Method for measuring target antigen, and insoluble particles and kit for target antigen measurement used therein

Abstract

To provide a method for measuring a target antigen using insoluble particles with high aggregation ability.SOLUTION: The present invention provides insoluble particles containing a granular carrier and an antibody to a target antigen carried on the granular carrier, with no sugar chain being bonded to the heavy chain of the antibody.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] One method for measuring a target antigen using an immune reaction is to use a granular carrier such as latex (for example, Patent Document 1). When the target antigen is present, an antigen-antibody reaction occurs on the granular carrier to which an antibody specific to the target antigen is bound. Then, due to the specificity and affinity, the antibodies bind to each other through the target antigen as a bridge, and agglutinate. The presence or absence and amount of the target antigen are measured based on the presence or absence of the resulting agglutinates and the degree of agglutination.

[0003] Measurement methods utilizing such 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. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2017-83440 A [Patent Document 2] JP 2009-162537 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, there is still room for improvement in the above-mentioned detection sensitivity. Here, if the particulate carrier (the insoluble particles containing the particulate carrier) to which the antibody specific to the target antigen is bound has high agglutination property, it is considered that even a trace amount of the target antigen can be detected, and therefore the detection sensitivity may be improved.

[0006] An object of the present invention is to provide insoluble particles with high agglutination ability, a kit for measuring a target antigen using the same, and a method for measuring a target antigen. [Means for solving the problem]

[0007] The inventors discovered that particulate carriers carrying antibodies that do not have a sugar chain bound to their heavy chains are more likely to aggregate than particulate carriers carrying antibodies that have a sugar chain bound to their heavy chains, and thus arrived at the present invention.

[0008] The present invention relates to insoluble particles containing a granular carrier and an antibody against a target antigen supported on the granular carrier, wherein no sugar chain is bound to the heavy chain of the antibody.

[0009] The present invention relates to a kit for measuring a target antigen, which comprises the insoluble particles, or which comprises an antibody and a particulate carrier for preparing the insoluble particles.

[0010] The present invention also relates to a method for measuring a target antigen using the above-mentioned insoluble particles. Effect of the Invention

[0011] According to the present invention, it is possible to provide insoluble particles having excellent agglutination ability when an antigen-antibody reaction occurs. By using the insoluble particles having excellent agglutination ability, it is expected that a target antigen can be detected with high sensitivity. [Brief description of the drawings]

[0012] [Figure 1] 1 is a graph showing the results of the agglutination reaction of anti-IgE antibody-sensitized latex. [Diagram 2] 1 is a graph showing the results of an agglutination reaction of anti-CRP antibody-sensitized latex. [Diagram 3] FIG. 1 is a schematic diagram showing the T-DNA regions of a TMV vector (pICH56122) and a PVX vector (pICH56131). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described in detail.

[0014] The insoluble particle according to one embodiment contains a granular carrier and an antibody against a target antigen supported on the granular carrier, and no sugar chain is bound to the heavy chain of the antibody. The antibody against the target antigen may be in a form in which not only no sugar chain is bound to the heavy chain, but also no sugar chain is bound to the light chain.

[0015] Examples of target antigens include protein markers such as CRP (C-reactive protein), prostate-specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, and creatinine; immunoglobulins such as IgG, IgE, IgA, and IgM; various tumor markers; lipoproteins such as LDL, HDL, and TG; influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, viral antigens such as HAV, HBs, HCV, HIV, and EBV; and chlamydia. Examples of such antibacterial agents include, but are not limited to, bacterial antigens such as Dia trachomatis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthracis, and MRSA, toxins produced by bacteria, and the like, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroids such as steroid hormones, physiologically active amines such as epinephrine and morphine, vitamins such as vitamin B, prostaglandins, antibiotics such as tetracycline, pesticides, and environmental hormones.

[0016] Examples of the granular carrier include latex particles, ceramic particles, alumina particles, silica-alumina particles, and carbon black particles. Among these particles, latex particles are preferred. Examples of the latex material include polystyrene and divinylbenzene, and polystyrene is 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 small particle size reaches 50% of the total in the volume-based particle size distribution curve obtained by dynamic light scattering.

[0017] The antibody is not particularly limited as long as it can specifically bind to the target antigen, and may be any of IgG, IgM, IgA, IgD, and IgE, but is preferably IgG. IgG is composed of two heavy chains (H chains) and two light chains (L chains). The heavy chain is composed of a variable region (VH), a first constant region (CH1), a second constant region (CH2), and a third constant region (CH3) in order from the N-terminus. The light chain is composed of a variable region (VL) and a constant region (CL) in order from the N-terminus. The portion composed of CH2 and CH3 is the Fc region. One heavy chain and one light chain are bonded by a disulfide bond between a cysteine ​​residue present in CH1 and a cysteine ​​residue present in CL. In addition, the heavy chains are bonded by a disulfide bond between cysteine ​​residues present in the hinge region located between CH1 and CH2. The antibody may be an IgG fragment having a heavy chain, an rIgG (reduced IgG) consisting of one heavy chain and one light chain, a fragment consisting of two heavy chains, or a fragment consisting of one heavy chain. In an antibody having two heavy chains, it is sufficient that at least one of the heavy chains does not have a sugar chain bound thereto, and it is also acceptable that neither of the heavy chains has a sugar chain bound thereto.

[0018] When the antibody is an IgG, its Fc region preferably has an amino acid sequence having 90% or more identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 13, but does not contain the glycosylation motif Asn-Xaa-Ser / Thr (Xaa is any amino acid).

[0019] Here, SEQ ID NO: 1 is Protein Accession No. P01857 (human Ig gamma-1 C region), SEQ ID NO: 2 is Protein Accession No. P01859 (human Ig gamma-2 C region), SEQ ID NO: 3 is Protein Accession No. P01860 (human Ig gamma-3 C region), SEQ ID NO: 4 is Protein Accession No. P01861 (human Ig gamma-4 C region), SEQ ID NO: 5 is Protein Accession No. P01868 (mouse Ig gamma-1 C region), SEQ ID NO: 6 is Protein Accession No. P01863 (mouse Ig gamma-2a C region), SEQ ID NO: 7 is Protein Accession No. P01867 (mouse Ig gamma-2b C region), and SEQ ID NO: 8 is Protein Accession No. P03987 (mouse Ig gamma-3 C region). SEQ ID NO: 9 is Protein Accession No. P20759 (rat Ig gamma-1 C region), SEQ ID NO: 10 is Protein Accession No. P20760 (rat Ig gamma-2a C region), SEQ ID NO: 11 is Protein Accession No. P20761 (rat Ig gamma-2b C region), SEQ ID NO: 12 is Protein Accession No. P20762 (rat Ig gamma-2c C region), and SEQ ID NO: 13 is Protein Accession No. P01870 (rabbit Ig gamma C region).

[0020] When the antibody is an IgG, its Fc region may have an amino acid sequence of any of SEQ ID NOs: 14 to 26 or an amino acid sequence having 90% or more identity to an amino acid sequence of SEQ ID NOs: 14 to 26, in which the Asp at position 180 of SEQ ID NO: 14, the Asp at position 176 of SEQ ID NO: 16, the Asp at position 177 of SEQ ID NO: 18, the Asp at position 174 of SEQ ID NO: 19, the Asp at position 180 of SEQ ID NO: 20, the Asp at position 185 of SEQ ID NO: 21, the Asp at position 179 of SEQ ID NO: 22, the Asp at position 172 of SEQ ID NO: 23, the Asp at position 183 of SEQ ID NO: 24, the Asp at position 179 of SEQ ID NO: 25, or the Asp at position 173 of SEQ ID NO: 26 is not mutated.

[0021] Here, SEQ ID NO:14 is the 180th position of SEQ ID NO:1, SEQ ID NO:15 is the 176th position of SEQ ID NO:2, SEQ ID NO:16 is the 227th position of SEQ ID NO:3, SEQ ID NO:17 is the 177th position of SEQ ID NO:4, SEQ ID NO:18 is the 174th position of SEQ ID NO:5, SEQ ID NO:19 is the 180th position of SEQ ID NO:6, SEQ ID NO:20 is the 185th position of SEQ ID NO:7, SEQ ID NO:21 is the 179th position of SEQ ID NO:8, SEQ ID NO:22 is the 176th position of SEQ ID NO:9, SEQ ID NO:23 is the 172nd position of SEQ ID NO:10, SEQ ID NO:24 is the 183rd position of SEQ ID NO:11, SEQ ID NO:25 is the 179th position of SEQ ID NO:12, and SEQ ID NO:26 is the 173rd position of SEQ ID NO:13, in which Asn is replaced with Asp.

[0022] When the antibody is an IgG, its Fc region may have an amino acid sequence of any of SEQ ID NOs: 27 to 39 or an amino acid sequence having 90% or more identity to an amino acid sequence of SEQ ID NOs: 27 to 39, in which the Gln at position 180 of SEQ ID NO: 27, the Gln at position 176 of SEQ ID NO: 28, the Gln at position 227 of SEQ ID NO: 30, the Gln at position 177 of SEQ ID NO: 31, the Gln at position 174 of SEQ ID NO: 32, the Gln at position 180 of SEQ ID NO: 33, the Gln at position 185 of SEQ ID NO: 34, the Gln at position 179 of SEQ ID NO: 35, the Gln at position 176 of SEQ ID NO: 36, the Gln at position 183 of SEQ ID NO: 37, the Gln at position 179 of SEQ ID NO: 38, or the Gln at position 173 of SEQ ID NO: 39 is not mutated.

[0023] Here, SEQ ID NO:27 is the 180th position of SEQ ID NO:1, SEQ ID NO:28 is the 176th position of SEQ ID NO:2, SEQ ID NO:29 is the 227th position of SEQ ID NO:3, SEQ ID NO:30 is the 177th position of SEQ ID NO:4, SEQ ID NO:31 is the 174th position of SEQ ID NO:5, SEQ ID NO:32 is the 180th position of SEQ ID NO:6, SEQ ID NO:33 is the 185th position of SEQ ID NO:7, SEQ ID NO:34 is the 179th position of SEQ ID NO:8, SEQ ID NO:35 is the 176th position of SEQ ID NO:9, SEQ ID NO:36 is the 172nd position of SEQ ID NO:10, SEQ ID NO:37 is the 183rd position of SEQ ID NO:11, SEQ ID NO:38 is the 179th position of SEQ ID NO:12, and SEQ ID NO:39 is the 173rd position of SEQ ID NO:13, in which Asn is replaced with Gln.

[0024] When the antibody is an IgG, its Fc region may have an amino acid sequence of any of SEQ ID NOs: 40 to 52 or an amino acid sequence having 90% or more identity to an amino acid sequence of SEQ ID NOs: 40 to 52, in which Ala at position 182 of SEQ ID NO: 40, Ala at position 178 of SEQ ID NO: 41, Ala at position 229 of SEQ ID NO: 42, Ala at position 179 of SEQ ID NO: 43, Ala at position 176 of SEQ ID NO: 44, Ala at position 182 of SEQ ID NO: 46, Ala at position 187 of SEQ ID NO: 45, Ala at position 181 of SEQ ID NO: 47, Ala at position 178 of SEQ ID NO: 48, Ala at position 174 of SEQ ID NO: 49, Ala at position 185 of SEQ ID NO: 50, Ala at position 181 of SEQ ID NO: 51, or Ala at position 175 of SEQ ID NO: 52 is not mutated.

[0025] Here, SEQ ID NO:40 is the 182nd position of SEQ ID NO:1, SEQ ID NO:41 is the 178th position of SEQ ID NO:2, SEQ ID NO:42 is the 229th position of SEQ ID NO:3, SEQ ID NO:43 is the 179th position of SEQ ID NO:4, SEQ ID NO:44 is the 176th position of SEQ ID NO:5, SEQ ID NO:45 is the 182nd position of SEQ ID NO:6, SEQ ID NO:46 is the 187th position of SEQ ID NO:7, SEQ ID NO:47 is the 181st position of SEQ ID NO:8, SEQ ID NO:48 is the 178th position of SEQ ID NO:9, SEQ ID NO:49 is the 174th position of SEQ ID NO:10, SEQ ID NO:50 is the 185th position of SEQ ID NO:11, SEQ ID NO:51 is the 181st position of SEQ ID NO:12, and SEQ ID NO:52 is the 175th position of SEQ ID NO:13, in which Thr is replaced with Ala.

[0026] An antibody having no sugar chain bound to its heavy chain can be produced by using a plant transient expression system, and can also be produced by using a genetically modified plant. As a plant transient expression system, for example, the method described in Japanese Patent No. 5015012 can be used. In addition, an antibody having no sugar chain bound to its heavy chain can also be obtained as a genetically modified organism using a mammalian cell as an expression host. In this case, examples of the mammalian cell include, but are not limited to, CHO (Chinese Hamster Ovary) cells and HEK293 (Human Embryonic Kidney cells 293) cells. Furthermore, the method of obtaining a genetically modified organism includes, but is not limited to, a transient expression system using a plasmid vector or a virus vector lacking autonomous replication ability, a semi-stable expression system using an episomal vector that has a nuclear transport signal and has autonomous replication ability, and a stable expression system in which a target gene is inserted into the genome of an expression host.

[0027] The antibody having no sugar chain bound to its heavy chain can be bound to the particulate carrier by a general method such as physical adsorption or chemical binding.

[0028] In one embodiment, the method for measuring a target antigen uses the insoluble particles. Such a measuring method can be performed 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.

[0029] In one embodiment, the measurement method includes a step of contacting insoluble particles with a test sample that may contain a target antigen, and a step of measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen. The insoluble particles may be already prepared, or the insoluble particles may be prepared by binding a granular carrier to an antibody having no sugar chain bound to its heavy chain when the measurement is performed.

[0030] By mixing a suspension containing insoluble particles with a test sample, the insoluble particles can be brought into contact with the test sample that may contain a target antigen. When the two are brought into contact, the insoluble particles are agglutinated by the interaction between the target antigen contained in the test sample and the antibody supported on the granular carrier, and the absorbance of the suspension changes. The amount of change in absorbance (endpoint method) or the rate of change (rate method) is measured. For the measurement, a turbidimetric method or a colorimetric method is preferably used. For example, the cell is irradiated with visible light to near-infrared light, usually 300 nm to 1000 nm, preferably 500 nm to 900 nm, from the outside, and the agglutination reaction of the insoluble particles is measured by detecting the change in absorbance or the change in the intensity of the scattered light.

[0031] The time for carrying out the agglutination reaction may be 1 to 30 minutes, preferably 1 to 10 minutes, but is not limited thereto. The temperature for carrying out the agglutination reaction may be 35°C to 40°C, or may be 36 to 38°C, but is not limited thereto.

[0032] 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 is measured for them by 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 amount or rate of change in the measured absorbance on the vertical axis. The amount or rate of change in absorbance of an unknown test sample is also measured by the same method, and the measurement results are applied to the above calibration curve, thereby quantitatively evaluating the target antigen in the test sample. In addition, a threshold value for the amount or rate of change in absorbance is set in advance, and it can be qualitatively evaluated that the target antigen is present in the test sample when the threshold value is exceeded.

[0033] The test sample is not particularly limited as long as it can contain the target antigen, and examples of the test sample include body fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swabs, etc., or dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, or dilutions thereof being preferred.

[0034] A kit for measuring a target antigen according to one embodiment includes the insoluble particles. The kit for measuring a target antigen 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 a target antigen in a test sample, and more specifically, can be used in a method for measuring the target antigen.

[0035] In one embodiment, the kit for measuring a target antigen may include a particulate carrier and an antibody that does not have a glycan bound to its heavy chain, and the two are bound together during measurement to prepare a particulate carrier carrying an antibody that does not have a glycan bound to its heavy chain.

[0036] The target antigen measurement kit 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, and a buffer solution for binding an antibody that does not have a glycan bound to its heavy chain with a particulate carrier. EXAMPLES

[0037] <Preparation of anti-IgE antibody (Anti-IgE Ab-Wild)> An antibody whose heavy chain constant region consists of SEQ ID NO:5 was produced by the following method.

[0038] The vector was prepared using the plant transient expression system described in Japanese Patent Publication No. 5015012. Tobacco mosaic virus (TMV) vector pICH56122 (Fig. 3) and synthetic DNA containing an anti-IgE antibody heavy chain coding sequence and a BsaI site were opened with BsaI and ligated by reacting with T4 DNA Ligase at 37°C overnight. The ligation reaction solution was added to E. coli competent cells, transformed by the heat shock method, and cultured at 37°C overnight. The anti-IgE antibody heavy chain coding vector was obtained from the culture using a QIAprep Spin Miniprep Kit (QIAGEN). Similarly, an anti-IgE antibody light chain coding vector was obtained using potato virus X (PVX) vector pICH56131 (Fig. 3) and synthetic DNA containing an anti-IgE antibody light chain coding sequence and a BsaI site. Each of these vectors was added to separate Agrobacterium competent cells, transformed by electroporation, and cultured at 28°C for 3 days. One infiltration buffer (10 mM MES 10 mM MgSO 4 The leaves of Nicotiana benthamiana were infiltrated with the culture medium at a ratio of 1 / 1000 and harvested after about one week. The harvested leaves (infected leaves) were frozen.

[0039] 200 g of frozen infected leaves were weighed and crushed in a cutter mixer. 200 mL of extraction solution (100 mM Tris, 250 mM NaCl, 40 mM sodium ascorbate) was added to the crushed material, and the infected leaves were repeatedly crushed. The mixture was centrifuged to recover the supernatant. The recovered liquid was filtered through a 0.22 μm filter. The filtrate was subjected to Protein A column chromatography to remove impurities.

[0040] <Preparation of anti-IgE antibody without glycan-binding motif (Anti-IgE Ab-NQ)> An antibody having a heavy chain constant region consisting of SEQ ID NO:31 was prepared in the same manner as the antibody having a heavy chain constant region consisting of SEQ ID NO:5 (Anti-IgE Ab-Wild).

[0041] <Preparation of anti-IgE antibody without glycan-binding motif (Anti-IgE Ab-TA)> An antibody having a heavy chain constant region sequence of SEQ ID NO: 44 was produced in the same manner as the antibody having a heavy chain constant region of SEQ ID NO: 5 (Anti-IgE Ab-Wild).

[0042] Western blotting using α1,3-fucose antibody confirmed that glycans were bound to the heavy chains of Anti-IgE Ab-Wild and that glycans were not bound to the heavy chains of Anti-IgE Ab-NQ and Anti-IgE Ab-TA.

[0043] <Latex agglutination test for anti-IgE antibodies> (1) Preparation of reagents Using an antibody against IgE, a measuring reagent for the immune agglutination method was prepared as follows. i) 0.07 mg of antibody was added to 1 mL of polystyrene latex suspension to prepare insoluble particles in which the antibody was supported on polystyrene latex particles. The insoluble particles were suspended in a buffer solution (glycine, pH 7.3) to a concentration of 0.125% by mass to prepare an insoluble particle suspension. ii) A buffer solution (glycine, pH 8.3) was prepared. (2) Measurement using an automatic analyzer The automatic analyzer was a Hitachi 7180 model automatic analyzer, and the agglutination state of insoluble particles via an antigen-antibody reaction between the target antigen (IgE) sample at each concentration and the antibody supported on the polystyrene latex particles described in (1) was measured by light transmittance. Specifically, 140 μL of the buffer prepared in (1)(ii) above was added to 3.5 μL of IgE solution, and the mixture was stirred and mixed at 37° C. After leaving it for 5 minutes, 70 μL of the insoluble particle suspension was added, and further stirred and mixed at 37° C. The agglutination reaction over about 5 minutes was measured as the amount of absorbance change.

[0044] The insoluble particles of Anti-IgE Ab-Wild, Anti-IgE Ab-NQ, and Anti-IgE Ab-TA prepared in (1) above were evaluated for the degree of agglutination due to reaction with the target antigen. As a result, it was confirmed that the insoluble particles of Anti-IgE Ab-NQ and Anti-IgE Ab-TA had a greater degree of agglutination than Anti-IgE Ab-Wild, enabling highly sensitive measurement.

[0045] <Preparation of anti-CRP antibody (Anti-CRP Ab-Wild)> An antibody whose heavy chain constant region consists of SEQ ID NO:1 was produced by the following method.

[0046] The vector was prepared using the plant transient expression system described in Japanese Patent Publication No. 5015012. Tobacco mosaic virus (TMV) vector pICH56122 (Figure 3) and synthetic DNA containing an anti-CRP antibody heavy chain coding sequence and a BsaI site were opened with BsaI and ligated by reacting with T4 DNA Ligase at 37°C overnight. The ligation reaction solution was added to E. coli competent cells, transformed by the heat shock method, and cultured at 37°C overnight. The anti-CRP antibody heavy chain coding vector was obtained from the culture using a QIAprepSpin Miniprep Kit (QIAGEN). Similarly, an anti-CRP antibody light chain coding vector was obtained using potato virus X (PVX) vector pICH56131 (Figure 3) and synthetic DNA containing an anti-CRP antibody light chain coding sequence and a BsaI site. Each of these vectors was added to separate Agrobacterium competent cells, transformed by electroporation, and cultured at 28°C for 3 days. One infiltration buffer (10 mM MES 10 mM MgSO 4 The leaves of Nicotiana benthamiana were infiltrated with the culture medium at a ratio of 1 / 1000 and harvested after about one week. The harvested leaves (infected leaves) were frozen.

[0047] 200 g of frozen infected leaves were weighed and crushed in a cutter mixer. 200 mL of extraction solution (100 mM Tris, 250 mM NaCl, 5 mM EDTA, 40 mM sodium ascorbate) was added to the crushed material, and the infected leaves were repeatedly crushed. The mixture was centrifuged to recover the supernatant. The recovered liquid was filtered through a 0.22 μm filter. The filtrate was subjected to Protein A column chromatography to remove impurities.

[0048] <Preparation of anti-CRP antibody without glycan-binding motif (Anti-CRP Ab-TA)> An antibody having a heavy chain constant region sequence consisting of SEQ ID NO: 40 was produced in the same manner as the antibody having a heavy chain constant region consisting of SEQ ID NO: 1 (Anti-CRP Ab-Wild).

[0049] The presence of glycan binding to the heavy chain of Anti-CRP Ab-Wild and the absence of glycan binding to the heavy chain of Anti-CRP Ab-TA were confirmed by Western blotting using α1,3-fucose antibody.

[0050] <Latex agglutination test for anti-CRP antibodies> (1) Preparation of reagents Using an antibody against CRP, a measuring reagent for the immunoagglutination method was prepared as follows. i) 0.06 mg of antibody was added to 1 mL of polystyrene latex suspension to prepare insoluble particles in which the antibody was supported on polystyrene latex particles. The insoluble particles were suspended in a buffer solution (glycine, pH 7.3) to a concentration of 0.110% by mass to prepare an insoluble particle suspension. ii) A buffer solution (glycine, pH 8.3) was prepared. (2) Measurement using an automatic analyzer The automatic analyzer was a Hitachi 3500 model automatic analyzer, and the agglutination state of insoluble particles via an antigen-antibody reaction between the target antigen (CRP) sample at each concentration and the antibody supported on the polystyrene latex particles described in (1) was measured by light transmittance. Specifically, 100 μL of the buffer prepared in (1)(ii) above was added to 2.0 μL of CRP solution, and the mixture was stirred and mixed at 37° C. After leaving it for 5 minutes, 100 μL of the insoluble particle suspension was added, and further stirred and mixed at 37° C. The agglutination reaction over about 5 minutes was measured as the amount of absorbance change.

[0051] The insoluble particles of Anti-CRP Ab-Wild and Anti-CRP Ab-TA prepared in (1) above were evaluated for the degree of agglutination due to reaction with the target antigen. As a result, it was confirmed that the insoluble particles of Anti-CRP Ab-TA had a greater degree of agglutination than Anti-CRP Ab-Wild, enabling highly sensitive measurement.

Claims

1. Using insoluble particles containing latex particles and an antibody against a target antigen supported by the latex particles, 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 and the target antigen; A method for measuring a target antigen by a latex agglutination method, comprising: The method, wherein the antibody has no glycosylation on its heavy chain and is a mouse IgG.

2. The method according to claim 1, wherein the IgG Fc region has an amino acid sequence having 90% or more identity to any one of the amino acid sequences of SEQ ID NOs: 5 to 8, but lacks the glycosylation motif Asn-Xaa-Ser / Thr.

3. The Fc region of the IgG is having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 18, in which Asp at position 174 is not mutated; having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 19, in which Asp at position 180 is not mutated; having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 20, in which Asp at position 185 is not mutated; or having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:21, in which Asp at position 179 is not mutated; The method of claim 1.

4. The Fc region of the IgG is having an amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 31, in which Gln at position 174 is not mutated; having the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 32, in which the 180th Gln is not mutated; having an amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 33, in which Gln at position 185 is not mutated; or having the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 34, in which Gln at position 179 is not mutated; The method of claim 1.

5. The Fc region of the IgG is having an amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 44, in which Ala at position 176 is not mutated; having an amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 45, in which Ala at position 182 is not mutated; having an amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 46, in which Ala at position 187 is not mutated; or having an amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 47, in which Ala at position 181 is not mutated; The method of claim 1.

6. 6. The method according to any one of claims 1 to 5, for qualitatively assessing the presence or absence of a target antigen in a test sample, or for quantitatively assessing the concentration of a target antigen in a test sample.

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