JP6982039B2 - Monoclonal antibody against cypress pollen antigen and its use - Google Patents

Description

The present invention relates to a monoclonal antibody against Cha o 1, which is a major allergen of cypress pollen.

Hinoki pollen is considered to be one of the causative substances that cause pollinosis symptoms, and Cha o 1 is known as a major allergen of hinoki pollen (Patent Document 1, Non-Patent Document 1). As other hinoki pollen allergens, Cha o 2 (Non-Patent Documents 2 and 3) and, in recent years, Cha o 3 (Patent Document 2 and Non-Patent Document 4) have been reported.

It is known that about one-third of the Japanese people have cedar pollinosis. It has been reported that hinoki pollen has common antigenicity with cedar pollen (Non-Patent Document 5), and 76.4% of patients with spring pollinosis are positive for both cedar pollen and hinoki pollen (non-patent). Patent Document 6), Japanese cedar pollen is considered to be one of the causative allergens that cause pollinosis seen in spring.

Antigen-specific monoclonal antibodies are useful for the analysis and research of pollen allergens in patients with hay fever, and enable highly sensitive detection and quantification of antigens. For example, for a monoclonal antibody against cedar pollen antigen Cry j 1 or Cry j 2 (Patent Document 3) and a monoclonal antibody against the mite antigen Der f 1 (Patent Document 4), the antibody, hybridoma, use and the like have already been described. .. On the other hand, there are no reports of fully verified antibodies against cypress pollen allergens or their uses. The only detection system that can be used on the market is the "ELISA kit for measuring hinoki pollen antigen" using Cha o 1 monoclonal antibody (Nichinichi Pharmaceutical Co., Ltd.), but at present, it is limited to limited sales as a prototype. The accuracy has not been determined, and the measurement range (25-1,600 ng / mL) is a tentative setting, so it cannot be said to be a reliable measurement system (Non-Patent Document 7).

Japanese Unexamined Patent Publication No. 8-176192 International release WO2012 / 105541 Japanese Unexamined Patent Publication No. 2005-289929 Japanese Unexamined Patent Publication No. 9-87298

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. 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 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 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 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. Okano et al., Allergy, 43, 1179-1184 (1994): Clinical study of hinoki-specific IgE antibody-positive patients: using the hinoki AlaSTAT test Kanto Chemical Co., Ltd., Nichinichi Pharmaceutical Co., Ltd. Reagent ELISA kit guide for measuring environmental allergens, 201604

There has been a demand for stable production of a monoclonal antibody against hinoki cypress pollen antigen, analysis of hinoki cypress pollen antigen using the monoclonal antibody, and development of a quantitative system.

As a result of diligent studies to solve the above problems, the present inventor has produced a monoclonal antibody that is specific to Cha o 1 and recognizes a specific site, and has completed the present invention.

That is, the present invention is as follows.
(1) The monoclonal antibody that is a monoclonal antibody against the hinoki pollen allergen Cha o 1 and does not react with Cha o 2 and the cedar pollen allergen Cry j 1.
(2) The monoclonal antibody according to (1), which recognizes all or part of the amino acid sequence represented by MRNVTDVWIDHNS as an epitope among the amino acid sequences of Cha o 1.
(3) The monoclonal antibody according to (1) or (2), wherein the minimum detection concentration of Cha o 1 is 5 ng / mL or less.
(4) The monoclonal antibody according to any one of (1) to (3), which has a coefficient of variation of 10% or less and has a reproducibility.
(5) An antibody that binds to an epitope to which the monoclonal antibody according to any one of (1) to (4) binds.
(6) The monoclonal antibody according to any one of (1) to (4), or the fragment of the antibody according to (5).
(7) A hybridoma that produces the monoclonal antibody according to any one of (1) to (4).
(8) A method for detecting Cha o 1 comprising reacting a sample with the antibody or fragment thereof according to any one of (1) to (6) to detect Cha o 1.
(9) A kit for detecting Cha o 1 containing the antibody or fragment thereof according to any one of (1) to (6).

The present invention solves the problems of antigen identification and quantitative system by using a monoclonal antibody that is specific to Cha o 1 and recognizes a specific site, and immunologically increases Cha o 1 in hinoki pollen extract. It has become possible to develop a test system that can be identified and quantified easily and sensitively.

It is a figure which shows the detectability of Cha o 1 by the combination of a capture antibody and a biotinylated detection antibody. It is a figure which shows the epitope mapping. It is a figure which shows the reactivity of the antibody in Indirect ELISA. It is a figure which shows the specificity of the anti-Cha o 1 antibody 3G12-A11 in Western blotting. It is a figure which shows the specificity of the detection of the antigen of Sandwich ELISA. It is a figure which shows the concentration range (1.25 to 50 ng / mL) which shows linearity in Sandwich ELISA. It is a figure which shows the crossing property to the cedar pollen extract. It is a figure which shows the comparison of the detection with the existing technology in Cha o 1 refined product.

The present invention relates to a plurality of monoclonal antibodies against the major allergen Cha o 1 of cypress pollen. In the present invention, a plurality of monoclonal antibodies against the hinoki cypress antigen were newly prepared, and their epitopes were successfully identified. In addition, by using these monoclonal antibodies, we have developed a highly sensitive and accurate detection method that is specific to Cha o 1 in hinoki cypress pollen extract.

In the study of hinoki pollen antigen, there is generally no verified specific measurement method, such as a test system using a polyclonal antibody or a test system using a monoclonal antibody against sugi antigen using the homology of the antigen. It is used. In the case of a polyclonal antibody against a cypress antigen, the epitope of the detected antigen cannot be determined, and it is generally inferior to a monoclonal antibody in terms of specificity. When a monoclonal antibody against Sugi antigen, which has homology to Hinoki antigen, is used, since Sugi antigen is also recognized, there are certain restrictions on the range of samples that can be measured and the interpretation of the results.
The Cha o 1 specific monoclonal antibody produced by the present invention has a feature of recognizing the hinoki pollen antigen Cha o 1 in a sample with high sensitivity, and is immunologically measured by Western blotting method, ELISA method, Sandwich ELISA method, etc. Since it can be widely applied to the method, it has a wide range of advantages and usefulness such as use in reagents and kits.

1. 1. Monochrome antibody of the present invention As a method for obtaining the monoclonal antibody of the present invention (also referred to as "anti-Cha o 1 antibody"), first, a non-human mammal is prepared using a Cha o 1 antigen protein or a partial peptide of the Cha o 1 protein. Immunize and collect antibody-producing cells (eg, B cells) from immune animals. The antibody-producing cells and myeloma cells are fused to prepare a hybridoma (fusion cell line). Then, by collecting the antibody produced from this hybridoma, the desired monoclonal antibody is obtained.

(1) Preparation of antigen In the present invention, the full-length protein of Cha o 1 is used as an immune antigen. The full-length protein of Cha o 1 can be obtained from the extract of cypress pollen by combining various purification methods by column chromatography. Further, in the present invention, a partial peptide can be synthesized and used as an immune antigen.

The basic amino acid sequence for synthesizing the antigenic peptide is a partial sequence of the full-length amino acid sequence of Cha o 1, and 10 to 50, preferably 10 to 30, and more preferably 15 are arbitrarily continuous from the amino acid sequence. Select an amino acid sequence of ~ 20 lengths.
The peptide that can be used as an antigen is the peptide in the 148th to 167th region of the full-length sequence of Cha o 1 (SEQ ID NO: 1) or the full-length amino acid sequence of Cha o 1 (SEQ ID NO: 2), or the 155th. It is a peptide in the region from the second to the 174th (SEQ ID NO: 3). Further, among the peptides in the above region, the 155th to 167th peptides having a common amino acid sequence (SEQ ID NO: 4) can also be used.

Partially synthesized peptides are small molecules, and it is difficult to obtain antibodies even if they are immunized with mice as they are. Therefore, a synthetic peptide and a carrier protein are disulfide-bonded to prepare an immune antigen.
The chemical synthesis of the peptide can be carried out by a method known to those skilled in the art, such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
Examples of the carrier protein include BSA (Bovin serum albumin), KLH (Keyhole Limpet Hemocyanin), OVA (Ovalbumin) and the like.

(2) Immunity of animals The type of non-human mammal to be immunized is not particularly limited, and examples thereof include mice, rats, guinea pigs, rabbits, dogs, goats and the like, and mice are preferable.
The total dose of antigen per animal is 10-150 μg. When immunizing an antigen, it is common to mix an adjuvant and an antigen solution, and examples of the type of adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, intramuscularly, subcutaneously into the footpad, etc. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks.

(3) Preparation of antibody-producing cells The antibody-producing cells are prepared from immunized non-human mammalian spleen cells or the like or regional lymph nodes. Examples of the lymph node include an iliac lymph node and an inguinal lymph node. Although it is not necessary to perform an operation for separating antibody-producing cells from the collected cell population, it is preferable to separate only antibody-producing cells from the cell population. However, when preparing antibody-producing cells, it is preferable to remove tissue debris and erythrocytes as much as possible.

(4) Cell fusion Cell fusion is a work performed to fuse the above antibody-producing cells with myeloma cells (myeloma cells) to produce cells (hybridoma) that continue to grow semi-permanently while producing antibodies. .. Those skilled in the art can fuse antibody-producing cells with myeloma cells using known cell fusion methods. Generally available cell lines of animals such as mice can be used as myeloma cells to be fused with antibody-producing cells. The cell line to be used is preferably one that cannot survive in a HAT selective medium (medium containing hypoxanthine, thymidine, and aminopterin) and can survive only in a state of being fused with antibody-producing cells. Examples of myeloma cells include SP 2/0, P3U1, NSI, P3-X63-Ag8.653 and the like.

Cell fusion is a commonly available medium such as DMEM or RPMI1640 medium that does not contain fetal bovine serum (FCS), etc., and is 1 × 10 ⁶ to 1 × 10 ⁷ cells / mL with spleen cells or lymph node cells 1 × 10 ⁵ to 1 × 10 ⁶ cells / mL of myeloma cells are mixed and fused in the presence of a cell fusion promoter. The cell ratio of spleen cells or lymph node cells to myeloma cells (spleen cells or lymph node cells: myeloma cells) is preferably 5: 1. As a cell fusion promoter, polyethylene glycol having an average molecular weight of 200 to 20000 daltons can be used.

Further, it can be fused by using a commercially available cell fusion device using electrical stimulation (for example, electroporation) or by using Sendai virus.
After fusion, dilute the cells in a HAT medium prepared with, for example, RPMI1640 medium containing 10 to 20% FCS, then seed 0.5 to 3 x 10 ⁵ cells in each well of a 96-well culture plate and culture in _{a CO 2 incubator.} do.

(5) Establishment of hybridomas Next, hybridomas that produce the desired antibody are selected from the cells after the cell fusion treatment. After 10-14 days of cell fusion, selected cells in HAT medium form colonies. The culture supernatant of each well of the colony-positive 96-well culture plate is collected to confirm the antibody titer against Cha o 1. As a confirmation method, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or the like is used. When a conjugate of a partial peptide and a carrier protein is used as an immune antigen, the antibody produced from the cells also includes an antibody against the carrier proteins KLH and BSA. Therefore, by measuring the antibody titer against KLH etc., KLH etc. KLH antibody-positive wells with high antibody titers to KLH antibody can be excluded.

After confirming antibody production positive wells for Cha o 1, transfer the cells to a 24-well or 12-well culture plate. Here, it is preferable to replace the medium with an HT medium from which aminopterin has been removed. The HT medium is used as a recovery medium for hybridomas that continue to supply precursors of purines and pyrimidines to the salvage pathway during the influence of aminopterin remaining in the cells. After culturing in HT medium for a while, check the antibody titer in the culture supernatant again.

As described above, in the present invention, it is necessary to obtain a hybridoma that does not cross-react with Cry j 1 and has high specificity for Cha o 1. Therefore, the cross-reactivity with Cry j 1 is confirmed by ELISA, RIA, etc. at the culture supernatant level.

The cells in the final selected well are cloned to form a single cell. For cloning, for example, the cell suspension is appropriately diluted with 10 to 20% FCS-containing (preferably 20%) RPMI1640 medium, and then the cells are seeded so that 0.3 to 2 cells are contained in each well of the 96-well culture plate. .. The number of cells to be placed in each well of the 96-well culture plate is preferably seeded so that one cell is placed in each well so that the probability of one cell in one well is high. Collect the culture supernatant of the colony-positive well 7 to 10 days after cell seeding. At this time, it is preferable to confirm that the colony is a single colony after 3 to 5 days. The antibody titer of the collected culture supernatant is confirmed. Again, select clones that have high specificity for Cha o 1 and low cross-reactivity with Cry j 1. Furthermore, the cells of the selected wells are increased to some extent to establish a hybridoma strain. Cloning may be performed several times as needed.
Five clones were obtained as hybridomas established in the present invention and named "2H4-F3", "3G12-A11", "4E11-F7", "5H3-F5" and "7B1-C5".

(6) Preparation of monoclonal antibody From the established hybridoma strain, a Cha o 1 specific monoclonal antibody is purified and collected by the following method. That is, a method of preparing an antibody from a culture supernatant cultured in a medium in which the serum concentration is suppressed, a method of preparing an antibody from a culture supernatant cultured in a commercially available serum-free medium, and a method of injecting a hybrid doma into the ascites of an animal. , There is a method of collecting ascites and preparing an antibody from the ascites. The culture supernatant is prepared from 0.1 to 4 x 10 ⁵ cells / mL and collected from the cells cultured for 1 to 2 weeks. ^{In the case of ascites, 0.1 to 1 × 10 7} hybridomas are intraperitoneally administered to the abdominal cavity of mammals derived from myeloma cells and homologous animals to proliferate a large amount of hybridomas. Then, collect ascites after 1 to 2 weeks.

Examples of the culture method include a method using a culture flask, a method using a spinner flask, a method using a shaker flask, and a method using a bioreactor. The antibody can be purified by a protein G affinity column or a protein A affinity column, a Cha o 1 affinity column, a sulfur salting out fraction by gel filtration chromatography, or ion exchange chromatography. There are methods for purification and the like, and these known methods can be appropriately selected or combined for purification.

2. 2. Epitope of anti-Cha o 1 antibody The epitope (antigen determinant) of the anti-Cha o 1 antibody of the present invention is not limited as long as it is at least a part of the antigen Cha o 1, but is shown in, for example, SEQ ID NO: 1. Of the full-length amino acid sequence of Cha o 1, the region consisting of the 148th to 167th amino acid sequence: QDGDAITMRNVTDVWIDHNS (SEQ ID NO: 2) or the 155th to 174th amino acid sequence: MRNVTDVWIDHNSLSDSSDG (SEQ ID NO: 3). It is preferably at least a part. Among them, a region consisting of the 155th to 167th amino acid sequences: MRNVTDVWIDHNS (SEQ ID NO: 4) or at least a part of the region is more preferable. An anti-Cha o 1 antibody that recognizes the region (binds to the region) is Cha o 1 specific and has high detection sensitivity, and is extremely useful for detecting Cha o 1.

Further, the antibody against Cha o 1 is not limited to the monoclonal antibody itself against Cha o 1 produced by the above hybridoma, and as long as it binds to the epitope recognized by the monoclonal antibody produced by these hybridomas, the anti-Cha o of the present invention. Included in 1 antibody. The term "epitope" as used herein refers to an epitope recognized by the monoclonal antibody produced by the hybridoma. This epitope may be a part of these regions as well as the amino acid residues 148th to 167th or 155th to 174th of the amino acid sequence of Cha o 1. Furthermore, in addition to the amino acid residues 155th to 167th in the region of Cha o 1, it may be a part of these regions.

3. 3. Recombinant antibody The monoclonal antibody of the present invention can also be a recombinant antibody or an antigen-binding fragment prepared and expressed by recombinant means. For example, the anti-Cha o 1 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 heavy chain and light chain.
To express a recombinant antibody, for example, a recombinant expression vector having a nucleic acid encoding a heavy chain and a light chain of the antibody is introduced into a host cell, and the host cell into which the vector has been introduced is cultured. Then, the target antibody is recovered from the culture of the host cell. To obtain the heavy and light chain genes of an antibody, integrate these nucleic acids into an expression vector, and introduce them into a host cell, a standard recombinant DNA method in the art (Sambroock et al., Molecular Cloning: A). Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012) can be adopted.

For example, in the case of a mouse-human chimeric antibody, an antibody gene is isolated from mouse cells that produce an antibody against the Cha o 1 protein, and the heavy chain (H chain) constant region and the light chain (L chain) constant region are respectively human. It can be prepared by recombining with the H chain constant region gene of IgG and the L chain constant region gene of human IgG and introducing them into mouse myeloma cells.
Nucleic acids encoding heavy chain variable regions (VHs) (eg, DNA) are constructed into full-length heavy chain genes by expressively binding to DNA encoding VHs and DNA encoding heavy chain constant regions. In addition, the nucleic acid encoding the light chain variable region (VL) (eg, DNA) is constructed into a full-length light chain gene by expressively binding to the DNA encoding VL and the DNA encoding the light chain constant region. Let me.
If the nucleic acid encoding the full-length light chain VH and VL is, for example, a mouse-derived nucleic acid, and the nucleic acid encoding the heavy chain and light chain constant region is a human-derived nucleic acid, a mouse-human chimeric antibody or antigen. It becomes a binding fragment. Nucleic acid sequences in heavy chain constant regions derived from humans, mice and the like are known in the art.

When producing a humanized antibody, a so-called CDR graphing (CDR transplantation) method can be adopted. CDR graphing involves transplanting complementarity determining regions (CDRs) from variable regions of a mouse antibody into human variable regions, where the framework regions (FR) are of human origin and the CDRs are of mouse origin. It is a method of producing a configured variable region. An antibody fragment (peptide) containing a CDR comprises at least one region of a CDR (CDR1-3) of VH or VL. Antibody fragments containing multiple CDRs can be bound directly or via a suitable peptide linker.

These humanized reconstructed human variable regions are then linked to human constant regions. Methods for producing such humanized antibodies are well known in the art (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)).

Human antibodies (fully human antibodies) generally have the same structure as human antibodies in the structure of the hypervariable region, which is the antigen binding site of the variable region (V region), other parts of the V region, and the constant region. It has. Techniques for producing human antibodies are also known, and methods for producing gene sequences common to humans have been established by genetic engineering techniques. The human antibody is, for example, a method using a human antibody-producing mouse having a human chromosomal fragment containing the H chain and L chain genes of the human antibody (Tomizuka, K. et al., Nature Genetics, (1977) 16, 133- 143; Kuroiwa, Y.et.al., Nuc. Acids Res., (1998) 26, 3447-3448, etc.) and methods for obtaining human antibodies derived from phage display selected from the human antibody library (Wormstone, IM). et.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 Etc.) can be obtained.

Further, in the present invention, a chimeric antibody, a humanized antibody, a humanized antibody can be produced by using a hybridoma or DNA or RNA extracted from the hybridoma as a raw material according to the above-mentioned well-known method.

4. Preparation of Antibody Fragment Fragment of antibody against Cha o 1 used in the present invention means a region of a part of the antibody of the present invention, for example, Fab, Fab', F (ab') 2, Fv, diabody (dibodies). ), DsFv, scFv (single chain Fv), etc. The antibody fragment can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes according to the purpose.
For example, Fab can be obtained by treating the antibody molecule with papain, and F (ab') 2 can be obtained by treating the antibody molecule with pepsin. Further, Fab'can be obtained by cleaving the disulfide bond in the hinge region of F (ab') 2 described above.

In the case of scFv, the cDNA encoding VH and VL of the antibody is obtained, and the DNA encoding scFv is constructed. ScFv can be produced by inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression.

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

5. Detection method and detection kit
Since Cha o 1 is an allergen for hinoki cypress pollinosis, Cha o 1 can be detected by reacting the anti-Cha o 1 antibody of the present invention with a sample and measuring Cha o 1 in the sample.
Therefore, the present invention provides a method for detecting Cha o 1, which comprises reacting an antibody of the present invention or a fragment thereof with Cha o 1 to detect Cha o 1.
Further, in the present invention, the anti-Cha o 1 antibody can be used as a reagent or kit for detecting Cha o 1.

When the anti-Cha o 1 antibody of the present invention is used as a kit, the anti-Cha o 1 antibody can be combined with other solvents or solutes to form a composition. For example, distilled water, pH buffering reagents, salts, proteins, surfactants and the like can be combined. In addition, the anti-Cha o 1 antibody can be labeled with an enzyme or biotin and used. As the labeling enzyme, in addition to HRP (horseradish peroxidase), alkaline phosphatase, malic acid dehydrating enzyme, α-glucosidase, β-galactosidase, colloidal gold and the like can be used.

When the present invention is used in a kit, in addition to the antibody of the present invention, the above solvent, solute, enzyme labeling reagent, antigen-immobilized microplate, antibody diluted solution, OPD (orthophenylenediamine) tablet, substrate solution, etc. Reaction stop solution, concentrated cleaning solution, instruction manual, etc. can be included. In addition, a reaction medium such as a buffer solution that provides optimum conditions for the reaction, a buffer solution that is useful for stabilizing the reaction product, and a stabilizer for the reaction substance may also be included in the kit of the present invention.

The anti-Cha o 1 antibody and kit of the present invention can measure Cha o 1 specifically and with high sensitivity, but can also achieve sufficient measurement accuracy. Therefore, the anti-Cha o 1 antibody and kit of the present invention can be used for quality evaluation of hinoki pollen extract used in clinical practice such as allergen immunotherapy and antibody titer measurement against hinoki pollen allergen in human serum. The measurement accuracy is an index showing how much each measured value varies when the same sample is divided into a plurality of test tubes or wells and one assay is performed, and is statistically. Is expressed as the Coefficient of variation (CV), that is, the ratio (%) of the standard deviation to the average value. In the present invention, this coefficient of variation (CV) is referred to as reproducibility. The reproducibility is 15% or less, preferably 10% or less, and more preferably 5% or less.

The minimum detection concentration of the kit of the present invention is 5 ng / mL or less, for example, 1 to 3 ng / mL. The simultaneous reproducibility is 10% or less, preferably around 5%, and the daily reproducibility is 10% or less, preferably about 8 to 10%. Both reproducibility is CV = 10% or less.

Examples Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

1 Materials and methods
1.1 Material
1.2 Reagent Hinoki pollen: Yamizo pollen study group or Torii Pharmaceutical Sugi pollen: Torii Pharmaceutical
HiTrap Butyl S FF: GE Healthcare
HiTrap Phenyl HP: GE Healthcare
Superdex 75 10/300 GL: GE Healthcare
ADJUVANT COMPLETE FREUND: DIFCO
Hybridoma-SFM: GIBCO
י3: Fujifilm Wako Pure Chemical Industries
HCl: Wako Pure Chemical Industries, Ltd.
NaOH: Wako Pure Chemical Industries, Ltd.
FBS: Biological Industry
Recombinant human IL-6: R & D Systems
D-MEM: Fuji Film Wako Pure Chemical Industries
Hypoxanthine: SIGMA-ALDRICH
Aminopterin: SIGMA-ALDRICH
Thymidine: Wako Pure Chemical Industries, Ltd.
PEG 1500: Roche
Penicillin-Streptomycin-Amphotericin B Suspension: Fujifilm Wako Pure Chemical Industries
Buffer Kit: Protenova Ammonium Sulfate: Fujifilm Wako Pure Chemical Industries, Ltd.
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: Junsei Chemical
BSA: Sigma-Aldrich
Tris-Glycine Sample Buffer (2X): TEFCO
DTT (1,4-Dithiothreitol): Junsei Chemical
Bullet PAGE One Precast Gel, 5-15% (17 well): Nacalai Tesque migration buffer: Fujifilm Wako Pure Chemical Industries, Ltd.
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: Genuine chemical anti-Cry j 1 antibody (2G2, 6D7 and 7A6): Torii Pharmaceutical Hinoki pollen antigen (Cha o 1) measurement kit: Nichinichi Pharmaceutical

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

1.4 Tissue solution
10% FBS, Hybridoma-SFM medium
To Hybridoma-SFM, 50 mL of deactivated FBS, 5 mL of penicillin-streptomycin-amphotericin B suspension, and 50 μL of 10 μg / mL IL-6 were added to make the total volume 500 mL.

Serum-free medium
To Hybridoma-SFM, 5 mL of penicillin-streptomycin-amphotericin B suspension and 50 μL of 10 μg / mL IL-6 were added to make the total volume 500 mL.

HAT medium
Hypoxanthine and Thymidine dissolved in water and Aminopterin dissolved in water and 1M NaOH were added to Hybridoma-SFM medium. At that time, 10% FBS and IL-6 (1 ng / mL) were added.

PBS-T
10 × PBS was diluted 10-fold to adjust Tween 20 to a final concentration of 0.05%.

1% BSA / PBS-T
BSA was prepared with PBS-T to a final concentration of 10% and stored at -20 ° C. It was dissolved before use and diluted 10-fold with PBS-T.
NH4HCO3 and NaCl were added to the extract water, and the final concentrations were adjusted to 40 mM and 0.5 M, respectively.

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
Bio-Image Analyzer: GE Healthcare, Image Quant LAS 4010
Transfer device (semi-dry type): BIO-RAD
Sample boil: Heat block Dry Thermo Unit (DTU-2B)

1.6 Method
1.6.1 Purification of Cha o 1 from hinoki cypress pollen 20 times the amount of 40 mM NH4HCO3 was added to hinoki cypress pollen, and the mixture was stirred at 4 ° C for 3 hours to extract Cha o 1. After stirring, ammonium sulfate was added to the supernatant which was centrifuged at 20,000 × g for 30 minutes so as to have 20% saturated ammonium sulfate, and the mixture was stirred overnight at 4 ° C. The supernatant, which was centrifuged at 20,000 × g for 30 minutes, was buffer-exchanged with 1.5 M ammonium sulfate and 50 mM Tris-hydrochloric acid buffer (pH 7.8) by dialysis. After dialysis, it was applied to a Butyl S FF column, and the obtained non-adsorbed fraction was further applied to a Phenyl HP column. A 1.5 M ammonium sulfate, 50 mM Tris-hydrochloric acid buffer (pH 7.8) was eluted with a gradient to 50 mM Tris-hydrochloric acid buffer (pH 7.8), and one Cha o fraction was recovered. The collected fraction was dialyzed against PBS, concentrated, and purified on a Superdex 75 10/300 GL column. The recovered fraction was purified Cha o 1 and stored at -20 ° C until use.

1.6.2 Preparation of anti-Cha o 1 monoclonal antibody-producing hybridoma
1.6.2.1 Preparation of immunity and hybridoma Anti-Cha o 1 monoclonal antibody was prepared by the mouse iliac lymph node method (Japanese Patent Laid-Open No. 2007-020547).
Cha o 1 purified from cypress pollen and Freund's Complete Adjuvant (FCA) were mixed at a volume ratio of 1: 2, and the ridge muscles of B6D2F1 / Slc mice (Japan SLC Co., Ltd., 8 weeks old at the time of arrival, 5 females) A dose of Cha o 1 was administered at 27 μg per animal. Seventeen days after the initial administration, Cha o 1 was administered at 16 μg per animal as booster immunity. Twenty-one days after the initial administration, the iliac lymph nodes were removed and used for cell fusion with mouse myeloma-derived cells (myeloma cells).

1.6.2.2 Cell fusion and antibody production hybridoma screening Suspend the iliac lymph nodes of mice that have been finely sterile in DMEM medium, and have a 5: 1 cell number (lymphocytes: myeloma) with myeloma cells (SP2 / W). The cells were fused so as to be as follows, and cell fusion was carried out by a conventional method using PEG. The cells were resuspended in HAT medium so that the number of lymphocytes was 0.9975 × 10 ⁶ cells / mL, and 100 μL / well was seeded on eight 96-well plates. After culturing at 5% CO2 at 37 ° C for 6 days, a part of the culture supernatant was collected and screened by ELISA and Western blotting. Wells that were reactive with Cha o 1 were selected, and monoclonal antibody-producing hybridomas were cloned by the limiting dilution method.

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

1.6.3 Selection of antibody combinations by Sandwich ELISA
50 μL of purified monoclonal antibody solution prepared to 20 μg / mL was added to each well of a 96-well microplate and left overnight at 25 ° C. After washing, the cells were blocked with 1% BSA / PBS-T solution, 50 μL of 100 μg / mL purified Cha o 1 solution was added, and the mixture was left at 25 ° C. for 1 hour. After washing with PBS-T, 50 μL of biotinylated purified monoclonal antibody solution (1 μg / ml) was added, and the mixture was reacted at 25 ° C. for 1 hour. After the reaction, the cells were washed with PBS-T and then reacted with avidin-HRP. After washing with PBS-T, TMB as a substrate was added and reacted at 25 ° C. for 15 minutes. After that, the reaction was stopped with 2N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

1.6.4 Identification of epitope
Amino acid sequence ( ¹ D to ³⁵⁴ S) ^{excluding the signal sequence (-21} M to ^-1 S) from the full-length amino acid sequence of Cha o 1 (UniProt No. Q96385: SEQ ID NO: 1) A 20-residue overlapping peptide library was prepared (SIGMA-ALDRICH, PEPscreen) in which 7 residues overlap for amino acid residues in single-letter notation.). Each Cha o 1 partial sequence peptide was prepared to 8 to 30 μM, 100 μL of the solution was added to a 96-well microplate, and the solution was allowed to stand overnight at room temperature. After washing, it was blocked with 1% BSA / PBS-T solution. 100 μL of a 2000-fold diluted purified monoclonal antibody solution was added to each well of the blocked plate, and the mixture was reacted at room temperature 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 15 minutes, the reaction was stopped with 2N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

1.6.5 Examination of reactivity by Indirect ELISA
Cha o 1 and Cha o 2 were prepared at 2 μg / mL each, and 100 μL of the solution was added to a 96-well microplate and left at 37 ° C. for 1 hour. After washing, it was blocked with 1% BSA / PBS-T solution. 50 μL of anti-Cha o 1 monoclonal antibody 3G12-A11 (0.5 μg / ml) was added to each well of the blocked plate, and the mixture was reacted 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 to the wells and reacted at room temperature for 5 minutes, then the reaction was stopped with 2N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

1.6.6 Preparation of pollen extract 10 mL (20 times the amount) of the extract was added to 0.5 g of cypress pollen or cedar pollen, and the mixture was stirred overnight at 4 ° C. After stirring, the supernatant obtained by centrifuging at 20,000 × g for 30 minutes was used as a pollen extract.

1.6.7 Examination of reactivity by Western blotting
A double amount of 50 mM DTT / 2 × SDS sample buffer was added to Cha o 1, Cry j 1 or cypress pollen extract, and the mixture was heated at 95 ° C for 5 minutes. After cooling, electrophoresis was performed on an SDS-polyacrylamide gel having a concentration of 5-15%, and protein staining and Western blotting were performed. Protein staining was performed with Oriole ^TM Fluorescent Gel Stain. Western blotting was performed by electrically transferring the separated protein on the gel to a PVDF membrane, blocking it with 1% skim milk, adding anti-Cha o 1 monoclonal antibody 3G12-A11, and reacting at room temperature for 1 hour. After the reaction, the cells 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 with a bioimage analyzer.

1.6.8 Examination of reactivity by Sandwich ELISA
In Section 1.6.3, a combination of anti-Cry j 1 monoclonal antibody 6D7 and anti-Cha o 1 antibody 3G12-A11 was selected as the capture antibody for Sandwich ELISA. 100 μL of anti-Cry j 1 antibody 6D7 solution (10 μg / mL) was added to a 96-well microplate and left overnight at 25 ° C. After washing, it was blocked with 1% BSA / PBS-T solution. Add 100 μL each of Cha o 1 prepared in the range of 1.25 to 1000 ng / mL or Cha o 2 and Cry j 1 solution prepared in the range of 100 to 2700 ng / mL to the wells of the blocked plate at 37 ° C. Left for 2 hours. After washing, 100 μl of a biotinylated anti-Cha o 1 antibody 3G12-A11 solution (0.5 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, TMB as a substrate was added, the reaction was carried out at 25 ° C. for 15 minutes, the reaction was stopped with 2N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. All measurements were performed by triple measurement.

1.6.9 Examination of reproducibility of Sandwich ELISA
Sandwich ELISA using purified Cha o 1 prepared at 1.25, 2.5, 5, 10, 20 and 50 ng / mL as a sample and purified Cha o 1 prepared at 2, 4, 10, 20 and 50 ng / mL as a sample. Simultaneous reproducibility and daily difference reproducibility were examined. The calibration curve was prepared for each experiment, and the same sample stored at -20 ° C was used. The calibration curve and sample were diluted with 1% BSA / PBS-T, and Sandwich ELISA was performed in the same manner as in Section 1.6.8. Simultaneous reproducibility was the coefficient of variation within the same measurement when 9-fold measurement was performed, and daily difference reproducibility was the coefficient of variation between measurements when the same sample was measured three times on different days in triple measurement.

1.6.10 Quantification of Cha o 1 in pollen extract The hinoki cypress pollen extract was diluted 10-fold to 3-fold, and measured by Sandwich ELISA. Sandwich ELISA was performed in the same manner as in Section 1.6.8.

1.6.11 Cross-reactivity to Japanese cedar pollen extract The cypress pollen extract was diluted 10 to 21870 times, and the cedar pollen extract was diluted 10 to 2430 times, and the measurement was performed by Sandwich ELISA. Sandwich ELISA was performed in the same manner as in Section 1.6.8.

1.6.12 Comparison with existing technology
Detection of purified Cha o 1 prepared in the range of 1.25 to 1000 ng / mL by Sandwich ELISA is performed by the same method as in Section 1.6.8 and existing technology (Cypress pollen antigen (Cha o 1) measurement kit, Item No .: 49500-19. , Nichinichi Pharmaceutical Co., Ltd.) and compared. All purified Cha o 1 was diluted using 1% BSA-PBS-T, and the measurement was performed by triple measurement. The quantification limit (QL) was calculated using the formula QL = 10σ / S (Pharmaceutical Trial No. 338: Text (implementation method) related to analytical method validation, Chief of Examination and Management Division, Pharmaceutical Safety Bureau, Ministry of Health and Welfare, October 28, 1997. Day). σ indicates the standard deviation of the blank, and S indicates the slope of the calibration curve. The existing technology used the slope of the calibration curve in the range of 1.25 to 1000 ng / mL.

2 [Result]
2.1 Preparation of anti-Cha o 1 monoclonal antibody-producing hybridoma As anti-Cha o 1 monoclonal antibody-producing hybridomas, 5 clones of 2H4-F3, 3G12-A11, 4E11-F7, 5H3-F5 and 7B1-C5 hybridomas were obtained.

2.2 Examination of antibody combination by Sandwich ELISA
Since it is highly possible that antibodies against the cedar pollen allergen Cry j 1, which has high homology with Cha o 1, also recognize Cha o 1, the five types of anti-Cha o 1 antibodies produced were combined with three types of anti-Cry j 1. We investigated the combination of antibodies that can detect Cha o 1 by Sandwich ELISA in all 8 types of monoclonal antibodies including antibodies (2G2, 6D7 and 7A6) (Fig. 1). As a result of examining 56 combinations of monoclonal antibodies, Cha o 1 can be detected by the combination of the prepared anti-Cha o 1 antibody 3G12-A11, 2H4-F3 and 4E11-F7 and the anti-Cry j 1 antibody 2G2, 6D7 and 7A6. Became clear.
Among these combinations, the highest reactivity was obtained when the capture antibody was anti-Cry j 1 antibody 6D7 and the detection antibody was anti-Cha o 1 antibody 3G12-A11. did.

2.3 Identification of epitopes
Epitopes were identified for the anti-Cha o 1 antibodies 3G12-A11 and 2H4-F3 that could detect Cha o 1 by Sandwich ELISA. Anti-Cha o 1 antibodies 3G12-A11 and 2H4-F3 recognize peptide No. 22 ( ¹⁴⁸ QDGDAITMRNVTDVWIDHNS ¹⁶⁷ (SEQ ID NO: 2)) and No. 23 ( ¹⁵⁵ MRNVTDVWIDHNSLSDSSDG ¹⁷⁴ (SEQ ID NO: 3)), and other peptides It did not react (Fig. 2). Since the anti-Cha o 1 antibodies 3G12-A11 and 2H4-F3 recognize peptides No. 22 and No. 23, they are ^{included in the epitope in the overlapping sequence 155} MRNVTDVWIDHNS ¹⁶⁷ (SEQ ID NO: 4) of peptides No. 22 and No. 23. Was thought to have.

2.4 Indirect ELISA
We investigated the detection of Cha o 1 by Indirect ELISA as a method of using the selected anti-Cha o 1 antibody 3G12-A11. The anti-Cha o 1 antibody 3G12-A11 was not reactive with Cha o 2, which is the major allergen of cypress pollen allergen, and was responsive to Cha o 1. It was found that the anti-Cha o 1 antibody 3G12-A11 can be used for the detection of Cha o 1 by Indirect ELISA (Fig. 3).

2.5 Western blotting We investigated the detection of Cha o 1 by Western blotting as a method of using the anti-Cha o 1 antibody 3G12-A11. The anti-Cha o 1 antibody 3G12-A11 was found to react only with purified Cha o 1 by Western blotting and Cha o 1 in the cypress pollen extract (Fig. 4). In addition, no reactivity with Cry j 1, which has high homology, was observed, demonstrating high specificity. In protein staining of hinoki pollen extract, many proteins other than Cha o 1 are present, but since the anti-Cha o 1 antibody 3G12-A11 has no reactivity with proteins other than Cha o 1, Western blotting It was revealed that it can be used for the specific detection of Cha o 1 in the hinoki pollen extract.

2.6 Sandwich ELISA
The reactivity by Sandiwich ELISA was investigated as a method of using the anti-Cha o 1 antibody 3G12-A11. In the set Sandwich ELISA, a Cha o 1 concentration-dependent increase in signal was observed (Fig. 5), and high linearity was observed in the range of 1.25 to 50 ng / mL (Fig. 6). On the other hand, Cry j 1, which is not reactive with Cha o 2 and has high homology with Cha o 1, showed slight reactivity at 2,700 ng / mL, but was more than 500 times more sensitive than the reaction with Cha o 1. There was a difference (Fig. 5).

2.7 Examination of reproducibility of Sandwich ELISA The reproducibility of the content measurement of 2 to 50 ng / mL purified Cha o 1 was examined using the calibration curve in the concentration range of 1.25 to 50 ng / mL where linearity was observed. .. As a result, the coefficient of variation for simultaneous reproducibility was 1.5% to 8%, and the coefficient of variation for daily difference reproducibility was 1.5% to 7.1%, showing good values. Sandwich ELISA using anti-Cha o 1 antibody 3G12-A11 showed good values. It was clarified that it is a Cha o 1 quantitative system with high reproducibility (Table 1).

2.8 Quantification of Cha o 1 in pollen extract We investigated whether Sandwich ELISA using anti-Cha o 1 antibody 3G12-A11 can be used for quantification of Cha o 1 in pollen extract containing a large amount of comorbidants. The absorbance obtained when the hinoki pollen extract was diluted 270, 810, 2430 times was within the calibration curve range (1.25-50 ng / mL). When the Cha o 1 concentration in the hinoki pollen extract was calculated from the absorbance at this time, it was 10.3, 10.4 and 9.6 μg / mL, respectively, and almost the same concentration was obtained (CV 4.2%). From this, Sandwich ELISA using anti-Cha o 1 antibody 3G12-A11 is a quantitative system that is not affected by comorbid substances regardless of the dilution ratio, and Cha o 1 in pollen extract can also be measured. It turned out that there was.

2.9 Crossing to Sugi pollen extract We examined whether the constructed ELISA intersects with Sugi pollen extract. There was a difference in sensitivity of 500 times or more from that of hinoki pollen extract (Fig. 7). The Cry j 1 in the Sugi pollen extract used was 28.1 μg / mL (using Nichinichi Pharmaceutical Co., Ltd., Cry j 1 measurement kit). From this, Sandwich ELISA using anti-Cha o 1 antibody 3G12-A11 has high specificity, and even if cedar pollen is mixed in cypress pollen, it is a measurement system that can measure only Cha o 1 content. It became clear that there was.

2.10 Comparison with existing technology
Reactivity to 1.25 to 1000 ng / mL purified Cha o 1 was compared between Sandwich ELISA using anti-Cha o 1 antibody 3G12-A11 and existing technology (Fig. 8). When the limit of quantification was determined, it was 0.368 ng / mL for Sandwich ELISA using the anti-Cha o 1 antibody 3G12-A11 and 15.323 ng / mL for the existing technology. From this, it is clear that the Sandwich ELISA using the anti-Cha o 1 antibody 3G12-A11 has 41 times the sensitivity of the existing Cha o 1 quantitative system and can be measured with a lower concentration sample. It became.

SEQ ID NOs: 2-4: Synthetic peptides

Claims (8)

A monoclonal antibody against the hinoki pollen allergen Cha o 1 , which recognizes all or part of the amino acid sequence represented by MRNVTDVWIDHNS as an epitope among the amino acid sequences of Cha o 1, and recognizes Cha o 2 and the cedar pollen allergen Cry j 1 The monoclonal antibody that does not react with. Cha lowest detectable concentration of o 1 is less than 5 ng / mL, the monoclonal antibody of claim 1. The monoclonal antibody according to claim 1 or 2 , which has a reproducibility of 10% or less. An antibody that binds to an epitope to which the monoclonal antibody according to any one of claims 1 to 3 binds. The monoclonal antibody according to any one of claims 1 to 3 , or the fragment of the antibody according to claim 4. A hybridoma that produces the monoclonal antibody according to any one of claims 1 to 3. A method for detecting Cha o 1 comprising reacting a sample with the antibody or fragment thereof according to any one of claims 1 to 5. A kit for detecting Cha o 1 containing the antibody or fragment thereof according to any one of claims 1 to 5.

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