Monoclonal antibodies reactive with glycopeptides and uses thereof

Abstract

The present invention relates to a monoclonal antibody reacting with a glycopeptide and use thereof. The present invention provides a monoclonal antibody which is an epitope of both a fucose moiety and an amino acid of a peptide moiety. The antibody of the present invention is a monoclonal antibody reacting with a glycopeptide containing a core fucose and an amino acid of 4 or more residues continuously from an asparagine of an additional sugar chain toward a C-terminal side, the antibody being an epitope of both the core fucose of the glycopeptide and an amino acid of the glycopeptide which is 3 or more residues from the asparagine of the additional sugar chain toward the C-terminal side.

Description

Technical Field

[0001] This invention relates to monoclonal antibodies that react with glycopeptides and their uses. Background Technology

[0002] It is claimed that during the progression from hepatitis and cirrhosis to liver cancer, the amount of alpha-fetoprotein (AFP) bound to lentil lectin (LCA) increases in biological samples (LCA-bound AFP). Patent Document 1 describes an antibody for detecting LCA-bound AFP. According to the document, the antibody in Patent Document 1 is reactive to bound AFP but not to LCA-unbound AFP.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 63-307900 Summary of the Invention

[0006] The problem that the invention aims to solve

[0007] Patent document 1 describes that the sugar chain of AFP bound by LCA contains fucose. The AFP fraction bound to lentil lectin in biological samples is called the AFP-L3 fraction. The AFP-L3 fraction is composed of fucosylated AFP (AFP with a core fucose (fucoose bound to the N-acetylglucosamine (GlcNAc) α1-6 at the reducing end of the N-type sugar chain) attached to the asparagine residue of the AFP).

[0008] The embodiments of Patent Document 1 yielded antibodies that bind to LCA-binding AFP (AFP-LCA-R) but not to LCA-non-binding AFP (AFP-LCA-NR) (see Example 1, Table 1). Figure 1 However, the epitope of this antibody is unknown. The examples indicate that LCA binding and non-binding, as described above, arise from the presence or absence of fucose; binding to the antigen may depend on the fucose moiety rather than the peptide sequence. In this case, it is possible to bind not only to AFP but also non-specifically to other proteins containing fucose. Therefore, it is desirable to develop monoclonal antibodies with both the fucose moiety and the amino acid sequence of the peptide as epitopes.

[0009] Solution for solving the problem

[0010] The present invention provides an antibody, which is a monoclonal antibody that reacts with a glycopeptide, wherein the glycopeptide contains a core fucose and an amino acid consisting of four or more consecutive residues from the asparagine of the attached sugar chain toward the C-terminus. The antibody uses the core fucose of the glycopeptide and the amino acid of the glycopeptide located three or more residues away from the C-terminus from the asparagine of the attached sugar chain as epitopes.

[0011] The present invention provides a method for producing the above-mentioned antibody, comprising the following steps: immunizing an animal with a glycopeptide antigen containing a core fucose and at least four amino acid residues continuously from the C-terminus of asparagine with an additional sugar chain.

[0012] The present invention provides a method for producing hybridomas that generate the above-mentioned antibodies, comprising the following steps: immunizing an animal with a glycopeptide antigen containing a core fucose and at least four amino acid residues continuously from the C-terminus of asparagine with an additional sugar chain.

[0013] This invention provides a monoclonal antibody that reacts with the following glycopeptides:

[0014] [Chemical Formula 1]

[0015]

[0016] It does not react with the following glycopeptides.

[0017] [Chemical Formula 2]

[0018]

[0019] It does not react with the following glycopeptides.

[0020] [Chemical Formula 3]

[0021]

[0022] This invention provides a method for producing the above-mentioned antibody, comprising the following steps: immunizing animals with a glycopeptide antigen containing the following structure.

[0023] [Chemical Formula 4]

[0024]

[0025] (X is any sugar chain).

[0026] This invention provides a method for producing hybridomas that generate the above-mentioned antibodies, comprising the following steps: immunizing animals with a glycopeptide antigen containing the following structure.

[0027] [Chemical Formula 5]

[0028]

[0029] (X is any sugar chain).

[0030] This invention provides a method for determining fucosylated AFP, which uses the aforementioned antibody.

[0031] The present invention provides a kit for detecting fucosylated AFP, which contains an antibody for capturing fucosylated AFP, an antibody for detecting fucosylated AFP, and a solid phase, wherein the antibody for capturing or the antibody for detecting AFP is the antibody described above.

[0032] This invention provides a hybridoma, with the international accession number NITE BP-02263 or NITE BP-02264.

[0033] Invention Effects

[0034] This invention provides a monoclonal antibody with both the fucose moiety of a glycopeptide and the amino acid of the peptide moiety as epitopes. Attached Figure Description

[0035] Figure 1 The diagram illustrates the protein imprinting results of clones 1F8-A4 and 2F11-2A9 obtained in Example 1 (lane 1: fucosylated AFP (AFP-L3 / recombinant) (positive antigen), lane 2: non-fucosylated AFP (LCA lectin non-adsorbed fraction of AFP (LEE biosolutions) from human serum) (negative antigen), lane 3: fucosylated ALP (Oriental yeast / 47787055) (negative antigen).

[0036] Figure 2 A graph showing the measured OD450 values ​​at various antigen concentrations when using clone I2-1F8 obtained in Example 1.

[0037] Figure 3 A graph showing OD450 measurements at various antigen concentrations when using clone I2-2F11 obtained in Example 1.

[0038] Figure 4 The diagram shows the results of protein imprinting of clone I2-1F8 obtained in Example 1 (lane 1: recombinant AFP-L3, lane 2: non-fucosylated AFP, lane 3: natural human AFP (standard 1 for MUTASUWAKO AFP-L3), lane 4: natural human AFP-L3 (standard 2 for MUTASUWAKO AFP-L3)). Detailed Implementation

[0039] The antibody of this embodiment only needs to exhibit specificity in the assay system using biological samples or the like employing the antibody of this embodiment. For example, even if non-specific binding occurs with substances not contained in the biological sample, the antibody of this embodiment will still exert the effects of the present invention if it exhibits specificity in a normally used environment. Specifically, when detecting substances in blood samples such as whole blood, serum, and plasma by ELISA, it is sufficient that the antibody shows specificity in the ELISA assay system; binding with substances not normally contained in blood samples or ELISA reagents is also acceptable.

[0040] The antibody in this embodiment is a monoclonal antibody that reacts with a glycopeptide. The glycopeptide contains a core fucose and four or more amino acids that are continuously extended from the asparagine of the attached sugar chain toward the C-terminus. The antibody uses the core fucose of the glycopeptide and the amino acid of the glycopeptide that is three or more residues away from the C-terminus from the asparagine of the attached sugar chain as epitopes.

[0041] The glycopeptide that reacts with the antibody in this embodiment only needs to contain a core fucose and at least four amino acid residues consecutively from the asparagine in the additional sugar chain toward the C-terminus.

[0042] The antibody of this embodiment is produced by a method comprising the following steps: immunizing an animal with a glycopeptide antigen containing a core fucose and at least four amino acid residues continuously extending from the C-terminus of asparagine with an additional sugar chain.

[0043] The N-terminus of glycopeptide antigens can be bound to biomolecules such as KLH and BSA via methods such as PEG. Furthermore, the C-terminus of these glycopeptide antigens can be amidated. Methods for binding biomolecules to the N-terminus can be employed using known techniques. Additionally, amidation of the C-terminus can also be performed using known methods.

[0044] The process of immunizing animals with glycopeptide antigens can utilize the immunization process in known monoclonal antibody production methods. Examples of monoclonal antibody production methods include, for example, the mouse spleen method and the mouse iliac lymph node method (see Japanese Patent No. 4098796).

[0045] There are no specific restrictions on the animals to be immunized; appropriate selection from non-human animals can be made based on the monoclonal antibody production method.

[0046] Specifically, when using the mouse iliac lymph node method as a monoclonal antibody production method, the glycopeptide is coupled with a carrier protein such as KLH, and the mouse tail root is immunized at least once with an emulsion mixed with Freund's adjuvant, etc., at a dose of 0.06 mL / mouse of glycopeptide solution (2.5 mg / mL carrier protein).

[0047] After immunizing the animals, the antibodies of this embodiment can be obtained by producing and screening hybridomas according to known methods.

[0048] When screening hybridomas, glycopeptides that serve as antigens, substances from which the core fucose has been removed from glycopeptides serving as antigens, and glycopeptides with the same glycan structure as antigens but different peptide chains can be appropriately used as positive or negative antigens. Glycopeptides that reduce the number of amino acid residues in the aforementioned antigenic glycopeptides are preferably, for example, glycopeptides with a C-terminus consisting of three consecutive residues starting from the asparagine in the attached glycan chain.

[0049] Regarding the screening criteria for hybridomas, for example, when using ELISA, the difference between the OD450 values ​​of the positive and negative antigens is greater than 0.05, and the OD450 value of the negative antigen is less than 0.05.

[0050] There is no particular limitation on the isotype of the antibody in this embodiment. In addition, the antibody in this embodiment also includes fragments such as peptides containing F(ab')2, Fab', Fab, and CDR.

[0051] The antibody in this embodiment can also be labeled with biotinylation, ALP, etc.

[0052] The antibody described above has the properties described above, and therefore, for example, it specifically reacts with glycoproteins containing a core fucose and four or more amino acids consecutively from the asparagine in the attached sugar chain toward the C-terminus.

[0053] The antibody described in this embodiment can be used for ELISA, protein blotting, immunohistochemical staining, immunoprecipitation, etc.

[0054] Another embodiment of the monoclonal antibody specifically reacts with the following glycopeptide (sequence number 13).

[0055] [Chemical Formula 1]

[0056]

[0057] This monoclonal antibody does not react with the following glycopeptide (sequence number 14).

[0058] [Chemical Formula 2]

[0059]

[0060] It does not react with the following glycopeptide (sequence number 15).

[0061] [Chemical Formula 3]

[0062]

[0063] That is, the antibody of this embodiment does not react with antigens that do not have a core fucose but have the amino acid "EIQ" (as described in (2) above), and does not react with antigens that have a core fucose but do not have the amino acid sequence "EIQ" (as described in (3) above). Therefore, it is believed that the epitope of the antibody of this embodiment contains both core fucose and the amino acid sequence "EIQ".

[0064] It should be noted that, in this specification, the antibody "reaction with glycopeptides" in this embodiment refers to the binding of glycopeptides and antibodies through an antigen-antibody reaction.

[0065] Furthermore, the above-mentioned antibody preferably does not react with the following glycopeptide (sequence number 16).

[0066] [Chemical Formula 4]

[0067]

[0068] Furthermore, the above-mentioned antibody preferably reacts with fucosylated AFP.

[0069] Furthermore, the aforementioned antibody can bind to denatured fucosylated AFP that has been pretreated with a denaturing agent such as SDS and by heating. When using a solution containing SDS as pretreatment, the concentration of SDS used to sufficiently denature the fucosylated AFP (hereinafter referred to as "SDS concentration during pretreatment") is not particularly limited, but is preferably 0.03% by mass (hereinafter referred to as "%)" or higher. On the other hand, if the antigen-antibody reaction contains an excessive amount of denaturing agent, the antibody will also denature, which may have an undesirable effect on the antigen-antibody reaction. Therefore, it is preferable to reduce the concentration of the denaturing agent by dilution or the like. When using a solution containing SDS as the denaturing agent, the concentration of SDS during the antigen-antibody reaction (hereinafter referred to as "final SDS concentration") is not particularly limited, but is preferably 0.025% or lower.

[0070] It should be noted that, in this specification, the antibody's "reaction with fucosylated AFP" in this embodiment refers to the binding of fucosylated AFP to the antibody through an antigen-antibody reaction. Fucosylated AFP can be any of the recombinant or natural substances. Natural fucosylated AFP is, for example, AFP present in human blood. The sequence of human AFP is, for example, registered with GenBank Accession No. NM_001134, and has amino acid sequence number 25.

[0071] Furthermore, the above-mentioned antibody is preferably reacted with fucosylated AFP denatured in the presence of SDS and DTT. The denaturation conditions are a reaction at room temperature (25°C) in the presence of 2% SDS and 50 mM DTT.

[0072] Furthermore, the aforementioned antibody preferably does not react with non-fucosylated AFP denatured in the presence of SDS and DTT. The denaturation conditions are a reaction at room temperature (25°C) in the presence of 2% SDS and 50 mM DTT.

[0073] Examples of CDRs used as antibodies in this embodiment include the following.

[0074] <CDR-A>

[0075] The heavy chain CDR contains the amino acid sequence shown in sequence number 1, the amino acid sequence shown in sequence number 2, and the amino acid sequence shown in sequence number 3.

[0076] The light chain CDR contains the amino acid sequences shown in sequence number 4, sequence number 5, and sequence number 6.

[0077] <CDR-B>

[0078] The heavy chain CDR contains the amino acid sequences shown in sequence number 7, sequence number 8, and sequence number 9.

[0079] The light chain CDR contains the amino acid sequence shown in sequence number 10, sequence number 11, and sequence number 12.

[0080] As CDRs, the hybridomas that produce antibodies of this embodiment having the above-mentioned CDR-A and CDR-B are named I2-1F8 and I2-2F11, respectively, and were internationally deposited on May 20, 2016, at the Patent Microbial Collection Center of the Technical Base for Product Evaluation (also known as NPMD - National Institute for Technology Evaluation) (Room 122, 2-5-8, Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) as NITE BP-02264 and NITE BP-02263.

[0081] The antibody of this embodiment is obtained by a method comprising the following steps: immunizing an animal with a glycopeptide antigen (sequence number 17) containing the following structure.

[0082] [Chemical Formula 5]

[0083]

[0084] X is any sugar chain, which can be any sugar chain that is usually bound to glycoproteins or glycopeptides, without any special restrictions.

[0085] The following glycopeptides are preferred as glycopeptide antigens (sequence number 18).

[0086] [Chemical Formula 6]

[0087]

[0088] X is any sugar chain, which can be any sugar chain that is usually bound to glycoproteins or glycopeptides, without any special restrictions.

[0089] The N-terminus of glycopeptide antigens can be bound to biomolecules such as KLH via PEG or similar methods. Furthermore, the C-terminus of these glycopeptide antigens can be amidated. Methods for binding biomolecules to the N-terminus can be employed using known techniques. Additionally, amidation of the C-terminus can also be performed using known methods.

[0090] The process of immunizing animals with glycopeptide antigens can utilize the immunization process in known monoclonal antibody production methods. Examples of monoclonal antibody production methods include, for example, the mouse spleen method and the mouse iliac lymph node method (see Japanese Patent No. 4098796).

[0091] There are no specific restrictions on the animals to be immunized; appropriate selection from non-human animals can be made based on the monoclonal antibody production method.

[0092] Specifically, when using the mouse iliac lymph node method as a method for producing monoclonal antibodies, the glycopeptide is coupled with a carrier protein such as KLH, and the mouse tail root is immunized at least once with an emulsion mixed with Freund's adjuvant, etc., at a dose of 0.06 mL / mouse of glycopeptide solution (2.5 mg / mL carrier protein).

[0093] After immunizing the animals, the antibodies of this embodiment can be obtained by producing and screening hybridomas according to known methods.

[0094] In hybridoma screening, glycopeptides serving as antigens, substances from which the core fucose has been removed from glycopeptides serving as antigens, glycopeptides that reduce the number of amino acid residues in glycopeptides serving as antigens, fucosylated AFP denatured in the presence of SDS and DTT, non-fucosylated AFP denatured in the presence of SDS and DTT, and glycopeptides or glycoproteins (e.g., fucosylated ALP) containing a core fucose and having an amino acid sequence different from that of AFP can be used as positive or negative antigens. Glycopeptides that reduce the number of amino acid residues in glycopeptides serving as antigens are preferably, for example, glycopeptides with a C-terminus consisting of three consecutive residues starting from the asparagine in the attached glycan chain.

[0095] Regarding the screening criteria for hybridomas, for example, when using ELISA, the difference between the OD450 values ​​of the positive and negative antigens is greater than 0.05, and the OD450 value of the negative antigen is less than 0.05.

[0096] There is no particular limitation on the isotype of the antibody in this embodiment. In addition, the antibody in this embodiment also includes fragments such as peptides containing F(ab')2, Fab', Fab, and CDR.

[0097] The antibody in this embodiment can also be labeled with biotinylation, ALP, etc.

[0098] The antibody of this embodiment possesses the aforementioned properties, and therefore reacts with fucosylated AFP but not with non-fucosylated AFP. Therefore, the antibody of this embodiment can, for example, determine fucosylated AFP in biological samples.

[0099] Examples of biological samples include whole blood, serum, and plasma collected from the subject. These biological samples can be pretreated by centrifugation, denaturation, etc. Denaturation treatment is preferred for biological samples. The denaturation treatment conditions are a reaction at room temperature (25°C) in the presence of 2% SDS and 50 mM DTT.

[0100] When using the antibody of this embodiment to determine fucosylated AFP, known immunological assay methods can be employed. Examples of immunological assay methods include enzyme-linked immunosorbent assay (ELISA), immune complex transfer assay (see Japanese Patent Application Publication No. 1-254868), immunoturbidimetry, immunochromatography, and latex agglutination. As an example of the assay procedure, the determination of fucosylated AFP concentration in a biological sample by sandwich ELISA will be described below.

[0101] First, a complex containing an antibody for capturing fucosylated AFP in a biological sample (hereinafter also referred to as "capture antibody"), an antibody for detecting fucosylated AFP (hereinafter also referred to as "detection antibody"), and fucosylated AFP is formed on a solid phase. When the biological sample contains fucosylated AFP, this complex can be formed by mixing the biological sample, the capture antibody, and the detection antibody. Then, the solution containing the complex is contacted with a solid phase capable of capturing the capture antibody, thereby forming the complex on the solid phase. Alternatively, a solid phase pre-immobilized with the capture antibody can be used. That is, by contacting a solid phase immobilized with the capture antibody, the biological sample, and the detection antibody, the complex can be formed on the solid phase. The antibody of this embodiment can be used as at least one of the capture antibody and the detection antibody.

[0102] There are no particular limitations on the method of immobilizing the capture antibody on the solid phase. For example, the capture antibody can be directly bound to the solid phase, or it can be indirectly bound to the solid phase through other substances. Examples of direct binding include physical adsorption. Examples of indirect binding include binding via a combination of biotin and avidin or streptavidin (hereinafter also referred to as "avidins"). In this case, by pre-modifying the capture antibody with biotin and pre-binding the avidin to the solid phase, the capture antibody can be indirectly bound to the solid phase through the binding of biotin and avidin.

[0103] There are no particular limitations on the materials used for the solid phase; selections can be made from, for example, organic polymers, inorganic compounds, and biopolymers. Examples of organic polymers include: latex, polystyrene, and polypropylene. Examples of inorganic compounds include: magnetic materials (iron oxide, chromium oxide, and ferrite, etc.), silicon dioxide, alumina, and glass. Examples of biopolymers include: insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these materials can be combined. There are no particular limitations on the shape of the solid phase; examples include: particles, membranes, microplates, microtubes, and test tubes.

[0104] By using methods known in this technical field to detect complexes formed on a solid phase, the fucosylated AFP level in a biological sample can be determined. For example, when using an antibody labeled with a marker as the detection antibody, the fucosylated AFP level can be obtained by detecting the signal generated by the marker. Alternatively, the fucosylated AFP level can also be obtained by using a labeled secondary antibody against the detection antibody.

[0105] In this embodiment, it is preferable to pretreat fucosylated AFP as described above. When using a solution containing SDS as the pretreatment, the concentration of SDS during pretreatment is not particularly limited, but is preferably 0.03% or higher. During the antigen-antibody reaction, as described above, it is preferable to reduce the concentration of the denaturing agent by dilution or the like. When using a solution containing SDS as the denaturing agent, the final SDS concentration is not particularly limited, but is preferably 0.025% or lower. In this embodiment, it is preferable to perform this type of treatment and react the antibody with the fucosylated AFP to obtain a measured value of fucosylated AFP.

[0106] In this embodiment, between the complex formation step and the complex detection step, a B / F (Bound / Free) separation can be performed to remove unreacted free components that do not form complexes. Unreacted free components refer to components that do not constitute complexes. Examples include antibodies that do not bind to fucosylated AFP, and substances other than fucosylated AFP (impurities) in biological samples. The method of B / F separation is not particularly limited. If the solid phase is particles, B / F separation can be performed by centrifuging to recover only the solid phase containing the complexes. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing unreacted free components. Alternatively, if the solid phase is magnetic particles, B / F separation can be performed by using a nozzle to aspirate and remove the liquid containing unreacted free components while the magnetic particles are magnetically bound by a magnet. After removing the unreacted free components, the solid phase containing the complexes can be washed with a suitable aqueous medium such as PBS.

[0107] In this specification, "detection signal" includes: qualitative detection of the presence or absence of a signal, quantitative detection of signal strength, and semi-quantitative detection of signal strength. Semi-quantitative detection refers to a method of representing signal strength in stages, such as "no signal generated," "weak," "medium," and "strong." In this embodiment, quantitative or semi-quantitative detection of signal strength is preferred.

[0108] There are no particular limitations on the labeling substance as long as it produces a detectable signal. For example, it can be a substance that generates a signal itself (hereinafter also referred to as a "signal-generating substance"), or a substance that catalyzes the reaction of other substances to produce a signal. Examples of signal-generating substances include: fluorescent substances, radioactive isotopes, etc. Examples of substances that catalyze the reaction of other substances to produce a detectable signal include: enzymes, etc. Examples of enzymes include: alkaline phosphatase, peroxidase, β-galactosidase, luciferase, etc. Examples of fluorescent substances include: fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), fluorescent proteins such as GFP, etc. Examples of radioactive isotopes include: 125 I, 14 C 32 P, etc. Among these, enzymes are preferred as labeling substances, with alkaline phosphatase and peroxidase being particularly preferred.

[0109] The method for detecting the signal is well known in the art. In this embodiment, the measurement method can be appropriately selected according to the type of signal from the labeled substance. For example, when the labeled substance is an enzyme, a known device such as a spectrophotometer can be used to measure the light, color, and other signals generated by the reaction between the enzyme and the substrate.

[0110] The substrate for an enzyme can be appropriately selected from a known pool of substrates, depending on the type of enzyme. For example, when using alkaline phosphatase as the enzyme, suitable substrates include: CDP-Star (registered trademark) (disodium 4-chloro-3-(methoxyspiro[1,2-dioxane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, CSPD (registered trademark) (disodium 3-(4-methoxyspiro[1,2-dioxane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, chemiluminescent substrates, 5-bromo-4-chloro-3-indole phosphate (BCIP), 5-bromo-6-chloro-indole phosphate, and p-nitrophenyl phosphate, among other chromogenic substrates. In addition, when using peroxidase as the enzyme, the following substrates can be used as examples: chemiluminescent substrates such as luminol and its derivatives, and chromogenic substrates such as 2,2'-adiazonobis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0111] When the labeled substance is a radioactive isotope, known devices such as a scintillation counter can be used to measure the radiation as a signal. Similarly, when the labeled substance is a fluorescent substance, known devices such as a fluorescence microplate reader can be used to measure the fluorescence as a signal. It should be noted that the excitation wavelength and fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

[0112] The signal detection results can be used as the determination value of fucosylated AFP. For example, when quantitatively detecting the signal intensity, the signal intensity measurement value itself or the value obtained from that measurement can be used as the determination value of fucosylated AFP. Examples of values ​​obtained from the signal intensity measurement include, for example, the value obtained by subtracting the measurement value of the negative control sample or the background value from the fucosylated AFP measurement value. The negative control sample can be appropriately selected, and examples include, for instance, biological samples obtained from healthy individuals.

[0113] In this embodiment, the fucosylated AFP concentration can be measured from multiple standard samples with known fucosylated AFP concentrations, and a standard curve showing the relationship between the fucosylated AFP concentration and the measured fucosylated AFP value can be created. By substituting the measured fucosylated AFP value obtained from the biological sample into this standard curve, the fucosylated AFP concentration in the biological sample can be obtained.

[0114] In this embodiment, the concentration of fucosylated AFP in a biological sample can also be determined using a sandwich ELISA method that combines a trapping antibody immobilized on magnetic particles with a detection antibody labeled with a marker substance. In this case, the determination can be performed using a commercially available fully automated immunoassay apparatus such as the HISCL series (manufactured by SYSMEX Corporation).

[0115] Furthermore, the antibody of this embodiment can be used in a fucosylated AFP detection kit. The fucosylated AFP detection kit of this embodiment contains a capture antibody, a detection antibody, and a solid phase. The antibody of this embodiment can be used as either the capture antibody or the detection antibody. In sandwich immunoassays, the antibody of this embodiment can be used in either the capture antibody or the detection antibody.

[0116] Regarding the fucosylated AFP detection kit of this embodiment, when the labeling substance of the detection antibody is an enzyme, the fucosylated AFP detection kit of this embodiment may further contain a substrate targeting the enzyme. There are no particular limitations on the form of the labeling substance and the substrate; they can be solids (e.g., powders, crystals, lyophilized products, etc.) or liquids (e.g., solutions, suspensions, emulsions, etc.).

[0117] In the fucosylated AFP detection kit of this embodiment, in order to pretreat the aforementioned fucosylated AFP, a pretreatment reagent containing 0.03% or more SDS may be further included. This reagent is the same as the solution containing 0.03% or more SDS described above.

[0118] The fucosylated AFP detection kit of this embodiment may further contain appropriate pretreatment solutions for biological samples, washing solutions for the solid phase, enzyme reaction stoppers, standards, etc.

[0119] Regarding the fucosylated AFP detection kit of this embodiment, the capture antibody, detection antibody, solid phase, etc., can be appropriately contained in a container in a binding kit form, or they can be packaged separately. In the fucosylated AFP detection kit of this embodiment, the capture antibody can be directly bound to the solid phase, or the capture antibody and the solid phase can be indirectly bound through other substances. When the capture antibody and the solid phase are indirectly bound, in the kit of this embodiment, the capture antibody and the solid phase can be contained in different containers. When the capture antibody and the solid phase are indirectly bound through, for example, biotin and avidin, the biotin-modified capture antibody can be contained in one container, and the solid phase bound to avidin can be contained in another container. It should be noted that the details of the biological sample, capture antibody, detection antibody, solid phase, etc., are the same as those described in the description of the above-described assay method.

[0120] The relationship between fucosylated AFP and liver cancer is known. Therefore, the fucosylated AFP detection kit of this embodiment can be used for the diagnosis of liver cancer.

[0121] Example

[0122] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples in any way.

[0123] Example 1

[0124] Obtaining antibodies: (1) Obtaining antibody groups

[0125] Hybridomas producing antibodies were obtained via the mouse iliac lymph node method (Japanese Patent No. 4098796). Specifically, glycopeptide A (sequence number 19) with the structure described in Table 1 below was synthesized, conjugated with KLH, mixed with Freund's complete adjuvant to a concentration of 2.5 mg / mL (carrier protein conversion), and the resulting emulsion was injected once into the tail base of mice at a dose of 0.06 mL / mouse. Additionally, 17 days after immunization, a booster immunization was performed by injecting 0.06 mL / mouse of the 2.5 mg / mL (carrier protein conversion) KLH-conjugated glycopeptide A solution into the tail base of mice. Furthermore, 4 days after the booster immunization, lymphocytes were isolated from the iliac lymph nodes and fused with myeloma cells to obtain hybridomas.

[0126] [Table 1]

[0127]

[0128] The circle represents mannose, the square represents N-acetylglucosamine, and the triangle represents fucose.

[0129] The N-terminus of the glycopeptide is KLH-PEG4, and the C-terminus is amidated.

[0130] (2) First screening

[0131] Antigen-based solid-phase ELISA using the positive antigen (glycopeptide A) or negative antigen (non-fucosylated glycopeptide A: sequence number 20) described in Table 2 was performed. Wells that showed a reaction with the positive antigen but a lesser reaction with the negative antigen were selected from the hybridomas obtained in (1) above. The antigen-based solid-phase ELISA was performed by the following method. The results are shown in Table 3.

[0132] [Table 2]

[0133]

[0134] The circle represents mannose, the square represents N-acetylglucosamine, and the triangle represents fucose.

[0135] The N-terminus of the glycopeptide is BSA-PEG4, and the C-terminus is amidated.

[0136] <Method>

[0137] (1) In a 96-well plate (nunc Maxisoap / 446612), add 1 μg / ml of each screening antigen (diluent, 10 mM phosphate buffer, pH 7) at 50 μl / well and incubate at 37°C for 1 hour.

[0138] (2) Wash each well with 300 μl of PBST 5 times.

[0139] (3) Seal each well by incubating 100 μl of 1% BSA-PBS at 4°C overnight.

[0140] (4) Wash each well with 300 μl of PBST 5 times.

[0141] (5) Dilute the antibody culture supernatant (primary antibody) 10 times with 1% BSA-PBS and add it at 50 μl / well. React at RT for 1 hour.

[0142] (6) Wash each well with 300 μl of PBST 5 times.

[0143] (7) Anti-mouse IgG-HRP (JIR / 715-035-151) and anti-mouse IgG Lchain-HRP (JIR / 115-035-174) were diluted 20,000 times with 1% BSA-PBS and added at 50 μl / well. The reaction was carried out at RT for 0.5 hours.

[0144] (8) Wash each well with 300 μl of PBST 5 times.

[0145] (9) Add 100 μl of HRP substrate per well for color development.

[0146] (10) Add 100 μl of stop solution per well to stop the color development.

[0147] (11) OD450 was measured.

[0148] (12) By visual inspection, select 7 wells with large differences in signals to positive antigens and negative antigens and low reaction to negative antigens for secondary screening.

[0149] [Table 3]

[0150]

[0151] (3) Secondary screening

[0152] Cloning was performed by selecting wells that recognize both the core fucose and amino acids from the wells obtained in (2) above using the following antigen protein imprinting. The protein imprinting was performed as follows.

[0153] <Materials>

[0154] • Screening antigens

[0155] Positive antigen: Fucosylated AFP (AFP-L3 / recombinant)

[0156] Negative antigen 1: Unfucosylated AFP (LCA lectin non-adsorbent fraction of AFP (LEE biosolutions) from human serum)

[0157] Negative antigen 2: Fucosylated ALP (Oriental yeast / 47787055)

[0158] ·First Antibody

[0159] Antibody screening: Culture supernatant from a single positive screening.

[0160] Negative control: Hybridoma culture medium

[0161] Secondary Antibody

[0162] Anti-mouse-IgG(Fc)Ab-HRP(BET / A90-131P)

[0163] Anti-mouse IgM Ab-HRP (SBA / 1020-05)

[0164] • Blocking agent: PVDF Blocking Reagent for Can Get Signal (TOYOBO / NYPBR01)

[0165] • Detergent: TBST

[0166] • Diluent: 1% BSA-TBST

[0167] • PVDF membrane (iBlot (registered trademark) 2 Transfer Stacks, PVDF, mini / IB24002)

[0168] • Imprinting device (iBlot2)

[0169] • ECL prime Western Blotting Detection Reagent (GEhealthcare / RPN2232)

[0170] <Method>

[0171] (1) Add 1 / 4 of NuPAGE LDS Sample Buffer (4X) (Thermo / NP0008) and 1 / 10 of NuPAGE Sample Reducing Agent (10X) (Thermo / NP0009) to each screening antigen and mix.

[0172] (2) Electrophoresis (SDS-PAGE) was performed on the molecular weight markers and each screening antigen in (1) using the following antigen amounts.

[0173] Lane 0: Molecular weight marker (MagicMark XP Western Protein Standard / LC5602)

[0174] Lane 1: Positive antigen: Fucosylated AFP (AFP-L3) 50 ng

[0175] Lane 2: Negative antigen 1: Non-fucosylated AFP 50ng

[0176] Lane 3: Negative antigen 2: Fucosylated ALP 50ng

[0177] (3) Imprinted on PVDF film.

[0178] (4) The signal is sealed by immersing it in PVDF Blocking Reagent for Can Get Signal at room temperature for 1 hour.

[0179] (5) Wash with TBST 3 times.

[0180] (6) Dilute the primary antibody with 1% BSA-TBST according to the following dilution ratio and react overnight at 4°C.

[0181] Antibody screening: The culture supernatant from the first positive screening was diluted 10-fold.

[0182] Negative control: Hybridoma culture medium diluted 10-fold

[0183] (7) Wash with TBST 3 times.

[0184] (8) Dilute the secondary antibody (2 types) with 1% BSA-TBST at the dilution ratios described below and react at room temperature for 1 hour.

[0185] Anti-mouse IgG (Fc)Ab-HRP 20,000-fold dilution

[0186] Anti-mouse IgM Ab-HRP 5,000-fold dilution

[0187] (9) Wash with TBST 3 times.

[0188] (10) Detection by chemiluminescence

[0189] (11) Five wells in which only a band was detected in the positive antigen were identified as positive and cloned.

[0190] (4) Cloning

[0191] Cells from the five wells selected in (3) were seeded using limiting dilution. The culture supernatant was then screened again according to the first screening procedure in (2) to obtain two clones. The ELISA data are shown in Table 4.

[0192] [Table 4]

[0193]

[0194] Similar to the secondary screening in (3), protein blotting was performed on the culture supernatant of the two clones to confirm their specific reaction with AFP-L3. The results of the protein blotting are shown in... Figure 1 . Figure 1 The antigens for each lane are as follows.

[0195] Lane 1: Fucosylated AFP (AFP-L3 / recombinant) (positive antigen)

[0196] Lane 2: Non-fucosylated AFP (LCA lectin non-adsorbed fraction of AFP (LEE biosolutions) from human serum) (negative antigen)

[0197] Lane 3: Fucosylated ALP (Oriental yeast / 47787055) (negative antigen)

[0198] (5) Confirmation of epitopes

[0199] To confirm the epitopes of the antibodies produced by these clones, the same steps as described in (2) above were followed to confirm whether they recognized glycopeptide B (sequence number 21) and non-fucosylated glycopeptide B (sequence number 22), which have fewer amino acid residues than the sequence of glycopeptide A used as an immunogen, as shown in Table 5. The results are shown in Table 6. Here, for the positive control (PC), AAL lectin recognizing fucose was used for glycopeptide B, and WGA lectin recognizing GlcNAc was used for non-fucosylated glycopeptide B. For the negative control (NC), buffer was used.

[0200] [Table 5]

[0201]

[0202] The circle represents mannose, the square represents N-acetylglucosamine, and the triangle represents fucose.

[0203] The N-terminus of the glycopeptide is BSA-PEG4, and the C-terminus is amidated.

[0204] [Table 6]

[0205] clone name Glycopeptide B Non-fucosylated glycopeptide B 1F8-A4 0.048 0.043 2F11-2A9 0.046 0.049 PC 0.335 3.0 NC 0.044 0.044

[0206] All antibodies reacted with glycopeptide A used in the antigen but not with glycopeptide B. This result indicates that the epitope sequences of the obtained antibodies are regions containing at least one of the sequences shown in Table 7.

[0207] [Table 7]

[0208]

[0209] (6) Confirmation of CDR sequence

[0210] For the five clones obtained in (4) above, CDR analysis of the antibodies was performed. The results showed that the CDR sequences were in the following two patterns. The CDR sequences of the antibodies 1F8-A4 (sequence numbers 1-6) are shown in Table 8. In addition, the CDR sequences of the antibodies 2F11-2A9 (sequence numbers 7-12) are shown in Table 9.

[0211] [Table 8]

[0212] Heavy chain Light chain CDR1 GFNIKDYY KSLLYRDGKTY CDR2 IDPEDGES LMS CDR3 TTFFN QQLVEYPFT

[0213] [Table 9]

[0214] Heavy chain Light chain CDR1 GFNINDYF KSLLYKDGKTY CDR2 IDPEDGET LMS CDR3 TGGYFV QQLVEYPFT

[0215] Of the clones obtained above, 1F8-A4 was named I2-1F8 and deposited internationally (NITE BP-02264). Additionally, 2F11-2A9 was named I2-2F11 and deposited internationally (NITE BP-02263).

[0216] Example 2

[0217] Construction of sandwich ELISA and the effect of SDS on reactivity:

[0218] The I2-1F8 or I2-2F11 obtained in Example 1 were detected by sandwich ELISA as follows. Additionally, the effect of SDS on reactivity was investigated.

[0219] <Materials>

[0220] • Antibody sensitization plates (I2-1F8, I2-2F11 / 2.5μg / mL 100μL / well)

[0221] • Recombinant AFP-L3 antigen

[0222] • Anti-AFP antibody: Anti-alpha-fetoprotein polyclonal antibody (WLS / #PAA153Hu01)

[0223] • Labeled antibody: Goat anti-rabbit immunoglobulin-HRP

[0224] • Buffer A: 150mM NaCl + 1% BSA / 10mM phosphate buffer (pH 7)

[0225] • Buffer B: 150mM NaCl + 0.05% Tween 20 / 10mM phosphate buffer (pH 7)

[0226] <Method>

[0227] (1) (Antigen Pretreatment) Add equal volumes of 2%, 1%, 0.5%, 0.25%, 0.13%, and 0.06% SDS solutions to a 20 μg / mL AFP-L3 antigen solution, mix, and let stand for at least 3 minutes. (SDS concentration during pretreatment: 0.03-1%)

[0228] (2) Dilute with buffer A to achieve antigen concentrations of 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. (Final SDS concentration: 0.00075%-0.1%)

[0229] (3) Add antigen solution to the antibody sensitization plate at 100 μL / well and react at room temperature for 60 minutes.

[0230] (4) After washing each well with buffer B, add 400-fold diluted anti-AFP antibody at 100 μL / well and react at room temperature for 60 minutes.

[0231] (5) After washing each well with buffer B, add 100 μL of labeled antibody diluted 4000 times per well and react at room temperature for 40 minutes.

[0232] (6) After washing each well with buffer B, add 100 μL of HRP substrate per well and develop for 20 minutes.

[0233] (7) Add 100 μl of stop solution per well to stop the color development and measure OD450.

[0234] <Results>

[0235] Table 10 shows the OD450 values ​​for various antigen concentrations, final SDS concentrations, and pretreated SDS concentrations when using I2-1F8. Table 11 shows the OD450 values ​​for various antigen concentrations, final SDS concentrations, and pretreated SDS concentrations when using I2-2F11.

[0236] [Table 10]

[0237]

[0238] [Table 11]

[0239]

[0240] In the case of I2-1F8, the signal intensity increases in a concentration-dependent manner when the final SDS concentration is below 0.013%, and in the case of I2-2F11, the signal intensity increases in a concentration-dependent manner when the final SDS concentration is below 0.025%.

[0241] Example 3

[0242] Specificity confirmation of sandwich ELISA:

[0243] The specificity of the sandwich ELISA constructed in Example 2 was confirmed as follows.

[0244] <Materials>

[0245] • Antibody sensitization plates (I2-1F8, I2-2F11 / 2.5μg / mL 100μL / well)

[0246] • Positive antigen: Recombinant AFP-L3 antigen

[0247] • Negative antigen 1: Unfucosylated AFP (LCA lectin non-adsorbent fraction of AFP (LEE biosolutions) from human serum)

[0248] • Negative antigen 2: ALP, a fucosylated protein other than AFP (Oriental yeast)

[0249] • Biotinylated anti-AFP antibody: Anti-AFP, Human (mouse) (ABV / H00000174-M01)

[0250] • Test reagent: HRP-Conjugated Streptavidin (Thermo / N100)

[0251] • Buffer A: 150mM NaCl + 1% BSA / 10mM phosphate buffer (pH 7)

[0252] • Buffer B: 150mM NaCl + 0.05% Tween 20 / 10mM phosphate buffer (pH 7)

[0253] <Method>

[0254] (1) Add an equal volume of 0.06% SDS solution (denaturing) or none (non-denaturing) to each antigen solution at a concentration of 20 μg / mL, mix, and let stand for at least 3 minutes. (SDS concentration during pretreatment: 0.03%)

[0255] (2) Dilute with buffer A according to the antigen concentrations of 1000ng / mL, 500ng / mL, 250ng / mL, 125ng / mL, 63ng / mL and 31ng / mL.

[0256] (3) Add antigen solution to the antibody sensitization plate at 100 μL / well and react at room temperature for 60 minutes.

[0257] (4) After washing each well with buffer B, add 480-fold diluted biotinylated anti-AFP antibody at 100 μL / well and react at room temperature for 60 minutes.

[0258] (5) After washing each well with buffer B, add 10000 times diluted detection reagent at 100 μL / well and react at room temperature for 60 minutes.

[0259] (6) After washing each well with buffer B, add 100 μL of HRP substrate per well and develop for 10 minutes.

[0260] (7) Add 100 μl of stop solution per well to stop the color development and measure OD450.

[0261] <Results>

[0262] The measured OD450 values ​​at various antigen concentrations using I2-1F8 are shown in Table 12 and... Figure 2 In addition, the OD450 values ​​at various antigen concentrations using I2-2F11 are shown in Table 13 and... Figure 3 .

[0263] [Table 12]

[0264]

[0265] [Table 13]

[0266]

[0267] I2-1F8 and I2-2F11 showed no reaction with non-fucosylated AFP or ALP, regardless of whether they were in denatured or non-denatured states. However, for I2-1F8 and I2-2F11, an antigen concentration-dependent signal increase was observed relative to AFP-L3. The reactivity of I2-1F8 and I2-2F11 to AFP-L3 was significantly enhanced, particularly in the denatured state. These results indicate that I2-1F8 and I2-2F11 simultaneously recognize both the fucosylate and peptide moieties of AFP-L3, thereby specifically reacting with AFP-L3 via sandwich ELISA. Furthermore, it was shown that I2-1F8 and I2-2F11 reacted strongly upon pretreatment of the antigen with 0.03% SDS.

[0268] Example 4

[0269] Reactivity with natural human AFP-L3:

[0270] The reaction between I2-1F8 and natural human AFP-L3 was confirmed by Western blotting as follows.

[0271] <Materials>

[0272] Primary antibody: I2-1F8 (10-fold dilution of hybridoma culture supernatant) 4℃ O / N

[0273] Secondary antibody: Anti-mouse-IgG (Fc)Ab-HRP (BET / #A90-131P) (20,000-fold dilution) RT 1 hour

[0274] <Method>

[0275] Except for changing the primary and secondary antibodies to the antibodies described above, protein blotting was performed in the same manner as in Example 1(3).

[0276] <Results>

[0277] The results of protein imprinting are shown in Figure 4 I2-1F8 also showed a reaction with natural human AFP-L3. Figure 4 The antigens for each lane are as follows.

[0278] Lane 1: Recombinant AFP-L3

[0279] Lane 2: Non-fucosylated AFP

[0280] Lane 3: Natural Human AFP (Muttasco AFP-L3 Standard Product 1)

[0281] Lane 4: Natural Human AFP-L3 (Muttasco AFP-L3 Standard Product 2)

[0282] Example 5

[0283] The effect of SDS on reactivity:

[0284] The effect of SDS on reactivity was investigated using the ELISA constructed in Example 2.

[0285] <Materials>

[0286] Except for using I2-1F8 as the antibody, the same materials as in Example 2 were used.

[0287] <Method>

[0288] (1) (Antigen pretreatment) Add equal amounts of 0.031%, 0.016%, 0.008%, and 0.004% SDS solution to a 20 μg / mL AFP-L3 antigen solution, mix, and let stand for at least 3 minutes.

[0289] (2) Dilute with buffer A to achieve antigen concentrations of 0.4 μg / mL, 0.2 μg / mL, and 0.1 μg / mL. (Final SDS concentration: 0.00032%-0.00001%)

[0290] (3) Subsequent operations shall be carried out in the same manner as in Example 2.

[0291] <Results>

[0292] Table 14 shows the measured values ​​of OD450 at various antigen concentrations, final SDS concentrations, and pretreated SDS concentrations when using I2-1F8.

[0293] [Table 14]

[0294]

[0295] Concentration-dependent signals can also be detected when the pretreated SDS concentration is below 0.016%. sequence list <110> Sysmex Corporation <120> Monoclonal antibodies that react with glycopeptides and their applications <130> IIC170517 <150> JP 2016-108461 <151> 2016-05-31 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 1 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 2 Ile Asp Pro Glu Asp Gly Glu Ser 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 3 Thr Thr Phe Phe Asn 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 4 Lys Ser Leu Leu Tyr Arg Asp Gly Lys Thr Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 5 Leu Met Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 6 Gln Gln Leu Val Glu Tyr Pro Phe Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 7 Gly Phe Asn Ile Asn Asp Tyr Phe 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 8 Ile Asp Pro Glu Asp Gly Glu Thr 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 9 Thr Gly Gly Tyr Phe Val 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 10 Lys Ser Leu Leu Tyr Lys Asp Gly Lys Thr Tyr 1 5 10 <210> 11 <211> 3 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 11 Leu Met Ser 1 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 12 Gln Gln Leu Val Glu Tyr Pro Phe Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by formula (1) <220> <221> CARBOHYD <222> (4)..(4) <400> 13 Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by equation (2) <220> <221> CARBOHYD <222> (4)..(4) <400> 14 Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by formula (3) <220> <221> CARBOHYD <222> (4)..(4) <400> 15 Thr Lys Val Asn Phe Thr 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by equation (4) <220> <221> CARBOHYD <222> (4)..(4) <400> 16 Thr Lys Val Asn Phe Thr 1 5 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by equations (5) and (7) <220> <221> CARBOHYD <222> (1)..(1) <400> 17 Asn Phe Thr Glu Ile Gln 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Glycopeptides represented by equations (6) and (8) <220> <221> CARBOHYD <222> (4)..(4) <400> 18 Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide A <220> <221> CARBOHYD <222> (5)..(5) <400> 19 Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Non-fucosylated glycopeptide A <220> <221> CARBOHYD <222> (5)..(5) <400> 20 Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide B <220> <221> CARBOHYD <222> (5)..(5) <400> twenty one Gly Thr Lys Val Asn Phe Thr 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Non-fucosylated glycopeptide B <220> <221> CARBOHYD <222> (5)..(5) <400> twenty two Gly Thr Lys Val Asn Phe Thr 1 5 <210> twenty three <211> 6 <212> PRT <213> Artificial sequence <220> <223> Table 1 <220> <221> CARBOHYD <222> (1)..(1) <400> twenty three Asn Phe Thr Glu Ile Gln 1 5 <210> twenty four <211> 10 <212> PRT <213> Artificial sequence <220> <223> Tablet 2 <220> <221> CARBOHYD <222> (5)..(5) <400> twenty four Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> 25 <211> 609 <212> PRT <213> Homo sapiens <300> <308> NM_001134 <309> 2016-09-10 <313> (1)..(609) <400> 25 Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr 1 5 10 15 Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30 Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45 Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60 Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp 65 70 75 80 Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95 Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110 Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125 Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140 Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn 145 150 155 160 Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175 Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190 Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205 Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220 Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val 225 230 235 240 Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255 Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270 Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285 Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300 Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp 305 310 315 320 Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335 Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350 Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365 Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380 Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu 385 390 395 400 Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415 Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430 Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445 Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460 Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile 465 470 475 480 Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495 Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525 Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540 Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys 545 550 555 560 Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575 Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590 Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605 Val

Claims

1. A monoclonal antibody which reacts with a glycopeptide shown below: …（1）， wherein the heavy chain of said monoclonal antibody contains: HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and the light chain contains: LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:

6.

2. A monoclonal antibody which reacts with a glycopeptide shown below: the heavy chain of said monoclonal antibody contains: …（1）， wherein HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9, and the light chain contains: LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:

12.

3. A monoclonal antibody produced by a hybridoma having an international deposit number of NITE BP-02263 or NITE BP-02264.

4. A hybridoma having an international deposit number of NITE BP-02263 or NITE BP-02264.

5. A method for measuring fucosylated AFP, which uses the antibody according to any one of claims 1 to 3.

6. The method according to claim 5, wherein, the fucosylated AFP is pretreated with a solution containing 0.03 mass / mass% or more of SDS, after the treatment, the fucosylated AFP is reacted with the antibody in the presence of 0.025 mass / mass% or less of SDS.

7. A kit for detecting fucosylated AFP, which contains a capture antibody for fucosylated AFP, a detection antibody for fucosylated AFP, and a solid phase, the capture antibody or the detection antibody is the antibody according to any one of claims 1 to 3. further contains a pretreatment reagent containing 0.03 mass / mass% or more of SDS. ​ 8. The kit of claim 7, wherein, ​

Images