Antibody against deoxynivalenol and acetylated deoxynivalenol

Abstract

To provide a monoclonal antibody that specifically binds to deoxynivalenol, a trichothecene toxin produced by Fusarium fungi when they infect grains such as wheat or barley and cereals such as corn, and / or acetylated deoxynivalenol.SOLUTION: The present invention provides an antibody that specifically binds to deoxynivalenol and / or acetylated deoxynivalenol having a specific CDR amino acid sequence, or an antigen-binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to monoclonal antibodies against deoxynivalenol and acetylated deoxynivalenol. [Background technology]

[0002] Fusarium graminearum ( Fusarium graminearum Fusarium fungi such as Fusarium cereale, ...

[0003] HPLC, GC / MS, LC / MS, etc. are used to detect trichothecene toxins such as DON, but these methods are not very suitable for processing many samples in a short time. Therefore, it is desirable to efficiently measure many samples using immunological quantitative methods such as ELISA. Currently, immunoassay kits targeting DON are commercially available, and standards and other criteria have been established. There have been many reports on antibodies against DON, including antibodies against its acetylated form. For example, there have been reports on polyclonal antibodies that recognize DON, 3-acetyldeoxynivalenol (3-Ac-DON), and 15-acetyldeoxynivalenol (15-Ac-DON) (Non-Patent Document 1), monoclonal antibodies that recognize DON, 3-Ac-DON, and several other trichothecene toxins (Non-Patent Document 2), and monoclonal antibodies that recognize DON and 3-Ac-DON (Non-Patent Document 3).

[0004] Although antibody-based detection kits for DON are currently available on the market, there is still a demand for anti-DON antibodies with improved reaction specificity and affinity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Usleber et al., J. Agric. Food Chem. 39:2091-2095 1991. [Non-Patent Document 2] Abouzied et al., J. Food Prot. 54:288-290 1991. [Non-Patent Document 3] Maragos et al., Food Agric. Immunol. 12:191-192 2000. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above circumstances, an objective of the present invention is to provide an antibody that specifically binds to DON and / or acetylated DON. [Means for solving the problem]

[0007] The inventors worked on producing the antibodies necessary for the development of an immunoassay for DON. DON has multiple OH groups for binding to proteins. In the structural formula of the trichothecene derivative below, R1 at position 3, R4 at position 7, and R3 at position 15 are -OH, and R2 at position 4 is -H. [ka] Therefore, when attempting to introduce a carrier protein to confer immunogenicity to DON, it was thought that the protein would bind randomly to the above-mentioned OH groups, making it difficult to bind the carrier protein while maintaining the original three-dimensional structure. Taking these circumstances into consideration, the inventors selectively bound a carrier protein to only position 15 of DON or 3-Ac-DON, produced a monoclonal antibody using this as an antigen, and clarified the amino acid sequences of the variable regions of the heavy and light chains of the antibody, as well as the amino acid sequences of the six CDRs.

[0008] That is, the present invention relates to the following (1) to (10). (1) An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (A-1) or (A-2) below: (A-1) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6; (A-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:8; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:9; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:12. (2) The antibody or antigen-binding fragment thereof according to (1) above, which satisfies either the following (a) or (b): (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. (3) An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (B-1) or (B-2) below: (B-1) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19, and Having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20; (B-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26. (4) The antibody or antigen-binding fragment thereof according to (3) above, which satisfies either the following (a) or (b): (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:28. (5) An antibody or an antigen-binding fragment thereof, characterized in that the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (C-1) or (C-2) below. (C-1) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 29; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 30; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 31; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 32; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 33, and Having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 34; (C-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 36; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 37; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:38; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:39, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:40. (6) The antibody or antigen-binding fragment thereof according to (5) above, which satisfies either the following (a) or (b): (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 41, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 42; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 42. (7) An antigen-binding fragment according to any one of (1), (3) or (5) above, which is a Fab, Fab', F(ab')2, Fv, a single-chain antibody, scFv, an scFv dimer or a dsFv. (8) A kit for detecting a trichothecene mycotoxin, comprising the antibody or antigen-binding fragment described in any one of (1), (3) or (5) above. (9) An antibody that binds to 3-acetyldeoxynivalenol (3-Ac-DON), which competitively inhibits the binding of the antibody described in (1) or (2) above to 3-Ac-DON, or an antigen-binding fragment thereof. (10) An antibody or an antigen-binding fragment thereof that binds to deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-Ac-DON), and competitively inhibits the binding of the antibody described in (3) above to DON and 15-Ac-DON. In this specification, the symbol "~" indicates a numerical range including the values ​​on either side of it. Effect of the Invention

[0009] INDUSTRIAL APPLICABILITY The present invention provides monoclonal antibodies that quantitatively and specifically bind to the trichothecene mycotoxin DON and its acetylated derivatives. [Brief description of the drawings]

[0010] [Figure 1] Examination of the reaction characteristics of anti-DON antibodies (MoAbs), MDN8 and MDN31, with DON and 15-Ac-DON. The vertical axis shows the ratio of the OD of the sample to the OD of the reaction without DON or 15-Ac-DON, and the horizontal axis shows the concentration of DON or 15-Ac-DON added to the reaction well. [Diagram 2] Examination of the reaction characteristics of anti-3-Ac-DON antibodies (MoAbs) M3ADN27 and M3ADN28 with DON, 3-Ac-DON, and 15-Ac-DON. The vertical axis indicates the ratio of the OD of the sample to the OD reacted without DON, 3-Ac-DON, or 15-Ac-DON, and the horizontal axis indicates the concentration of DON, 3-Ac-DON, or 15-Ac-DON added to the reaction wells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described. Note that, when the term "the present embodiment" is used, it refers to all embodiments described in this specification unless otherwise specified. The first embodiment is an antibody that binds to DON and / or acetylated DON (3-Ac-DON or 15-Ac-DON) (hereinafter, in this specification, an antibody that binds to DON and / or acetylated DON is also referred to as an "anti-DON antibody according to this embodiment"), or an antigen-binding fragment of the antibody, characterized in that the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 of the antibody satisfy any of the following (A-1) or (A-2), (B-1) or (B-2), or (C-1) or (C-2). The CDR sequences were analyzed using the IgBLAST (National Center for Biotechnology Information: NCBI) algorithm based on information from the Kabat database or the IMGT database.

[0012] (A-1) CDR sequence of M3ADN27 antibody (referred to as "M3ADN27 MoAb" in the Examples) (using Kabat database) The heavy chain CDR1 amino acid sequence is YYAMS (SEQ ID NO: 1); The heavy chain CDR2 amino acid sequence is SISAGGRKYNSDNVEG (SEQ ID NO: 2). The heavy chain CDR3 amino acid sequence is GGHDYDGSLLAY (SEQ ID NO:3). The light chain CDR1 amino acid sequence is RSSTGTVTTTNYAN (SEQ ID NO: 4). the light chain CDR2 amino acid sequence is GTNNRVP (SEQ ID NO:5), and The light chain CDR3 amino acid sequence has the sequence ALWYRNYWV (SEQ ID NO:6). (A-2) CDR sequence of M3ADN27 antibody (using IMGT database) The heavy chain CDR1 amino acid sequence is GFTFSYYA (SEQ ID NO: 7). The heavy chain CDR2 amino acid sequence is ISAGGRK (SEQ ID NO: 8). The heavy chain CDR3 amino acid sequence is VRGGHDYDGSLLAY (SEQ ID NO: 9). The light chain CDR1 amino acid sequence is TGTVTTTNY (SEQ ID NO: 10). the light chain CDR2 amino acid sequence is GTN (SEQ ID NO: 11), and The light chain CDR3 amino acid sequence has the sequence ALWYRNYWV (SEQ ID NO: 12).

[0013] (B-1) CDR sequence of M3ADN28 antibody (referred to as "M3ADN28 MoAb" in the Examples) (using Kabat database) The heavy chain CDR1 amino acid sequence is TYAMS (SEQ ID NO: 15). The heavy chain CDR2 amino acid sequence is SISSGGKEYPSDSVKG (SEQ ID NO: 16). The heavy chain CDR3 amino acid sequence is GGFDYDGSLLAY (SEQ ID NO: 17). The light chain CDR1 amino acid sequence is RSSTGAVTTSNYAN (SEQ ID NO: 18). the light chain CDR2 amino acid sequence is GTNNRVP (SEQ ID NO: 19), and The light chain CDR3 amino acid sequence has the sequence ALWYSNYWV (SEQ ID NO:20). (B-2) CDR sequence of M3ADN28 antibody (using IMGT database) The heavy chain CDR1 amino acid sequence is GFTFSTYA (SEQ ID NO: 21). The heavy chain CDR2 amino acid sequence is ISSGGKE (SEQ ID NO: 22); The heavy chain CDR3 amino acid sequence is VRGGFDYDGSLLAY (SEQ ID NO: 23). The light chain CDR1 amino acid sequence is TGAVTTSNY (SEQ ID NO: 24). the light chain CDR2 amino acid sequence is GTN (SEQ ID NO: 25), and The light chain CDR3 amino acid sequence has the sequence ALWYSNYWV (SEQ ID NO:26).

[0014] (C-1) CDR sequence of MDN31 antibody (referred to as "MDN31 MoAb" in the Examples) (using Kabat database) The heavy chain CDR1 amino acid sequence is NYWIG (SEQ ID NO: 29); The heavy chain CDR2 amino acid sequence is DIYPGGGYSNNNEKFKG (SEQ ID NO: 30). The heavy chain CDR3 amino acid sequence is SHYGNYGDY (SEQ ID NO: 31). The light chain CDR1 amino acid sequence is RASSSVNFMH (SEQ ID NO: 32). the light chain CDR2 amino acid sequence is DTSKLAS (SEQ ID NO: 33), and The light chain CDR3 amino acid sequence has the sequence LQWISNPLT (SEQ ID NO:34). (C-2) CDR sequence of MDN31 antibody (using IMGT database) The heavy chain CDR1 amino acid sequence is GYTFTNYW (SEQ ID NO: 35). The heavy chain CDR2 amino acid sequence is IYPGGGYS (SEQ ID NO: 36). The heavy chain CDR3 amino acid sequence is VRSHYGNYGDY (SEQ ID NO: 37). The light chain CDR1 amino acid sequence is SSVNF (SEQ ID NO: 38). the light chain CDR2 amino acid sequence is DTS (SEQ ID NO: 39), and The light chain CDR3 amino acid sequence has the sequence LQWISNPLT (SEQ ID NO:40).

[0015] It has been confirmed that the above-mentioned M3ADN27 and M3ADN28 antibodies bind to 3-Ac-DON with high specificity, and that the MDN31 MoAb binds to DON and 15-Ac-DON with high specificity.

[0016] Furthermore, the anti-DON antibodies and antigen-binding fragments thereof according to this embodiment include antibodies comprising any one of heavy chain variable regions comprising the amino acid sequence represented by SEQ ID NO: 13 (M3ADN27 antibody), SEQ ID NO: 27 (M3ADN28 antibody), or SEQ ID NO: 41 (MDN31 antibody), or antibodies comprising any one of light chain variable regions comprising the amino acid sequence represented by SEQ ID NO: 14 (M3ADN27 antibody), SEQ ID NO: 28 (M3ADN28 antibody), or SEQ ID NO: 42 (MDN31 antibody), and antigen-binding fragments thereof, as well as the heavy chain variable regions and / or light chain variable regions constituting these antibodies. The antibody comprises an amino acid sequence having an amino acid sequence identity of about 70% or more, preferably about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, more preferably about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and most preferably about 99% or more with each amino acid sequence of the chain variable region, and includes an antibody that binds to DON or acetylated DON and an antigen-binding fragment thereof. The amino acid sequences represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 41, and SEQ ID NO: 42 are shown below.

[0017] Heavy chain variable region of M3ADN27 antibody GGLVKPGGSLKLSCAASGFTFSYYAMSGIRQTPEKRLERVTSISAGGRKYNSDNVEGRFTISRDNARNILYLQMSSLRSEDSAMYYCVRGGHDYDGSLLAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPA (SEQ ID NO: 13) Light chain variable region of M3ADN27 antibody MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGTVTTTNYANWVQEKPDHLFTGLIGGTNNRVPGVPARFSGSLIGGKAALTITGAQTEDEAMYFCALWYRNYWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHT (SEQ ID NO: 14)

[0018] Heavy chain variable region of M3ADN28 antibody MNFGFSLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWIRQTPEKRLERVTSISSGGKEYPSDSVKGRFTISRDNVRSILYLQMSSLRSEDTAMYYCVRGGFDYDGSLLAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPA (SEQ ID NO: 27) Light chain variable region of M3ADN28 antibody MAWISLILSLLALSSGAISQTVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNYWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHT (SEQ ID NO: 28)

[0019] Heavy chain variable region of MDN31 antibody MEWSGVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKMSCKAGGYTFTNYWIGWVKQRPGHGLEWIGDIYPGGGYSNNNEKFKGKARLTADTSSSTAYMQLSRLTSEDSAIYYCVRSHYGNYGDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPAS (SEQ ID NO: 41) Light chain variable region of MDN31 antibody MDFQVQIFSFLLISASVIISRGQNVLIQSPAIMSASPGEKVTMTCRASSSVNFMHWVQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSFSLTISMEAEDAATYYCLQWISNPLTFGTGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP (SEQ ID NO: 42)

[0020] The nucleic acid sequences of the heavy chain variable region and light chain region of the M3ADN27 antibody are shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively; the nucleic acid sequences of the heavy chain variable region and light chain region of the M3ADN28 antibody are shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively; and the nucleic acid sequences of the heavy chain variable region and light chain region of the MDN31 antibody are shown in SEQ ID NO: 47 and SEQ ID NO: 48, respectively.

[0021] Nucleic acid sequence of the heavy chain variable region of the M3ADN27 antibody GGAGGCTTGGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTATTATGCCATGTCTGGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGAGGGTCACATCCATTAGTGCTGGTGGTAGAAAATATAATTCAGACAATGTGGAGGGCCGATTCACCATCTCCAGAGATAATGCCCGGAACATCCTATACCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTGTAAGAGGCGGGCATGATTACGACGGTTCCCTGCTTGCTTACTGGGGCCAAGGGACTCTGGTCACGGTCTCTGCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAA(SEQ ID NO: 43) Nucleic acid sequence of the light chain variable region of the M3ADN27 antibody ATTATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGACTGTTACAACTACTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGCGGTACCAACAACCGAGTTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGGCAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATGTATTTCTGTGCTCTATGGTACAGAAATTACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCTTGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACT(SEQ ID NO: 44)

[0022] Nucleic acid sequence of the heavy chain variable region of the M3ADN28 antibody AGACACCTCACCATGAACTTCGGGTTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAATGTGAAGTGAAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGCCATGTCTTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGAGGGTCACATCCATTAGTAGTGGTGGTAAAGAGTATCCTTCAGACAGTGTGAAGGGCCGATTCACCATATCTAGAGATAATGTCAGGAGCATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGAGGCGGTTTTGATTACGACGGTTCCCTGCTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAA(SEQ ID NO:45) Nucleic acid sequence of the light chain variable region of the M3ADN28 antibody ATTATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAGACTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGCGGTACCAACAACCGAGTTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATTTATTTCTGTGCTCTATGGTACAGCAATTACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCTTGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACT(SEQ ID NO: 46)

[0023] Nucleic acid sequence of the heavy chain variable region of the MDN31 antibody CTCACCATGGAATGGAGCGGGGTCTTTATCTTTCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATGTCCTGTAAGGCTGGTGGATACACCTTCACTAACTACTGGATAGGTTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGATATTTACCCTGGAGGTGGTTATAGTAACAATAATGAGAAGTTCAAGGGCAAGGCCAGACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGTAGGCTGACATCTGAGGACTCTGCCATCTATTACTGTGTTAGATCCCACTATGGTAACTACGGGGACTACTGGGGCCAGGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCA(SEQ ID NO:47) Nucleic acid sequence of the light chain variable region of the MDN31 antibody GACAGAATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGACAAAATGTTCTCATCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCCGTGCCAGCTCAAGTGTAAATTTCATGCACTGGGTCCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTTCTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCTGCAGTGGATTAGTAACCCGCTCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCC(SEQ ID NO:48)

[0024] The anti-DON antibody according to this embodiment may be produced by injecting an antigen into an animal to be immunized, or may be produced by genetic engineering, and any method may be used for the production. When the anti-DON antibody according to this embodiment is a recombinant antibody, its form is not particularly limited, and examples thereof include chimeric antibodies. Chimeric antibodies are, for example, antibodies in which variable regions and constant regions derived from different animal species are linked (for example, antibodies in which the variable region of a rat-derived antibody is bound to a constant region derived from a human) (for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 1984.), and can be easily constructed by recombinant technology.

[0025] A humanized antibody is an antibody that has a human-derived sequence in the framework region (FR) and a complementarity determining region (CDR) consisting of a sequence derived from another animal species (e.g., mouse, etc.). A humanized antibody can be produced by first transplanting the CDR from the variable region of an antibody derived from another animal species (here, mouse is used for explanation) into a human antibody variable region, reconstructing the heavy and light chain variable regions, and then linking these humanized reconstructed human antibody variable regions to a human antibody constant region. Methods for producing such humanized antibodies are well known in the art (e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033 1989, etc.).

[0026] The antigen-binding fragment of this embodiment is a partial region of the antibody of this embodiment, and refers to an antibody fragment that binds to a desired antigen. Examples of the fragment include Fab, Fab', F(ab')2, Fv (variable fragment of antibody), single-chain antibodies (heavy chain, light chain, heavy chain variable region, light chain variable region, nanobody, etc.), scFv (single chain Fv), diabody (scFv dimer), dsFv (disulfide-stabilized Fv), and peptides comprising at least a portion of the CDR of the antibody of this embodiment.

[0027] Fab is an antibody fragment having antigen-binding activity, in which about the N-terminal half of the heavy chain and the entire light chain are bound by a disulfide bond, among fragments obtained by treating an antibody molecule with the protease papain. Fab can be produced by treating an antibody molecule with papain to obtain a fragment, or, for example, by constructing an appropriate expression vector into which DNA encoding Fab is inserted, introducing this into an appropriate host cell (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.), and then expressing Fab in the cell.

[0028] F(ab')2 is an antibody fragment that has antigen-binding activity and is slightly larger than the fragments obtained by treating an antibody molecule with the protease pepsin, in which Fab is bonded via a disulfide bond in the hinge region. In addition to obtaining fragments by treating an antibody molecule with pepsin, F(ab')2 can also be produced by bonding Fab via a thioether bond or disulfide bond, and can also be produced by genetic engineering techniques in the same way as Fab.

[0029] Fab' is an antibody fragment having antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2. Fab' can also be prepared by genetic engineering techniques, like Fab.

[0030] An scFv is an antibody fragment having antigen-binding activity, which is a VH-linker-VL or VL-linker-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker. The scFv can be produced by genetic engineering techniques using cDNAs encoding the heavy and light chain variable regions of an antibody.

[0031] A diabody is an antibody fragment in which scFv is dimerized and has bivalent antigen-binding activity. The bivalent antigen-binding activity may be the same antigen-binding activity or one of the antigens may be different. A diabody can be produced by genetic engineering techniques by obtaining cDNAs encoding the heavy and light chain variable regions of an antibody and constructing cDNAs encoding scFvs in which the heavy and light chain variable regions are linked by a peptide linker.

[0032] dsFv refers to a polypeptide in which one amino acid residue in each of the heavy chain variable region and the light chain variable region is replaced with a cysteine ​​residue, and the cysteine ​​residues are linked via a disulfide bond. The amino acid residue to be replaced with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody. dsFv can be produced by genetic engineering techniques by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of an antibody and constructing DNA encoding dsFv.

[0033] The peptide containing CDR is configured to include at least one region of the CDR (CDR1 to 3) of the heavy chain or the light chain. The peptide containing multiple CDRs can be linked directly or via a suitable peptide linker. The peptide containing CDR is prepared by constructing a DNA encoding the CDR of the heavy chain or the light chain of an antibody and inserting it into an expression vector. There is no particular limitation on the type of vector, and it may be appropriately selected depending on the type of host cell to be introduced therein. These can be introduced into a suitable host cell (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.) to be expressed as an antibody, and produced. The peptide containing CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0034] A human antibody (fully human antibody) generally has the same structure as a human antibody in the hypervariable region, which is the antigen-binding site of the V region, other parts of the V region, and the constant region. A person skilled in the art can easily prepare a human antibody using known techniques. A human antibody can be obtained, for example, by a method using a human antibody-producing mouse having a human chromosome fragment containing the H-chain and L-chain genes of a human antibody (e.g., Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97, 722-727 2000., etc.), or by a method of obtaining a human antibody derived from phage display selected from a human antibody library (e.g., Siriwardena et al., Opthalmology, 109, 427-431 2002., etc.).

[0035] A multispecific antibody may be constructed using the antigen-binding fragment according to the present embodiment. Multispecificity means having binding specificity for two or more antigens, and examples of such include monoclonal antibodies or protein forms containing antigen-binding fragments having binding specificity for two or more antigens. This can be performed by a person skilled in the art using known techniques. As a method for constructing multispecificity, several techniques have been developed, which are classified into a technique for constructing asymmetric IgG in which two different types of antibody heavy chain molecules are subjected to protein engineering so as to form a heterodimer, a technique for linking low molecular weight antigen-binding fragments obtained from an antibody to each other, or a technique for linking them to another antibody molecule, and the like. For specific examples of construction methods, the following literature can be referred to, for example. Kontermann et al., Drug Discovery Today, 20, 838-847 2015.

[0036] The second embodiment is an antibody that competitively inhibits the binding of an antibody that binds to DON or an acetylated form of DON, characterized in that the amino acid sequence of CDRs (complementarity determining regions) 1 to 3 satisfies any of the above (A-1) or (A-2), (B-1) or (B-2), or (C-1) or (C-2), and DON or an acetylated form of DON (hereinafter also referred to as a "competitive antibody according to this embodiment"). The competitive antibody according to this embodiment can be prepared and obtained by a competitive experiment well known to those skilled in the art. Specifically, when the binding of a first anti-DON antibody (the anti-DON antibody according to this embodiment) to DON or an acetylated form of DON is competitively inhibited by a second anti-DON antibody, it is determined that the first anti-DON antibody and the second anti-DON antibody are substantially identical or bind to antigen sites in close proximity. The second anti-DON antibody is a competitive antibody according to this embodiment. As a method for the above-mentioned competitive experiment, for example, a method using a Fab fragment or the like is usually performed in the technical field. See, for example, WO95 / 11317, WO94 / 07922, WO2003 / 064473, WO2008 / 118356 and WO2004 / 046733.

[0037] The third embodiment is a kit for detecting trichothecene mycotoxins, which comprises at least an anti-DON antibody according to this embodiment (an antibody according to the first embodiment) or a competitive antibody according to this embodiment (an antibody according to the second embodiment). The kit of this embodiment may include, in addition to the above-mentioned antibody, reagents, equipment (e.g., immunochromatography or immunosensors for detecting antigens), and instruments (such as ELISA plates) necessary for detecting trichothecene mycotoxins.

[0038] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include the plural as well as the singular, unless the context clearly indicates otherwise. In addition, in this specification, "to the same extent" and "about" refer to a numerical range of ±10%. The present invention will be further described below with reference to examples. However, these examples are merely illustrative of embodiments of the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0039] 1. Cultivation of deoxynivalenol (DON)-producing strains Stock strains producing DON, Fusarium graminearum TSY 0341 was inoculated into potato dextrose agar and cultured at 20°C for one week. To prepare the toxin production medium, 50 g of commercially available rolled barley was placed in a 500 mL Erlenmeyer flask, and an equal amount of purified water was added, followed by standing overnight in a refrigerator. After standing, the flask was heated at 121°C for 60 minutes and returned to room temperature (medium volume approximately 100 g), after which the bacteria cultured in the potato dextrose agar was inoculated into the toxin production medium, and the culture that had grown in the toxin production medium after two weeks was used for extracting DON.

[0040] 2. Preparation of DON and 3-acetyl-DON 2-1. Purification of DON DON was extracted from the culture (approximately 400 g) grown in the toxin production medium with 85% acetonitrile (v / v) (400 mL, three times). The amount of DON contained in this extract (approximately 1.2 L) was approximately 244 mg. This extract (approximately 1.2 L) was concentrated under reduced pressure and then washed three times with an equal volume (approximately 900 mL) of n-hexane. The aqueous layer (approximately 900 mL) was applied to a synthetic adsorption resin (DIAION HP20, 300 mL, 6.0 cm id × 9.5 cm, Mitsubishi Chemical), washed with water (900 mL), and DON was eluted with methanol (450 mL). The eluate was concentrated under reduced pressure. DON was extracted from this concentrate three times with an equal volume (approximately 100 mL) of ethyl acetate. The ethyl acetate layer was dried under reduced pressure, and then loaded onto a silica gel column (2 cm id x 20 cm; Silica gel 60, Merck) and developed with a dichloromethane / methanol mixture (20:1, 10:1) and methanol in that order. The resulting eluates (dichloromethane:methanol = 10:1) containing DON were combined and concentrated under reduced pressure. The resulting solution was subjected to ODS-HPLC. The chromatographic conditions for ODS-HPLC were as follows: pump: LC-6AD (Shimadzu), column: InertSustein C18 (20 mm id x 250 mm, 5 μm, GL Sciences), mobile phase: acetonitrile:water = 5:95 (v / v), flow rate: 10 mL / min, column temperature: 40 °C, detector: SPD-20A (Shimadzu), detection wavelength: 220 nm. The fraction containing DON was dried under reduced pressure to obtain 69 mg of DON (colorless candy-like solid) represented by the following formula (1). [ka]

[0041] Synthesis of 2-2.3-acetyl-DON To a solution of DON (145 mg) in pyridine (10 mL), n-butylboronic acid (498 mg) was added and stirred at room temperature overnight. Then, a solution of acetic anhydride (0.92 mL) in pyridine (4 mL) was added and stirred at 55°C for 3.5 hours. Methanol (2 mL) was added to the reaction solution, which was then concentrated and purified by reversed-phase HPLC. The fraction containing the target product was freeze-dried to obtain 3-acetyl-DON (142 mg) represented by the following formula (5) as a white powder. [ka] [Table 1]

[0042] 3. Synthesis of derivatives in which a carboxy group has been introduced via a linker to DON or 3-acetyl-DON 3-1.DON derivative (DON-Linker-COOH) The modification reaction to DON was carried out in two separate vessels. "First container": DON (21 mg) and 1-methylimidazole (1.7 μL) were added to a solution of glutaric anhydride (8.2 mg) in CHCl3 (1.8 mL). "Second vessel": DON (200 mg) and 1-methylimidazole (16 μL) were added to a solution of glutaric anhydride (77 mg) in CHCl3 (17 mL). Both vessels were stirred at room temperature for 10 days, and the reaction solutions were combined, concentrated, and purified by reversed-phase HPLC. The fraction containing the target product was freeze-dried to obtain DON-linker-COOH (15-O-(4-carboxybutyryl)DON) (51 mg) represented by the following formula (3) as a white powder. [ka] [Table 2]

[0043] 3-2.3-acetyl-DON derivatives (3-acetyl-DON -Linker-COOH) A solution of glutaric anhydride (206 mg) and N,N-dimethylaminopyridine (49 mg) in CHCl3 (20 mL) was added to 3-acetyl-DON (122 mg) and stirred at room temperature for 3 hours. Then, glutaric anhydride (206 mg) and N,N-dimethylaminopyridine (49 mg) were added and stirred for another 2.5 hours. The reaction solution was concentrated and purified by reversed-phase HPLC. The fraction containing the target product was freeze-dried to obtain 3-acetyl-DON-Linker-COOH (105 mg) represented by the following formula (6) as a white powder. [ka] [Table 3]

[0044] 4. Preparation of conjugates of DON derivatives or 3-acetyl-DON (3-Ac-DON) derivatives with proteins The synthesized DON-Linker-COOH and 3-Ac-DON-Linker-COOH were covalently linked to the primary amine at the ε position of L-lysine residues of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and horseradish peroxidase (HRP) by the activated ester method to obtain immunogens (DON-KLH conjugates, 3-Ac-DON-KLH conjugates), antigens for indirect competitive ELISA (DON-BSA conjugates, 3-Ac-DON-BSA conjugates), and antigens for direct competitive ELISA (HRP-labeled DON, HRP-labeled 3-Ac-DON), respectively. First, 5.0 μmol of each derivative was dissolved in 100 μL of DMSO. Next, 6.0 μmol of N-hydroxysuccinimide (NHS) dissolved in 5 μL of DMSO was added to these solutions, and 6.0 μmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dissolved in 10 μL of DMSO was added and mixed. After standing at room temperature for 1.5 hours to react, 10 mg each of KLH, BSA, and HRP dissolved in 1 mL of 10 mmol / L phosphate buffer (pH 7.0) was added to this reaction solution, and the reaction was again carried out at room temperature for 1.5 hours. After the reaction was completed, the mixture was dialyzed against phosphate buffer (PBS) containing 150 mmol of sodium chloride to prepare DON-KLH conjugate, 3-Ac-DON-KLH conjugate, DON-BSA conjugate, and 3-Ac-DON-BSA conjugate. On the other hand, HRP-labeled DON and HRP-labeled 3-Ac-DON were purified by gel filtration (Sephadex G-25, fine grade, Cytiva).

[0045] 5. Immunization with DON-KLH conjugate or 3-Ac-DON-KLH conjugate Immunization was performed by preparing the DON-KLH conjugate and the 3-Ac-DON-KLH conjugate at a concentration of 2 mg / mL (PBS), emulsifying and mixing with an equal amount of Freund's complete adjuvant, and then injecting 100 μL of the mixture intraperitoneally into mice (Balb / c, 7-week-old, female). Thereafter, booster immunization was performed three times every two weeks, with 1 / 4 the amount (25 μg) of the initial immunization amount emulsified and mixed with Freund's incomplete adjuvant. One week after the booster immunization, partial blood was collected from the tail vein to prepare antiserum. Antiserum obtained by immunization with the DON-KLH conjugate was used as anti-DON polyclonal antibody (PoAb), and antiserum obtained by immunization with the DON-KLH conjugate was used as anti-NIV PoAb. In addition, mice three days after the booster immunization were used for the production of monoclonal antibodies (MoAb). For anti-3-Ac-DON antibodies, only MoAb production was performed.

[0046] 6. Construction of direct ELISA and confirmation of antibody titer of antiserum The antibody titers of anti-DON antibodies (PoAb, MoAb) were confirmed by direct ELISA. Anti-mouse IgG goat antibody (Thermo Fisher Scientific) diluted to 4 μg / mL with PBS was added to each well of a 96-well microtiter plate at 100 μL / well, and then the plate was left to stand overnight at 4°C to immobilize the antibody. After removing the liquid by aspiration, 300 μL / well of PBS containing 0.4% BSA (hereinafter referred to as blocking solution) was added for blocking at room temperature for 1 hour. The wells were washed once with PBS containing 0.02% Tween 20 (hereinafter referred to as washing solution), and anti-DON antibodies serially diluted with PBS containing 0.2% BSA (hereinafter referred to as antibody dilution solution) were added at 100 μL / well and reacted at room temperature for 1 hour. After washing three times with washing solution, HRP-labeled DON diluted to 20 ng / mL with antibody dilution solution was added at 100 μL / well and reacted at room temperature for 1 hour. After washing three times with the washing solution, the plate was colored with 100 μL of 0.1 mol / L sodium acetate buffer (pH 5.5) containing 100 μg / mL 3,3',5,5'-tetramethylbenzidine and 0.006% hydrogen peroxide (hereinafter referred to as the color-developing substrate solution), and after 10 minutes, the color development was stopped with an equal volume of 0.5 mol / L sulfuric acid, after which the absorbance at 450 nm was measured. From the results obtained, the dilution ratio of the anti-DON antibody that resulted in half the absorbance at saturation was defined as the antibody titer. For anti-3-Ac-DON MoAb, the same procedure was carried out except that HRP-labeled 3-Ac-DON was used at 20 ng / mL instead of HRP-labeled DON, and the antibody titer was obtained.

[0047] 7. Constructing a Direct Competitive ELISA In the direct competitive ELISA using anti-DON antibody, anti-mouse IgG goat antibody was immobilized and blocked in the same manner as in the direct ELISA. Anti-DON antibody prepared to a dilution factor showing the antibody titer was added at 100 μL / well and reacted at room temperature for 1 hour. Meanwhile, DON or its analogue standard solution serially diluted with 10% methanol was mixed with an equal amount of HRP-labeled DON (40 ng / mL). This mixture was added to each well after washing three times at 100 μL / well. After reacting at room temperature for 1 hour, washing, color development, and absorbance were measured in the same manner as in the direct ELISA. The anti-3-Ac-DON MoAb was also performed in the same manner, except that HRP-labeled 3-Ac-DON was used at 40 ng / mL instead of HRP-labeled DON.

[0048] 8. Indirect ELISA and indirect competitive ELISA Indirect ELISA and indirect competitive ELISA were used for screening when producing MoAbs. Indirect ELISA for the preparation of anti-DON MoAb was carried out as follows. First, the DON-BSA conjugate was prepared at 1 μg / mL in PBS, added to a 96-well microtiter plate at 50 μL / well, and then immobilized by standing overnight at 4°C. After removing the liquid by suction, 200 μL / well of blocking solution was added and blocked at room temperature for 1 hour. After washing with a washing solution, 50 μL / well of hybridoma culture supernatant was added and reacted at room temperature for 1 hour. After washing three times with a washing solution, 50 μL / well of HRP-labeled anti-mouse IgG(γ) goat antibody (American Qualex) diluted 5000-fold with antibody diluent was added and reacted for 1 hour. After washing three times with a washing solution again, a color-developing substrate solution was added to develop color, and the absorbance at 450 nm was measured.

[0049] On the other hand, indirect competitive ELISA for the preparation of anti-DON MoAb was carried out as follows. First, the DON-BSA conjugate was prepared in PBS at 25 ng / mL and BSA at 100 ng / mL, and added to a 96-well microtiter plate at 50 μL / well, and then immobilized by standing at 4°C overnight. After removing the liquid by suction, 200 μL / well of blocking solution was added and blocked at room temperature for 1 hour. After washing with washing solution, an equal mixture of hybridoma culture supernatant and DON dissolved in 10% methanol was added at 50 μL / well and competitively reacted at room temperature for 1 hour. After washing three times with washing solution, 50 μL / well of HRP-labeled anti-mouse IgG(γ) goat antibody (American Qualex) diluted 5000-fold with antibody diluent was added and reacted for 1 hour. After washing three times with washing solution again, a color-developing substrate solution was added to develop color, and the absorbance at 450 nm was measured.

[0050] 9. Generation of Anti-DON MoAb Monoclonal antibodies were produced by conventional methods. First, spleen cells taken from mice 3 days after the final immunization were used for cell fusion. The spleen cells taken out in RPMI1640 medium were washed three times with RPMI1640 medium while removing large solids with a mesh, and then mixed with mouse myeloma cells P3U1 at a cell number ratio of 5:1 (spleen cells: myeloma cells), and centrifuged (1300 rpm, 5 minutes) to collect the cell sediment. 1 mL of 50% polyethylene glycol (molecular weight 1500) pre-warmed to 37°C was added to this cell sediment to fuse the cells. The cell fusion was stopped by gradually adding 30 mL of RPMI1640 medium supplemented with 10% fetal bovine serum (hereinafter referred to as FBS) (hereinafter referred to as "RPMI1640 / 10% FBS medium"). The fused cells were suspended in HAT medium (RPMI1640 / 10% FBS medium supplemented with 100 μmol / L hypoxanthine, 0.4 μmol / L aminopterin, and 16 μmol / L thymidine), then dispensed into four 96-well microplates and cultured at 37°C in the presence of 5% CO2 for 10-14 days. After culture, the antibody reactivity in the wells of the hybridomas that had produced colonies was examined using indirect ELISA and indirect competitive ELISA.

[0051] 10. Reactivity of anti-DON MoAb There were many hybridomas that produced antibodies reactive to DON, but among them, hybridomas that showed high reactivity in indirect competitive ELISA were cloned by limiting dilution method to produce monoclonal antibody-producing cells (MDN8 and MDN31). The culture supernatants of these cells were used as monoclonal antibodies derived from MDN8 (hereafter referred to as MDN8 MoAb) and MDN31 (hereafter referred to as MDN31 MoAb). The subclasses of both antibodies were MDN8: IgG2a λ, MDN31: IgG2a κ. The reactivity of MDN8 MoAb and MDN31 MoAb with DON was examined by direct competitive ELISA. As shown in Fig. 1, MDN8 MoAb reacted quantitatively with DON in the range of 3-200 ng / mL and with 15-Ac-DON in the range of 3-85 ng / mL, while MDN31 MoAb reacted quantitatively with DON in the range of 2-200 ng / mL and with 15-Ac-DON in the range of 3-120 ng / mL. These results showed high reactivity with DON and 15-Ac-DON, similar to anti-DON PoAb. On the other hand, they showed no reactivity with 3-Ac-DON or nivalenol (NIV), another trichothecene mycotoxin.

[0052] 11. Preparation and reactivity of anti-3-Ac-DON MoAb Anti-3-Ac-DON MoAb was also produced under the same conditions as anti-DON MoAb. Screening was performed using direct ELISA and direct competitive ELISA. Hybridomas that showed high reactivity to 3-Ac-DON in direct competitive ELISA were cloned by limiting dilution method to obtain MoAb-producing cells (M3ADN27 and M3ADN28). The culture supernatants of these cells were used as M3ADN27 MoAb and M3ADN28 MoAb, and their reactivity with 3-Ac-DON, DON, and 15-Ac-DON was examined by direct competitive ELISA. As shown in Figure 2, M3ADN27 MoAb reacted quantitatively with 3-Ac-DON in the range of 11-120 ng / mL, 15-Ac-DON in the range of 210-2300 ng / mL, and DON in the range of 3300-10000 ng / mL, while M3ADN28 MoAb reacted quantitatively with 3-Ac-DON in the range of 13-140 ng / mL, 15-Ac-DON in the range of 180-1900 ng / mL, and DON in the range of 2000-10000 ng / mL, and showed particularly high reactivity with 3-Ac-DON. However, they showed no reactivity with NIV.

[0053] 12. Determination of the amino acid sequence of the antibody CDR region The amino acid sequences of the variable regions of the heavy and light chains of the anti-DON monoclonal antibody MDN31 MoAb and the anti-3-Ac-DON monoclonal antibodies M3ADN27 MoAb and M3ADN28 MoAb were determined. Details were as follows: The procedures were carried out according to the instructions for each kit used. mRNA was extracted from monoclonal antibody-producing cells using an RNeasy mini kit (QIAGEN). Then, cDNA fragments of the light and heavy chains of each monoclonal antibody were amplified by the 5'RACE method using a 5' / 3' RACE Kit, 2nd Generation version 10 (Roche). The primers used in the 5'RACE method are shown in Table 4. The amplified single-stranded cDNA fragments were purified using a High Pure PCR Product Purification Kit (Roche). After purification, poly(A) was added again using a 5' / 3' RACE Kit, 2nd Generation, and 1st PCR and 2nd PCR were performed. After agarose gel electrophoresis, the corresponding DNA fragments were excised and purified using the illustra GFX PCR DNA and Gel Band Purification Kit. The purified DNA fragments were cloned into pGEM-T Easy Vector (Promega), and the DNA sequence was analyzed to determine the nucleotide sequences of the light and heavy chains of the antibodies. From these sequences, the amino acid sequences of the CDRs (CDR1, CDR2, CDR3) of the heavy chain variable region and the CDRs (CDR1, CDR2, CDR3) of the light chain variable region of each monoclonal antibody, as well as the amino acid sequences of the heavy chain variable region and the light chain variable region were determined. [Table 4] [Industrial Applicability]

[0054] The antibody of the present invention is useful for investigating the actual state of grain contamination by trichothecene fungi, and is therefore expected to be used in the agricultural field.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (A-1) or (A-2) below: (A-1) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6; (A-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:8; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:9; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, which satisfies either (a) or (b) below: (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:

14.

3. An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (B-1) or (B-2) below: (B-1) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19, and Having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20; (B-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:22; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:23; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:25, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

26.

4. The antibody or antigen-binding fragment thereof according to claim 3, which satisfies either (a) or (b) below: (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:27, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:

28.

5. An antibody or an antigen-binding fragment thereof, characterized in that the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (C-1) or (C-2) below. (C-1) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:29; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 30; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:31; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 32; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:33, and Having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 34; (C-2) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 36; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:37; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:38; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:39, and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

40.

6. The antibody or antigen-binding fragment thereof according to claim 5, which satisfies either (a) or (b) below: (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 41, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 42; (b) having a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:41, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:

42.

7. Fab, Fab', F(ab') 2 10. The antigen-binding fragment of claim 1, 3 or 5, which is an Fv, a single chain antibody, an scFv, an scFv dimer or a dsFv.

8. A kit for detecting a trichothecene mycotoxin, comprising an antibody or antigen-binding fragment of any one of claims 1, 3 or 5.

9. An antibody that binds to 3-acetyldeoxynivalenol (3-Ac-DON), which competitively inhibits the binding of the antibody described in claim 1 or claim 2 to 3-Ac-DON, or an antigen-binding fragment thereof.

10. An antibody or an antigen-binding fragment thereof that binds to deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-Ac-DON), and competitively inhibits the binding of the antibody described in claim 3 to DON and 15-Ac-DON.

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