Antibodies against Marek's disease virus Meq protein

Abstract

To provide an anti-Meq antibody that can detect, without nonspecific reaction, Meq protein in a formalin-fixed paraffin-embedded sample.SOLUTION: The present invention provides a monoclonal antibody against Meq protein produced by a hybridoma identified with the accession number NITE P-03455, NITE P-03456, or NITE P-03457.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody against Meq protein, a hybridoma producing the same, a method for diagnosing Marek's disease using the same, and a diagnostic kit for Marek's disease containing the same. [Background technology]

[0002] Marek's disease is a notifiable infectious disease caused by avian alphaherpesvirus 2 (Marek's disease virus: MDV), which causes virus-induced lymphoma in chickens. Despite widespread use of vaccines, 20,000 to 180,000 cases of Marek's disease are reported annually, significantly impacting productivity in the poultry industry. Furthermore, the emergence of field virus strains that are resistant to vaccines overseas is becoming a problem.

[0003] A definitive diagnosis of Marek's disease is made by confirming lymphoma in histopathological examination. However, a correct diagnosis may not be made because a direct relationship between MDV infection and lymphoma formation cannot be proven by simple morphological examination alone.

[0004] To establish accurate diagnostic techniques for Marek's disease, the oncogenic protein MDV-EcoRI-Q (Meq), encoded by MDV, has been attracting attention. Meq is involved in the tumorigenesis of T lymphocytes by acting on transcriptional regulators and tumor suppressor genes, and is thought to be specifically expressed in tumorigenic lymphocytes. Therefore, if antibodies against Meq can be produced, it will be possible to immunohistochemically demonstrate Meq expression in lymphoma cells, leading to the establishment of a new diagnostic method for Marek's disease with higher accuracy than conventional methods.

[0005] Literature related to anti-Meq antibodies includes Non-Patent Documents 1 to 4. Non-Patent Document 1 describes the production of a mouse anti-Meq monoclonal antibody (clone 23B46) by immunizing mice with a recombinant fowlpox virus into which an open reading frame of Meq had been inserted. Non-Patent Document 2 describes the production of a rabbit anti-Meq polyclonal antibody using an Meq antigen produced using an Escherichia coli protein expression system. Non-Patent Document 3 describes the production of a mouse anti-Meq monoclonal antibody (clone FD7). Non-Patent Document 4 describes the use in immunohistochemistry of a mouse anti-Meq monoclonal antibody produced using an Meq antigen produced using a prokaryotic protein expression system. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Liu et al., 1996. Biological properties of the Marek's disease latent protein Meq: subcellular localization and transforming potential. Current Research on Marek's Disease (pp. 271-277)Kennett Square, PA: American Association of Avian Pathologists. [Non-patent document 2] Liu et al., 1997. Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ. J Virol. 71(4):3188-3196. [Non-patent document 3] Brown et al., 2006. Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek's disease virus. Proc. Natl. Acad. Sci. 103:1687-1692. [Non-patent document 4] Wen et al., 2018. Characterizing the histopathology of natural co-infection with Marek's disease virus and subgroup J avian leucosis virus in egg-laying hens. Avian Pathol. 47(1):83-89. Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, several publications regarding anti-Meq antibodies are already known. However, Non-Patent Documents 1 and 3 describe that the prepared anti-Meq monoclonal antibodies (clone 23B46, FD7) were able to detect Meq by immunohistochemistry using frozen specimens, but do not describe whether they can also be used in immunohistochemistry using formalin-fixed, paraffin-embedded specimens. Furthermore, Non-Patent Documents 2 and 4 describe that the prepared anti-Meq antibodies were able to detect Meq by immunohistochemistry using formalin-fixed, paraffin-embedded specimens, but also describe that a reaction against non-tumor cells was observed.

[0008] The present invention has been made under these circumstances, and aims to provide an anti-Meq antibody that can detect Meq protein in formalin-fixed, paraffin-embedded specimens without non-specific reactions. [Means for solving the problem]

[0009] Hybridomas are generally selected by ELISA, but the present inventors used both ELISA and immunohistochemistry to select antibodies that can be used in immunohistochemistry using formalin-fixed, paraffin-embedded Marek's disease tumors. As a result, they successfully established multiple hybridoma strains that secrete anti-Meq antibodies that can detect Meq protein in formalin-fixed, paraffin-embedded specimens without nonspecific reactions, thereby completing the present invention.

[0010] That is, the present invention provides the following [1] to

[12] . [1] An antibody-producing hybridoma identified by accession number NITE P-03455, NITE P-03456, or NITE P-03457.

[0011] [2] A monoclonal antibody against Meq protein, produced by the antibody-producing hybridoma according to [1].

[0012] [3] A monoclonal antibody against Meq protein, the monoclonal antibody having a heavy chain variable region and a light chain variable region selected from any one of the following (a) to (c): (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0013] [4] A monoclonal antibody against Meq protein, the monoclonal antibody having a heavy chain variable region and a light chain variable region described in any one of (d) to (f) below: (d) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, in which one or more amino acids have been substituted, deleted, inserted, and / or added; (e) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4, in which one or more amino acids have been substituted, deleted, inserted, and / or added; (f) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, in which one or more amino acids have been substituted, deleted, inserted, and / or added.

[0014] [5] A monoclonal antibody against Meq protein, the monoclonal antibody having a heavy chain variable region and a light chain variable region described in any one of (g) to (i) below: (g) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; (h) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (i) A heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6.

[0015] [6] A monoclonal antibody against Meq protein, the monoclonal antibody having a heavy chain variable region and a light chain variable region described in any one of (j) to (l) below: (j) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12; (k) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (l) A heavy chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a light chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 24.

[0016] [7] A method for diagnosing Marek's disease, comprising the following steps (1) and (2): (1) contacting a sample collected from an animal to be diagnosed with the monoclonal antibody according to any one of [2] to [6] to react the Meq protein in the sample with the monoclonal antibody; (2) A step of detecting the reaction product between the Meq protein and the monoclonal antibody.

[0017] [8] The method for diagnosing Marek's disease according to [7], wherein the animal to be diagnosed is a bird.

[0018] [9] A method for diagnosing Marek's disease according to [7] or [8], wherein the sample is tumor tissue.

[0019]

[10] The method for diagnosing Marek's disease described in [9], wherein the tumor tissue is a tumor tissue section.

[0020]

[11] The method for diagnosing Marek's disease according to

[10] , wherein the tumor tissue section is a formalin-fixed, paraffin-embedded tumor tissue section.

[0021]

[12] A Marek's disease diagnostic kit comprising the monoclonal antibody according to any one of [2] to [6]. [Effects of the Invention]

[0022] The present invention provides a novel anti-Meq antibody that can detect Meq protein in formalin-fixed, paraffin-embedded specimens without nonspecific reactions. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of detecting rMeq by Western blotting. [Figure 2] FIG. 1 shows the results of immunohistochemical detection of Meq using three anti-Meq monoclonal antibodies (3A3-112, 5F7-82, and 6B5-128). [Figure 3] A diagram showing the amino acid sequences of the heavy chain variable region, light chain variable region, and CDR of the monoclonal antibodies derived from each hybridoma. [Figure 4] FIG. 1 shows the results of detecting Meq in MSB-1 cells by Western blotting using three types of anti-Meq monoclonal antibodies (3A3-112, 5F7-82, and 6B5-128). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in detail below. (A) Hybridoma The antibody-producing hybridoma of the present invention is identified by accession number NITE P-03455, NITE P-03456, or NITE P-03457.

[0025] The hybridoma clones identified by accession numbers NITE P-03455, NITE P-03456, and NITE P-03457 are named 3A3-112, 5F7-82, and 6B5-128, respectively. These hybridomas were generated by immunizing mice with the Meq protein, which was engineered based on the meq gene sequence of the Md5 strain, a virulent strain of Marek's disease virus, and fusing the spleen cells of the immunized mice with a mouse myeloma cell line. These hybridomas have been deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation. Detailed information on the deposit is as follows:

[0026] (1)3A3-112 (1-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (1-2) Deposit date: April 6, 2021 (1-3) Accession number: NITE P-03455

[0027] (2)5F7-82 (2-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (2-2) Deposit date: April 6, 2021 (2-3) Accession number: NITE P-03456

[0028] (3)6B5-128 (3-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (3-2) Deposit date: April 6, 2021 (3-3) Accession number: NITE P-03457

[0029] (B) Monoclonal antibody The monoclonal antibody against the Meq protein of the present invention may be produced by the antibody-producing hybridoma of the present invention.

[0030] The monoclonal antibody against Meq protein of the present invention may have a heavy chain variable region and a light chain variable region described in any one of (a) to (c) below: (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0031] The heavy chain variable region and light chain variable region of (a) above are those of a monoclonal antibody produced by an antibody-producing hybridoma (3A3-112) identified by accession number NITE P-03455; the heavy chain variable region and light chain variable region of (b) above are those of a monoclonal antibody produced by an antibody-producing hybridoma (5F7-82) identified by accession number NITE P-03456; and the heavy chain variable region and light chain variable region of (c) above are those of a monoclonal antibody produced by an antibody-producing hybridoma (6B5-128) identified by accession number NITE P-03457.

[0032] The monoclonal antibody against Meq protein of the present invention may have a heavy chain variable region and a light chain variable region described in any one of (d) to (f) below: (d) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, in which one or more amino acids have been substituted, deleted, inserted, and / or added; (e) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4, in which one or more amino acids have been substituted, deleted, inserted, and / or added; (f) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, in which one or more amino acids have been substituted, deleted, inserted, and / or added.

[0033] The number of amino acids substituted, deleted, inserted, and / or added in (d), (e), and (f) above may be "one or more," but is preferably "one or several," more preferably "one to five," and even more preferably "one to three."

[0034] The monoclonal antibody against Meq protein of the present invention may have a heavy chain variable region and a light chain variable region described in any one of (g) to (i) below: (g) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; (h) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (i) A heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6.

[0035] In the above (g), (h), and (i), the sequence identity may be 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0036] The monoclonal antibody against Meq protein of the present invention may have a heavy chain variable region and a light chain variable region described in any one of (j) to (l) below: (j) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12; (k) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (l) A heavy chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a light chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 24.

[0037] The antibodies of the present invention include antibodies produced by genetic engineering, as well as antibody fragments (eg, Fab, F(ab')2, Fc, etc.), single-chain antibodies (scFv), and the like. The antibodies of the present invention are characterized by their ability to detect Meq protein in formalin-fixed, paraffin-embedded specimens without reacting with normal tissues (Table 2 and Figure 2). The subclass of these antibodies is IgG1. Each of the three antibodies may be used alone, or two or more may be mixed and used as a cocktail antibody.

[0038] The antibody of the present invention is useful for the definitive diagnosis of Marek's disease, particularly for the differentiation of Marek's disease from similar diseases such as avian leukosis and reticuloendotheliosis, although the use of the antibody is not limited thereto.

[0039] (C) Method for diagnosing Marek's disease The method for diagnosing Marek's disease of the present invention comprises the following steps (1) and (2):

[0040] In step (1), a sample collected from an animal to be diagnosed is contacted with the monoclonal antibody of the present invention to allow the Meq protein in the sample to react with the monoclonal antibody.

[0041] Since Marek's disease is an infectious disease of birds, animals to be diagnosed are usually birds, but animals other than birds (excluding humans) may also be the subject of diagnosis. There are no particular limitations on the birds to be diagnosed, and poultry such as chickens, quails, turkeys, geese, and ducks are usually the subject of diagnosis, but wild birds such as white-fronted geese may also be the subject of diagnosis.

[0042] Although the sample is not particularly limited, tumor tissue is preferably used as the sample. In the present invention, Meq protein is usually detected by immunohistochemistry, so it is preferable that the tumor tissue be sectioned so that it can be detected by such a technique.

[0043] Tissue sections include frozen tissue sections and formalin-fixed, paraffin-embedded tissue sections, but in the present invention, formalin-fixed, paraffin-embedded tissue sections are preferred because they can be stored for long periods at room temperature and are easy to organize and handle. Formalin-fixed, paraffin-embedded tissue sections denature antigens, making them more difficult to detect than frozen tissue sections. However, as described above, the monoclonal antibody of the present invention can detect Meq protein without any problems.

[0044] When detecting antigens in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry, paraffin removal and antigen retrieval are usually performed before reacting the antigen with an antibody, and these treatments are also preferably performed in the present invention. Antigen retrieval methods include methods using protease and methods using heat, and either method can be used in the present invention.

[0045] In step (2), the reaction product between the Meq protein and the monoclonal antibody is detected.

[0046] The reaction product can be detected using a label. In detecting the reaction product, the anti-Meq antibody itself may be labeled, or an antibody (secondary antibody) against the anti-Meq antibody (primary antibody) may be labeled.

[0047] The label may be any label commonly used in antibody-based antigen detection methods, such as an enzyme, an enzyme substrate, a radioisotope, a luminescent substance, a fluorescent substance, biotin, or a coloring substance. Among these, an enzyme or a fluorescent substance is preferred. Examples of enzymes include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase. Examples of fluorescent substances include fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, and phycoerythrin.

[0048] (D) Marek's disease diagnostic kit. The Marek's disease diagnostic kit of the present invention comprises the monoclonal antibody of the present invention.

[0049] The monoclonal antibody (anti-Meq antibody) of the present invention contained in the kit may be labeled or unlabeled. The Marek's disease diagnostic kit of the present invention may also contain other components in addition to the monoclonal antibody of the present invention. Such components may include a labeled secondary antibody, a washing buffer, a blocking solution, and operating instructions. [Example]

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0051] Example 1: Construction of recombinant Marek's disease virus EcoRI-Q (rMeq) The meq gene sequence (GenBank AF243438.1) of the highly virulent Md5 strain of Marek's disease virus (MDV) was obtained from the National Center for Biotechnology Information database. Based on the full 339-bp sequence of this meq gene, rMeq was constructed using the Bac to Bac baculovirus recombinant protein expression system (Thermo Fisher Scientific Inc.). Specifically, a His-tag sequence was first inserted into the N-terminus of the meq gene, and the artificially synthesized meq gene was cloned into the pFastbac1 baculovirus transfer vector. The resulting vector was transfected into an Escherichia coli strain (DH10Bac) to generate a bacmid. Recombinant baculovirus was obtained by transfecting this bacmid into Sf9 insect cells. Sf9 insect cells were then infected with this recombinant baculovirus to generate His-tagged rMeq. The expressed protein was purified by affinity purification using a Ni column. The expression of rMeq was confirmed by Western blotting using an anti-His tag antibody (clone 3D5, Thermo Fisher Scientific Inc.). The results of Western blotting are shown in Figure 1.

[0052] Example 2: Preparation of anti-Meq monoclonal antibody Two 7-week-old BALB / c mice were immunized three times at two-week intervals with rMeq (0.3 mg / mL) mixed with aluminum hydroxide adjuvant. Three days after the final immunization, mouse spleens were harvested, and splenocytes were fused with the mouse myeloma cell line P3U1 using Hybri-Max (Sigma-Aldrich Inc.) to generate hybridomas. Hybridomas secreting anti-rMeq IgG antibodies were selected by enzyme-linked immunosorbent assay (ELISA) (method described in Example 3 below) and cloned by limiting dilution. After cloning, culture supernatants containing anti-rMeq monoclonal antibodies from three hybridoma lines (3A3-112, 5F7-82, and 6B5-128) were collected for use in immunohistochemistry (IHC) as described in Example 4 below. The subclass of the anti-Meq monoclonal antibody was determined using a rapid mouse antibody isotyping kit (Thermo Fisher Scientific Inc.). The results of the subclass determination are shown in Table 1. [Table 1]

[0053] Example 3: Screening of anti-Meq antibodies by ELISA rMeq (0.3 mg / mL) was mixed with an equal volume of 0.1% Triton X-100 (Nacalai Tesque, Inc.) and then dispensed into a 96-well plate at 100 μl per well. The antigen was immobilized at 37°C for 1 hour. After washing, 20% Block Ace solution (UK-B80, DS Pharma Biomedical Co., Ltd.) was dispensed at 100 μl per well and blocked overnight at 4°C. After washing, 100 μl of culture supernatant containing anti-Meq antibody collected from each well of an antibody-secreting hybridoma cell culture plate was added to each well and incubated at 37°C for 1 hour. After washing, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG gamma chain (61-6020, Zymed Laboratories Inc.) was added and incubated at 37°C for 1 hour. After washing, 2,2′-Azino-di-(3-ethylbenzthiazoline sulfonic acid) (Sigma-Aldrich Inc.) was added as an HRP substrate, and the absorbance at 405 nm was measured using a microplate reader (Multiskan FC basic, Thermo Fisher Scientific Inc.).

[0054] Example 4: Detection of Meq in formalin-fixed, paraffin-embedded (FFPE) sections by IHC using anti-Meq monoclonal antibody We evaluated the feasibility of IHC detection of Meq antigen present in FFPE sections using the generated anti-Meq monoclonal antibody. Two types of FFPE sections were used: 1) MDCC-MSB-1 (MSB-1) cells, a tumor cell line derived from Marek's disease (MD), and 2) tumor tissues from specific pathogen-free (SPF) chickens experimentally inoculated with the highly virulent MS1 strain of MDV. Furthermore, nonspecific reactivity of the anti-Meq monoclonal antibody in IHC was evaluated using FFPE sections of 3) normal tissues (brain, heart, lung, thymus, pancreas, liver, kidney, spleen, duodenum, cecum, bursa of Fabricius, and skin) from MDV-uninfected SPF chickens.

[0055] 1) MSB-1 cells, 2) tumor tissues, and 3) normal tissues were fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.), embedded in paraffin, and prepared as FFPE sections. After deparaffinization, sections were immersed in 0.3% H2O2-containing methanol for 20 minutes at room temperature to inhibit endogenous peroxidase activity. Four antigen retrieval conditions were tested: no treatment, heat treatment with citrate buffer (pH 6) or ethylenediaminetetraacetic acid (EDTA) buffer (pH 9), and enzyme treatment with actinase E. Heat treatment was performed by heating sections immersed in each buffer in a microwave oven (500 W) for 15 minutes. Enzyme treatment was performed by immersing sections in 0.1% actinase E (Kaken Pharmaceutical Co., Ltd.) for 10 minutes at 35°C. Nonspecific antibody binding was inhibited by incubation with 5% skim milk solution for 20 minutes at room temperature. The anti-Meq monoclonal antibodies from the three strains described above were diluted 4-fold in phosphate buffered saline (PBS) and incubated overnight at 4°C. After washing the sections with PBS, they were incubated with Histofine Simple Stain MAX-PO(M) (Nichirei Biosciences) as a secondary antibody for 45 minutes at room temperature. The reaction was visualized under a microscope using a DAB substrate kit (Nichirei Biosciences). Counterstaining was performed with Mayer's hematoxylin (Muto Chemical Industries, Ltd.).

[0056] The positive IHC reaction to Meq was graded on a four-point scale: -: absent, +: weak, ++: moderate, +++: strong. Nonspecific reactions to normal tissues were graded on a -: absent, +: present. The results are shown in Table 2 and Figure 2. [Table 2]

[0057] Example 5: Determination of amino acid sequences of heavy chain variable region, light chain variable region, and CDR Approximately 2 × 10 hybridomas 5mRNA was extracted from each hybridoma using Quick-RNA Microprep (Zymo Research Corp.), and cDNA was synthesized. The cDNA was amplified using the 5'-RACE method and cloned into a vector for sequence analysis. Ten clones from each hybridoma were sequenced for the heavy and light chains to determine the consensus sequence. CDRs were identified from the resulting sequences.

[0058] The amino acid sequences of the heavy chain variable region, light chain variable region, and CDRs of each hybridoma (3A3-112, 5F7-82, 6B5-128) are shown in Figure 3. Each amino acid sequence is also shown in the sequence listing. The relationship between the amino acid sequence and the SEQ ID NO: is as shown in the table below. [Table 3]

[0059] Example 6: Detection of Meq in MSB-1 cells by Western blotting [Materials and methods] 1 × 10 MSB-1 cells were incubated in RIPA buffer (Nacalai Tesque, Inc.) supplemented with 0.1% SDS solution. 6Proteins were extracted from the cells. Proteins extracted under reducing conditions using NuPAGE MOPS SDS Running Buffer (Thermo Fisher Scientific, Inc.) were subjected to SDS-PAGE electrophoresis using NuPAGE 4-12% Bis-Tris acrylamide electrophoresis gels (Thermo Fisher Scientific, Inc.) and transferred to a nitrocellulose membrane. After washing, the nitrocellulose membrane was blocked overnight at 4°C with 0.8% Block Ace solution (UK-B80, DS Pharma Biomedical Co., Ltd.). After washing, the membrane was incubated for 1 hour at 37°C with culture supernatant containing a 5-fold diluted anti-Meq antibody. After washing, the membrane was incubated for 1 hour at 37°C with HRP-conjugated goat anti-mouse IgG+A+M(H+L) (61-6420, Zymed Laboratories Inc.). After washing, 2,2′-Azino-di-(3-ethylbenzthiazoline sulfonic acid) (Sigma-Aldrich Inc.) was added as an HRP substrate to visualize the reaction.

[0060] 〔result〕 The antibody of the present invention detected Meq of approximately 55-60 kDa in MSB-1 cells (FIG. 4). [Industrial Applicability]

[0061] The present invention can be used in industries related to antibodies.

Claims

1. An antibody-producing hybridoma identified by accession number NITE P-03455, NITE P-03456, or NITE P-03457.

2. A monoclonal antibody against Meq protein, produced by the antibody-producing hybridoma of claim 1.

3. A monoclonal antibody against Meq protein, which has a heavy chain variable region and a light chain variable region described in any one of (a) to (c) below: (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

6.

4. A monoclonal antibody against Meq protein, which has a heavy chain variable region and a light chain variable region described in any one of (j) to (l) below: (j) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12; (k) a heavy chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (l) A heavy chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a light chain variable region having CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:

24.

5. A method for diagnosing Marek's disease, comprising the following steps (1) and (2): (1) contacting a sample collected from a subject animal (excluding humans) with the monoclonal antibody according to any one of claims 2 to 4 to react the Meq protein in the sample with the monoclonal antibody; (2) A step of detecting the reaction product between the Meq protein and the monoclonal antibody.

6. 6. The method for diagnosing Marek's disease according to claim 5, wherein the animal to be diagnosed is a bird.

7. The method for diagnosing Marek's disease according to claim 5 or 6, wherein the sample is tumor tissue.

8. The method for diagnosing Marek's disease according to claim 7, wherein the tumor tissue is a tumor tissue section.

9. The method for diagnosing Marek's disease according to claim 8, wherein the tumor tissue section is a formalin-fixed, paraffin-embedded tumor tissue section.

10. A Marek's disease diagnostic kit comprising the monoclonal antibody described in any one of claims 2 to 4.

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