WO2023106250A1 - Antigen for preparing antibody against trichothecene mycotoxin, and use thereof - Google Patents

Abstract

The present invention addresses the problem of providing an antigen suitable for the preparation of an antibody recognizing a trichothecene derivative, a method of producing an antibody against this antigen, and this antibody. More specifically, the present invention pertains to a method for producing an antibody, said method comprising immunization with a trichothecene derivative represented by formula (I) as an antigen. [In formula (I): R₂ represents a hydrogen or a hydroxyl group; and X represents a carrier protein.]

Description

Antigens for the preparation of antibodies against trichothecene mycotoxins and uses thereof

The present invention relates to the preparation of antibodies against trichothecene mycotoxins, particularly deoxynivalenol, nivalenol, and their acetylated forms and glycosides.

Fusarium genus fungi such as Fusarium graminearum infect barley such as wheat and barley, and cereals such as corn, and cause Fusarium mold, which is difficult to control. In addition, these Fusarium mold fungi can produce trichothecene toxins such as deoxynivalenol (DON) and nivalenol (NIV), so humans who consume Fusarium-contaminated grains and It is known to cause poisoning symptoms such as diarrhea, vomiting, and disorders of the digestive tract, etc. in livestock, and to cause health problems such as weight loss when the symptoms become chronic. Therefore, rapid detection of Fusarium mold is required at production sites.

HPLC, GC/MS, and LC/MS methods are used to detect trichothecene toxins such as DON and NIV, but these methods are not suitable for processing many samples in a short time. Not suitable. Therefore, it is desirable to efficiently measure many samples by immunological quantification methods such as ELISA. Immunoassay kits for DON are currently on the market, and specifications and standards have been established. On the other hand, although the toxicity of NIV is known to be equal to or higher than that of DON, no kit capable of detecting NIV is available on the market, and standards and the like have not been established. Therefore, it is important to develop a simple immunoassay especially for NIV. As for DON, it is still important to obtain more optimized antibodies in terms of reaction specificity and the like.

There are many reports on antibodies against DON, including antibodies against its acetylated form. So far, for example, polyclonal antibodies recognizing DON, 3-Ac-DON and 15-Ac-DON (Non-Patent Document 1), monoclonal antibodies recognizing DON, 3-Ac-DON and some trichothecene toxins There are reports on antibodies (Non-Patent Document 2), monoclonal antibodies recognizing DON and 3-Ac-DON (Non-Patent Document 3), and the like.
For NIV, antibodies that cross-react with DON and 3-Ac-DON (Non-Patent Document 4), antibodies that recognize 3,4,15-triAc-NIV, 3,4,15-triAc-NIV and 3 Although there is a report on an antibody that recognizes ,15-diAc-NIV (Patent Document 1), there is no report on an antibody that specifically recognizes NIV.

In light of the above circumstances, although DON detection kits using antibodies are currently on the market, there is still a need for improved anti-DON antibodies in terms of reaction specificity and reaction affinity. ing. On the other hand, as for NIV, since there are no NIV-specific antibodies yet, the need for development of anti-NIV antibodies is very high.

WO2001/018196

In view of the above circumstances, the present invention provides a method for preparing an antibody that recognizes DON, NIV, and trichothecene derivatives including their acetylated forms and glycosides, an antigen suitable for use in the method, and an antibody against the antigen. The challenge is to provide

Among the trichothecene derivatives, the inventors first worked on the production of antibodies necessary for the development of DON and NIV immunoassays. DON and NIV have multiple OH groups for protein binding. In the structural formula of the trichothecene derivative below, DON is R ₁ at position 3, R ₄ at position 7, R ₃ at position 15 is -OH, R ₂ at position 4 is -H, and NIV is at position 3. , R ₂ at position 4, R ₄ at position 7 _, and R ₃ at position 15 are all -OH.

Therefore, when trying to introduce a carrier protein for imparting immunogenicity to trichothecene derivatives such as DON and NIV, it randomly binds to the OH group, and the carrier protein binds while maintaining the original three-dimensional structure. was considered difficult.
In consideration of these circumstances, the present inventors developed a synthetic method of introducing a linker (a linker containing a terminal carboxyl group) for selectively binding a carrier protein only to the 15-position of DON and NIV. Found it. Furthermore, DON and NIV having a linker at position 15 synthesized by this selective synthesis method and DON and NIV having a carrier protein introduced into the linker portion at position 15 of NIV are prepared, and monoclonal antibodies are prepared using these DON and NIV as antigens. As a result, we succeeded in producing antibodies specific to DON and NIV.

That is, the present invention is a method for producing an antibody, comprising immunizing with a trichothecene derivative represented by the following formula (I) as an antigen.

(In formula (I), _R2 represents a hydrogen or hydroxyl group, and X represents a carrier protein.)
The present invention also provides an antibody against the trichothecene derivative represented by formula (I) above, and a kit for detecting a trichothecene mycotoxin containing the antibody.
Furthermore, the present invention is an immunogenic composition comprising the trichothecene derivative represented by the above formula (I) as an antigen.

Furthermore, the present invention is a trichothecene derivative represented by the following formula (III).

(In formula (III), _R2 represents a hydrogen or hydroxyl group, and Y represents a functional group for cross-linking proteins or a linker containing the functional group.)

The present invention provides a more efficient method than the conventionally reported methods for producing antibodies against trichothecene mycotoxins.

In addition, according to the present invention, it is possible to provide antibodies that exhibit different specificity and/or affinity from known antibodies against trichothecene mycotoxins.

Investigation of the reaction characteristics of anti-DON PoAb to DON and 15-Ac-DON. The vertical axis indicates the ratio of the OD of the sample to the OD of the reaction without DON, and the horizontal axis indicates the concentration of DON or 15-Ac-DON added to the reaction wells. Investigation of reaction characteristics of anti-NIV PoAb to NIV. The vertical axis indicates the ratio of the OD of the sample to the OD of the reaction without NIV and 15-Ac-NIV, and the horizontal axis indicates the concentration of NIV or 15-Ac-NIV added to the reaction wells. Investigation of the reaction characteristics of anti-DON MoAb MDN8 and MDN31 to DON and 15-Ac-DON. The vertical axis indicates the ratio of the OD of the sample to the OD of the reaction without DON and 15-Ac-DON, and the horizontal axis indicates the concentration of DON or 15-Ac-DON added to the reaction wells. Investigation of the reaction characteristics of anti-NIV MoAb MNV80 and MNV87 to NIV and 15-Ac-NIV. The vertical axis indicates the ratio of the OD of the sample to the OD of the reaction without NIV and 15-Ac-NIV, and the horizontal axis indicates the concentration of NIV or 15-Ac-NIV added to the reaction wells. Investigation of the reaction characteristics of anti-NIV MoAb NIV150 and NIV464 to NIV and 15-Ac-NIV. The vertical axis indicates the ratio of the OD of the sample to the OD of the reaction without NIV and 15-Ac-NIV, and the horizontal axis indicates the concentration of NIV or 15-Ac-NIV added to the reaction wells. Investigation of the reaction characteristics of anti-3-Ac-DON MoAb M3ADN27 and M3ADN28 to DON, 3-Ac-DON and 15-Ac-DON. The vertical axis shows the ratio of sample OD to the OD of reactions without DON, 3-Ac-DON and 15-Ac-DON, and the horizontal axis shows DON, 3-Ac-DON or 15-acid added to the reaction wells. - Indicates the concentration of Ac-DON.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated.
The first embodiment is a method for producing an antibody, which comprises immunizing a trichothecene derivative represented by the following formula (I) as an antigen (hereinafter referred to as "antibody producing method of the present embodiment" also described).

(In formula (I), _R2 represents a hydrogen or hydroxyl group, and X represents a carrier protein.)

An antibody produced by the method for producing an antibody according to this embodiment is an antibody that specifically binds to a trichothecene mycotoxin. Here, the trichothecene mycotoxin is a general term for fungal toxins having a trichothecene ring. For example, in the following formula (II),

DON where _R2 is hydrogen, _R1 and _R3 are hydroxyl groups, 3-Ac-DON where _{R2 is} hydrogen, R1 is an acetoxy group, _R3 is a hydroxyl group, _R2 is hydrogen, _R1 is a hydroxyl group, R 15-Ac-DON in which ₃ is an acetoxy group, _R2 is hydrogen, _R1 is a glucosyloxy group, _R3 is a hydroxyl group, DON-3-O-glucoside, _R2 , _R1 and _R3 are hydroxyl groups Examples include NIV, fusalenone-X (4-Ac-NIV) in which _R2 is an acetoxy group and _R1 and _R3 are hydroxyl groups.

In addition, the method for producing an antibody according to this embodiment may include a step of binding carrier protein X via Y to position 15 of the trichothecene derivative represented by the following formula (III).

Here, in formula (III), R ₂ represents hydrogen or a hydroxyl group, and Y represents a functional group for cross-linking proteins or a linker containing the functional group.

This embodiment is characterized in that an antibody is produced using, as an antigen, a trichothecene derivative in which a carrier protein (represented by X in formula (I)) is bound to position 15, as represented by formula (I). ing. In this embodiment, a carrier protein is a protein that is used to conjugate a hapten to confer immunogenicity. The carrier protein used in the present embodiment may be any protein that can be appropriately selected by those skilled in the art, and is not particularly limited. Examples include keyhole limpet hemocyanin (KLH), ovalbumin ( OVA), rabbit serum albumin (RSA), bovine serum albumin (BSA), human serum albumin (HSA), thyroglobulin (TG), and immunoglobulins.

In addition to the carrier protein, X in formula (I) has a functional group for binding the carrier protein to position 15 of the trichothecene skeleton corresponding to Y in formula (III), or a linker containing the functional group. may be included. Here, for example, when a carboxyl group is selected as the functional group, Y corresponds to a linker containing the carboxyl group as well as the carboxyl group. In this embodiment, Y may be a carboxyl group, a maleimide group, a thiol group, an amino group, an azide group, or a linker containing these functional groups, but a preferred functional group is a carboxyl group. can. The linker containing a carboxyl group includes, for example, a linear, branched or cyclic C2-C10 acyloxy group containing a carboxyl group, preferably a 3-carboxypropionyloxy group, a 4-carboxybutyryloxy group. , a carboxy C3-C5 alkoxy group such as a 5-carboxypentanoyloxy group, more preferably a carboxy C4 alkoxy group such as a 4-carboxybutyryloxy group.

The antibody production method according to the present embodiment is characterized by using the trichothecene derivative represented by the above formula (I) as an antigen, and specific antibody preparation methods are well known in the art. It can be easily obtained by those skilled in the art by using the method.

A second embodiment is an antibody against a trichothecene derivative represented by the following formula (II) or an antigen-binding fragment thereof (hereinafter also referred to as an "antibody according to this embodiment" or an "antigen-binding fragment according to this embodiment"). do).

(In formula (II), _R1 represents a hydroxyl group, an acetoxy group or a glucosyloxy group, _R2 represents a hydrogen group, a hydroxyl group or an acetoxy group, and _R3 represents a hydroxyl group or an acetoxy group.)
More specifically, the antibody according to this embodiment is an antibody against DON, 3-Ac-DON and/or 15-Ac-DON, or NIV and/or fusalenone-X (4-Ac-NIV), DON It is an antibody against -3-O-glucoside.
The antibody according to this embodiment may be produced by any method, and may be an antibody produced by the antibody production method according to the first embodiment.

When the antibody according to this embodiment is a polyclonal antibody, for example, a mixture of an antigen and an adjuvant is administered to an immunized animal (such as, but not limited to, rabbits, goats, sheep, chickens, guinea pigs, mice, rats or pigs) can be prepared by injecting Typically, the antigen and/or adjuvant are injected subcutaneously or intraperitoneally multiple times into the immunized animal. Adjuvants include, but are not limited to, Freund's complete and monophosphoryl lipid A synthetic-trehalose dicorynomycolate (MPL-TMD). After immunization with the antigen, the antibody of interest can be purified from serum derived from the immunized animal by a standard method (for example, a method using Protein A-retained Sepharose or the like).

Moreover, when the antibody according to the present embodiment is a monoclonal antibody, it can be produced, for example, as follows. As used herein, the term "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population (an antibody population in which the amino acid sequences of the heavy and light chains that constitute the antibody are the same). and should not be construed as being limited to antibodies produced by a specific method (eg, hybridoma method, etc.).
Methods for producing monoclonal antibodies include, for example, the hybridoma method (Kohler and Milstein, Nature 256 495-497 1975) or the recombinant method (US Pat. No. 4,816,567). Furthermore, antibodies according to this embodiment may be isolated from phage antibody libraries (eg, Clackson et al., Nature 352 624-628 1991; Marks et al., J. Mol. Biol. 222 581-597 1991, etc.). More specifically, in the case of preparation using the hybridoma method, the preparation method includes, for example, the following four steps: (i) immunizing an immunized animal with an antigen, (ii) monoclonal antibody (iii) fusing the lymphocytes to immortalized cells; (iv) selecting cells that secrete the desired monoclonal antibody. Examples of animals to be immunized include mice, rats, guinea pigs, hamsters, and rabbits. After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusing agent such as polyethylene glycol or an electrofusion method to establish hybridoma cells. As fusion cells, for example, rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. A common technique uses parental cells that lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, aminopterin is added to the medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hybridomas. Hybridomas producing the desired antibody can be selected from the hybridomas thus obtained, and the monoclonal antibody of interest can be obtained from the culture medium in which the selected hybridoma grows according to conventional methods.
The hybridoma thus prepared can be cultured in vitro or cultured in vivo in the ascites fluid of mice, rats, guinea pigs, hamsters, etc., and the antibody of interest can be prepared from the culture supernatant or the ascites fluid.

A nanobody is a polypeptide consisting of the variable domain of the heavy chain of heavy chain antibody (VHH). Generally, human antibodies are composed of heavy and light chains, but camelids such as llamas, alpacas, and camels produce single-chain antibodies (heavy-chain antibodies) composed only of heavy chains. A heavy chain antibody can recognize and bind to a target antigen in the same way as a normal antibody consisting of heavy and light chains. The variable region of a heavy chain antibody is the smallest unit that has binding affinity for an antigen, and this variable region fragment is called a "nanobody". Nanobodies have high heat resistance, digestion resistance, and room temperature stability, and can be easily prepared in large amounts by genetic engineering techniques.
Nanobodies can be produced, for example, as follows. A camelid animal is immunized with an antigen, the presence or absence of the antibody of interest is detected in the collected serum, and cDNA is prepared from RNA derived from peripheral blood lymphocytes of the immunized animal in which a desired antibody titer is detected. A VHH-encoding DNA fragment is amplified from the resulting cDNA and inserted into a phagemid to prepare a VHH phagemid library. Desired nanobodies can be produced through several rounds of screening from the produced VHH phagemid library.

The antibody according to this embodiment may be a genetically engineered antibody. Examples of genetically modified antibodies include, but are not limited to, humanized antibodies and chimeric antibodies with human antibodies. A chimeric antibody is, for example, an antibody in which a variable region and a constant region derived from different animal species are linked (for example, an antibody in which a rat-derived antibody variable region is linked to a human-derived constant region) (for example, Morrison et al., Proc. Natl. Acad. Sci.

A humanized antibody is an antibody that has a human-derived sequence in the framework region (FR) and a complementarity-determining region (CDR) that consists of sequences derived from other animal species (eg, mouse). Humanized antibodies are first described in other animal species, here mouse, by grafting the CDRs from mouse-derived antibody variable regions into human antibody variable regions to reconstitute the heavy and light chain variable regions. Later, these humanized reshaped human antibody variable regions can be made by joining them to human antibody constant regions. Methods for producing such humanized antibodies are well known in the art (eg, Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033 1989).

The antigen-binding fragment according to this embodiment is a partial region of the antibody according to this embodiment, and is an antibody fragment that binds to a desired antigen. Examples of fragments include Fab, Fab', F (ab') ₂ , Fv (variable fragment of antibody), single chain antibody (heavy chain, light chain, heavy chain variable region, light chain variable region, nano antibody, etc.), scFv (single chain Fv), diabody (scFv dimers), dsFv (disulfide-stabilized Fv), and peptides at least partially containing the CDRs of the antibody according to this embodiment.

Fab is an antibody fragment with antigen-binding activity in which about half of the N-terminal side of the heavy chain and the entire light chain are bound by disulfide bonds, among the fragments obtained by treating the antibody molecule with the proteolytic enzyme papain. Preparation of Fab is carried out by treating antibody molecules with papain to obtain fragments, for example, constructing an appropriate expression vector into which DNA encoding Fab is inserted, and inserting this into an appropriate host cell (e.g., CHO cell, etc.). (mammalian cells, yeast cells, insect cells, etc.), and then expressing Fab in the cells.

F(ab') ₂ is an antibody fragment having antigen-binding activity that is slightly larger than a fragment obtained by treating an antibody molecule with a proteolytic enzyme pepsin and having Fab bound via a disulfide bond in the hinge region. is. F(ab') ₂ can be prepared by treating an antibody molecule with pepsin to obtain a fragment, or by forming a thioether bond or a disulfide bond with Fab. can be made.

Fab' is an antibody fragment having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab') ₂ . Fab' can also be produced by a genetic engineering technique like Fab and the like.

scFv 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. It is an antibody fragment having antigen-binding activity. scFv can be produced by obtaining cDNAs encoding the heavy chain variable region and light chain variable region of an antibody and using genetic engineering techniques.

A diabody is an antibody fragment in which scFv is dimerized and has bivalent antigen-binding activity. The bivalent antigen-binding activities may be the same antigen-binding activity or one of which may be different. Diabody obtains cDNAs encoding the heavy and light chain variable regions of an antibody, constructs a scFv-encoding cDNA by linking the heavy and light chain variable regions with a peptide linker, and genetically engineered method.

dsFv refers to polypeptides in which one amino acid residue in each of the heavy chain variable region and light chain variable region is substituted with a cysteine residue, and are linked via a disulfide bond between the cysteine residues. Amino acid residues to be substituted for cysteine residues can be selected based on antibody tertiary structure prediction. A dsFv can be produced by genetic engineering techniques by obtaining cDNAs encoding the heavy chain variable region and light chain variable region of an antibody and constructing a DNA encoding the dsFv.

A peptide containing CDRs is constructed to contain at least one region or more of CDRs (CDR1-3) of a heavy or light chain. Peptides containing multiple CDRs can be joined directly or via suitable peptide linkers. The CDR-containing peptides are inserted into an expression vector by constructing DNA encoding the heavy or light chain CDRs of the antibody. The type of vector is not particularly limited, and may be appropriately selected depending on the type of host cell into which it is subsequently introduced. In order to express these as antibodies, they can be produced by introducing them into suitable host cells (eg, mammalian cells such as CHO cells, yeast cells, insect cells, etc.). Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

Human antibodies (fully human antibodies) generally have the same structures as human antibodies in the structure of the hypervariable region, which is the antigen-binding site of the V region, other parts of the V region, and the constant region. . Human antibodies can be easily produced by those skilled in the art using known techniques. Human antibodies can be obtained, for example, by methods using human antibody-producing mice that have human chromosome fragments containing human antibody H chain and L chain genes (for example, Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97, 722 -727 2000., etc.) or a method of obtaining phage display-derived human antibodies selected from a human antibody library (see, for example, Siriwardena et al., Opthalmology, 109, 427-431 2002.). .

A multispecific antibody may be constructed using the antigen-binding fragment according to this embodiment. Multispecific means having binding specificities for two or more antigens, for example, protein forms comprising monoclonal antibodies or antigen-binding fragments that have binding specificities for two or more antigens mentioned. This is done by a person skilled in the art according to known techniques. Methods for constructing multispecificity include the technique of constructing asymmetric IgG by protein engineering so that two different types of antibody heavy chain molecules form a heterodimer, and the low-molecular-weight antigen-binding technique obtained from antibodies. A number of techniques have been developed that can be classified into techniques for linking fragments together or linking to another antibody molecule. Examples of specific construction methods can be referred to, for example, the following documents. Kontermann et al., Drug Discovery Today, 20, 838-847 2015.

A third embodiment is an immunogenic composition comprising a trichothecene derivative represented by the following formula (I) as an antigen.

(In formula (I), _R2 represents a hydrogen or hydroxyl group, and X represents a carrier protein.)
Immunogenicity in this embodiment means the ability to induce a specific immune response in vivo against the trichothecene derivative represented by the above formula (I). In addition to the trichothecene derivative represented by the above formula (I), the immunogenic composition according to this embodiment includes any substance, such as an adjuvant, as long as it is useful for inducing an immune response in vivo. It may be

The adjuvant contained in the immunogenic composition of the present embodiment is not particularly limited. ), oil-in-water emulsion, monophosphoryl lipid A (MPL), potassium alum, adjuvants containing saponin, and the like.

A fourth embodiment is a kit for detecting a trichothecene mycotoxin, the kit comprising at least the antibody produced by the method according to the first embodiment or the antibody according to the second embodiment. be.
In addition to the above antibodies, the kit according to the present embodiment includes reagents necessary for detecting trichothecene mycotoxins, devices (for example, immunochromatographs and immunosensors for detecting antigens), instruments (plates for ELISA etc.) may be included.

A fifth embodiment is a trichothecene derivative represented by the following formula (III).

(In formula (III), _R2 represents a hydrogen or hydroxyl group, and Y represents a functional group for cross-linking proteins or a linker containing the functional group.)
A fifth embodiment is a trichothecene derivative in which a functional group for cross-linking proteins or a linker containing the functional group (Y in formula (III)) is bound to position 15 of formula (III) above.
In the above formula (III), Y is a functional group that is reactive with specific functional groups such as primary amines and sulfhydryls of proteins, or reactive with specific functional groups such as primary amines and sulfhydryls. It is a linker with a certain functional group at the end. Here, the functional group reactive with a specific functional group such as primary amine or sulfhydryl is not particularly limited, but examples thereof include carboxyl group, maleimide group, amino group and azide group. In addition, Y may be a linker having these functional groups at the end (that is, the terminal side that forms a crosslink with the protein), and the linker portion is not particularly limited. It may be a hydrogen chain (which may optionally contain oxygen, nitrogen, etc.), or the like.

When this specification is translated into English and contains the words "a", "an" and "the" in the singular, the singular as well as the plural unless the context clearly indicates otherwise. shall also include those of
EXAMPLES The present invention will be further described below with reference to Examples, but these Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Cultivation of strains producing deoxynivalenol (DON) and nivalenol (NIV) Potato dextrose agar for the DON-producing stock strain Fusarium graminearum TSY 0341 and the NIV-producing stock strain Fusarium kyushuense Fn-2B-1, respectively. and cultured at 20°C for 1 week. As a medium for toxin production, 50 g of commercially available rolled barley was placed in a 500 mL triangular kolben, an equal amount of purified water was further added, and the mixture was allowed to stand overnight in a refrigerator. After standing, heat at 121 ° C for 60 minutes and return to room temperature (medium volume: about 100 g), inoculate the bacteria cultured in the potato dextrose agar above into the toxin production medium, and after 2 weeks grow on the toxin production medium. cultures were used for the extraction of DON and NIV.

2. Preparation of DON, NIV and 3-acetyl-DON 2-1. Purification of DON DON was extracted with 85% acetonitrile (v/v) (400 mL, 3 times) from the culture (approximately 400 g) grown in the medium for toxin production. The amount of DON contained in this extract (about 1.2 L) was about 244 mg. This extract (about 1.2 L) was concentrated under reduced pressure and then washed three times with an equal volume (about 900 mL) of n-hexane. The aqueous layer (about 900 mL) was applied to a synthetic adsorption resin (DIAION HP20, 300 mL, 6.0 cm i.d. × 9.5 cm, Mitsubishi Chemical), washed with water (900 mL), and DON was eluted with methanol (450 mL). let me This eluate was concentrated under reduced pressure. From this concentrate, DON was extracted three times with equal volumes (approximately 100 mL) of ethyl acetate. After the ethyl acetate layer was dried under reduced pressure, it was loaded onto a silica gel column (2 cm id x 20 cm; Silica gel 60, Merck), and washed with dichloromethane/methanol mixture (20:1, 10:1) and then with methanol. Expanded. The resulting DON-containing eluate (dichloromethane:methanol=10:1) was combined and concentrated under reduced pressure. The liquid was subjected to ODS-HPLC. Chromatographic conditions for ODS-HPLC are pump: LC-6AD (Shimadzu), column: InertSustein C18 (20 mm id × 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. By drying the fraction containing DON under reduced pressure, 69 mg of DON (a colorless candy-like solid substance) represented by the following formula (1) was obtained.

2-2. Purification of NIV NIV was extracted by adding 5 volumes of purified water (3.5 L) to the culture (approximately 700 g) grown in toxin production medium. The centrifuged supernatant was mixed with HP20 (300 mL) for adsorption. Thereafter, washing by adding 300 mL of purified water was repeated 10 times. The washed HP20 was packed in a column (6.0 cm i.d. × 9.5 cm), washed with 300 mL of purified water, and NIV was eluted with 450 mL of methanol. After concentrating to dryness, it was suspended in 1 mL of ethyl acetate/methanol mixed solution (8:1), 1 mL of silica gel was added, and ultrasonic waves were applied to disperse and adsorb NIV to the gel. This silica gel was packed in a silica gel column and developed with an ethyl acetate/methanol mixture (8:1). The resulting fractions containing NIV were combined and concentrated to dryness. It was dissolved in 800 µL of 5% acetonitrile (water: acetonitrile = 95:5) and allowed to stand at room temperature for precipitation to obtain 140 mg of NIV represented by the following formula (2).

2-3. Synthesis of 3-acetyl-DON To a solution of DON (145 mg) in pyridine (10 mL) was added n-butylboronic acid (498 mg), stirred overnight at room temperature, and then acetic anhydride (0.92 mL) was added. in pyridine (4 mL) was added, and the mixture was stirred at 55°C for 3.5 hours. Methanol (2 mL) was added to the reaction mixture, and the mixture was concentrated and purified by reverse-phase HPLC. Fractions containing the desired product were freeze-dried to obtain 3-acetyl-DON (142 mg) represented by the following formula (5) as a white powder.

3. Synthesis of derivatives in which a carboxyl group is introduced into DON, NIV or 3-acetyl-DON via a linker 3-1. DON derivative (DON-Linker-COOH)
The modification reaction to DON was performed in two separate vessels.
"First container"; DON (21 mg) and 1-methylimidazole (1.7 µL) were added to a CHCl ₃ (1.8 mL) solution of glutaric anhydride (8.2 mg).
"Second container"; DON (200 mg) and 1-methylimidazole (16 μL) were added to a CHCl ₃ (17 mL) solution of glutaric anhydride (77 mg). Both vessels were stirred at room temperature for 10 days and the combined reactions were concentrated and purified by reverse phase HPLC. Fractions containing the desired product were freeze-dried to obtain DON-linker-COOH (15-O-(4-carboxybutyryl)DON) (51 mg) represented by the following formula (3) as a white powder.

3-2. NIV derivative (NIV-Linker-COOH)
Glutaric anhydride (23 mg) and 4-dimethylaminopyridine (2.4 mg) were added to a solution of NIV (20 mg) in dimethylsulfoxide (1 mL)-CHCl ₃ (10 mL) and stirred at room temperature for 3 hours. bottom. 1% aqueous acetic acid was added to the reaction mixture for liquid separation, and the aqueous layer was purified by reverse-phase HPLC. Fractions containing the desired product were freeze-dried to obtain NIV-linker-COOH (15-O-(4-carboxybutyryl)NIV) (2.0 mg) represented by the following formula (4) as a white powder. This process was repeated to obtain 22 mg of NIV-linker-COOH.

3-3. 3-acetyl-DON derivative (3-acetyl-DON-Linker-COOH)
A CHCl ₃ (20 mL) solution of glutaric anhydride (206 mg) and N,N-dimethylaminopyridine (49 mg) was added to 3-acetyl-DON (122 mg), and the mixture was stirred at room temperature for 3 hours. After that, glutaric anhydride (206 mg) and N,N-dimethylaminopyridine (49 mg) were added, and the mixture was further stirred for 2.5 hours. The reaction was concentrated and purified by reverse phase HPLC. Fractions containing the desired product were freeze-dried to obtain 3-acetyl-DON-Linker-COOH (105 mg) represented by the following formula (6) as a white powder.

4. Preparation of conjugates of DON derivatives, NIV derivatives and 3-acetyl-DON (3-Ac-DON) derivatives with proteins Synthesized DON-Linker-COOH, NIV-Linker-COOH and 3-Ac-DON-Linker-COOH are covalently attached to the primary amines at the epsilon position of the L-lysine residues of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and horseradish peroxidase (HRP) by the activated ester method, yielding immunogens, respectively. (DON-KLH conjugate, NIV-KLH conjugate, 3-Ac-DON-KLH conjugate), antigen for indirect competitive ELISA (DON-BSA conjugate, NIV-BSA conjugate, 3-Ac-DON-BSA conjugate antibody) and antigens for direct competitive ELISA (HRP-labeled DON, HRP-labeled NIV, HRP-labeled 3-Ac-DON).
First, 5.0 μmol of each derivative were dissolved in 100 μL of DMSO. To these solutions was then added 6.0 μmol N-hydroxysuccinimide (NHS) dissolved in 5 μL DMSO and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) 6.0 dissolved in 10 μL DMSO. μmol was added and mixed. After reacting at room temperature for 1.5 hours, 10 mg each of KLH, BSA, and HRP dissolved in 1 mL of 10 mmol/L phosphate buffer (pH 7.0) was added to the reaction solution, and the mixture was cooled to room temperature again. and reacted for 1.5 hours. After completion of the reaction, dialyze against phosphate buffered saline (PBS) containing 150 mmol of sodium chloride to obtain DON-KLH conjugate, NIV-KLH conjugate, 3-Ac-DON-KLH conjugate and DON-BSA. Conjugates, NIV-BSA conjugate and 3-Ac-DON-BSA conjugate were prepared. On the other hand, HRP-labeled DON, HRP-labeled NIV, and HRP-labeled 3-Ac-DON were purified by gel filtration (Sephadex G-25, fine grade, Cytiva).

5. Immunization with DON-KLH Conjugate and NIV-KLH Conjugate After preparing and emulsifying and mixing with an equal amount of Freund's complete adjuvant, 100 μL of this was intraperitoneally inoculated into mice (Balb/c, 7 weeks old, female). After that, 1/4 amount (25 μg) of the initial immunization was emulsified and mixed with incomplete Freund's adjuvant for booster immunization, 3 times in total every 2 weeks. One week after the booster immunization, partial blood was collected from the tail vein and antiserum was prepared. Antiserum obtained by immunization with DON-KLH conjugate was designated as anti-DON polyclonal antibody (PoAb), and antiserum obtained by immunization with NIV-KLH conjugate was designated as anti-NIV PoAb. In addition, mice 3 days after the booster immunization were subjected to monoclonal antibody (MoAb) production. For the anti-3-Ac-DON antibody, only MoAb production was performed.

6. Construction of Direct ELISA and Confirmation of Antiserum Antibody Titers Anti-DON antibody (PoAb, MoAb) antibody titers were confirmed by direct ELISA. Add 100 μL/well of anti-mouse IgG goat antibody (Thermo Fisher Scientific) diluted to 4 μg/mL in PBS to each well of a 96-well microtiter plate, and leave overnight at 4°C. The antibody was immobilized by this. After removing the liquid by aspiration, 300 µL/well of PBS containing 0.4% BSA (hereinafter referred to as blocking solution) was added to block at room temperature for 1 hour. After washing the wells once with 0.02% tween20-added PBS (hereinafter referred to as washing solution), serially diluted anti-DON antibody with 0.2% BSA-added PBS (hereinafter referred to as antibody diluent) was added to 100 μL/well. Added in wells and allowed to react for 1 hour at room temperature. After washing three times with washing solution, 100 μL/well of HRP-labeled DON diluted to 20 ng/mL with antibody diluent was added and allowed to react at room temperature for 1 hour. After washing three times with washing solution, add 100 μg/ mL 3,3',5,5'-tetramethylbenzidine and 0.006% hydrogen peroxide in 0.1 mol/L sodium acetate buffer (pH 5.5) ( The color was developed with 100 μL of a chromogenic substrate solution, 10 minutes later, the color was stopped with an equal volume of 0.5 mol/L sulfuric acid, and the absorbance at 450 nm was measured. Based on the results obtained, the antibody titer was defined as the dilution factor of the anti-DON antibody that reduces the absorbance to 1/2 of the saturation.
Anti-NIV antibodies (PoAb, MoAb) were similarly tested except that HRP-labeled NIV was used at 50 ng/mL instead of HRP-labeled DON to obtain antibody titers. Anti-3-Ac-DON MoAb was also tested in the same manner except that HRP-labeled 3-Ac-DON was used at 20 ng/mL in place of HRP-labeled DON to obtain an antibody titer.

7. Construction of Direct Competitive ELISA In direct competitive ELISA using an anti-DON antibody, an anti-mouse IgG goat antibody was immobilized and blocked in the same manner as in the direct ELISA. An anti-DON antibody prepared at a dilution factor that indicates the antibody titer was added at 100 μL/well and allowed to react at room temperature for 1 hour. On the other hand, a standard solution of DON, NIV, or analogs thereof serially diluted with 10% methanol was mixed with HRP-labeled DON (40 ng/mL) in an equal amount. This mixed solution was added to each well after washing three times at 100 μL/well. After reacting at room temperature for 1 hour, the cells were washed and developed in the same manner as in direct ELISA, and the absorbance was measured. Anti-NIV antibodies were tested in the same manner, except that HRP-labeled NIV was used at 100 ng/mL instead of HRP-labeled DON. Anti-3-Ac-DON MoAb was also tested in the same manner, except that HRP-labeled 3-Ac-DON was used at 40 ng/mL instead of HRP-labeled DON.

8. Indirect ELISA and indirect competitive ELISA
Indirect ELISA and indirect competitive ELISA were used for screening during MoAb production.
Indirect ELISA during anti-DON MoAb generation was performed as follows. First, a DON-BSA conjugate was prepared with PBS to 1 μg/mL, added to a 96-well microtiter plate at an amount of 50 μL/well, and allowed to stand overnight at 4° C. for immobilization. After removing the liquid by aspiration, blocking was performed at room temperature for 1 hour by adding 200 μL/well of blocking liquid. After washing with a washing solution, 50 μL/well of hybridoma culture supernatant was added and allowed to react 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 (manufactured by American Qualex) diluted 5000-fold with antibody diluent was added and allowed to react for 1 hour. After washing again with the washing solution three times, a coloring substrate solution was added to develop color, and the absorbance at 450 nm was measured.
For indirect ELISA during anti-NIV MoAb production, the NIV-BSA conjugate was adjusted to 4 μg/mL in PBS and added to a 96-well microtiter plate at a volume of 100 μL/well. In addition, 100 μL/well of HRP-labeled anti-mouse IgG (H+L) goat antibody (manufactured by Thermo Fisher Scientific) diluted 8000-fold with an antibody diluent was added. Color development was performed according to direct ELISA, and absorbance at 450 nm was measured. Other steps were the same as indirect ELISA for anti-DON MoAb production.

On the other hand, indirect competitive ELISA during anti-DON MoAb production was performed as follows. First, prepare 25 ng/mL DON-BSA conjugate and 100 ng/mL BSA in PBS, add 50 μL/well to a 96-well microtiter plate, and allow to stand overnight at 4°C. It was solid-phased by this. After removing the liquid by aspiration, blocking was performed at room temperature for 1 hour by adding 200 μL/well of blocking liquid. After washing with a washing solution, an equal volume mixture of hybridoma culture supernatant and DON dissolved in 10% methanol was added at 50 μL/well and allowed to react competitively 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 (manufactured by American Qualex) diluted 5000-fold with antibody diluent was added and allowed to react for 1 hour. After washing again with the washing solution three times, a coloring substrate solution was added to develop color, and the absorbance at 450 nm was measured.
For indirect competitive ELISA for anti-NIV MoAb production, the NIV-BSA conjugate was adjusted to 4 μg/mL in PBS and added to a 96-well microtiter plate at a volume of 100 μL/well. In addition, 8000-fold diluted HRP-labeled anti-mouse IgG (H+L) goat antibody (manufactured by Thermo Fisher Scientific) was added in an amount of 100 µL/well. Color development was performed according to direct ELISA, and absorbance at 450 nm was measured. Other steps were the same as indirect competitive ELISA for anti-DON MoAb production.

9. Reactivity of anti-DON PoAbs with DON and its analogues The reactivity of anti-DON antibodies with DON and its analogues was examined by direct competitive ELISA. At that time, the range of DON capable of reacting quantitatively was defined as the concentration of DON showing 0.2 to 0.8 by competitive inhibition with respect to the absorbance in the reaction without DON. As is clear from FIG. 1, the anti-DON antibody quantitatively reacted with DON at 4.0-300 ng/mL, and 1.0-40 ng with 15-Ac-DON, in which the linker site of the DON derivative was acetylated. /mL showed high reactivity. On the other hand, as shown in Table 5, it showed no reactivity with 3-Ac-DON and NIV. Thus, the synthesized DON derivatives were effective in preparing highly reactive antisera against DON and 15-Ac-DON.

10. Reactivity of Anti-NIV PoAbs with NIV and Its Analogues The reactivity of anti-NIV PoAbs with NIV and its analogues was examined by direct competitive ELISA. As a result, as is clear from FIG. 2, anti-NIV PoAb reacted quantitatively with NIV at 12-950 ng/mL, and with 15-Ac-NIV, in which the linker site of the NIV derivative is acetylated, 1.7-950 ng/mL. It showed high reactivity at 85 ng/mL. On the other hand, as shown in Table 5, it showed no reactivity with DON, 3-Ac-DON, 15-Ac-DON and 4-Ac-NIV. Thus, the synthesized NIV derivatives were effective in preparing PoAbs exhibiting high reactivity against NIV.

11. Preparation of Anti-DON MoAb Monoclonal antibodies were prepared by a conventional method. First, for cell fusion, spleen cells removed from mice three days after the final immunization were used. While removing large solids with a mesh, the spleen cells taken out in RPMI1640 medium were washed three times with RPMI1640 medium, and then the cells were added to 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) prewarmed to 37° C. was added to the cell sediment to fuse the cells. 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"). Fused cells were suspended in HAT medium supplemented with 100 μmol/L hypoxanthine, 0.4 μmol/L aminopterin, and 16 μmol/L thymidine in RPMI1640/10% FBS medium, and placed in four 96-well microplates. and cultured at 37°C in the presence of 5% CO ₂ for 10-14 days. After culturing, the antibody reactivity in colony-bearing hybridoma wells was examined using indirect ELISA and indirect competitive ELISA.

12. Reactivity of anti-DON MoAb There were many hybridomas that produced antibodies that showed reactivity to DON. and MDN31). The culture supernatant of these cells was used as an MDN8-derived monoclonal antibody (hereinafter referred to as MDN8 MoAb) and an MDN31-derived monoclonal antibody (hereinafter 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 well as anti-DON PoAb, which is a polyclonal antibody. As shown in Figure 3, MDN8 MoAb was 3-200 ng/mL with DON, 3-85 ng/mL with 15-Ac-DON, MDN31 MoAb was 2-200 ng/mL with DON, 15 It quantitatively reacted with -Ac-DON in the range of 3-120 ng/mL, and showed high reactivity with DON and 15-Ac-DON like the anti-DON PoAb. On the other hand, as shown in Table 5, it showed no reactivity with 3-Ac-DON and NIV. The DON derivatives synthesized in this way were effective in producing monoclonal antibodies exhibiting high reactivity against DON and 15-Ac-DON.

13. Production of anti-NIV MoAb and its reactivity Anti-NIV MoAb was also produced under the same conditions as the anti-DON MoAb. However, for screening, a combination of indirect ELISA and indirect competitive ELISA and a combination of direct ELISA and direct competitive ELISA were used. There were also many hybridomas that produced antibodies that showed reactivity to NIV. Among them, hybridomas that showed high reactivity in direct competitive ELISA were cell-cloned by the limiting dilution method and MoAb-producing cells (MNV80, MNV87, NIV150, NIV464) were obtained. and The culture supernatants of these cells were used as MNV80 MoAb, MNV87 MoAb, NIV150 MoAb and NIV464 MoAb, and their reactivity with NIV was examined by direct competitive ELISA. The results, as shown in Figures 4 and 5, were 6.9-180 ng/mL for MNV80 MoAb, 7.1-170 ng/mL for MNV87 MoAb, 0.7-14 ng/mL for NIV150 MoAb, and 6.2-185 ng for NIV464 MoAb. /mL ranged quantitatively reacted with NIV. On the other hand, 15-acetyl-NIV (15-Ac-NIV) is 2.8-100 ng/mL for MNV80 MoAb, 2.4-95 ng/mL for MNV87 MoAb, 0.1-1.2 ng/mL for NIV150 MoAb, and 0.1-1.2 ng/mL for NIV464 MoAb. It reacted quantitatively in the range of 0.4-8.5 ng/mL. As shown in Table 5, MNV80 MoAb and MNV87 MoAb showed no reactivity with DON, 3-Ac-DON, 15-Ac-DON and 4-Ac-NIV. NIV150 MoAb and NIV464 MoAb showed little reactivity with DON and 4-Ac-NIV. Since the contamination of barley with 15-Ac-NIV is scarcely known, the synthesized NIV derivative was thought to be effective in producing MoAb showing high reactivity against NIV.

14. Preparation of anti-3-Ac-DON MoAb and its reactivity Anti-3-Ac-DON MoAb was also prepared under the same conditions as anti-DON MoAb. Screening used direct ELISA and direct competitive ELISA. Hybridomas that showed high reactivity to 3-Ac-DON in direct competitive ELISA were cloned by limiting dilution to obtain MoAb-producing cells (M3ADN27 and M3ADN28).
The culture supernatants of these cells were used as M3ADN27 MoAb and M3ADN28 MoAb, and their reactivities with 3-Ac-DON, DON and 15-Ac-DON were examined by direct competitive ELISA. As shown in Fig. 6, M3ADN27 MoAb was 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. , M3ADN28 MoAb quantitatively reacted with 3-Ac-DON at 13-140 ng/mL, with 15-Ac-DON at 180-1900 ng/mL, and with DON at 2000-10000 ng/mL. It showed high reactivity with Ac-DON. On the other hand, as shown in Table 5, it showed no reactivity with NIV. The NIV derivatives thus synthesized were effective in producing MoAbs with high reactivity to 3-Ac-DON.

15. Reference value of DON, comparison of DON and tolerable daily intake of NIV Antibodies obtained using DON derivatives are anti-DON PoAb, MDN8 MoAb and MDN31 MoAb. The quantification ranges for DON in these direct competitive ELISAs were 4.0-300 ng/mL for the anti-DON PoAb, 3-200 ng/mL for the MDN8 MoAb, and 2-200 ng/mL for the MDN31 MoAb. In 2020, the Food Sanitation Subcommittee of the Pharmaceutical Affairs and Food Sanitation Council of the Ministry of Health, Labor and Welfare (Japan) established the DON standard for wheat (brown barley) as 1.0 mg/kg. Considering that DON is extracted from wheat and diluted appropriately before measurement, a direct competitive ELISA constructed using these antibodies is effective for measuring DON in foods near or below the reference value. It was considered suitable sensitivity.

On the other hand, although standards and standards have not been established for NIV, the Food Safety Commission of the Cabinet Office (Japan) has established a tolerable daily intake of 1 μg/kg body weight for DON alone in the food health assessment of DON and NIV. /day, NIV alone was set at 0.4 μg/kg body weight/day. In other words, there is a high possibility that NIV will require a measurement method that is as sensitive as or slightly higher than DON. Quantification range for anti-NIV PoAb was 12-950 ng/mL, MNV80 MoAb 6.9-180 ng/mL, MNV87 MoAb 7.1-170 ng/mL, NIV150 MoAb 0.7-14 ng/mL, NIV464 MoAb 6.2-185 ng/mL, the direct competitive ELISA constructed using anti-NIV MoAb was also considered to have suitable sensitivity for NIV measurement.
Furthermore, for 3-Ac-DON, which is a major analogue of DON, we were able to generate MoAb capable of constructing a measurement method showing the same level of sensitivity.
Based on the above, we were able to provide and design a technique for synthesizing derivatives suitable for producing antibodies for measuring DON, NIV, or their analogues that contaminate wheat, etc.

INDUSTRIAL APPLICABILITY The present invention is useful in conducting fact-finding surveys of grain contamination by mycotoxins, and is therefore expected to be used in the agricultural field.

Claims (24)

1. A method for producing an antibody, comprising immunizing with a trichothecene derivative represented by the following formula (I) as an antigen.
   

   

   (In formula (I), _R2 represents a hydrogen or hydroxyl group, and X represents a carrier protein.)
2. 2. A method according to claim 1, comprising the step of binding a carrier protein X via Y to a compound of formula (III) below.
   

   

   (In formula (III), _R2 represents a hydrogen or hydroxyl group, and Y represents a functional group for cross-linking proteins or a linker containing the functional group.)
3. 3. The method according to claim 1 or 2, wherein the antibody is an antibody against a trichothecene derivative represented by the following formula (II).
   

   

   (In formula (II), _R1 represents a hydroxyl group, an acetoxy group or a glucosyloxy group, _R2 represents a hydrogen group, a hydroxyl group or an acetoxy group, and _R3 represents a hydroxyl group or an acetoxy group.)
4. 3. An antibody or an antigen-binding fragment thereof against a trichothecene derivative represented by the following formula (II), which is produced by the method according to claim 1 or 2.
   

   

   (In formula (II), _R1 represents a hydroxyl group, an acetoxy group or a glucosyloxy group, _R2 represents a hydrogen group, a hydroxyl group or an acetoxy group, and _R3 represents a hydroxyl group or an acetoxy group.)
5. An antibody or an antigen-binding fragment thereof against DON and 15-Ac-DON represented by the following formula (II).
   

   

   (In formula (II), _R1 represents a hydroxyl group, _R2 represents hydrogen, and _R3 represents a hydroxyl group or an acetoxy group.)
6. The antibody or antigen-binding fragment thereof according to claim 5, which reacts quantitatively with 4.0-300 ng/mL DON and 1.0-40 ng/mL 15-Ac-DON.
7. The antibody or antigen-binding fragment thereof according to claim 5, which reacts quantitatively with 3.0-200 ng/mL of DON and 3.0-85 ng/mL of 15-Ac-DON.
8. The antibody or antigen-binding fragment thereof according to claim 5, which reacts quantitatively with 2.0-200 ng/mL of DON and 3.0-120 ng/mL of 15-Ac-DON.
9. An antibody or an antigen-binding fragment thereof against 3-Ac-DON represented by the following formula (II).
   

   

   (In formula (II), _R1 represents an acetoxy group, _R2 represents hydrogen, and _R3 represents a hydroxyl group.)
10. The antibody or antigen-binding fragment thereof according to claim 9, which reacts quantitatively with 11 to 120 ng/mL of 3-Ac-DON.
11. The antibody or antigen-binding fragment thereof according to claim 9, which reacts quantitatively with 13 to 140 ng/mL of 3-Ac-DON.
12. An antibody or an antigen-binding fragment thereof against NIV and 15-Ac-NIV represented by the following formula (II).
    

    

    (In formula (II), R ₁ and R ₂ represent a hydroxyl group, and R ₃ represents a hydroxyl group or an acetoxy group.)
13. The antibody or antigen-binding fragment thereof according to claim 12, which reacts quantitatively with 12-950 ng/mL of NIV and 1.7-85 ng/mL of 15-Ac-NIV.
14. The antibody or antigen-binding fragment thereof according to claim 12, which reacts quantitatively with 6.9-180 ng/mL of NIV and 2.8-100 ng/mL of 15-Ac-NIV.
15. The antibody or antigen-binding fragment thereof according to claim 12, which reacts quantitatively with 7.1-170 ng/mL of NIV and 2.4-95 ng/mL of 15-Ac-NIV.
16. The antibody or antigen-binding fragment thereof according to claim 12, which reacts quantitatively with 0.7-14 ng/mL of NIV and 0.1-1.2 ng/mL of 15-Ac-NIV.
17. The antibody or antigen-binding fragment thereof according to claim 12, which reacts quantitatively with 6.2-185 ng/mL of NIV and 0.4-8.5 ng/mL of 15-Ac-NIV.
18. An immunogenic composition comprising a trichothecene derivative represented by the following formula (I) as an antigen.
    

    

    (In formula (I), _R2 represents a hydrogen or hydroxyl group, and X represents a carrier protein.)
19. The immunogenic composition according to claim 18, wherein said carrier protein is bound to position 15 of said formula (I) via a linker.
20. A kit for detecting a trichothecene mycotoxin, comprising the antibody or antigen-binding fragment according to claim 4.
21. A kit for detecting a trichothecene mycotoxin, comprising the antibody or antigen-binding fragment of claim 5, 9 or 12.
22. A trichothecene derivative represented by the following formula (III).
    

    

    (In formula (III), R ₂ represents hydrogen or a hydroxyl group, and Y represents a functional group (excluding a hydroxyl group) for cross-linking proteins or a linker containing the functional group.)
23. The trichothecene derivative according to claim 22, wherein said Y is a carboxyl group or a linker containing a carboxyl group.
24. The trichothecene derivative according to claim 22, wherein said Y is a 4-carboxybutyryloxy group.
    
    

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