medium To hybridoma-SFM, 5 mL of penicillin-streptomycin-amphotericin B suspension and 50 μL of 10 μg / mL IL-6 were added, bringing the total volume to 500 mL.

[0051] HAT medium Hypoxanthine and thymidine dissolved in water, and aminopterin dissolved in water and 1M NaOH were added to Hybridoma-SFM medium. 10% FBS and IL-6 (1 ng / mL) were then added.

[0052] PBS-T 10×PBS was diluted 10-fold to prepare Tween 20 to a final concentration of 0.05%.

[0053] 1% BSA / PBS-T BSA was prepared in PBS-T to a final concentration of 10% and stored at -20°C. Before use, it was dissolved and diluted 10-fold with PBS-T. NH4HCO3 and NaCl were added to the extract solution to prepare it so that the final concentrations were 40 mM and 0.5 M, respectively.

[0054] 1.5 Equipment Cool Incubator: Mitsubishi Electric Engineering Co., Ltd., CN-40A Personal Incubator: EYELA (Model: LTI2000) Microplate reader: Molecular Devices, SPECTRAmaxPLUS 384 Power supply: BIO-RAD POWER PAC 300 Bioimage analyzer: GE Healthcare, Image Quant LAS 4010 Transfer device (semi-dry type): BIO-RAD Sample boil: Heat block Dry Thermo Unit (DTU-2B)

[0055] 1.6 Method 1.6.1 Purification of Cha o 2 from cypress pollen Hinoki pollen was mixed with 20 times the volume of 40 mM NH4HCO3 and stirred at 4°C for 3 hours to extract. After stirring, the precipitate was centrifuged at 20,000 × g for 30 minutes, and 20 times the volume of 40 mM NH4HCO3 and 0.5 M NaCl were added, and the mixture was stirred at 4°C for 3 hours to extract Cha o 2. Ammonium sulfate was added to achieve a 20% saturated ammonium sulfate solution, and the mixture was stirred at 4°C overnight. Ammonium sulfate was added to the supernatant after centrifuging at 20,000 × g for 30 minutes to achieve an 80% saturated ammonium sulfate solution, and the mixture was stirred at 4°C for 4 hours. The precipitate obtained by centrifugation at 20,000 × g for 30 minutes was buffer-changed by dialysis with 2 M ammonium sulfate and 50 mM Tris-Cl (pH 7.8). After dialysis, the solution was applied to an Octyl FF column. The Cha o 2 fraction was collected by eluting from 2 M ammonium sulfate, 50 mM Tris-HCl buffer (pH 7.8) to 50 mM Tris-HCl buffer (pH 7.8) using a gradient elution method. The collected fraction was dialyzed to 0.5 M NaCl, 30 mM phosphate buffer (pH 7.8) and applied to a HiTrap IMAC HP. The Cha o 2 fraction was collected by eluting from 0.5 M NaCl, 30 mM phosphate buffer (pH 7.8) to 0.5 M NaCl, 30 mM acetate buffer (pH 4.0) using a gradient elution method. The collected fraction was dialyzed to 0.2 M NaCl, 30 mM acetate buffer (pH 4.0) and applied to a HiTrap SP HP. The Cha o 2 fraction was collected by eluting in a gradient from 0.2 M NaCl, 30 mM acetate buffer (pH 4.0) to 1 M NaCl, 30 mM acetate buffer (pH 4.0). The collected fraction was purified as Cha o 2 and stored at -20°C until use.

[0056] 1.6.2 Production of anti-Chao2 monoclonal antibody-producing hybridomas 1.6.2.1 Immunization and Hybridoma Production Anti-Cha o 2 monoclonal antibodies were produced using the mouse iliac lymph node method (Japanese Patent Publication No. 2007-020547). Cha o 2 purified from cypress pollen and Freund's Complete Adjuvant (FCA) were mixed in a volume ratio of 1:2, and 27 μg of Cha o 2 was administered per mouse to the tail muscle of B6D2F1 / Slc mice (5 female mice, 8 weeks old at arrival, from Nippon SLC Co., Ltd.). Seventeen days after the initial administration, 16 μg of Cha o 2 was administered per mouse as an additional immunization. Twenty-one days after the initial administration, iliac lymph nodes were excised and used for cell fusion with mouse myeloma-derived cells (myeloma cells).

[0057] 1.6.2.2 Screening of cell fusion and antibody-producing hybridomas Sterile mouse iliac lymph nodes were suspended in DMEM medium and mixed with myeloma cells (SP2 / W) in a 5:1 ratio (lymphocytes:myeloma). Cell fusion was performed using a standard method with PEG. 0.9975 × 10⁶ lymphocytes were added to HAT medium. 6 The cells were resuspended to a concentration of cells / mL and seeded at 100 μL / well in eight 96-well plates. After incubation at 5% CO2 and 37°C for 6 days, a portion of the culture supernatant was collected and screened by ELISA and Western blotting. Wells that showed reactivity to Cha o 2 were selected, and monoclonal antibody-producing hybridomas were cloned using the limiting dilution method.

[0058] 1.6.2.3 Preparation of Monoclonal Antibodies Cloned monoclonal antibody-producing hybridomas were cultured overnight in 10% FBS, Hybridoma-SFM medium, then replaced with serum-free medium and cultured for 5-7 days. The culture supernatant was collected. Ammonium sulfate was added to the collected culture supernatant to a 50% saturation, and the precipitate obtained by centrifugation was dissolved in PBS. Monoclonal antibodies were purified from this solution using an affinity purification column (Ab-Rapid PuRe EX: Protenova). Biotinylated monoclonal antibodies were biotin-labeled using a commercially available biotin labeling kit (Dojin Chemical, biotin labeling Kit-NH2).

[0059] 1.6.3 Selection of antibody combinations by sandwich ELISA A 50-μL aliquot of a purified monoclonal antibody solution prepared at 20 μg / mL was added to each well of a 96-well microplate and left overnight at 25°C. After washing, blocking was performed with a 1% BSA / PBS-T solution. Then, a 50-μL aliquot of a purified Chao 2 solution at 50 ng / mL was added, and the mixture was left at 25°C for 1 hour. After washing with PBS-T, a 50-μL aliquot of a biotinylated purified monoclonal antibody solution (1 μg / ml) was added, and the reaction was carried out at 25°C for 1 hour. After the reaction, washing with PBS-T was performed, followed by reaction with avidin-HRP. After washing with PBS-T, the substrate TMB was added, and the reaction was carried out at 25°C for 5 minutes. Then, the reaction was stopped with 2 N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. <(

[0060] 1.6.4 Epitope identification From the full-length amino acid sequence of Chao 2 (UniProt No. Q7M1E7: SEQ ID NO: 1), an amino acid sequence excluding the signal sequence ( -50 M~ -1 H) ( 1 S~ 464 [[ID=!7]]P) (the numbers above indicate the positions of amino acid residues, and the alphabet represents the amino acid residues in one-letter notation. The same applies hereinafter), a 20-residue overlapping peptide library with 7-residue overlap was prepared (SIGMA-ALDRICH, PEPscreen). Each Chao 2 partial sequence peptide was prepared at 20 μM, and a 100-μL aliquot of the solution was added to a 96-well microplate and left overnight at 25°C. After washing, blocking was performed with a l% BSA / PBS-T solution. A 100-μL aliquot of a purified monoclonal antibody solution prepared at 0.5 μg / mL was added to each well of the blocked plate, and the reaction was carried out at 25°C for 1 hour. After washing with PBS-T, a goat-derived peroxidase-labeled anti-mouse IgG antibody was reacted. After washing with PBS-T, the substrate TMB was added, and the reaction was carried out at room temperature for 5 minutes. Then, the reaction was stopped with 2 N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

[0061] 1.6.5 Examination of reactivity using indirect ELISA Cha o 2 and Cry j 2 were each prepared at 1 μg / mL, and 50 μL of each solution was added to a 96-well microplate and left at 37°C for 1 hour. After washing, the wells were blocked with 1% BSA / PBS-T solution. 50 μL of anti-Cha o 2 monoclonal antibody 4H5-3C3, 9A11-1F8, or anti-Cry j 2 monoclonal antibody 8D6 (1 or 10 μg / ml) was added to each well of the blocked plate and reacted at 25°C for 1 hour. After washing with PBS-T, goat-derived peroxidase-labeled anti-mouse IgG antibody was reacted. After washing with PBS-T, the substrate TMB was added to the wells and reacted at room temperature for 5 minutes, then the reaction was stopped with 2 N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

[0062] 1.6.6 Preparation of pollen extract 0.5 g of cypress or cedar pollen was mixed with 10 mL (20 times the volume) of extract and stirred overnight at 4°C. After stirring, the supernatant was obtained by centrifugation at 20,000 × g for 30 minutes and used as the pollen extract.

[0063] 1.6.7 Examination of reactivity by Western blotting Equal volumes of 50 mM DTT / 2×SDS sample buffer were added to Cha o 2, Cry j 2, or cypress pollen extract, and heated at 95°C for 5 minutes. After cooling, electrophoresis was performed on a 5-15% SDS-polyacrylamide gel, followed by protein staining and Western blotting. Protein staining was performed using Oriole. TMThe samples were stained with Fluorescent Gel Stain. Western blotting was performed by electrically transferring the isolated proteins from the gel to a PVDF membrane, blocking with 1% skim milk, and adding either anti-Cha o 2 monoclonal antibody 4H5-3C3 or anti-Cha o 2 antibody 9A11-1F8. The mixture was reacted at room temperature for 1 hour. After the reaction, the samples were washed with PBS-T and reacted with goat-derived peroxidase-labeled anti-mouse IgG antibody. After washing with PBS-T, ECLprime detection reagent was added, and chemiluminescence was detected using a bioimage analyzer.

[0064] 1.6.8 Examination of reactivity using Sandwich ELISA In section 1.6.3, a combination of anti-Cha o 2 monoclonal antibody 4H5-3C3 as the capture antibody and anti-Cha o 2 antibody 9A11-1F8 as the detection antibody was selected for the Sandwich ELISA. 100 μL of anti-Cha o 2 monoclonal antibody 4H5-3C3 solution (10 μg / mL) was added to each of the 96 wells of a microplate and left overnight at 25°C. After washing, the wells were blocked with 1% BSA / PBS-T solution. 100 μL of Cha o 2 solution prepared in the range of 0.625 to 1000 ng / mL, or Cha o 1 and Cry j 2 solution prepared in the range of 50 to 3000 ng / mL, was added to each well of the blocked plate and left for 2 hours at 25°C. After washing, 100 μl of biotinylated anti-Cha o 2 antibody 9A11-1F8 solution (1 μg / ml) was added and the mixture was reacted at 25°C for 1 hour. After washing with PBS-T, avidin-HRP was reacted. After washing with PBS-T, the substrate TMB was added and the mixture was reacted at 25°C for 5 minutes. The reaction was then stopped with 2N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. All measurements were performed in duplicate.

[0065] 1.6.9 Examination of the reproducibility of the Sandwich ELISA The simultaneous and intermittent reproducibility of sandwich ELISA was investigated using purified Cha o 2 prepared at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 50 ng / mL as a calibration curve, and purified Cha o 2 prepared at concentrations of 2, 4, 10, 20, and 50 ng / mL as samples. Calibration curves were prepared for each experiment, and the same sample stored at -20°C was used. Dilution of the calibration curve and samples was performed with 1% BSA / PBS-T, and sandwich ELISA was performed in the same manner as described in section 1.6.8. Simultaneous reproducibility was measured using the coefficient of variation within the same measurement when nine copies were made, and intermittent reproducibility was measured using the coefficient of variation between measurements when the same sample was measured three times on different days using triple copies.

[0066] 1.6.10 Determination of Cha o 2 in pollen extract Hinoki pollen extract was diluted 27-fold to a 3-fold common ratio and analyzed by sandwich ELISA. The sandwich ELISA was performed using the same method as described in section 1.6.8.

[0067] 1.6.11 Cross-reactivity with cedar pollen extract Cypress pollen extract was diluted 27 to 59049 times, and cedar pollen extract was diluted 7.49 to 1820 times, and measurements were performed using sandwich ELISA. Sandwich ELISA was performed using the same method as described in section 1.6.8.

[0068] 2 [Results] 2.1 Production of anti-Chao2 monoclonal antibody-producing hybridomas Eleven hybridoma clones were obtained as anti-Chao2 monoclonal antibody-producing hybridomas: "2D6-2C3", "9A11-1F8", "3A8-2F2", "4H5-3C3", "8G10-1H7", "4G6-1D6", "9E12-2B7", "4F6-4B11", "5A8-4B2", "3E1-1C8", and "6H5-2F2".

[0069] 2.2 Examination of antibody combinations using Sandwich ELISA We investigated antibody combinations that could detect Cha o 2 using sandwich ELISA with the 11 types of anti-Cha o 2 monoclonal antibodies we prepared (Figure 1). After examining 110 combinations of monoclonal antibodies, it was found that 12 combinations using the seven types of anti-Cha o 2 antibodies we prepared—3A8-2F2, 3E1-1C8, 4G6-1D6, 4H5-3C3, 5A8-4B2, 6H5-2F2, and 9A11-1F8—could detect Cha o 2 with high sensitivity, achieving a signal-to-noise ratio of 10 or higher.

[0070] 2.3 Identification of Epitope For anti-Cha o 2 antibodies 3A8-2F2, 3E1-1C8, 4G6-1D6, 4H5-3C3, 5A8-4B2, 6H5-2F2, and 9A11-1F8, which were capable of highly sensitive detection of Cha o 2 by sandwich ELISA, two clones (4H5-3C3 and 9A11-1F8) that did not contain the epitopes of the 13 polymorphisms reported (Tateno M. et al., PLoS ONE 16, e0261327 (2021)) were selected (Figure 2). Anti-Cha o 2 antibody 4H5-3C3 contains peptide No. 17( 113 WWAGQCKVVNGRTVCNDRNR 132 (Sequence ID 3)) was recognized. Anti-Cha o 2 antibody 9A11-1F8 is peptide No. 42( 288 NPILINQFYCTSASACQNQR 307 (Sequence No. 4) and No. 43 ( 295 FYCTSASACQNQRSAVQIQG 314 The antibody (SEQ ID NO: 5) recognized Cha o 2 and did not react with other peptides (Figure 2). Since the anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8 do not contain amino acid polymorphisms in their epitopes, it is thought that they can recognize Cha o 2 without amino acid substitution.

[0071] 2.4 Indirect ELISA As a method of utilizing the selected anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8, we investigated the detection of Cha o 2 using indirect ELISA. Anti-Cha o 2 antibody 4H5-3C3 showed reactivity to Cha o 2, the major allergen of cypress pollen, but not to Cry j 2, the major allergen of cedar pollen. Anti-Cha o 2 antibody 9A11-1F8 showed comparable reactivity to both Cha o 2, the major allergen of cypress pollen, and Cry j 2, the major allergen of cedar pollen. It was found that both anti-Cha o 2 antibodies 9A11-1F8 and 4H5-3C3 can be used for the detection of Cha o 2 using indirect ELISA (Figure 4). The anti-Chao2 antibody 4H5-3C3 was found to be usable for the specific detection of Chao2 in indirect ELISA (Figure 4).

[0072] 2.5 Western blotting As a method of utilizing the anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8, we investigated the detection of Cha o 2 by Western blotting. Anti-Cha o 2 antibody 4H5-3C3 showed reaction only with purified Cha o 2 by Western blotting (Figure 5-1). Furthermore, it showed no reactivity to Cry j 2, which has high homology, revealing its high specificity. Anti-Cha o 2 antibody 9A11-1F8 showed comparable reactivity to both purified Cha o 2 and Cry j 2, a major allergen of Japanese cedar pollen, by Western blotting (Figure 5-1). Protein staining of cypress pollen extract revealed the presence of numerous proteins other than Cha o 2. However, the anti-Cha o 2 antibodies 9A11-1F8 and 4H5-3C3 did not react to proteins other than Cha o 2, indicating their potential use in the specific detection of Cha o 2 in cypress pollen extract by Western blotting (Figure 5-2).

[0073] When the epitope sequences of the anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8 were compared on the homologous amino acid sequence of Cry j 2, it was found that the epitope sequence of 4H5-3C3 showed low conservation between Cha o 2 and Cry j 2 (Figure 6). On the other hand, the epitope sequence of 9A11-1F8 was almost identical between Cha o 2 and Cry j 2. This is consistent with the fact that the anti-Cha o 2 monoclonal antibody 9A11-1F8 was able to recognize Cha o 2 and Cry j 2 with equivalent intensity in indirect ELISA and Western blotting, and that the anti-Cha o 2 monoclonal antibody 4H5-3C3 was unable to recognize Cry j 2 in indirect ELISA and Western blotting.

[0074] 2.6 Sandwich ELISA The reactivity of the anti-Chao2 antibodies 4H5-3C3 and 9A11-1F8 was investigated using sandwich ELISA. In the prepared sandwich ELISA, a Chao2 concentration-dependent increase in signal was observed (Figure 7), with R occurring in the range of 0.313 to 50 ng / mL. 2 A four-parameter logistic regression with a response of =1 was generated (Figure 8). On the other hand, Cha o 1 and Cry j 2 showed no response (Figure 7).

[0075] 2.7 Examination of the reproducibility of the Sandwich ELISA Using concentrations of 0.625–50 ng / mL, which were within the quantitative range, as a calibration curve, the reproducibility of content measurements of purified Cha o 2 at concentrations of 2–50 ng / mL was investigated. As a result, the coefficient of variation for simultaneous reproducibility was 3.0%–7.3%, and the coefficient of variation for day-to-day reproducibility was 7.1%–14.8%, showing good values. This revealed that the sandwich ELISA using anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8 is a highly reproducible Cha o 2 quantitative system (Table 1). [Table 1]

[0076] 2.8 Determination of Cha o 2 in pollen extract We investigated whether sandwich ELISA using anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8 could be used for the quantitative determination of Cha o 2 in pollen extracts containing many contaminants. When cypress pollen extract was diluted 729, 2187, and 6561 times, the absorbances obtained were within the calibration curve range (0.625-50 ng / mL). Estimating the Cha o 2 concentrations in the cypress pollen extract from these absorbances yielded values ​​of 6.44, 6.55, and 6.40 μg / mL, respectively, with almost identical concentrations obtained (CV 1.2%). From this, it was found that sandwich ELISA using anti-Cha o 2 antibodies 4H5-3C3 and 9A11-1F8 is a quantitative system that is unaffected by contaminants regardless of the dilution ratio, and can measure Cha o 2 in pollen extracts.

[0077] 2.9 Cross-reactivity with cedar pollen extract We investigated whether the constructed ELISA cross-referenced with cedar pollen extract. There was a sensitivity difference of more than 86 times compared to cypress pollen extract (Figure 9). From this, it became clear that the sandwich ELISA using anti-Cha o 2 antibodies 9A11-1F8 and 4H5-3C3 has high specificity and is a measurement system that can measure only the Cha o 2 content even when cedar pollen is mixed in with cypress pollen.

[0078] 2.10 Comparison with existing technologies Furthermore, the "Cha o 2 polyclonal antibody" sold by Agresera is polyclonal, so its affinity may change if the epitope contains polymorphisms. In addition, polyclonal antibodies are limited in terms of production volume. In contrast, the monoclonal antibody of the present invention has high specificity, can detect Cha o 2 polymorphisms, and can be produced indefinitely. [Sequence Listing Free Text]

[0079] Sequence IDs 2-6: Synthetic peptides

Claims

1. A monoclonal antibody against the cypress pollen allergen Cha o 2, wherein the monoclonal antibody recognizes any of the amino acid sequences shown in SEQ ID NOs. 3 to 6 as an epitope and does not react with Cha o 1.

2. A monoclonal antibody according to claim 1 that does not react with the cedar pollen allergen Cry j 2.

3. A monoclonal antibody according to claim 1, which reacts with the cedar pollen allergen Cry j 2.

4. The monoclonal antibody according to claim 3, wherein the minimum detection concentration of Cha o 2 when combined with the monoclonal antibody according to claim 2 is 5 ng / mL or less.

5. The monoclonal antibody according to claim 2, wherein the minimum detection concentration of Cha o 2 when combined with the monoclonal antibody according to claim 3 is 5 ng / mL or less.

6. A monoclonal antibody according to claim 1, having reproducibility with a coefficient of variation of 20% or less.

7. An antibody that binds to an epitope to which the monoclonal antibody described in claim 1 binds.

8. A monoclonal antibody according to claim 1, or an antigen-binding fragment of an antibody according to claim 7.

9. A hybridoma that produces the monoclonal antibody described in claim 1.

10. A method for detecting Cha o 2, characterized by reacting a sample with a monoclonal antibody according to claim 1 or an antigen-binding fragment of an antibody according to claim 7.

11. A kit for detecting Cha o 2, comprising the monoclonal antibody described in claim 1, or an antigen-binding fragment of the antibody described in claim 7.

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