Reagents containing antibodies with partial deletion of the Fc region

Abstract

The present invention provides a reagent that contains an antibody in which a portion of CH3 or CH2 in the Fc region has been deleted.

Description

[Technical field]

[0001] The present invention broadly relates to reagents and the like comprising antibodies in which part of CH3 or CH2 in the Fc region is deleted. [Background technology]

[0002] In the field of immunological diagnostics, antigen-antibody reactions are used to detect and quantify target substances in samples. Immune reactions that use antigen-antibody reactions can be affected by a wide variety of substances other than the target substance present in the sample, resulting in non-specific reactions. If non-specific reactions occur, the diagnostic result may be false positive or false negative.

[0003] Conventionally, a method for reducing non-specific reactions has been known to involve removing the entire Fc region of an antibody (Patent Document 1). However, when antibody fragments such as F(ab) and F(ab')2 are bound to a carrier such as a microplate or latex, there is a problem in that the binding efficiency to the carrier and the binding activity to the antigen are lower than those of IgG. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Unexamined Patent Publication No. 54-119292 Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above circumstances, an object of the present invention is to provide a reagent or the like containing an antibody capable of reducing non-specific reactions. [Means for solving the problem]

[0006] The present inventors discovered that non-specific reactions in antigen-antibody reactions can be reduced simply by deleting a portion of the Fc region of an antibody, and thus completed the present invention.

[0007] That is, the present application includes the following inventions. [1] A reagent comprising an antibody lacking a portion of CH3 or CH2 in the Fc region. [2] The reagent described in [1], in which all of CH3 or CH2 is missing. [3] The reagent described in [1], wherein the entire CH2 is deleted and CH3 is directly or indirectly bound to the hinge region of the antibody. [4] The reagent according to any one of [1] to [3], wherein the antibody is an IgG. [5] The reagent according to any one of [1] to [4], wherein non-specific reactions of an antibody are suppressed by deleting a portion of CH3 or CH2. [6] The reagent described in [5], wherein the non-specific reaction is caused by a non-specific reaction factor that binds to the Fc region. [7] The reagent described in [6], wherein the non-specific reaction factor is complement C1q, Fcγ receptor or rheumatoid factor in the sample. [8] The reagent according to any one of [1] to [7], wherein the antigen-antibody reaction between the antibody and the antigen to be diagnosed is equal to or greater than that of an antibody that does not lack the Fc domain. [9] The reagent according to any one of [1] to [8], wherein the antibody is derived from a mouse, rabbit, rat, guinea pig, hamster, goat, sheep or camel.

[10] The reagent according to any one of [1] to [9], wherein the antibody is a human antibody, a humanized antibody, or a human chimeric antibody.

[11] The reagent according to any one of [1] to [9], wherein the antibody is a non-human chimeric antibody.

[12] The reagent according to any one of [1] to

[11] for diagnosis.

[13] A reagent according to any one of [1] to

[11] for research purposes.

[14] A kit comprising the reagent according to any one of [1 to

[11] .

[15] A kit comprising the reagent according to any one of [1] to

[11] and a blocking agent.

[16] The kit according to

[14] or

[15] , further comprising a carrier.

[17] The kit according to

[16] , wherein the antibody is bound to a carrier surface.

[18] The kit according to

[16] or

[17] , wherein the carrier is a microplate or latex.

[19] The kit according to any one of

[16] to

[18] , wherein the antibody is immobilized on a carrier via a linker.

[20] A method for suppressing non-specific antigen-antibody reactions using an antibody lacking part of CH3 or CH2 in the Fc region. Effect of the Invention

[0008] According to the present invention, it is possible to provide a reagent capable of suppressing non-specific reactions by simply deleting a portion of the Fc region of an antibody. Furthermore, an antibody deleting a portion of the Fc region can have improved carrier binding efficiency and antigen binding activity compared to F(ab')2. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of each ΔCH3 used in the examples. [Diagram 2] FIG. 2 is a schematic diagram of each ΔCH2 used in the examples. [Diagram 3] FIG. 3 shows the results of binding of the ΔCH3 antibody to FcγRI. [Figure 4] FIG. 4 shows the results of binding of the ΔCH2 antibody to FcγRI. [Diagram 5] FIG. 5 shows the results of binding of the ΔCH3 antibody to FcγRII. [Figure 6] FIG. 6 shows the results of binding of the ΔCH2 antibody to FcγRII. [Figure 7] FIG. 7 shows the results of binding of the ΔCH3 antibody to C1q. [Figure 8] FIG. 8 shows the results of binding of the ΔCH2 antibody to C1q. [Figure 9] FIG. 9 shows the binding results of full-length antibodies against Rf. [Figure 10] FIG. 10 shows the binding results of the ΔCH3 antibody to Rf. [Figure 11] FIG. 11 shows the results of binding of the ΔCH2 antibody to Rf. [Figure 12] Figure 12 shows the results of measuring the IgE concentration in human samples. The black circles indicate the measured values ​​for full-length antibodies, and the white circles indicate the measured values ​​for CH3-deficient antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] (reagent) In a first embodiment, the present invention provides a reagent comprising an antibody lacking a portion of CH3 or CH2 in the Fc region.

[0011] An antibody has a structure in which a total of four polypeptide chains, consisting of two identical heavy chains and two identical light chains, are linked by disulfide bonds in a Y-shaped configuration. As used herein, "antibody" refers to a natural or wholly or partially synthetically produced artificial immunoglobulin or fragment thereof. The antibody may be of any isotype, IgG, IgM, IgA, IgE, or IgD. Nine classes (isotypes) of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. It is preferable to delete IgG antibodies. The definition of IgG antibodies in this specification is broad, and includes heavy chain antibodies (HCAbs) consisting only of heavy chains, which are found in camelids such as llamas and alpacas.

[0012] The antibody may be of any origin, including any mammal, such as a human, or a non-human animal, such as a mouse, rabbit, rat, guinea pig, hamster, goat, sheep, or camel. Not only human antibodies, but also recombinant antibodies, such as humanized antibodies and chimeric antibodies, may be used. The antibody may be a polyclonal antibody or a monoclonal antibody.

[0013] Antibodies are largely divided into the Fab region, which is involved in antigen binding, and the Fc region, which is involved in non-specific reactions, etc. When IgG is digested with papain, two Fab fragments and one Fc fragment are obtained. The Fc region is further divided into a part of the hinge region, and, in order of proximity to the hinge region, the CH2 region (also simply called "CH2") and the CH3 region (also simply called "CH3").

[0014] As used herein, "a part of CH3 or CH2 in the Fc region" means at least a part of either the CH2 region or the CH3 region. Antibodies lacking a part of CH3 or CH2 in the Fc region (hereinafter collectively referred to as "Fc-deficient antibodies", or "CH2-deficient antibodies" or "CH3-deficient antibodies" depending on the region deleted) do not lack both the entire CH2 region and the entire CH3 region. Therefore, Fc-deficient antibodies do not include F(ab) or F(ab')2.

[0015] Those skilled in the art can produce the desired Fc-deficient antibody by known techniques, such as genetic recombination techniques or denaturation by physical or chemical techniques. For example, as described in Japanese Patent No. 5015012, the gene of the desired antibody may be expressed in a plant body, plant tissue, or plant cell. Alternatively, the defective antibody may be produced directly, or a full-length antibody may be produced first and then the defective antibody may be obtained from the full-length antibody. For example, a part of the Fc region may be chemically or physically denatured by shifting the pH of the buffer in which the full-length antibody is dissolved to an acidic or alkaline pH or by heating, and the resulting antibody may be purified through a column or the like to obtain an Fc-deficient antibody.

[0016] It is preferable that either the CH2 or CH3 region is substantially entirely deleted. When CH2 is deleted, CH3 is directly bound to the hinge region or indirectly bound via a linker or the like. A known linker can be used. For example, an amino acid sequence of about 5 to 20 residues may be interposed between the hinge region and CH3. An example of such an amino acid sequence is a peptide (GS linker) consisting of four consecutive glycines and one serine. The same linker may be repeated. For example, multiple GS linkers, for example 2 to 5, preferably 4, linked together may be placed between the hinge region and CH3, thereby increasing the expression efficiency of the antibody.

[0017] Furthermore, as long as the desired effect is achieved, the antibody may have one or more, for example 1 to 20, preferably several, for example 1 to 10, for example 1 to 5 amino acids deleted or substituted in the amino acid sequence, or one or more, for example 1 to 20, preferably several, for example 1 to 10, for example 1 to 5 other amino acids added. For example, one or more amino acids may be inserted in the cleavage region (on the C-terminal side of CH2 when CH3 is deleted, or between the hinge region and CH3 when CH2 is deleted), or one or more amino acids may be deleted from the cleavage region. The type of amino acid to be added is not particularly limited, but an amino acid located on the N-terminal side of the deletion site can be selected. Mutations such as deletions are preferably performed within a range that allows the three-dimensional structure of the antibody to be maintained.

[0018] The Fc-deficient antibody may be modified with a label or the like required for antibody detection. Examples of the label include fluorescent substances, fluorescent proteins, enzymes, or labels with biotin or streptavidin. The Fc-deficient antibody may also be modified in other required functions.

[0019] As used herein, the term "reagent" broadly refers to a reagent used in an immunoassay for detecting and quantifying a biologically relevant substance (hereinafter also referred to as an "analyte") to which an Fc-deficient antibody binds, and includes diagnostic agents and test agents such as in vitro diagnostic pharmaceuticals. The reagent may contain a buffer or the like for stably retaining the antibody. Other components can be appropriately determined by one skilled in the art depending on the immunoassay used.

[0020] Immunological assays include, for example, immunostaining methods (including fluorescent antibody methods, enzyme antibody methods, heavy metal-labeled antibody methods, and radioisotope-labeled antibody methods), methods that combine electrophoretic separation with detection methods using fluorescence, enzymes, radioisotopes, etc. (including Western blotting and fluorescent two-dimensional electrophoresis), enzyme-linked immunosorbent assay (ELISA), dot blotting, latex agglutination (LA), and immunochromatography.

[0021] The substances to be detected and quantified by the reagent are not limited to antigens such as proteins and polysaccharides, haptens including low molecular weight physiologically active substances such as monosaccharides, oligosaccharides, amino acids, peptides, and substances having a steroid skeleton, as well as pathogens such as bacteria, viruses, and parasites that are composed of these substances, as long as they are substances to which Fc-deficient antibodies bind. For example, when the substance to be measured is an antigen, protein markers such as CRP (C-reactive protein), prostate specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, and creatinine, immunoglobulins such as IgG, IgA, and IgM, various tumor markers, lipoproteins such as LDL, HDL, and TG, viral antigens such as influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, and EBV, Examples of the antigens include, but are not limited to, bacterial antigens such as Chlamydia trachomatis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthracis, and MRSA, toxins produced by bacteria, etc., mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroids such as steroid hormones, bioactive amines such as epinephrine and morphine, vitamins such as vitamin B, prostaglandins, antibiotics such as tetracycline, agricultural chemicals, and environmental hormones. Preferred examples include antigens such as CRP, prostate specific antigen, ferritin, β-2 microglobulin, and hemoglobin.

[0022] When the substance to be measured is an antibody, examples of the antibody include antibodies that specifically react with antigens such as the above-mentioned protein markers, various tumor markers, lipoproteins, viral antigens, bacterial antigens, toxins produced by bacteria, etc., peptide hormones, steroids, physiologically active amines, vitamins, antibiotics, pesticides, and environmental hormones.

[0023] The reagent is added to a specimen from a subject for which it is necessary to confirm the presence or absence of a substance to be measured, etc. The specimen is not particularly limited as long as it contains the substance to be measured, and examples thereof include body fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, and swabs, or dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, and dilutions thereof being preferred.

[0024] (kit) In a second embodiment, the present invention provides a kit comprising a reagent.

[0025] The kit can be used for diagnosis, research, and the like. The kit may contain not only an antibody as a reagent, but also a carrier for immobilizing an Fc-deficient antibody and a secondary antibody depending on the purpose of use. Such a carrier is preferably an insoluble carrier, and examples thereof include latex particles such as polyethylene and polystyrene, alumina particles, silica particles, gold colloids, and magnetic particles. More specifically, a microplate can be used as the insoluble carrier in the case of the ELISA method (direct method, indirect method, sandwich method, etc.), and latex can be used as the insoluble carrier in the case of the latex agglutination method. From the viewpoint of preventing non-specific reactions between antibodies and antigens, it is preferable to include a blocking agent in the kit.

[0026] The Fc-deficient antibody may be directly immobilized on the carrier by physical adsorption or the like, or may be indirectly immobilized via chemical crosslinking, for example via a linker.

[0027] A kit used in a direct method includes, for example, an enzyme-labeled antibody that binds to the antigen, a substrate for the enzyme reaction, and a standard antigen for use in preparing a calibration curve or in a control experiment, as reagents including an Fc-deficient antibody.

[0028] A kit used in the indirect method includes, for example, a primary antibody that binds to an antigen, a secondary antibody that binds to the primary antibody, a substrate for an enzyme reaction, a standard antigen for use in preparing a calibration curve or in a control experiment, etc. as a reagent containing an Fc-deficient antibody. The secondary antibody may be labeled for detection.

[0029] A kit used in the sandwich method includes, for example, an antibody bound to a solid phase as a reagent containing an Fc-deficient antibody, an antibody for enzyme labeling, a substrate for the enzyme reaction, a standard antigen for use in preparing a calibration curve or in a control experiment, etc. The antibody for enzyme labeling may be an Fc-deficient antibody.

[0030] A kit for use in the latex agglutination assay includes, for example, a reagent containing the Fc-deficient antibody, a detection antibody to be bound, a standard antigen for use in preparing a calibration curve or in control experiments, and a blocking agent to prevent non-specific reactions between the antibody and the antigen.

[0031] (Non-specific reaction inhibition method) In a third embodiment, the present invention provides a method for suppressing non-specific antigen-antibody reactions using an Fc-deficient antibody.

[0032] By deleting a portion of the Fc region, it is possible to suppress nonspecific reactions that occur when nonspecific reaction factors such as complement (C1q), Fc receptors, rheumatoid factors, and heterophilic antibodies bind to the Fc region, i.e., CH2 and / or CH3. Nonspecific reactions can also occur when substances unrelated to the desired antigen-antibody reaction form antigen-antibody reactants or exhibit cross-reactions, so it is preferable to use the method in combination with a method for suppressing nonspecific reactions known to those skilled in the art.

[0033] Here, Eur J Biochem., 267(24):7246-57 examines the effect of Fcγ receptor (FcγR) binding on scFv-Fc and its CH3-deleted scFv-hCH2. From the results in Fig. 7, it is considered that scFv-hCH2 has a higher non-specific reaction, so it is considered preferable not to delete CH3 for single-chain antibodies.

[0034] The antigen-binding activity of an Fc-deficient antibody, i.e., the antigen-antibody reaction with the antigen of interest and the binding efficiency to a carrier, may be increased compared to that of an antibody having a non-Fc region.

[0035] The method for inhibiting a non-specific reaction comprises the step of contacting an Fc-deficient antibody with a sample, and may comprise any step before or after the contacting step.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0037] 1. Vector construction and protein expression As antibodies to delete the Fc region, we selected an anti-H5N1 hemagglutinin antibody (Xueyong Zhu et al., "A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A / Goose / Guangdong / 1 / 96 hemagglutinin", Journal of virology.2013;87(23):12619-35. PMID:24049169) and an anti-butyrylcholinesterase antibody (Peng H.et al., "Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences", Chemico-Biological Interactions.2015;240: 336-345. DOI: 10.1016 / j.cbi.2015.08.024;PMID:26343001).

[0038] Anti-H5N1 hemagglutinin antibody Light chain DIVLTQSPGSLTVSLGQRATISCRASESVDNFGKSFMHWYQQKPGQSPKLLIYRASNREFGIPARFNGSGSGTDFALTINPVEADDVATYFCQQSNEDPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 1: GenBank, KF499999) Heavy chain EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGT TVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 2: GenBank, KF500000)

[0039] Anti-butylcholinesterase antibody Light chain DILLTQSPAILSVSPGERVRLSCRASQSCGTSIYWYQQRTNGSPRLLIMYASEPFSGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTRLEIKRADAAPT VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 3: GenBank, KT189148) Heavy chain QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVP EVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 4: GenBank, KT189147)

[0040] Following the method of Example 4 of Japanese Patent No. 5015012, the light chain and heavy chain (sequences with partial deletions) of each IgG were expressed using separate vectors.

[0041] 1.3.ΔCH3 ΔCH3 represents an antibody lacking CH3 of the heavy chain of each antibody. CH3 is the region from 341 to 447 in the EU numbering notation. Therefore, the corresponding construct can be said to be a protein composed of a constant region (Fc region) consisting of amino acids from 118 to 340 in the EU numbering notation. To reflect these characteristics, ΔCH3 is also called Δ341-447 or Fc118-340. Hereafter, the other deleted antibodies are named according to the same rule. In addition, a GS linker (GGGGS) and a His tag (HHHHHH) were linked to the C-terminus of ΔCH3.

[0042] 1.3.1.ΔCH3=Δ341-447=Fc118-340: 1.3.1.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGGGGSHHHHHH (SEQ ID NO: 5)

[0043] 1.3.1.2. Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGGGGSHHHHHH (SEQ ID NO: 6)

[0044] To confirm the effect of the deletion, four different types of ΔCH3 were constructed, one with five amino acids deleted or added from the cleavage site as shown in Fig. 1, and one with 10 amino acids deleted or added. The specific sequences are shown below.

[0045] 1.3.2.Δ331-447=Fc118-330(-10 amino acids): 1.3.2.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAGGGGSHHHHHH (SEQ ID NO: 7)

[0046] 1.3.2.2. Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAGGGGSHHHHHH (SEQ ID NO: 8)

[0047] 1.3.3.Δ336-447=Fc118-335(-5 amino acids): 1.3.3.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTGGGGSHHHHHH (SEQ ID NO: 9)

[0048] 1.3.3.2. Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTGGGGSHHHHHH (SEQ ID NO: 10)

[0049] 1.3.4.Δ346-447=Fc118-345(+5 amino acids): 1.3.4.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAGGGGSHHHHHH (SEQ ID NO: 11)

[0050] 1.3.4.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAGGGGSHHHHHH (SEQ ID NO: 12)

[0051] 1.3.5.Δ351-447=Fc118-350(+10 amino acids): 1.3.5.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTGGGGSHHHHH (SEQ ID NO: 13)

[0052] 1.3.5.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTF (SEQ ID NO: 14)

[0053] 1.4.ΔCH2 ΔCH2 represents an anti-IgE antibody heavy chain in which CH2 has been deleted and the front and rear hinge regions and CH3 have been linked. CH2 is the region from 231 to 340 in the EU numbering notation. Therefore, the construct can be said to be a protein composed of amino acids from 118 to 230 in the EU numbering notation and a constant region (Fc region) that links amino acids from 341 to 447.

[0054] Considering some effect of CH2 deletion, we also prepared a peptide with a GS linker (GGGGS) inserted into the linking portion. GS linkers were used that were repeated one to four times. The specific amino acid sequences were: 1) no linker, 2) GGGGS (SEQ ID NO: 15), 3) GGGGSGGGGS (SEQ ID NO: 16), 4) GGGGSGGGGSGGGGS (SEQ ID NO: 17), and 5) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). To obtain data such as evaluation of non-specific reactions, the GS linker in 5) was repeated four times. The specific sequences are shown below.

[0055] 1.4.1.ΔCH2=Δ231-340=Fc118-230+341-447(no linker): 1.4.1.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 19)

[0056] 1.4.1.2. Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 20)

[0057] 1.4.2.ΔCH2=Δ231-340=Fc118-230+341-447(1×GS linker): 1.4.2.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 21)

[0058] 1.4.2.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 22)

[0059] 1.4.3.ΔCH2=Δ231-340=Fc118-230+341-447(2×GS linker): 1.4.3.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 23)

[0060] 1.4.3.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 24)

[0061] 1.4.4.ΔCH2=Δ231-340=Fc118-230+341-447(3×GS linker): 1.4.4.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 25)

[0062] 1.4.4.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 26)

[0063] 1.4.5.ΔCH2=Δ231-340=Fc118-230+341-447(4×GS linker): 1.4.5.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 27)

[0064] 1.4.5.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (SEQ ID NO: 28)

[0065] To confirm the effect of the deletion, four different types of ΔCH2 were created, one in which 5 amino acids were deleted or added from the C-terminal cleavage site as shown in Figure 2, and one in which 10 amino acids were deleted or added. The specific sequences are shown below.

[0066] 1.4.6.ΔCH2=Δ231-330=Fc118-230+331-447(4×GS linker)(−10 amino acids): 1.4.6.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 29)

[0067] Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 30)

[0068] 1.4.7.ΔCH2=Δ231-335=Fc118-230+336-447(4×GS linker)(−5 amino acids): 1.4.7.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 31)

[0069] 1.4.7.2. Anti-Butylcholinesterase Antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 32)

[0070] 1.4.8.ΔCH2=Δ231-345=Fc118-230+346-447(4×GS linker)(+5 amino acids): 1.4.8.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 33)

[0071] Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGGSGGGGSPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 34)

[0072] 1.4.9.ΔCH2=Δ231-350=Fc118-230+351-447(4×GS linker)(+10 amino acids): 1.4.9.1. Anti-H5N1 hemagglutinin antibodies EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATTLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 35)

[0073] Anti-butylcholinesterase antibodies QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 36)

[0074] The IgG light chain was cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics), and each heavy chain (including CH3 deletion or CH2 deletion) was cloned into a potato virus X (PVX) vector (Icon Genetics). After introducing these vectors into separate Agrobacterium for transformation and cultivation, the mixture of culture fluids was infiltrated into Nicotiana benthamiana leaves and harvested about one week later. The harvested leaves (infected leaves) were frozen.

[0075] 2. Purification 500 g of frozen infected leaves were weighed and crushed in a cutter mixer. 250 mL of extraction solution (100 mM Tris, 250 mM NaCl, 40 mM sodium ascorbate) was added to the crushed material, and the infected leaves were repeatedly crushed.

[0076] The crushed infected leaves collected from the cutter mixer were mixed with the crushed infected leaves obtained by washing the inside of the mixer with 250 mL of new extraction solution. A portion was sampled and centrifuged at 12,000 × g for 5 minutes to collect the supernatant (extraction process fraction).

[0077] After adjusting the pH of the extraction fraction, it was filtered through a 0.45 μm filter and then a 0.22 μm filter. The filtrate was subjected to nickel column chromatography (GE Healthcare, HisPrep FF 16 / 10 column, catalog number 28936551) and HAP column chromatography to remove plant-derived impurities and antibody-derived impurities.

[0078] 3. Expression test The infected leaves were added with three times the volume of extraction buffer (100 mM Tris, 250 mM NaCl, 5 mM EDTA) and crushed in a bead mill. The leaves were then left on ice for 30 minutes and centrifuged (8000xg, 4°C, 10 minutes) to collect the supernatant, which was then analyzed by SDS-PAGE and Western blotting.

[0079] The expression of the antibodies described in Tables 1 and 2 was evaluated by Western blotting using HRP-labeled anti-His tag antibody. The expression of all antibodies was confirmed.

Table 1

[0080]

Table 2

[0081] The expression of the camel ΔCH3 antibody was also confirmed.

[0082] 4. Non-specific evaluation The interaction between the purified antibody and non-specific factors was evaluated by surface plasmon resonance (SPR). The BIAcore X-100 (manufactured by Biacore) was used as the instrument. As non-specific factors, Fc receptors (Fc gamma receptor I, Fc gamma receptor II), C1q, and rheumatoid factor were used.

[0083] <Fc receptor> Biotin-labeled human-derived FcγRI or FcγRII (both are purified products, manufactured by Sino Biological) was captured on Sensor Chip CAP (manufactured by GE Healthcare), and the increase in the signal (resonance unit: RU) associated with the interaction with the antibody was observed. The running buffer used was 0.01M HEPES pH7.4, 0.15M NaCl, 3mM EDTA, 0.05% Tween20, and the measurement was performed at 25°C with a flow rate of 30 μL / min. All measurements were carried out with n = 3 (the same applies hereinafter). The binding results of the antibody to FcγRI are shown in Figures 3 and 4, and the binding results of the antibody to FcγRII are shown in Figures 5 and 6.

[0084] When 1 μM of the antibody was added for 1 minute, the full-length antibody showed an increase in signal, indicating an interaction, whereas ΔCH3 showed no change in signal, indicating no interaction (Figures 3 and 5). Similarly, when 0.25 μM of the ΔCH2 antibody was added for 1 minute, the full-length antibody showed an interaction, whereas ΔCH2 antibody showed no change in signal, indicating no interaction (Figures 4 and 6).

[0085] <c1q> The antibody was captured with a Sensor chip Protein L (GE Healthcare), and the increase in signal (resonance unit: RU) due to interaction with human C1q (Abcam ab96363, purified product) was observed. The running buffer was 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% Tween 20, and measurements were performed at a flow rate of 30μL / min at 25℃. The results of antibody binding to C1q are shown in Figures 7 and 8.

[0086] Human C1q diluted 100-fold with running buffer was added for 1 minute, and it was confirmed that neither ΔCH3 nor ΔCH2 had any interaction with C1q.

[0087] <rf> The antibody was bound to a Sensor chip CM5 (GE Healthcare) by the amine coupling method, and the increase in signal (resonance unit: RU) due to interaction with Rf-positive human serum or plasma was observed. The running buffer was 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% Tween 20, and measurements were performed at a flow rate of 30μL / min at 25℃.

[0088] We evaluated 10 types of human sera or plasma that were Rf positive and known to contain factors that interact with full-length antibodies. When serum or plasma diluted 30-fold with running buffer was added for 1 minute, an increase in signal was observed with the full-length antibody, indicating interaction (Figure 9), but no interaction was observed with either ΔCH3 or ΔCH2 (Figures 10 and 11).

[0089] 5.Latex agglutination test The binding efficiency of the obtained antibody to latex was evaluated according to the following procedure.

[0090] (1) Preparation of reagents Using an antibody against IgE, a measuring reagent for the immune agglutination method was prepared as follows. i) Sensitized particles having an antibody supported thereon at 0.1 mg per 1 mL of polystyrene latex suspension having an average particle size of 200 nm were suspended in a buffer solution (glycine, pH 7.3) to a concentration of 0.1%, to prepare a latex suspension. ii) A buffer solution (glycine, pH 8.3) was prepared.

[0091] (2) Measurement using an automatic analyzer The automated analyzer was a Hitachi 7180-type automated analyzer, and the end-point method was used to perform automatic measurements. Using the reagent prepared in (1) i) above, measurements were performed on 4 human specimens known to show false positive reactions due to non-specific reactions and 15 specimens that did not show non-specific reactions. 140 μL of the buffer solution prepared in (1) ii) above was added to 3.5 μL of specimen solution, and the mixture was stirred and mixed at 37°C. After leaving it for 5 minutes, 70 μL of latex suspension was added, and the mixture was further stirred and mixed at 37°C. The agglutination reaction over about 5 minutes was measured as the amount of absorbance change, and the IgE concentration of each specimen was calculated from the calibration curve.

[0092] (3) Comparison of false positive sample measurements The results of the measurement in (2) above are shown in FIG. 12 and Table 3. [Table 3]

[0093] If there were no false positives or false negatives, the IgE concentration in the sample and the measured value should be equivalent, but clear false positives were observed when using the reagent with full-length antibodies for samples No. 1, 5, 9, and 18. On the other hand, the reagent with CH3-deficient antibodies generally showed measured values ​​equivalent to the IgE concentration, and no clear false positives were confirmed. [Industrial Applicability]

[0094] According to the present invention, it is possible to provide a reagent capable of suppressing non-specific reactions simply by deleting a portion of the Fc region of an antibody.< / rf>

Claims

1. A reagent for suppressing non-specific antigen-antibody reactions, comprising an antibody lacking at least a portion of CH3 in the Fc region.

2. The reagent of claim 1 , wherein all of CH3 is deleted.

3. The reagent according to claim 1 or 2, wherein the antibody is an IgG.

4. The reagent according to any one of claims 1 to 3, wherein non-specific reactions of the antibody are suppressed by the deletion of CH3.

5. The reagent according to claim 4, wherein the non-specific reaction is caused by a non-specific reaction factor that binds to the Fc region.

6. The reagent according to claim 5, wherein the non-specific reaction factor is complement C1q, an Fcγ receptor or a rheumatoid factor in a specimen.

7. The reagent according to any one of claims 1 to 6, wherein the antigen-antibody reaction between the antibody and an antigen to be diagnosed is equal to or greater than that of an antibody that does not have an Fc region deleted.

8. The reagent according to any one of claims 1 to 7, wherein the antibody is derived from a mouse, rabbit, rat, guinea pig, hamster, goat, sheep or camel.

9. The reagent according to any one of claims 1 to 8, wherein the antibody is a human antibody, a humanized antibody, or a human chimeric antibody.

10. The reagent according to any one of claims 1 to 8, wherein the antibody is a non-human chimeric antibody.

11. The reagent according to any one of claims 1 to 10 for diagnosis.

12. A reagent according to any one of claims 1 to 10 for research purposes.

13. A kit comprising the reagent according to any one of claims 1 to 10.

14. A kit comprising the reagent according to any one of claims 1 to 10 and a blocking agent.

15. 15. The kit of claim 13 or 14, comprising a carrier.

16. The kit of claim 15, wherein the antibody is bound to a carrier surface.

17. 17. The kit according to claim 15 or 16, wherein the carrier is a microplate or latex.

18. The kit according to any one of claims 15 to 17, wherein the antibody is immobilized on a carrier via a linker.

19. A method for suppressing non-specific antigen-antibody reactions using an antibody lacking a portion of CH3 in the Fc region.

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