Insoluble particles, kit for measuring a target antigen or a target antibody, method for measuring a target antigen or a target antibody, and method for producing insoluble particles

Abstract

Disclosed are insoluble particles containing a granular carrier and a protein or a peptide carried on the granular carrier, wherein the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected so that the nucleophilicity and / or electron donating property of the α-amino group is lost. Such insoluble particles do not cause the problem of steric hindrance when reacting with a target substance.

Description

Technical Field

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

Background Art

[0002] As a method for measuring a target antigen using an immune reaction, there is a method using a particulate carrier such as latex (for example, Patent Document 1). Insoluble particles having a particulate carrier and an antibody that recognizes the target antigen carried on the particulate carrier cause an antigen-antibody reaction when the target antigen is present. Then, due to their specificity and affinity, they bridge the target antigens and bind to each other, and the insoluble particles aggregate. The presence or absence and the amount of the target antigen are measured based on the presence or absence and the degree of aggregation of the formed aggregates. Similarly, by using insoluble particles having a particulate carrier and an antigen recognized by the target antibody carried on the particulate carrier, the presence or absence and the amount of the target antibody are measured.

[0003] When an antibody or an antigen is carried on a particulate carrier, a technique of forming a covalent bond between the amino group of the antibody or antigen and the carboxy group on the surface of the particulate carrier is widely used. The amino group at the α-position of the N-terminal amino acid residue of the antibody or antigen can be an amino group that forms a covalent bond with the carboxy group on the surface of the particulate carrier.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] When measuring a target antigen using insoluble particles having a granular carrier and an antibody that recognizes the target antigen supported on the granular carrier, since the antigen-binding site of the antibody is conformationally located on the N-terminal side, if the antibody is supported on the granular carrier via the amino group at the α-position of the N-terminal amino acid residue of the antibody, the reaction between the antigen-binding site and the antigen may be sterically inhibited. When measuring a target antibody using insoluble particles having a granular carrier and an antigen recognized by the target antibody supported on the granular carrier, if the density of the antigen supported on the granular carrier is high, the reactivity with the target antibody may be low due to the steric hindrance.

[0006] As a means to avoid these problems of steric hindrance, it is conceivable to use insoluble particles with a low density of antibody or antigen supported on the granular carrier. However, when using such insoluble particles, the measurement sensitivity (S / N ratio) of the target antigen or antibody decreases. Therefore, steric hindrance and measurement sensitivity are in a trade-off relationship.

[0007] As another means to avoid these problems of steric hindrance, it is conceivable to insert a spacer between the carboxy group of the granular carrier and the amino group at the α-position of the N-terminal amino acid residue of the antibody or antigen. However, there is a possibility that the antibody or antigen may bind non-specifically to the spacer. Also, since it is necessary to optimize the spacer length according to the type of antibody and / or antigen, there is a drawback in that it lacks versatility.

[0008] When the antibody or antigen has a lysine residue, the amino group of the side chain (4-aminobutyl group) of the lysine residue can also be an amino group that reacts with the carboxy group on the surface of the granular carrier. When an antibody or antigen in which one of the amino groups at the α-position of the N-terminal amino acid residue and the amino group of the side chain of the lysine residue is not protected is used in the loading reaction with the granular carrier, there arises a problem that the position of the amino group forming a covalent bond with the carboxy group on the surface of the granular carrier cannot be controlled.

[0009] An object of the present invention is to provide insoluble particles having a protein or peptide supported on a granular carrier, which do not cause a steric hindrance problem when reacting with a target substance. Another object of the present invention is to provide a method for controlling the position of an amino group of a protein or peptide that forms a covalent bond with a carboxy group of a granular carrier when the protein or peptide is supported on the granular carrier, and to provide insoluble particles obtained by the method.

Means for Solving the Problems

[0010] The inventors of the present invention selectively protected the amino group of the N-terminal amino acid residue of a protein or peptide, and subjected the protected protein or peptide to a reaction with a granular carrier, thereby controlling the binding position between the protein or peptide and the granular carrier, and thus completed the present invention. Further, it is considered that the insoluble particles obtained by such a method do not cause a steric hindrance problem when reacting with a target substance.

[0011] One aspect of the present invention is insoluble particles containing a granular carrier and a protein or peptide supported on the granular carrier, wherein the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected so that the nucleophilicity and / or electron-donating property of the α-amino group is lost.

[0012] Another aspect of the present invention is a kit for measuring a target antigen or a target antibody containing the insoluble particles.

[0013] Another aspect of the present invention is a kit for measuring a target antigen or a target antibody containing a granular carrier and a protein or peptide for preparing the insoluble particles.

[0014] Another aspect of the present invention is a method for measuring a target antigen using the insoluble particles.

[0015] Another aspect of the present invention is a method for measuring a target antibody using the insoluble particles.

[0016] Another aspect of the present invention is a method for producing the above insoluble particles.

Advantages of the Invention

[0017] According to the present invention, when a protein or peptide is supported on a particulate carrier, the position of the amino group of the protein or peptide that forms a covalent bond with the carboxy group of the particulate carrier can be controlled. Further, according to the present invention, it is possible to provide insoluble particles having a protein or peptide supported on a particulate carrier and not causing a problem of steric hindrance when reacting with a target substance.

Brief Description of the Drawings

[0018]

Figure 1

Modes for Carrying Out the Invention

[0019] Hereinafter, embodiments of the present invention will be described in detail.

[0020] The insoluble particles according to one embodiment contain a particulate carrier and a protein or peptide supported on the particulate carrier, and the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected so that the nucleophilicity and / or electron-donating property of the α-amino group disappear.

[0021] Examples of the particulate carrier include particles such as 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, divinylbenzene, etc., and polystyrene is preferred. The average particle diameter of the particulate carrier can be 0.05 to 5 μm. The average particle diameter of the particulate carrier can be measured by the dynamic light scattering method. In this specification, the "average particle diameter" means the particle diameter (median diameter) when the integrated value from the small particle diameter reaches 50% of the whole in the volume-based particle diameter distribution curve obtained by the dynamic light scattering method.

[0022] A protein or peptide is a macromolecular compound in which a plurality of amino acids are linked in a chain. Generally, a substance with more than approximately 50 constituent amino acids is understood as a protein, and a substance with 50 or fewer amino acids is understood as a peptide. The amino acids that make up a protein or peptide are 20 types including alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), and tyrosine (Tyr). The amino acids may be L-amino acids or D-amino acids.

[0023] The protein or peptide supported on the particulate carrier is not particularly limited as long as it can specifically bind to the target substance to be detected by the insoluble particles. For example, insoluble particles containing a particulate carrier carrying an antigen, an antigen fragment containing an epitope, or a hapten can be used as a reagent for detecting an antibody or an antibody fragment. Further, for example, insoluble particles containing a particulate carrier carrying an antibody or an antibody fragment having a paratope can be used as a reagent for detecting an antigen, an antigen fragment containing an epitope, or a hapten.

[0024] Antigens supported by particulate carriers or antigens recognized by antibodies supported by particulate carriers include, for example, protein markers such as CRP (C-reactive protein), prostate-specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, creatinine, immunoglobulins such as IgG, IgE, IgA, IgM, various tumor markers, lipoproteins such as LDL, HDL, TG, influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, EBV and other viral antigens, Chlamydia trachomatis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthracis, MRSA and other bacterial antigens, toxins produced by bacteria and the like, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroids such as steroid hormones, bioactive amines such as epinephrine and morphine, vitamins such as vitamin Bs, prostaglandins, antibiotics such as tetracycline, pesticides, environmental hormones, etc., but are not limited thereto.

[0025] An epitope refers to a specific structural unit of an antigen that an antibody recognizes and binds to, and a hapten refers to a substance that has the ability to bind to an antibody but does not show immunogenicity alone. Also, a paratope refers to a part of an antibody that recognizes and binds to an antigen.

[0026] The antibody is not particularly limited as long as it can specifically bind to an antigen, and for example, it can be 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 bound by a disulfide bond between a cysteine residue present in CH1 and a cysteine residue present in CL. Also, the heavy chains are bound by a disulfide bond between cysteine residues present in the hinge region located between CH1 and CH2. The antibody may be a fragment of IgG having a heavy chain, may be rIgG (reduced IgG) composed of one heavy chain and one light chain, may be a fragment composed of two heavy chains, or may be a fragment composed of one heavy chain.

[0027] The mode of loading of the above protein or peptide and the above particulate carrier is a covalent bond, and examples thereof include an amide bond and a disulfide bond. The amide bond is preferably formed between an amino group in the side chain of a lysine residue, which the above protein or peptide has, and a carboxy group on the surface of the above particulate carrier.

[0028] The α-amino group of at least one N-terminal amino acid residue of the above protein or peptide is protected so that the nucleophilicity and / or electron-donating property of the α-amino group is lost. Since the α-amino group of the N-terminal amino acid residue has nucleophilicity and / or electron-donating property, that is, high reactivity, when the protein or peptide is supported on the granular carrier, the α-amino group of the N-terminal amino acid residue can become a reaction point. Therefore, there is a possibility of obtaining insoluble particles in which the protein or peptide is supported on the granular carrier via the α-amino group of its N-terminal amino acid residue. In the insoluble particles of the present embodiment, since the α-amino group of the N-terminal amino acid residue is protected so that the nucleophilicity and / or electron-donating property of the α-amino group is lost, it does not become a reaction point during the supporting reaction. Therefore, insoluble particles in which the protein or peptide is supported on the granular carrier at a desired position can be obtained.

[0029] The second amino acid residue from the N-terminus of the above protein or peptide can be an amino acid other than proline, that is, 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.

[0030] When a protein or peptide forms a multimer and there are multiple N-terminal amino acid residues, it is sufficient that the α-amino group of at least one N-terminal amino acid residue is protected. However, from the viewpoint of high binding efficiency with the target substance, it is preferable that the α-amino groups of all N-terminal amino acid residues are protected. For example, when the protein supported on the granular carrier is IgG, IgG has four N-terminal amino acid residues. It is sufficient that the α-amino group of at least one of the four N-terminal amino acid residues of IgG is protected, but the α-amino groups of two or three N-terminal amino acid residues may be protected, and it is preferable that the α-amino groups of the four N-terminal amino acid residues are protected.

[0031] Protecting groups for protecting the α-amino group of the N-terminal amino acid residue so that the nucleophilicity and / or electron-donating property of the α-amino group is lost include protecting groups generally used as protecting groups for amino groups. Such protecting groups are well-known to those skilled in the art, and appropriate protecting groups can be selected with reference to Wuts and Greene, “Greene’s Protective Groups in Organic Synthesis”, Wiley-Interscience, 2006 April, etc.

[0032] In one embodiment, the protected protein or peptide has the formula (1):

Chemical formula

[0033] In one embodiment, the protected protein or peptide has the formula (2):

Chemical formula

[0034] In one embodiment, the protected protein or peptide has the formula (3):

Chemical formula

[0035] The compound represented by formula (3) may be either a compound represented by formula (3a) or a compound represented by formula (3b), and preferably it is a compound represented by formula (3a).

Chemical formula

[0036] R 1 、R 3 、R 4 and R 9The organic group in is not particularly limited as long as it is a group derived from an organic molecule or an organic molecular complex, for example, a group obtained by removing one atom or multiple atoms from an organic molecule or an organic molecular complex. The organic molecule is not particularly limited and may be natural, synthetic, or artificial. The organic molecular complex is not particularly limited, but may be, for example, a complex (or a living organism) formed by linking multiple molecules including organic molecules. The mode of the linkage is not particularly limited, but may be, for example, a hydrogen bond, an electrostatic force, a van der Waals force, a hydrophobic bond, a covalent bond, a coordinate bond, or the like. These bonds may be made via a linker (for specific examples, see the linker described below). The organic molecule or organic molecule complex is preferably a functional substance, and specific examples thereof include medicinal compounds, luminescent molecules, polymer compounds, ligands, molecules to which ligands are bound, antigenic proteins, antibodies, proteins, nucleic acids, sugars, lipids, cells, viruses, labels (e.g., radioisotope labels), carbon electrodes, carbon nanomaterials, linkers, spacer molecules (e.g., polyethylene glycol or derivatives thereof, peptides (e.g., peptides containing an amino acid sequence that is cleaved by an enzyme within a cell), etc.), complexes thereof, linking molecules, etc.

[0037] R 1 , R 3 , R 4 and R 9 The inorganic material in the above is a material that may or may not contain metal atoms, and is not particularly limited. Examples of the inorganic material include electrode materials, metal particles, metal oxide particles, semiconductor particles, magnetic particles, etc. The inorganic material may hold organic molecules or organic molecule complexes.

[0038] R 1 , R 3 , R 4 and R 9 The organic group in the formula (C) may have one or more substituents selected from the substituent group A. 1-6It can be a 6- to 10-membered aryl group which may have one or more substituents selected from an alkyl group or substituent group B, where substituent group A consists of a halogen atom and a 6- to 10-membered aryl group, and substituent group B consists of a halogen atom, a nitro group, a C 1-6 alkyl group, a C 1-6 alkoxy group, a carboxy group, and a di(C 1-6 alkyl)amino group.

[0039] R 1 、R 3 、R 4 and R 9 The organic groups in are preferably a methyl group, a trifluoromethyl group, a phenyl group, a tolyl group, a nitrophenyl group, a carboxyphenyl group, a dicarboxyphenyl group, a diethylaminophenyl group, a methoxyphenyl group, a benzyl group, or a naphthylmethyl group.

[0040] The halogen atom is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0041] C 1-6 Examples of the alkyl group include linear or branched ones such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, an s-pentyl group, a t-pentyl group, a 2-methylbutyl group, a 1-methylbutyl group, a 2-methylbutyl group, a neopentyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 1-ethylpropyl group, an n-hexyl group, an isohexyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 1-ethylbutyl group, a 2-ethylbutyl group, a 1,1,2-trimethylpropyl group, a 1,2,2,-trimethylpropyl group, a 1-ethyl-1-methylpropyl group, or a 1-ethyl-2-methylpropyl group, etc. C 1-6The alkyl group is preferably a methyl group, an ethyl group or a t-butyl group, more preferably a methyl group or an ethyl group.

[0042] C 1-6 Examples of the alkoxy group include linear or branched ones such as methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, pentoxy group, isopentoxy group, 2-methylbutoxy group, neopentoxy group, hexyloxy group, 4-methylpentoxy group, 3-methylpentoxy group, 2-methylpentoxy group, 3,3-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1,1-dimethylbutoxy group, 1,2-dimethylbutoxy group, 1,3-dimethylbutoxy group or 2,3-dimethylbutoxy group. C 1-6 The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group or an isopropoxy group, more preferably a methoxy or ethoxy group.

[0043] The 6- to 10-membered aryl group means an aromatic hydrocarbon cyclic group having 6 to 10 carbon atoms (in the case of a condensed ring, at least one of the rings may show aromaticity). Examples of the 6- to 10-membered aryl group include a phenyl group, 1-naphthyl group, 2-naphthyl group, indenyl group, indanyl group, azulenyl or heptalenyl group. The 6- to 10-membered aryl group is preferably a phenyl group, 1-naphthyl group or 2-naphthyl group, more preferably a phenyl group.

[0044] The above-protected protein or peptide is preferably a compound represented by formula (3a).

Chemical formula

[0045] In the compound represented by formula (3a), R 4 and R 9 The preferred combinations are as shown in Table 1 below.

[0046]

Table 1

[0047] In the compound represented by formula (2), R 4 and R 9 The preferred combinations are as shown in Table 2 below.

[0048]

Table 2

[0049] Formula (1):

Chemical formula

Chemical formula

[0050] Formula (2):

Chemical formula

Chemical formula

[0051] Formula (3):

Chemical formula

Chemical formula

[0052] In one embodiment, in the insoluble particles, the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is deprotected. The protein or peptide with the α-amino group of the N-terminal amino acid residue deprotected is likely to retain its original function and structure. Therefore, since such insoluble particles are insoluble particles obtained by loading a protein or peptide with the α-amino group of the N-terminal amino acid residue protected onto a particulate carrier, the binding efficiency with the target substance is higher compared to the prior art. Such insoluble particles can be produced by deprotecting the α-amino group of at least one N-terminal amino acid residue of the protein or peptide contained in the insoluble particles obtained by loading a protein or peptide with the α-amino group of the N-terminal amino acid residue protected onto a particulate carrier. The reaction conditions for deprotection may be the reaction conditions generally used for deprotection of the protected amino group. Such reaction conditions are well known to those skilled in the art, and appropriate reaction conditions can be selected with reference to Wuts and Greene, “Greene’s Protective Groups in Organic Synthesis”, Wiley-Interscience, 2006 April, etc.

[0053] In one embodiment, a kit for measuring a target antigen or a target antibody contains the above insoluble particles. The kit for measuring a target antigen or a target antibody can be used for qualitatively evaluating the presence or absence of a target antigen or a target antibody in a test sample, or for quantitatively evaluating the concentration of a target antigen or a target antibody in a test sample, and more specifically, can be used in the following methods for measuring a target antigen or a target antibody.

[0054] In one embodiment, a kit for measuring a target antigen or a target antibody contains a particulate carrier for preparing insoluble particles and a protein or a peptide. The kit for measuring a target antigen or a target antibody according to this embodiment prepares the insoluble particles by binding the protein or the peptide and the particulate carrier during the measurement.

[0055] The kit for measuring a target antigen or a target antibody may further contain a target antigen or a target antibody for use as a positive control or for preparing a calibration curve, and may also contain a buffer for diluting a test sample, a buffer for mixing an insoluble particle and a test sample, a buffer for binding a protein or a peptide to a particulate carrier, etc.

[0056] In one embodiment, the method for measuring a target antigen uses the above insoluble particles. Such a measurement 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 a target antigen in a test sample.

[0057] In one embodiment, the method for measuring a target antigen includes a step of bringing the above insoluble particles into contact with a test sample that may contain the target antigen, and a step of measuring an aggregation reaction of the above insoluble particles due to an antigen-antibody reaction between an antibody or an antibody fragment contained in the above insoluble particles and the above target antigen. As the above insoluble particles, those that have been prepared may be used, or they may be prepared by binding a particulate carrier and an antibody or an antibody fragment during measurement.

[0058] By mixing a suspension containing the above insoluble particles and a test sample, the above insoluble particles can be brought into contact with the test sample that may contain the target antigen. When the two are brought into contact, the insoluble particles aggregate due to the interaction between the target antigen contained in the test sample and the antibody or antibody fragment contained in the insoluble particles, and the absorbance of the suspension changes. The amount of change (endpoint method) or the rate of change (rate method) of this absorbance is measured. For the measurement, a turbidimetry or a colorimetry is preferably used. For example, by irradiating light from visible light to the near-infrared region, usually light of 300 nm to 1000 nm, preferably 500 nm to 900 nm, from outside the cell and detecting the change in absorbance or the change in the intensity of scattered light, the aggregation reaction of the above insoluble particles is measured.

[0059] The time for performing the agglutination reaction can be from 1 minute to 30 minutes, preferably from 1 minute to 10 minutes, but is not limited thereto. The temperature for performing the agglutination reaction can be from 35°C to 40°C, and can also be from 36°C to 38°C, but is not limited thereto.

[0060] Prepare a plurality of standard samples containing the target antigen to be measured at various known concentrations, and measure the change amount or change rate of the absorbance for them by the above method. Plot the concentration of the antigen to be measured in the standard sample on the horizontal axis and the measured change amount or change rate of the absorbance on the vertical axis to draw a calibration curve. For an unknown test sample, also measure the change amount or change rate of the absorbance by the same method, and apply the measurement result to the above calibration curve, whereby the target antigen in the test sample can be quantitatively evaluated. Further, a threshold value of the change amount or change rate of the absorbance can be set in advance, and when it exceeds the threshold value, it can be qualitatively evaluated that the target antigen is present in the test sample.

[0061] The test sample is not particularly limited as long as it can contain the target antigen, and examples include body fluids such as blood, serum, plasma, urine, feces, saliva, tissue fluid, cerebrospinal fluid, sweat, etc. or dilutions thereof, and blood, serum, plasma, urine, feces, cerebrospinal fluid or dilutions thereof are preferred.

[0062] In one embodiment, the method for measuring the target antibody uses the above insoluble particles. Such a measurement method can be performed to qualitatively evaluate the presence or absence of the target antibody in the test sample or to quantitatively evaluate the concentration of the target antibody in the test sample.

[0063] In one embodiment, the method for measuring the target antibody includes a step of contacting the above insoluble particles with a test sample that may contain the target antibody, and a step of measuring the agglutination reaction of the above insoluble particles due to the antigen-antibody reaction between the antigen, antigen fragment or hapten contained in the above insoluble particles and the above target antibody. The above insoluble particles may be those that have been prepared, or may be prepared by binding a particulate carrier to an antigen, antigen fragment or hapten during measurement.

[0064] In the method for measuring a target antibody, the measurement of the aggregation reaction of insoluble particles, the conditions for performing the aggregation reaction, the method for quantitatively or qualitatively evaluating the target antibody, and the test sample are the same as those described in the method for measuring a target antigen.

Example

[0065] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[0066] Example 1: Preparation of an influenza virus detection reagent by latex agglutination method (1-1) N-terminal protection of anti-influenza A virus antibody 1H-1,2,3-triazole-4-carboxaldehyde:

Chemical formula

[0067] (1-2) Preparation of antibody-conjugated latex particles The N-terminal protected anti-influenza A antibody prepared in (1-1) was diluted with an MES buffer (pH 5.0) to a concentration of 0.5 mg / mL, and latex particles were added thereto to a concentration of 0.25% (w / v). After stirring, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added thereto to a concentration of 1% (w / v), and further stirred. The supernatant was removed by centrifugation and resuspended in a 5 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.8), 0.01% (w / v) casein to obtain N-terminal protected anti-influenza A virus antibody-conjugated latex particles (hereinafter referred to as N-terminal protected anti-influenza A antibody-conjugated latex).

[0068] Detection of Influenza A virus antigen of subtype (1-3) The N-terminal protected anti-influenza A antibody-conjugated latex or antibody-unconjugated latex prepared in 1-2 was diluted with a reaction solution (50 mM Tris buffer (pH 8.0), 2% Triton® X-100) to a final concentration of 0.01% (w / v), and mixed in equal volume with a reaction solution containing Influenza A virus antigen at a concentration of 113.4 pfu / mL. The reaction solution was irradiated with light at a wavelength of 635 nm, and the aggregability of the latex was evaluated by measuring the scattered light every 1 minute for 15 minutes. The samples were compared by calculating the slope of the plot up to the 6-minute time point when the signal of the scattered light reached a plateau. The measurement of the scattered light was performed using a nephelometric reader NEPHELOstar Plus (BMG LABTECH). The results are shown in Fig. 1.

Claims

1. Insoluble particles composed of a protein or peptide supported on a granular carrier, wherein the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected, and the protected protein or peptide has the formula (2): 【Chemical 1】 The compound represented by [in formula (2), R ３ and R ４ represent a hydrogen atom, a hydroxy group, an organic group or a group derived from an inorganic material, and R ５ represents a group obtained by removing the at least one N-terminal amino acid residue and the adjacent -NH- from the protein or peptide, and R ６ represents the side chain of the at least one N-terminal amino acid residue of the protein or peptide.], an insoluble particle.

2. Insoluble particles composed of a protein or peptide supported on a granular carrier, wherein the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected, and the protected protein or peptide has the formula (3): 【Chemical 2】 The compound represented by [in formula (3), R ４ represents a hydrogen atom, a hydroxy group, a group derived from an organic group or an inorganic material, and among R ７ and R ８ , one is -N(-R ９ )(R ９ represents a hydrogen atom, a hydroxy group, a group derived from an organic group or an inorganic material, and the other represents =N-, and R ５ represents a group obtained by removing the at least one N-terminal amino acid residue and the adjacent -NH- from the protein or peptide, and R ６ represents the side chain of the at least one N-terminal amino acid residue of the protein or peptide.], an insoluble particle.

3. The insoluble particles according to claim 1 or 2, wherein the second amino acid residue from the N-terminus of the protein or peptide is an amino acid other than proline.

4. The insoluble particles according to any one of claims 1 to 3, wherein the protein or peptide is supported on the granular carrier by a covalent bond.

5. The insoluble particles according to any one of claims 1 to 4, wherein the α-amino groups of all N-terminal amino acid residues of the protein or peptide are protected.

6. The insoluble particles according to any one of claims 1 to 5, wherein the granular carrier is latex particles.

7. Insoluble particles, wherein the α-amino group of at least one N-terminal amino acid residue of the protein or peptide contained in the insoluble particles according to any one of claims 1 to 6 is deprotected.

8. The insoluble particles according to any one of claims 1 to 7, wherein the protein or peptide is an antigen, an antigen fragment containing an epitope, or a hapten.

9. The insoluble particles according to any one of claims 1 to 7, wherein the protein or peptide is an antibody or an antibody fragment having a paratope.

10. The insoluble particles according to claim 9, wherein the antibody or antibody fragment is IgG or a fragment thereof.

11. A kit for measuring a target antigen or a target antibody, comprising the insoluble particles according to any one of claims 8 to 10.

12. A kit for measuring a target antigen or a target antibody, comprising a granular carrier and a protein or peptide for preparing the insoluble particles according to any one of claims 8 to 10, and preparing the insoluble particles when measuring the target antigen or the target antibody.

13. A method for measuring a target antigen, using the insoluble particles according to claim 9 or 10.

14. A method for measuring a target antibody, using the insoluble particles according to claim 8.

15. A step of contacting a test sample that may contain the insoluble particles according to claim 9 or 10 with a target antigen, and a step of measuring an aggregation reaction of the insoluble particles by an antigen-antibody reaction between an antibody or antibody fragment contained in the insoluble particles and the target antigen. A method for measuring a target antigen, comprising the steps.

16. A step of contacting a test sample that may contain the insoluble particles according to claim 8 with a target antibody, and a step of measuring an aggregation reaction of the insoluble particles by an antigen-antibody reaction between an antigen, antigen fragment or hapten contained in the insoluble particles and the target antibody. A method for measuring a target antibody, comprising the steps.

17. The method according to claim 13 or 15, for qualitatively evaluating the presence or absence of a target antigen in a test sample or for quantitatively evaluating the concentration of a target antigen in a test sample.

18. The method according to claim 14 or 16, for qualitatively evaluating the presence or absence of a target antibody in a test sample or for quantitatively evaluating the concentration of a target antibody in a test sample.

19. A method for producing insoluble particles, wherein the insoluble particles are insoluble particles composed of a protein or peptide supported on a particulate carrier, and the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected. The protected protein or peptide is of formula (1): 【Chemical Formula 3】 The compound represented by [in formula (1), R １ represents a hydrogen atom, a hydroxy group, a group derived from an organic group or an inorganic material, and R ２ represents a group in which the α-amino group of the at least one N-terminal amino acid residue has been removed from the protein or peptide.], and The protein or peptide and formula (4): [Chemical Formula 4] A method for producing insoluble particles, comprising a step of reacting a compound represented by [in formula (4), R １ represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material.]

20. A protein or peptide and formula (5): 【Chemical Formula 5】 The compound represented by [in formula (5), R ３ as well as R ４ represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material.], and a step of reacting them, a method for producing the insoluble particles according to claim 1.

21. A protein or peptide and formula (6): 【Chemical Formula 6】 The compound represented by [Formula (6) wherein R ４ represents any one of a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, and R ７ and R ８ of which, one represents -N(-R ９ )(R ９ represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material.), and the other represents =N-.], and a process for producing the insoluble particles according to claim 2, comprising reacting the same.

22. A method for producing the insoluble particles according to claim 7, comprising a step of deprotecting the α-amino group of at least one N-terminal amino acid residue of the protein or peptide contained in the insoluble particles according to any one of claims 1 to 9.

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