Monoclonal antibodies reactive with glycopeptides and uses thereof

Abstract

The present invention relates to monoclonal antibodies reactive with glycopeptides and uses thereof. The present invention provides monoclonal antibodies that have as epitopes both the fucose moiety and the amino acids of the peptide moiety of a glycopeptide. The monoclonal antibodies are reactive with (a) glycopeptides of the formula: 【Chemical 1】 but not with (b) glycopeptides of the formula: 【Chemical 2】

Description

[Technical Field]

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

[0002] As hepatitis progresses from cirrhosis to liver cancer, an increase in alpha-fetoprotein (AFP) bound to lentil lectin (LCA) (LCA-bound AFP) has been reported in biological samples. Patent Document 1 describes an antibody for detecting LCA-bound AFP. The antibody in Patent Document 1 is described as reactive to bound AFP but not reactive 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 technical problem that the invention aims to solve]

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

[0008] In the embodiments of Patent Document 1, antibodies that bind to LCA-binding AFP (AFP-LCA-R) but do not bind to LCA-non-binding AFP (AFP-LCA-NR) were obtained (see Example 1, Table 1). Figure 1 However, the epitope of this antibody is unknown. In the examples, LCA binding and non-binding, as described above, are considered to be due to the presence or absence of fucose, and the binding to the antigen may depend on the fucose moiety rather than the peptide sequence. In this case, not only AFP, but also proteins with additional fucose may bind nonspecifically. Therefore, the development of monoclonal antibodies using both the fucose moiety of the glycopeptide and the amino acid of the peptide moiety as epitopes is desirable.

[0009] [Technical Solution to the Problem]

[0010] This invention provides a monoclonal antibody, which

[0011] Reaction with the glycopeptide shown in (a):

[0012]

Chemistry 1

[0013]

[0014] It does not react with the glycopeptide shown in (b):

[0015]

Chemistry 2

[0016]

[0017] This invention provides hybridomas with international accession numbers NITE BP-02349 or NITE BP-02350.

[0018] This invention provides a method for determining fucosylated AFP using the above-described antibody.

[0019] 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 aforementioned antibody.

[0020] [Invention Effects]

[0021] The antibody of the present invention can recognize both the glycan and peptide moieties with a single antibody and with high specificity. [Attached Image Description]

[0022]

【 Figure 1 The graph shows the results of solid-phase ELISA performed using the culture supernatant of antibody-produced cells, with either the positive antigen (glycopeptide A) or the negative antigen (non-fucosylated glycopeptide A) listed in Table 2 as the antigen.

[0023]

【 Figure 2 The graph shows the results of solid-phase ELISA performed on the culture supernatant of antibody-produced cells, using recombinant AFP-L3, non-fucosylated AFP, and fucosylated ALP as antigens.

[0024]

【 Figure 3 The graph shows the results of solid-phase ELISA using hybridoma culture supernatant with recombinant AFP-L3, non-fucosylated AFP, and fucosylated ALP as antigens.

[0025]

【 Figure 4The image shows the results of Western blotting of clones S4-1F8 and S4-4F9 obtained in Example 1 (lane 1: fucosylated AFP (AFP-L3 / recombinant) (positive antigen), lane 2: non-fucosylated AFP (LCA lectin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions)) (negative antigen), lane 3: fucosylated ALP (ORIENTAL yeast / 47787055)) (negative antigen).

[0026]

【 Figure 5 The graph shows the results of solid-phase ELISA using positive glycopeptides (Fuc+) and negative glycopeptides (Fuc-) as antigens.

[0027]

【 Figure 6 The image shows the results of Western blot analysis of S4-1F8 (lane 1: recombinant AFP-L3, lane 2: unfucosylated AFP, lane 3: natural human AFP (μTAS WAKO AFP-L3 calibrator 1), lane 4: natural human AFP-L3 (μTASWAKO AFP-L3 calibrator 2)).

Implementation Method

[0028] The antibody of this embodiment can exhibit specificity in an assay system using biological samples or the like that employing the antibody of this embodiment. For example, even if it binds non-specifically to substances not present in the biological sample, it can still exhibit specificity in an environment where the antibody of this embodiment is typically used, thus achieving the effects of the present invention. Specifically, when detecting substances in blood samples such as whole blood, serum, and plasma by ELISA, it is sufficient that it exhibits specificity in the ELISA assay system, and it can also bind to substances not normally present in the blood sample or ELISA reagent.

[0029] The antibody used in this embodiment is a monoclonal antibody.

[0030] Reaction with the glycopeptide (SEQ ID NO:13) shown in (a):

[0031]

Transformation 3

[0032]

[0033] It does not react with the glycopeptide (SEQ ID NO:14) shown in (b):

[0034]

Chemistry 4

[0035]

[0036] Furthermore, in this specification, the antibody "reacting with glycopeptides" in this embodiment refers to the binding of glycopeptides and antibodies due to an antigen-antibody reaction.

[0037] Furthermore, the aforementioned antibody preferably reacts with fucosylated AFP.

[0038] Furthermore, the aforementioned antibody can bind to denatured fucosylated AFP pretreated with a denaturing agent such as SDS or heat. When using an SDS-containing solution as pretreatment, the concentration of SDS used to sufficiently denature the fucosylated AFP (hereinafter referred to as the "pretreatment SDS concentration") is not particularly limited, but preferably 0.03% by mass (hereinafter simply "%)" or more, more preferably 0.25%. On the other hand, if an excessive amount of denaturing agent is used during antigen-antibody reaction, there is a possibility that the denaturation of the antibody may also have an adverse 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 a denaturing agent, the concentration of SDS during antigen-antibody reaction (hereinafter referred to as the "final SDS concentration") is not particularly limited, but preferably 0.025% or less, more preferably 0.0015%.

[0039] Furthermore, in this specification, the antibody's "reaction with fucosylated AFP" in this embodiment refers to the binding of fucosylated AFP and the antibody due to an antigen-antibody reaction. Fucosylated AFP can also be either recombinant or natural. Natural fucosylated AFP is, for example, AFP present in human blood. The sequence of human AFP, for example, is registered with GenBank Accession No. NM_001134, and has the amino acid sequence SEQ ID NO:26.

[0040] Furthermore, the aforementioned antibody is preferably reacted with denatured fucosylated AFP 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.

[0041] Furthermore, the aforementioned antibody preferably does not react with denatured non-fucosylated AFP 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.

[0042] Examples of CDRs for antibodies used in this embodiment include the following CDRs.

[0043] <cdr-a>

[0044] The heavy chain CDR contains the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0045] The CDR of the light chain contains the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence shown in SEQ ID NO:6.

[0046] <cdr-b>

[0047] The CDR of the heavy chain contains the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0048] The CDR of the light chain contains the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0049] The hybridomas that produced antibodies of this embodiment with the aforementioned CDR-A and CDR-B as CDRs were named S4-1F8 and S4-4F9, respectively, and were internationally deposited on September 8, 2016, at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation (Room 8122, 2-5-8, Kazusa-Kamazoshi, Kisarazu City, Chiba Prefecture, Japan 292-0818) as NITE BP-02349 and NITE BP-02350.

[0050] The antibody of this embodiment is obtained by a method comprising the step of immunizing animals with a glycopeptide antigen (SEQ ID NO:15) containing the following structure:

[0051]

Transformation 5

[0052]

[0053] X is any sugar chain, as long as it is a sugar chain that is generally bound to glycoproteins or glycopeptides, without any particular restrictions.

[0054] Biomolecules such as KLH and BSA can also be bound to the N-terminus of glycopeptide antigens via PEG or similar methods. Furthermore, the C-terminus of these glycopeptide antigens can also 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.

[0055] The process of immunizing animals with glycopeptide antigens can utilize the animal immunization process in known monoclonal antibody production methods. Examples of monoclonal antibody production methods include, for instance, the mouse spleen method and the mouse intestinal lymph node method (see Japanese Patent No. 4098796).

[0056] There are no particular restrictions on the animals used for immunization; any non-human animal can be selected based on a suitable method for producing monoclonal antibodies.

[0057] Specifically, when using the mouse spleen method as a method for producing monoclonal antibodies, the animals can be immunized according to known methods.

[0058] After immunizing the animals, hybridomas are prepared and screened according to known methods to obtain the antibodies of this embodiment.

[0059] In hybridoma screening, glycopeptides that serve as antigens, substances from which the core fucose has been removed from glycopeptides that serve as antigens, substances containing a portion of the amino acid residues of glycopeptides that serve 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 of polypeptides with a core fucose and an amino acid sequence different from that of AFP (e.g., fucosylated ALP) can be used as suitable positive or negative antigens.

[0060] The screening criteria for hybridomas are, for example, when using ELISA, a difference of OD450 values ​​between the positive and negative antigens greater than 0.05, and an OD450 value of the negative antigen less than 0.05.

[0061] The isotype of the antibody in this embodiment is not particularly limited. In addition, the antibody in this embodiment also includes fragments containing peptides such as F(ab')2, Fab', Fab, and CDR.

[0062] The antibody in this embodiment can also be labeled with biotinylation, ALP, etc.

[0063] The antibody of this embodiment, due to the aforementioned properties, reacts with fucosylated AFP but not with non-fucosylated AFP. Therefore, the antibody of this embodiment, for example, can be used to measure fucosylated AFP in biological samples.

[0064] Examples of biological samples include whole blood, serum, and plasma collected from the subject. These biological samples may also undergo pretreatment such as centrifugation or denaturation. Denaturation treatment is preferred for the biological samples. The denaturation treatment is performed at room temperature (25°C) in the presence of 2% SDS and 50 mM DTT.

[0065] To determine fucosylated AFP using the antibody of this embodiment, known immunological assay methods can be employed. Examples of such immunological assays 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 using a sandwich ELISA method will be described below.

[0066] 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. This complex, when the biological sample contains fucosylated AFP, can be formed by mixing the biological sample, the capture antibody, and the detection antibody. Furthermore, by contacting the solution containing the complex with a solid phase capable of capturing the capture antibody, the aforementioned complex can be formed on the solid phase. Alternatively, a solid phase pre-immobilized with the capture antibody can also be used. That is, by contacting a solid phase immobilized with the capture antibody, the biological sample, and the detection antibody, the aforementioned complex can be formed on the solid phase. The antibody of this embodiment can be used for at least one of the capture antibody and the detection antibody.

[0067] The method of immobilizing the capture antibody onto the solid phase is not particularly limited. For example, the capture antibody and the solid phase can be directly bound, or they can be indirectly bound via another substance. 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 and the solid phase can be indirectly bound via the binding of biotin and the avidin.

[0068] The raw materials for the solid phase are not particularly limited; for example, they can be selected from organic polymers, inorganic compounds, and biological polymers. Examples of organic polymers include latex, polystyrene, and polypropylene. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, and ferrite, etc.), silicon oxide, alumina, and glass. Examples of biological polymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these can also be used in combination. The shape of the solid phase is not particularly limited; for example, it can be particles, membranes, microplates, microtubes, and test tubes.

[0069] The fucosylated AFP value in a biological sample can be obtained by detecting the complex formed on the solid phase using methods known in the art. For example, when an antibody labeled with a labeling substance is used as the detection antibody, the fucosylated AFP value can be obtained by detecting the signal generated by the labeling substance. Alternatively, the fucosylated AFP value can also be obtained similarly when a second antibody labeled with the detection antibody is used.

[0070] In this embodiment, it is preferable to pretreat fucosylated AFP as described above. When using an SDS-containing solution as pretreatment, the concentration of SDS is not particularly limited, but preferably 0.03% or more, more preferably 0.25%. 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 an SDS-containing solution as a denaturing agent, the final SDS concentration is not particularly limited, but preferably 0.025% or less, more preferably 0.0015%. In this embodiment, it is preferable to perform such treatment to react the antibody and the fucosylated AFP to obtain a measured value of fucosylated AFP.

[0071] In this embodiment, B / F (Bound / Free) separation to remove unreacted free components that do not form a complex can also be performed between the complex formation step and the complex detection step. Unreacted free components refer to components that do not constitute a complex. 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 particulate, B / F separation can be performed by centrifuging to recover only the solid phase containing the complex. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. Alternatively, when the solid phase is magnetic particles, B / F separation can be performed by suctioning the liquid containing the unreacted free components through a nozzle while the magnetic particles are magnetically confined by a magnet. After removing the unreacted free components, the solid phase containing the complex can also be washed with a suitable aqueous medium such as PBS.

[0072] 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 representing the signal strength in stages, such as "no signal," "weak," "medium," and "strong." In this embodiment, quantitative or semi-quantitative detection of signal strength is preferred.

[0073] The labeling substance is not particularly limited as long as it generates 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 generates a signal by catalyzing the reaction of other substances. Examples of signal-generating substances include fluorescent substances and radioactive isotopes. Examples of substances that generate a detectable signal by catalyzing the reaction of other substances include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, and luciferase. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and AlexaFluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioactive isotopes include… 125 I, 14 C 32 P, etc. Among them, enzymes are preferred as markers, especially alkaline phosphatase and peroxidase.

[0074] The method for detecting the signal is known in the prior art. In this embodiment, it is appropriate to select a measurement method based on the type of signal derived from the labeled substance. For example, when the labeled substance is an enzyme, the signal, such as light or color, generated by reacting with the substrate of the enzyme can be measured using a known device such as a spectrophotometer.

[0075] The substrate for an enzyme can be appropriately selected from known substrates depending on the type of enzyme. For example, when using alkaline phosphatase as an enzyme, suitable substrates include chemiluminescent substrates such as CDP-Star (4-chloro-3-(methoxyspiro[1,2-dioxane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), CSPD (3-(4-methoxyspiro[1,2-dioxane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. In addition, when peroxidase is used as an enzyme, examples of substrates include chemiluminescent substrates such as LUMINOR and its derivatives, and chromogenic substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylene diamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0076] When the labeled substance is a radioactive isotope, the radiation signal can be measured using a known device such as a scintillation counter. Similarly, when the labeled substance is a fluorescent substance, the fluorescence signal can be measured using a known device such as a fluorescence microplate reader. Furthermore, the excitation wavelength and fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.

[0077] The signal detection results can also be used as the determination value of fucosylated AFP. For example, when the signal intensity is also quantitatively detected, the signal intensity determination value itself or the value obtained from that determination value can be used as the determination value of fucosylated AFP. As a value obtained from the signal intensity determination value, for example, the value obtained by subtracting the determination value of the negative control sample or the background value from the determination value of fucosylated AFP can be given. The negative control sample can be appropriately selected, for example, a biological sample obtained from a healthy individual.

[0078] In this embodiment, fucosylated AFP concentrations can be measured using multiple standard samples with known fucosylated AFP concentrations, and a standard curve representing the relationship between fucosylated AFP concentration and fucosylated AFP measurement values ​​can be generated. The fucosylated AFP measurement values ​​obtained from biological samples can be applied to this standard curve to obtain the fucosylated AFP concentration in the biological samples.

[0079] In this embodiment, the concentration of fucosylated AFP in a biological sample can also be determined by a sandwich ELISA method using a capture antibody immobilized on magnetic particles and a detection antibody labeled with a labeling substance. In this case, the determination can also be performed using commercially available fully automated immunoassay apparatus such as the HISCL series (manufactured by Sysmex Corporation).

[0080] 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.

[0081] Furthermore, when the labeling substance of the antibody used for detection is an enzyme, the fucoidylated AFP detection kit of this embodiment may also contain a substrate for the enzyme. The form of the labeling substance and the matrix is ​​not particularly limited, and may be solid (e.g., powder, crystals, freeze-dried products, etc.) or liquid (e.g., solution, suspension, emulsion, etc.).

[0082] To pretreat the fucosylated AFP described above, the fucosylated AFP detection kit of this embodiment may also contain a pretreatment reagent containing 0.03% or more SDS, preferably 0.25% SDS. This reagent is the same as the solution containing 0.03% or more SDS described above.

[0083] The fucosylated AFP detection kit of this embodiment is also suitable for pretreatment solutions containing biological samples, cleaning solutions for solid phases, enzyme reaction stoppers, calibrators, etc.

[0084] The fucoidosylated AFP detection kit of this embodiment contains the capture antibody, detection antibody, and solid phase in a suitable container, or individually packaged, according to the kit's form. In this fucoidosylated AFP detection kit, the capture antibody can bind directly to the solid phase, or the capture antibody and solid phase can bind indirectly via another substance. When the capture antibody and solid phase bind indirectly, they can be contained in different containers in this kit. When the capture antibody and solid phase bind indirectly, for example via biotin and avidin, the biotin-modified capture antibody can be contained in one container, and the avidin-bound solid phase in another container. Furthermore, the details regarding the biological sample, capture antibody, detection antibody, and solid phase are the same as described in the above description of the assay method.

[0085] The association 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.

Example

[0086] The present invention will now be described in detail using specific embodiments, but the present invention is not limited to these embodiments in any way.

[0087] [Example 1: Obtaining Antibodies]

[0088]

(1) Obtaining the antibody group

[0089] Hybridomas producing antibodies were obtained from mouse spleens. Specifically, glycopeptide A (SEQ ID NO: 16) with the structure described in Table 1 below was synthesized, conjugated with KLH, and immunized in three mice. After confirming an increase in antibody titer, hybridomas were obtained by fusing isolated lymphocytes from the mouse spleen with myeloma cells.

[0090] Table 1

[0091]

[0092] The dots represent mannose, the squares represent N-acetylglucosamine, and the triangles represent fucose. The N-terminus of the glycopeptide is KLH-PEG4, and the C-terminus is amidated.

[0093]

(2) First screening

[0094] The hybridomas obtained in (1) above were selected by antigen solid-phase ELISA using the positive antigen (glycopeptide A) or negative antigen (non-fucosylated glycopeptide A: SEQ ID NO: 17) listed in Table 2, with wells showing minimal reaction to the positive antigen and minimal reaction to the negative antigen. Antigen solid-phase ELISA was performed using the following method. Results are shown in... Figure 1 .

[0095] Table 2

[0096]

[0097] The dots represent mannose, the squares represent N-acetylglucosamine, and the triangles represent fucose. The N-terminus of the glycopeptide is KLH-PEG4, and the C-terminus is amidated.

[0098] <Method>

[0099] (1) Add 1 μg / ml of each screening antigen (diluent 10mM phosphate buffer pH 7) to each well of a 96-well plate (nunc Maxisoap / 446612) at 50 μl / well and incubate at 37°C for 1 hour.

[0100] (2) Wash each well with 300 μl PBST per well × 5 times.

[0101] (3) Block each well with 100 μl of 1% BSA-PBS overnight at 4°C.

[0102] (4) Wash each well with 300 μl PBST per well × 5 times.

[0103] (5) Dilute the antibody culture supernatant (primary antibody) 10 times with 1% BSA-PBS, add 50 μl / well, and react at RT for 1 hour.

[0104] (6) Wash each well with PBST 300μl / well × 5 times.

[0105] (7) Anti-mouse IgG-HRP (JIR / 715-035-151) and anti-mouse IgG L-chain-HRP (JIR / 115-035-174) were diluted 20,000 times with 1% BSA-PBS and added at 50 μl / well, and reacted at RT for 0.5 hr.

[0106] (8) Wash each well with 300 μl PBST per well × 5 times.

[0107] (9) Add HRP substrate at 100 μl / well and develop color.

[0108] (10) Add 100 μl of stop solution per well to stop the color development.

[0109] (11) OD450 was measured.

[0110]

(3) Second screening

[0111] Similar to the first screening, antibody-produced cell culture supernatant was used, and antigen-solid phase ELISA was performed using AFP-L3, AFP, and fucose-modified ALP as antigens. Results are shown in... Figure 2 .

[0112] Antibodies were selected from 1F8 and 4F9 clones based on the results of the first and second screenings. Of the clones obtained above, 1F8 clone was named S4-1F8 and deposited internationally (NITE BP-02349). Similarly, 4F9 clone was named S4-4F9 and deposited internationally (NITE BP-02350).

[0113] [Example 2: Confirmation of the specificity of the obtained antibody]

[0114] To investigate the response specificity of antibodies S4-1F8 and S4-4F9 produced from the clones selected in Example 1, the following investigation was conducted.

[0115]

(1) Antigen solid-phase ELISA

[0116] <Materials>

[0117] Solid-phase antigens: recombinant AFP-L3, non-fucosylated AFP, fucosylated ALP

[0118] Primary antibodies: S4-1F8, S4-4F9 (hybridoma culture supernatant)

[0119] Second antibody: Anti-mouse IgG-HRP (Institute of Medical Biology / IM-0817)

[0120] <Method>

[0121] (1) Add 0.5 μg / ml of each antigen (10 mM Tris buffer, pH 7.4) to a 96-well plate (nunc Maxisoap / 446612) at 100 μl / well and incubate on RT for 1 hour.

[0122] (2) Clean each well with 300 μl TBST per well × 5 times.

[0123] (3) Seal each well with 300 μl of 1% BSA-TBS per well and incubate overnight at 4°C.

[0124] (4) Clean each well with 300 μl TBST per well × 5 times.

[0125] (5) Dilute the antibody culture supernatant (primary antibody) 10 times with 1% BSA-TBST, add 100 μl / well, and react at RT for 1 hour.

[0126] (6) Clean each well with 300 μl TBST per well × 5 times.

[0127] (7) Dilute the second antibody 10,000 times with 1% BSA-TBST, add 100 μl / well, and react at RT for 0.5 hr.

[0128] (8) Clean each well with 300 μl TBST per well × 5 times.

[0129] (9) Add HRP substrate at 100 μl / well and develop color.

[0130] (10) Add 100 μl of stop solution per well to stop the color development.

[0131] (11) OD450 was measured.

[0132] <Results>

[0133] The results of antigen solid-phase ELISA are shown in Figure 3 Both S4-1F8 and S4-4F9 showed a specific response to AFP-L3.

[0134]

(2) Western blot

[0135] <Materials>

[0136] Electrophoretic antigens: 0.05 μg each of recombinant AFP-L3 (lane 1), non-fucosylated AFP (lane 2), and fucosylated ALP (lane 3).

[0137] Primary antibody: S4-1F8, S4-4F9 (diluted 10-fold to hybridoma culture supernatant) 4℃ O / N

[0138] Second antibody: A mixture of the following two, incubated at RT for 1 hour.

[0139] Anti-mouse-IgG (Fc)Ab-HRP (BET / cat#A90-131P) (diluted 20,000 times)

[0140] Anti-mouse IgM Ab-HRP (SBA / cat#1020-05) (diluted 5,000 times)

[0141] <Method>

[0142] (1) Add NuPAGE LDS sample buffer (4×)(Thermo / NP0008) to each antigen at 1 / 4 volume and NuPAGE sample reducing agent (10×)(Thermo / NP0009) at 1 / 10 volume and mix.

[0143] (2) Electrophoresis (SDS-PAGE) was performed on the molecular weight markers and each antigen in (1).

[0144] (3) Imprinted onto PVDF film.

[0145] (4) The PVDF Blocking Reagent for Can Get Signal was sealed by immersing it at room temperature for 1 hour.

[0146] (5) Clean with TBST 3 times.

[0147] (6) Dilute the first antibody with 1% BSA-TBST to the dilution ratios shown below and react overnight at 4°C.

[0148] (7) Clean with TBST 3 times.

[0149] (8) Dilute the second antibody with 1% BSA-TBST to the dilution ratio described in the above <Materials> and react at room temperature for 1 hour.

[0150] (9) Clean with TBST 3 times.

[0151] (10) Detection using chemiluminescence.

[0152] <Results>

[0153] The results of the protein blot are shown in Figure 4 S4-1F8 and S4-4F9 both showed that they reacted only with AFP-L3.

[0154]

(3) Confirmation of epitopes

[0155] To investigate the epitope ranges of antibodies S4-1F8 and S4-4F9, the following exploration was conducted.

[0156] <Materials>

[0157] Solid-phase antigen: Use the glycopeptides (SEQ ID NO: 18-25) (Fuc+, Fuc-) shown in Table 3 below.

[0158] Primary antibodies: S4-1F8, S4-4F9 (hybridoma culture supernatant)

[0159] Second antibody: Anti-mouse IgG-HRP (Institute of Medical Biology / IM-0817)

[0160] Table 3

[0161]

[0162] <Method>

[0163] (1) Add 2 μg / ml of each antigen (10 mM Tris buffer pH 7.4) to a 96-well plate (nunc Maxisoap / 446612) at 100 μl / well and incubate at RT for 1 hour.

[0164] (2) Clean each well with 300 μl TBST per well × 5 times.

[0165] (3) Seal each well overnight at 4°C with 300 μl of 1% BSA-TBS.

[0166] (4) Clean each well with 300 μl TBST per well × 5 times.

[0167] (5) Dilute the antibody culture supernatant (primary antibody) 10 times with 1% BSA-TBST, add 100 μl / well, and react at RT for 1 hour.

[0168] (6) Clean each well with 300 μl TBST per well × 5 times.

[0169] (7) Dilute the second antibody 10,000 times with 1% BSA-TBST, add 100 μl / well, and react at RT for 0.5 hr.

[0170] (8) Clean each well with 300 μl TBST per well × 5 times.

[0171] (9) Add HRP substrate at 100 μl / well and develop color.

[0172] (10) Add 100 μl of stop solution per well to stop the color development.

[0173] (11) OD450 was measured.

[0174] <Results>

[0175] The results of antigen solid-phase ELISA are shown in Figure 5 S4-1F8 and S4-4F9 are reactive to any positive glycopeptide (Fuc+) but not to negative glycopeptides (Fuc-).

[0176] The reactivity of any glycopeptide differs significantly depending on the presence or absence of fucose, suggesting the presence of fucose in the epitope.

[0177] The lack of reactivity with other proteins containing fucose (ALP) suggests that the epitope contains not only fucose but also a peptide moiety. Since it also reacts with the shortest glycopeptide (7a.a.3 sugar), the peptide moiety of the epitope is believed to be contained within "TKVNFT".

[0178]

(4) Confirmation of CDR sequence

[0179] CDR sequences of S4-1F8 and S4-4F9 were analyzed. The CDR sequences of the S4-1F8 antibody (SEQ ID NO: 1-6) are shown in Table 4. The CDR sequences of the S4-4F9 antibody (SEQ ID NO: 7-12) are shown in Table 5.

[0180] Table 4

[0181] Heavy chain Light chain CDR1 GFNIKDYY GNIHNY CDR2 IDPEDGES DAK CDR3 ARPLYSTYDVDWYFDV QHFWTTPLT

[0182] Table 5

[0183] Heavy chain Light chain CDR1 GFNIKDYY GNIHNY CDR2 IDPEDGES DVK CDR3 ARPLYSTYDVDWYFDV QHFWTTPLT

[0184] [Example 3: Construction of Sandwich ELISA and the Effect of SDS on Reactivity]

[0185] Using S4-1F8 or S4-4F9 obtained in Example 1, the construction of sandwich ELISAs as described below was attempted. Additionally, the effect of SDS on reactivity was investigated.

[0186] <Materials>

[0187] Antibody sensitization plates (S4-1F8, S4-4F9 / 2.5μg / mL 100μL / well)

[0188] Recombinant AFP-L3 antigen anti-AFP antibody: a polyclonal antibody against alpha-fetoprotein (WLS / #PAA153Hu01)

[0189] Labeled antibody: Goat anti-rabbit immunoglobulin-HRP

[0190] Buffer A: 150mM NaCl + 1% BSA / 10mM phosphate buffer (pH 7)

[0191] Buffer B: 150mM NaCl + 0.05% Tween 20 / 10mM phosphate buffer (pH 7)

[0192] <Method>

[0193] (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. (Pretreatment SDS concentration: 0.03-1%)

[0194] (2) Dilute with buffer A to antigen concentrations of 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. (Final SDS concentration: 0.00075%-0.1%)

[0195] (3) Add antigen solution to the antibody sensitization plate at 100 μL / well and react at room temperature for 60 minutes.

[0196] (4) After washing each well with buffer B, add 100 μL of anti-AFP antibody diluted 400 times per well and react at room temperature for 60 minutes.

[0197] (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.

[0198] (6) After washing each well with buffer B, add 100 μl of HRP substrate per well and develop for 20 minutes.

[0199] (7) Add 100 μl of stop solution per well to stop the color development and measure OD450.

[0200] <Results>

[0201] The OD450 values ​​for various antigen concentrations, final SDS concentrations, and pretreated SDS concentrations when using S4-1F8 are shown in Table 6. Additionally, the OD450 values ​​for various antigen concentrations, final SDS concentrations, and pretreated SDS concentrations when using S4-4F9 are shown in Table 7.

[0202] Table 6

[0203]

[0204] Table 7

[0205]

[0206] Signals were detected at all SDS concentrations, indicating the presence of AFP-L3. Strong signals were detected at SDS concentrations below 0.05% during the antigen-antibody reaction, and even stronger signals were detected at concentrations below 0.025%.

[0207] [Example 4: Specificity Confirmation of Sandwich ELISA]

[0208] For sandwich ELISA using the S4-1F8 constructed in Example 3, specificity was confirmed as follows.

[0209] <Materials>

[0210] Antibody sensitization plate (S4-1F8 / 2.5μg / mL 100μL / well)

[0211] Positive antigen: Recombinant AFP-L3 antigen

[0212] Negative antigen 1: Non-fucosylated AFP (LCA lectin non-adsorbent fraction of human serum-derived AFP (LEE biosolutions))

[0213] Negative antigen 2: ALP, a fucosylated protein other than AFP (Oriental yeast)

[0214] Biotinylated anti-AFP antibody: Anti-AFP, human (mouse) (ABV / H00000174-M01)

[0215] Test reagent: HRP-conjugated streptavidin (Thermo / N100)

[0216] Buffer A: 150mM NaCl + 1% BSA / 10mM phosphate buffer (pH 7)

[0217] Buffer B: 150mM NaCl + 0.05% Tween 20 / 10mM phosphate buffer (pH 7)

[0218] <Method>

[0219] (1) Add equal volumes 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. (Pretreatment SDS concentration: 0.03%)

[0220] (2) Dilute with buffer A to the antigen concentrations of 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 63 ng / mL, and 31 ng / mL.

[0221] (3) Add antigen solution to the antibody sensitization plate at 100 μL / well and react at room temperature for 60 minutes.

[0222] (4) After washing each well with buffer B, add 100 μL of biotinylated anti-AFP antibody diluted 480 times per well and react at room temperature for 60 minutes.

[0223] (5) After washing each well with buffer B, add 100 μL of the detection reagent diluted 10,000 times per well and react at room temperature for 60 minutes.

[0224] (6) After washing each well with buffer B, add 100 μl of HRP substrate per well and develop for 10 minutes.

[0225] (7) Add 100 μl of stop solution per well to stop the color development and measure OD450.

[0226] <Results>

[0227] The measured values ​​of OD450 for various antigen concentrations when using S4-1F8 are shown in Table 8.

[0228] Table 8

[0229] Antigen concentration (ng / mL) 1000 500 250 125 63 31 Transgenic AFP-L3 ※ ※ 3.32 2.77 1.89 1.17 Non-denatured AFP-L3 3.15 2.57 1.77 1.13 0.71 0.47 Transgender AFP 0.23 0.22 0.22 0.21 0.21 0.22 Non-denatured AFP 0.24 0.23 0.23 0.21 0.22 0.22 Transgender ALP 0.25 0.24 0.24 0.23 0.23 0.25 Non-denatured ALP 0.26 0.25 0.27 0.25 0.26 0.28

[0230] ※No data available due to exceeding the upper limit of the measurement.

[0231] In both the denatured and non-denatured states, the S4-1F8 antibody showed no response to non-fucosylated AFP and ALP, but an antigen concentration-dependent increase in signal was observed for AFP-L3. In particular, the reactivity to AFP-L3 was significantly enhanced in the denatured state. This indicates that, in sandwich ELISA, the S4-1F8 antibody specifically reacts for AFP-L3 by simultaneously recognizing both the fucosylated and peptide moieties of AFP-L3. Furthermore, pretreatment of the antigen with 0.03% SDS enhanced the reaction.

[0232] [Example 5: Reactivity with natural human AFP-L3]

[0233] Use Western blotting as follows to confirm whether S4-1F8 reacts with natural human AFP-L3.

[0234] <Materials>

[0235] Electrophoretic antigen:

[0236] Recombinant AFP-L3 (lane 1) 50 ng,

[0237] Non-fucosylated AFP (lane 2) 50 ng

[0238] μTAS WAKO AFP-L3 calibrator 1 (lane 3) 0.5 ng,

[0239] μTAS WAKO AFP-L3 with calibrator 2 (lane 4) 0.5 ng

[0240] Primary antibody: S4-1F8 (diluted 10-fold to hybridoma culture supernatant) 4℃ O / N

[0241] Secondary antibody: Anti-mouse-IgG (Fc)Ab-HRP (BET / #A90-131P) (diluted 20,000 times) RT 1hr

[0242] <Method>

[0243] Except that the electrophoretic antigen, first antibody and second antibody are replaced with those described above, the protein blotting is performed in the same manner as in (2) of Example 2.

[0244] <Results>

[0245] The results of the protein blot are shown in Figure 6 The results show that S4-1F8 also reacts with μTAS WAKO AFP-L3 using calibrator 2, namely natural human AFP-L3. sequence list <110> Sysmex Corporation <120> Monoclonal antibodies that react with glycopeptides and their applications <130> 16-025CN <160> 26 <170> SIPOSequenceListing 1.0 <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> 16 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 3 Ala Arg Pro Leu Tyr Ser Thr Tyr Asp Val Asp Trp Tyr Phe Asp Val 1 5 10 15 <210> 4 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 4 Gly Asn Ile His Asn Tyr 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 5 Asp Ala Lys 1 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-A <400> 6 Gln His Phe Trp Thr Thr Pro Leu Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 7 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 8 Ile Asp Pro Glu Asp Gly Glu Ser 1 5 <210> 9 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 9 Ala Arg Pro Leu Tyr Ser Thr Tyr Asp Phe Asp Trp Tyr Phe Asp Val 1 5 10 15 <210> 10 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 10 Gly Asn Ile His Asn Tyr 1 5 <210> 11 <211> 3 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 11 Asp Val Lys 1 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-B <400> 12 Gln His Phe Trp Thr Thr Pro Leu Thr 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial sequence <220> <223> The glycopeptide shown in formula (a) <220> <221> CARBOHYD <222> (4)..(4) <400> 13 Thr Lys Val Asn Phe Thr 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial sequence <220> <223> The glycopeptide shown in formula (b) <220> <221> CARBOHYD <222> (4)..(4) <400> 14 Thr Lys Val Asn Phe Thr 1 5 <210> 15 <211> 16 <212> PRT <213> Artificial sequence <220> <223> The glycopeptide shown in formula (c) <220> <221> CARBOHYD <222> (5)..(5) <400> 15 Ala Thr Lys Val Asn Phe Thr Glu Ala Gln Lys Ala Ala Leu Asp Val 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide A <220> <221> CARBOHYD <222> (5)..(5) <400> 16 Ala Thr Lys Val Asn Phe Thr Glu Ala Gln Lys Ala Ala Leu Asp Val 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Non-fucosylated glycopeptide A <220> <221> CARBOHYD <222> (5)..(5) <400> 17 Ala Thr Lys Val Asn Phe Thr Glu Ala Gln Lys Ala Ala Leu Asp Val 1 5 10 15 <210> 18 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc+ <220> <221> CARBOHYD <222> (5)..(5) <400> 18 Gly Thr Lys Val Asn Phe Thr 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc- <220> <221> CARBOHYD <222> (5)..(5) <400> 19 Gly Thr Lys Val Asn Phe Thr 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc+ <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> 10 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc- <220> <221> CARBOHYD <222> (5)..(5) <400> twenty one Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> twenty two <211> 10 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc+ <220> <221> CARBOHYD <222> (5)..(5) <400> twenty two Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc- <220> <221> CARBOHYD <222> (5)..(5) <400> twenty three Gly Thr Lys Val Asn Phe Thr Glu Ile Gln 1 5 10 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc+ <220> <221> CARBOHYD <222> (5)..(5) <400> twenty four Ala Thr Lys Val Asn Phe Thr Glu Ala Gln Lys Ala Ala Leu Asp Val 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Glycopeptide Fuc- <220> <221> CARBOHYD <222> (5)..(5) <400> 25 Ala Thr Lys Val Asn Phe Thr Glu Ala Gln Lys Ala Ala Leu Asp Val 1 5 10 15 <210> 26 <211> 609 <212> PRT <213> Homo sapiens <300> <308> NM_001134 <309> 2016-09-10 <313> (1)..(609) <400> 26 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. Monoclonal antibodies that react with the following glycopeptides: …（a）， The monoclonal antibody of Heavy chains include: HCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 1, HCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3, which consists of the amino acid sequence shown in SEQ ID NO: 3, and Light chains contain: LCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 4, LCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

6.

2. Monoclonal antibodies that react with the following glycopeptides: …（a）， The monoclonal antibody of Heavy chains include: HCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 7, HCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 8, and HCDR3, which consists of the amino acid sequence shown in SEQ ID NO: 9, and Light chains contain: LCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 10, LCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

12.

3. Monoclonal antibodies produced from hybridomas with international accession numbers NITE BP-02349 or NITE BP-02350.

4. Hybridoma, with international accession numbers NITE BP-02349 or NITE BP-02350.

5. Use of the antibody according to any one of claims 1 to 3 for manufacturing a kit for detecting fucosylated AFP.

6. The use as described in claim 5, wherein Fucosylated AFP was pretreated with a solution containing 0.03% by mass or more SDS. After the above treatment, the antibody was reacted with the above-mentioned fucosylated AFP in the presence of SDS at a concentration of less than 0.025% by mass.

7. Kit for detecting fucosylated AFP Its components include antibodies for capturing fucosylated AFP, antibodies for detecting fucosylated AFP, and a solid phase. The aforementioned capture antibody or detection antibody is the antibody described in any one of claims 1 to 3.

8. The kit according to claim 7, further comprising a pretreatment reagent containing 0.03% by mass or more of SDS.

Images