Reagents for antibodies containing a portion of the Fc region deleted

Abstract

The present invention provides an agent comprising an antibody with a portion of CH3 or CH2 in the Fc region missing.

Description

Technical Field

[0001] This invention broadly relates to reagents containing antibodies that have partially or completely missing CH3 or CH2 in the Fc region. Background Technology

[0002] In the field of diagnostics using immunological methods, antibody-based antigen-antibody reactions are used to detect and quantify target substances in a sample. However, these immune reactions are affected by a wide variety of substances present in the sample, other than the target substance, and sometimes nonspecific reactions occur. If nonspecific reactions occur, there is a concern about false positives and false negatives in the diagnosis.

[0003] As a means to reduce nonspecific reactions, methods such as removing the entire Fc region of the antibody have been known (Patent Document 1). However, when antibody fragments such as F(ab) and F(ab')2 are bound to support carriers such as microplates or latex, there is a problem that the binding efficiency with the support carrier and the binding activity with the antigen are reduced compared to IgG.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 54-119292 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] In view of the above facts, the object of the present invention is to provide reagents, etc., containing antibodies that can reduce nonspecific reactions.

[0009] Methods for solving problems

[0010] The inventors of this application discovered that by deleting only a portion of the Fc region in an antibody, the non-specific reaction in the antigen-antibody reaction is reduced, thus completing this invention.

[0011] That is, this application includes the following inventions. [1]

[0013] A reagent containing an antibody that is missing a portion of CH3 or CH2 in the Fc region. [2]

[0015] According to the reagent described in [1], all portions of CH3 or CH2 are missing. [3]

[0017] According to the reagent described in [1], all parts of CH2 are missing, and CH3 binds directly or indirectly to the hinge region of the antibody. [4]

[0019] The reagent according to any one of [1] to [3], wherein the antibody is IgG. [5]

[0021] The reagent according to any one of [1] to [4], wherein the nonspecific reaction of the antibody is inhibited by the deletion of a portion of CH3 or CH2. [6]

[0023] According to the reagent described in [5], the nonspecific reaction occurs through a nonspecific reaction factor bound to the Fc region. [7]

[0025] According to the reagent described in [6], the non-specific reaction factor is complement C1q, Fcγ receptor or rheumatoid factor in the sample. [8]

[0027] The reagent according to any one of [1] to [7], wherein the antigen-antibody reaction between the antibody and the antigen of the diagnostic object is equivalent to or better than the antigen-antibody reaction of the antibody in which the Fc region is not missing. [9]

[0029] The reagent according to any one of [1] to [8], wherein the antibody is derived from mouse, rabbit, rat, guinea pig, hamster, goat, sheep or camel.

[10]

[0031] 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]

[0033] The reagent according to any one of [1] to [9], wherein the antibody is a non-human chimeric antibody.

[12]

[0035] The reagent according to any one of [1] to

[11] is used for diagnosis.

[13]

[0037] The reagent according to any one of [1] to

[11] is used for research.

[14]

[0039] A kit comprising any one of the reagents described in [1] to

[11] .

[15]

[0041] A kit comprising a blocking agent and any one of the reagents described in [1] to

[11] .

[16]

[0043] The kit described in

[14] or

[15] contains a support carrier.

[17]

[0045] According to the kit described in

[16] , the antibody binds to the surface of the carrier.

[18]

[0047] According to the kit described in

[16] or

[17] , the support is a microplate or latex.

[19]

[0049] The kit according to any one of

[16] to

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

[20]

[0051] A method for inhibiting a nonspecific reaction of antigen-antibody reaction, wherein an antibody with a portion of CH3 or CH2 missing in the Fc region is used.

[0052] Invention Effects

[0053] According to the present invention, a reagent can be provided that inhibits nonspecific reactions by deleting only a portion of the Fc region in an antibody. Furthermore, antibodies with a partial deletion of the Fc region exhibit improved binding efficiency with the support and enhanced antigen-binding activity compared to F(ab')2. Attached Figure Description

[0054] [ Figure 1 ] Figure 1 This is a schematic diagram of each ΔCH3 used in the embodiments.

[0055] [ Figure 2 ] Figure 2 This is a schematic diagram of each ΔCH2 used in the embodiment.

[0056] [ Figure 3 ] Figure 3 The binding results of the ΔCH3 antibody against FcγRI are shown.

[0057] [ Figure 4 ] Figure 4 The binding results of the ΔCH2 antibody against FcγRI are shown.

[0058] [ Figure 5 ] Figure 5 The binding results of the ΔCH3 antibody against FcγRII are shown.

[0059] [ Figure 6 ] Figure 6 The binding results of the ΔCH2 antibody against FcγRII are shown.

[0060] [ Figure 7 ] Figure 7 The binding results of the ΔCH3 antibody against C1q are shown.

[0061] [ Figure 8 ] Figure 8 The binding results of the ΔCH2 antibody against C1q are shown.

[0062] [ Figure 9 ] Figure 9 The binding results of the full-length antibody against Rf are shown.

[0063] [ Figure 10 ] Figure 10 The binding results of the ΔCH3 antibody against Rf are shown.

[0064] [ Figure 11 ] Figure 11 The binding results of the ΔCH2 antibody against Rf are shown.

[0065] [ Figure 12 ] Figure 12 This shows the results of measuring the IgE concentration in human samples. Black dots represent the measured values ​​of full-length antibodies, and white dots represent the measured values ​​of CH3-deficient antibodies. Detailed Implementation

[0066] (Reagents)

[0067] In a first embodiment, the present invention provides a reagent containing an antibody that is partially missing from the CH3 or CH2 region of the Fc region.

[0068] Antibodies have the following structure: 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-shape. In the context of this specification, "antibody" refers to a natural antibody, or an artificial immunoglobulin or fragment thereof, manufactured wholly or partially through synthesis. Antibodies can be any isotype of IgG, IgM, IgA, IgE, and IgD. As human immunoglobulins, nine subclasses (isotypes) are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Preferably, IgG antibodies are absent. The definition of IgG antibody in this specification is broad, encompassing heavy chain antibodies (HCAbs) consisting solely of heavy chains, which can be found in camel-like animals such as llamas and alpacas.

[0069] The source of antibodies is unrestricted and can be any mammal, such as humans or non-human animals like mice, rabbits, rats, guinea pigs, hamsters, goats, sheep, and camels. Not only human antibodies can be used, but also recombinant antibodies such as humanized antibodies and chimeric antibodies. Antibodies can be polyclonal or monoclonal.

[0070] Antibodies can be broadly divided into Fab regions, which are associated with antigen binding, and Fc regions, which are associated with non-specific reactions. When IgG is broken down with papain, two Fab fragments and one Fc fragment are obtained. The Fc region can be further divided into: a portion of the hinge region, and, in order of proximity to the hinge region, the CH2 region (also simply referred to as "CH2") and the CH3 region (also simply referred to as "CH3").

[0071] As used in this specification, "a portion of CH3 or CH2 in the Fc region" means at least a portion of either the CH2 or CH3 region. Antibodies lacking a portion of CH3 or CH2 in the Fc region (hereinafter collectively referred to as "Fc-deficient antibodies," and also as "CH2-deficient antibodies" or "CH3-deficient antibodies" depending on the region missing) will not completely lack both the CH2 and CH3 regions. Therefore, Fc-deficient antibodies do not contain F(ab) or F(ab')2.

[0072] Those skilled in the art can use known methods such as gene recombination technology and physical / chemical modification to manufacture desired Fc-deficient antibodies. For example, as described in Japanese Patent No. 5015012, the gene for the desired antibody can be expressed in plant tissues or plant cells. Alternatively, the deficient antibody can be manufactured directly, or a full-length antibody can be manufactured first, and then the deficient antibody can be obtained. For example, the pH of the buffer solution containing the full-length antibody can be shifted to acidic or alkaline, or the solution can be heated, thereby chemically or physically modifying a portion of the Fc region, and then purifying it on a column or similar surface to obtain the Fc-deficient antibody.

[0073] Ideally, either region of CH2 or CH3 should be substantially completely deleted. In the case of CH2 deletion, CH3 binds directly to the hinge region or indirectly via a linker. Known linkers can be used. For example, an amino acid sequence of approximately 5 to 20 residues may exist between the hinge region and CH3. Examples of such amino acid sequences include peptides consisting of four consecutive glycine residues and one serine residue (GS linkers). The same linker can be repeated. For example, by configuring a linker consisting of multiple, for example, 2 to 5, preferably 4 GS linkers, between the hinge region and CH3, the antibody expression efficiency can be increased.

[0074] Furthermore, as long as the antibody can achieve the desired effect, one or more, for example 1 to 20, preferably several, for example 1 to 10, or for example 1 to 5 amino acids in the amino acid sequence can be deleted or substituted, or one or more, for example 1 to 20, preferably several, for example 1 to 10, or for example 1 to 5 other amino acids can be added. For example, one or more amino acids can be inserted into the cleavage region (the C-terminal side of CH2 in the case of CH3 deletion, and between the hinge region and CH3 in the case of CH2 deletion), or one or more amino acids can be deleted from the cleavage region. The types of added amino acids are not particularly limited, and amino acids located on the N-terminal side of the deletion site can be selected. Deletions and other mutations are preferably performed within a range that maintains the three-dimensional structure of the antibody.

[0075] Fc-deficient antibodies can be modified with labels necessary for antibody detection. Examples of labels include those using fluorescent substances, fluorescent proteins, enzymes, or biotin or streptavidin. Other necessary functional modifications can also be made to Fc-deficient antibodies.

[0076] In the context of this specification, "reagent" refers to a broad range of reagents used in immunological assays for the detection and quantification of biologically related substances (hereinafter also referred to as "analytes") bound by Fc-deficient antibodies, including diagnostic and testing drugs such as in vitro diagnostic reagents. Reagents may contain buffer solutions for stably maintaining antibodies. Other components may be appropriately determined by those skilled in the art based on the immunological assay used.

[0077] Examples of immunological assays include immunostaining methods (including fluorescent antibody methods, enzyme antibody methods, heavy metal labeled antibody methods, and radioisotope labeled antibody methods), methods that combine separation using electrophoresis with detection using fluorescence, enzymes, radioisotopes, etc. (including Western blotting and fluorescent two-dimensional electrophoresis), enzyme immunoassay (ELISA), dot blot, latex agglutination-turbidimetric immunoassay (LA), and immunochromatography.

[0078] As substances to be detected and quantified using reagents, there are antigens such as proteins and polysaccharides; haptens, which are low-molecular-weight physiologically active substances including monosaccharides, oligosaccharides, amino acids, peptides, and substances with steroidal backbones; and pathogens such as bacteria, viruses, and parasites composed of these substances. There are no limitations as long as the substance is bound by an Fc-deficient antibody. For example, when the substance to be detected is an antigen, examples include 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; influenza A virus, influenza B virus, RSV virus, rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBsAg, and HBeAg. Viral antigens such as CV, HIV, and EBV; bacterial antigens such as Chlamydia trachomatis, hemolytic streptococci, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Treponema pallidum, Legionella, Bacillus anthracis, and MRSA; toxins produced by bacteria; mycoplasma lipid antigens; peptide hormones such as human chorionic gonadotropin; steroid hormones such as steroids; bioactive amines such as adrenaline and morphine; vitamins such as vitamin B; prostaglandins; antibiotics such as tetracycline; pesticides; and environmental hormones, but not limited to these. Preferred examples include antigens such as CRP, prostate-specific antigen, ferritin, β-2 microglobulin, and hemoglobin.

[0079] When the substance being measured is an antibody, examples include antibodies that specifically react with the aforementioned protein markers, various tumor markers, lipoproteins, viral antigens, bacterial antigens, toxins produced by bacteria, peptide hormones, steroids, bioactive amines, vitamins, antibiotics, pesticides, environmental hormones, and other antigens.

[0080] The reagent is added to a sample from the subject to confirm the presence of the substance to be measured. The sample is not particularly limited as long as it contains the substance to be measured; examples include bodily fluids such as blood, serum, plasma, urine, feces, saliva, tissue fluid, cerebrospinal fluid, and swabs, or dilutions thereof, with blood, serum, plasma, urine, feces, cerebrospinal fluid, or dilutions thereof being preferred.

[0081] (Reagent test kit)

[0082] In a second embodiment, the present invention provides a reagent kit containing reagents.

[0083] The kit can be used for diagnostics, research, and other applications. Depending on its intended use, the kit may contain not only antibodies as reagents but also a support for immobilizing Fc-deficient antibodies and a secondary antibody. Insoluble support is preferred, and examples include latex particles from polyethylene or polystyrene, alumina particles, silica particles, colloidal gold, and magnetic particles. More specifically, microplates can be used for ELISA methods (direct, indirect, sandwich, etc.), and latex can be used as an insoluble support for latex agglutination methods. From the viewpoint of preventing non-specific reactions between antibodies and antigens, it is preferable to include a blocking agent in the kit.

[0084] Fc-deficient antibodies can be directly immobilized on a support through physical adsorption or other means, or indirectly immobilized through chemical cross-linking, such as through a linker.

[0085] The kits used in the direct method may contain, for example, enzyme-labeled antibodies that bind to the antigen as reagents containing Fc-deficient antibodies, substrates for the enzyme reaction, and standard antigens for use in the preparation of calibration curves or control experiments.

[0086] The kits used in indirect methods may contain, for example, a primary antibody that binds to the antigen as a reagent containing an Fc-deficient antibody, a secondary antibody that binds to the primary antibody, a substrate for the enzyme reaction, and a standard antigen for use in the preparation of calibration curves or control experiments. The secondary antibody may also be labeled based on the detection purpose.

[0087] The kits used in sandwich assays may contain, for example, antibodies bound to a solid phase as reagents containing Fc-deficient antibodies, enzyme-labeled antibodies, substrates for the enzyme reaction, and standard antigens for use in calibration curves or control experiments. The enzyme-labeled antibody may be an Fc-deficient antibody.

[0088] Kits used in latex agglutination assays may contain, for example, an antibody for detection that binds to an Fc-deficient antibody, a standard antigen for use in creating calibration curves or control experiments, and a blocking agent to prevent non-specific reactions between the antibody and the antigen.

[0089] (Non-specific reaction inhibition method)

[0090] In a third embodiment, the present invention provides a method for inhibiting a nonspecific antigen-antibody reaction using an Fc-deficient antibody.

[0091] By partially deleting the Fc region, nonspecific reactions that occur due to the binding of nonspecific reactive factors such as complement (C1q), Fc receptors, rheumatoid factor, and heterophile antibodies to the Fc region, i.e., CH2 and / or CH3, can be suppressed. Nonspecific reactions may also occur due to the formation of antigen-antibody reactants from other substances unrelated to the desired antigen-antibody reaction, or due to cross-reactivity; therefore, it is preferable to use this method in combination with nonspecific reaction suppression methods known to those skilled in the art.

[0092] Here, in Eur J Biochem., 267(24): 7246-57, the effects of binding to the Fcγ receptor (FcγR) on scFv-Fc and its CH3-deficient scFv-hCH2 were investigated. Based on the results in Fig. 7, it can be considered that scFv-hCH2 has a higher non-specific response; therefore, it can be considered that CH3 deletion is more preferred for single-chain antibodies.

[0093] The binding activity of Fc-deficient antibodies to antigens, i.e., antigen-antibody reactions with antigens of the diagnostic subject, and the binding efficiency with the support vector, is increased compared to antibodies without Fc region deletion.

[0094] Non-specific reaction inhibition methods include the step of contacting the sample with the Fc-deficient antibody. Any step can be included before or after the contact step.

[0095] The present invention will be described in more detail below with examples, but the present invention is not limited thereto.

[0096] Example

[0097] 1. Vector fabrication and protein expression

[0098] As antibodies for Fc region deletion, 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 / 96hemagglutinin", Journal of virology. 2013; 87(23):12619?35.PMID:24049169) and anti-butyrylcholinesterase antibody (Peng H. et al., "Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and aminoacid sequences", Chemico-Biological) were selected. Interactions.2015;240:336-345.DOI:10.1016 / j.cbi.2015.08.024;PMID:26343001).

[0099] Anti-H5N1 hemagglutinin antibody

[0100] Light chain

[0101] DIVLTQSPGSLTVSLGQRATISCRASESVDNFGKSFMHWYQQKPGQSPKLLIYRASNREFGIPARFNGSGSGTDFALTINPVEADDVATYFCQQSNEDPRTFGGGTKLEIKRADAAPTVS IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (Serial Number 1: GenBank, KF499999)

[0102] Heavy chain

[0103] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:2; GenBank, KF500000)

[0104] Anti-butyrylcholinesterase antibody

[0105] Light chain

[0106] DILLTQSPAILSVSPGERVRLSCRASQSCGTSIYWYQQRTNGSPRLLIMYASEPFSGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:3; GenBank, KT189148)

[0107] Heavy chain

[0108] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSA KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPP KEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (Serial number 4: GenBank, KT189147)

[0109] Following the method of Example 4 in Japanese Patent No. 5015012, the light chain and heavy chain (partially missing sequences) of each IgG were expressed using different vectors.

[0110] 1.3.ΔCH3

[0111] ΔCH3 indicates antibodies that lack CH3 in their heavy chain. CH3 is the region designated by EU numbers 341–447. Therefore, the corresponding construct can also be described as a protein composed of a constant region (Fc region) consisting of amino acids designated by EU numbers 118–340. As a name reflecting their characteristics, ΔCH3 is referred to as Δ341-447 or Fc118-340. The same naming convention applies to other deletion antibodies. It should be noted that ΔCH3 has a GS linker (GGGGS) and a His tag (HHHHHH) attached to its C-terminus.

[0112] 1.3.1.ΔCH3=Δ341-447=Fc118-340:

[0113] 1.3.1.1. Anti-H5N1 hemagglutinin antibody

[0114] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVL QSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGGGGSHHHHHH (serial number 5)

[0115] 1.3.1.2. Anti-butyrylcholinesterase antibody

[0116] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGGGGSHHHHHH (serial number 6)

[0117] To confirm the effect of the missing material, four different ΔCH3 types were prepared, namely, [the types described above]. Figure 1 The diagram shows ΔCH3 obtained by deleting or adding 5 amino acids at the cut site, and ΔCH3 obtained by deleting or adding 10 amino acids.

[0118] The specific sequence is shown below.

[0119] 1.3.2.Δ331-447=Fc118-330(-10 amino acids):

[0120] 1.3.2.1. Anti-H5N1 hemagglutinin antibody

[0121] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAGGGGSHHHHHH (serial number 7)

[0122] 1.3.2.2. Anti-butyrylcholinesterase antibody

[0123] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPA VLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKDVLTITPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAGGGGSHHHHHH (serial number 8)

[0124] 1.3.3.Δ336-447=Fc118-335(-5 amino acids):

[0125] 1.3.3.1. Anti-H5N1 hemagglutinin antibody

[0126] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKDVLTITPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTGGGGSHHHHHH (serial number 9)

[0127] 1.3.3.2. Anti-butyrylcholinesterase antibody

[0128] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQ SDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTGGGGSHHHHHH (serial number 10)

[0129] 1.3.4.Δ346-447=Fc118-345(+5 amino acids):

[0130] 1.3.4.1. Anti-H5N1 hemagglutinin antibody

[0131] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAGGGGSHHHHHH (Serial Number 11)

[0132] 1.3.4.2. Anti-butyrylcholinesterase antibody

[0133] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAGGGGSHHHHHH (Serial Number 12)

[0134] 1.3.5.Δ351-447=Fc118-350(+10 amino acids):

[0135] 1.3.5.1. Anti-H5N1 hemagglutinin antibody

[0136] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTGGGGSHHHHHH (Serial Number 13)

[0137] 1.3.5.2. Anti-butyrylcholinesterase antibody

[0138] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE PVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTF (serial number 14)

[0139] 1.4.ΔCH2

[0140] ΔCH2 indicates the deletion of CH2 in the heavy chain of the anti-IgE antibody, with the hinge regions before and after it connected to CH3. CH2 is the region labeled 231–340 by EU numbers. Therefore, the construct can also be described as a protein composed of a constant region (Fc region) connecting amino acids labeled 118–230 by EU numbers and amino acids 341–447.

[0141] Considering the potential impact of CH2 deletion, constructs were also created with the GS adapter (GGGGS) inserted into the linker. The GS adapter was repeated 1–4 times. The specific amino acid sequences are: 1) no adapter, 2) GGGGS (Sequence No. 15), 3) GGGGSGGGGS (Sequence No. 16), 4) GGGGSGGGGSGGGGS (Sequence No. 17), and 5) GGGGSGGGGSGGGGSGGGGS (Sequence No. 18). When obtaining data such as evaluations of nonspecific reactions, the GS adapter repeated 4 times (Sequence No. 5) was used. The specific sequence is shown below.

[0142] 1.4.1.ΔCH2=Δ231-340=Fc118-230+341-447 (no connector):

[0143] 1.4.1.1. Anti-H5N1 hemagglutinin antibody

[0144] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVL QSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 19)

[0145] 1.4.1.2. Anti-butyrylcholinesterase antibody

[0146] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 20)

[0147] 1.4.2. ΔCH2=Δ231-340=Fc118-230+341-447(1×GS connector):

[0148] 1.4.2.1. Anti-H5N1 hemagglutinin antibody

[0149] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 21)

[0150] 1.4.2.2. Anti-butyrylcholinesterase antibody

[0151] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 22)

[0152] 1.4.3. ΔCH2=Δ231-340=Fc118-230+341-447 (2×GS connector):

[0153] 1.4.3.1. Anti-H5N1 hemagglutinin antibody

[0154] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 23)

[0155] 1.4.3.2. Anti-butyrylcholinesterase antibody

[0156] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS SSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 24)

[0157] 1.4.4.ΔCH2=Δ231-340=Fc118-230+341-447(3×GS connector):

[0158] 1.4.4.1. Anti-H5N1 hemagglutinin antibody

[0159] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS SSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 25)

[0160] 1.4.4.2. Anti-butyrylcholinesterase antibody

[0161] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 26)

[0162] 1.4.5. ΔCH2=Δ231-340=Fc118-230+341-447 (4×GS connector):

[0163] 1.4.5.1. Anti-H5N1 hemagglutinin antibody

[0164] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 27)

[0165] 1.4.5.2. Anti-butyrylcholinesterase antibody

[0166] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV PSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSHHHHHH (serial number 28)

[0167] To confirm the effect of the missing material, four different ΔCH2 types were prepared, namely, [the types described above]. Figure 2 The C-terminal cleavage site shown illustrates ΔCH3, obtained by deleting or adding 5 amino acids, and ΔCH2, obtained by deleting or adding 10 amino acids. The specific sequences are shown below.

[0168] 1.4.6. ΔCH2=Δ231-330=Fc118-230+331-447(4×GS adapter)(-10 amino acids):

[0169] 1.4.6.1. Anti-H5N1 hemagglutinin antibody

[0170] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 29)

[0171] 1.4.6.2. Anti-butyrylcholinesterase antibody

[0172] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV PSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 30)

[0173] 1.4.7. ΔCH2=Δ231-335=Fc118-230+336-447(4×GS adapter)(-5 amino acids):

[0174] 1.4.7.1. Anti-H5N1 hemagglutinin antibody

[0175] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTL SSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (Serial No. 31)

[0176] 1.4.7.2. Anti-butyrylcholinesterase antibody

[0177] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 32)

[0178] 1.4.8. ΔCH2=Δ231-345=Fc118-230+346-447(4×GS connector)(+5 amino acids):

[0179] 1.4.8.1. Anti-H5N1 hemagglutinin antibody

[0180] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQS DLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 33)

[0181] 1.4.8.2. Anti-butyrylcholinesterase antibody

[0182] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 34)

[0183] 1.4.9. ΔCH2=Δ231-350=Fc118-230+351-447(4×GS connector)(+10 amino acids):

[0184] 1.4.9.1. Anti-H5N1 hemagglutinin antibody

[0185] EVHLQQSGPELVKPGASVKMSCKTSGYTFTEYTIHWMKQSHGKSLEWIGGIFPNNGDTTYNQKFKVRATLTVGRSSSTAYMDLRSLTSEDSAVYYCVRNYGSSYGYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVL QSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 35)

[0186] 1.4.9.2. Anti-butyrylcholinesterase antibody

[0187] QVQLQQSGAELARPGASVKLSCKASGYTFTSQWLQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLTNLAPEDSAVYYCARSSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTGGGGSGGGGSGGGGSGGGGSIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (serial number 36)

[0188] The light chain of IgG was cloned into a tobacco mosaic virus (TMV) vector (manufactured by Icon Genetics), and each heavy chain (including the CH3 or CH2 deleter) was cloned into a potato virus X (PVX) vector (manufactured by Icon Genetics). These vectors were introduced into different Agrobacterium species for transformation and culture. The resulting culture mixture was then used to soak leaves of Nicotiana abenthamiana, which were harvested after approximately one week. The harvested leaves (infected leaves) were then frozen.

[0189] 2. Purification

[0190] Take 500g of frozen infected leaves and crush them using a cutting mixer. Add the crushed material to 250mL of extraction solution (100mM Tris, 250mM NaCl, 40mM sodium ascorbate), and repeat the crushing of infected leaves.

[0191] The pulverized infected leaves recovered from the cutting mixer and the pulverized infected leaves obtained by rinsing the mixer with 250 mL of fresh extraction solution were mixed. A portion of the sample was centrifuged at 12000 × g for 5 minutes, and the supernatant (extraction step component) was recovered.

[0192] After adjusting the pH of the components from the extraction step, the solution was filtered through a 0.45 μm filter, followed by a 0.22 μm filter. The filtrate was then subjected to nickel column chromatography (GE Healthcare HisPrep FF 16 / 10 column, catalog number 28936551) and HAP column chromatography to remove impurities from the plant and from the antibody.

[0193] 3. Expression test

[0194] Add 3 times the volume of extraction buffer (100 mM Tris, 250 mM NaCl, 5 mM EDTA) to the infected leaves, break them up with a bead mill, place them on ice for 30 minutes, and then centrifuge (8000 x g, 4 °C, 10 min). Collect the supernatant and analyze it by SDS-PAGE and Western blotting.

[0195] The expression of the antibodies described in Tables 1 and 2 was evaluated using Western blotting with HRP-labeled anti-His-tagged antibodies. Expression of each antibody was confirmed.

[0196] [Table 1]

[0197]

[0198] [Table 2]

[0199]

[0200] Expression of the camel ΔCH3 antibody was also confirmed.

[0201] 4. Non-specific evaluation

[0202] The interaction between purified antibodies and nonspecific factors was evaluated using surface plasmon resonance (SPR). The apparatus used was a BIAcore X-100 (manufactured by Biacore). Fc receptors (Fcγ receptor I, Fcγ receptor II), C1q, and rheumatoid factor were used as nonspecific factors.

[0203] <Fc receptor>

[0204] Biotin-labeled human-derived FcγRI or FcγRII (manufactured by Sino Biological, both purified) were captured using a Sensor Chip CAP (manufactured by GE Healthcare), and the rise in the signal (resonance unit: RU) accompanying the antibody interaction was observed. The running buffer consisted of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20, and the assay was performed at 25 °C at a flow rate of 30 μL / min. All assays were performed with n=3 (the same applies below). The antibody binding results against FcγRI are shown below. Figure 3 and Figure 4 The results of antibody binding to FcγRII are shown in Figure 5 and Figure 6 .

[0205] When 1 μM of antibody was added for 1 minute, an increase in signal was observed in the full-length antibody, indicating an interaction, while no signal change was observed in ΔCH3, indicating no interaction. Figure 3 and Figure 5 Similarly, with the ΔCH2 antibody, after adding 0.25 μM of antibody for 1 minute, interactions were observed in the full-length antibody, while no signal change or interaction was observed in the ΔCH2 antibody. Figure 4 and Figure 6 ).

[0206] <C1q>

[0207] Antibody was captured using a Sensor chip Protein L (GE Healthcare), and the increase in signal (resonance unit: RU) accompanying the interaction with human-derived C1q (Abcam, ab96363, purified) was observed. The run buffer consisted of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20, and the assay was performed at 25 °C at a flow rate of 30 μL / min. The antibody binding results for C1q are shown below. Figure 7 and Figure 8 .

[0208] Adding human-derived C1q diluted 100-fold with running buffer for 1 minute confirmed that neither ΔCH3 nor ΔCH2 interacted with C1q.

[0209] <Rf>

[0210] The antibody was bound to a CM5 sensor chip (manufactured by GE Healthcare) using an amine coupling method, and the increase in the signal (resonance unit: RU) accompanying the interaction with Rf-positive human serum or plasma was observed. The run buffer consisted of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20, and the assay was performed at 25 °C at a flow rate of 30 μL / min.

[0211] Ten human sera or plasmas that were Rf positive and identified as containing factors interacting with the full-length antibody were evaluated. The results showed that, upon addition of serum or plasma diluted 30-fold with run buffer for 1 minute, an increase in signal intensity and an observable interaction were observed in the full-length antibody. Figure 9 Interactions were not identified for ΔCH3 and ΔCH2. Figure 10 and Figure 11 ).

[0212] 5. Latex Aggregation Test

[0213] The binding efficiency of the obtained antibody to latex was evaluated by following these steps.

[0214] (1) Preparation of reagents

[0215] Using antibodies against IgE, the assay reagent based on immunoagglutination was prepared as described below.

[0216] i) Prepare a latex suspension by suspending 0.1 mg of antibody in 1 mL of a polystyrene latex suspension with an average particle size of 200 nm in a buffer solution (glycine, pH 7.3) at a concentration of 0.1%.

[0217] ii) Prepare the buffer solution (glycine, pH 8.3).

[0218] (2) Measurement using an automatic analysis device

[0219] The automated analyzer used was a Hitachi 7180 model automated analyzer, employing an endpoint method for automated determination. Using the reagents prepared in (1)i) above, four samples known to show false positives due to nonspecific reactions and 15 samples that did not exhibit nonspecific reactions were measured. For 3.5 μL of sample solution, 140 μL of the buffer solution prepared in (1)ii) above was added, and the mixture was stirred at 37°C. After standing for 5 minutes, 70 μL of latex suspension was added, and the mixture was further stirred at 37°C. The agglutination reaction over approximately 5 minutes was measured as a change in absorbance, and the IgE concentration of each sample was calculated using a calibration curve.

[0220] (3) Comparison of the measured values ​​of false positive samples

[0221] The results of the measurements in (2) above are shown in Figure 12 And Table 3.

[0222] [Table 3]

[0223]

[0224] (Unit: IU / mL)

[0225] If there are no false positives or false negatives, the IgE concentration in the sample should be equal to the measured value. However, for samples No. 1, 5, 9, and 18, clear false positives were observed when using reagents with full-length antibodies. On the other hand, reagents using CH3-deficient antibodies generally showed measured values ​​at the same level as the IgE concentration, and no clear false positives were confirmed.

[0226] Industrial availability

[0227] According to the present invention, a reagent can be provided that can inhibit nonspecific reactions by deleting only a portion of the Fc region in an antibody.

Claims

1. The use of antibodies with all portions of CH3 missing in the Fc region in the manufacture of reagents for inhibiting nonspecific reactions of antigen-antibody reactions. in, The nonspecific reaction of the antibody is inhibited by the deletion of CH3. The nonspecific reaction occurs through nonspecific reaction factors that bind to the Fc region, such as complement C1q, Fcγ receptor or rheumatoid factor in the sample.

2. The use according to claim 1, wherein, The antibody is IgG.

3. The use according to claim 1 or 2, wherein, The antigen-antibody reaction between the antibody and the antigen of the diagnostic subject was at least equal to the antigen-antibody reaction of the antibody with the Fc region not missing.

4. The use according to claim 1 or 2, wherein, The antibodies are derived from mice, rabbits, rats, guinea pigs, hamsters, goats, sheep, or camels.

5. The use according to claim 1 or 2, wherein, The antibody is a human antibody, a humanized antibody, or a human chimeric antibody.

6. The use according to claim 1 or 2, wherein, The antibody is a non-human chimeric antibody.

7. The use according to claim 1 or 2, wherein, The reagent is used for diagnosis.

8. The use according to claim 1 or 2, wherein, The reagents are used for research.

9. Use of antibodies with all portions of CH3 missing in the Fc region in the manufacture of kits containing reagents for inhibiting nonspecific reactions of antigen-antibody reactions. in, The nonspecific reaction of the antibody is inhibited by the deletion of CH3. The nonspecific reaction occurs through nonspecific reaction factors that bind to the Fc region, such as complement C1q, Fcγ receptor or rheumatoid factor in the sample.

10. The use of antibodies with all portions of CH3 missing in the Fc region in the manufacture of kits containing reagents for inhibiting nonspecific reactions of antigen-antibody reactions and blocking agents. in, The nonspecific reaction of the antibody is inhibited by the deletion of CH3. The nonspecific reaction occurs through nonspecific reaction factors that bind to the Fc region, such as complement C1q, Fcγ receptor or rheumatoid factor in the sample.

11. The use according to claim 9 or 10, wherein, The kit contains a support vector.

12. The use according to claim 11, wherein, Antibodies bind to the surface of the carrier.

13. The use according to claim 11, wherein, The carrier is a microplate or latex.

14. The use according to claim 11, wherein, Antibodies are immobilized on the support via linkers.

15. A method for inhibiting a nonspecific reaction of antigen-antibody reaction, wherein, Using an antibody that has all of the CH3 regions missing in the Fc region, the non-specific reaction of the antibody is inhibited by the absence of CH3. The non-specific reaction occurs by non-specific reaction factors that bind to the Fc region, such as complement C1q, Fcγ receptor, or rheumatoid factor in the sample.

Images