Antigen processing method

Abstract

There is a need for a method for preparing a selective binding substance such as an antibody that specifically reacts with the sugar chain of a glycoprotein without previously treating the glycoprotein in a biological sample with a denaturant, a selective binding substance prepared by the method, and a method for specifically detecting glycoproteins using the selective binding substance. In order to solve the above problems, the present invention prepares a selective binding substance that specifically reacts with the sugar chain of a glycoprotein by treating the glycoprotein with a sugar chain-specific sugar chain cleaving enzyme.

Description

Technical Field

[0001] The present invention relates to: a method for treating an antigen for producing a selective binding substance such as an antibody, a method for producing a selective binding substance such as an antibody using the treated antigen, a selective binding substance such as an antibody produced by the method, a method for detecting a glycoprotein using the selective binding substance such as the antibody, and a kit for use in these methods. Background Art

[0002] Glycoproteins have sugar chains of various structures, and if antibodies that recognize these individual sugar chains can be used, various information on the state of the organism can be obtained.

[0003] On the other hand, it is known that it is difficult to obtain antibodies specific to the sugar chains of glycoproteins, and one of the reasons is believed to be that similar sugar chains exist in the host animal that produces the antibody. Various efforts have been made to overcome this difficulty. For example, Patent Document 1 discloses a method for preparing antibodies using an antigen composed of both the fucose part and the peptide part of a glycopeptide as epitopes.

[0004] However, in order to detect glycoproteins in a biological sample using antibodies prepared by this method, the biological sample must be treated with a denaturing agent such as SDS in advance to denature the glycoproteins, which is not practical.

[0005] Furthermore, Patent Document 2 discloses a method of increasing the detection level of a reaction product obtained by reacting a glycoprotein with a sugar-binding compound by treating the glycoprotein with a protease.

[0006] However, in this method, since the activity of the protease is non-specific to the sugar chain structure, glycoproteins having specific sugar chains cannot be specifically detected.

[0007] Therefore, there is a need for a general method for preparing an antibody that specifically reacts with sugar chains of glycoproteins without previously treating a biological sample with a denaturing agent.

[0008] Prior art literature

[0009] Patent Literature

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-214348

[0011] [Patent Document 2] International Publication No. 2017 / 131022 Summary of the invention

[0012] Technical problem solved by the invention

[0013] The present invention provides: a method for preparing a selective binding substance such as an antibody that specifically reacts with the sugar chain of a glycoprotein without previously treating a biological sample with a denaturing agent, an antibody such as a selective binding substance prepared by the method, and a method for specifically detecting a glycoprotein using the antibody such as a selective binding substance.

[0014] Technical means of solving problems

[0015] The present inventors have conducted intensive studies to solve the above problems and have found that antibodies that specifically react with sugar chains of glycoproteins can be prepared by treating glycoproteins with a sugar chain-specific sugar chain cleaving enzyme under mild conditions, thereby completing the present invention.

[0016] Although not limited to this in theory, the reason is considered to be as follows: since sugar chains treated with sugar chain-specific sugar chain cleaving enzymes produce sugar chain structures that are extremely rare in animals, such as fucosylated glycopeptide structures, they do not inhibit antibody production in antibody-producing animals.

[0017] That is, the present invention provides the following in one embodiment.

[0018] Item 1. A method for treating a glycoprotein, wherein the method is a method for treating a glycoprotein when the glycoprotein is used as an antigen, wherein the method comprises:

[0019] A step in which sugar chains are cleaved by sugar chain-specific sugar chain cleaving enzymes.

[0020] Item 2. The method for treating glycoprotein according to Item 1, wherein:

[0021] The step of cleaving the sugar chain is a step of making the epitope inherent in the sugar chain in a state that can be recognized by the selective binding substance through the cleavage.

[0022] Item 3. The method for treating glycoprotein according to Item 1 or 2, wherein:

[0023] The sugar chain-specific sugar chain cleaving enzyme is selected from Endo-A, Endo-M, Endo-H, Endo-D, Endo-F1, Endo-F2, and Endo-F3.

[0024] Item 4. The method for treating an antigen according to any one of Items 1 to 3, wherein

[0025] The glycoprotein is AFP.

[0026] Item 5. A method for producing a selective binding substance, comprising using the method for treating a glycoprotein according to any one of Items 1 to 4.

[0027] Item 6. A method for detecting a glycoprotein in a biological sample, comprising using the method for treating a glycoprotein according to any one of Items 1 to 4.

[0028] Item 7. A glycoprotein treated by the method for treating a glycoprotein according to any one of Items 1 to 4.

[0029] Item 8. A selective binding substance that binds to a glycoprotein treated by the method for treating an antigen according to any one of Items 1 to 4 and does not bind to the same glycoprotein that has not been treated.

[0030] Item 9. The selective binding substance according to Item 8, which is an antibody or an antigen-binding fragment of an antibody.

[0031] Item 10. A method for producing the selective binding substance according to Item 8 or 9, wherein the method comprises:

[0032] The glycoprotein treated by the method for treating an antigen according to any one of items 1 to 4 is used as an antigen.

[0033] Item 11. A method for producing the selective binding substance according to Item 8 or 9, wherein the method comprises:

[0034] Glycoproteins obtained by chemical synthesis or gene recombination technology are used as antigens.

[0035] Item 12. A method for detecting glycoproteins, comprising the following steps:

[0036] A step of treating a sample that may contain the glycoprotein with a sugar chain cleaving enzyme;

[0037] a step of allowing the selective binding substance according to item 8 to act on the glycoprotein treated with the sugar chain cleaving enzyme to obtain a reaction product between the glycoprotein and the selective binding substance, and

[0038] The step of detecting the reaction product.

[0039] Item 13. The method according to Item 12, wherein the following steps are performed simultaneously:

[0040] A step of treating the glycoprotein with a sugar chain-specific sugar chain cleaving enzyme; and

[0041] A step of obtaining a reaction product between the glycoprotein and a selective binding substance.

[0042] Item 14. A kit, which is a kit for the method described in Item 12 or 13, wherein the kit contains the following two reagents in the same composition:

[0043] A reagent for treating glycoprotein with a sugar chain-specific sugar chain cleaving enzyme; and

[0044] A reagent for obtaining a reaction product of the glycoprotein and a selective binding substance.

[0045] Item 15. A method for producing an antibody, using an antigen having a sugar chain structure that does not exist in a living body or exists only in extremely rare amounts.

[0046] Item 16. The method according to Item 15, wherein:

[0047] The antigen is a glycoprotein processed by a sugar chain-specific sugar chain cleaving enzyme.

[0048] Item 17. The method according to Item 15, wherein:

[0049] The antigen is obtained by chemical synthesis or gene recombination technology.

[0050] Effects of the Invention

[0051] By utilizing the method for treating glycoproteins of the present invention, it is possible to prepare a selective binding substance such as an antibody that can detect glycoproteins by only treating under mild conditions without denaturing the glycoproteins in a sample.

[0052] In addition, the method of detecting glycoproteins using selective binding substances such as antibodies prepared by the method of the present invention can be applied without denaturing the biological sample. Therefore, it can be used in combination with other tests that cannot be used under denaturing conditions, thereby obtaining more detailed information about the biological sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] [ Figure 1 ] Figure 1 This is a diagram showing the outline of a method for producing an antibody by treating a glycoprotein with a sugar chain-specific sugar chain cleaving enzyme.

[0054] [ Figure 2 ] Figure 2 This is a diagram showing that sugar chains are cleaved by treating glycoprotein with a sugar chain-specific sugar chain cleaving enzyme.

[0055] [ Figure 3 ] Figure 3 It is a diagram showing a schematic diagram of a glycopeptide.

[0056] [ Figure 4 ] Figure 4 This is a diagram showing the results of treating AFP (alpha-fetoprotein) with Endo-F2 or Endo-F3. Both enzymes were shown to be able to digest the sugar chain of AFP.

[0057] [ Figure 5 ] Figure 5 This is a diagram showing the results of an antigen solid-phase ELISA method using antisera from immunized animals when an immunogen formed by cross-linking a disaccharide peptide with a protein was used. Antisera from immunized animals showed a higher concentration of antibodies against the disaccharide peptide than sera from non-immunized animals.

[0058] [ Figure 6 ] Figure 6 The graph shows the results of an antigen-immobilized ELISA method using a culture supernatant of a hybridoma cell producing an anti-AFP antibody, showing that a variety of strains producing antibodies that react with disaccharide peptides but not with monosaccharide peptides can be confirmed from animals immunized with disaccharide peptides.

[0059] [ Figure 7 ] Figure 7 This is a diagram showing the results of an antigen solid phase ELISA method using antiserum from immunized animals when AFP purified from the culture supernatant of HepG2 was treated with Endo-F3 and the enzyme was removed to obtain an immunogen. Antiserum from immunized animals showed a higher concentration of antibodies against disaccharide peptides than sera from non-immunized animals.

[0060] [ Figure 8 ] Figure 8 This is a diagram showing the results of an antigen solid-phase ELISA method for hybridoma cells producing anti-AFP antibodies, showing that a variety of strains producing antibodies that react with disaccharide peptides but not with monosaccharide peptides were confirmed from animals immunized with disaccharide peptides.

[0061] [ Fig. 9 ] Fig. 9 This is a diagram showing the results of the specificity analysis of the monoclonal antibody, showing that the S20205R antibody or the S20206R antibody recognizes only the glycolytic AFP produced by the enzyme treatment.

[0062] [ Fig.10 ] Fig.10 It is a figure which shows the result of treating AFP by Endo-F3 or PNGase F. Both enzymes showed digestion of AFP.

[0063] [ Fig.11 ] Fig.11 This is a diagram showing the results of specificity analysis of monoclonal antibodies. The S20205R antibody and the S20206R antibody were shown to contain a sugar chain produced by Endo-F3 as a part of the recognition site.

[0064] [ Fig.12a ] Fig.12aThis is a graph showing the results of sandwich ELISA using the antibody of the present invention and a conventional antibody. It is shown that when the S20205R antibody was used, the absorbance of the enzyme-treated AFP increased in a concentration-dependent manner.

[0065] [ Figure 12b ] Figure 12b This is a graph showing the results of sandwich ELISA using the antibody of the present invention and a conventional antibody. It shows that when the S20206R antibody is used as a labeled detection antibody, the absorbance of the enzyme-treated AFP increases in a concentration-dependent manner.

[0066] [ Fig.13a ] Fig.13a This is a graph showing the results of reactivity analysis of each enzyme-treated product, showing that when a conventional anti-AFP antibody is used as a labeled detection antibody in ELISA analysis, the absorbance increases in an AFP concentration-dependent manner, regardless of whether or not enzyme treatment is performed.

[0067] [ Fig.13b ] Fig.13b This is a graph showing the results of reactivity analysis of each enzyme-treated product. It shows that when the S20206R antibody was used, the absorbance of only the AFP treated with Endo-F2 and Endo-F3 increased in a concentration-dependent manner.

[0068] [ Fig.14 ] Fig.14 This is a graph showing the results of enzyme treatment in serum, indicating that Endo-F3 acts on AFP even in serum and that enzyme-treated AFP can be detected by the antibody of the present invention.

[0069] [ Fig.15 ] Fig.15 This is a graph showing the results of studying the pH conditions for enzyme treatment, which shows that the enzyme used in the present invention can be used regardless of the pH.

[0070] [ Fig.16 ] Fig.16 This is a diagram showing the results of the on-plate enzyme treatment, showing that the antigen-antibody reaction and the antigen-enzyme reaction can be performed simultaneously. DETAILED DESCRIPTION

[0071] The following description is for illustrative purposes only, and the scope of the present invention is not limited to the following description, and the present invention can be implemented with appropriate changes without impairing the gist of the present invention.

[0072] (definition)

[0073] Unless otherwise specified, all technical and scientific terms used in the present specification have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0074] In the present specification, when a plurality of numerical ranges are shown, a combination of the lower limit values ​​and the upper limit values ​​of the plurality of ranges is also meaningful.

[0075] In this specification, "glycoprotein" refers to a protein containing one or more carbohydrate groups attached to a polypeptide chain by covalent bonds. For example, a glycoprotein contains 1 wt% to 60 wt% of carbohydrates in the form of short branched oligosaccharide chains of various compositions.

[0076] Most natural peptides and proteins contain carbohydrate moieties that are bound to the peptide or protein through specific binding to amino acids, which are a specific number of amino acids along the length of the main peptide or protein chain. The variability of the glycosylation pattern on any peptide or protein can affect the function of the peptide or protein. For example, the structure of N-bound glycans on a peptide or protein can affect various properties of the peptide or protein, including: protease sensitivity, intracellular transport, secretion, tissue targeting, biological half-life and antigenicity of the peptide or protein in a cell or organism.

[0077] Changes in one or more of these properties can affect the effectiveness of the peptide or protein in its natural environment.

[0078] In addition, for example, glycoprotein AFP having a peptide containing 590 amino acids is known to exist in vivo in a state having different sugar chain structures and called AFP-L1 and AFP-L3. Among them, only AFP-L3 is known to exist specifically in liver cancer.

[0079] Therefore, the presence or absence of liver cancer in a living body can be examined by using an antibody that specifically detects the sugar chain of AFP-L3.

[0080] In this specification, a specific biological molecule "does not exist in the body or is extremely rare even if it exists" means that the molecular weight or weight of the biological molecule existing in the body in a normal state cannot be detected by normal detection means, or is less than similar biological molecules. The organism in a normal state refers to, for example, an organism that does not have a specific disease, condition, etc. and is in a quiet state. "Few" means, for example, that the molecular weight or weight of the molecule in the body is 1 / 100, 1 / 1000 or 1 / 10000 less than that of similar biological molecules. For example, in the case of glycoproteins, "does not exist in the body or is extremely rare even if it exists" means, for example, that the amount present in the body is less than that of glycoproteins that contain the same protein in the structure and have different sugar chain structures. For example, glycoproteins of hexasaccharides, tetrasaccharides or disaccharides (e.g., fucosylated glycopeptides of disaccharides) are extremely rare in the body.

[0081] In this specification, "detection" or "detection" is used in a broad sense, and includes both qualitative and quantitative determination of target molecules. Detection includes direct and indirect detection, and includes any means of detection.

[0082] In one embodiment, the antibody of the present invention is used to detect the presence of a glycoprotein in a biological sample. In a specific embodiment, it includes: under conditions that allow the antibody of the present invention to bind to the glycoprotein, the antibody of the present invention is contacted with the biological sample, and the process of detecting whether a complex is formed between the antibody and the glycoprotein. Such a method can be an in vitro or in vivo method. In one embodiment, the antibody of the present invention is used to detect a biomarker representing a patient's status.

[0083] In this specification, when a selective binding substance such as an antibody "reacts", "shows reactivity", "has reactivity", "binds" or a selective binding substance such as an antibody "recognizes" a certain compound, the meanings commonly used in the field of the present invention are included, and all can be used interchangeably. As for whether a selective binding substance such as an antibody "reacts" with a certain compound, it can be carried out by antigen solid-phase ELISA method, competitive ELISA method, sandwich ELISA method, etc. known to those skilled in the art, and can be carried out by a method (SPR method) based on the principle of surface plasmon resonance, etc. The SPR method can be carried out using devices, sensors, and reagents commercially available under the name of Biacore (registered trademark).

[0084] In this specification, the selective binding substance such as the antibody of the present invention "does not react" with a certain compound means that the selective binding substance such as the antibody of the present invention does not actually react with the certain compound. "Does not actually react" means, for example, in the antigen solid-phase ELISA method, the binding of the selective binding substance such as the antibody to the solid-phase antigen is not actually affected by the addition of the compound. "Does not actually react" can also be confirmed by methods and means well known to those skilled in the art other than the antigen solid-phase ELISA method.

[0085] In the present specification, "specific reaction" of a selective binding substance such as an antibody or "specificity" of a selective binding substance such as an antibody refers to the ability of a selective binding substance such as an antibody to react with an epitope present on an antigen in a detectable manner, while on the other hand, the detectable reactivity with other antigens is relatively small or no reactivity is actually detected. For example, when a selective binding substance such as an antibody "specifically reacts" with a specific antigen, the selective binding substance such as an antibody reacts with the antigen, but on the other hand, does not react with other antigens. In a preferred embodiment, when a selective binding substance such as an antibody "specifically reacts" with a specific antigen, for example, in an antigen solid-phase ELISA method, the interaction between the immobilized antigen and the selective binding substance such as the antibody is inhibited by the free antigen, but on the other hand, is not inhibited by other free antigens.

[0086] In the present specification, the "reaction product" between the selective binding substance and the antigen refers to a substance produced by the specific reaction between the selective binding substance and the antigen, and refers to a substance that can be detected by a common detection method.

[0087] In this specification, "selective binding substance" or "antigen-binding substance" refers to a natural or non-natural substance that specifically binds to a specific substance, and can be used in the same manner as antibodies. For example, "selective binding substance" includes aptamers such as peptide aptamers, DNA aptamers, and RNA aptamers, lectins, receptors, variants thereof, and combinations thereof.

[0088] In this specification, "antigen" refers to a molecule or a part of a molecule that can be bound by a selective binding substance such as an antibody. An antigen may have more than one epitope, and each of them may interact with different selective binding substances, such as antibodies. "Antigen" can be used to prepare selective binding substances such as antibodies, and can be used as a standard when measuring glycoproteins.

[0089] In the present specification, an "epitope" is a region of an antigen where a selective binding substance targeting the antigen binds to the antigen.

[0090] In this specification, "antibody" is an immunoglobulin molecule comprising four polypeptide chains, namely two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (including CH1, CH2 and CH3 domains). Each light chain comprises a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions named complementarity determining regions (CDRs), which can be interspersed with a large number of conserved regions named framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged in the following order from the amine terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The term "antibody" also includes all genetic recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies.

[0091] In this specification, "antibody" refers to an immunoglobulin molecule containing four polypeptide chains and two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain includes a variable region of a heavy chain ("HCVR" or "VH") and a constant region of a heavy chain (including CH1, CH2 and CH3 domains). Each light chain includes a variable region of a light chain ("LCVR" or "VL") and a constant region of a light chain (CL). The VH and VL regions can be further divided into hypervariable regions named complementarity determining regions (CDRs), and can be dispersed in conserved regions named frameworks (FRs). Each VH and VL includes three CDRs and four FRs, and is configured from the amine terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains include binding domains that interact with antigens. The term "antibody" also includes all recombinant genes of antibodies, such as antibodies expressed in prokaryotes and non-glycosylated antibodies.

[0092] In addition, as shown in Padlan (1995 FASEB J. 9: 133-139), Vajdos et al. 2002 J Mol Biol 320: 415-428, it is known that only a portion of the CDR residues are actually in contact with the antigen, and the CDR residues that are not in contact with the antigen can be determined by molecular modeling or empirically from the CDR region of Kabat that exists outside the CDR of Chothia. In the case of removing a CDR or one or more of its residues, it is usually replaced by other human antibody sequences or amino acids occupying the corresponding position in the consensus sequence of such sequences. In addition, the replacement position in the CDR and the amino acid can be selected empirically. The empirical replacement can be a conservative or non-conservative replacement.

[0093] In this specification, the "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that has the ability to specifically bind to an antigen. Non-limiting examples of binding fragments contained in the "antigen-binding fragment" of an antibody include the following fragments: (i) Fab fragment, which is a monovalent fragment comprising VL, VH, CL and CH domains; (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments bound by a disulfide bridge in the hinge region; (iii) Fd fragment, which is composed of VH and CH domains; (iv) Fv fragment, which is composed of VL and VH domains of a single antibody arm; (v) dAb fragment composed of VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementary determining regions (CDRs); and (vii) a combination of two or more isolated CDRs optionally bound by a synthetic linker. In addition, as known as single-chain Fv (scFv), VL and VH regions can also be paired as a single protein chain using genetic recombination methods through synthetic linkers (Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). In addition, an "antigen-binding fragment" can be a binding domain immunoglobulin fusion protein, which comprises: (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. These antibody fragments can be obtained using existing techniques known to those skilled in the art.

[0094] The antibodies or antigen-binding fragments thereof that can be used in the present invention can be from all animals including birds and mammals. Preferably, the antibodies or fragments are from humans, chimpanzees, rodents (e.g., mice, rats, guinea pigs or rabbits), chickens, turkeys, pigs, sheep, goats, camels, cattle, horses, donkeys, cats or dogs. The antibodies of the present invention include chimeric molecules composed of a constant region of an antibody derived from a certain species and an antigen-binding site derived from another species. Furthermore, the antibodies of the present invention include humanized molecules obtained by combining an antigen-binding site of an antibody derived from a non-human species (e.g., from a mouse) with a constant region and framework region of human origin.

[0095] The antibody of the present invention can be obtained from a hybridoma expressing the antibody or a host cell expressing the antibody by gene recombination. Examples of host cells that can be used include CHO cells, lymphocytes, bacterial cells such as Escherichia coli, and fungal cells such as yeast.

[0096] In addition, the antibodies of the present invention can be produced in non-human animals or plants that have been genetically modified using genetic recombination technology. In one embodiment, the genes of hybridoma cells that produce the desired antibody are analyzed, so that antibodies having the same amino acid sequences of the light chain variable region and the heavy chain variable region as the antibody can be expressed by other host cells using genetic recombination technology.

[0097] As the antibody in the present invention, for example, a monoclonal antibody produced by hybridoma cell S20205R or S20206R is preferred. The hybridoma cell is internationally deposited under the Budapest Treaty as described below.

[0098] Name of the depository: NITE International Patent Microorganism Collection Center, address of the depository: Room 122, 2-5-8, Kazusa Kamata, Kisarazu City, Chiba Prefecture (Postal Code 292-0818), deposit date: March 22, 2019.

[0099] Deposit number: NITE BP-02926 (hybridoma cell S20205R)

[0100] Deposit number: NITE BP-02927 (hybridoma cell S20206R)

[0101] The sugar chain-specific sugar chain cleaving enzyme used in the present invention is not particularly limited, and various known enzymes that cleave sugar chains of glycoproteins in vivo can be used, and multiple enzymes can be used in combination. In one embodiment, with respect to the sugar chain-specific sugar chain cleaving enzyme of the present invention or a combination thereof, it is preferred that a sugar chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 in length is generated as a result of the treatment.

[0102] In one embodiment, as such a sugar chain-specific sugar chain cleaving enzyme, it is preferred to use a sugar chain cleaving enzyme that selectively recognizes a structure containing an N,N'-diacetylchitobiose structure (GlcNA cβ1,4GlcNAc) constituting a basic skeleton of an N-bound sugar chain, and cleaves and releases the sugar chain while leaving one N-acetylglucosamine residue (to which fucose may be attached) on the protein side. It is particularly preferred to use an enzyme that recognizes core fucose in the sugar chain of a glycoprotein, and selectively cleaves the sugar chain while leaving a disaccharide containing core fucose on the protein side.

[0103] In one embodiment, as such a sugar chain-specific sugar chain shearing enzyme, for example, endoglycosidase can be used. In particular, it is preferred to use an enzyme selected from β-N-acetylglucosaminidase (EC3.2.1.96). With regard to the selection of enzymes, it can be carried out by exploring the enzyme reaction conditions of the range usually performed by those skilled in the art. Whether the desired sugar chain shearing is carried out by the enzyme reaction can be confirmed by a general analytical method, for example, by analyzing the substance produced by the enzyme reaction using a mass spectrometer, etc.

[0104] As non-limiting examples of specific sugar chain-specific sugar chain cleaving enzymes, β-N-acetylglucosaminidase such as Endo-A, Endo-M, and Endo-H derived from Streptomyces plicatus, Endo-D derived from Streptococcus pneumoniae, and Endo-F1, Endo-F2, and Endo-F3 derived from Flavobacterium meningosepticum can be used. In one embodiment, Endo-F1, Endo-F2, and Endo-F3 derived from Flavobacterium meningosepticum are preferably used, and Endo-F3 is more preferably used.

[0105] The conditions for treating the glycoprotein in the sample with a sugar chain-specific sugar chain cleaving enzyme can be appropriately changed according to the enzyme used, for example, by treating in the following manner: mixing the glycoprotein with the sugar chain-specific sugar chain cleaving enzyme at a pH of 5 to 8 or 6 to 7, at 25 to 40°C, 28 to 39°C, 30 to 37°C, 32°C to 36°C, 33°C to 35°C or 34°C for 5 to 60 minutes, 7 to 50 minutes, 10 to 40 minutes, or 15 to 30 minutes.

[0106] The glycoprotein having the sugar chain structure of the present invention can be used as an antigen in the production of selective binding substances such as antibodies, and can be used as a standard in the measurement of glycoproteins.

[0107] (Method for preparing an antibody that specifically reacts with the sugar chain structure of the glycoprotein of the present invention)

[0108] The glycoprotein of the present invention having a sugar chain structure that does not exist in the body or exists only in extremely rare amounts, obtained by the above method, can be used as an antigen to prepare an antibody that specifically reacts with the sugar chain structure. The outline of the method for preparing an antibody that specifically reacts with the sugar chain of the glycoprotein of the present invention is shown in Figure 1 .

[0109] Specifically, by treating glycoproteins with sugar chain-specific sugar chain shearing enzymes, a sugar chain structure that does not exist in the body or is extremely rare even if it exists is produced, and by using this structure as an antigen, antibodies are easily prepared. The antigen is used to immunize mammals such as mice by known methods described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988)). Hybridoma cells are prepared by removing spleen cells or lymph node cells from the animal and performing cell fusion with myeloma cells. A substance that reacts with the antigen can be isolated from the prepared hybridoma cell population to obtain a monoclonal antibody.

[0110] When an antigen having a sugar chain structure of the present invention is used as an immunogen, the antigen obtained by the treatment with the sugar chain cleaving enzyme can be used as it is, or the antigen can be bound to a general carrier and used. Such a carrier and a method for binding an antigen to a carrier are well known to those skilled in the art, for example, a protein such as KLH (Thermo 77600) as a carrier can be bound to an antigen using a linker such as Sulfo-SMCC (Thermo 22322).

[0111] As the antigen having the sugar chain structure of the present invention, an antigen obtained by treating glycoprotein with a sugar chain-specific sugar chain cleaving enzyme as described above can be used, and a compound having the same structure as the antigen obtained by the present invention obtained by a known method such as chemical synthesis and gene recombination technology can be used as an antigen. In addition, a part other than the epitope can also be modified and used as an antigen. For example, a glycopeptide having the same amino acid and sugar chain sequence as the site that binds to the selective binding substance and present in the glycoprotein obtained by treating the glycoprotein with a sugar chain-specific sugar chain cleaving enzyme can be used as an antigen. These glycopeptides that can be used as antigens or compounds obtained by chemical synthesis or gene recombination can be used not only for the manufacture of selective binding substances such as antibodies, but also as standards when measuring glycoproteins.

[0112] The preparation of hybridoma cells can be carried out according to methods known in the art, for example, the polyethylene glycol method, the method using Sendai virus, the method using electric current, etc. The obtained hybridoma cells can be propagated according to known methods, and the desired hybridoma cells can be selected while confirming the properties of the produced antibodies. The cloning of hybridoma cells can be carried out by known methods such as limiting dilution method and soft agar method.

[0113] The obtained antibody can be directly produced by the hybridoma cell, or a host cell expressing the antibody by gene recombination can be prepared and obtained from the host cell. As the host cell, for example, CHO cells, lymphocytes, bacterial cells such as Escherichia coli, and fungal cells such as yeast can be used.

[0114] (Other methods for producing selective binding substances)

[0115] The glycoprotein obtained by the glycoprotein treatment method of the present invention can be used to produce antibodies, and can be used to produce other selective binding substances that specifically select the glycoprotein, for example, aptamers such as peptide aptamers, DNA aptamers, RNA aptamers, lectins, receptors, variants thereof, and combinations thereof. For the same purpose, as glycoproteins, glycoproteins obtained by a method of treating with a sugar chain-specific sugar chain cleaving enzyme can be used, and compounds having the same structure obtained by known methods such as chemical synthesis and gene recombination technology or variants thereof can be used.

[0116] For nucleic acid aptamers such as DNA aptamers and RNA aptamers, aptamers that specifically bind to the glycoprotein obtained by the glycoprotein treatment method of the present invention can be obtained by using methods well known to those skilled in the art, such as repeated rounds of in vitro selection or exponentially enriched ligands (SELEX) method (Archemix, Cambridge, MA, USA) (Sampson, 2003), but are not limited to these.

[0117] Peptide aptamers that specifically bind to the glycoprotein obtained by the glycoprotein treatment method of the present invention can be obtained by known methods such as the method disclosed in Lu et al., Chem Rev 2009: 109(5): 1948-1998, but are not limited thereto.

[0118] In the case where lectins are used as selective binding substances, the lectins used can be obtained from any organisms including plants, animals, yeasts, bacteria, protozoa, etc. Lectins can be isolated from these naturally occurring sources or recombinantly expressed and purified by methods well known to those skilled in the art. In addition, purified commercially available lectins can be used. As a selective binding substance that specifically binds to sugar chains having fucose, for example, fucose-specific lectins can be used (see Mansour MH et al. (2005) Immunobiology. 210: 335-48, etc.). In order to obtain lectins that specifically bind to the glycoprotein obtained by the method for treating the glycoprotein of the present invention, lectins that specifically bind to the glycoprotein of the present invention can be screened from currently available lectins, or modified lectins that specifically bind to the glycoprotein of the present invention can be screened using genetic recombination technology. For the same purpose as lectins, proteins having a lectin-like domain can be isolated and used (see Drickamer K (1999) Curr. Opin. Struct. Biol. 9: 585-90, etc.).

[0119] When a receptor is used as a selective binding substance, a receptor that specifically binds to the glycoprotein of the present invention can be screened from known glycoprotein binding receptors, or a modified receptor that specifically binds to the glycoprotein of the present invention can be screened using genetic recombination technology. It is also possible to isolate and use a modified receptor obtained by binding a portion of the receptor to other proteins such as the Fc region of an antibody for the same purpose as the receptor.

[0120] (Uses of the Selective Binding Substance of the Present Invention)

[0121] The selective binding substance such as the antibody obtained by the method of the present invention can be used to detect glycoproteins that function as biomarkers in biological samples.

[0122] (Method for specifically detecting glycoprotein in a sample)

[0123] When performing the method of the present invention for specifically detecting glycoproteins in a sample, the sample that may contain glycoproteins may be treated with a sugar chain-specific sugar chain cleaving enzyme. The sugar chain-specific sugar chain cleaving enzyme may be the same as or different from the sugar chain-specific sugar chain cleaving enzyme used when preparing a selective binding substance such as an antibody. In one embodiment, the same enzyme or a combination of enzymes is preferably used.

[0124] In the case of using a sugar chain shearing enzyme, an enzyme treatment can be performed by adding a sugar chain-specific sugar chain shearing enzyme to the sample that may contain glycoproteins, or the glycoprotein can be purified to a certain extent from the sample and then subjected to an enzyme treatment using a sugar chain shearing enzyme. In addition, after the process of enzymatic treatment of the glycoprotein with a sugar chain shearing enzyme, a process of obtaining a reaction product of a selective binding substance and a glycoprotein can be applied. In addition, the process of enzymatic treatment of the glycoprotein with a sugar chain shearing enzyme and the process of obtaining a reaction product of the glycoprotein and a selective binding substance can be performed simultaneously. In one embodiment, it is preferred to simultaneously perform a process of enzymatic treatment of the glycoprotein with a sugar chain shearing enzyme and a process of obtaining a reaction product of the glycoprotein and a selective binding substance. In the case of simultaneously performing a process of enzymatic treatment of the glycoprotein with a sugar chain shearing enzyme and a process of obtaining a reaction product of the glycoprotein and a selective binding substance, as a selective binding substance, it is preferred to select a substance that is not affected by the sugar chain shearing enzyme.

[0125] As a method for detecting the reaction product, a known method can be used, wherein the reaction product is obtained by a selective binding substance reacting specifically with an antigen. For example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, immunoagglutination assay, immunoturbidimetry, particle immunoassay and other methods can be used, but are not limited thereto (for example, refer to Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, DM, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston). In one embodiment, the selective binding substance such as the antibody of the present invention can be used as: a fixed (solid phase) substance fixed on an insoluble carrier, a labeled substance labeled with a labeling substance (for example, a detection antibody). Such immobilized selective binding substances and labeled selective binding substances are all included in the scope of the present invention. For example, the selective binding material of the present invention can be physically adsorbed on an insoluble carrier or chemically bound to an insoluble carrier (via an appropriate spacer) to produce an immobilized selective binding material. As an insoluble carrier, an insoluble carrier comprising a polymer substrate such as a polystyrene resin, an inorganic substrate such as a glass, a polysaccharide substrate such as cellulose, agarose, etc. can be used. Its shape is not particularly limited, and any shape such as a plate (e.g., microplate, film), a bead or a particulate (e.g., latex particles, magnetic particles), a tubular (e.g., a test tube) can be selected.

[0126] As a labeling substance for manufacturing a labeled selective binding substance, for example, enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin or radioisotopes, gold colloid particles, colored latex, etc. can be cited. As a method for combining a labeling substance with a selective binding substance, methods such as glutaraldehyde method, maleimide method, pyridyl disulfide method or periodic acid method available to those skilled in the art can be used. The types of immobilized selective binding substances and labeled selective binding substances and their manufacturing methods are not particularly limited. For example, enzymes such as peroxidase and alkaline phosphatase (hereinafter, sometimes referred to as ALP) can be used as labeling substances. In this case, the specific substrate of the enzyme (when the enzyme is horseradish peroxidase (Horse Radish Peroxidase) (hereinafter, sometimes referred to as HRP), for example, 1,2-phenylenediamine (hereinafter, sometimes referred to as OPD) or 3,3',5,5'-tetramethylbenzidine, in the case of ALP, p-nitrophenyl phosphate, etc.) can be used to measure the enzyme activity. When biotin is used as a labeling substance, it is usually reacted with avidin or enzyme-modified avidin.

[0127] The sample used in the detection using the selective binding substance of the present invention is not particularly limited, and various samples can be used. In one embodiment, biological samples such as liquid biological samples such as blood, plasma, serum, urine, and saliva, and solid biological samples such as skin, feces, and biopsy tissue can be used.

[0128] The selective binding substance of the present invention can be provided as a measuring reagent for the method for detecting glycoproteins. In addition to the selective binding substance of the present invention, the reagent may also contain other components required for carrying out the detection method, such as a buffer, a preservative, and the like.

[0129] In one embodiment, for example, when the selective binding substance of the present invention contains an enzyme as a labeling substance, it can be provided in the form of a kit together with a reagent containing a specific substrate thereof.

[0130] When providing a kit for use in a method for detecting a glycoprotein using a selective binding substance of the present invention, the reagent for performing an enzyme treatment using a sugar chain cleaving enzyme and the reagent for the step of obtaining a reaction product of a glycoprotein and a selective binding substance may be provided as separate compositions, respectively. Alternatively, the reagent for performing an enzyme treatment using a sugar chain cleaving enzyme and the reagent for the step of obtaining a reaction product of a glycoprotein and a selective binding substance may be provided as the same composition. In one embodiment, it is preferred that the reagent for performing an enzyme treatment using a sugar chain cleaving enzyme and the reagent for the step of obtaining a reaction product of a glycoprotein and a selective binding substance are provided as the same composition.

[0131] Example

[0132] Hereinafter, the present invention will be described in further detail by way of examples, but the present invention is not limited thereto.

[0133] [Example 1]

[0134] 1. Processing of glycoprotein AFP-L3 by Endo-F3

[0135] Glycoprotein AFP affinity purified from the culture supernatant of liver cancer cell line HepG2 cells was treated with sugar chain cleaving enzyme Endo-F3 (38.8 kDa, manufactured by New England BioLabs) under the following conditions. The purified AFP contained AFP-L3.

[0136] [Materials and conditions]

[0137] For (1) AFP: 8 μg,

[0138] Mix (2) Endo-F3: 16U (0.1M MES buffer (pH 5.5) 20μL),

[0139] Treatment (enzyme reaction) time: 10 minutes or 30 minutes

[0140] Treatment (enzyme reaction) temperature: 37°C

[0141] The enzyme treatment was carried out under the conditions of to obtain an enzyme-treated AFP liquid.

[0142] Equal amounts of the enzyme-treated AFP solution and Tris SDS sample treatment solution (manufactured by COSMO BIO, 423420) were added and boiled for 10 minutes. The boiled sample was subjected to electrophoresis at 30 mA for 70 minutes using MULTIGEL (registered trademark) II Mini 4 / 20 (13W) (manufactured by COSMO BIO, 414879), and the gel was stained using conventional CBB staining.

[0143] Figure 2 The results of electrophoresis (SDS-PAGE) of samples that were not treated, treated for 10 minutes, and treated for 30 minutes are shown. The band detected between 52 kDa and 76 kDa in the untreated lane is a band derived from AFP, and the band detected near 38 kDa in lanes 1 and 2 is a band derived from Endo-F3. It was confirmed that for the band derived from AFP, the detection position of the band for 10 minutes and 30 minutes of treatment time moved in the direction of decreasing molecular weight compared to the detection position of the band for the untreated band. From the above results, it can be seen that the fucose-containing sugar chain bound to AFP-L3 was cleaved by the treatment.

[0144] [Example 2]

[0145] 2. Preparation of Monoclonal Antibodies

[0146] AFP having the cleaved sugar chain is used as an antigen (immunogen) to immunize mice or rats by a known method, and hybridoma cells are prepared from B cells obtained from the spleen of the mice or rats.

[0147] AFP having the cleaved sugar chain is used to screen hybridoma cells, thereby obtaining a monoclonal antibody that specifically detects AFP having the cleaved sugar chain.

[0148] In more detail, the following experiment was conducted.

[0149] [Material]

[0150] (1) Freund's complete adjuvant: manufactured by Wako Pure Chemical Industries, Ltd., 014-09541

[0151] (2) Myeloma cells (SP2 / O)

[0152] (3) RPMI1640, GlutaMAX: manufactured by GIBCO, 61870-036

[0153] (4) Fetal bovine serum (FBS): manufactured by BIOLOGICAL INDUSTRIES, 04-001-1A

[0154] (5) HAT medium: manufactured by COSMO BIO, 16213004

[0155] (6) 96-well plate: NUNC, 167008

[0156] (7) HRP-labeled goat anti-mouse IgG (H&L) and HRP-labeled goat anti-rat IgG (H&L) antibodies: manufactured by Southern Biotech, 1031-05 and 3050-05

[0157] (8) MULTIGEL (registered trademark) II Mini 4 / 20 (13W): Made by COSMO BIO, 414879

[0158] (9) Tris SDS sample treatment solution: manufactured by COSMO BIO, 423420

[0159] (10) Glycopeptide: Synthesized disaccharide 8a.a. and monosaccharide 8a.a. (sequences as Figure 3 )

[0160] [Test Example 1] Confirmation of enzyme treatment

[0161] 1. Test methods

[0162] 2 μL (16 U) of Endo-F2 (39.8 kD, manufactured by New England BioLabs) or 2 μL (2 μg) of Endo-F3 was added to 2 μL (5-7 μg) of AFP affinity purified from the culture supernatant of HepG2, and 16 μL of 0.1 M MES buffer (pH 5.5) was further added. After these samples were subjected to enzyme reaction at 37°C for 10 minutes, an equal amount of Tris SDS sample treatment solution was added and boiled for 10 minutes. These samples were added to MULTIGEL (registered trademark) II Mini4 / 20 (13W) in an amount of 10 μL per lane, and electrophoresis was performed at 30 mA for 70 minutes. The gel was then stained with conventional CBB staining.

[0163] 2. Test results

[0164] The results are shown in Figure 4 In the case of AFP without added sugar chain cleaving enzyme and AFP with added sugar chain cleaving enzyme Endo-F2 and Endo-F3, the patterns of detected bands were different. In the case of AFP treated with enzyme, the concentration of the band on the low molecular weight side was particularly high ( Figure 4 ). This indicates that part of the modified sugar chains in AFP are digested and cleaved by sugar chain cleaving enzymes, making the molecular weight smaller. This result shows that regardless of the type of sugar chain cleaving enzyme, the sugar chains of AFP can be digested.

[0165] [Test Example 2] Method for producing monoclonal antibody-1

[0166] 1. Preparation of Immunogen

[0167] The immunogen solution was prepared by cross-linking a disaccharide peptide that mimics the amino acid and sugar chain sequence generated when AFP is treated with a sugar chain cleaving enzyme with various proteins such as KLH (Thermo 77600) using a commercially available linker reagent.

[0168] 2. Preparation of hybridoma cells producing monoclonal antibodies against AFP treated with sugar chain cleavage enzymes

[0169] (1) Immunization of animals

[0170] Using an emulsion prepared by mixing the immunogen solution (0.2-2 mg / mL) with Freund's complete adjuvant in equal volumes, 10-40 μg of the emulsion was injected into each Balb / cAJcl mouse or F344 rat as an immunogen. In addition, the injection of the emulsion was repeated 3-8 times at weekly intervals. The antibody titer in the antiserum obtained from tail vein blood was measured by the antigen solid-phase ELISA method described later.

[0171] (2) Primary screening (antigen solid-phase ELISA method)

[0172] The presence of antibodies against the sugar chain peptide in the antiserum of the immunized animal is confirmed by ELISA method in which the disaccharide peptide conjugate prepared in the same manner as the immunogen is solid-phased (antigen solid-phased ELISA method). The details of the antigen solid-phased ELISA method are as follows.

[0173] (2-1) Preparation of antigen-immobilized ELISA plate

[0174] The conjugate solution formed by the disaccharide peptide and a protein different from the protein used to prepare the immunogen was dissolved in 20 mM phosphate buffer (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride to make the conjugate solution 1 μg / mL, and 50 μL of the dissolved solution was distributed to each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.

[0175] After washing each well three times with 400 μL of PBS containing 0.05% Tween (registered trademark) 20 (hereinafter referred to as PBST), 100 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added and blocked at room temperature for 1 hour. This was used as an ELISA plate.

[0176] (2-2) Antigen solid phase ELISA method

[0177] After washing each well of the ELISA plate with 400 μL of PBST three times, 50 μL of antiserum of immune animals and serum of non-immune animals diluted stepwise with BSA-PBST were added to each well and left to stand at room temperature for 1 hour. After washing each well with 400 μL of PBST three times, 50 μL of HRP-labeled anti-mouse IgG (H&L) diluted 10,000 times with BSA-PBST or HRP-labeled rat IgG (H&L) diluted 5,000 times was distributed to each well and left to stand at room temperature for 1 hour. After washing each well with 400 μL of PBST three times, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured. Based on the results of the measurement, the spleen and lymph nodes are removed from mice or rats with high antibody titers, and cells derived from the spleen and lymph nodes are prepared and used for cell fusion.

[0178] (2-3) Test results

[0179] As an example of the results, the case where mice were immunized is shown in Figure 5 Compared with the serum of non-immune animals, the antiserum of immune animals shows a higher absorbance at a lower dilution factor when using solid-phase disaccharide glycopeptide. In this measurement system, the absorbance measured depends on the concentration of antibodies against disaccharide peptides contained in the serum of the animal from which the blood was collected ( Figure 5 ). Thus, the results show that the antisera from immunized animals contain a higher concentration of antibodies against the disaccharide peptide than the sera from non-immunized animals, and therefore the antibody titer is higher.

[0180] (3) Cell fusion

[0181] The spleen-derived cells or lymph node-derived cells were mixed with myeloma cells at a ratio of 1:1, and cell fusion was performed by electric pulse method. The fused cells were suspended in HAT medium and cultured in a CO2 incubator at 37°C and 5% CO2 for 8 days to obtain fused cells (hybridoma cells).

[0182] (4) Selection of hybridoma cells (antigen solid-phase ELISA method)

[0183] (4-1) Method

[0184] In the antigen solid-phase ELISA method, the same method is performed except that the culture supernatant of the fused cells is used instead of the antiserum of the immunized animal, and as the solid-phase antigen, in addition to the disaccharide peptide conjugate, the monosaccharide peptide, the AFP not subjected to enzyme treatment, the AFP treated with Endo-F3, and the respective conjugates are used. As a result of the measurement, the wells in which the absorbance for the disaccharide peptide is higher than that for the monosaccharide peptide or the wells in which the absorbance for the AFP treated with Endo-F3 is higher than that for the AFP not subjected to enzyme treatment are selected as the wells in which the hybridoma cells producing the anti-disaccharide peptide or anti-enzyme treated AFP antibodies exist (positive wells).

[0185] (4-2) Test results

[0186] As an example of the results, the case where a disaccharide peptide or a monosaccharide peptide was solid-phased is shown in Figure 6 From animals immunized with disaccharide peptides, it was possible to identify a variety of strains that produce antibodies that react with disaccharide peptides but not with monosaccharide peptides ( Figure 6 ). This result indicates that hybridoma cells that produce antibodies in the vicinity of the sugar chain modification site of the disaccharide of enzyme-treated AFP can be obtained by using the disaccharide peptide as an immunogen.

[0187] [Test Example 3] Method for producing monoclonal antibody-2

[0188] 1. Test methods

[0189] AFP purified from the culture supernatant of HepG2 was treated with Endo-F3 and the enzyme was removed as in Test Example 1, and the immunogen was used in the same manner as in [Test Example 2]. In the antigen solid phase ELISA method, AFP treated with Endo-F3 was used as the antigen.

[0190] 2. Test results

[0191] As an example of the results, the case where rats were immunized is shown in Figure 7 , 8 Compared with the serum of non-immune animals, the lower the dilution factor of the antiserum of immune animals, the higher the absorbance of the immobilized enzyme-treated AFP ( Figure 7 ). In this measurement system, the absorbance measured depends on the concentration of antibodies against enzyme-treated AFP contained in the serum of the animal from which the blood was collected. That is, the results show that the antiserum of the immunized animal contains a higher concentration of antibodies against enzyme-treated AFP than the serum of the non-immunized animal, and the antibody titer is higher.

[0192] like Figure 8As shown, various strains that produce antibodies that react with enzyme-treated AFP but do not react with untreated AFP can be confirmed from animals immunized with enzyme-treated AFP. That is, it is possible to obtain hybridoma cells that produce antibodies specific to enzyme-treated AFP using enzyme-treated AFP as an immunogen.

[0193] [Test Example 4] Cloning

[0194] Using the strain hybridoma cells producing antibodies selected by the primary screening and secondary screening, the hybridoma cells were monoclonalized and the monoclonal antibodies were purified. The cloning was performed by conventional methods (limiting dilution method, for example, see "Laboratory Medicine Section Protein Experiment Manual" ISBN4-89706-369-8), and the positive wells were selected by the same method as the antigen solid phase ELISA method, and finally 2 hybridoma cells (S20205R and S20206R) producing monoclonal antibodies were obtained.

[0195] [Test Example 5] Specificity analysis of the monoclonal antibody of the present invention 1

[0196] First, immunoprecipitation and Western blotting were used to confirm how the antibody produced by S20205R and the antibody produced by S20206R reacted with the enzyme-treated AFP specifically.

[0197] 1. Test methods

[0198] 400 μL of 0.1M MES buffer was added to 400 μL of culture supernatant of HepG2 cells, and 7.5 μg of Endo-F3 was further added. After reacting for 30 minutes at 37°C, 800 μL of BSA-PBST was added to prepare the sample. Then, 400 μL of the sample was added to 100 μL of agarose gel (GEHEALTHCARE: CNBr activated Sepharose 4 Fast Flow (17098101)) to which 4 antibodies were fixed by conventional methods, respectively, and stirred at room temperature for 2 hours. The 4 antibodies were S20205R antibody, S20206R antibody, existing anti-AFP antibody (02211 manufactured by our company) and an antibody that does not react with AFP. They were centrifuged at 10000g for 2 minutes, the supernatant was removed, PBST 1mL was used to fully suspend, and centrifuged at 10000g for 2 minutes again. After repeating this operation twice, the supernatant was removed, 60 μL of TrisSDS sample treatment solution was added, and the mixture was boiled for 10 minutes. Then, centrifugation was performed at 10,000 g for 2 minutes, 15 μL was added to 1 lane, and SDS-PAGE was performed in the same manner as [Test Example 1]. The operation so far was set as immunoprecipitation. The gel after electrophoresis was transferred to a PVDF membrane by conventional methods, and the PVDF membrane was shaken at room temperature for 1 hour in 1% BSA-PBST. Then, the existing anti-AFP antibody labeled with biotin or an antibody that does not react with AFP was diluted to 1 μg / mL in 1% BSA-PBST as a detection antibody, and the PVDF membrane was shaken at room temperature for 1 hour. Next, the PVDF membrane was shaken at room temperature for 5 minutes in PBST, and the supernatant was removed. After repeating this operation 3 times, the PVDF membrane was shaken at room temperature for 30 minutes in 1% BSA-PBST in which HRP-labeled streptavidin was diluted to 0.33 μg / mL. Next, the PVDF membrane was shaken in PBST at room temperature for 5 minutes, and the supernatant was removed. This operation was repeated 3 times, and then immersed in 50 mM Tris hydrochloric acid buffer (pH 7.4) containing 0.2 mg / mL diaminobenzidine and 0.0012% hydrogen peroxide, and after the bands could be fully confirmed by naked eye observation, the PVDF membrane was washed with RO water.

[0199] 2. Test results

[0200] The results are shown in Fig. 9. In the case where immunoprecipitation was performed using an existing anti-AFP antibody and detection was performed using an anti-AFP antibody (left lane), bands were detected regardless of whether or not enzyme treatment was performed (- and + lanes in the left lane). It was also confirmed that, in the case of enzyme treatment, the bands became double on the high molecular weight side and the low molecular weight side (+ lane in the left lane). On the other hand, in the case where immunoprecipitation was performed using an S20205R antibody (central left lane) or an S20206R antibody (central right lane) and detection was performed using an anti-AFP antibody, in the case of enzyme treatment (each + lane in the central left and central right lanes), bands were detected only on the lower molecular weight side ( Fig. 9 ). In this measurement system, the band of AFP bound by the antibody used in the immunoprecipitation was detected. That is, as shown in [Test Example 1], in addition to the increase in the band on the low molecular weight side by enzyme treatment, it was also shown that the S20205R antibody or the S20206R antibody recognized the glycolytic AFP produced by enzyme treatment. It should be noted that in the case of an antibody that does not react with AFP (right lane), no band was actually detected.

[0201] [Test Example 6] Specificity analysis of the monoclonal antibody of the present invention 2

[0202] 1. Test methods

[0203] The specificity of the S20205R antibody and the S20206R antibody was confirmed by the same method as the antigen immobilized ELISA method. Figure 3 At the same time, SDS-PAGE of each AFP was performed in the same manner as in [Test Example 1].

[0204] 2. Test results

[0205] The results are shown in Fig.10 , Fig.11 In SDS-PAGE, AFP without enzyme treatment, AFP treated with Endo-F3, and AFP treated with PNGaseF all showed different band patterns. AFP treated with Endo-F3 had double bands on the high molecular weight side and the low molecular weight side, while in the case of AFP treated with PNGaseF, a band was confirmed on the low molecular weight side ( Fig.10 ). It was shown that Endo-F3 cleaved part of the sugar chains of AFP into disaccharides, while PNGaseF digestion cleaved all N-type sugar chains, producing AFP without sugar chains. Fig.11As shown, the absorbance of the existing AFP antibody increased regardless of whether the enzyme treatment was performed, while in the case of the S20205R antibody and the S20206R antibody, the absorbance of the AFP treated only by Endo-F3 increased. In this measurement system, the stronger the reactivity of the antibody to the AFP fixed on the plate, the higher the absorbance. That is, it is shown that the S20205R antibody and the S20206R antibody contain the sugar chain produced by Endo-F3 as part of the recognition site.

[0206] [Test Example 7] Sandwich ELISA of existing antibodies

[0207] So far, the reactivity to antigens immobilized on a plate has been evaluated, and the reactivity to antigens present in a liquid phase has also been evaluated. The test method is as follows.

[0208] 1. Test methods

[0209] (1) Preparation of sandwich ELISA plates

[0210] Purified AFP was prepared as an immunogen, and a solution containing an existing anti-AFP monoclonal antibody (in-house product) that reacted regardless of whether or not it was subjected to enzyme treatment was dissolved in a 20 mM phosphate buffer (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride, and the concentration of the existing anti-AFP monoclonal antibody was adjusted to 5 μg / mL. 50 μL of the dissolved solution was distributed to each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.

[0211] After washing each well three times with 400 μL of PBS containing 0.05% Tween (registered trademark) 20 (hereinafter referred to as PBST), 100 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added and blocked at room temperature for 1 hour. This was used as a sandwich ELISA plate.

[0212] (2) Sandwich ELISA

[0213] In the same manner as in [Test Example 1], AFP treated with Endo-F3 was gradually diluted with BSA-PBST. This was used as an antigen-containing liquid. After removing the liquid from each well of the sandwich ELISA plate, 50 μL of the antigen-containing liquid was added to each well and allowed to stand at room temperature for 15 minutes. After washing each well three times with 400 μL of PBST, 50 μL of HRP-labeled S20205R antibody or HRP-labeled S20206R antibody diluted 1500 times with BSA-PBST was distributed to each well of the plate and allowed to stand at room temperature for 15 minutes. After washing each well three times with 400 μL of PBST, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added. After standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

[0214] 2. Test results

[0215] The results are shown in Fig.12a , b. For enzyme-treated AFP, the absorbance increased in a concentration-dependent manner ( Fig.12a , b). In this assay system, the absorbance increases by reacting the HRP-labeled S20205R antibody and the HRP-labeled S20206R antibody with the enzyme-treated AFP in the liquid phase that is bound to the existing anti-AFP antibody fixed on the plate. That is, these antibodies specifically react with the enzyme-treated AFP in the liquid phase.

[0216] Based on the results so far, since the properties of the S20205R antibody and the S20206R antibody are roughly the same, only the S20206R antibody will be used in subsequent evaluations.

[0217] [Test Example 8] Reactivity analysis of each enzyme-treated product by sandwich ELISA

[0218] 1. Test methods

[0219] The test method was the same as that of [Test Example 7]. AFP prepared in the same manner as in [Test Example 1] and treated with Endo-F2 or Endo-F3 was used as the antigen. In addition, in the step of adding the HRP-labeled antibody in [Test Example 7], an existing anti-AFP polyclonal antibody labeled with HRP diluted 6000 times with BSA-PBST was also used for the test.

[0220] 2. Test results

[0221] The results are shown in Fig.13a, b. When the measurement is performed using the existing AFP antibody, the absorbance increases in an AFP concentration-dependent manner regardless of whether the enzyme treatment is performed ( Fig.13a On the other hand, when the S20206R antibody was used, the absorbance of AFP treated only with Endo-F2 and Endo-F3 increased in a concentration-dependent manner ( Fig.13b ). That is, it was shown that when Endo-F2 was used, glycolytic AFP similar to that produced when Endo-F3 was used was produced, and even when an enzyme other than Endo-F3 was used, the same measurement as in the previous test examples could be performed.

[0222] [Test Example 9] Study on Enzyme Treatment in Serum

[0223] Until now, enzymes and buffers have been added to purified AFP to perform enzyme treatment. However, in actual clinical tests, the sample used for measurement is assumed to be serum, and in addition to AFP, serum is also expected to contain a very high concentration of N-type sugar chain proteins. Therefore, a test was conducted to determine whether the enzyme acts on AFP in serum.

[0224] 1. Test methods

[0225] The test method was the same as that of [Test Example 7]. However, as for the antigen used, 25 μL of 3 healthy subject sera were added to 200 ng of AFP derived from HepG2, and 25 μL of 0.1 M MES buffer (pH 5.5) and 2 μg of Endo-F3 were added. After these were reacted at 37°C for 10 minutes, 1 mL of BSA-PBST was added to prepare a solution with an AFP concentration of 200 ng / mL as the antigen-containing solution.

[0226] 2. Test results

[0227] The results are shown in Fig.14 When AFP is added to the serum of healthy subjects, as in the case of adding to the buffer, in the sandwich ELISA performed according to the present invention, the enzyme treatment of AFP specifically increases the absorbance ( Fig.14 ). That is, it means that Endo-F3 acts on AFP even in serum.

[0228] [Test Example 10] Study on pH conditions for enzyme treatment

[0229] 1. Test methods

[0230] So far, 0.1 M MES buffer (pH 5.5) has been mainly used as a buffer, and whether other buffers (pH) can be used is investigated. The test method is the same as [Test Example 7]. The buffers used in the enzyme treatment are shown in Table 1.

[0231] [Table 1]

[0232]

[0233] After the enzyme treatment, the sample was diluted with BSA-PBST to a concentration of 100 ng / mL of AFP and used for sandwich ELISA.

[0234] 2. Test results

[0235] The results are shown in Fig.15 In the range of about pH 0.5 to about pH 12.5, the absorbance becomes above 0.1, and it can be said that a reaction has occurred ( Fig.15 ). This indicates that the pH range of a common buffer used in molecular biology is fully satisfied and the enzyme used in the present invention can be used regardless of the pH.

[0236] [Test Example 11] Study on on-board enzyme treatment

[0237] Until now, AFP has been diluted with BSA-PBST after enzyme treatment and then measured by sandwich ELISA. Therefore, it was investigated whether the enzyme treatment of AFP can be performed simultaneously with the antigen-antibody reaction in sandwich ELISA.

[0238] 1. Test methods

[0239] Prior to the preparation of the plate, the same procedures as in [Test Example 7] were followed.

[0240] (1) On-board enzyme treatment

[0241] Endo-F3 was diluted to 0.33 mg / mL with 0.1 M MES buffer (pH 5.5) to prepare an enzyme-containing solution. In addition, the HepG2 culture supernatant containing AFP was diluted stepwise with 0.1 M MES buffer (pH 5.5) to prepare an antigen-containing solution.

[0242] After removing the liquid from each well of the ELISA plate, the enzyme-containing liquid was added to each well with 30 μL. Then, 30 μL of antigen-containing liquid was added to each well and reacted at 37 ° C for 20 minutes. After washing each well with PBST 400 μL 3 times, 50 μL of HRP-labeled S20206R antibody diluted 1500 times with BSA-PBST was distributed to each well of the plate and left to stand at room temperature for 15 minutes. After washing each well with PBST 400 μL 3 times, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

[0243] 2. Test results

[0244] The results are shown in Fig.16 Similar to the results so far, when the enzyme was added, the absorbance increased in a concentration-dependent manner ( Fig.16 ). In this assay system, the solid-phase antibody reacts with the antigen, and the antigen reacts with the enzyme at the same time. That is, it is shown that the antigen-antibody reaction and the enzyme treatment reaction of the antigen can be carried out simultaneously.

[0245] Although the present invention is described using the above specific examples, the scope of the present invention is not limited to the specific examples. It is understood that those skilled in the art can adopt various other configurations without departing from the gist of the present invention.

Claims

1. A method for treating a glycoprotein, wherein the method comprises: The method comprises: The process of cleaving sugar chains by sugar chain-specific sugar chain cleaving enzymes. The sugar chain-specific sugar chain cleaving enzyme is Endo-F2 or Endo-F3, The glycoprotein is AFP-L3.

2. The method for treating glycoprotein according to claim 1, wherein: The step of cleaving the sugar chain is a step of making the epitope inherent in the sugar chain in a state that can be recognized by the selective binding substance through the cleavage.

3. A glycoprotein treated by the method for treating a glycoprotein according to any one of claims 1 to 2.

4. A selective binding substance which binds to a glycoprotein treated by the antigen treatment method according to any one of claims 1 to 2 and does not bind to the same glycoprotein which has not been treated. The selective binding substance is the monoclonal antibody S20205R or S20206R produced by the hybridoma cell with the deposit number NITE BP-02926 or the hybridoma cell with the deposit number NITE BP-02927. The selective binding substance according to claim 4 , which is an antibody or an antigen-binding fragment of an antibody.

6. A kit for use in a method for detecting a glycoprotein using a selective binding substance, wherein: The selective binding substance is the monoclonal antibody S20205R or S20206R produced by the hybridoma cell with the deposit number NITE BP-02926 or the hybridoma cell with the deposit number NITE BP-02927, The kit contains the following two reagents in the same composition: Reagents for treating glycoproteins with a sugar chain-specific sugar chain cleaving enzyme; and a reagent for obtaining a reaction product of the glycoprotein and a selective binding substance, The sugar chain-specific sugar chain cleaving enzyme is Endo-F2 or Endo-F3, The glycoprotein is AFP-L3.

7. A hybridoma cell, which is the hybridoma cell with the deposit number NITE BP-02926 or the hybridoma cell with the deposit number NITE BP-02927.

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