Method for measuring target antigen, and antibody, insoluble particles and target antigen measurement kit used in said method

Abstract

Provided is an IgG antibody having a Fab region and an Fc region connected to the Fab region, wherein four or more N-type sugar chains are bound to the Fc region (excluding a human IgG).

Description

METHOD FOR MEASURING TARGET ANTIGEN, AND ANTIBODY, INSOLUBLE PARTICLES, AND TARGET ANTIGEN MEASUREMENT KIT USED THEREIN

[0001] The present invention relates to a method for measuring a target antigen, and to an antibody, insoluble particles, and a kit for measuring a target antigen used therein.

[0002] One method for measuring target antigens using immune reactions is to use granular carriers such as latex (see, for example, Patent Document 1). When the target antigen is present, the granular carriers, to which antibodies specific to the target antigen are bound, undergo an antigen-antibody reaction. Due to their specificity and affinity, the antibodies bind to each other, using the target antigen as a bridge, and agglutinate. The presence or absence of the resulting agglutinates and the degree of agglutination determine the presence or absence of the target antigen and its quantity.

[0003] Such measurement methods utilizing immune reactions can have the problem of insufficient detection sensitivity. For example, Patent Document 2 discloses polysaccharide-modified composite particles in which the occurrence of nonspecific adsorption is reduced so as not to reduce the sensitivity of the measurement.

[0004] JP 2017-83440 A JP 2009-162537 A

[0005] However, since the above-mentioned polysaccharide-modified composite particles are produced by utilizing the adsorption of polysaccharides onto the surfaces of silica particles, the types of polysaccharide-modified composite particles are limited, and their applications are restricted.

[0006] In view of the above circumstances, the present invention aims to provide an antibody that, when supported on a granular carrier, can suppress nonspecific agglutination of insoluble particles containing the granular carrier, insoluble particles containing the antibody, a kit for measuring a target antigen using the antibody or the insoluble particles, or a method for measuring a target antigen using the insoluble particles.

[0007] As a result of extensive research, the present inventors discovered that insoluble particles containing a granular carrier carrying an IgG antibody, wherein the IgG antibody has a Fab region and an Fc region connected to the Fab region, and wherein four or more sugar chains are bound to the Fc region of the IgG antibody, can suppress nonspecific aggregation, and thus completed the present invention.

[0008] The present invention provides, for example, the following inventions: [1] An IgG antibody (excluding human IgG) having a Fab region and an Fc region linked to the Fab region, wherein four or more N-glycans are bound to the Fc region. [2] The antibody according to [1], wherein four N-glycans are bound. [3] The antibody according to [1] or [2], wherein two N-glycans are bound to the second constant domain and two N-glycans are bound to the third constant domain. [3a] The antibody according to [1] or [2], wherein two of the four or more N-glycans are bound to the second constant domain and two N-glycans different from the two N-glycans of the four or more N-glycans are bound to the third constant domain. [4] The amino acid sequence of the Fc region is: (1) 90% or more identical to the amino acid sequence of SEQ ID NO: 2, wherein the residue at position 174 is an asparagine residue, the residue at position 175 is an amino acid residue other than proline, the residue at position 176 is a serine or threonine residue, the residue at position 301 is an asparagine residue, the residue at position 302 is an amino acid residue other than proline, and the residue at position 303 is a serine or threonine residue; (2) 90% or more identical to the amino acid sequence of SEQ ID NO: 4, wherein the residue at position 180 is an asparagine residue, the residue at position 181 is an amino acid residue other than proline, the residue at position 182 is a serine or threonine residue, the residue at position 307 is an asparagine residue, the residue at position 308 is an amino acid residue other than proline, and the residue at position 309 is a serine or threonine residue; (3) 90% or more identical to the amino acid sequence of SEQ ID NO: 6, wherein the amino acid sequence is: Residue 185 is an asparagine residue, residue 186 is an amino acid residue other than proline, residue 187 is a serine or threonine residue, residue 312 is an asparagine residue, residue 313 is an amino acid residue other than proline, and residue 314 is a serine or threonine residue;(4) having 90% or more identity with the amino acid sequence of SEQ ID NO: 8, wherein the 179th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue; or (5) having 90% or more identity with the amino acid sequence of SEQ ID NO: 16, wherein the 173rd residue is an asparagine residue, the 174th residue is an amino acid residue other than proline, the 175th residue is a serine or threonine residue, the 300th residue is an asparagine residue, the 301st residue is an amino acid residue other than proline, and the 302nd residue is a serine or threonine residue;The antibody according to any one of [1] to [3a]. [5] Insoluble particles comprising a particulate carrier and an IgG antibody against a target antigen supported on the particulate carrier, wherein the antibody has a Fab region and an Fc region linked to the Fab region, and four or more N-glycans are bound to the Fc region. [6] The insoluble particle according to [5], wherein four N-glycans are bound to the antibody. [7] The insoluble particle according to [5] or [6], wherein, in the antibody, two N-glycans are bound to the second constant domain and two N-glycans are bound to the third constant domain. [7a] The insoluble particle according to [5] or [6], wherein, in the antibody, two N-glycans of the four or more N-glycans are bound to the second constant domain and two N-glycans different from the two N-glycans of the four or more N-glycans are bound to the third constant domain. [8] The insoluble particles according to any one of [5] to [7a], wherein the granular carrier is a latex particle. [9] The amino acid sequence of the Fc region of the antibody: (1) has 90% or more identity with the amino acid sequence of SEQ ID NO: 2, wherein the residue at position 174 is an asparagine residue, the residue at position 175 is an amino acid residue other than proline, the residue at position 176 is a serine or threonine residue, the residue at position 301 is an asparagine residue, the residue at position 302 is an amino acid residue other than proline, and the residue at position 303 is a serine or threonine residue; (2) has 90% or more identity with the amino acid sequence of SEQ ID NO: 4, wherein the residue at position 180 is an asparagine residue, the residue at position 181 is an amino acid residue other than proline, the residue at position 182 is a serine or threonine residue, the residue at position 307 is an asparagine residue, the residue at position 308 is an amino acid residue other than proline, and the residue at position 309 is a serine or threonine residue;(3) having 90% or more identity with the amino acid sequence of SEQ ID NO: 6, wherein the 185th residue is an asparagine residue, the 186th residue is an amino acid residue other than proline, the 187th residue is a serine or threonine residue, the 312th residue is an asparagine residue, the 313th residue is an amino acid residue other than proline, and the 314th residue is a serine or threonine residue; (4) having 90% or more identity with the amino acid sequence of SEQ ID NO: 8, wherein the 179th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue; (5) having 90% or more identity with the amino acid sequence of SEQ ID NO: 10, wherein the 189th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue; (6) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 12, wherein the residue at position 176 is an asparagine residue, the residue at position 177 is an amino acid residue other than proline, the residue at position 178 is a serine or threonine residue, the residue at position 303 is an asparagine residue, the residue at position 304 is an amino acid residue other than proline, and the residue at position 305 is a serine or threonine residue; (7) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 14, wherein the residue at position 176 is an asparagine residue, the residue at position 177 is an amino acid residue other than proline, the residue at position 178 is a serine or threonine residue, the residue at position 303 is an asparagine residue, the residue at position 304 is an amino acid residue other than proline, and the residue at position 305 is a serine or threonine residue; the 177th residue is an asparagine residue, the 178th residue is an amino acid residue other than proline, the 179th residue is a serine or threonine residue, the 304th residue is an asparagine residue, the 305th residue is an amino acid residue other than proline, and the 306th residue is a serine or threonine residue;or (8) an insoluble particle according to any one of [5] to [8], which has 90% or more identity with the amino acid sequence of SEQ ID NO: 16, wherein the residue at position 173 is an asparagine residue, the residue at position 174 is an amino acid residue other than proline, the residue at position 175 is a serine or threonine residue, the residue at position 300 is an asparagine residue, the residue at position 301 is an amino acid residue other than proline, and the residue at position 302 is a serine or threonine residue;

[10] A kit for measuring a target antigen, which includes the insoluble particle according to any one of [5] to [9], or includes an antibody and a granular carrier for preparing the insoluble particle according to any one of [5] to [9].

[11] A method for measuring a target antigen, which uses the insoluble particle according to any one of [5] to [9].

[12] A method for measuring a target antigen, comprising: a step of contacting the insoluble particles according to any one of [5] to [9] with a test sample that may contain the target antigen; and a step of measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody contained in the insoluble particles and the target antigen.

[0009] According to the present invention, it is possible to provide an antibody that, when supported on a granular carrier, can suppress nonspecific agglutination of insoluble particles containing the granular carrier, insoluble particles containing the antibody, a kit for measuring a target antigen using the antibody or the insoluble particles, or a method for measuring a target antigen using the insoluble particles.

[0010] 1 is a graph showing the amount of change in absorbance due to the agglutination reaction of anti-human BNP antibody-sensitized latex in the presence of 0 pg / mL or 1000 pg / mL of human BNP. 2 is a graph showing the ratio of the amount of change in absorbance in the presence of 1000 pg / mL of human BNP to the amount of change in absorbance in the presence of 0 pg / mL of human BNP in FIG. 1. 3 is a graph showing the amount of change in absorbance due to the nonspecific agglutination reaction of anti-human BNP antibody-sensitized latex in a human BNP-free plasma sample. 4 is a graph showing the results of evaluating the antigen binding activity of anti-human BNP antibodies.

[0011] The present invention will be described in detail below.

[0012] An IgG antibody according to one embodiment has four or more N-glycans bound to the Fc region. The IgG antibody is not particularly limited as long as it can specifically bind to the target antigen described below.

[0013] According to one embodiment of the IgG antibody, nonspecific aggregation of insoluble particles containing a granular carrier carrying the IgG antibody can be suppressed without impairing the antigen-binding activity of the IgG antibody.

[0014] Examples of IgG antibodies according to one embodiment include mouse IgG and rabbit IgG (excluding human IgG). The IgG antibodies also include genetically modified IgG antibodies, such as those having a glycosylation motif Asn-Xaa-Ser / Thr (Xaa is an amino acid other than proline; hereinafter, also referred to as a "glycolysis motif") introduced into the Fc region of the IgG antibody. An amino acid other than proline refers to an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. Preferred IgG antibodies according to one embodiment include mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, and rabbit IgG, each of which has a glycosylation motif introduced into the Fc region.

[0015] An IgG antibody has a Fab region and an Fc region linked to the Fab region. In an IgG antibody, the Fab region and Fc region may be linked via a hinge region. An IgG antibody is composed of two heavy chains (H chains) and two light chains (L chains). The heavy chain has, from the N-terminus, a variable region (VH), a first constant domain (CH1), a second constant domain (CH2), and a third constant domain (CH3). The light chain has, from the N-terminus, a variable region (VL) and a constant region (CL). The portion composed of VH, CH1, VL, and CL is the Fab region. The portion composed of CH2 and CH3 is the Fc region.

[0016] In one embodiment of the IgG antibody, it is preferred that two of the four or more N-glycans bound to the Fc region are bound to the second constant domain, and two N-glycans different from the two N-glycans among the four or more N-glycans are bound to the third constant domain.

[0017] In an IgG antibody according to one embodiment, it is preferred that four N-glycans are bound to the Fc region, and it is more preferred that two N-glycans are bound to the second constant domain and two N-glycans are bound to the third constant domain.

[0018] In an IgG antibody according to one embodiment, the number of N-glycans bound to the Fc region is preferably even. In this case, the even number of N-glycans is preferably bound to corresponding amino acid residues in the Fc regions of the two heavy chains.

[0019] In an IgG antibody according to one embodiment, the N-glycans are preferably bound to asparagine residues in the glycobinding motif, and more preferably, all N-glycans are bound to asparagine residues in the glycobinding motif.

[0020] In one embodiment, an IgG antibody has an Fc region that has 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 16, and in the amino acid sequences, SEQ ID NO: 2, the 174th residue is an asparagine residue, the 175th residue is an amino acid residue other than proline, the 176th residue is a serine or threonine residue, the 301st residue is an asparagine residue, the 302nd residue is an amino acid residue other than proline, and the 303rd residue is a serine or threonine residue. SEQ ID NO: 4, 18 SEQ ID NO: 6, in which the residue at position 0 is an asparagine residue, the residue at position 181 is an amino acid residue other than proline, the residue at position 182 is a serine or threonine residue, the residue at position 307 is an asparagine residue, the residue at position 308 is an amino acid residue other than proline, and the residue at position 309 is a serine or threonine residue, is an amino acid residue at position 185 is an asparagine residue, the residue at position 186 is an amino acid residue other than proline, the residue at position 187 is a serine or threonine residue, and the residue at position 312 is an asparagine residue. In SEQ ID NO: 8, the 179th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue; or in SEQ ID NO: 16, the 173rd residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue. residue, the 174th residue is an amino acid residue other than proline, the 175th residue is a serine or threonine residue, the 300th residue is an asparagine residue, the 301st residue is an amino acid residue other than proline, and the 302nd residue is a serine or threonine residue. In the above-mentioned preferred embodiment, SEQ ID NO: 2 is an amino acid residue other than positions 174 to 176 and 301 to 303; SEQ ID NO: 4 is an amino acid residue other than positions 180 to 182 and 307 to 309; and SEQ ID NO: 6 is an amino acid residue other than positions 185 to 187 and 312 to 314.It is more preferable that SEQ ID NO: 8 has no sugar chain binding motifs other than those at positions 179 to 181 and 306 to 308, or that SEQ ID NO: 16 has no sugar chain binding motifs other than those at positions 173 to 175 and 300 to 302.

[0021] Here, SEQ ID NO: 2 is a sequence in which the threonine residue at position 301 (i.e., position 424 in EU numbering) of the amino acid sequence of the Fc region of mouse IgG1 (SEQ ID NO: 1) has been substituted with an asparagine residue. In SEQ ID NOs: 1 and 2, residues 174 to 176 are located in the second constant domain, and residues 301 to 303 are located in the third constant domain. SEQ ID NO: 1 has a carbohydrate binding motif at residues 174 to 176, and SEQ ID NO: 2 has carbohydrate binding motifs at residues 174 to 176 and residues 301 to 303.

[0022] SEQ ID NO:4 has the amino acid sequence of the Fc region of mouse IgG2a (SEQ ID NO:3) in which the serine residue at position 307 has been substituted with an asparagine residue. In SEQ ID NOs:3 and 4, residues 180 to 182 are located in the second constant domain, and residues 307 to 309 are located in the third constant domain. SEQ ID NO:3 has a carbohydrate-binding motif at residues 180 to 182, and SEQ ID NO:4 has carbohydrate-binding motifs at residues 180 to 182 and residues 307 to 309.

[0023] SEQ ID NO: 6 is a sequence in which the serine residue at position 312 in the amino acid sequence of the Fc region of mouse IgG2b (SEQ ID NO: 5) is substituted with an asparagine residue and the asparagine residue at position 314 is substituted with a serine residue. In SEQ ID NOs: 5 and 6, residues 185 to 187 are located in the second constant domain, and residues 312 to 314 are located in the third constant domain. SEQ ID NO: 5 has a carbohydrate-binding motif at residues 185 to 187, and SEQ ID NO: 6 has carbohydrate-binding motifs at residues 185 to 187 and residues 312 to 314.

[0024] SEQ ID NO: 8 corresponds to the amino acid sequence of the Fc region of mouse IgG3 (SEQ ID NO: 7) in which the threonine residue at position 306 has been substituted with an asparagine residue. In SEQ ID NOs: 7 and 8, residues 179 to 181 are located in the second constant domain, and residues 306 to 308 are located in the third constant domain. SEQ ID NO: 7 has a carbohydrate-binding motif at residues 179 to 181, and SEQ ID NO: 8 has carbohydrate-binding motifs at residues 179 to 181 and residues 306 to 308.

[0025] SEQ ID NO: 16 corresponds to the amino acid sequence of the Fc region of rabbit IgG (SEQ ID NO: 15) in which the threonine residue at position 300 is substituted with an asparagine residue. In SEQ ID NOs: 15 and 16, residues 173 to 175 are located in the second constant domain, and residues 300 to 302 are located in the third constant domain. SEQ ID NO: 15 has a carbohydrate-binding motif at residues 173 to 175, and SEQ ID NO: 16 has carbohydrate-binding motifs at residues 173 to 175 and residues 300 to 302.

[0026] The N-glycan is not particularly limited and may be, for example, a high-mannose, hybrid, or complex N-glycan. The four or more N-glycans bound to the Fc region of an IgG antibody according to one embodiment may be one type of N-glycan or two or more types of N-glycans.

[0027] An IgG antibody according to one embodiment may be produced by artificially introducing a glycosylation motif into the Fc region of an IgG antibody, thereby increasing the number of glycosylation motifs in the Fc region to four or more. The IgG antibody according to one embodiment can be obtained as a genetic recombinant using mammalian cells as an expression host. Examples of mammalian cells include, but are not limited to, CHO (Chinese Hamster Ovary) cells and HEK293 (Human Embryonic Kidney cells 293) cells. Furthermore, the IgG antibody according to one embodiment can be produced using a plant transient expression system, or can be produced using a genetically modified plant. For example, the method described in Japanese Patent No. 5,015,012 can be used as a plant transient expression system. Furthermore, methods for obtaining recombinant proteins include, but are not limited to, transient expression systems using plasmid vectors lacking autonomous replication ability, viral vectors, etc., semi-stable expression systems using episomal vectors that are provided with a nuclear localization signal and have autonomous replication ability, and stable expression systems in which a gene of interest is inserted into the genome of an expression host. An IgG antibody having a glycosylation motif in the Fc region can have a glycosylation chain bound to the asparagine residue of the glycosylation motif in a host cell by glycosyltransferases such as N-acetylglucosaminyltransferase, mannosyltransferase, galactosyltransferase, and fucosyltransferase.

[0028] Whether or not a glycan is bound to an IgG antibody may be confirmed by liquid chromatography mass spectrometry (hereinafter also referred to as "LC-MS"). In LC-MS, for example, an IgG antibody is first fragmented with trypsin and subjected to LC-MS. Next, mass spectra derived from fragments containing a glycosylation motif to which an N-glycan is bound may be detected. In this case, the percentage of the total peak area of ​​the mass spectra derived from the fragments to which an N-glycan is bound may be calculated relative to the total peak area of ​​the mass spectra derived from fragments containing the glycosylation motif (including fragments to which an N-glycan is not bound to the glycosylation motif), thereby determining the percentage of glycosylation motifs to which an N-glycan is bound among the glycosylation motifs.

[0029] In one embodiment, the insoluble particles contain a granular carrier and an IgG antibody supported on the granular carrier, the IgG antibody having four or more N-glycans bound to the Fc region against a target antigen.

[0030] The antibody contained in the insoluble particles may be, in addition to the IgG antibody according to the embodiment described above, a human IgG having four or more N-glycans bound to the Fc region. Human IgG is preferably human IgG1, human IgG2, or human IgG4, each of which has a glycan-binding motif introduced into the Fc region. The mode of binding of the N-glycan to the Fc region of human IgG is preferably the same as that of the IgG antibody according to the embodiment described above. Preparation of human IgG and confirmation of whether or not a glycan is bound to the human IgG may be performed in the same manner as that of the IgG antibody according to the embodiment described above.

[0031] Human IgG is an IgG antibody whose Fc region has 90% or more identity to the amino acid sequence of SEQ ID NO: 10, 12, or 14, and in the amino acid sequence, SEQ ID NO: 10, the 180th residue is an asparagine residue, the 181st residue is an amino acid residue other than proline, the 182nd residue is a serine or threonine residue, the 307th residue is an asparagine residue, the 308th residue is an amino acid residue other than proline, and the 309th residue is a serine or threonine residue; and in SEQ ID NO: 12, the 176th residue is an asparagine residue, the 177th residue is an amino acid residue other than proline, the 178th residue is a serine or threonine residue, the 303rd residue is an asparagine residue, and the 304th residue is proline. or in SEQ ID NO: 14, the residue at position 177 is an asparagine residue, the residue at position 178 is an amino acid residue other than proline, the residue at position 179 is a serine or threonine residue, the residue at position 304 is an asparagine residue, the residue at position 305 is an amino acid residue other than proline, and the residue at position 306 is a serine or threonine residue. In the above-mentioned preferred embodiment, it is more preferable that SEQ ID NO: 10 does not have a glycosylation motif other than positions 180 to 182 and 307 to 309, SEQ ID NO: 12 does not have a glycosylation motif other than positions 176 to 178 and 303 to 305, or SEQ ID NO: 14 does not have a glycosylation motif other than positions 177 to 179 and 304 to 306.

[0032] Here, SEQ ID NO: 10 is the amino acid sequence of the Fc region of human IgG1 (SEQ ID NO: 9) in which the serine residue at position 307 has been substituted with an asparagine residue. In SEQ ID NOs: 9 and 10, residues 180 to 182 are located in the second constant domain, and residues 307 to 309 are located in the third constant domain. SEQ ID NO: 9 has a carbohydrate-binding motif at residues 180 to 182, and SEQ ID NO: 10 has carbohydrate-binding motifs at residues 180 to 182 and residues 307 to 309.

[0033] SEQ ID NO: 12 is the amino acid sequence of the Fc region of human IgG2 (SEQ ID NO: 11) in which the serine residue at position 303 is substituted with an asparagine residue. In SEQ ID NOs: 11 and 12, residues 176 to 178 are located in the second constant domain, and residues 303 to 305 are located in the third constant domain. SEQ ID NO: 11 has a carbohydrate-binding motif at residues 176 to 178, and SEQ ID NO: 12 has carbohydrate-binding motifs at residues 176 to 178 and residues 303 to 305.

[0034] SEQ ID NO: 14 corresponds to the amino acid sequence of the Fc region of human IgG4 (SEQ ID NO: 13) in which the serine residue at position 304 has been substituted with an asparagine residue. In SEQ ID NOs: 13 and 14, residues 177 to 179 are located in the second constant domain, and residues 304 to 306 are located in the third constant domain. SEQ ID NO: 13 has a carbohydrate-binding motif at residues 177 to 179, and SEQ ID NO: 14 has carbohydrate-binding motifs at residues 177 to 179 and residues 304 to 306.

[0035] Target antigens include, for example, protein markers such as CRP (C-reactive protein), prostate-specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, and creatinine; immunoglobulins such as IgG, IgE, IgA, and IgM; various tumor markers; lipoproteins such as LDL, HDL, and TG; viral antigens such as influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, and EBV; Chlamydia trachomatis, streptococcus, and Hepatitis B virus. Examples of the antibacterial agent include, but are not limited to, bacterial antigens such as cough bacteria, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthrax, and MRSA, toxins produced by bacteria, etc., mycoplasma lipid antigens, peptide hormones such as human brain natriuretic peptide (hereinafter also referred to as "human BNP") and human chorionic gonadotropin, steroids such as steroid hormones, physiologically active amines such as epinephrine and morphine, vitamins such as B vitamins, prostaglandins, antibiotics such as tetracycline, pesticides, and environmental hormones.

[0036] Examples of granular carriers include latex particles, ceramic particles, alumina particles, silica-alumina particles, and carbon black particles. Among these particles, latex particles are preferred. Examples of latex materials include polystyrene and divinylbenzene, with polystyrene being preferred. The average particle size of the granular carrier can be 0.1 to 5 μm. The particle size of the granular carrier can be measured by dynamic light scattering. In this specification, the term "average particle size" refers to the particle size (median diameter) when the integrated value from the smallest particle size reaches 50% of the total in a volume-based particle size distribution curve obtained by dynamic light scattering.

[0037] The IgG antibody having four or more N-glycans bound to the Fc region of the particulate carrier can be bound by a general method, such as physical adsorption or chemical binding.

[0038] In one embodiment, a method for measuring a target antigen uses the insoluble particles described above, and the measurement method can be performed to qualitatively evaluate the presence or absence of the target antigen in a test sample, or to quantitatively evaluate the concentration of the target antigen in a test sample.

[0039] In one embodiment, the measurement method includes the steps of contacting the insoluble particles with a test sample that may contain a target antigen, and measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody contained in the insoluble particles and the target antigen. The insoluble particles may be pre-prepared, or the insoluble particles may be prepared by binding a granular carrier to an IgG antibody having four or more N-glycans bound to its Fc region.

[0040] By mixing a suspension containing insoluble particles with a test sample, the insoluble particles can be brought into contact with the test sample, which may contain a target antigen. When the two are brought into contact, the insoluble particles aggregate due to an interaction between the target antigen contained in the test sample and the antibody supported on the granular carrier, resulting in a change in the absorbance of the suspension. The amount of change in absorbance (endpoint method) or the rate of change (rate method) is measured. For measurement, turbidimetry or colorimetric method is preferably used. For example, the agglutination reaction of insoluble particles can be measured by irradiating the cell from outside with light in the visible to near-infrared range, typically 300 nm to 1000 nm, and preferably 500 nm to 900 nm, and detecting the change in absorbance or the change in the intensity of scattered light.

[0041] The time for carrying out the agglutination reaction may be, but is not limited to, 1 to 30 minutes, preferably 1 to 10 minutes, and the temperature for carrying out the agglutination reaction may be, but is not limited to, 30°C to 40°C, or 33 to 37°C.

[0042] A plurality of standard samples containing the target antigen to be measured at various known concentrations are prepared, and the amount or rate of change in absorbance for each is measured using the above method. A calibration curve is drawn by plotting the concentration of the antigen to be measured in the standard samples on the horizontal axis and the measured amount or rate of change in absorbance on the vertical axis. The amount or rate of change in absorbance for an unknown test sample is also measured using the same method, and the measurement results are applied to the above calibration curve, allowing for quantitative evaluation of the target antigen in the test sample. Alternatively, a threshold value for the amount or rate of change in absorbance can be set in advance, and the presence of the target antigen in the test sample can be qualitatively determined if the threshold value is exceeded.

[0043] The test sample is not particularly limited as long as it can contain the target antigen, and examples thereof include body fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, and swabs, or dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, and dilutions thereof being preferred.

[0044] A target antigen measurement kit according to one embodiment includes the insoluble particles. The target antigen measurement kit can be used to qualitatively evaluate the presence or absence of a target antigen in a test sample or to quantitatively evaluate the concentration of the target antigen in a test sample, and more specifically, can be used in the target antigen measurement method.

[0045] In one embodiment, the kit for measuring a target antigen may comprise a particulate carrier and an IgG antibody having four or more N-glycans bound to its Fc region, and may be in a form in which the two are bound together during measurement to prepare a particulate carrier carrying an IgG antibody having four or more N-glycans bound to its Fc region.

[0046] The kit for measuring a target antigen may further include a target antigen for use as a positive control or for preparing a calibration curve, and may also include a buffer solution for diluting the test sample, a buffer solution for mixing the insoluble particles with the test sample, a buffer solution for binding an IgG antibody having four or more N-glycans bound to the Fc region with a particulate carrier, etc.

[0047] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0048] <Preparation of pre-modification IgG1 antibodies> Two types of expression plasmids (produced in-house) for anti-human brain natriuretic peptide antibodies (hereinafter also referred to as "anti-human BNP antibodies"), which are mouse-derived IgG1 antibodies, were transformed into CHO (Chinese Hamster Ovary) cells to express the pre-modification IgG1 antibodies, and the culture supernatant was then purified using a Protein A (GE) column to obtain two types of pre-modification IgG1 antibodies. Note that the two types of pre-modification IgG1 antibodies recognize different epitopes on human BNP.

[0049] <Preparation of IgG1 Antibody Having Four N-Glycans Binding to the Fc Region> An IgG1 antibody having four N-glycans bound to the Fc region (hereinafter also referred to as "glycosylated antibody") was prepared by artificially introducing a glycosylation motif Asn-X-Ser / Thr (X is any amino acid; hereinafter also referred to as "glycosylated motif") into the Fc region (SEQ ID NO: 1) of an anti-human BNP antibody, which is a mouse-derived IgG1 antibody. Specifically, two types of expression plasmids (produced in-house) for the anti-human BNP antibody, which is a mouse-derived IgG1 antibody, were prepared, and a mutation was introduced into each of them by PCR to replace Thr at position 424 of the EU numbering (position 301 in SEQ ID NO: 1) with Asn, thereby obtaining two types of glycosylated antibody expression plasmids. The glycosylated antibody has two pairs of glycosylation motifs, Asn-Ser-Thr (174-176) and Asn-Cys-Ser (301-303), in the Fc region (SEQ ID NO: 2). Asn-Ser-Thr (174-176) is present in the second constant domain, and Asn-Cys-Ser (301-303) is present in the third constant domain. Each of the two obtained plasmids was transformed into CHO (Chinese Hamster Ovary) cells to express the glycosylated antibody, and the culture supernatant was purified using a Protein A (GE) column to obtain two types of glycosylated antibodies.

[0050] The presence of N-glycosylation of the artificially introduced glycosylation motif in the two glycosylated antibodies was confirmed by liquid chromatography mass spectrometry (LC-MS) of peptide fragments of the glycosylated antibodies containing Asn 424 of Eu numbering (hereinafter also referred to as "mutant peptide fragments"). Specifically, each of the two glycosylated antibodies was first heated sequentially using RapiGest SF solution (Waters), dithiothreitol solution, and iodoacetamide solution, then heated overnight in trypsin solution, and finally fragmented using formic acid solution. Next, the fragmented glycosylated antibodies were analyzed by LC-MS to obtain the sum of the peak areas of the mass spectra derived from the mutant peptide fragments and the sum of the peak areas of the mass spectra derived from the mutant peptide fragments in which an N-glycan was bound to Asn 424 of Eu numbering. The percentage of the total peak area of ​​the latter relative to the total peak area of ​​the former was calculated to determine the percentage of Asn bound to an N-glycan among the above Asn. The percentages for the two types of glycosylated antibodies were 99.6% and 100%, respectively. This indicates that an N-glycan is bound to Asn at position 424 of the Eu numbering in the Fc region of almost all glycosylated antibodies.

[0051] <Preparation of antibody-immobilized polystyrene beads> Two types of unmodified anti-human BNP antibody solutions and two types of glycosylated antibody solutions were each mixed with polystyrene beads (latex particles, average particle size: 0.3 μm) and allowed to react at 25°C for 1 hour. Subsequently, the mixture was centrifuged, the supernatant was removed, and a 1% BSA solution was added for blocking, thereby obtaining antibody-immobilized polystyrene beads for each of the two types of unmodified anti-human BNP antibodies and the two types of glycosylated antibodies. Subsequently, the mixture was centrifuged, the supernatant was removed, and a dispersion solution containing 5 mM 3-morpholinopropanesulfonic acid (pH 7.0), 5% by mass sucrose, and 0.4% by mass BSA was added, followed by ultrasonication to disperse the beads. Equal amounts of antibody-immobilized polystyrene beads of the two types of unmodified anti-human BNP antibodies and antibody-immobilized polystyrene beads of the two types of glycated antibodies were mixed together to adjust the final concentration to 0.125 wt%, and antibody-immobilized polystyrene bead solutions were prepared for each of the unmodified anti-human BNP antibodies and the glycated antibodies.

[0052] <Preparation of stabilizing solution> A stabilizing solution containing 120 mM Tris (pH 8.5), 300 mM KCl, and 0.4 mass % Lipidure (registered trademark)-BL405 (NOF Corporation) was prepared.

[0053] <Evaluation of the Reactivity of Antibody-Immobilized Polystyrene Beads> The effect of glycosylated antibodies in the presence and absence of human BNP was examined using a two-reagent latex agglutination immunoassay consisting of the prepared stabilizing solution and antibody-immobilized polystyrene bead solution. Specifically, 100 μL of the prepared stabilizing solution was added to 10 μL of a diluted solution (2% by mass BSA solution) containing 1000 pg / mL of human BNP or not containing human BNP, mixed, and then allowed to stand at 37°C for 5 minutes. Subsequently, 30 μL of the prepared antibody-immobilized polystyrene bead solution of the unmodified anti-human BNP antibody or glycosylated antibody was further added, stirred, and allowed to react for 5 minutes at 37°C. The absorbance was continuously measured for 5 minutes after the addition of the antibody-immobilized polystyrene bead solution using a clinical chemistry analyzer TBA-120FR, with photometric points 21-23 and 31-33 and a dominant wavelength of 628 nm, and the change in absorbance over that 5-minute period was calculated. The results are shown in Figure 1. In Fig. 1, "ΔAbs." indicates the change in absorbance. The ratio of the change in absorbance at 1000 pg / ml of human BNP to the change in absorbance at 0 pg / ml of human BNP (hereinafter also referred to as "S / N") was evaluated and the results are shown in Fig. 2.

[0054] Figure 1 confirms that when a glycosylated antibody is used, the value at 0 pg / ml of human BNP is significantly lower than when the unmodified anti-human BNP antibody is used. This is presumably because the presence of N-glycans improves the dispersibility of the glycosylated antibody-immobilized polystyrene beads, thereby suppressing nonspecific aggregation of the antibody-immobilized polystyrene beads. Furthermore, Figure 2 shows that when a glycosylated antibody is used, the S / N ratio is significantly higher than when the unmodified anti-human BNP antibody is used. From the above, it has been shown that by increasing the number of N-glycans bound to the Fc region of an IgG1 antibody to four or more, it is possible to improve the performance of antibody-immobilized polystyrene beads, such as by suppressing nonspecific reactions in latex agglutination immunoassays.

[0055] <Evaluation of nonspecific reactions in plasma samples> The change in absorbance over 5 minutes was calculated in the same manner as described in "Evaluation of the reactivity of antibody-immobilized polystyrene beads" above, except that three types of plasma-derived samples (samples A to C) containing no human BNP (below the detection limit) were used instead of the diluted solution (2% by mass BSA solution) containing 1000 pg / ml of human BNP or no human BNP. The results are shown in Figure 3. In Figure 3, "ΔAbs." indicates the change in absorbance.

[0056] As can be seen from Figure 3, non-specific reactions were detected with the unmodified anti-human BNP antibody in all of samples A to C, but the non-specific reactions were significantly suppressed with the glycosylated antibody. This suggests that the glycosylated antibody is also effective in suppressing non-specific reactions in actual samples such as plasma-derived samples.

[0057] <Evaluation of Antibody Antigen-Binding Activity> To confirm that the antigen-binding activity of the glycosylated antibody was not reduced by the binding of N-glycans, the antigen-binding activity of the glycosylated antibody and the unmodified anti-human BNP antibody was evaluated by antibody immobilization using a Biacore® 8K (GE). Specifically, various antibodies were first immobilized on a Series S Sensor Chip CM5 according to the manufacturer's protocol to prepare a measurement sensor chip. Using this sensor chip, binding to 0.01 μg / ml and 0.1 μg / ml human BNP solutions was evaluated according to the manufacturer's protocol. The results are shown in Figure 4. In Figure 4, the horizontal axis represents time (seconds), and the vertical axis represents reactivity (RU). In Figure 4, the binding phase was flowed through a flow cell during the time period marked "binding," and the dissociation phase was flowed through a flow cell during the time period marked "dissociation," to contact the sensor chip on which the glycosylated antibody or the unmodified anti-human BNP antibody was immobilized.

[0058] As shown in Figure 4, no difference was observed in the antigen-binding activity of the glycosylated antibody and the anti-human BNP antibody before modification, indicating that the attachment of N-glycans does not affect the antigen-binding activity of the antibody.

Claims

1. An IgG antibody (excluding human IgG) having a Fab region and an Fc region linked to the Fab region, with four or more N-glycans bound to the Fc region.

2. The antibody described in claim 1, wherein two of the four or more N-glycans are bound to a second constant domain, and two N-glycans different from the two N-glycans are bound to a third constant domain.

3. The amino acid sequence of the Fc region is: (1) 90% or more identical to the amino acid sequence of SEQ ID NO:2, wherein the 174th residue is an asparagine residue, the 175th residue is an amino acid residue other than proline, the 176th residue is a serine or threonine residue, the 301st residue is an asparagine residue, the 302nd residue is an amino acid residue other than proline, and the 303rd residue is a serine or threonine residue; (2) 90% or more identical to the amino acid sequence of SEQ ID NO:4, wherein the 180th residue is an asparagine residue, the 181st residue is an amino acid residue other than proline, the 182nd residue is a serine or threonine residue, the 307th residue is an asparagine residue, the 308th residue is an amino acid residue other than proline, and the 309th residue is a serine or threonine residue; (3) 90% or more identical to the amino acid sequence of SEQ ID NO:6, wherein the (4) an amino acid sequence having an identity of 90% or more with the amino acid sequence of SEQ ID NO:8, wherein the 179th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue;or (5) the antibody according to claim 2, which has 90% or more identity with the amino acid sequence of SEQ ID NO: 16, wherein in the amino acid sequence, the 173rd residue is an asparagine residue, the 174th residue is an amino acid residue other than proline, the 175th residue is a serine or threonine residue, the 300th residue is an asparagine residue, the 301st residue is an amino acid residue other than proline, and the 302nd residue is a serine or threonine residue; 4. An insoluble particle comprising a particulate carrier and an IgG antibody against a target antigen supported on the particulate carrier, wherein the antibody has a Fab region and an Fc region connected to the Fab region, and four or more N-glycans are bound to the Fc region.

5. An insoluble particle as described in claim 4, wherein in the antibody, two of the four or more N-glycans are bound to a second constant domain, and two N-glycans different from the two N-glycans are bound to a third constant domain.

6. The insoluble particles according to claim 4, wherein said particulate carrier is a latex particle.

7. The amino acid sequence of the Fc region of the antibody: (1) has 90% or more identity with the amino acid sequence of SEQ ID NO:2, in which the 174th residue is an asparagine residue, the 175th residue is an amino acid residue other than proline, the 176th residue is a serine or threonine residue, the 301st residue is an asparagine residue, the 302nd residue is an amino acid residue other than proline, and the 303rd residue is a serine or threonine residue; (2) has 90% or more identity with the amino acid sequence of SEQ ID NO:4, in which the 180th residue is an asparagine residue, the 181st residue is an amino acid residue other than proline, the 182nd residue is a serine or threonine residue, the 307th residue is an asparagine residue, the 308th residue is an amino acid residue other than proline, and the 309th residue is a serine or threonine residue; (3) having 90% or more identity with the amino acid sequence of SEQ ID NO:6, wherein the 185th residue is an asparagine residue, the 186th residue is an amino acid residue other than proline, the 187th residue is a serine or threonine residue, the 312th residue is an asparagine residue, the 313th residue is an amino acid residue other than proline, and the 314th residue is a serine or threonine residue; (4) having 90% or more identity with the amino acid sequence of SEQ ID NO:8, wherein the 179th residue is an asparagine residue, the 180th residue is an amino acid residue other than proline, the 181st residue is a serine or threonine residue, the 306th residue is an asparagine residue, the 307th residue is an amino acid residue other than proline, and the 308th residue is a serine or threonine residue;(5) having 90% or more identity with the amino acid sequence of SEQ ID NO: 10, wherein the 180th residue is an asparagine residue, the 181st residue is an amino acid residue other than proline, the 182nd residue is a serine or threonine residue, the 307th residue is an asparagine residue, the 308th residue is an amino acid residue other than proline, and the 309th residue is a serine or threonine residue; (6) having 90% or more identity with the amino acid sequence of SEQ ID NO: 12, wherein the 176th residue is an asparagine residue, the 177th residue is an amino acid residue other than proline, the 178th residue is a serine or threonine residue, the 303rd residue is an asparagine residue, the 304th residue is an amino acid residue other than proline, and the 305th residue is a serine or threonine residue; (7) having 90% or more identity with the amino acid sequence of SEQ ID NO: 14, wherein the 176th residue is an asparagine residue, the 177th residue is an amino acid residue other than proline, the 178th residue is a serine or threonine residue, the 303rd residue is an asparagine residue, the 304th residue is an amino acid residue other than proline, and the 305th residue is a serine or threonine residue; The insoluble particle according to claim 5, wherein the 177th residue is an asparagine residue, the 178th residue is an amino acid residue other than proline, the 179th residue is a serine or threonine residue, the 304th residue is an asparagine residue, the 305th residue is an amino acid residue other than proline, and the 306th residue is a serine or threonine residue; or (8) having 90% or more identity with the amino acid sequence of SEQ ID NO: 16, wherein the 173rd residue is an asparagine residue, the 174th residue is an amino acid residue other than proline, the 175th residue is a serine or threonine residue, the 300th residue is an asparagine residue, the 301st residue is an amino acid residue other than proline, and the 302nd residue is a serine or threonine residue.

8. A kit for measuring a target antigen, comprising the insoluble particle according to any one of claims 4 to 7, or comprising an antibody and a particulate carrier for preparing the insoluble particle according to any one of claims 4 to 7.

9. A method for measuring a target antigen, which uses the insoluble particles according to any one of claims 4 to 7.

10. A method for measuring a target antigen, comprising: a step of contacting an insoluble particle according to any one of claims 4 to 7 with a test sample that may contain the target antigen; and a step of measuring an agglutination reaction of the insoluble particle due to an antigen-antibody reaction between an antibody contained in the insoluble particle and the target antigen.

Images