Anti-agalactosylated human immunoglobulin-g antibody, method for measuring agalactosylated human immunoglobulin-g using said antibody, and measurement reagent therefor

Abstract

Provided are: an anti-agalactosylated human IgG antibody or an antibody fragment thereof; a method for measuring agalactosylated human IgG in a sample using the anti-agalactosylated human IgG antibody or the antibody fragment thereof; and a measurement reagent for measuring agalactosylated human IgG in a sample.

Description

Anti-agalactosylated human immunoglobulin G antibody, method for measuring agalactosylated human immunoglobulin G using said antibody, and reagent for the measurement

[0001] The present invention relates to an anti-agalactosylated human immunoglobulin G antibody, a method for measuring agalactosylated human immunoglobulin G using the antibody, and a reagent for the measurement.

[0002] Human immunoglobulin G (hereinafter sometimes referred to as IgG) is a glycoprotein secreted into the blood by mature B cells during adaptive immunity. Human IgG is a tetramer consisting of two light chains (L chains) and two heavy chains (H chains), and exists in four subclasses (IgG1, 2, 3, and 4). In all subclasses, a sugar chain is attached to asparagine (N) at position 297 of the heavy chain contained in the Fc region (also called the constant region) composed of the C-terminal domains (CH2 domain and CH3 domain) of the two heavy chains as a post-translational modification (Figure 1). The sugar chain attached to asparagine (N) at position 297 of the heavy chain contained in the IgG Fc region is called an N-linked sugar chain. The N-linked sugar chains of the human IgG Fc region are complex-type biantennary sugar chains with a pentasaccharide core consisting of two N-acetylglucosamines and three mannose residues to which N-acetylglucosamine, galactose, sialic acid, and fucose are attached. These sugar chains exhibit structural diversity depending on the presence or absence, number, and location of each monosaccharide addition (Non-Patent Document 1). Sixteen types of such sugar chains are known, including the 12 shown in Figure 2 (Patent Document 1). Of these, human immunoglobulin G containing galactose-free N-linked sugar chains in the Fc region is called agalactosylated human immunoglobulin G (hereinafter sometimes referred to as "agalactosylated human IgG"), and human immunoglobulin G containing galactose-linked N-linked sugar chains in the Fc region is called galactosylated human immunoglobulin G (hereinafter sometimes referred to as "galactosylated human IgG").

[0003] Diagnostic methods for various diseases have been proposed based on changes in IgG sugar chains, and it has long been reported that agalactosylated IgG increases in inflammatory diseases in general (Non-Patent Document 2). Patent Document 1 discloses that Crohn's disease and ulcerative colitis can be differentiated by measuring the relative ratio of the amount of specific agalactosylated sugar chains to the amount of galactosylated sugar chains in patient serum using HPLC. Furthermore, Non-Patent Document 3 discloses that when agalactosylated IgG in serum was measured using lectin-EIA (lectin-enzyme immunoassay) using an anti-human IgG antibody and a specific lectin, the amount of agalactosylated IgG in the serum of Crohn's disease patients was significantly higher than the amount of agalactosylated IgG in the serum of healthy subjects and patients with various inflammatory bowel diseases. Patent Document 2 discloses a method for differentiating inflammatory bowel disease using a specific lectin to differentiate between healthy subjects, ulcerative colitis, and Crohn's disease.

[0004] International Publication No. 2007 / 136001 Japanese Patent Application Laid-Open No. 2011-247665

[0005] Mahan et al., "A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis", J Immunol Methods. 2015 Feb:417:34-44. doi: 10.1016 / j.jim.2014.12.004. Epub 2014 Dec 15.Rademacher et al., "The role of IgG glycoforms in the pathogenesis of rheumatoid arthritis", Springer Semin Immunopathol.1988;10(2-3):231-49. doi: 10.1007 / BF01857227.Shinzaki et al., "Lectin-based immunoassay for aberrant IgG glycosylation as the biomarker for Crohn's disease", Inflamm Bowel Dis. 2013Feb;19(2):321-31. doi: 10.1097 / MIB.0b013e318280eade.Riou et al., "Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells", medRxiv 2020Nov3;2020.10.30.20223099.

[0006] However, methods for measuring the relative ratio of the amount of a specific agalactosylated sugar chain to the amount of a galactosylated sugar chain in serum by HPLC involve isolating human IgG from human serum using protein A or protein G, etc., liberating sugar chains from the isolated human IgG using N-glycanase, and developing the fluorescently labeled released sugar chains by HPLC. These methods are cumbersome and make it difficult to measure a large number of samples in a short period of time. Furthermore, methods for measuring the amount of agalactosylated IgG in serum using lectin-EIA also require a step of isolating human IgG from human serum using protein A or protein G, etc., making the procedure cumbersome and making it difficult to measure a large number of samples in a short period of time. Therefore, there has been a demand for a method for measuring agalactosylated human IgG in a sample using the sample itself as a measurement sample, without including a step of isolating human IgG from the sample. To achieve this method, an antibody that specifically reacts with agalactosylated human IgG was needed.

[0007] An object of the present invention is to provide an anti-agalactosylated human IgG antibody or antibody fragment thereof that reacts with agalactosylated human IgG but not with galactosylated human IgG. Another object of the present invention is to provide a method for measuring agalactosylated human IgG in a sample using the anti-agalactosylated human IgG antibody or antibody fragment thereof, and a measurement reagent for measuring agalactosylated human IgG in a sample.

[0008] As a result of extensive research, the present inventors have succeeded in obtaining an anti-agalactosylated human IgG antibody or an antibody fragment thereof that is characterized by reacting with agalactosylated human IgG but not with galactosylated human IgG, thereby completing the present invention.

[0009] That is, the present disclosure relates to the following [1] to

[55] . [1] An anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof, which reacts with human immunoglobulin G containing a galactose-free N-linked sugar chain in the Fc region (hereinafter referred to as agalactosylated human immunoglobulin G) but does not react with human immunoglobulin G containing a galactose-linked N-linked sugar chain in the Fc region (hereinafter referred to as galactosylated human immunoglobulin G). [2] The antibody or antibody fragment thereof according to [1], whose reactivity with galactosylated human immunoglobulin G in ELISA is less than 20% of the reactivity with agalactosylated human immunoglobulin G. [3] The galactose-free N-linked sugar chain is represented by the following general formula (I), (II), (III), or (IV): [4] The antibody or antibody fragment thereof according to any one of [1] to [3], wherein the agalactosylated human immunoglobulin G is a human immunoglobulin G that binds to at least one antigen selected from the group consisting of an autoantigen, an alloantigen, a virus-derived antigen, and a bacteria-derived antigen. [5] The antibody or antibody fragment thereof according to any one of [1] to [4], wherein the anti-agalactosylated human immunoglobulin G antibody is a monoclonal antibody. [6] A hybridoma producing the monoclonal antibody of [5].

[0010] [7] A method for producing an anti-agalactosylated human immunoglobulin G antibody, comprising: (1) immunizing a non-human animal with agalactosylated human immunoglobulin G or an Fc region fragment thereof as an immunogen; (2) fusing antibody-producing cells obtained from the non-human animal immunized in step (1) with myeloma cells to obtain hybridomas; (3) culturing the hybridomas obtained in step (2); (4) selecting, from the culture obtained in step (3), hybridomas that produce anti-agalactosylated human immunoglobulin G antibodies that react with agalactosylated human immunoglobulin G but do not react with galactosylated human immunoglobulin G; and (5) culturing the hybridomas selected in step (4) and collecting, from the culture, anti-agalactosylated human immunoglobulin G antibodies that react with agalactosylated human immunoglobulin G but do not react with galactosylated human immunoglobulin G. [8] The method according to [7], wherein the non-human animal is an autoimmune disease model animal. [9] The N-linked sugar chain to which no galactose is bound is represented by the following general formula (I), (II), (III), or (IV): The method according to [7] or [8], wherein the N-linked sugar chain comprises a sugar chain represented by the formula:

[0011]

[10] A method for measuring agalactosylated human immunoglobulin G in a sample, using the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[11] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] to produce an immune complex A1 containing the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof and agalactosylated human immunoglobulin G; (2) reacting the immune complex A1 produced in step (1) with an antibody or antibody fragment thereof that binds to human immunoglobulin G to produce an immune complex A2 containing the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof, agalactosylated human immunoglobulin G, and an antibody or antibody fragment thereof that binds to human immunoglobulin G; and (3) measuring the immune complex A2 produced in step (2).

[12] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with an antibody or antibody fragment thereof that binds to human immunoglobulin G to produce an immune complex B1 containing an antibody or antibody fragment thereof that binds to human immunoglobulin G and agalactosylated human immunoglobulin G; (2) reacting the immune complex B1 produced in step (1) with an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] to produce an immune complex B2 containing an antibody or antibody fragment thereof that binds to human immunoglobulin G, agalactosylated human immunoglobulin G, and the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof; and (3) measuring the immune complex B2 produced in step (2).

[0012]

[13] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with a labeled competitor, which is a competitor of agalactosylated human immunoglobulin G to which a label is bound, and an anti-agalactosylated human immunoglobulin G antibody or an antibody fragment thereof according to any one of [1] to [5] immobilized on an insoluble carrier, to form, on the insoluble carrier, an immune complex C1 comprising the anti-agalactosylated human immunoglobulin G antibody or an antibody fragment thereof and the labeled competitor, and an immune complex C2 comprising the anti-agalactosylated human immunoglobulin G antibody or an antibody fragment thereof and the agalactosylated human immunoglobulin G; and (2) measuring the labeled competitor in the immune complex C1 comprising the anti-agalactosylated human immunoglobulin G antibody or an antibody fragment thereof and the labeled competitor, which is formed on the insoluble carrier in step (1).

[14] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] and a competitor of the agalactosylated human immunoglobulin G immobilized on an insoluble carrier to form an immune complex D1 on the insoluble carrier, the immune complex D1 comprising the competitor and the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof; and (2) measuring the immune complex D1 formed on the insoluble carrier in step (1).

[0013]

[15] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with an antigen to which agalactosylated human immunoglobulin G reacts to produce an immune complex E1 containing the antigen and agalactosylated human immunoglobulin G; (2) reacting the immune complex E1 produced in step (1) with the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of [1] to [5] to produce an immune complex E2 containing the antigen, agalactosylated human immunoglobulin G, and the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment; and (3) measuring the immune complex E2 produced in step (2).

[16] A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of [1] to [5] to produce an immune complex F1 containing the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment and the agalactosylated human immunoglobulin G; (2) reacting the immune complex F1 produced in step (1) with an antigen to which the agalactosylated human immunoglobulin G reacts to produce an immune complex F2 containing the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment, the agalactosylated human immunoglobulin G, and the antigen; and (3) measuring the immune complex F2 produced in step (2).

[17] The measurement method according to

[15] or

[16] , wherein the antigen to which agalactosylated human immunoglobulin G reacts is at least one antigen selected from the group consisting of an autoantigen, an alloantigen, a virus-derived antigen, and a bacteria-derived antigen.

[0014]

[18] A method for measuring agalactosylated human IgG in a sample, comprising: (1) reacting the sample with latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof according to any one of [1] to [5] is bound, to produce an immune complex G1 containing the agalactosylated human IgG and the latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is bound; and (2) measuring the agglutination of latex particles caused by the binding of the agalactosylated human IgG to the latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is bound, to obtain a measurement value.

[0015]

[19] A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[20] A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], and an antibody or antibody fragment thereof that binds to human immunoglobulin G.

[0016]

[21] A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] immobilized on an insoluble carrier, and a labeled competitor in which a label is bound to a competitor of agalactosylated human immunoglobulin G.

[22] A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the competitor of agalactosylated human immunoglobulin G immobilized on an insoluble carrier, and the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[0017]

[23] A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], and an antigen to which agalactosylated human immunoglobulin G reacts.

[24] The measurement reagent according to

[23] , wherein the antigen to which agalactosylated human immunoglobulin G reacts is at least one antigen selected from the group consisting of an autoantigen, an alloantigen, a virus-derived antigen, and a bacteria-derived antigen.

[0018]

[25] A reagent for measuring agalactosylated human IgG in a sample, comprising latex particles to which the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] is bound.

[26] A reagent for measuring agalactosylated human IgG in a sample, comprising latex particles to which the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] is bound, and latex particles to which an antibody that binds to human IgG or an antibody fragment thereof is bound.

[0019]

[27] A method for collecting data for determining whether or not a subject is affected by inflammatory bowel disease, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject using the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[28] A method for diagnosing or assisting in the diagnosis of inflammatory bowel disease, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject using the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[29] A method for diagnosing inflammatory bowel disease, comprising: obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from a subject using the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5]; and determining that the subject is highly likely to be affected by inflammatory bowel disease if the measurement value of agalactosylated human immunoglobulin G obtained is equal to or greater than a reference value, and determining that the subject is highly likely not affected by inflammatory bowel disease if the measurement value is less than the reference value.

[30] A method for collecting data for determining whether or not a subject has inflammatory bowel disease or a method for assisting in the diagnosis of inflammatory bowel disease, comprising: obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from a subject using the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5]; and determining that the subject is likely to have inflammatory bowel disease if the measurement value of agalactosylated human immunoglobulin G obtained is equal to or greater than a reference value, and determining that the subject is likely not to have inflammatory bowel disease if the measurement value is less than the reference value.

[31] The method according to any one of

[27] to

[30] , wherein obtaining a measurement value of agalactosylated human immunoglobulin G is performed by the method according to any one of

[10] to

[18] .

[32] A kit for performing the method of any one of

[27] to

[31] , comprising: (a) the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], and (b) a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human immunoglobulin G is equal to or greater than a reference value, the subject is determined to have inflammatory bowel disease, and if the measured value of the agalactosylated human immunoglobulin G is less than the reference value, the subject is determined to not have inflammatory bowel disease.

[33] A diagnostic agent for inflammatory bowel disease, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[34] Use of the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] for the manufacture of a diagnostic agent for inflammatory bowel disease.

[35] The anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], which is used for diagnosing inflammatory bowel disease.

[0020]

[36] A method for collecting data for determining whether a subject is suffering from chronic pancreatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject.

[37] A method for diagnosing or assisting in the diagnosis of chronic pancreatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject.

[38] A method for diagnosing chronic pancreatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject, and determining that the subject is likely to be suffering from chronic pancreatitis if the obtained measurement value of agalactosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is likely not suffering from chronic pancreatitis if the measurement value is less than the reference value.

[39] A method for collecting data for determining whether or not a subject has chronic pancreatitis or a method for assisting in the diagnosis of chronic pancreatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject, and determining that the subject is likely to have chronic pancreatitis if the obtained measurement value of agalactosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is likely not to have chronic pancreatitis if the measurement value is less than the reference value.

[40] The method of any one of

[36] to

[39] , wherein the measurement value of agalactosylated human immunoglobulin G is obtained using an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof described in any one of [1] to [5].

[41] The method of any one of

[36] to

[40] , wherein the measurement value of agalactosylated human immunoglobulin G is obtained by the method described in any one of

[10] to

[18] .

[42] A kit for carrying out the method according to any one of

[36] to

[41] , comprising: (a) the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5]; and (b) a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human immunoglobulin G is equal to or greater than the reference value, the subject is determined to have chronic pancreatitis; and if the measured value of the agalactosylated human immunoglobulin G is less than the reference value, the subject is determined not to have chronic pancreatitis.

[43] A diagnostic agent for chronic pancreatitis, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[44] Use of the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] for the manufacture of a diagnostic agent for chronic pancreatitis.

[45] The anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], for use in diagnosing chronic pancreatitis.

[0021]

[46] A method for collecting data for determining whether a subject is suffering from chronic hepatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject.

[47] A method for diagnosing or assisting in the diagnosis of chronic hepatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject.

[48] A method for diagnosing chronic hepatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject, and determining that the subject is likely to be suffering from chronic hepatitis if the obtained measurement value of agalactosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is likely not suffering from chronic hepatitis if the measurement value is less than the reference value.

[49] A method for collecting data for determining whether or not a subject has chronic hepatitis or a method for assisting in the diagnosis of chronic hepatitis, comprising obtaining a measurement value of agalactosylated human immunoglobulin G in a biological sample from the subject, and determining that the subject is likely to have chronic hepatitis if the obtained measurement value of agalactosylated human immunoglobulin G is equal to or greater than a reference value, and determining that the subject is likely not to have chronic hepatitis if the measurement value is less than the reference value.

[50] The method of any one of

[46] to

[49] , wherein the measurement value of agalactosylated human immunoglobulin G is obtained using an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof described in any one of [1] to [5].

[51] The method of any one of

[46] to

[50] , wherein the measurement value of agalactosylated human immunoglobulin G is obtained by the method described in any one of

[10] to

[18] .

[52] A kit for carrying out the method according to any one of

[46] to

[51] , comprising: (a) the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5]; and (b) a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human immunoglobulin G is equal to or greater than a reference value, the subject is determined to have chronic hepatitis; and if the measured value of the agalactosylated human immunoglobulin G is less than the reference value, the subject is determined not to have chronic hepatitis.

[53] A diagnostic agent for chronic hepatitis, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5].

[54] Use of the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5] for the manufacture of a diagnostic agent for chronic hepatitis.

[55] The anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of [1] to [5], for use in diagnosing chronic hepatitis.

[0022] The present invention provides an anti-agalactosylated human IgG antibody or antibody fragment thereof, which reacts with agalactosylated human IgG but not with galactosylated human IgG. The present invention also provides a method for measuring agalactosylated human IgG in a sample using the anti-agalactosylated human IgG antibody or antibody fragment thereof, and a measurement reagent or measurement kit for measuring agalactosylated human IgG in a sample. This method or kit enables a method for measuring agalactosylated human IgG in a sample using the sample itself as a measurement sample, without the step of isolating human IgG from the sample. Furthermore, the present invention provides a method for diagnosing or assisting in the diagnosis of inflammatory bowel disease, which comprises measuring agalactosylated human immunoglobulin G in a biological sample from a subject using the anti-agalactosylated human IgG antibody or antibody fragment thereof. Furthermore, the present invention provides a method for diagnosing or assisting in the diagnosis of chronic pancreatitis, and a method for diagnosing or assisting in the diagnosis of chronic hepatitis, which comprises obtaining a measurement of agalactosylated human immunoglobulin G in a biological sample from a subject.

[0023] The structure of human IgG is shown schematically. The 12 types of sugar chains of IgG antibodies are shown. Black circles represent galactose, triangles represent fucose, white circles represent mannose, and squares represent N-acetylglucosamine (GlcNAc). This figure shows the reactivity of human IgG Fc (agalactosylated human IgG Fc) in which galactose attached to the non-reducing end of the N-linked sugar chain has been digested using β-galactosidase, with RCA120 lectin. This figure shows the reactivity of human IgG (N-linked sugar chain-removed human IgG) from which N-linked sugar chains have been removed by PNGase F treatment, with RCA120 lectin and ConA lectin.

[0033] Figure 1 shows the reactivity of monoclonal antibodies produced by hybridoma strains 11E8, 15D12, 20G2, 22F12, 42B1, and 50A1 with agalactosylated human IgG or galactosylated human IgG. Figure 2 compares wells in which agalactosylated human IgG Fc was immobilized with wells in which galactosylated human IgG Fc was immobilized, evaluating that approximately the same amount of antibody was immobilized in each well. Figure 3 shows a standard curve obtained by measuring a standard substance (agalactosylated human IgG) in a sandwich ELISA using a fragmented anti-human IgG antibody and an anti-agalactosylated human IgG antibody. Figure 4 compares the agalactosylated human IgG concentration in the serum of healthy subjects (HV) with that of patients with inflammatory bowel disease (IBD). Also shown is the p-value obtained by Wilcoxon rank-sum test. This figure shows a comparison of the measured values ​​(absorbance) of agalactosylated human IgG in the serum of healthy subjects with those of patients with chronic pancreatitis. Also shown is the p-value obtained by a Wilcoxon rank-sum test. This figure shows a comparison of the measured values ​​(absorbance) of agalactosylated human IgG in the serum of healthy subjects with those of patients with chronic hepatitis. Also shown is the p-value obtained by a Wilcoxon rank-sum test.

[0024] Hereinafter, embodiments for carrying out the present disclosure will be described in detail. Note that the embodiment described below shows an example of a typical embodiment of the present disclosure, and the scope of the disclosure should not be interpreted narrowly. Note that a numerical range indicated using "to" indicates a range that includes the numerical values ​​written before and after "to" as the minimum and maximum values, respectively. Furthermore, when multiple substances corresponding to each component are present in the solution, the amount of each component in the solution means the total amount of the multiple substances present in the reaction or reagent, unless otherwise specified.

[0025] In the present disclosure, when an antibody is expressed as "reacting with," "binding to," or "recognizing" agalactosylated human immunoglobulin G, these terms include the meanings commonly used in the field of the present disclosure and are used interchangeably. Methods for confirming the "reaction" of an antibody with agalactosylated human immunoglobulin G include methods utilizing the principles of antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, surface plasmon resonance, immunochromatography, and quartz crystal microbalance, which are well known to those skilled in the art.

[0026] In the present disclosure, the expression "non-reactive" of an antibody of the present disclosure with galactosylated human immunoglobulin G means, for example, that in an antigen-immobilized ELISA, the measured value when galactosylated human immunoglobulin G is immobilized is not detectable (below the detection limit), or that the measured value when galactosylated human immunoglobulin G is immobilized is relatively smaller than the measured value when agalactosylated human immunoglobulin G is immobilized. Furthermore, the "non-reactive" of an antibody of the present disclosure with galactosylated human immunoglobulin G can also be evaluated relatively using the "cross-reactivity with galactosylated human immunoglobulin G" described below as an indicator.

[0027] In the present disclosure, the term "specifically reacting" with an antibody or the "specificity" of an antibody refers to the ability of the antibody to detectably bind to an epitope presented on an antigen. Also, in the present disclosure, the term "specifically reacting" with an antibody or the "specificity" of an antibody means that the detectable reactivity with other antigens is relatively small or no reactivity is detectable. For example, when an antibody "specifically reacts" with agalactosylated human immunoglobulin G, the antibody reacts with agalactosylated human immunoglobulin G but does not react with galactosylated human immunoglobulin G. Whether an antibody specifically reacts with agalactosylated human immunoglobulin G can be evaluated, for example, by relative evaluation using "cross-reactivity with galactosylated human immunoglobulin G" as an indicator. For example, in an antigen-immobilized ELISA, a measurement value (measured value A) when agalactosylated human immunoglobulin G is immobilized and a measurement value (measured value B) when galactosylated human immunoglobulin G is immobilized are measured, and cross-reactivity (%) is calculated as (measured value B) / (measured value A) × 100. The closer the cross-reactivity is to 0%, the lower the cross-reactivity, and the antibody can be evaluated as "specifically reacting" with agalactosylated human immunoglobulin G. The preferred value of antibody cross-reactivity varies depending on the application of the antibody. For example, even if the cross-reactivity with galactosylated human immunoglobulin G is 50%, if a method for measuring agalactosylated human IgG using the antibody can detect agalactosylated human IgG in a sample without isolating human IgG from the sample, or if a useful application or effect, such as the ability to diagnose a specific disease, is observed, the cross-reactivity of the antibody can be said to be suitable.The cross-reactivity is preferably less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%, and more preferably less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0028] [1] Anti-agalactosylated human immunoglobulin G antibody In the present disclosure, the anti-agalactosylated human IgG antibody refers to an antibody that reacts with human immunoglobulin G containing non-galactose-linked N-linked sugar chains in the Fc region (sometimes referred to as "agalactosylated human immunoglobulin G" or "agalactosylated human IgG"), but does not react with human immunoglobulin G containing galactose-linked N-linked sugar chains in the Fc region (sometimes referred to as "galactosylated human immunoglobulin G" or "galactosylated human IgG").

[0029] Galactosylation in human IgG refers to modification of asparagine (N) at position 297 of the heavy chain of the Fc region of human IgG with an N-linked sugar chain in which all or part of the sugar chain terminus is galactose. Examples of the modified sugar chain include N-linked sugar chains containing sugar chains represented by formulas (V) to (XVI) shown below.

[0030]

[0031] Agalactosylation in human IgG refers to modification of asparagine (N) 297 of the heavy chain of the Fc region of human IgG with a galactose-deficient N-linked sugar chain. Examples of the sugar chain to be modified include N-linked sugar chains containing sugar chains represented by the following formulas (I) to (IV).

[0032]

[0033] The agalactosylated human IgG of the present disclosure is a human IgG containing an N-linked sugar chain to which no galactose is bound in the Fc region, and examples thereof include the human IgG shown in FIG.

[0034] The antigen to which agalactosylated human IgG of the present disclosure binds is not particularly limited as long as it is an antigen to which human IgG containing non-galactose-bound N-linked glycans in the Fc region binds, and examples thereof include autoantigens, alloantigens, virus-derived antigens, and bacterial-derived antigens. Autoantigens include intranuclear antigens, etc. Alloantigens include major histocompatibility complex antigens (MHC antigens), etc. Viral-derived antigens include HBs antigen of hepatitis B virus and core antigen of hepatitis C virus, etc. Bacterial-derived antigens include O antigen of the outer membrane of gram-negative bacteria and pertussis toxin of Bordetella pertussis, etc.

[0035] The anti-agalactosylated human IgG antibody of the present disclosure may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Examples of monoclonal antibodies include monoclonal antibodies produced by hybridomas and recombinant monoclonal antibodies produced by transformants transformed with expression vectors containing antibody genes. Examples of monoclonal antibodies produced by hybridomas include clones 11E8, 15D12, 20G2, 22F12, 42B1, and 50A1.

[0036] The hybridoma of the present disclosure is not particularly limited as long as it produces the anti-agalactosylated human IgG monoclonal antibody of the present disclosure, and examples thereof include hybridomas that produce the above-mentioned clone antibodies. Examples of hybridomas that produce the above-mentioned clone antibodies include clones 11E8, 15D12, 20G2, 22F12, 42B1, and 50A1. The hybridoma of the present disclosure can be produced by known methods, for example, the production method described below based on the description in "Monoclonal Antibodies" (Yodosha, 1996).

[0037] The anti-agalactosylated human IgG antibody fragment of the present disclosure is not particularly limited as long as it reacts with agalactosylated human IgG but does not react with galactosylated human IgG. Examples of such fragments include Fab obtained by treating IgG with papain, and F(ab') obtained by treating IgG with pepsin. ２ Examples of such antibodies include Fab' obtained by treating IgG with pepsin followed by reduction, single-chain antibodies (scFv) in which the variable region of the antibody light chain (VL region) and the variable region of the antibody heavy chain (VH region) are artificially linked as a single polypeptide, and fragments of the variable region of the antibody heavy chain (VHH region) of single-chain antibodies that do not have the antibody light chain, such as those found in camelids.

[0038] [2] Method for producing anti-agalactosylated human IgG antibody There are no particular limitations on the method for producing the anti-agalactosylated human IgG antibody of the present disclosure, as long as it is a method that can produce the anti-agalactosylated human IgG antibody of the present disclosure. For example, a production method comprising the following steps may be used. (1) immunizing a non-human animal with agalactosylated human IgG or an Fc region fragment thereof as an immunogen; (2) fusing antibody-producing cells obtained from the non-human animal immunized in step (1) with myeloma cells to obtain hybridomas; (3) culturing the hybridomas obtained in step (2); (4) selecting, from the culture obtained in step (3), hybridomas that produce anti-agalactosylated human IgG antibodies that react with agalactosylated human IgG but do not react with galactosylated human IgG; and (5) culturing the hybridomas selected in step (4) and collecting, from the culture, anti-agalactosylated human IgG antibodies that react with agalactosylated human IgG but do not react with galactosylated human IgG.

[0039] Step (1) is a step of immunizing a non-human animal with agalactosylated human IgG or an Fc region fragment thereof as an immunogen. Methods for immunizing a non-human animal include administering the prepared immunogen subcutaneously, intravenously, or intraperitoneally to the non-human animal together with an appropriate adjuvant.

[0040] The immunogen is not particularly limited as long as it is capable of producing the anti-agalactosylated human IgG antibody of the present disclosure, and examples thereof include agalactosylated human IgG, an Fc region fragment of agalactosylated human IgG, and the like. Agalactosylated human IgG or an Fc region fragment of agalactosylated human IgG may also be used in which a carrier protein such as KLH (keyhole limpet hemocyanin) or albumin is bound to the immunogen. Examples of albumin include bovine serum albumin (BSA) and ovalbumin.

[0041] The Fc region fragment is not particularly limited as long as it contains an N-linked glycan lacking galactose, and examples include the full-length heavy chain Fc region of human IgG, the full-length CH2 region containing asparagine (N) at position 297 of the heavy chain of the IgG Fc region, and a peptide fragment containing asparagine (N) at position 297 of the heavy chain of the IgG Fc region.

[0042] The non-human animal is not particularly limited as long as it is an animal other than a human that can produce the anti-agalactosylated human IgG antibody of the present disclosure in vivo, and examples thereof include mice, rats, hamsters, rabbits, camels, and alpacas. In particular, autoimmune disease model animals that are expected to easily produce antibodies against agalactosylated human IgG are preferably used. An example of an autoimmune disease model animal is the SKG mouse (manufactured by CLEA Japan, Inc.), a mouse model of human rheumatoid arthritis.

[0043] The adjuvant is not particularly limited as long as it is an adjuvant that can produce the anti-agalactosylated human IgG antibody of the present disclosure, and examples thereof include Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), Ribi adjuvant, etc.

[0044] The method for preparing the immunogen is not particularly limited as long as it can produce an immunogen capable of generating the aforementioned anti-agalactosylated human IgG antibody. For example, a method can be used in which human IgG is treated with β-galactosidase to digest the galactose attached to the non-reducing end of the N-linked glycan, and then agalactosylated human IgG is purified using a gel filtration column. Human IgG may be purified from a human blood sample or commercially available human IgG. Examples of the Fc region fragment of agalactosylated human IgG include the Fc region fragments described above. The Fc region fragment of agalactosylated human IgG may be a commercially available Fc region fragment, or may be prepared by treating human IgG with a protease, or may be produced using genetic engineering techniques. Examples of proteases include pepsin and papain.

[0045] Step (2) Step (2) is a step of preparing hybridomas by fusing antibody-producing cells obtained from the non-human animal immunized in step (1) with myeloma cells.

[0046] The antibody-producing cells are not particularly limited as long as they are capable of producing the anti-agalactosylated human IgG antibodies of the present disclosure, and examples include B cells obtained from the spleen, lymph nodes, or peripheral blood of a non-human animal immunized with an immunogen.

[0047] The myeloma cells are not particularly limited as long as they are capable of producing the anti-agalactosylated human IgG antibody of the present disclosure. Examples thereof include 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell lines P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, vol. 18, p. 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. Immunology, vol. 6, p. 511 (1976)], SP2 / O-Ag14 (SP-2) [Nature, vol. 276, p. 269 (1978)], and P3-X63-Ag8653(653) [J. Immunology, vol. 123, p. 1548 (1979)], P3-X63-Ag8(X63) [Nature, vol. 256, p. 495 (1975)], and the like.

[0048] The method for cell fusion is not particularly limited as long as it is a method that can fuse antibody-producing cells and myeloma cells, and examples thereof include a method using polyethylene glycol (PEG) and an electric pulse method.

[0049] Step (3) is a step of culturing the hybridomas prepared in step (2). The hybridomas can be cultured, for example, in a selective medium [GIT medium (manufactured by Thermo Fisher Scientific) containing 10% (v / v) BM-conditioned H1 and 2% (v / v) HAT supplement] or a culture medium [GIT medium (manufactured by Kohjin Bio Co., Ltd.) containing 10% (v / v) BM-conditioned H1 (manufactured by Roche)] at 37°C and 5% CO ２ Examples include a method of culturing in an incubator.

[0050] Step (4) Step (4) is a step of selecting only hybridomas that produce anti-agalactosylated human IgG antibodies that react with agalactosylated human IgG but do not react with galactosylated human IgG. Examples of methods for selecting only these hybridomas include the following methods.

[0051] The culture supernatant obtained by the hybridoma culture method described in step (3) is dispensed into each well of a microtiter plate on which agalactosylated human IgG or galactosylated human IgG has been solidified, and the mixture is allowed to react at 25° C. After washing each well with a washing solution [10 mmol / L phosphate buffer (pH 7.4) containing 0.05% (v / v) Tween 20 and 150 mmol / L sodium chloride], an anti-non-human animal immunoglobulin antibody labeled with an enzyme or the like is dispensed and allowed to react. After washing each well with a washing solution, the amount of label in each well is measured to detect wells in which immune complex I containing agalactosylated human IgG, anti-agalactosylated human IgG antibody in the culture supernatant, and labeled anti-non-human animal IgG antibody, or immune complex II containing galactosylated human IgG, anti-galactosylated human IgG antibody in the culture supernatant, and labeled anti-non-human animal immunoglobulin antibody has formed, allowing for determination of whether the monoclonal antibody contained in the culture supernatant binds to agalactosylated human IgG or galactosylated human IgG. From these results, only hybridomas producing anti-agalactosylated human IgG antibodies that react with agalactosylated human IgG but do not bind to galactosylated human IgG can be selected. Examples of non-human animals in anti-non-human animal immunoglobulin antibodies include the non-human animals described above. When the non-human animal is a mouse, examples of anti-non-human animal immunoglobulin antibodies include anti-mouse IgG antibodies.

[0052] The agalactosylated human IgG and galactosylated human IgG used for hybridoma selection can be the above-mentioned agalactosylated human IgG and galactosylated human IgG.

[0053] Step (5) Step (5) is a step of collecting the anti-agalactosylated human IgG antibody of the present disclosure. Examples of methods for collecting anti-agalactosylated human IgG antibodies include culturing hybridomas selected by the method described in step (4) above using the method in step (3), and collecting the antibody from the resulting culture supernatant using a molecule that binds to the antibody. Alternatively, anti-agalactosylated human IgG antibodies can be collected from the culture supernatant obtained by the above method using a gel filtration column or ion exchange column.

[0054] The molecule that binds to the antibody in step (5) is not particularly limited as long as it can bind to the anti-agalactosylated human IgG antibody, and examples thereof include protein A, protein G, and antibodies that bind to the Fc region of non-human animal immunoglobulin. Examples of the non-human animal include the non-human animals described above.

[0055] [3] Method for measuring agalactosylated human IgG The method for measuring agalactosylated human IgG of the present disclosure is a method for measuring agalactosylated human IgG using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. Specific embodiments of the measurement method of the present disclosure are shown below.

[0056] Measurement method 1 (sandwich method 1): A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with an anti-agalactosylated human IgG antibody or an antibody fragment thereof to form an immune complex A1 containing the anti-agalactosylated human IgG antibody or an antibody fragment thereof and the agalactosylated human IgG; (2) reacting the immune complex A1 formed in step (1) with an antibody or antibody fragment thereof that binds to human IgG to form an immune complex A2 containing the anti-agalactosylated human IgG antibody or antibody fragment thereof, the agalactosylated human IgG, and an antibody or antibody fragment thereof that binds to human IgG; and (3) measuring the immune complex A2 formed in step (2).

[0057] The following step (4) can be carried out after step (3) or in parallel with step (3), and the following step (5) can be carried out after steps (3) and (4). The "agalactosylated human IgG of known concentration" below can be, for example, agalactosylated human IgG prepared by the above-described immunogen preparation method. The concentration of agalactosylated human IgG in the "agalactosylated human IgG of known concentration" can be calculated, for example, from the absorbance and molar extinction coefficient of the agalactosylated human IgG. Human IgG can also be used as the "agalactosylated human IgG of known concentration" below. In this case, the concentration of agalactosylated human IgG in the "agalactosylated human IgG of known concentration" can be expressed as the ratio of "agalactosylated human IgG / total human IgG" in the human IgG. This ratio can be determined by performing glycan analysis of human IgG using liquid chromatography-mass spectrometry (LC-MS) or the like and calculating the ratio of "agalactosylated human IgG / total human IgG" in the human IgG. Furthermore, when human IgG is used as the "agalactosylated human IgG of known concentration," the concentration of agalactosylated human IgG in the "agalactosylated human IgG of known concentration" can also be expressed as a concentration such as "mass of agalactosylated human IgG per unit volume," taking into account the ratio of "agalactosylated human IgG / total human IgG" obtained from the results of the glycan analysis. As the human IgG, for example, the human IgG described above can be used. (4) performing steps (1) to (3) above using agalactosylated human IgG of known concentration instead of the sample, and preparing a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of immune complex A2; (5) determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (4) and the measured value of immune complex A2 measured in step (3).

[0058] Steps (1) and (2) may be performed sequentially or simultaneously. A washing step may be inserted between steps (1) and (2). Furthermore, a washing step may be inserted between steps (2) and (3). In step (1), the anti-agalactosylated human IgG antibody or antibody fragment thereof may or may not be immobilized on an insoluble carrier, but is preferably immobilized. The antibody or antibody fragment thereof that binds to human IgG may or may not be labeled with a labeling substance, but is preferably labeled. When a labeled antibody or antibody fragment thereof that binds to human IgG is used, the labeling substance of the immune complex A2 may be measured in step (3).

[0059] When an anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized on an insoluble carrier, the insoluble carrier on which the anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized may be produced in the reaction solution for the antigen-antibody reaction. In this case, the anti-agalactosylated human IgG antibody or an antibody fragment thereof bound to one of a pair of affinity substances (A) is reacted with the insoluble carrier on which the other of the pair of affinity substances (a) is bound in the reaction solution for the antigen-antibody reaction, thereby producing the insoluble carrier on which the anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized in the reaction solution for the antigen-antibody reaction. Examples of combinations of A-a include the following combinations. Biotin-avidins (avidin, neutravidin, streptavidin, etc.); Avidins (avidin, neutravidin, streptavidin, etc.)-biotin; Fc region of anti-agalactosylated human IgG antibody or antibody fragment thereof - an antibody that binds to the Fc region.

[0060] The antibody fragment of the present disclosure that binds to human IgG is not particularly limited as long as it is an antibody fragment that reacts with human IgG, and examples thereof include Fab obtained by treating IgG with papain, F(ab') obtained by treating IgG with pepsin, and the like. ２Examples of such antibodies include Fab' obtained by treating IgG with pepsin followed by reduction, single-chain antibodies (scFv) in which the variable region of the antibody light chain (VL region) and the variable region of the antibody heavy chain (VH region) are artificially linked as a single polypeptide, and fragments of the variable region of the antibody heavy chain (VHH region) of single-chain antibodies that do not have the antibody light chain, such as those found in camelids.

[0061] Measurement method 2 (sandwich method 2): A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with an antibody or antibody fragment thereof that binds to human IgG to produce an immune complex B1 containing the antibody or antibody fragment thereof that binds to human IgG and agalactosylated human IgG; (2) reacting the immune complex B1 produced in step (1) with an anti-agalactosylated human IgG antibody or antibody fragment thereof to produce an immune complex B2 containing the antibody or antibody fragment thereof that binds to human IgG, agalactosylated human IgG, and the anti-agalactosylated human IgG antibody or antibody fragment thereof; and (3) measuring the immune complex B2 produced in step (2).

[0062] The following step (4) can be performed after step (3) or in parallel with step (3), and the following step (5) can be performed after steps (3) and (4). The "agalactosylated human IgG of known concentration" described below can be, for example, the "agalactosylated human IgG of known concentration" described above. (4) A step of performing steps (1) to (3) above using agalactosylated human IgG of known concentration instead of the sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of immune complex B2; (5) A step of determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (4) and the measured value of immune complex B2 measured in step (3).

[0063] Steps (1) and (2) may be performed sequentially or simultaneously. A washing step may be inserted between steps (1) and (2). Furthermore, a washing step may be inserted between steps (2) and (3). In step (1), the antibody or antibody fragment thereof that binds to human IgG may or may not be immobilized on an insoluble carrier, but is preferably immobilized. The anti-agalactosylated human IgG antibody or antibody fragment thereof may or may not be labeled with a labeling substance, but is preferably labeled. When a labeled anti-agalactosylated human IgG antibody or antibody fragment thereof is used, the labeling substance of the immune complex B2 may be measured in step (3).

[0064] When an antibody or antibody fragment thereof that binds to human IgG is immobilized on an insoluble support, the insoluble support to which the antibody or antibody fragment thereof that binds to human IgG is immobilized may be produced in the reaction solution of the antigen-antibody reaction. In this case, the antibody or antibody fragment thereof that binds to human IgG, bound to one of a pair of affinity substances (B), is reacted with an insoluble support to which the other of the pair of affinity substances (b) is bound in the reaction solution of the antigen-antibody reaction, thereby producing an insoluble support to which the antibody or antibody fragment thereof that binds to human IgG is immobilized in the reaction solution of the antigen-antibody reaction. Examples of B-b combinations include the following: biotin-avidins (avidin, neutravidin, streptavidin, etc.); avidins (avidin, neutravidin, streptavidin, etc.)-biotin; the Fc region of an antibody or antibody fragment thereof that binds to human IgG-antibody that binds to the Fc region.

[0065] The fragment of the antibody that binds to human IgG is not particularly limited as long as it is an antibody fragment that reacts with human IgG, and examples thereof include the aforementioned fragments of the antibody that bind to human IgG.

[0066] Measurement method 3 (competitive method 1): A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with a labeled competitor, in which a label is bound to a competitor of agalactosylated human IgG, and an anti-agalactosylated human IgG antibody or an antibody fragment thereof immobilized on an insoluble carrier, to form, on the insoluble carrier, an immune complex C1 comprising the anti-agalactosylated human IgG antibody or an antibody fragment thereof and the labeled competitor, and an immune complex C2 comprising the anti-agalactosylated human IgG antibody or an antibody fragment thereof and agalactosylated human IgG; (2) measuring the labeled competitor in the immune complex C1 comprising the anti-agalactosylated human IgG antibody or an antibody fragment thereof and the labeled competitor formed on the insoluble carrier in step (1).

[0067] The following step (3) can be performed after step (2) or in parallel with step (2), and the following step (4) can be performed after steps (2) and (3). The "agalactosylated human IgG of known concentration" below can be, for example, the "agalactosylated human IgG of known concentration" described above. (3) A step of performing steps (1) and (2) above using agalactosylated human IgG of known concentration instead of a sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of the labeled substance; (4) A step of determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (3) and the measured value of the labeled substance measured in step (2).

[0068] A washing step may be inserted between step (1) and step (2). In step (1), the sample is reacted with an anti-agalactosylated human IgG antibody or its antibody fragment immobilized on an insoluble carrier, and then the labeled competitor may be added to the reaction solution for the reaction. Alternatively, the insoluble carrier on which the anti-agalactosylated human IgG antibody or its antibody fragment is immobilized may be produced in the reaction solution for the antigen-antibody reaction. In this case, the insoluble carrier on which the anti-agalactosylated human IgG antibody or its antibody fragment is immobilized can be produced in the reaction solution for the antigen-antibody reaction by reacting an anti-agalactosylated human IgG antibody or its antibody fragment bound to one of a pair of affinity substances (C) with an insoluble carrier bound to the other of the pair of affinity substances (c) in the reaction solution for the antigen-antibody reaction. Examples of combinations of C-c include the following: Biotin-avidins (avidin, neutravidin, streptavidin, etc.); Avidins (avidin, neutravidin, streptavidin, etc.)-biotin; Fc region of anti-agalactosylated human IgG antibody or antibody fragment thereof - an antibody that binds to the Fc region.

[0069] Measurement method 4 (competitive method 2): A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with an anti-agalactosylated human IgG antibody or an antibody fragment thereof and a competitor of the agalactosylated human IgG immobilized on an insoluble carrier to form an immune complex D1 on the insoluble carrier, the immune complex D1 comprising the competitor and the anti-agalactosylated human IgG antibody or an antibody fragment thereof; and (2) measuring the immune complex D1 formed on the insoluble carrier in step (1).

[0070] The following step (3) can be performed after step (2) or in parallel with step (2), and the following step (4) can be performed after steps (2) and (3). The "agalactosylated human IgG of known concentration" described below can be, for example, the "agalactosylated human IgG of known concentration" described above. (3) A step of performing steps (1) and (2) above using agalactosylated human IgG of known concentration instead of a sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of the immune complex D1; (4) A step of determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (3) and the measured value of the immune complex D1 measured in step (2).

[0071] A washing step may be inserted between step (1) and step (2). Furthermore, the anti-agalactosylated human IgG antibody or its antibody fragment may be labeled with a labeling substance. When a labeled anti-agalactosylated human IgG antibody or its antibody fragment is used, the labeling substance of the immune complex D1 may be measured in step (2).

[0072] In step (1), after reacting the sample with the anti-agalactosylated human IgG antibody or its antibody fragment, a competitor of the agalactosylated human IgG immobilized on an insoluble carrier may be added to the reaction solution. Alternatively, the insoluble carrier to which the competitor of the agalactosylated human IgG is immobilized may be generated in the reaction solution of the antigen-antibody reaction. In this case, the insoluble carrier to which the competitor of the agalactosylated human IgG is immobilized can be generated in the reaction solution of the antigen-antibody reaction by reacting, in the reaction solution of the antigen-antibody reaction, a competitor of the agalactosylated human IgG bound to one of a pair of affinity substances (D) with an insoluble carrier to which the other of the pair of affinity substances (d) is bound. Examples of combinations of D-d include the following: Biotin-avidins (avidin, neutravidin, streptavidin, etc.); Avidins (avidin, neutravidin, streptavidin, etc.)-biotin.

[0073] Measurement method 5 (Method 1 for measuring antigen-specific agalactosylated human IgG) A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with an antigen to which the agalactosylated human IgG reacts to produce an immune complex E1 containing the antigen and the agalactosylated human IgG; (2) reacting the immune complex E1 produced in step (1) with an anti-agalactosylated human IgG antibody or an antibody fragment thereof to produce an immune complex E2 containing the antigen, the agalactosylated human IgG, and the anti-agalactosylated human IgG antibody or an antibody fragment thereof; and (3) measuring the immune complex E2 produced in step (2).

[0074] The following step (4) can be performed after or in parallel with step (3), and the following step (5) can be performed after steps (3) and (4). The "agalactosylated human IgG of known concentration" described below can be, for example, the "agalactosylated human IgG of known concentration" described above. (4) Performing steps (1) to (3) using agalactosylated human IgG of known concentration instead of the sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of immune complex E2; (5) Determining the concentration of agalactosylated human IgG in the sample from the calibration curve prepared in step (4) and the measured value of immune complex E2 measured in step (3).

[0075] Steps (1) and (2) may be performed sequentially or simultaneously. A washing step may be inserted between steps (1) and (2). Furthermore, a washing step may be inserted between steps (2) and (3). In step (1), the antigen with which the agalactosylated human IgG reacts may or may not be immobilized on an insoluble carrier, but is preferably immobilized. The anti-agalactosylated human IgG antibody or antibody fragment thereof may or may not be labeled with a labeling substance, but is preferably labeled. When a labeled anti-agalactosylated human IgG antibody or antibody fragment thereof is used, the labeling substance of the immune complex E2 may be measured in step (3).

[0076] When the antigen to which agalactosylated human IgG reacts is immobilized on an insoluble carrier, the insoluble carrier on which the antigen to which agalactosylated human IgG reacts may be immobilized in the reaction solution for the antigen-antibody reaction. In this case, the antigen to which agalactosylated human IgG reacts, bound to one of a pair of affinity substances (E), is reacted with an insoluble carrier to which the other of the pair of affinity substances (e) is bound in the reaction solution for the antigen-antibody reaction, thereby producing the insoluble carrier on which the antigen to which agalactosylated human IgG reacts immobilized in the reaction solution for the antigen-antibody reaction. Examples of combinations of E-e include the following: biotin-avidin (avidin, neutravidin, streptavidin, etc.); avidin (avidin, neutravidin, streptavidin, etc.)-biotin.

[0077] Measurement method 6 (Method 2 for measuring antigen-specific agalactosylated human IgG) A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with an anti-agalactosylated human IgG antibody or an antibody fragment thereof to form an immune complex F1 containing the anti-agalactosylated human IgG antibody or an antibody fragment thereof and the agalactosylated human IgG; (2) reacting the immune complex F1 formed in step (1) with an antigen to which the agalactosylated human IgG reacts to form an immune complex F2 containing the anti-agalactosylated human IgG antibody or an antibody fragment thereof, the agalactosylated human IgG, and the antigen; and (3) measuring the immune complex F2 formed in step (2).

[0078] The following step (4) can be performed after step (3) or in parallel with step (3), and the following step (5) can be performed after steps (3) and (4). The "agalactosylated human IgG of known concentration" described below can be, for example, the "agalactosylated human IgG of known concentration" described above. (4) A step of performing steps (1) to (3) above using agalactosylated human IgG of known concentration instead of the sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of immune complex F2; (5) A step of determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (4) and the measured value of immune complex F2 measured in step (3).

[0079] Steps (1) and (2) may be performed sequentially or simultaneously. A washing step may be inserted between steps (1) and (2). Furthermore, a washing step may be inserted between steps (2) and (3). In step (1), the anti-agalactosylated human IgG antibody or its antibody fragment may or may not be immobilized on an insoluble carrier, but is preferably immobilized. The antigen with which the agalactosylated human IgG reacts may or may not be labeled with a labeling substance, but is preferably labeled. When a labeled antigen with which the agalactosylated human IgG reacts is used, the labeling substance of the immune complex F2 may be measured in step (3).

[0080] When an anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized on an insoluble carrier, the insoluble carrier on which the anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized may be produced in the reaction solution for the antigen-antibody reaction. In this case, the anti-agalactosylated human IgG antibody or an antibody fragment thereof bound to one of a pair of affinity substances (F) is reacted with the insoluble carrier on which the other of the pair of affinity substances (f) is bound in the reaction solution for the antigen-antibody reaction, thereby producing the insoluble carrier on which the anti-agalactosylated human IgG antibody or an antibody fragment thereof is immobilized in the reaction solution for the antigen-antibody reaction. Examples of combinations of F-f include the following: Biotin-avidins (avidin, neutravidin, streptavidin, etc.); Avidins (avidin, neutravidin, streptavidin, etc.)-biotin; Fc region of anti-agalactosylated human IgG antibody or antibody fragment thereof - an antibody that binds to the Fc region.

[0081] Measurement Method 7 (Latex Agglutination Immunoassay): A method for measuring agalactosylated human IgG in a sample, comprising the following steps: (1) reacting the sample with latex particles bound to anti-agalactosylated human IgG antibodies or antibody fragments thereof to generate an immune complex G1 containing agalactosylated human IgG and latex particles bound to the anti-agalactosylated human IgG antibodies or antibody fragments thereof; and (2) measuring the agglutination of latex particles caused by the binding of agalactosylated human IgG to the latex particles bound to the anti-agalactosylated human IgG antibodies or antibody fragments thereof to obtain a measurement value. The following step (Z) may be included before step (1): (Z) mixing the sample with an aqueous medium. Examples of aqueous media include the aqueous media described below.

[0082] The following step (3) can be performed after step (2) or in parallel with step (2), and the following step (4) can be performed after steps (2) and (3). The "agalactosylated human IgG of known concentration" below can be, for example, the "agalactosylated human IgG of known concentration" described above. (3) A step of performing the above steps (1) and (2) using agalactosylated human IgG of known concentration instead of a sample to prepare a calibration curve showing the relationship between the agalactosylated human IgG concentration and the measured value of agglutination; (4) A step of determining the agalactosylated human IgG concentration in the sample from the calibration curve prepared in step (3) and the measured value of agglutination of latex particles measured in step (2).

[0083] In measurement method 7, one type of anti-agalactosylated human IgG antibody or its antibody fragment may be used, or two or more types of anti-agalactosylated human IgG antibodies or their antibody fragments may be used in combination. Furthermore, in step (1) of measurement method 7, latex particles bound to an anti-human IgG antibody or its antibody fragment may be further added to generate immune complex G2 containing agalactosylated human IgG, latex particles bound to the anti-agalactosylated human IgG antibody or its antibody fragment, and latex particles bound to the anti-human IgG antibody or its antibody fragment. In this case, step (2) is a step of measuring the agglutination of latex particles caused by binding between the agalactosylated human IgG, the latex particles bound to the anti-agalactosylated human IgG antibody or its antibody fragment, and the latex particles bound to the anti-human IgG antibody or its antibody fragment, to obtain a measurement value.

[0084] The aggregation in step (2) can be detected by measuring absorbance, transmitted light intensity, scattered light intensity, or the like.

[0085] Examples of latex particles in Measurement Method 7 include fine particles of organic polymeric substances, fine particles of inorganic oxides, and fine particles whose surfaces as core particles have been surface-treated with an organic substance or the like. Specific examples include synthetic resins such as polystyrene, copolymers containing styrene as a main component, polyvinyl chloride, polypropylene, (meth)acrylic resin, and polymethyl methacrylate. In Measurement Method 7, one type of latex particle may be used alone, or two or more types may be used in combination.

[0086] In measurement method 7, the method for binding "anti-agalactosylated human IgG antibody or antibody fragment thereof" or "anti-human IgG antibody or antibody fragment thereof" to latex particles is not particularly limited as long as it enables the measurement method of the present disclosure, and may be, for example, binding by physical adsorption or chemical bonding. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonding include covalent bonding and coordinate bonding. Alternatively, binding may be performed by utilizing the specific bond between a pair of affinity substances such as biotin and avidins (avidin, streptavidin, neutravidin, etc.).

[0087] The sample used in the measurement method of the present disclosure is not particularly limited as long as it allows the measurement method of the present disclosure to be used, and examples thereof include biological samples and buffer solutions containing agalactosylated human IgG. Biological samples include, for example, whole blood (blood), blood cells, serum, plasma, cerebrospinal fluid, urine, seminal plasma, amniotic fluid, saliva, feces, tissue, and cultured cells, with serum and plasma being preferred. The buffer solution for the "buffer solution containing agalactosylated human IgG" is not particularly limited as long as it allows the measurement method of the present disclosure to be used, and examples thereof include the buffer solutions described below.

[0088] The competitor for agalactosylated human IgG of the present disclosure is not particularly limited as long as it enables measurement by the above-described competitive method 1 or 2 of the present disclosure, and a substance having the same structure as the epitope recognized by the anti-agalactosylated human IgG antibody of the present disclosure is preferred. Furthermore, the binding strength of the anti-agalactosylated human IgG antibody of the present disclosure to the competitor is preferably similar to the binding strength of the anti-agalactosylated human IgG antibody of the present disclosure to the agalactosylated human IgG. Examples of such competitors include agalactosylated human IgG itself and the Fc fragment of agalactosylated human IgG.

[0089] The antigen to which agalactosylated human IgG reacts is not particularly limited as long as it can be bound to the antigen, and examples thereof include the antigens mentioned above.

[0090] The measurement method of the present disclosure can be applied to both dry chemistry and reactions in solution. In the measurement method of the present disclosure, the reaction temperature for the antigen-antibody reaction, i.e., step (1) and step (2) in measurement methods 1, 2, 5, and 6, and step (1) in measurement methods 3, 4, and 7, is not particularly limited as long as it is a reaction temperature that enables the measurement method of the present disclosure, and is typically 0 to 50°C, preferably 4 to 40°C. The reaction time is not particularly limited as long as it is a reaction time that enables the measurement method of the present disclosure, and is typically 1 minute to 72 hours, and preferably 5 minutes to 20 hours.

[0091] The washing solution used in the washing step is not particularly limited as long as it is capable of removing the reaction solution of the antigen-antibody reaction of the present disclosure, and examples thereof include phosphate-buffered saline [10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2 (hereinafter referred to as PBS)], PBS containing a surfactant, an aqueous medium described below, etc. Examples of such surfactants include nonionic surfactants such as Tween 20.

[0092] The insoluble carrier in the measurement method of the present disclosure is not particularly limited as long as it is an insoluble carrier that enables the measurement method of the present disclosure, and examples thereof include glass slides, ELISA plates (microtiter plates), beads, latex particles, magnetic particles, filters, films, and membranes. Materials for the insoluble carrier include those made of inorganic substrates, polysaccharide substrates, polymer substrates, etc., such as glass, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and polyurethane.

[0093] The binding of the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure to an insoluble carrier, the binding of an antibody or antibody fragment thereof that binds to human IgG to an insoluble carrier, the binding of an antigen with which agalactosylated human IgG reacts to an insoluble carrier, and the binding of a competitor of agalactosylated human IgG to an insoluble carrier can be exemplified by physical adsorption, chemical bonding, etc. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonding include covalent bonding and coordinate bonding. Note that "binding of an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure to an insoluble carrier, binding of an antibody that binds to human IgG or an antibody fragment thereof to an insoluble carrier, binding of an antigen with which agalactosylated human IgG reacts to an insoluble carrier, and binding of a competitor of agalactosylated human IgG to an insoluble carrier" is synonymous with "immobilization of an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure to an insoluble carrier, immobilization of an antibody that binds to human IgG or an antibody fragment thereof to an insoluble carrier, immobilization of an antigen with which agalactosylated human IgG reacts to an insoluble carrier, and immobilization of a competitor of agalactosylated human IgG to an insoluble carrier."

[0094] The anti-agalactosylated human IgG antibody or antibody fragment thereof, the antibody or antibody fragment thereof that binds to human IgG, the antigen with which agalactosylated human IgG reacts, or the competitor of agalactosylated human IgG of the present disclosure may be immobilized directly or indirectly to an insoluble carrier using the aforementioned physical adsorption and / or chemical binding. Examples of indirect binding methods include a method that utilizes the specific binding between a pair of affinity substances such as biotin and avidin (avidin, streptavidin, neutravidin, etc.), and a method that binds to an insoluble carrier by a covalent bond via a linker.

[0095] When the indirect binding method utilizes a pair of affinity substances, an insoluble support having an anti-agalactosylated human IgG antibody or its antibody fragment bound thereto can be obtained by binding an anti-agalactosylated human IgG antibody or its antibody fragment bound to one of the pair of affinity substances (Y) with an insoluble support having the other of the pair of affinity substances (y) bound thereto. In this case, the insoluble support having an anti-agalactosylated human IgG antibody or its antibody fragment bound thereto may be produced in the reaction solution of the antigen-antibody reaction.

[0096] Examples of a combination of affinity substances Y-y include the following: a combination of biotin and an avidin (avidin, neutravidin, streptavidin, etc.); a combination of an avidin (avidin, neutravidin, streptavidin, etc.) and biotin;

[0097] When the indirect binding method involves binding to an insoluble carrier by a covalent bond via a linker, the linker can be a molecule that can covalently bond both a functional group on the surface of the insoluble carrier and a functional group possessed by the anti-agalactosylated human IgG antibody or its antibody fragment. For example, a molecule that simultaneously has a first reactive group that can react with a functional group possessed by the anti-agalactosylated human IgG antibody or its antibody fragment, and a second reactive group that can react with a functional group on the surface of the insoluble carrier, wherein the first reactive group and the second reactive group are different groups, is preferably used.

[0098] Examples of functional groups possessed by an anti-agalactosylated human IgG antibody or antibody fragment thereof, and functional groups carried on the surface of an insoluble carrier include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinyl group, a maleimide group, etc. Examples of reactive groups in a linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen, pyridyl disulfide, vinyl sulfone, etc.

[0099] In step (3) of Measurement Methods 1, 2, 5, and 6, the method for measuring each immune complex (immune complex A2; immune complex B2; immune complex E2; immune complex F2) formed in step (2) of each measurement method is not particularly limited as long as it is a method that can measure each immune complex formed in step (2) of each measurement method, and examples thereof include a method using a quartz crystal oscillator, a method using an electrode, a method for measuring aggregation, and the like. Furthermore, when the antibody or antibody fragment thereof that binds to human IgG in step (2) of Measurement Method 1 is an antibody that binds to human IgG labeled with a labeling substance, when the anti-agalactosylated human IgG antibody or antibody fragment of the present disclosure in step (2) of Measurement Method 2 and Measurement Method 5 is an anti-agalactosylated human IgG antibody or antibody fragment labeled with a labeling substance, and when the antigen with which agalactosylated human IgG reacts in step (2) of Measurement Method 6 is an antigen with which agalactosylated human IgG labeled with a labeling substance reacts, in step (3), examples of a method for measuring the immune complex produced in step (2) of each measurement method include a method for measuring the labeling substance in each immune complex produced in step (2) of each measurement method.

[0100] In step (2) of measurement method 3, the method for measuring the immune complex C1 produced in step (1) is not particularly limited as long as it is a method that can measure the immune complex C1 produced in step (1). Examples of the method include a method using a quartz crystal oscillator, a method using an electrode, a method for measuring aggregation, a method for measuring a labeled substance, and the like.

[0101] In step (2) of measurement method 4, the method for measuring the immune complex D1 produced in step (1) is not particularly limited as long as it is a method that can measure the immune complex D1 produced in step (1), and examples include a method using a quartz crystal oscillator, a method using an electrode, a method for measuring aggregation, etc. Furthermore, when the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure in step (1) is an anti-agalactosylated human IgG antibody or antibody fragment thereof labeled with a labeling substance, an example of a method for measuring the immune complex D1 produced in step (1) in step (2) is a method of measuring the labeling substance in the immune complex produced in step (1), etc.

[0102] Examples of labeling substances include enzymes, radioisotopes, fluorescent substances, luminescent substances, DNA, RNA, coenzymes or substances that specifically bind to coenzymes (biotin, avidin), tags, substances that absorb in the ultraviolet to infrared region, chromogenic fine particles, fluorescent fine particles, metallic fine particles (gold colloid particles, etc.), magnetic substances, substances that have properties as spin labeling agents, and colored latex.

[0103] Examples of the enzyme include alkaline phosphatase, peroxidase, glucuronidase, luciferase, etc. Examples of the radioisotope include: ３ H. １４ C. ３５ S. ３２ P. １２５ I, １３１ Examples of fluorescent substances include FITC (fluorescein isothiocyanate), RITC (rhodamine B isothiocyanate), etc. Examples of luminescent substances include acridinium and its derivatives, ruthenium complex compounds, lophine, etc.

[0104] The immune complex can be measured by detecting the signal generated from these labeling substances. The method for measuring the signal generated from the labeling substance can be appropriately selected depending on the labeling substance used. When the labeling substance is a chromogenic substance, i.e., a substance that absorbs light of a certain wavelength, the labeling substance can be measured by measuring absorbance using a spectrophotometer, a multiwell plate reader, or the like. When the labeling substance is a fluorescent substance, the labeling substance can be measured by measuring fluorescence intensity using a fluorometer, a fluorescent multiwell plate reader, or the like. When the labeling substance is a luminescent substance, the labeling substance can be measured by measuring luminescence intensity using a luminescence photometer, a luminescence multiwell plate reader, or the like. When the labeling substance is a radioisotope, the labeling substance can be measured by measuring radioactivity using a scintillation counter, a γ-well counter, or the like. When the labeling substance is an enzyme, the labeling substance can be measured by measuring enzymatic activity. For example, the labeling substance can be measured by reacting the enzyme's substrate with the enzyme and measuring the resulting substance.

[0105] When the enzyme is peroxidase, peroxidase activity can be measured by, for example, absorbance, fluorescence, or luminescence. Examples of methods for measuring peroxidase activity by absorbance include reacting peroxidase with a combination of its substrates, hydrogen peroxide, and an oxidative color-developing chromogen, and measuring the absorbance of the reaction solution using a spectrophotometer or a multiwell plate reader. Examples of oxidative color-developing chromogens include leuco chromogens and oxidative coupling color-developing chromogens.

[0106] When the enzyme substance is alkaline phosphatase, alkaline phosphatase activity can be measured, for example, by a luminescence method, etc. Examples of methods for measuring alkaline phosphatase activity by a luminescence method include a method in which alkaline phosphatase is reacted with its substrate and the intensity of the generated luminescence is measured using a luminescence photometer, a luminescence multiwell plate reader, or the like.

[0107] Examples of the substrate for alkaline phosphatase include 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro-5-{4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 ３．７ ]decane]-4-yl}phenyl phosphate disodium salt (CDP-Star ＴＭ ), 3-[4'-methoxy-5-chlorospiro[tricyclo[3.3.1.1 ３．７ ]decane-2,3'-[1,2]dioxetan]-4'-yl]phenyl phosphate disodium salt (CSPD ＴＭ ), 9-[(phenyloxy)(phosphoryloxy)methylidene]-10-methylacridan disodium salt, 9-[(4-chlorophenylthio)(phosphoryloxy)methylidene]-10-methylacridan disodium salt (Lumigen) ＴＭ APS-5) and the like.

[0108] In the measurement methods of the present disclosure, the antigen-antibody reaction, i.e., step (1) and step (2) of measurement methods 1, 2, 5, and 6, and step (1) of measurement methods 3, 4, and 7, may be carried out in an aqueous medium. Furthermore, in the measurement methods of the present disclosure, the detection step may be carried out in an aqueous medium. The aqueous medium used in the measurement methods of the present disclosure is not particularly limited as long as it is an aqueous medium that enables the measurement methods of the present disclosure. Examples include deionized water, distilled water, and buffer solutions, and an aqueous medium containing a buffer solution is preferred. The concentration of the buffer solution is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and particularly preferably 0.01 to 0.1 mol / L. The buffer used to prepare the buffer solution is not particularly limited as long as it has buffering capacity, and examples thereof include lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbiturate buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, Tris buffer, Good's buffer, etc., each having a pH of 1 to 11. One type of buffer may be used alone, or two or more types may be used in combination.

[0109] Examples of the Good's buffer include 2-morpholinoethanesulfonic acid (MES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, 2-[N-(2-acetamido)amino]ethanesulfonic acid (ACES) buffer, 3 ...sulfonic acid (ACES) buffer, 3-morpholinoethanesulfonic acid (MES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminomethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminomethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminomethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminomethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminomethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)aminom N-(2-hydroxyethyl)-N'-(2-sulfoethyl)piperazine (HEPES) buffer, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2-[N,N-bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2-{N-[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer, N-(2-hydroxyethyl)-N'-(2-sulfoethyl)piperazine (HEPES) buffer, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (MOPS) buffer, Dimethylaminopropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPSO) buffer, piperazine-N,N'-bis(2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N-(2-hydroxyethyl)-N'-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO) buffer, N-(2-hydroxyethyl)-N'-(3-sulfopropyl)piperazine (EPPS) buffer, Tricine [ [N-tris(hydroxymethyl)methylglycine] buffer, bicine [N,N-bis(2-hydroxyethyl)glycine] buffer, 3-[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS) buffer, 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer, 3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) buffer, 3-(N-cyclohexylamino)propanesulfonic acid (CAPS) buffer, and the like.

[0110] The aqueous medium according to the present disclosure may further contain metal ions, salts, sugars, preservatives, proteins, protein stabilizers, etc. Examples of metal ions include magnesium ions, manganese ions, zinc ions, etc. Examples of salts include sodium chloride and potassium chloride, etc. Examples of sugars include mannitol and sorbitol, etc. Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), BioAce, Proclin 300, Proxel GXL, etc. Examples of proteins include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, Block Ace (manufactured by KAC Corporation), etc. Examples of protein stabilizers include peroxidase stabilizing buffer (manufactured by DakoCytomation), etc.

[0111] According to the method for measuring agalactosylated human IgG in a sample of the present disclosure, the concentration of agalactosylated human IgG in the sample can be obtained. In the present disclosure, "agalactosylated human IgG concentration" is used synonymously with the "measured value of agalactosylated human IgG" described below. In the present disclosure, "agalactosylated human IgG concentration" can also be quantified and displayed as signal intensity (e.g., absorbance) based on the amount of agalactosylated human IgG present per unit volume, depending on the measurement method. The agalactosylated human IgG concentration refers to the amount of agalactosylated human IgG present per unit amount of sample, and is typically expressed as the mass or moles of agalactosylated human IgG per unit volume of sample. Furthermore, the agalactosylated human IgG concentration is not necessarily quantified as the amount of agalactosylated human IgG per unit volume of sample; it can also be, for example, the amount of agalactosylated human IgG per unit weight of sample.

[0112] [4] Agalactosylated human IgG measurement reagent The agalactosylated human IgG measurement reagent of the present disclosure is a reagent used in the agalactosylated human IgG measurement method of the present disclosure, and is a reagent characterized by containing the anti-agalactosylated human IgG antibody or an antibody fragment thereof of the present disclosure. Specific embodiments of the measurement reagent of the present disclosure are shown below.

[0113] Measurement Reagent 1: A reagent for measuring agalactosylated human IgG in a sample, comprising the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure.

[0114] Measurement Reagent 2: A reagent for measuring agalactosylated human IgG in a sample, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier. The insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, instead of the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized, the measurement reagent of the present disclosure includes an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (G) and an insoluble carrier to which the other of a pair of affinity substances (g) is bound. Examples of combinations of a pair of affinity substances, i.e., combinations of G and g, include the following combinations. Biotin-avidins (avidin, neutravidin, streptavidin, etc.); Avidins (avidin, neutravidin, streptavidin, etc.)-biotin; Fc region of anti-agalactosylated human IgG antibody or antibody fragment thereof - an antibody that binds to the Fc region.

[0115] Measurement Reagent 3: A reagent for measuring agalactosylated human IgG in a sample, comprising the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and an antibody that binds to human IgG or an antibody fragment thereof.

[0116] Measurement Reagent 4: A reagent for measuring agalactosylated human IgG in a sample, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and an antibody or antibody fragment thereof that binds to human IgG. The antibody or antibody fragment thereof that binds to human IgG is preferably labeled with a labeling substance. Furthermore, the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, instead of the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized, the measurement reagent of the present disclosure includes an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (A), and an insoluble carrier to which the other of a pair of affinity substances (a) is bound. Examples of combinations of affinity substances, i.e., the combination of A and a, include the combination of A and a described above.

[0117] Measurement Reagent 5: A reagent for measuring agalactosylated human IgG in a sample, comprising an antibody or antibody fragment thereof that binds to human IgG and an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, immobilized on an insoluble carrier. The anti-agalactosylated human IgG antibody or antibody fragment thereof is preferably labeled with a labeling substance. Furthermore, the insoluble carrier to which the antibody or antibody fragment thereof that binds to human IgG is immobilized may be generated in the reaction solution of the sample and the antibody or antibody fragment thereof that binds to human IgG. In this case, instead of an insoluble carrier to which an antibody or antibody fragment thereof that binds to human IgG is immobilized, the measurement reagent of the present disclosure includes an antibody or antibody fragment thereof that binds to human IgG, bound to one of a pair of affinity substances (B), and an insoluble carrier to which the other of a pair of affinity substances (b) is bound. Examples of combinations of affinity substances, i.e., the combination of B and b, include the combination of B and b described above.

[0118] Measurement Reagent 6: A reagent for measuring agalactosylated human IgG in a sample, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and a labeled competitor in which a label is bound to a competitor for agalactosylated human IgG. The insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the measurement reagent of the present disclosure includes, instead of the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized, an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (C), and an insoluble carrier to which the other of a pair of affinity substances (C) is bound. Examples of combinations of a pair of affinity substances, i.e., the combination of C and c, include the combination of C and c described above.

[0119] Measurement Reagent 7: A reagent for measuring agalactosylated human IgG in a sample, comprising an agalactosylated human IgG competitor immobilized on an insoluble carrier, and an anti-agalactosylated human IgG antibody or its antibody fragment of the present disclosure. The anti-agalactosylated human IgG antibody or its antibody fragment may be labeled with a labeling substance. Furthermore, the insoluble carrier to which the agalactosylated human IgG competitor is immobilized may be generated in the reaction solution of the agalactosylated human IgG competitor and the anti-agalactosylated human IgG antibody or its antibody fragment. In this case, the measurement reagent of the present disclosure includes, instead of an insoluble carrier to which an agalactosylated human IgG competitor is immobilized, an agalactosylated human IgG competitor bound to one of a pair of affinity substances (D) and an insoluble carrier to which the other of a pair of affinity substances (d) is bound. A combination of a pair of affinity substances, that is, a combination of D and d, includes, for example, the combination of D and d described above.

[0120] Measurement Reagent 8: A reagent for measuring agalactosylated human IgG in a sample, comprising the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure and an antigen to which agalactosylated human IgG reacts.

[0121] Measurement Reagent 9: A reagent for measuring agalactosylated human IgG in a sample, comprising an antigen to which agalactosylated human IgG reacts, immobilized on an insoluble carrier, and an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. The anti-agalactosylated human IgG antibody or antibody fragment thereof is preferably labeled with a labeling substance. Furthermore, the insoluble carrier to which the antigen to which agalactosylated human IgG reacts may be immobilized may be generated in the reaction solution between the sample and the antigen to which agalactosylated human IgG reacts. In this case, the measurement reagent of the present disclosure includes, instead of an insoluble carrier to which an antigen to which agalactosylated human IgG reacts immobilized is immobilized, an antigen to which agalactosylated human IgG reacts, bound to one of a pair of affinity substances (E), and an insoluble carrier to which the other of a pair of affinity substances (e) is bound. Examples of combinations of affinity substances, i.e., the combination of E and e, include the combination of E and e described above.

[0122] Measurement Reagent 10: A reagent for measuring agalactosylated human IgG in a sample, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and an antigen to which the agalactosylated human IgG reacts. The antigen to which the agalactosylated human IgG reacts is preferably labeled with a labeling substance. Furthermore, the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the measurement reagent of the present disclosure includes, instead of an insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized, an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (F) and an insoluble carrier to which the other of a pair of affinity substances (f) is bound. An example of a combination of affinity substances, that is, a combination of F and f, is the combination of F and f described above.

[0123] Measurement Reagent 11: A reagent for measuring agalactosylated human IgG in a sample, comprising latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure is bound.

[0124] Measurement Reagent 12: A reagent for measuring agalactosylated human IgG in a sample, comprising latex particles bound to the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and latex particles bound to an antibody that binds to human IgG or an antibody fragment thereof.

[0125] In the measurement reagent of the present disclosure, examples of the method for immobilizing an anti-agalactosylated human IgG antibody or its antibody fragment to an insoluble carrier, the method for immobilizing an antibody that binds to human IgG or its antibody fragment to an insoluble carrier, the method for binding an anti-agalactosylated human IgG antibody or its antibody fragment to latex particles, and the method for binding an antibody that binds to human IgG or its antibody fragment to latex particles include the methods described above. Furthermore, examples of the latex particles in the measurement reagent of the present disclosure include the latex particles described above. In the above "method for immobilizing an antibody or antibody fragment to an insoluble carrier" and "method for binding an antibody or antibody fragment to latex particles," "immobilize" and "bind" are synonymous.

[0126] [5] Agalactosylated Human IgG Measurement Kit The measurement reagent of the present disclosure can be in the form of a kit from the viewpoints of storage, transportation, distribution, etc. Examples of the kit form include a two-reagent kit and a three-reagent kit. Specific examples of the measurement kit of the present disclosure are shown below.

[0127] Measurement Kit 1: A kit comprising a first reagent containing the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and a second reagent containing an antibody that binds to human IgG or an antibody fragment thereof.

[0128] Assay Kit 2: A kit comprising a first reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and a second reagent containing an antibody or antibody fragment thereof that binds to human IgG. The antibody or antibody fragment thereof that binds to human IgG is preferably labeled with a labeling substance. The insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be produced in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (A) and an insoluble carrier bound to the other of the pair of affinity substances (a), as shown in the example of Assay Kit 3. Examples of combinations of affinity substances, i.e., the combination of A and a, include the aforementioned combination of A and a.

[0129] Measurement Kit 3: A kit comprising a first reagent containing an anti-agalactosylated human IgG antibody or its antibody fragment bound to one of a pair of affinity substances (A), a second reagent containing an insoluble carrier to which the other of the pair of affinity substances (a) is bound, and a third reagent containing an antibody that binds to human IgG or its antibody fragment. Examples of the combination of a pair of affinity substances, i.e., the combination of A and a, include the combination of A and a described above.

[0130] Assay Kit 4: A kit comprising a first reagent containing an antibody or antibody fragment thereof that binds to human IgG immobilized on an insoluble carrier, and a second reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. The anti-agalactosylated human IgG antibody or antibody fragment thereof is preferably labeled with a labeling substance. The insoluble carrier to which the antibody or antibody fragment thereof that binds to human IgG is immobilized may be generated in the reaction solution of the sample and the antibody or antibody fragment thereof that binds to human IgG. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, an antibody or antibody fragment thereof that binds to human IgG bound to one of a pair of affinity substances (B), and an insoluble carrier to which the other of the pair of affinity substances (b) is bound, as shown in Assay Kit 5. Examples of combinations of affinity substances, i.e., the combination of B and b, include the combination of B and b described above.

[0131] Measurement Kit 5: A kit comprising: a first reagent containing an antibody or antibody fragment thereof that binds to human IgG, bound to one of a pair of affinity substances (B); a second reagent containing an insoluble carrier to which the other of the pair of affinity substances (b) is bound; and a third reagent containing the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. Examples of the combination of a pair of affinity substances, i.e., the combination of B and b, include the combination of B and b described above.

[0132] Assay Kit 6: A kit comprising a first reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and a second reagent containing a labeled competitor in which a label is bound to a competitor of the agalactosylated human IgG. The insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the reaction between the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (C), and an insoluble carrier bound to the other of the pair of affinity substances (c), as shown in Assay Kit 7. Examples of combinations of affinity substances, i.e., the combination of C and c, include the aforementioned combination of C and c.

[0133] Measurement Kit 7: A kit comprising: a first reagent containing an anti-agalactosylated human IgG antibody or its antibody fragment bound to one of a pair of affinity substances (C); a second reagent containing an insoluble carrier bound to the other of the pair of affinity substances (c); and a third reagent containing a labeled competitor in which a label is bound to a competitor of agalactosylated human IgG. Examples of the combination of a pair of affinity substances, i.e., the combination of C and c, include the combination of C and c described above.

[0134] Assay Kit 8: A kit comprising a first reagent containing a competitor for agalactosylated human IgG immobilized on an insoluble carrier, and a second reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. The anti-agalactosylated human IgG antibody or antibody fragment thereof may be labeled with a labeling substance. Furthermore, the insoluble carrier to which the competitor for agalactosylated human IgG is immobilized may be generated in the reaction solution of the agalactosylated human IgG competitor and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, the competitor for agalactosylated human IgG bound to one of the pair of affinity substances (D) and the insoluble carrier bound to the other of the pair of affinity substances (d), as shown in Assay Kit 9. Examples of combinations of affinity substances, i.e., the combination of D and d, include the aforementioned combination of D and d.

[0135] Measurement Kit 9: A kit comprising: a first reagent containing a competitor of agalactosylated human IgG bound to one of a pair of affinity substances (D), a second reagent containing an insoluble carrier bound to the other of the pair of affinity substances (d), and a third reagent containing an anti-agalactosylated human IgG antibody or an antibody fragment thereof. Examples of the combination of a pair of affinity substances, i.e., the combination of D and d, include the combination of D and d described above.

[0136] Measurement Kit 10: A kit comprising a first reagent containing the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and a second reagent containing an antigen with which the agalactosylated human IgG reacts.

[0137] Assay Kit 11: A kit comprising a first reagent containing an antigen with which agalactosylated human IgG reacts, immobilized on an insoluble carrier, and a second reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. The anti-agalactosylated human IgG antibody or antibody fragment thereof is preferably labeled with a labeling substance. Furthermore, the insoluble carrier on which the antigen with which agalactosylated human IgG reacts may be immobilized may be generated in the reaction solution of the sample and the antigen with which agalactosylated human IgG reacts. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, one of a pair of affinity substances (E) bound to the antigen with which agalactosylated human IgG reacts, and an insoluble carrier with the other of the pair of affinity substances (e) bound to the insoluble carrier. For example, an example of the assay kit 12 is an example of a combination of affinity substances, i.e., the combination of E and e, such as the combination of E and e described above.

[0138] Measurement Kit 12: A kit comprising: a first reagent containing an antigen to which agalactosylated human IgG reacts, bound to one of a pair of affinity substances (E); a second reagent containing an insoluble carrier to which the other of the pair of affinity substances (e) is bound; and a third reagent containing an anti-agalactosylated human IgG antibody or an antibody fragment thereof. Examples of the combination of affinity substances, i.e., the combination of E and e, include the combination of E and e described above.

[0139] Assay Kit 13: A kit comprising a first reagent containing an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure immobilized on an insoluble carrier, and a second reagent containing an antigen to which the agalactosylated human IgG reacts. The antigen to which the agalactosylated human IgG reacts is preferably labeled with a labeling substance. Furthermore, the insoluble carrier to which the anti-agalactosylated human IgG antibody or antibody fragment thereof is immobilized may be generated in the reaction solution of the sample and the anti-agalactosylated human IgG antibody or antibody fragment thereof. In this case, the assay kit of the present disclosure preferably contains, in separate reagents, an anti-agalactosylated human IgG antibody or antibody fragment thereof bound to one of a pair of affinity substances (F) and an insoluble carrier bound to the other of the pair of affinity substances (f), as shown in the embodiment of Assay Kit 14. Examples of combinations of affinity substances, i.e., the combination of F and f, include the aforementioned combination of F and f.

[0140] Measurement Kit 14: A kit comprising: a first reagent containing an anti-agalactosylated human IgG antibody or its antibody fragment bound to one of a pair of affinity substances (F); a second reagent containing an insoluble carrier bound to the other of the pair of affinity substances (f); and a third reagent containing an antigen with which the agalactosylated human IgG reacts. Examples of the combination of a pair of affinity substances, i.e., the combination of F and f, include the combination of F and f described above. Examples of the anti-agalactosylated human IgG antibody or antibody fragment thereof, antibody or antibody fragment thereof that binds to human IgG, competitor of agalactosylated human IgG, labeling substance, antigen with which agalactosylated human IgG reacts, and insoluble carrier in the measurement reagent and measurement kit of the present disclosure include the above-mentioned anti-agalactosylated human IgG antibody or antibody fragment thereof, antibody or antibody fragment thereof that binds to human IgG, competitor of agalactosylated human IgG, labeling substance, antigen with which agalactosylated human IgG reacts, and insoluble carrier, respectively.

[0141] Measurement Kit 15: A kit comprising a first reagent containing an aqueous medium and a second reagent containing latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure is bound.

[0142] Measurement kit 16: A kit comprising a first reagent containing an aqueous medium, latex particles to which the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure is bound, and a second reagent containing latex particles to which an antibody that binds to human IgG or an antibody fragment thereof is bound. Examples of the aqueous medium in measurement kits 15 and 16 include the aqueous media described above.

[0143] The measurement reagent and measurement kit of the present disclosure may contain, as necessary, an aqueous medium, metal ions, salts, sugars, preservatives, proteins, protein stabilizers, etc. Examples of the aqueous medium, metal ions, salts, sugars, preservatives, proteins, and protein stabilizers include the aforementioned aqueous medium, metal ions, salts, sugars, preservatives, proteins, protein stabilizers, etc. The form of the measurement reagent and measurement kit of the present disclosure is not particularly limited as long as it is a form that enables the measurement method of the present disclosure, and may be in the form of, for example, a solution, a lyophilized form, or the like.

[0144] [6] Method for diagnosing inflammatory bowel disease using an anti-agalactosylated human IgG antibody Another aspect of the present disclosure is a method for measuring agalactosylated human IgG in a subject's biological sample, or a method comprising obtaining a measured value of agalactosylated human IgG in the subject's biological sample, using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, thereby collecting data for determining whether the subject has inflammatory bowel disease (hereinafter also referred to as a "data collection method"). Another aspect of the present disclosure is a method for diagnosing or assisting in the diagnosis of inflammatory bowel disease, using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, or a method comprising obtaining a measured value of agalactosylated human IgG in the subject's biological sample (hereinafter also referred to as a "diagnostic method" and a "diagnostic assisting method," respectively).

[0145] Methods for measuring agalactosylated human IgG in a biological sample from a subject using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, or methods comprising obtaining a measured value of agalactosylated human IgG in a biological sample from a subject, include, for example, the above-mentioned Measurement Methods 1 to 7. Biological samples used in data collection methods, diagnostic methods, or diagnostic support methods include the above-mentioned biological samples. Examples of inflammatory bowel diseases include ulcerative colitis and Crohn's disease.

[0146] In the present disclosure, the term "subject" refers to a concept that includes both humans suffering from inflammatory bowel disease and humans not suffering from inflammatory bowel disease, and may also refer to a human who may have inflammatory bowel disease. That is, the data collection method, diagnostic method, or diagnostic assistance method according to one embodiment may be a method used to determine or diagnose a human who exhibits symptoms that suggest the patient may have inflammatory bowel disease, or may be a method used to discover (screen) a human who may have inflammatory bowel disease in a health checkup or the like.

[0147] In one embodiment, the data collection method, diagnostic method, or diagnostic assistance method may include, in addition to obtaining a measured value of agalactosylated human IgG in a biological sample from a subject, determining that the subject has inflammatory bowel disease if the obtained measured value of agalactosylated human IgG is equal to or greater than a reference value, and determining that the subject does not have inflammatory bowel disease if the measured value is less than the reference value. Note that the above-mentioned "measured value of agalactosylated human IgG" is used synonymously with the agalactosylated human IgG concentration or agalactosylated human IgG amount obtained by the above-mentioned Measurement Methods 1 to 7.

[0148] The data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure makes a determination, for example, by comparing the amount of agalactosylated human IgG obtained by measuring agalactosylated human IgG in a biological sample from a subject with a reference value. That is, the data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure determines, for example, that the subject is likely to have inflammatory bowel disease if the amount of agalactosylated human IgG is elevated compared to the reference value, and determines that the subject is likely not to have inflammatory bowel disease if the measured value is decreased compared to the reference value.

[0149] The reference values ​​for the data collection method, diagnostic method, or diagnostic support method of the present disclosure can be determined by referring to the amount of agalactosylated human IgG in healthy individuals and patients who have been definitively diagnosed as suffering from inflammatory bowel disease.

[0150] Another aspect of the present disclosure is a measurement reagent for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Reagents 1 to 12. Also, another aspect of the present disclosure is a measurement kit for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Kits 1 to 16.

[0151] Another aspect of the present disclosure is a measurement reagent or measurement kit for performing a data collection method, diagnostic method, or diagnostic support method according to an aspect of the present disclosure, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human IgG is equal to or greater than a reference value, the subject is determined to be suffering from inflammatory bowel disease; and if the measured value of the agalactosylated human IgG is less than the reference value, the subject is determined not to be suffering from inflammatory bowel disease.

[0152] [7] Method for diagnosing chronic pancreatitis. Another aspect of the present disclosure is a method for collecting data for determining whether a subject has chronic pancreatitis by measuring agalactosylated human IgG in a biological sample from the subject (hereinafter also referred to as a "data collection method"). Another aspect of the present disclosure is a method for diagnosing or assisting in the diagnosis of chronic pancreatitis by measuring agalactosylated human IgG in a biological sample from the subject (hereinafter also referred to as a "diagnostic method" and a "diagnostic assisting method," respectively).

[0153] Examples of methods for measuring agalactosylated human IgG in a biological sample from a subject include immunoassay methods using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. Examples of immunoassay methods using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure include the above-mentioned measurement methods 1 to 7. Examples of biological samples used in the data collection method, diagnostic method, or diagnostic support method include the above-mentioned biological samples.

[0154] In the present disclosure, the term "subject" refers to a concept that includes both humans suffering from chronic pancreatitis and humans not suffering from chronic pancreatitis, and may also refer to humans who may have chronic pancreatitis. That is, the data collection method, diagnostic method, or diagnostic assistance method according to one embodiment may be a method used to determine or diagnose humans who exhibit symptoms that suggest chronic pancreatitis, or may be a method used to discover (screen) humans who may have chronic pancreatitis in a health checkup or the like.

[0155] In one embodiment, the data collection method, diagnostic method, or diagnostic assistance method may include, in addition to obtaining a measured value of agalactosylated human IgG in a biological sample from a subject, determining that the subject has chronic pancreatitis if the obtained measured value of agalactosylated human IgG is equal to or greater than a reference value, and determining that the subject does not have chronic pancreatitis if the measured value is less than the reference value. Here, the "measured value of agalactosylated human IgG" is synonymous with the agalactosylated human IgG concentration or amount obtained by the above-mentioned Measurement Methods 1 to 7.

[0156] The data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure makes a determination, for example, by comparing the amount of agalactosylated human IgG obtained by measuring agalactosylated human IgG in a biological sample from a subject with a reference value. That is, the data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure determines, for example, that a subject is likely to have chronic pancreatitis if the amount of agalactosylated human IgG is elevated compared to the reference value, and determines that a subject is likely not to have chronic pancreatitis if the measured value is decreased compared to the reference value.

[0157] The reference values ​​for the data collection method, diagnostic method, or diagnostic support method of the present disclosure can be determined by referring to the amount of agalactosylated human IgG in healthy individuals and patients who have been definitively diagnosed with chronic pancreatitis.

[0158] Another aspect of the present disclosure is a measurement reagent for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Reagents 1 to 12. Also, another aspect of the present disclosure is a measurement kit for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Kits 1 to 16.

[0159] Another aspect of the present disclosure is a measurement reagent or measurement kit for performing a data collection method, diagnostic method, or diagnostic support method according to an aspect of the present disclosure, including an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and a package insert explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human IgG is equal to or greater than a reference value, the subject is determined to be suffering from chronic pancreatitis; and if the measured value of the agalactosylated human IgG is less than the reference value, the subject is determined to not be suffering from chronic pancreatitis.

[0160] [8] Method for diagnosing chronic hepatitis Another aspect of the present disclosure is a method for collecting data for determining whether a subject has chronic hepatitis by measuring agalactosylated human IgG in a biological sample from the subject (hereinafter also referred to as a "data collection method"). Another aspect of the present disclosure is a method for diagnosing or assisting in the diagnosis of chronic hepatitis by measuring agalactosylated human IgG in a biological sample from the subject (hereinafter also referred to as a "diagnostic method" and a "diagnostic assisting method", respectively).

[0161] Examples of methods for measuring agalactosylated human IgG in a biological sample from a subject include immunoassay methods using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure. Examples of immunoassay methods using the anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure include the above-mentioned measurement methods 1 to 7. Examples of biological samples used in the data collection method, diagnostic method, or diagnostic support method include the above-mentioned biological samples.

[0162] In the present disclosure, the term "subject" refers to a concept that includes both humans suffering from chronic hepatitis and humans not suffering from chronic hepatitis, and may also refer to a human who may have chronic hepatitis. That is, the data collection method, diagnostic method, or diagnostic assistance method according to one embodiment may be a method used to determine or diagnose a human who exhibits symptoms that suggest chronic hepatitis, or may be a method used to discover (screen) a human who may have chronic hepatitis in a health checkup or the like.

[0163] In one embodiment, the data collection method, diagnostic method, or diagnostic assistance method may include, in addition to obtaining a measured value of agalactosylated human IgG in a biological sample from a subject, determining that the subject has chronic hepatitis if the obtained measured value of agalactosylated human IgG is equal to or greater than a reference value, and determining that the subject does not have chronic hepatitis if the measured value is less than the reference value. Here, the "measured value of agalactosylated human IgG" is used synonymously with the agalactosylated human IgG concentration or agalactosylated human IgG amount obtained by the above-mentioned Measurement Methods 1 to 7.

[0164] The data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure makes a determination, for example, by comparing the amount of agalactosylated human IgG obtained by measuring agalactosylated human IgG in a biological sample from a subject with a reference value. That is, the data collection method, diagnostic method, or diagnostic auxiliary method according to the present disclosure determines, for example, that the subject is likely to have chronic hepatitis if the amount of agalactosylated human IgG is elevated compared to the reference value, and determines that the subject is likely not to have chronic hepatitis if the measured value is decreased compared to the reference value.

[0165] The reference values ​​for the data collection method, diagnostic method, or diagnostic support method of the present disclosure can be determined by referring to the amount of agalactosylated human IgG in healthy individuals and patients who have been definitively diagnosed as suffering from chronic hepatitis.

[0166] Another aspect of the present disclosure is a measurement reagent for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Reagents 1 to 12. Also, another aspect of the present disclosure is a measurement kit for carrying out the data collection method, diagnostic method, or diagnostic assistance method according to an aspect of the present disclosure, which includes any one of the above-mentioned Measurement Kits 1 to 16.

[0167] Another aspect of the present disclosure is a measurement reagent or measurement kit for performing a data collection method, diagnostic method, or diagnostic support method according to an aspect of the present disclosure, comprising an anti-agalactosylated human IgG antibody or antibody fragment thereof of the present disclosure, and an attached document explaining that the determination is made in accordance with the following criteria: if the measured value of the agalactosylated human IgG is equal to or greater than a reference value, the subject is determined to have chronic hepatitis; and if the measured value of the agalactosylated human IgG is less than the reference value, the subject is determined not to have chronic hepatitis.

[0168] The anti-agalactosylated human IgG antibodies and antibody fragments thereof in the diagnostic agent and kit according to one aspect of the present disclosure can be those described in the above-mentioned measurement methods 1 to 7, and can be produced by the above-mentioned methods. Furthermore, antibodies or antibody fragments thereof that bind to human IgG can be obtained by the above-mentioned methods. Furthermore, the diagnostic agent and kit according to one aspect of the present disclosure can be used, for example, by the methods described above in the data collection method, diagnostic method, and diagnostic support method according to one aspect of the present disclosure.

[0169] The present disclosure will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present disclosure in any way.

[0170] Example 1 Production of Anti-Agalactosylated Human IgG Antibodies (1) Preparation of Immunizing Antigen Human IgG Fc (Abcam) (hereinafter referred to as "galactosylated human IgG Fc") was reacted with β-galactosidase (New England Biolabs) at 37°C for 16 hours to digest the galactose attached to the non-reducing end of the N-linked sugar chain. The mixture was then heated at 75°C for 10 minutes to inactivate the β-galactosidase, and the buffer was then exchanged with PBS buffer using a PD-10 column (Cytiva) to prepare human IgG with galactose-deficient N-linked sugar chains (hereinafter referred to as "agalactosylated human IgG Fc"). To confirm the presence or absence of galactose in agalactosylated human IgG Fc, the reactivity of RCA120 lectin with agalactosylated human IgG Fc was analyzed. Agalactosylated human IgG Fc and galactosylated human IgG Fc were adjusted to 0.2 μg / mL in PBS buffer, dispensed into a 96-well EIA plate (Thermo Fisher Scientific) at 50 μL / well, and allowed to stand at 25°C for 12 hours to prepare wells on which agalactosylated human IgG Fc or galactosylated human IgG Fc was immobilized. Next, 200 μL of blocking solution [10 mmol / L phosphate buffer (pH 7.4) containing 1% (w / v) BSA, 0.05% (v / v) Tween 20, and 150 mmol / L sodium chloride] was dispensed into each well, and the plate was left to stand at 25°C for 2 hours to block the plate. The blocking solution was then discarded, and the plate was washed three times with 350 μL of washing solution [containing 0.05% (v / v) Tween 20, 150 mmol / L sodium chloride, and 10 mmol / L phosphate buffer (pH 7.4)]. Then, peroxidase-labeled RCA120 lectin (manufactured by J-Oil Mills) diluted 1,000-fold with sample diluent [a solution in which the blocking solution was diluted 10-fold with washing solution], or peroxidase-labeled anti-human IgG antibody (manufactured by Thermo Fisher Scientific) diluted 10,000-fold, was dispensed at 50 μL / well and allowed to stand at 25 ° C. for 1 hour. After washing three times with 350 μL of washing solution, TMB-ONE (manufactured by Kementec) was added at 50 μL / well, and the plate was allowed to stand at 25 ° C. for 20 minutes.50 μL / well of 0.5 mol / L sulfuric acid was added as a stop solution, and the absorbance of the reaction was measured using a plate reader at a dominant wavelength of 450 nm and a subordinate wavelength of 650 nm. The results are shown in Figure 3. As shown in Figure 3, the reactivity of RCA120 lectin with agalactosylated human IgG Fc was significantly reduced compared to the reactivity of RCA120 lectin with galactosylated human IgG Fc, confirming that the agalactosylated human IgG Fc prepared by β-galactosidase treatment is human IgG Fc with N-linked sugar chains lacking galactose.

[0171] (2) Immunization of Animals and Preparation of Antibody-Producing Cells Three SKG mice (manufactured by CLEA Japan), a mouse model of human rheumatoid arthritis, were prepared as immunized animals. Mannan (20 mg / individual) was administered to the SKG mice, which were then housed for three weeks to induce arthritis. The agalactosylated human IgG Fc (250 μg) prepared in (1) above was administered together with an adjuvant. Two and three weeks later, agalactosylated human IgG Fc (250 μg) was administered once each, for a total of three administrations of agalactosylated human IgG Fc. Three days after the final immunization, the spleens were removed from the mice. The spleens were minced in RPMI medium (manufactured by Thermo Fisher Scientific), disaggregated with tweezers, and centrifuged at 1200 rpm for 5 minutes at 25°C. After removing the supernatant, the precipitate was treated with erythrocyte lysis buffer (Sigma-Aldrich) for 1 to 2 minutes to remove erythrocytes, and then washed three times with RPMI medium to obtain spleen cells, which were used in the cell fusion described below.

[0172] (3) Preparation of Mouse Myeloma Cells 8-azaguanine-resistant mouse myeloma cell line P3-U1 was prepared as mouse myeloma cells. 2 × 10 cells were cultured in GIT medium (Kohjin Bio Co., Ltd.). ７ The cells were cultured until at least 10 cells were obtained.

[0173] (4) Cell Fusion: The spleen cells obtained in (2) above and the myeloma cells obtained in (3) above were mixed at a cell number ratio of 10:1, and the cell mixture was centrifuged at 1200 rpm for 5 minutes at 25°C. The supernatant was removed from the cell mixture, and the precipitated cells were thoroughly loosened. While stirring the cell mixture at 25°C, 1 mL of PEG1500 (Roche) was added to the cell mixture over 1 minute. Thereafter, RPMI medium (1-2 mL) was added four times every minute, and then RPMI medium was added to bring the total volume to 50 mL. The cell suspension was centrifuged at 900 rpm for 5 minutes at 25°C, and the supernatant was discarded. The cells were then gently suspended in 100 mL of selective medium (GIT medium (Kohjin Bio) containing 10% (v / v) BM-condition H1 (Roche) and 2% (v / v) HAT supplement (50x) (Thermo Fisher Scientific)) pre-warmed to 37°C. 100 μL of this suspension was dispensed into a 96-well culture plate at a volume of 100 μL per well, and the cells were incubated in 5% CO ２ The cells were cultured in an incubator at 37°C for 10 to 14 days.

[0174] (5) Preparation of Absorption Antigen for Screening Human IgG (Abcam) was heated at 75°C for 5 minutes, and then the human IgG was mixed with PNGase F (New England Biolabs) and reacted at 50°C for 10 minutes to digest the N-linked glycans. The reaction solution was then heated at 75°C for 10 minutes to inactivate the PNGase F in the reaction solution. Using a PD-10 column (Cytiva), the aqueous medium of the reaction solution was replaced with PBS buffer to prepare IgG from which the N-linked glycans, the absorption antigen, had been removed. To confirm that the N-linked glycans had been removed, the reactivity of PNGase F-treated human IgG with ConA lectin, RCA120 lectin, and anti-human IgG antibody was analyzed. PNGase F-treated human IgG and human IgG were adjusted to 0.2 μg / mL in PBS buffer and dispensed at 50 μL / well into a 96-well EIA plate (Thermo Fisher Scientific) and left to stand at 25°C for 12 hours to prepare wells with immobilized agalactosylated human IgG or wells with immobilized galactosylated human IgG. Next, 200 μL / well of blocking solution [1% (w / v) BSA, 0.05% (v / v) Tween 20, 150 mmol / L sodium chloride, 10 mmol / L phosphate buffer (pH 7.4)] was dispensed into each well and left to stand at 25°C for 2 hours to block the plate. The blocking solution was then discarded, and the plate was washed three times with 350 μL of washing solution [10 mmol / L phosphate buffer (pH 7.4) containing 0.05% (v / v) Tween 20 and 150 mmol / L sodium chloride]. Then, 50 μL / well of peroxidase-labeled ConA lectin (J-Oil Mills), peroxidase-labeled RCA120 lectin (J-Oil Mills), or peroxidase-labeled anti-human IgG antibody (Thermo Fisher Scientific) diluted 1,000-fold with sample diluent [a solution prepared by diluting the blocking solution 10-fold with the washing solution] was dispensed at 50 μL / well and allowed to stand at 25°C for 1 hour. After washing three times with 350 μL of washing solution, 50 μL / well of TMB-ONE was added, and the plate was allowed to stand at 25°C for 20 minutes.50 μL / well of 0.5 mol / L sulfuric acid was added as a stop solution, and the absorbance of each well (main wavelength 450 nm, secondary wavelength 650 nm) was measured using a plate reader. The results are shown in Figure 4. The results in Figure 4 show that the reactivity of RCA120 lectin or ConA lectin with PNGase F-treated human IgG was significantly reduced compared to the reactivity of RCA120 lectin or ConA lectin with human IgG not treated with PNGase F, confirming that human IgG free of N-linked glycans (hereinafter also referred to as "N-linked glycan-depleted human IgG") was successfully prepared.

[0175] (6) Selection of Hybridoma Strains: Agalactosylated human IgG Fc and galactosylated human IgG Fc were diluted with PBS buffer to prepare antigen solutions (final concentration: 0.25 μg / mL). 50 μL of each antigen solution was dispensed into a 96-well EIA plate at each well. The plate was then incubated at 25°C for 12 hours to prepare wells with immobilized agalactosylated human IgG Fc and wells with immobilized galactosylated human IgG Fc. After removing the antigen solution, 200 μL of blocking solution [10 mmol / L phosphate buffer (pH 7.4) containing 1% (w / v) BSA, 0.05% (v / v) Tween 20, and 150 mmol / L sodium chloride] was dispensed into each well. The plate was then incubated at 25°C for 2 hours to block the antibody. After removing the blocking solution, hybridoma culture supernatant diluted two-fold with sample diluent [10 mmol / L phosphate buffer (pH 7.4) containing 60 μg / mL N-linked glycosylated IgG, 0.1% (w / v) BSA, 0.05% (v / v) Tween 20, and 150 mmol / L sodium chloride] was dispensed at 50 μL / well and allowed to stand at 25°C for 2 hours. The diluted hybridoma culture supernatant was removed, and the plate was washed three times with 350 μL of washing solution [10 mmol / L phosphate buffer (pH 7.4) containing 0.05% (v / v) Tween 20 and 150 mmol / L sodium chloride]. Then, peroxidase-labeled anti-mouse IgG antibody (Thermo Fisher Scientific) diluted 10,000-fold with labeled antibody diluent [10 mmol / L phosphate buffer (pH 7.4) containing 0.1% (w / v) BSA, 0.05% (v / v) Tween 20, and 150 mmol / L sodium chloride] was dispensed at 50 μL / well and allowed to stand at 25°C for 1 hour. The labeled antibody solution was removed, and the plate was washed three times with 350 μL of washing solution. Then, 50 μL / well of TMB-ONE was added, and the plate was allowed to stand at 25°C for 20 minutes. After adding 50 μL / well of 0.5 mol / L sulfuric acid as a stop solution, the absorbance of the reaction solution (main wavelength 450 nm, secondary wavelength 650 nm) was measured using a plate reader.Hybridoma strains that showed strong color development in wells immobilized with agalactosylated human IgG Fc and weak or no color development in wells immobilized with galactosylated human IgG Fc were selected and cloned twice by limiting dilution. As a result, six hybridoma strains, 11E8, 15D12, 20G2, 22F12, 42B1, and 50A1, were obtained from three mice. The reactivity of the culture supernatant of each hybridoma strain to agalactosylated human IgG Fc immobilized on a plate, as well as the reactivity of the culture supernatant of each hybridoma strain to galactosylated human IgG Fc immobilized on a plate, are shown in Figure 5. The results in Figure 5 demonstrate that anti-agalactosylated human IgG antibodies that specifically reacted with agalactosylated human IgG were obtained for all six clones. A confirmation test was also conducted to evaluate whether the amount of solid-phase agalactosylated human IgG Fc or the amount of solid-phase galactosylated human IgG Fc in each well was comparable. This confirmation test was performed using the same method as the ELISA test described above, except that an anti-human IgG antibody was used instead of the hybridoma strain supernatant. The results are shown in Figure 6. The results in Figure 6 confirmed that approximately the same amount of antibody was solid-phased in each well, comparing the wells in which agalactosylated human IgG Fc was solid-phased with the wells in which galactosylated human IgG Fc was solid-phased.

[0176] (7) Cross-reactivity with galactosylated human IgG The cross-reactivity (%) with galactosylated human IgG was calculated based on the results of Figure 5, i.e., the reactivity of each antibody clone with galactosylated human IgG (absorbance A) and the reactivity of each antibody clone with galactosylated human IgG (absorbance B), and the results are shown in Table 1. The cross-reactivity was calculated according to the formula "cross-reactivity (%) = (measured value B) / (measured value A) × 100." As shown in Table 1, the cross-reactivity was 10.9 to 17.9%.

[0177] The clones obtained in the above test (6) were obtained from three mice (mouse 1, mouse 2, and mouse 3), and the information on these clones is also listed in Table 1. This time, three SKG mice were prepared and tests to obtain monoclonal antibodies were independently conducted according to the methods (1) to (6) above. When any of the individual mice was used, monoclonal antibodies that specifically reacted with agalactosylated human IgG were obtained.

[0178] (8) Identification of antibody class, subtype, and light chain type Among the obtained hybridoma strains, the culture supernatant of the 42B1 strain was used to determine the antibody class (isotype), subclass, and light chain type of the produced antibody using an IsoStrip Mouse Monoclonal Antibody Isotyping Kit (manufactured by Roche). The result was that the antibody was mouse IgG2aκ.

[0179] (9) Purification of anti-agalactosylated IgG antibody Among the obtained hybridoma strains, the 42B1 strain was cultured in 150 mL of medium [GIT medium (Kohjin Bio Co., Ltd.) containing 10% (v / v) BM-condition H1 (Roche)], and the culture supernatant was collected. After centrifugation at 3,000 rpm for 10 minutes at 25 ° C, the supernatant was collected and filtered using a 0.22 μm pore size cellulose acetate membrane. Sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 3 mol / L, and the pH was adjusted to 8.9. After equilibrating an rProtein A GraviTrap column (Cytiva) with PBS buffer, the filtered supernatant was passed through the column. The column was then washed with PBS buffer, and the antibody derived from the 42B1 strain bound to rProtein A (hereinafter referred to as the 42B1 antibody) was eluted by passing it through a 0.1 mol / L glycine-HCl solution (pH 2.7). The solvent of the eluate containing the 42B1 antibody was then replaced with PBS buffer using a PD-10 column (manufactured by Cytiva), and the absorbance at 280 nm of the 42B1 antibody solution replaced with PBS buffer was measured using an absorptiometer (U-3900, manufactured by Hitachi, Ltd.), and the 42B1 antibody concentration was calculated assuming a molar extinction coefficient of 1.4.

[0180] [Example 2] Construction of an agalactosylated human IgG measurement system (1) Anti-human IgG antibody F(ab') ２ Preparation of purified fragments: Rabbit anti-human IgG antibody (Abcam) was purified by Pierce ＴＭ F(ab') ２ F(ab') was prepared using a Preparation Kit (manufactured by Thermo Fisher Scientific) according to the attached manual. ２ The Fc was removed using an rProtein A GraviTrap column (manufactured by Cytiva), and the rabbit anti-human IgG antibody F(ab') was purified using a PD-10 column (manufactured by Cytiva). ２ The solvent of the eluate containing the purified fragment was replaced with PBS buffer. ２ The absorbance of the purified fragment at 280 nm was measured using an absorptiometer (U-3900, Hitachi, Ltd.), and the molar extinction coefficient was set to 1.48, which corresponds to the rabbit anti-human IgG antibody F(ab') ２ The concentration of purified fragments was calculated.

[0181] (2) Anti-human IgG antibody F(ab') ２ Preparation of Immobilized Fragment Plates Anti-human IgG antibody F(ab') diluted to 3 μg / mL with PBS buffer was placed on a 96-well EIA plate. ２ The antibody fragment was dispensed at 50 μL / well and allowed to stand at 25° C. for 12 hours to immobilize the antibody on the plate. After removing the antibody solution, 200 μL / well of blocking solution was dispensed and the plate was blocked by allowing to stand at 25° C. for 2 hours. After removing the blocking solution, the plate was used for measuring agalactosylated human IgG.

[0182] (3) Preparation of peroxidase-labeled anti-agalactosylated human IgG antibody: The purified 42B1 antibody was labeled with peroxidase using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The peroxidase-labeled 42B1 antibody was suspended in the storage buffer provided with the kit, and this was used as a peroxidase-labeled anti-agalactosylated human IgG antibody solution.

[0183] (4) Preparation of Standards Serum collected from healthy individuals was passed through an rProtein A GraviTrap column (manufactured by Cytiva) equilibrated with PBS buffer. The column was then washed with PBS buffer, and human IgG bound to rProtein A was eluted by passing it through a 0.1 mol / L glycine-HCl solution (pH 2.7). Subsequently, using a PD-10 column (manufactured by Cytiva), the solvent of the eluate containing human IgG was replaced with PBS buffer. The absorbance at 280 nm of the human IgG solution replaced with PBS buffer was measured using an absorptiometer (U-3900, manufactured by Hitachi, Ltd.), and the human IgG concentration was calculated using a molar extinction coefficient of 1.4. The resulting human IgG was reacted with β-galactosidase (New England Biolabs) overnight at 37°C to digest galactose. The β-galactosidase was then inactivated by heating at 75°C for 10 minutes, and the buffer was then replaced with PBS buffer using a PD-10 column (Cytiva) to prepare agalactosylated human IgG. Agalactosylated human IgG was diluted with sample diluent [10 mmol / L phosphate buffer (pH 7.4) containing 0.1% (w / v) BSA, 0.05% (v / v) Tween 20, and 150 mmol / L sodium chloride] to concentrations of 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.3 ng / mL, 15.6 ng / mL, 7.8 ng / mL, and 3.9 ng / mL. This two-fold dilution series was used as a standard substance to obtain a standard curve and to measure agalactosylated human IgG.

[0184] (5) Measurement of agalactosylated human IgG Anti-human IgG antibody F(ab') prepared in (2) above ２ The standard substance prepared in (4) above was dispensed at 50 μL / well onto the plate with the immobilized fragments. ２Samples (human serum samples from healthy volunteers [HV], inflammatory bowel disease [IBD], chronic pancreatitis, or chronic hepatitis patients) diluted 100,000-fold or 200,000-fold with sample diluent were dispensed at 50 μL / well onto the plate. After standing at 25°C for 1 hour, the samples were removed, and the wells were washed three times with 350 μL of the washing solution described in (6) above. Next, the peroxidase-labeled anti-agalactosylated human IgG antibody solution prepared in (3) was diluted 10,000-fold with sample diluent, and this solution was dispensed at 50 μL / well. After standing at 25°C for 1.5 hours, the labeled antibody solution was removed, and the wells were washed four times with 350 μL of washing solution. TMB-ONE was added at 50 μL / well, and the plate was allowed to stand at 25°C for 20 minutes. 50 μL / well of 0.5 mol / L sulfuric acid was added as a stop solution, and the absorbance of each well (dominant wavelength 450 nm, minor wavelength 650 nm) was measured using a plate reader. The resulting standard curve is shown in Figure 7. For serum samples from healthy subjects and inflammatory bowel disease patients, the absorbance was converted using the standard curve, and the dilution rate using the sample diluent was corrected to calculate the serum agalactosylated human IgG concentration. The serum agalactosylated human IgG concentrations in inflammatory bowel disease patients and healthy subjects are shown in Figure 8. A Wilcoxon rank-sum test was performed on the results in Figure 8, and the serum agalactosylated human IgG concentrations in inflammatory bowel disease patients were significantly higher than those in healthy subjects. The serum agalactosylated human IgG concentrations (absorbance) of chronic pancreatitis patients, chronic hepatitis patients, and healthy subjects are also shown in Figures 9 and 10. When the Wilcoxon rank sum test was performed on the results of Figures 9 and 10, the measured values ​​of agalactosylated human IgG in the serum of patients with chronic pancreatitis and patients with chronic hepatitis were significantly higher than the measured values ​​of agalactosylated human IgG in the serum of healthy subjects.

Claims

1. An anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof, characterized in that it reacts with human immunoglobulin G containing N-linked glycans without galactose in the Fc region (hereinafter referred to as agalactosylated human immunoglobulin G), but does not react with human immunoglobulin G containing N-linked glycans with galactose in the Fc region (hereinafter referred to as galactosylated human immunoglobulin G).

2. The N-linked sugar chain to which no galactose is bound is represented by the following general formula (I), (II), (III) or (IV): The antibody or antibody fragment thereof according to claim 1, wherein the N-linked sugar chain comprises a sugar chain represented by the formula:

3. The antibody or antibody fragment thereof according to claim 1, wherein the agalactosylated human immunoglobulin G is a human immunoglobulin G that binds to at least one antigen selected from the group consisting of an autoantigen, an alloantigen, a virus-derived antigen, and a bacteria-derived antigen.

4. The antibody or antibody fragment thereof according to claim 1, wherein the anti-agalactosylated human immunoglobulin G antibody is a monoclonal antibody.

5. A hybridoma producing the monoclonal antibody of claim 4.

6. (1) immunizing a non-human animal with a human immunoglobulin G containing a galactose-free N-linked sugar chain in the Fc region (hereinafter referred to as agalactosylated human immunoglobulin G) or an Fc region fragment thereof as an immunogen; (2) fusing antibody-producing cells obtained from the non-human animal immunized in step (1) with myeloma cells to obtain hybridomas; (3) culturing the hybridomas obtained in step (2); (4) selecting, from the culture obtained in step (3), hybridomas that produce anti-agalactosylated human immunoglobulin G antibodies that react with agalactosylated human immunoglobulin G but do not react with human immunoglobulin G containing a galactose-linked N-linked sugar chain in the Fc region (hereinafter referred to as galactosylated human immunoglobulin G); and (5) culturing the hybridoma selected in step (4) and collecting from the culture an anti-agalactosylated human immunoglobulin G antibody that reacts with agalactosylated human immunoglobulin G but does not react with galactosylated human immunoglobulin G.

7. The method according to claim 6, wherein the non-human animal is an animal model for an autoimmune disease.

8. The N-linked sugar chain to which no galactose is bound is represented by the following general formula (I), (II), (III) or (IV): The method according to claim 6 or 7, wherein the N-linked sugar chain comprises a sugar chain represented by the formula:

9. A method for measuring agalactosylated human immunoglobulin G in a sample, using the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of claims 1 to 4.

10. A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of claims 1 to 4 to produce an immune complex A1 comprising the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment and agalactosylated human immunoglobulin G; (2) reacting the immune complex A1 produced in step (1) with an antibody or its antibody fragment that binds to human immunoglobulin G to produce an immune complex A2 comprising the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment, agalactosylated human immunoglobulin G, and an antibody or its antibody fragment that binds to human immunoglobulin G; and (3) measuring the immune complex A2 produced in step (2).

11. A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with an antibody or antibody fragment thereof that binds to human immunoglobulin G to produce an immune complex B1 containing an antibody or antibody fragment thereof that binds to human immunoglobulin G and agalactosylated human immunoglobulin G; (2) reacting the immune complex B1 produced in step (1) with an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof described in any one of claims 1 to 4 to produce an immune complex B2 containing an antibody or antibody fragment thereof that binds to human immunoglobulin G, agalactosylated human immunoglobulin G, and an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof; and (3) measuring the immune complex B2 produced in step (2).

12. A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with an antigen to which agalactosylated human immunoglobulin G reacts to produce an immune complex E1 containing the antigen and agalactosylated human immunoglobulin G; (2) reacting the immune complex E1 produced in step (1) with an anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof described in any one of claims 1 to 4 to produce an immune complex E2 containing the antigen, agalactosylated human immunoglobulin G, and the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof; and (3) measuring the immune complex E2 produced in step (2).

13. A method for measuring agalactosylated human immunoglobulin G in a sample, comprising: (1) reacting the sample with the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of claims 1 to 4 to produce an immune complex F1 comprising the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment and the agalactosylated human immunoglobulin G; (2) reacting the immune complex F1 produced in step (1) with an antigen to which the agalactosylated human immunoglobulin G reacts to produce an immune complex F2 comprising the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment, the agalactosylated human immunoglobulin G, and the antigen; and (3) measuring the immune complex F2 produced in step (2).

14. The measurement method described in claim 12 or 13, wherein the antigen to which agalactosylated human immunoglobulin G reacts is at least one antigen selected from the group consisting of autoantigens, alloantigens, virus-derived antigens, and bacteria-derived antigens.

15. A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or its antibody fragment according to any one of claims 1 to 4.

16. A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of claims 1 to 4, and an antibody or antibody fragment thereof that binds to human immunoglobulin G.

17. A reagent for measuring agalactosylated human immunoglobulin G in a sample, comprising the anti-agalactosylated human immunoglobulin G antibody or antibody fragment thereof according to any one of claims 1 to 4 and an antigen to which agalactosylated human immunoglobulin G reacts.

18. The assay reagent according to claim 17, wherein the antigen to which agalactosylated human immunoglobulin G reacts is at least one antigen selected from the group consisting of autoantigens, alloantigens, virus-derived antigens, and bacteria-derived antigens.

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