JP7710028B2 - Insoluble particles, kit for measuring target antigen or target antibody, method for measuring target antigen or target antibody, and method for producing insoluble particles - Google Patents

Description

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.

One method for measuring target antigens using immune reactions is to use granular carriers such as latex (see, for example, Patent Document 1). In the presence of target antigens, insoluble particles having granular carriers and antibodies that recognize the target antigens supported on the granular carriers undergo an antigen-antibody reaction. Due to their specificity and affinity, the insoluble particles bind to each other using the target antigens as a bridge, causing agglutination. The presence or absence and amount of target antigens present are measured based on the presence or absence of the resulting agglutinates and the degree of agglutination. Similarly, the presence or absence and amount of target antibodies are measured by using insoluble particles having granular carriers and antigens recognized by the target antibodies supported on the granular carriers.

When an antibody or antigen is supported on a granular carrier, a method of forming a covalent bond between the amino group of the antibody or antigen and the carboxy group on the surface of the granular 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 the amino group that forms a covalent bond with the carboxy group on the surface of the granular carrier.

JP 2017-83440 A

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, the antigen-binding site of the antibody is located on the N-terminus side, so 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 hindered. When measuring a target antibody using insoluble particles having a granular carrier and an antigen recognized by a 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 steric hindrance.

One way to avoid these problems of steric hindrance is to use insoluble particles with a low density of antibodies or antigens supported on a granular carrier. However, the use of such insoluble particles reduces the measurement sensitivity (S/N ratio) of the target antigen or antibody. Thus, there is a trade-off between steric hindrance and measurement sensitivity.

Another method for avoiding these steric hindrance problems is 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 nonspecifically to the spacer. In addition, there is a disadvantage that it is not very versatile because the spacer length needs to be optimized depending on the type of antibody and/or antigen.

When an antibody or antigen has a lysine residue, the amino group in the side chain of the lysine residue (4-aminobutyl group) can also be an amino group that reacts with a carboxy group on the surface of the granular carrier. If 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 in the side chain of the lysine residue is not protected is used in a support reaction with a granular carrier, a problem arises in that the position of the amino group that forms a covalent bond with the carboxy group on the surface of the granular carrier cannot be controlled.

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

The inventors of the present invention believed that by selectively protecting the amino group of the N-terminal amino acid residue of a protein or peptide and reacting the protected protein or peptide with a particulate carrier, it would be possible to control the binding position between the protein or peptide and the particulate carrier, and thus completed the present invention. Furthermore, it is believed that the insoluble particles obtained by this method do not cause steric hindrance problems when reacting with a target substance.

One aspect of the present invention is an insoluble particle comprising 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 as to eliminate the nucleophilicity and/or electron donating property of the α-amino group.

Another aspect of the present invention is a kit for measuring a target antigen or a target antibody, comprising the above-mentioned insoluble particles.

Another aspect of the present invention is a kit for measuring a target antigen or a target antibody, comprising a particulate carrier for preparing the insoluble particles and a protein or peptide.

Another aspect of the present invention is a method for measuring a target antigen using the above-mentioned insoluble particles.

Another aspect of the present invention is a method for measuring target antibodies using the above-mentioned insoluble particles.

Another aspect of the present invention is a method for producing the above-mentioned insoluble particles.

According to the present invention, when a protein or peptide is supported on a granular carrier, the position of the amino group of the protein or peptide that forms a covalent bond with the carboxy group of the granular carrier can be controlled. Furthermore, according to the present invention, it is possible to provide insoluble particles that have a protein or peptide supported on a granular carrier and do not cause steric hindrance problems when reacting with a target substance.

FIG. 1 shows a graph illustrating the results of Example 1.

The following describes in detail an embodiment of the present invention.

In one embodiment, the insoluble particles contain a granular carrier and a protein or peptide supported on the granular carrier, and the α-amino group of at least one N-terminal amino acid residue of the protein or peptide is protected so as to eliminate the nucleophilicity and/or electron donating property of the α-amino group.

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, and polystyrene is preferred. The average particle size of the granular carrier can be 0.05 to 5 μm. The average particle size of the granular carrier can be measured by dynamic light scattering. In this specification, "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.

A protein or peptide is a polymeric compound in which multiple amino acids are linked in a chain. Generally, a substance with more than about 50 constituent amino acids is understood to be a protein, and a substance with 50 or fewer amino acids is understood to be a peptide. There are 20 types of amino acids that make up a protein or peptide: 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.

There are no particular limitations on the proteins or peptides carried by the granular carriers, and any substances may be used as long as they can specifically bind to the target substance to be detected by the insoluble particles. For example, insoluble particles containing granular carriers carrying antigens, antigen fragments containing epitopes, or haptens can be used as reagents for detecting antibodies or antibody fragments. Also, for example, insoluble particles containing granular carriers carrying antibodies or antibody fragments having paratopes can be used as reagents for detecting antigens, antigen fragments containing epitopes, or haptens.

Antigens supported on granular carriers or antigens recognized by antibodies supported on granular carriers 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, influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, and HIV. Examples of the antigens include, but are not limited to, viral antigens such as EBV, bacterial antigens such as Chlamydia trachomatis, Streptococcus hemolyticus, 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 B vitamins, prostaglandins, antibiotics such as tetracycline, pesticides, and environmental hormones.

An epitope is a specific structural unit of an antigen that an antibody recognizes and binds to, a hapten is a substance that has the ability to bind to an antibody but is not immunogenic by itself, and a paratope is a part of an antibody that recognizes and binds to an antigen.

The antibody is not particularly limited as long as it can specifically bind to an antigen, and can be, for example, 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 present, or rIgG (reduced IgG) consisting of one heavy chain and one light chain, or a fragment consisting of two heavy chains, or a fragment consisting of one heavy chain.

The protein or peptide is supported on the particulate carrier by a covalent bond, such as an amide bond or a disulfide bond. The amide bond is preferably formed between an amino group in the side chain of a lysine residue of the protein or peptide and a carboxy group on the surface of the particulate carrier.

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. The α-amino group of the N-terminal amino acid residue is nucleophilic and/or electron donating, i.e., highly reactive, and therefore, when the protein or peptide is supported on the granular carrier, the α-amino group of the N-terminal amino acid residue can be a reaction site. Therefore, it is possible to obtain insoluble particles in which the protein or peptide is supported on the granular carrier via the α-amino group of the N-terminal amino acid residue. In the insoluble particles of this embodiment, 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, and therefore does not become a reaction site during the support reaction. Therefore, insoluble particles in which the protein or peptide is supported on the granular carrier at the desired position can be obtained.

The second amino acid residue from the N-terminus of the protein or peptide may be an amino acid other than proline, i.e., 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.

When a protein or peptide forms a multimer and has 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, but 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.

Examples of protecting groups for protecting the α-amino group of the N-terminal amino acid residue so as to eliminate the nucleophilicity and/or electron donating property of the α-amino group include protecting groups that are commonly 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 by referring to Wuts and Greene, "Greene's Protective Groups in Organic Synthesis", Wiley-Interscience, April 2006, and the like.

In one embodiment, the protected protein or peptide has the formula (1):
In formula (1), ^R1 represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, and ^R2 represents a group obtained by removing the α-amino group of at least one N-terminal amino acid residue from the protein or peptide.

In one embodiment, the protected protein or peptide has the formula (2):
In formula (2), ^R3 and ^R4 each represent a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, ^R5 represents a group obtained by removing the at least one N-terminal amino acid residue and the -NH- adjacent thereto from the protein or peptide, and ^R6 represents the side chain of the at least one N-terminal amino acid residue of the protein or peptide.

In one embodiment, the protected protein or peptide has the formula (3):
In formula (3), R ⁴ represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, one of R ⁷ and R ⁸ 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-), R ⁵ represents a group obtained by removing the at least one N-terminal amino acid residue and the -NH- adjacent thereto 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.

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

The organic group in R ¹ , R ³ , R ⁴ and R ⁹ 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 or synthetic/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, etc. These bonds may be made via a linker (see the linker described below for specific examples). 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.

The inorganic materials in R ¹ , R ³ , R ⁴ and R ⁹ may or may not contain metal atoms, and are not particularly limited. Examples of the inorganic materials include electrode materials, metal particles, metal oxide particles, semiconductor particles, magnetic particles, etc. The inorganic materials may hold organic molecules or organic molecule complexes.

The organic group in R ¹ , R ³ , R ⁴ and R ⁹ may be a C _1-6 alkyl group which may have one or more substituents selected from Substituent Group A or a 6-10 membered aryl group which may have one or more substituents selected from Substituent Group B, where Substituent Group A consists of halogen atoms and 6-10 membered aryl groups, and Substituent Group B consists of halogen atoms, nitro groups, C _1-6 alkyl groups, C _1-6 alkoxy groups, carboxy groups and di(C _1-6 alkyl)amino groups.

The organic group in R ¹ , R ³ , R ⁴ and R ⁹ is 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.

A halogen atom is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

Examples of the _C1-6 alkyl group include 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, Examples of the C 1-6 alkyl group include linear or branched ones such as methyl, ethyl or t-butyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl or 1-ethyl-2-methylpropyl. The _{C 1-6} alkyl group is preferably a methyl group, an ethyl group or a t-butyl group, and more preferably a methyl group or an ethyl group.

Examples of the C _1-6 alkoxy group include linear or branched groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, s-butoxy, t-butoxy, pentoxy, isopentoxy, 2-methylbutoxy, neopentoxy, hexyloxy, 4-methylpentoxy, 3-methylpentoxy, 2-methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, and 2,3-dimethylbutoxy. The C _1-6 alkoxy group is preferably a methoxy, ethoxy, propoxy, or isopropoxy group, and more preferably a methoxy or ethoxy group.

The 6-10 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 needs to be aromatic). Examples of the 6-10 membered aryl group include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, an indenyl group, an indanyl group, an azulenyl group, or a heptalenyl group. The 6-10 membered aryl group is preferably a phenyl group, a 1-naphthyl group, or a 2-naphthyl group, and more preferably a phenyl group.

The protected protein or peptide is preferably a compound represented by formula (3a).

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

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

Formula (1):
The compound represented by the formula (4):
For example, the compound represented by formula (4) is dissolved in dimethyl sulfoxide (DMSO) and added to an aqueous solution containing the protein or peptide to react with the compound represented by formula (1). The aqueous solution may be a buffer solution that does not contain primary or secondary amines, and the pH of the buffer solution is 4.5 to 9.0, preferably 7.0 to 8.0. The compound represented by formula (4) may be used in an amount equal to or greater than the number of moles of the protein or peptide. The reaction temperature may be 4° C. to 37° C., and the reaction time may be 30 minutes or more.

Formula (2):
The compound represented by the formula (5):
The reaction conditions can be, for example, those described in Onoda et al., "Triazolecarbaldehyde Reagents for One-Step N-Terminal Protein Modification", Chembiochem. 2020 May 4; 21(9): 1274-1278. and WO 2020/175680.

Formula (3):
The compound represented by the formula (6):
The reaction conditions can be, for example, those described in Onoda et al., "Triazolecarbaldehyde Reagents for One-Step N-Terminal Protein Modification", Chembiochem. 2020 May 4; 21(9): 1274-1278. and WO 2020/175680.

In one embodiment, the insoluble particles have the α-amino group of at least one N-terminal amino acid residue of the protein or peptide 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 in which a protein or peptide with a protected α-amino group of the N-terminal amino acid residue is supported on a granular carrier, the binding efficiency with the target substance is higher than that of conventional techniques. Such insoluble particles can be produced by deprotecting the α-amino group of at least one N-terminal amino acid residue of a protein or peptide contained in insoluble particles in which a protein or peptide with a protected α-amino group of the N-terminal amino acid residue is supported on a granular carrier. The reaction conditions for deprotection may be reaction conditions commonly used for deprotection of a protected amino group. Such reaction conditions are well known to those skilled in the art, and appropriate reaction conditions can be selected by referring to Wuts and Greene, "Greene's Protective Groups in Organic Synthesis", Wiley-Interscience, April 2006, etc.

In one embodiment, a kit for measuring a target antigen or a target antibody contains the insoluble particles. The kit for measuring a target antigen or a target antibody can be used to qualitatively evaluate the presence or absence of a target antigen or a target antibody in a test sample, or to quantitatively evaluate the concentration of a target antigen or a target antibody in a test sample, and more specifically, can be used in the method for measuring a target antigen or a target antibody described below.

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

The kit for measuring a target antigen or a target antibody may further include a target antigen or a target antibody for use as a positive control or for creating a calibration curve, and may also include a buffer for diluting the test sample, a buffer for mixing the insoluble particles with the test sample, a buffer for binding proteins or peptides with the particulate carrier, etc.

In one embodiment, the method for measuring a target antigen uses the 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 the target antigen in a test sample.

In one embodiment, the method for measuring a target antigen includes a step of contacting the insoluble particles 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 or antibody fragment contained in the insoluble particles and the target antigen. The insoluble particles may be pre-prepared, or may be prepared by binding a granular carrier to an antibody or antibody fragment at the time of measurement.

By mixing the suspension containing the insoluble particles with the 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 or antibody fragment contained in the insoluble particles, 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.

The time for carrying out the agglutination reaction may be 1 to 30 minutes, preferably 1 to 10 minutes, but is not limited to these. 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 to these.

A number 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 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 of an unknown test sample is also measured using the same method, and the measurement results are applied to the above calibration curve, allowing the target antigen in the test sample to be quantitatively evaluated. 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 if the threshold value is exceeded.

The test sample is not particularly limited as long as it can contain the target antigen, but examples 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.

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

In one embodiment, the method for measuring a target antibody includes a step of contacting the insoluble particles with a test sample that may contain the target antibody, and a step of measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antigen, antigen fragment, or hapten contained in the insoluble particles and the target antibody. The insoluble particles may be pre-prepared, or may be prepared by binding a granular carrier with an antigen, antigen fragment, or hapten at the time of measurement.

In the method for measuring a target antibody, the measurement of the agglutination reaction of insoluble particles, the conditions for performing the agglutination 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.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the examples below.

Example 1: Preparation of influenza virus test reagent by latex agglutination method (1-1) N-terminal protection of anti-type A influenza virus antibody 1H-1,2,3-triazole-4-carbaldehyde:
A dimethyl sulfoxide (DMSO) aqueous solution (200 mM) of the above was diluted with phosphate buffer (10 mM, pH 7.5) to a final concentration of 20 mM, and an anti-type A influenza virus antibody was added thereto to a final concentration of 2 mg/mL, followed by shaking at 37° C. for 16 hours. The solution after the reaction was replaced with 5 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) by centrifugal ultrafiltration. The obtained antibody is hereinafter referred to as an N-terminal protected anti-type A antibody.

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

(1-3) Detection of influenza A virus antigen The N-terminal protected anti-type A antibody-bound latex or antibody-unbound latex prepared in 1-2 was diluted with a reaction solution (50 mM Tris buffer (pH 8.0), 2% Triton (registered trademark) X-100) to a final concentration of 0.01% (w/v), and mixed in equal amounts with a reaction solution containing influenza A virus antigen at a concentration of 113.4 pfu/mL. The reaction solution was irradiated with light of a wavelength of 635 nm, and the scattered light was measured every minute for 15 minutes to evaluate the latex aggregation. Comparison between samples was performed by calculating the slope of the plot up to the 6-minute point when the scattered light signal reached a plateau. The scattered light was measured using a turbidimetric measurement reader NEPHELOstar Plus (BMG LABTECH). The results are shown in FIG. 1.

Claims (22)

Insoluble particles comprising a protein or peptide supported on a particulate carrier, the α-amino group of at least one N-terminal amino acid residue of the protein or peptide being protected;
The protected protein or peptide has the formula (2):

[In formula (2), ^R3 and ^R4 represent a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, ^R5 represents a group obtained by removing the at least one N-terminal amino acid residue and the -NH- adjacent thereto from the protein or peptide, and ^R6 represents a side chain of the at least one N-terminal amino acid residue of the protein or peptide.]. Insoluble particles comprising a protein or peptide supported on a particulate carrier, the α-amino group of at least one N-terminal amino acid residue of the protein or peptide being protected;
The protected protein or peptide has the formula (3):

[in formula (3), R ⁴ represents a hydrogen atom, a hydroxy group, an organic group, or a group derived from an inorganic material, one of R ⁷ and R ⁸ 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-, R ⁵ represents a group in which the at least one N-terminal amino acid residue and the -NH- adjacent thereto have been removed from the protein or peptide, and R ⁶ represents a side chain of the at least one N-terminal amino acid residue of the protein or peptide]. The insoluble particle 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. The insoluble particles according to any one of claims 1 to 3, wherein the protein or peptide is covalently bonded to the particulate carrier. The insoluble particle 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. The insoluble particles according to any one of claims 1 to 5, wherein the granular carrier is a latex particle. An insoluble particle in which the α-amino group of at least one N-terminal amino acid residue of a protein or peptide contained in the insoluble particle according to any one of claims 1 to 6 has been deprotected. The insoluble particle 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. The insoluble particle according to any one of claims 1 to 7, wherein the protein or peptide is an antibody or an antibody fragment having a paratope. The insoluble particle according to claim 9, wherein the antibody or antibody fragment is IgG or a fragment thereof. A kit for measuring a target antigen or a target antibody, comprising the insoluble particles according to any one of claims 8 to 10. A kit for measuring a target antigen or a target antibody, comprising a particulate carrier for preparing the insoluble particles according to any one of claims 8 to 10 and a protein or a peptide, and preparing the insoluble particles when measuring a target antigen or a target antibody. A method for measuring a target antigen using the insoluble particles according to claim 9 or 10. A method for measuring a target antibody using the insoluble particles according to claim 8. A method for measuring a target antigen, comprising the steps of: contacting the insoluble particles according to claim 9 or 10 with a test sample that may contain the target antigen; and measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody or antibody fragment contained in the insoluble particles and the target antigen. A method for measuring a target antibody, comprising the steps of: contacting the insoluble particles according to claim 8 with a test sample that may contain a target antibody; and measuring an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antigen, antigen fragment, or hapten contained in the insoluble particles and the target antibody. The method according to claim 13 or 15 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. The method according to claim 14 or 16 for qualitatively assessing the presence or absence of a target antibody in a test sample, or for quantitatively assessing the concentration of a target antibody in a test sample. 1. A method for producing insoluble particles, comprising:
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 has the formula (1):

[In formula (1), R ¹ represents a hydrogen atom, a hydroxyl group, an organic group, or a group derived from an inorganic material, and R ² represents a group in which the α-amino group of at least one N-terminal amino acid residue has been removed from the protein or peptide],
The protein or peptide and a compound of formula (4):

[In the formula (4), R ¹ represents a hydrogen atom, a hydroxyl group, an organic group, or a group derived from an inorganic material.]. A protein or peptide and a compound of formula (5):

The method for producing the insoluble particles according to claim 1, comprising a step of reacting a compound represented by the formula (5): wherein R ³ and R ⁴ each represent a hydrogen atom, a hydroxyl group, an organic group, or a group derived from an inorganic material. A protein or peptide and a compound of formula (6):

with a 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 one of R ⁷ and R ⁸ 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-). A method for producing the insoluble particle 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 particle according to any one of claims 1 to 9.