JP6935184B2 - Monoclonal antibodies that react with glycopeptides and their uses - Google Patents

Description

The present invention relates to monoclonal antibodies that react with glycopeptides and their uses.

It is said that α-fetoprotein (AFP) (LCA-binding AFP) that binds to lentil lectins (LCA) increases in biological samples when transitioning from hepatitis / cirrhosis to liver cancer. Patent Document 1 describes an antibody for detecting LCA-binding AFP. It is described that the antibody of Patent Document 1 is reactive with binding AFP and not with LCA non-binding AFP.

JP-A-63-307900

Patent Document 1 describes that the sugar chain of AFP to which LCA binds has fucose. The AFP fraction that binds to the lentil lectin in the biological sample is called the AFP-L3 fraction. The AFP-L3 fraction is produced by fucosylated AFP (AFP in which core fucose (AFP in which α1-6 is bound to N-acetylglucosamine (GlcNAc) at the reducing end of N-type sugar chain) is added to the asparagine residue of AFP). It is composed.

In the examples of Patent Document 1, an antibody that binds to LCA-binding AFP (AFP-LCA-R) and does not bind to LCA non-binding AFP (AFP-LCA-NR) has been obtained (Example 1, Table). 1, see Figure 1). However, the epitope of this antibody is unknown. In the examples, LCA binding and non-binding are attributed to the presence or absence of fucose as described above, and the binding to the antigen may depend on the fucose moiety rather than the sequence of the peptide moiety. be. In that case, it may bind not only to AFP but also to proteins having other fucose in a non-specific manner. Therefore, it is desired to develop a monoclonal antibody having both the fucose portion of the glycopeptide and the amino acid of the peptide portion as epitopes.

The present invention is a monoclonal antibody that reacts with a glycopeptide, wherein the glycopeptide contains a core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side, and the core fucose of the glycopeptide. Provided are the above-mentioned antibody having both amino acids having 3 or more residues separated from the glycosylated asparagine of the glycopeptide on the C-terminal side as epitopes.

The present invention is a method for producing the above-mentioned antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing core fucose and amino acids having 4 or more residues continuous from sugar chain-added asparagine to the C-terminal side. Provide a method.

The present invention is a method for producing a hybridoma that produces the above-mentioned antibody, which comprises immunizing an animal with a glycopeptide antigen containing core fucose and a glycopeptide antigen containing 4 or more consecutive amino acids on the C-terminal side from glycosylated asparagine. The method is provided, including.

以下の糖ペプチドに反応せず、

以下の糖ペプチドに反応しない、

モノクローナル抗体を提供する。 The present invention reacts with the following glycopeptides and

Does not react with the following glycopeptides

Does not react with the following glycopeptides,

Provided is a monoclonal antibody.

（Xは、任意の糖鎖である）
前記方法を提供する。 The present invention is a method for producing the above antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing the following structure.

(X is any sugar chain)
The method is provided.

（Xは、任意の糖鎖である）
前記方法を提供する。 The present invention is a method for producing a hybridoma that produces the above antibody, which comprises a step of immunizing an animal with a glycopeptide antigen containing the following structure.

(X is any sugar chain)
The method is provided.

The present invention provides a method for measuring fucosylated AFP using the above antibody.

The present invention provides a fucosylated AFP detection kit comprising a capture antibody for fucosylated AFP, a detection antibody for fucosylated AFP, and a solid phase, wherein the capture antibody or the detection antibody is the antibody.

The present invention provides a hybridoma having an international deposit number of NITE BP-02263 or NITE BP-02264.

The present invention provides a monoclonal antibody in which both the fucose portion of the glycopeptide and the amino acid of the peptide portion are epitopes.

It is a figure which shows the result of the Western blotting of the clone 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 human serum-derived AFP (LEE biosolutions)) (negative antigen), Lane 3: Fucosylated ALP (Oriental yeast / 47787055)) (negative antigen). It is a figure which shows the measured value of OD450 at various antigen concentrations when the clone I2-1F8 obtained in Example 1 was used. It is a figure which shows the OD450 measurement value at various antigen concentrations when the clone I2-2F11 obtained in Example 1 was used. It is a figure which shows the result of the Western blotting of the clone I2-1F8 obtained in Example 1 (lane 1: recombinant AFP-L3, lane 2: non-fucosylated AFP, lane 3: natural human AFP (Mutaswako AFP-). Calibrator for L3 1), Lane 4: Natural human AFP-L3 (calibrator for Mutasuwako AFP-L3 2)).

The antibody of the present embodiment may exhibit specificity in a measurement system using a biological sample or the like in which the antibody of the present embodiment is used. For example, even if it binds non-specifically to a substance not contained in a biological sample, the action and effect of the present invention can be obtained if the antibody of the present embodiment exhibits specificity in an environment where it is usually used. Specifically, when a substance in a blood sample such as whole blood, serum, or plasma is detected by ELISA, it is sufficient to show specificity in the ELISA measurement system, and the substance is bound to a substance that is not normally contained in the blood sample or the ELISA reagent. You may.

The antibody of the present embodiment is a monoclonal antibody that reacts with a glycopeptide, wherein the glycopeptide contains core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side, and is the glycopeptide. Both core fucose and amino acids separated by 3 or more residues on the C-terminal side from glycosylated asparagine of glycopeptides are used as epitopes.

The glycopeptide with which the antibody of the present embodiment reacts may contain core fucose and amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side.

The antibody of the present embodiment is produced by a method including a step of immunizing an animal with a glycopeptide antigen containing core fucose and amino acids having 4 or more residues contiguously on the C-terminal side from glycosylated asparagine.

A biopolymer such as KLH or BSA may be bound to the N-terminal of the glycopeptide antigen via PEG or the like. Further, the C-terminal of these glycopeptide antigens may be amidated or the like. As a method for binding a biopolymer to the N-terminal, a known method can be used. Also, a known method can be used for amidation of C-terminal.

As a step of immunizing an animal with a glycopeptide antigen, a step of immunizing an animal in a known method for producing a monoclonal antibody can be utilized. Examples of the method for producing a monoclonal antibody include a mouse spleen method and a mouse iliac lymph node method (see Japanese Patent No. 4098796).

The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals according to the method for producing the monoclonal antibody.

Specifically, when the mouse iliac lymph node method is used as a method for producing a monoclonal antibody, an emulsion obtained by conjugating a glycopeptide with a carrier protein such as KLH and mixing it with Freund's adjuvant is used as a glycopeptide solution ((2.5 mg /)). mL) Carrier protein equivalent) 0.06 mL / animal may be immunized at least once on the ridge of the mouse.

After immunizing an animal, a hybridoma may be prepared, sorted, or the like according to a known method to obtain an antibody of the present embodiment.

When selecting hybridomas, a glycopeptide used as an antigen, a glycopeptide obtained by removing core fucose from the glycopeptide used as an antigen, or a peptide having the same sugar chain structure as the glycopeptide used as an antigen and having a different peptide chain are positive or negative. It may be appropriately used as an antigen. As for the shortened amino acid residues of the glycopeptide used as the antigen, for example, it is preferable that the C-terminal side of the glycopeptide has three consecutive residues from the glycosylated asparagine.

The criteria for selecting hybridomas are, for example, when ELISA is used, the difference in OD450 value between the positive antigen and the negative antigen is 0.05 or more, and the OD450 value of the negative antigen is 0.05 or less.

The isotype of the antibody of this embodiment is not particularly limited. The antibody of the present embodiment also includes fragments of peptides and the like containing _{F (ab') 2, Fab', Fab, and CDR.}

The antibody of the present embodiment may be labeled such as biotinylated and ALPylated.

Since the antibody of the present embodiment described above has the above-mentioned properties, it specifically reacts with, for example, core fucose and a glycoprotein whose partial sequence contains amino acids having 4 or more residues continuous from the glycosylated asparagine to the C-terminal side. do.

The antibody of this embodiment can be used for ELISA, Western blotting, immunohistochemical staining, immunoprecipitation and the like.

Another monoclonal antibody of the present embodiment specifically reacts with the following glycopeptide (SEQ ID NO: 13).

以下の糖ペプチド（配列番号15）に反応しない。

This monoclonal antibody did not react with the following glycopeptide (SEQ ID NO: 14) and

Does not react with the following glycopeptides (SEQ ID NO: 15).

That is, the antibody of the present embodiment does not react with the antigen having no core fucose and having the amino acid "EIQ" (the above (2)), and has the core fucose and does not have the amino acid sequence "EIQ" (the above). Does not react to (3)). From this, it is considered that the epitope of the antibody of the present embodiment contains both core fucose and the amino acid sequence “EIQ”.

In addition, in this specification, "reacting with a glycopeptide" by an antibody of this embodiment means that a glycopeptide and an antibody bind to each other by an antigen-antibody reaction.

Furthermore, the antibody preferably does not react with the following glycopeptide (SEQ ID NO: 16).

Further, the antibody is preferably one that reacts with fucosylated AFP.

Furthermore, the antibody can bind to a denaturing agent such as SDS or a modified fucosylated AFP pretreated with heat or the like. When a solution containing SDS is used as the pretreatment, the SDS concentration for sufficiently modifying the fucosylated AFP (hereinafter, this is referred to as “pretreatment SDS concentration”) is not particularly limited, but is 0.03% by mass / mass%. (Hereinafter, simply referred to as "%") or more is preferable. On the other hand, if an excessive amount of the denaturing agent is contained during the antigen-antibody reaction, the antibody may also be denatured and an unfavorable effect on the antigen-antibody reaction may occur. Therefore, it is preferable to reduce the concentration of the denaturing agent by dilution or the like. When a solution containing SDS is used as the denaturing agent, the SDS concentration at the time of the antigen-antibody reaction (hereinafter, this is referred to as "final SDS concentration") is not particularly limited, but is preferably 0.025% or less.

In addition, in this specification, "reacting with fucosylated AFP" by the antibody of this embodiment means that fucosylated AFP and an antibody bind to each other by an antigen-antibody reaction. Fucosylated AFP may be recombinant or natural. Natural fucosylated AFP is, for example, AFP present in human blood. The sequence of human AFP is registered in, for example, GenBank Accession No. NM_001134 and has the amino acid sequence of SEQ ID NO: 25.

Further, the antibody is preferably one that reacts with fucosylated AFP denatured in the presence of SDS and DTT. The conditions for denaturation are the reaction at room temperature (25 ° C) in the presence of 2% SDS and 50 mM DTT.

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

Examples of the CDR of the antibody of this embodiment include the following.

<CDR-A>
The CDR of the heavy chain contains the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6.

<CDR-B>
The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid sequence set forth in SEQ ID NO: 9.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12.

The hybridomas that produce the antibodies of this embodiment having the above CDR-A and CDR-B as CDRs were named I2-1F8 and I2-2F11, respectively, and were named I2-1F8 and I2-2F11 on May 20, 2016 by the National Institute of Technology and Evaluation. International deposits were made as NITE BP-02264 and NITE BP-02263 at the Patent Microorganisms Depositary Center (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818).

Xは、任意の糖鎖であり、一般的に糖タンパクや糖ペプチドに結合している糖鎖であれば特に限定されない。 The antibody of the present embodiment is obtained by a method including a step of immunizing an animal with a glycopeptide antigen (SEQ ID NO: 17) containing the following structure.

X is an arbitrary sugar chain, and is not particularly limited as long as it is a sugar chain generally bound to a glycoprotein or a glycopeptide.

Xは、任意の糖鎖であり、一般的に糖タンパクや糖ペプチドに結合している糖鎖であれば特に限定されない。 The following are more preferable as the glycopeptide antigen (SEQ ID NO: 18).

X is an arbitrary sugar chain, and is not particularly limited as long as it is a sugar chain generally bound to a glycoprotein or a glycopeptide.

A biopolymer such as KLH may be bound to the N-terminal of the glycopeptide antigen via PEG or the like. Further, the C-terminal of these glycopeptide antigens may be amidated or the like. As a method for binding a biopolymer to the N-terminal, a known method can be used. Also, a known method can be used for amidation of C-terminal.

As a step of immunizing an animal with a glycopeptide antigen, a step of immunizing an animal in a known method for producing a monoclonal antibody can be utilized. Examples of the method for producing a monoclonal antibody include a mouse spleen method and a mouse iliac lymph node method (see Japanese Patent No. 4098796).

The animal to be immunized is not particularly limited, and may be appropriately selected from non-human animals according to the method for producing the monoclonal antibody.

Specifically, when the mouse iliac lymph node method is used as a method for producing a monoclonal antibody, an emulsion obtained by conjugating a glycopeptide with a carrier protein such as KLH and mixing it with Freund's adjuvant is used as a glycopeptide solution ((2.5 mg /)). mL) Carrier protein equivalent) 0.06 mL / animal may be immunized at least once on the ridge of the mouse.

After immunizing an animal, a hybridoma may be prepared, sorted, or the like according to a known method to obtain an antibody of the present embodiment.

When selecting hybridomas, glycopeptides used as antigens, those in which core fucose was removed from glycopeptides used as antigens, those in which the amino acid residues of glycopeptides used as antigens were shortened, and fucosyl modified in the presence of SDS and DTT. Non-fucosylated AFP denatured in the presence of AFP, SDS and DTT, glycopeptides or glycoproteins having a core fucose and a polypeptide having an amino acid sequence different from that of AFP (for example, fucosylated ALP) and the like as positive antigens. Alternatively, it may be appropriately used as a negative antigen. As for the shortened amino acid residues of the glycopeptide used as the antigen, for example, it is preferable that the C-terminal side of the glycopeptide has three consecutive residues from the glycosylated asparagine.

The criteria for selecting hybridomas are, for example, when ELISA is used, the difference in OD450 value between the positive antigen and the negative antigen is 0.05 or more, and the OD450 value of the negative antigen is 0.05 or less.

The isotype of the antibody of this embodiment is not particularly limited. The antibody of the present embodiment also includes fragments of peptides and the like containing _{F (ab') 2, Fab', Fab, and CDR.}

The antibody of the present embodiment may be labeled such as biotinylated and ALPylated.

Since the antibody of the present embodiment has the above-mentioned properties, it reacts with fucosylated AFP and does not react with non-fucosylated AFP. Therefore, the antibody of the present embodiment can measure, for example, fucosylated AFP in a biological sample.

Examples of the biological sample include whole blood, serum, plasma and the like collected from the subject. This biological sample may be subjected to pretreatment such as centrifugation or denaturation. The biological sample is preferably denatured. The conditions for the denaturation treatment are a reaction at room temperature (25 ° C) in the presence of 2% SDS and 50 mM DTT.

A known immunological measurement method can be used to measure fucosylated AFP using the antibody of the present embodiment. Examples of the immunological measurement method include an enzyme-linked immunosorbent assay (ELISA method), an immune complex transfer measurement method (see JP-A 1-254868), an immunoturbidimetry method, an immunochromatography method, and a latex aggregation method. Can be mentioned. As an example of the measurement process, the case where the fucosylated AFP concentration in the biological sample is measured by the sandwich ELISA method will be described below.

First, an antibody for capturing fucosylated AFP in a biological sample (hereinafter, also referred to as “capture antibody”) and an antibody for detecting fucosylated AFP (hereinafter, also referred to as “detection antibody”). A complex containing fucosylated AFP is formed on the 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, by contacting the solution containing the complex with a solid phase capable of capturing the capture antibody, the above complex can be formed on the solid phase. Alternatively, a solid phase in which a capture antibody is immobilized in advance may be used. That is, the above complex can be formed on the solid phase by contacting the solid phase on which the capture antibody is immobilized, the biological sample, and the detection antibody. The antibody of the present embodiment can be used for at least one of a capture antibody and a detection antibody.

The mode of fixing the capture antibody to the solid phase is not particularly limited. For example, the capture antibody and the solid phase may be directly bound, or the capture antibody and the solid phase may be indirectly bound via another substance. Examples of the direct bond include physical adsorption and the like. Examples of the indirect binding include binding via a combination of biotin and avidin or streptavidin (hereinafter, also referred to as "avidins"). In this case, by modifying the capture antibody with biotin in advance and binding the avidins to the solid phase in advance, the capture antibody and the solid phase are indirectly bound via the binding between biotin and the avidins. Can be made to.

The material of the solid phase is not particularly limited, and can be selected from, for example, an organic polymer compound, an inorganic compound, a biopolymer and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene and the like. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, and test tubes.

By detecting the complex formed on the solid phase by a method known in the art, the measured value of fucosylated AFP in the biological sample can be obtained. For example, when an antibody labeled with a labeling substance is used as the detection antibody, the measured value of fucosylated AFP can be obtained by detecting the signal generated by the labeling substance. Alternatively, when a labeled secondary antibody against the detection antibody is used, the measured value of fucosylated AFP can be obtained in the same manner.

In this embodiment, the fucosylated AFP is preferably pretreated as described above. When a solution containing SDS is used as the pretreatment, the SDS concentration during the pretreatment is not particularly limited, but is preferably 0.03% or more. At the time of the antigen-antibody reaction, it is preferable to reduce the concentration of the denaturing agent by dilution or the like as described above. When a solution containing SDS is used as the denaturing agent, the final SDS concentration is not particularly limited, but is preferably 0.025% or less. In the present embodiment, it is preferable to carry out such a treatment to react the antibody with the fucosylated AFP to obtain a measured value of the fucosylated AFP.

In the present embodiment, B / F (Bound / Free) separation for removing unreacted free components that do not form a complex may be performed between the complex formation step and the complex detection step. .. The unreacted free component refers to a component that does not form a complex. For example, an antibody that does not bind to fucosylated AFP, a substance other than fucosylated AFP (contaminant substance) in a biological sample, and the like can be mentioned. The means for B / F separation is not particularly limited, but if the solid phase is particles, B / F separation can be performed by recovering only the solid phase in which the complex is captured by centrifugation. 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. When the solid phase is magnetic particles, B / F separation can be performed by suctioning and removing the liquid containing the unreacted free component with a nozzle while the magnetic particles are magnetically constrained by a magnet. After removing the unreacted free components, the solid phase trapped in the complex may be washed with a suitable aqueous medium such as PBS.

As used herein, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means that the signal intensity is indicated stepwise, such as "no signal generated", "weak", "medium", "strong", and the like. In this embodiment, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.

The labeling substance is not particularly limited as long as a detectable signal is generated. For example, it may be a substance that generates a signal by itself (hereinafter, also referred to as a “signal generating substance”), or may be a substance that catalyzes the reaction of another substance to generate a signal. Examples of the signal generating substance include a fluorescent substance, a radioisotope and the like. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes and the like. Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, luciferase and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Radioisotopes include ¹²⁵ I, ¹⁴ C, ³² P and the like. Among them, enzymes are preferable as labeling substances, and alkaline phosphatase and peroxidase are particularly preferable.

The method itself for detecting a signal is known in the art. In the present embodiment, the measuring method according to the type of the signal derived from the above-mentioned labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate with the enzyme can be measured using a known device such as a spectrophotometer.

The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, CDP-Star® (4-chloro-3- (methoxyspiro [1, 2-dioxetane-3, 2'-(5'-chloro) trixhiro] [ 3. 3. 1. 13, 7] Decane] -4-yl) disodium phenylphosphate), CSPD® (3- (4-methoxyspiro [1, 2-dioxetane-3, 2- (5) Chemically luminescent substrates such as'-chloro) tricyclo [3. 3. 1. 13, 7] decane] -4-yl) disodium phenylphosphate), 5-bromo-4-chloro-3-indrill phosphate ( BCIP), 2-sodium 5-bromo-6-chloro-indrill phosphate, p-nitrophenyl phosphate and other color-developing substrates can be mentioned. When peroxidase is used as the enzyme, the substrate is a chemiluminescent substrate such as luminol and its derivatives, 2, 2'-azinobis (3-ethylbenzothiazolin-6-ammonium sulfonate) (ABTS), 1, 2- Color-developing substrates such as phenylenediamine (OPD), 3, 3', 5, 5'-tetramethylbenzidine (TMB) can be mentioned.

When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.

The signal detection result may be used as a measured value of fucosylated AFP. For example, when the signal intensity is quantitatively detected, the measured value of the signal intensity itself or the value obtained from the measured value can be used as the measured value of fucosylated AFP. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control sample or the background value from the measured value of fucosylated AFP. The negative control sample can be appropriately selected, and examples thereof include biological samples obtained from healthy subjects.

In the present embodiment, even if the measured values of fucosylated AFP are acquired for a plurality of standard samples having known fucosylated AFP concentrations and a calibration curve showing the relationship between the fucosylated AFP concentration and the measured values of fucosylated AFP is created. good. By applying the measured value of fucosylated AFP obtained from the biological sample to this calibration curve, the value of the fucosylated AFP concentration in the biological sample can be obtained.

In the present embodiment, the fucosylated AFP concentration in a biological sample may be measured 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 measurement may be performed using a commercially available fully automatic immunoassay device such as the HISCL series (manufactured by Sysmex Corporation).

In addition, the antibody of the present embodiment can be used in a kit for detecting fucosylated AFP. The fucosylated AFP detection kit of the present embodiment includes a capture antibody, a detection antibody, and a solid phase. The antibody of the present embodiment can be used as a capture antibody or a detection antibody. In the sandwich immunoassay, the antibody of the present embodiment can be used as either a capture antibody or a detection antibody.

In the fucosylated AFP detection kit of the present embodiment, when the labeling substance of the detection antibody is an enzyme, the fucosylated AFP detection kit of the present embodiment may further contain a substrate for the enzyme. The form of the labeling substance and the substrate is not particularly limited, and may be a solid (for example, powder, crystal, lyophilized product, etc.) or a liquid (for example, solution, suspension, emulsion, etc.). May be good.

The fucosylated AFP detection kit of the present embodiment may further contain a pretreatment reagent containing 0.03% or more of SDS in order to pretreat the fucosylated AFP described above. The reagent is the same as the above-mentioned solution containing 0.03% or more of SDS.

The fucosylated AFP detection kit of the present embodiment may further appropriately contain a pretreatment liquid for a biological sample, a solid phase cleaning liquid, an enzyme reaction terminator, a calibrator and the like.

In the fucosylated AFP detection kit of the present embodiment, the capture antibody, the detection antibody, the solid phase and the like may be appropriately contained in a container or individually packaged according to the form of the kit. In the fucosylated AFP detection kit of the present embodiment, the capture antibody may be directly bound to the solid phase, or the capture antibody and the solid phase may be indirectly bound via another substance. .. When the capture antibody and the solid phase are indirectly bound, the capture antibody and the solid phase may be contained in separate containers in the kit of the present embodiment. When the capture antibody and the solid phase are indirectly bound to each other via, for example, biotin and avidins, the capture antibody modified with biotin is contained in one container, and the solid phase to which the avidins are bound is contained in another container. It may be contained in a container. The details of the biological sample, the capture antibody, the detection antibody, the solid phase, and the like are the same as those described in the above description of the measurement method.

Fucosylated AFP is known to be associated with liver cancer. Therefore, the fucosylated AFP detection kit of the present embodiment can be used for the diagnosis of liver cancer.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1
Acquisition of antibody:
(1) Acquisition of antibody group A hybridoma producing an antibody group was obtained by the mouse iliac lymph node method (Patent No. 4098796). Specifically, glycopeptide A (SEQ ID NO: 19) having the structure shown in Table 1 below is synthesized and conjugated with KLH so that it becomes 2.5 mg / mL (carrier protein equivalent) with Freund's complete adjuvant. The mixed emulsion was made by injecting 0.06 mL / animal once into the ridge of the mouse. In addition, 17 days after immunization, booster immunization was performed by injecting 2.5 mg / mL (carrier protein equivalent) KLH conjugated glycopeptide A solution at 0.06 mL / animal once into the ridge of the mouse. Furthermore, 4 days after booster immunization, lymphocytes were isolated from the iliac lymph nodes and fused with myeloma to obtain hybridomas.

丸はMannoses、四角はN-acetylglucosamine、三角はfucoseを表す。
糖ペプチドのN末は、KLH-PEG4であり、C末はアミド化されている。

Circles represent Mannoses, squares represent N-acetylglucosamine, and triangles represent fucose.
The N-terminal of the glycopeptide is KLH-PEG4, and the C-terminal is amidated.

(2) Primary screening By antigen solid phase ELISA using the positive antigen (glycopeptide A) or negative antigen (non-fucosylated glycopeptide A: SEQ ID NO: 20) shown in Table 2 from the hybridoma obtained in (1) above. Wells that responded to positive antigens and responded less to negative antigens were selected. Antigen solid phase ELISA was performed by the following method. The results are shown in Table 3.

丸はMannoses、四角はN-acetylglucosamine、三角はfucoseを表す。
糖ペプチドのN末は、BSA-PEG4であり、C末はアミド化されている。

Circles represent Mannoses, squares represent N-acetylglucosamine, and triangles represent fucose.
The N-terminal of the glycopeptide is BSA-PEG4, and the C-terminal is amidated.

<Method>
(1) To a 96-well plate (nunc Maxisoap / 446612), add 1 μg / ml of each screening antigen (diluted solution 10 mM phosphate buffer pH 7) at 50 μl / well, and solidify at 37 ° C. for 1 hr.
(2) Wash each well with PBST 300 μl / well × 5 times.
(3) Block each well at 1% BSA-PBS 100 μl / well, 4 ° C, Overnight.
(4) Wash each well with PBST 300 μl / well × 5 times.
(5) The antibody culture supernatant (primary antibody) is diluted 10-fold with 1% BSA-PBS, 50 μl / well is added, and RT is reacted for 1 hr.
(6) Wash each well with PBST 300 μl / well × 5 times.
(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 50 μl / well. , RT, 0.5 hr reaction.
(8) Wash each well with PBST 300 μl / well × 5 times.
(9) Add 100 μl / well of HRP color-developing substrate to develop color.
(10) Add 100 μl / well of stop solution to stop color development.
(11) Measure OD450.
(12) 7 wells with a large difference between the signal for the positive antigen and the signal for the negative antigen and less reaction to the negative antigen were selected for the secondary screening.

(3) Secondary screening From the wells obtained in (2) above, wells that recognize both core fucose and amino acids were selected by Western blotting using the following antigens and proceeded to cloning. Western blotting was performed as follows.

<Material>
-Screening antigen Positive antigen: Fucosylated AFP (AFP-L3 / recombinant)
Negative antigen 1: Non-fucosylated AFP (LCA lectin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions))
Negative antigen 2: Fucosylated ALP (Oriental yeast / 47787055)
-Primary antibody Screening antibody: Culture supernatant positive for primary screening Negative control: Hybridoma medium-Secondary antibody
anti mouse-IgG (Fc) Ab -HRP (BET / A90-131P)
anti mouse-IgM Ab -HRP (SBA / 1020-05)
・ Blocking agent: PVDF Blocking Reagent for Can Get Signal (TOYOBO / NYPBR01)
・ Cleaning liquid: TBST
・ Diluted solution: 1% BSA-TBST
・ PVDF membrane (iBlot (registered trademark) 2 Transfer Stacks, PVDF, mini / IB24002)
・ Blotting device (iBlot2)
・ Emission detection reagent ECL prime Western Blotting Detection Reagent (GE Healthcare / RPN2232)

<Method>
(1) Add 1/4 amount of NuPAGE LDS Sample Buffer (4X) (Thermo / NP0008) to each screening antigen.
Add 1/10 amount of NuPAGE Sample Reducing Agent (10X) (Thermo / NP0009) and mix.
(2) Molecular weight markers and each screening antigen of (1) are electrophoresed (SDS-PAGE) with the following antigen amounts.
Lane0: Molecular Weight Marker (MagicMark ™ XP Western Protein Standard / LC5602)
Lane1: Positive antigen: Fucosylated AFP (AFP-L3) 50ng
Lane2: Negative antigen 1: Non-fucosylated AFP 50ng
Lane 3: Negative antigen 2: Fucosylated ALP 50ng
(3) Blotting on PVDF membrane.
(4) Immerse in PVDF Blocking Reagent for Can Get Signal at room temperature for 1 hour to block.
(5) Wash with TBST 3 times.
(6) The primary antibody is diluted with 1% BSA-TBST to the following dilution ratio and reacted at 4 ° C. overnight.
Screening antibody: 1st screening positive culture supernatant 10-fold dilution Negative control: Hybridoma medium 10-fold dilution
(7) Wash with TBST 3 times.
(8) Dilute the secondary antibody (mixture of 2 types) with 1% BSA-TBST to the following dilution ratio and react at room temperature for 1 hour.
anti mouse-IgG (Fc) Ab -HRP 20,000 times diluted
anti mouse-IgM Ab -HRP 5,000 times diluted
(9) Wash with TBST 3 times.
(10) Detected by chemiluminescence
(11) The 5 wells in which the band was detected only in the positive antigen were judged to be positive, and cloning proceeded.

(4) Cloning The 5-well cells selected in (3) above were seeded by the limiting dilution method. The culture supernatant was screened again in the same manner as in the primary screening of (2) to obtain 2 clones. The Elisa data are shown in Table 4.

Western blotting was performed on the culture supernatants of the above two clones in the same manner as in the secondary screening of (3), and it was confirmed that they specifically react with AFP-L3. The results of Western blotting are shown in Fig. 1. In FIG. 1, the antigens in each lane are as follows.
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)

(5) Confirmation of epitopes In order to confirm the epitopes of the antibodies produced by these clones, glycopeptide B (SEQ ID NO: 21) and glycopeptide B (SEQ ID NO: 21) shown in Table 5 having fewer amino acid residues than the sequence of glycopeptide A used as an immunogen. It was confirmed by the same procedure as in (2) above whether the non-fucosylated glycopeptide B (SEQ ID NO: 22) was recognized. The results are shown in Table 6. Here, as a positive control (PC), an AAL lectin that recognizes fucose for glycopeptide B and a WGA lectin that recognizes GlcNAc for non-fucosylated glycopeptide B were used. A buffer solution was used for negative control (NC).

丸はMannoses、四角はN-acetylglucosamine、三角はfucoseを表す。
糖ペプチドのN末は、BSA-PEG4であり、C末はアミド化されている。

Circles represent Mannoses, squares represent N-acetylglucosamine, and triangles represent fucose.
The N-terminal of the glycopeptide is BSA-PEG4, and the C-terminal is amidated.

All antibodies reacted with glycopeptide A used as the antigen and not with glycopeptide B. From this result, it was found that the obtained epitope sequence of the antibody was a region containing at least one of the sequences shown in Table 7.

(6) Confirmation of CDR sequence The CDR analysis of the antibody was performed on the 5 clones obtained in (4) above. As a result, the CDR sequence was an array with the following two patterns. The CDR sequences of the 1F8-A4 antibody (SEQ ID NOs: 1 to 6) are shown in Table 8. The CDR sequences of the 2F11-2A9 antibody (SEQ ID NOs: 7 to 12) are shown in Table 9.

Of the clones obtained above, 1F8-A4 was named I2-1F8 and deposited internationally (NITE BP-02264). In addition, 2F11-2A9 was named I2-2F11 and made an international deposit (NITE BP-02263).

Example 2
Construction of sandwich ELISA and impact of SDS on reactivity:
Using I2-1F8 or I2-2F11 obtained in Example 1, detection by sandwich ELISA was performed as follows. We also examined the effect of SDS on reactivity.
<Material>
・ Antibody sensitization plate (I2-1F8, I2-2F11 / 2.5 μg / mL 100 μL / well)
・ Recombinant AFP-L3 antigen ・ Anti-AFP antibody: Polyclonal Antibody to Alpha-Fetoprotein (WLS / # PAA153Hu01)
・ Labeled antibody: Goat anti rabbit immunoglobulin-HRP
・ Buffer A: 150 mM NaCl + 1% BSA / 10 mM phosphate buffer (pH 7)
・ Buffer B: 150 mM NaCl + 0.05% Tween20 / 10 mM phosphate buffer (pH 7)

<Method>
(1) (Antigen pretreatment) Add equal amounts of 2%, 1%, 0.5%, 0.25%, 0.13%, 0.06% SDS solution to 20 μg / mL AFP-L3 antigen solution, and allow to stand for 3 minutes or more after mixing. do. (SDS concentration during pretreatment: 0.03-1%)
(2) Dilute with Buffer A so that the antigen concentration becomes 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL. (Final SDS concentration: 0.00075% --0.1%)
(3) Add 100 μL / well of the antigen solution to the antibody sensitized plate and react at room temperature for 60 minutes.
(4) After washing each well with Buffer B, add 100 μL / well of anti-AFP antibody diluted 400-fold, and react at room temperature for 60 minutes.
(5) After washing each well with Buffer B, add 100 μL / well of the labeled antibody diluted 4000 times, and react at room temperature for 40 minutes.
(6) After washing each well with Buffer B, add 100 μl / well of HRP coloring substrate and let the color develop for 20 minutes.
(7) Add 100 μl / well of stop solution to stop color development, and measure OD450.

<Result>
Table 10 shows the measured values of OD450 at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-1F8 was used. Table 11 shows the OD450 measured values at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-2F11 was used.

In I2-1F8, the final SDS concentration was 0.013% or less, and in I2-2F11, the final SDS concentration was 0.025% or less, and the signal increased in an antigen concentration-dependent manner.

Example 3
Confirmation of sandwich ELISA specificity:
The specificity of the sandwich ELISA constructed in Example 2 was confirmed as follows.
<Material>
・ Antibody sensitization plate (I2-1F8, I2-2F11 / 2.5 μg / mL 100 μL / well)
-Positive antigen: Recombinant AFP-L3 antigen-Negative antigen 1: Non-fucosylated AFP (LCA lek-tin non-adsorbed fraction of human serum-derived AFP (LEE biosolutions))
-Negative antigen 2: Fucosylated protein ALP (Oriental yeast) other than AFP
-Biotinylated anti-AFP antibody: anti AFP, Human (mouse) (ABV / H00000174-M01)
-Detection reagent: HRP-Conjugated Streptavidin (Thermo / N100)
・ Buffer A: 150 mM NaCl + 1% BSA / 10 mM phosphate buffer (pH 7)
・ Buffer B: 150 mM NaCl + 0.05% Tween20 / 10 mM phosphate buffer (pH 7)

<Method>
(1) Add an equal amount of 0.06% SDS solution to each 20 μg / mL antigen solution (denaturation) or no addition (non-denaturation), and let stand for 3 minutes or more after mixing. (SDS concentration during pretreatment: 0.03%)
(2) Dilute with Buffer A so that the antigen concentration is 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 63 ng / mL, 31 ng / mL.
(3) Add 100 μL / well of the antigen solution to the antibody sensitized plate and react at room temperature for 60 minutes.
(4) After washing each well with Buffer B, add 100 μL / well of biotinylated anti-AFP antibody diluted 480 times, and react at room temperature for 60 minutes.
(5) After washing each well with Buffer B, add 100 μL / well of the detection reagent diluted 10000 times, and react at room temperature for 60 minutes.
(6) After washing each well with Buffer B, add 100 μl / well of HRP coloring substrate and let the color develop for 10 minutes.
(7) Add 100 μl / well of stop solution to stop color development, and measure OD450.

<Result>
Table 12 and FIG. 2 show the measured values of OD450 at various antigen concentrations when I2-1F8 was used. In addition, Table 13 and FIG. 3 show OD450 measured values at various antigen concentrations when I2-2F11 was used.

I2-1F8 and I2-2F11 did not respond to non-fucosylated AFP and ALP in either the denatured or non-modified state. On the other hand, I2-1F8 and I2-2F11 showed an increase in signal for AFP-L3 in an antigen concentration-dependent manner. In particular, I2-1F8 and I2-2F11 had significantly improved reactivity to AFP-L3 in the denatured state. These results indicate that I2-1F8 and I2-2F11 specifically react with AFP-L3 in sandwich ELISA by simultaneously recognizing the fucose and peptide moieties of AFP-L3. It was also shown that I2-1F8 and I2-2F11 react strongly by pretreating the antigen with 0.03% SDS.

Example 4
Reactivity with natural human AFP-L3:
Whether or not I2-1F8 reacts with natural human AFP-L3 was confirmed by Western blotting as follows.
<Material>
Primary antibody: I2-1F8 (10-fold diluted hybridoma culture supernatant) 4 ° C O / N
Secondary antibody: anti mouse-IgG (Fc) Ab-HRP (BET / # A90-131P) (20,000-fold dilution) RT 1hr

<Method>
Western blotting was performed in the same manner as in Example 1 (3) except that the primary antibody and the secondary antibody were replaced with the following.

<Result>
The results of Western blotting are shown in FIG. I2-1F8 also responded to natural human AFP-L3. In FIG. 4, the antigens in each lane are as follows.
Lane 1: Recombinant AFP-L3
Lane 2: Non-fucosilized AFP
Lane 3: Natural human AFP (calibrator 1 for Mutaswaco AFP-L3)
Lane 4: Natural Human AFP-L3 (Mutaswako AFP-L3 Calibrator 2)

Example 5
Impact of SDS on reactivity:
The effect of SDS on reactivity was examined using the Elisa constructed in Example 2.
<Material>
The same material as in Example 2 was used except that I2-1F8 was used as the antibody.

<Method>
(1) (Antigen pretreatment) Add equal amounts of 0.031%, 0.016%, 0.008%, 0.004% SDS solution to 20 μg / mL AFP-L3 antigen solution, and allow to stand for 3 minutes or more after mixing.
(2) Dilute with Buffer A so that the antigen concentration becomes 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL. (Final SDS concentration: 0.00032% --0.00001%)
The following operations were performed in the same manner as in Example 2.

<Result>
Table 14 shows the measured values of OD450 at various antigen concentrations, final SDS concentration, and pretreated SDS concentration when I2-1F8 was used.

A concentration-dependent signal could be detected even when the pretreated SDS concentration was 0.016% or less.

Claims (17)

Reacts with the following glycopeptides

Does not react with the following glycopeptides

Does not react with the following glycopeptides,

Monoclonal antibody. Does not react with the following glycopeptides,

The antibody according to claim 1. The antibody according to claim 1, which further reacts with fucosylated AFP. Any one of claims 1 to 3 that reacts with fucosylated AFP obtained by pretreatment with a solution containing 0.03% by mass or more of SDS in the presence of SDS of 0.025% by mass or less. The antibody described. The antibody according to claim 1, which reacts with fucosylated AFP denatured in the presence of 2% by weight SDS and 50 mM DTT. The antibody according to claim 1 or 2, which does not react with denatured non-fucosylated AFP in the presence of 2% by weight SDS, 50 mM DTT. The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in SEQ ID NO: 2, and the amino acid sequence set forth in SEQ ID NO: 3.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6.
The antibody according to any one of claims 1 to 6. The heavy chain CDR comprises the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 9.
The CDR of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12.
The antibody according to any one of claims 1 to 6. A method for producing an antibody according to any one of claims 1 to 6.
Including the step of immunizing a non-human animal with a glycopeptide antigen containing the following structure,

(X is any sugar chain)
The method. The method of claim 9, wherein the glycopeptide antigen comprises the following structure.

(X is any sugar chain) A method for producing a hybridoma that produces the antibody according to any one of claims 1 to 6.
Including the step of immunizing a non-human animal with a glycopeptide antigen containing the following structure,

(X is any sugar chain)
The method. 11. The method of claim 11, wherein the glycopeptide antigen comprises the following structure.

(X is any sugar chain) A hybridoma with an international deposit number of NITE BP-02263 or NITE BP-02264. A method for measuring fucosylated AFP using the antibody according to any one of claims 1 to 8. The fucosylated AFP is pretreated with a solution containing 0.03% by mass / mass% or more of SDS, and after the treatment, the antibody is used to react with the fucosylated AFP in the presence of 0.025% by mass / mass% or less of SDS. The method according to claim 14. Includes an antibody for capturing fucosylated AFP, an antibody for detecting fucosylated AFP, and a solid phase.
The capture antibody or the detection antibody is the antibody according to any one of claims 1 to 8.
Fucosilized AFP detection kit. 16. The kit of claim 16, further comprising a pretreatment reagent comprising 0.03% by weight or more of SDS.

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