Affinity particle, test reagent, and detection method for detecting target immunoglobulin g in human specimen

Abstract

To provide a test reagent for a latex agglutination method, the reagent being excellent in sensitivity of detection of target immunoglobulin G in a human specimen and being capable of sufficiently securing a range (measurement range) in which a detection amount increases with an increase in amount of the target immunoglobulin G, and to provide a detection method therefor.SOLUTION: An affinity particle for detecting immunoglobulin G in a human specimen by a latex agglutination method, the affinity particle having a volume-average particle diameter of 400 nm or less, comprising a latex particle and a protein carried on a surface of the latex particle, wherein the protein contains an antigen having a molecular weight of 10,000 or more, and wherein the mass of the protein carried on the surface of the latex particle is 1.0 μg or more and 20.0 μg or less per 1 mg of the affinity particle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to affinity particles, test reagents, and detection methods for detecting target immunoglobulin G in human specimens. [Background technology]

[0002] Tests for specific immunoglobulins present in human samples (antibody tests) are widely used in infectious disease-related tests, such as antibody tests for hepatitis viruses such as HCV (hepatitis C) and HBV (hepatitis B), and antibody tests for Treponema pallidum antigens. Simple and rapid testing is desirable, particularly when used for population screening tests or as infection confirmation tests to prevent infection during medical procedures such as surgery.

[0003] A simple and rapid immunoassay method is the immuno-latex agglutination assay (hereinafter referred to as latex agglutination). In this method, when the target substance is a specific antibody (immunoglobulin) present in a human specimen as mentioned above, the target substance (immunoglobulin) in the specimen is reacted with latex particles carrying an antigen that has affinity for the target substance. The particle agglutination reaction mediated by the antigen-antibody reaction is then optically detected as changes in scattered light intensity, transmitted light intensity, absorbance, etc., for diagnosis. For example, HCV-specific immunoglobulins in human samples have been measured by latex agglutination assay.

[0004] In Patent Document 1, HCV-specific immunoglobulins are detected using sensitized particles that are sensitized with recombinant HCV antigens and then blocked with bovine serum albumin (BSA).The technology disclosed involves freeze-drying the sensitized particles to prevent a decrease in the detection amount due to peeling or oxidation of the sensitized antigen, thereby improving the stability of the test reagent during long-term storage. Patent document 2 discloses a technology in which a test reagent using HCV antigen-sensitized polystyrene latex particles uses a specific fatty acid (salt) in the measurement system, which can strongly suppress non-specific reactions even when the polystyrene particles are not coated with a blocking agent. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 11-258241 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-148496 Summary of the Invention [Problem to be solved by the invention]

[0006] However, Patent Documents 1 and 2 do not mention the range of HCV-specific immunoglobulin levels that can be measured (measurement range). In HCV antibody testing, distinguishing between negative and positive test results is extremely effective as a means of quickly confirming a negative result, but a positive result also includes a medical history, and cannot be used alone to confirm current infection. A definitive diagnosis of infection requires subsequent nucleic acid testing and other methods. Meanwhile, if the antibody "abundance" of positive samples could be further classified during the antibody testing process, it would be possible to predict current infection to a certain degree, contributing to the efficiency of definitive diagnosis. Therefore, it is desirable to have an HCV antibody test reagent that can classify the amount of HCV antibody present in a sample. However, particularly in the case of test reagents that use the latex agglutination method as their measurement principle, it has been difficult to ensure a measurement range while improving detection sensitivity, which has been a challenge.

[0007] When latex particles with a large particle size are used to improve detection sensitivity, the amount of absorbance change is large even when the amount of antibody in the sample is small, i.e., the number of agglutination reactions is small, and this is expected to improve detection sensitivity. However, because the absorbance of the original particles (before agglutination) is large and the specific surface area of ​​the particles carrying the antigen is reduced, the range (measurement range) over which the amount of absorbance change increases with increasing antibody amount may be narrow. The aforementioned Patent Documents 1 and 2 disclose test agents using 0.48 μm polystyrene latex. The inventors' studies have found that while using particles of this size provides excellent detection sensitivity, it is difficult to sufficiently ensure the range (measurement range) over which the amount of detection increases with increasing antibody amount.

[0008] The present invention has been made in consideration of these background technologies and problems. An object of the present invention is to provide a test reagent for latex agglutination assay that has excellent detection sensitivity for target immunoglobulin G in human samples and can sufficiently ensure a range (measurement range) in which the detection amount increases as the amount of target immunoglobulin G increases. A further object of the present invention is to provide a method for detecting target immunoglobulin G in human samples. [Means for solving the problem]

[0009] The present invention relates to affinity particles for detecting immunoglobulin G in a human sample by latex agglutination, which have a volume average particle size of 400 nm or less, and comprise latex particles and a protein supported on the surface of the latex particles, the protein including an antigen with a molecular weight of 10,000 or more, and the amount of protein supported on the surface of the latex particles is 1.0 μg or more and 20.0 μg or less per 1 mg of the affinity particles. The present invention also provides a test reagent comprising a first reagent solution in which the affinity particles are dispersed. The present invention also relates to a method for detecting immunoglobulin G in a human specimen by a latex agglutination method using a test reagent comprising a first reagent solution in which affinity particles are dispersed and a second reagent solution which is a buffer solution, the method comprising: a first step of obtaining a mixture containing the human specimen and the second reagent solution; a second step of mixing the mixture with the first reagent solution and reacting them to obtain a reaction solution; and a third step of measuring the concentration of immunoglobulin G in the reaction solution, wherein the affinity particles have a volume average particle size of 400 nm or less, the affinity particles comprise latex particles and a protein supported on the surface of the latex particles, the protein comprising an antigen with a molecular weight of 10,000 or more, and the amount of protein supported on the surface of the latex particles is 1.0 μg or more and 20.0 μg or less per 1 mg of the affinity particles. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a test reagent for a latex agglutination assay that has excellent detection sensitivity for target immunoglobulin G in a human specimen and can sufficiently ensure a range (measurement range of immunoglobulin G amount) in which the detection amount increases as the amount of target immunoglobulin G increases, and further to provide a method for detecting target immunoglobulin G in a human specimen. [Brief explanation of the drawings]

[0011] [Figure 1-1] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 1 according to an example of the present invention. [Figure 1-2] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 2 according to an example of the present invention. [Figure 1-3] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 3 according to an example of the present invention. [Figure 1-4]1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 4 according to an example of the present invention. [Figure 1-5] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 5 according to an example of the present invention. [Figure 1-6] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 6 according to an example of the present invention. [Figure 1-7] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 7 according to an example of the present invention. [Figure 1-8] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 8 according to an example of the present invention. [Figure 1-9] 1 shows the results of measuring six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using test reagent 9 according to an example of the present invention. [Figure 1-10] The graph shows the results of measurements of six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using comparative reagent 1 of the prior art. [Figure 1-11] The graph shows the results of measurements of six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using comparative reagent 2 in the prior art. [Figure 1-12] The graph shows the results of measurements of six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using comparative reagent 3 of the prior art. [Figure 1-13] The graph shows the results of measurements of six serogroups from healthy individuals (Evaluation 1) and a positive serum P1 (low positive value) (Evaluation 2) using comparative reagent 4 of the prior art. [Figure 2-1] 1 shows the results of measuring serum N (negative control) (evaluation 1) and positive sera P1 to P5 (evaluation 2) using test reagents 1 to 5 according to an example of the present invention. [Figure 2-2]1 shows the results of measuring serum N (negative control) (evaluation 1) and positive sera P1 to P5 (evaluation 2) using test reagents 6 to 9 according to an example of the present invention. [Figure 2-3] The graph shows the results of measuring serum N (negative control) (evaluation 1) and positive sera P1 to P5 (evaluation 2) using comparative reagents 2 to 4 according to the prior art. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments. In addition, in this specification, the term "(meth)acrylate" refers to methacrylate and / or acrylate, and for example, the term "glycidyl (meth)acrylate" refers to glycidyl methacrylate and / or glycidyl acrylate.

[0013] <Amount of protein supported on the surface of latex particles> The affinity particles according to one embodiment of the present invention are affinity particles for detecting immunoglobulin G in a human sample by latex agglutination, and have a volume average particle size of 400 nm or less. They comprise latex particles and a protein carried on the surface of the latex particles, the protein comprising an antigen with a molecular weight of 10,000 or more for detecting immunoglobulin G, and the amount of protein carried on the surface of the latex particles is 1.0 μg or more and 20.0 μg or less per 1 mg of affinity particles. The proteins carried on the surface of the latex particles include an antigen for detecting immunoglobulin G.

[0014] Immunoglobulin G, the target substance in the present invention, is a substance that has two antigen-recognition sites within one molecule. When measuring this substance by latex agglutination, it is important for improving detection sensitivity that the two antigen-recognition sites of the immunoglobulin G molecule react with different particles, thereby efficiently causing particle agglutination via immunoglobulin G. Therefore, it is preferable that the antigen molecules for detecting immunoglobulin G, which are supported on the surface of latex particles, are present in a state where they are sufficiently separated from each other.

[0015] When antigen molecules are densely packed on particles, it is expected that the binding of the target substance, immunoglobulin G, will be completed within each particle. This reaction does not lead to particle agglutination, resulting in a reduced amount of detection, making it difficult to achieve excellent detection sensitivity. Furthermore, when antigens are present on particles in an aggregated state, it is likely to cause nonspecific adsorption of proteins other than the target substance. Nonspecific adsorption causes unwanted particle agglutination, increasing the detection value of samples that do not contain the target substance, even though the detection amount should be zero, and increasing the variability in the detection amount. In this case, it becomes difficult to detect the difference between samples that do not contain the target substance and samples with low amounts of the target substance, making it difficult to achieve excellent detection sensitivity.

[0016] In the present invention, the reagent solution in which affinity particles are dispersed may contain proteins other than the antigens (such as albumin used for particle blocking treatment), but the amount is preferably small. Specifically, it is preferable that the proportion of antigens among the proteins supported on the surface of latex particles is 70 mass% or more, as this increases the reactivity with immunoglobulin G, which is the target substance.

[0017] The amount of protein supported on the surface of latex particles can be determined as the amount of protein converted into bovine serum albumin (BSA) by the measurement method described below.

[0018] The affinity particles of the present invention for detecting immunoglobulin G in human samples by the latex agglutination method preferably have a volume-average particle size of 400 nm or less, contain latex particles, and an antigen with a molecular weight of 10,000 or more supported on the surface of the latex particles, with the amount of antigen supported on the surface of the latex particles being 1.0 μg to 20.0 μg per mg of affinity particles. This allows for efficient particle agglutination via the immunoglobulin G of the target substance, resulting in excellent detection sensitivity. For example, when the reagent solution containing dispersed affinity particles does not contain any proteins other than the antigen for detecting immunoglobulin G, the amount of antigen supported on the surface of the latex particles is 1.0 μg to 20.0 μg per 1 mg of affinity particles. In other words, it is more preferable that the proportion of the antigen among the proteins supported on the surface of the latex particles is 100% by mass.

[0019] <Volume average particle size of affinity particles> The antigen-loaded affinity particles of the present invention have a volume-average particle size of 400 nm or less. A particle size of 400 nm or less provides sufficient particle specific surface area, which, as described above, allows for the appropriate spacing of antigens on the particles while optimizing the antigen loading amount. This reduces nonspecific adsorption in latex agglutination assays and reduces the increase and variability in the detection amount of samples containing no target substance, thereby enabling highly sensitive detection of the difference between samples containing no target substance and samples containing low amounts of target substance. A sufficient particle specific surface area is also highly advantageous because it broadens the range in which the detection amount increases with increasing amounts of target immunoglobulin G in samples (the measurement range of immunoglobulin G levels). On the other hand, a volume-average particle size greater than 400 nm may result in reduced absorbance in regions with high concentrations of target immunoglobulin G. Furthermore, the volume average particle diameter is preferably 100 nm or more. If the particle aggregates after the reaction are small, the difference in absorbance before and after aggregation will also be small, and it may be difficult to improve sensitivity in areas with low concentrations of the target substance. The volume average particle diameter of the affinity particles according to the present invention can be 200 nm or more. The volume average particle size of the affinity particles dispersed in the reagent solution can be measured by the measurement method described below.

[0020] <Latex particles before antigen loading> The pre-antigen-loaded latex particles of the present invention are polymer particles capable of loading antigens against target substances, and any of the latex particles used in conventional latex agglutination reagents can be used. Pre-antigen-loaded latex particles can be synthesized by polymerizing a polymerizable monomer. For example, core particles are synthesized by polymerizing a polymerizable monomer, and then a polymerizable monomer (such as glycidyl (meth)acrylate) is reacted with the core particles to form a shell, synthesizing latex particles having a core-shell structure, which can be used as pre-antigen-loaded latex particles. That is, latex particles have a skeletal structure composed of structural units derived from the polymerizable monomer. Examples of pre-antigen-loaded latex particles include polystyrene particles, polystyrene particles containing siloxane, and polystyrene particles containing polyglycidyl (meth)acrylate. These particles have advantages such as the relative ease of obtaining nano-sized particles and the ability to chemically modify the particle surface according to the purpose, making them suitable for use in the affinity particles and latex agglutination test reagents of the present invention. Among these, latex particles having a structure derived from polyglycidyl (meth)acrylate are preferred because the particle surface can be chemically hydrophilized by reacting a specific compound with the glycidyl group. In particular, it is preferred to load an antigen using particles containing a polymer having a structural unit derived from glycidyl (meth)acrylate, as the latex particles before loading the antigen, which is represented by the following formula (1):

[0021] Furthermore, it is preferable that the latex particles further have structural units derived from, for example, styrenes, 1-vinylnaphthalene, 2-vinylnaphthalene, etc. as part of the skeletal structure consisting of structural units derived from polymerizable monomers, in order to improve sensitivity from the viewpoint of the particle refractive index. In addition to these, the latex particles preferably further have a crosslinked structure as a skeletal structure. The crosslinked structure is obtained by polymerization using a crosslinkable monomer having two or more radically polymerizable unsaturated bonds in one molecule. Examples of crosslinkable monomers include divinylbenzene, diethynylnaphthalene, diallyl phthalate, allyl acrylate, allyl methacrylate, multifunctional (meth)acrylate, and conjugated diolefin. The crosslinked structure makes the particles physically strong, eliminating the risk of cracking or chipping even when subjected to repeated centrifugation during purification.

[0022] The affinity particles according to the present invention are latex particles having a skeleton structure composed of structural units derived from a polymerizable monomer, and the skeleton structure is composed of structural units represented by the following formula (1) in the form of R 2 The antigen contains a structure corresponding to the structure obtained by removing R in formula (1). 2 By bonding to a group represented by formula (1), the affinity particles can be supported on the surface of latex particles. 2 The content of the structure corresponding to the structure excluding R in formula (1) can be 5% by mass or more and less than 50% by mass. That is, for latex particles before antigen loading, which contain a polymer having a structural unit represented by the following formula (1), 2 The affinity particles can be prepared by carrying an antigen on the group represented by the formula (I). [ka] (In formula (1), R 1 represents a hydrogen atom or a methyl group. R 2 represents a group having an epoxy group, a group having a hydroxy group, or a group having a carboxy group. R 1 and R 2 may be different for each structural unit.)

[0023] The structural unit represented by formula (1) in the skeletal structure of the latex particle, which is composed of structural units derived from the polymerizable monomer, is converted into R 2When the content of the structure corresponding to the structure excluding is 5% by mass or more, the hydrophobicity of the particle surface is reduced, and nonspecific adsorption can be suppressed.

[0024] The structural unit represented by formula (1) preferably contains a structural unit represented by the following formula (1-A): The structural unit represented by formula (1-A) has either a hydroxy group or a carboxy group, and is preferred because it has the same or greater ability to suppress nonspecific adsorption than a structure having an epoxy group. [ka] (In formula (1-A), R 3 represents a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C):

[0025] [ka] (In formula (1-B), R 4 represents a single bond or a methylene group. R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and the group represented by formula (1-B) has at least one hydroxy group. Y 1 represents a sulfur atom or an imino group. *1 indicates the bond position. At least one hydroxy group in the group represented by formula (1-B) may be a hydroxy group contained in a hydroxymethyl group. [ka] (In formula (1-C), R 8 represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y 2 represents a sulfur atom or an imino group. Y 3 represents a single bond or a methylene group. *2 indicates the bond position.)

[0026] Specific examples of the structure represented by formula (1-A) include, but are not limited to, the structure represented by the following formula (1-A-1), the structure represented by the following formula (1-A-2), the structure represented by the following formula (1-A-3), the structure represented by the following formula (1-A-4), the structure represented by the following formula (1-A-5), the structure represented by the following formula (1-A-6), the structure represented by the following formula (1-A-7), the structure represented by the following formula (1-A-8), the structure represented by the following formula (1-A-9), the structure represented by the following formula (1-A-10), the structure represented by the following formula (1-A-11), and the structure represented by the following formula (1-A-12). [ka]

[0027] To obtain pre-antigen-loaded latex particles containing a polymer having a structural unit represented by formula (1) (particularly, a structural unit represented by formula (1-A)), the surfaces of the pre-antigen-loaded latex particles may be surface-treated with a surface treatment agent, such as mercaptosuccinic acid, 3-mercapto-1,2-propanediol, 3-amino-1,2-propanediol, and 2-amino-2-hydroxymethyl-1,3-propanediol.

[0028] <Antigen loading onto latex particles> In the present invention, the antigen for detecting the target immunoglobulin G may be supported on the surface of the latex particles by chemical bonding or physical adsorption, but is preferably supported by chemical bonding.

[0029] By supporting the affinity particles through chemical bonding, antigen detachment from the affinity particles and antigen denaturation can be suppressed even when the affinity particles are dispersed in a reagent solution and stored for long periods of time, making it easier to obtain a liquid affinity particle dispersion reagent that can be used stably for a long period of time. Antigen detachment and denaturation can also be suppressed during the test reagent manufacturing process. For example, to remove undesirable unreacted antigens and other components remaining in the reaction system, centrifugal washing with detergent-containing wash solutions or wash solutions with different pH values ​​may be repeated after antigen loading. Even in such cases, antigen detachment and denaturation are unlikely to occur, making it possible to select a process appropriate for the purpose, such as increasing detection sensitivity.

[0030] Antigen loading by chemical bonding can be carried out according to standard methods, but functional groups on the latex particles before antigen loading can be reacted with primary amines, secondary amines, carboxy groups, thiol groups, etc. on the antigen. Examples of functional groups on latex particles before antigen loading include carboxy groups, amino groups, aldehyde groups, epoxy groups, thiol groups, and maleimide groups. Latex particles before antigen loading may have only one type of these functional groups, or two or more types. It is preferable that the antigen is chemically bonded via a carboxy group in the side chain structure represented by formula (1-C) on the latex particles before antigen loading.

[0031] Conventional methods can be used for the chemical reaction of binding antigens to carboxy groups on latex particles prior to antigen loading. For example, carbodiimide-mediated reactions and NHS ester activation reactions are suitable examples of such reactions. Alternatively, avidin may be bound to the carboxy groups, and then a biotin-modified ligand (antigen) may be bound to the carboxy groups. The antigen to be supported is preferably in a dispersed state, avoiding aggregation or multimerization of antigen molecules, from the viewpoint of reducing non-specific adsorption and improving reactivity.

[0032] <Antigen for detecting target immunoglobulin G> In the present invention, the antigen carried on the surface of latex particles for detecting target immunoglobulin G is a polypeptide having a molecular weight of 10,000 or more and containing an epitope sequence specifically recognized by the target immunoglobulin G. For example, recombinant antigens produced by genetic engineering (antigens expressed by introducing a gene encoding the antigen sequence into Escherichia coli or mammalian cells) or antigens extracted and purified from human specimens can be used.

[0033] Using an antigen with a molecular weight of 10,000 or greater reduces the possibility of substances present near the surface of latex particles inhibiting the antigen-antibody reaction. Furthermore, combined with the aforementioned optimal range of antigen loading, epitopes within the antigen can be present on the particle surface at an appropriate density, ensuring sufficient reactivity between the antigen and the target immunoglobulin G. Furthermore, the interparticle distance required for particle aggregation via immunoglobulin G is increased, promoting particle aggregation.

[0034] In the present invention, two or more types of antigens may be used in combination. In this case, multiple types of antigens may be simultaneously reacted with latex particles, or each antigen may be individually supported on a latex particle and then mixed together for use. For example, in the case of affinity particles in which HCV antigens are supported on latex particles for detecting HCV antibodies, it is preferable to use multiple types of epitope regions to react with various types of HCV antibodies in human samples. An antigen having the amino acid sequence of the HCV virus itself may be used, or a chimeric antigen combining epitope regions may be used. To maintain the reactivity and dispersion state of the antigen, the antigen may be treated with a reducing agent, surfactant, or the like before being supported on the latex particles.

[0035] When affinity particles, in which HBc antigen is carried on latex particles, are used to detect HBc antibodies, an antigen containing the full-length sequence of the HBV core protein (HBc) or an antigen containing a major epitope may be used. Pretreatment with a surfactant or the like may also be used.

[0036] <Test reagents and detection methods> The test reagent according to the present invention can be a test reagent containing a first reagent solution in which the affinity particles according to the present invention are dispersed. The test reagent according to the present invention can be a test reagent composed of one liquid or two or more liquids, but a test reagent composed of two liquids is particularly preferred from the viewpoint of measurement in a general-purpose biochemical device. An example of a test reagent consisting of two or more liquids is a test reagent containing a liquid (first reagent liquid) in which affinity particles carrying a protein (antigen) are dispersed and a buffer solution (second reagent liquid). In this case, affinity particles according to the present invention are dispersed in a liquid (first reagent liquid) in which affinity particles are dispersed. The affinity particles have a volume average particle size of 400 nm or less, and contain latex particles and a protein supported on the surface of the latex particles, the protein containing an antigen with a molecular weight of 10,000 or more, and the amount of protein supported on the surface of the latex particles is 1.0 μg to 20.0 μg per mg of affinity particles. The buffer solution (second reagent liquid) may not contain the affinity particles.

[0037] An example of a method for detecting immunoglobulin G in a human specimen by the latex agglutination method according to the present invention using a test reagent consisting of two or more liquids will be described. That is, this is a method for detecting immunoglobulin G in a human specimen by a latex agglutination method using a test reagent comprising a first reagent solution in which affinity particles are dispersed and a second reagent solution which is a buffer solution, and the method comprises: a first step of obtaining a mixture containing the human specimen and the buffer solution (second reagent solution); a second step of mixing and reacting the mixture obtained in the first step with a solution in which affinity particles are dispersed (first reagent solution) to obtain a reaction solution; and a third step of measuring the concentration of immunoglobulin G in the reaction solution, wherein the affinity particles have a volume average particle size of 400 nm or less, the affinity particles comprise latex particles and a protein supported on the surface of the latex particles, the protein comprising an antigen with a molecular weight of 10,000 or more for detecting immunoglobulin G, and the amount of protein supported on the surface of the latex particles per 1 mg of affinity particles is 1.0 μg or more and 20.0 μg or less.

[0038] In the first step, a human sample is mixed with a buffer solution (second reagent solution) to obtain a mixed solution. The reaction in the second step is preferably carried out at a pH range of 3.0 to 11.0. The mixing temperature is 20°C to 50°C, and the mixing time is 1 to 20 minutes. The concentration of affinity particles in the reaction solution is preferably 0.001% to 5% by mass, more preferably 0.01% to 1% by mass.

[0039] The detection method of the present invention optically detects inter-particle agglutination that occurs as a result of mixing the affinity particles of the present invention with a sample, thereby enabling detection of the target immunoglobulin G in the sample. The method for optically detecting the agglutination reaction between affinity particles simply involves measuring changes in scattered light intensity, transmitted light intensity, absorbance, etc. using an optical device capable of detecting these values.

[0040] The test reagent according to the present invention preferably contains a nonionic surfactant and a zwitterionic surfactant, and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride. The test reagent according to the present invention may also be a test reagent containing a first reagent solution in which affinity particles according to the present invention are dispersed and a second reagent solution that is a buffer solution, where the second reagent solution does not contain affinity particles according to the present invention, but contains a nonionic surfactant and a zwitterionic surfactant, and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride. The test reagent according to the present invention may also contain at least one of a nonionic surfactant and a zwitterionic surfactant, and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride. The test reagent according to the present invention may also contain a nonionic surfactant and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride.

[0041] <Surfactant> The test reagent according to the present invention preferably contains a nonionic surfactant and a zwitterionic surfactant. In the case of a test reagent consisting of two liquids, these components may be contained in either the buffer solution (second reagent liquid) or the liquid in which affinity particles are dispersed (first reagent liquid), or may be contained in both. The primary purpose of using nonionic and / or zwitterionic surfactants is to reduce nonspecific agglutination caused by substances other than the target substance present in human specimens.

[0042] Nonionic surfactants are surfactants whose polar moieties are nonionic (uncharged) and are known to suppress interactions between lipids and proteins. In the test reagent of the present invention, the use of nonionic surfactants improves the dispersibility of affinity particles (moderately weakening their agglutination). Furthermore, nonspecific adsorption to the affinity particle surface (especially hydrophobic components such as polystyrene) is reduced, allowing the detection amount of negative samples to approach zero.

[0043] Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters (registered trademarks: Tween 20, Tween 80, etc.), polyoxyethylene octylphenyl ethers (registered trademarks: Triton X-100, Triton X-114, etc.), polyoxyethylene alkyl ethers (registered trademarks: Brij 35, Brij 58, etc.), alkyl glycosides (n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, etc.), and n-D-gluco-n-methylalkanamides (MEGA 9, MEGA 10, etc.). Among these, polyoxyethylene sorbitan monolaurate (registered trademark: Tween 20) and n-octyl-β-D-glucopyranoside are preferred.

[0044] The content of the nonionic surfactant in the test reagent is preferably such that it is equal to or greater than the critical micelle concentration in the step of detecting immunoglobulin G in a human sample. The content of the nonionic surfactant in the reaction solution in the third step (i.e., the amount relative to the total including, for example, the human sample, buffer solution (second reagent solution), and solution in which affinity particles are dispersed (first reagent solution)) is preferably as follows: In the case of polyoxyethylene sorbitan monolaurate (Tween 20), the content is preferably 0.005% by mass or more and 5% by mass or less, and more preferably 0.03% by mass or more and 0.5% by mass or less. In the case of n-octyl-β-D-glucopyranoside, the content is preferably 0.3% by mass or more and 5% by mass or less, and even more preferably 0.5% by mass or more and 2% by mass or less. The amount of Tween 20 relative to the total of the buffer solution (second reagent solution) and the solution in which the affinity particles are dispersed (first reagent solution) is preferably 0.006% by mass or more and 5.6% by mass or less, and more preferably 0.03% by mass or more and 0.6% by mass or less.

[0045] Zwitterionic surfactants have both positively and negatively charged ions in their polar moieties. Unlike nonionic surfactants, they are known to be effective in suppressing some protein-protein interactions. The use of zwitterionic surfactants in the test reagents of the present invention reduces nonspecific adsorption, primarily to antigens supported on latex particles, enabling the detection amount of negative samples to approach zero. While zwitterionic surfactants have a stronger effect than nonionic surfactants, they are less effective at denaturing proteins themselves than anionic and cationic surfactants. Therefore, they are effective in reducing nonspecific adsorption without significantly impairing the reactivity of antibodies, which are important for detection.

[0046] Examples of amphoteric surfactants include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (hereinafter also referred to as CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxysulfonic acid (hereinafter also referred to as CHAPSO), n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (Zwittergent 3-12), N,N-dimethyldodecylamine N-oxide, etc. Among them, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxysulfonic acid (CHAPSO) are preferred.

[0047] The content of the zwitterionic surfactant in the reaction solution in the third step (i.e., the amount relative to the total including the human specimen, buffer solution (second reagent solution), and liquid in which affinity particles are dispersed (first reagent solution)) is preferably as follows: 0.05% by mass or more and 5.0% by mass or less, and more preferably 0.1% by mass or more and 3.0% by mass or less. The amount relative to the total including the buffer solution (second reagent solution) and liquid in which affinity particles are dispersed (first reagent solution) is preferably 0.06% by mass or more and 5.6% by mass or less, and more preferably 0.1% by mass or more and 3.3% by mass or less.

[0048] <Metal salts> The test reagent of the present invention preferably contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride. In the case of a test reagent consisting of two or more liquids, the above metal salt may be contained in either the buffer solution (second reagent solution) or the liquid in which affinity particles are dispersed (first reagent solution), or may be contained in both, but it is preferable that the buffer solution (second reagent solution) contains the above metal salt.

[0049] In the test reagent and detection method according to the present invention, it is preferable that the buffer solution (second reagent solution) contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride, and that the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride in the buffer solution (second reagent solution) is 120 mM to 500 mM. Also, in the test reagent and detection method according to the present invention, it is preferable that at least one of the first reagent solution and the second reagent solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride, and that the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride in the first reagent solution and the second reagent solution is 90 mM to 400 mM. Furthermore, in the detection method of the present invention, in the third step of measuring the concentration of immunoglobulin G in a reaction solution obtained by mixing and reacting a mixture containing a human sample and a buffer solution (second reagent solution) with a solution in which affinity particles are dispersed (first reagent solution), it is preferable that the reaction solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride that is not derived from the human sample, and that the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride that is not derived from the human sample in the reaction solution in the third step is 80 mM or more and 360 mM or less. Furthermore, the test reagent of the present invention is a test reagent comprising a first reagent solution in which affinity particles are dispersed and a second reagent solution which is a buffer solution, wherein the second reagent solution does not contain affinity particles but contains a nonionic surfactant and a zwitterionic surfactant, and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride, and the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride in the second reagent solution is 120 mM or more and 500 mM or less.

[0050] By keeping the amount of metal salt derived from the test reagent within the above range, the electrical conductivity of the measurement system is appropriately increased. Therefore, when affinity particles with a surface charge are used, electrostatic nonspecific adsorption to the affinity particles by substances other than the target substance present in the human sample can be reduced, suppressing the variation in the detected amount of negative samples. Furthermore, by appropriately thinning the electric double layer on the surface of the affinity particles, particle aggregation can be increased, improving detection sensitivity. If the test reagent contains an ionic substance other than the metal salt, it is preferable that in the third step of detecting immunoglobulin G, the electrical conductivity resulting from the ionic substances in the test reagent, including the metal salt, is 15 mS / cm or more and 60 mS / cm or less.

[0051] <Other ingredients> The test reagent according to the present invention may contain substances such as a buffer, a sensitizer, a surfactant, a sugar, albumin, and immunoglobulin G, within the scope that allows the object of the present invention to be achieved. In the case of a test reagent consisting of two or more liquids, these components may be contained in either the buffer solution (second reagent liquid) or the liquid in which affinity particles are dispersed (second reagent liquid), or may be contained in both. At least the buffer solution (second reagent solution) contains a buffering agent. Examples of buffer solutions containing a buffering agent include various aqueous buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, Hepes buffer, Meth buffer, and ammonia buffer, but the buffer solution contained in the test reagent of the present invention is not limited to these.

[0052] Sensitizers for latex agglutination assays include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, alginic acid, and the like.

[0053] [Method for calculating the amount of protein supported on the surface of latex particles] The amount of protein supported on the surface of latex particles in affinity particles (the amount of protein supported on the surface of latex particles (μg) per 1 mg of affinity particles) can be calculated as the amount of protein converted into bovine serum albumin (BSA). First, the reagent solution containing the dispersed affinity particles is centrifuged to precipitate the affinity particles and remove the supernatant. Next, pure water is added to disperse the affinity particles. Centrifugation is repeated again to obtain the precipitated particle component (referred to as "particle component 1"). This is then dried at 130°C, after which the dry weight is measured and this is taken as the mass "W" of the protein-loaded latex particles (affinity particles). Similarly, "particle component 1" is separately collected from the same amount of reagent solution. This is dispersed again in pure water, and using the particle mass "W" measured as described above, the particle concentration is adjusted to 0.2 mass% (referred to as "particle aqueous dispersion 1"). Protein quantification is carried out using the following method.

[0054] First, solutions A and B from the Protein Assay BCA Kit (Wako Pure Chemical Industries) were mixed at a ratio of 50:1 (v / v), resulting in a solution designated as solution AB. 200 μL of solution AB was added to 25 mg (50 μg particle weight) of "particle aqueous dispersion 1" (0.2% by mass solution) and incubated at 60°C. The solution was centrifuged, and the supernatant was placed in a 96-well microwell. The absorbance at 562 nm was measured using a microplate reader along with standard protein samples (several 0-200 μg / mL solutions of bovine serum albumin (BSA) standard diluted with pure water), and the amount of protein converted to BSA was calculated from the standard curve. The calculated BSA-equivalent protein amount is divided by the particle mass (0.05 mg when evaluating using 25 mg of a 0.2% by mass solution as described above) to determine the amount of protein (μg) supported on the surface of the latex particles per mg of affinity particles.

[0055] [Method for measuring the volume average particle size of affinity particles] Measurements are performed after diluting a reagent solution containing affinity particles with ion-exchange water (electrical conductivity of 10 μS / cm or less). Dynamic light scattering is used to measure the volume average particle size. Specifically, measurements are performed at 25°C using a Zetasizer (Zetasizer Nano-ZS, Zetasizer Ultra, etc.: Spectris Inc.). The analysis parameters are latex (n≒1.59) as the particle refractive index and pure water as the solvent. Three measurements are performed, and the average of the measured volume average particle size values ​​is used. [Example]

[0056] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0057] [Synthesis of latex particles 1] (Step 1 / Preparation of core particles A) A 2 L four-neck separable flask was charged with 23.5 g of styrene (hereinafter also referred to as St; Kishida Chemical Co., Ltd.), 0.43 g of divinylbenzene (hereinafter also referred to as DVB; Kishida Chemical Co., Ltd.), and 800 g of ion-exchanged water to prepare a mixture. The mixture was stirred at 100 rpm and maintained at 70 °C. A nitrogen flow at a flow rate of 200 ml / min was introduced to deoxygenate the four-neck separable flask. Next, a separately prepared solution of 1.02 g of V-50 (Fujifilm Wako Pure Chemical Corporation) dissolved in 50 g of ion-exchanged water was added to the mixture to initiate soap-free emulsion polymerization. The reaction was allowed to proceed for 48 hours from the start of polymerization, yielding a dispersion of core particles A containing a copolymer of St and DVB. A portion of the mixture was sampled and the volume average particle size of core particles A was evaluated, which was found to be 280 nm.

[0058] (Step 2 / Preparation of mother particles A) A 150 g dispersion of core particles A with a solids concentration of 2.0% was prepared using ion-exchanged water. Next, 1.54 g of glycidyl methacrylate (hereinafter also referred to as GMA; Kishida Chemical Co., Ltd.) was added, and the mixture was stirred at 100 rpm while maintaining the temperature at 70°C. A nitrogen flow was introduced at a flow rate of 200 mL / min to deoxygenate the four-neck separable flask. A separately prepared solution of 0.018 g of V-50 (Fujifilm Wako Pure Chemical Corporation) dissolved in 1 g of ion-exchanged water was then added to the mixture to initiate shell formation. Stirring was continued for 17 hours after the start of the reaction, yielding a dispersion containing base particles A with a core-shell structure. After slowly cooling the dispersion to room temperature, a portion was sampled and the polymerization conversion was evaluated using gas chromatography, confirming that it was essentially 100%. The polymerization conversion was calculated from the amount of monomer charged in the polymerization process. The solution after the polymerization reaction was analyzed using gas chromatography or the like to quantify the remaining monomer, and the value of the degree to which the monomer had been polymerized by the polymerization reaction (polymerization conversion rate) was calculated from the remaining amount of monomer. In this case, when the remaining amount of monomer was below the detection limit, the polymerization conversion rate was considered to be substantially 100% (all the monomer had polymerized).

[0059] (Step 3 / Preparation of latex particles 1) The following materials were added to the aqueous dispersion containing the base particles A, and triethylamine (Kishida Chemical Co., Ltd.) was further added to adjust the pH to 10. A previously prepared aqueous solution of mercaptosuccinic acid (hereinafter also referred to as MSA: Wako Pure Chemical Industries, Ltd.) and 3-mercapto-1,2-propanediol (hereinafter also referred to as 3MPD: Wako Pure Chemical Industries, Ltd.) dissolved therein (MSA:3MPD = 7:3 (molar fraction), the total number of moles of MSA and 3MPD being twice the number of moles of GMA used in step 2). Next, the mixture was heated to 70°C while stirring at 200 rpm and maintained at this temperature for an additional 18 hours for surface treatment, yielding a dispersion of latex particles 1. Latex particles 1 were separated from the dispersion by centrifugation, and the latex particles 1 were redispersed in ion-exchanged water eight times to purify the latex particles 1. The final aqueous dispersion was adjusted to a latex particle 1 concentration of 1.0% by mass, and then stored. The volume-average particle size of latex particles 1 was evaluated and found to be 320 nm. The physical properties of latex particles 1 were evaluated and the results are shown in Table 1.

[0060] [Synthesis of latex particles 2] A dispersion of latex particles 2 was obtained by the same experimental procedure as in Example 1, except that the base particles A prepared in Example 1 were used and 3-amino-1,2-propanediol (hereinafter also referred to as 3APD: Tokyo Chemical Industry Co., Ltd.) was used instead of 3MPD in step 3 of Example 1. The physical properties of latex particles 2 were evaluated and the results are shown in Table 1.

[0061] [Synthesis of latex particles 3] Using the base particles A produced in Example 1, 2-amino-2-hydroxymethyl-1,3-propanediol (trishydroxymethylaminomethane, hereinafter also referred to as Tris: Kishida Chemical Co., Ltd.) was used instead of 3MPD in step 3 of Example 1. Except for this, a dispersion of latex particles 3 was obtained by the same experimental procedure as in Example 1. The physical properties of latex particles 3 were evaluated and the results are shown in Table 1.

[0062] [Synthesis of latex particles 4] In step 1 of Example 1, the amounts of St used were 71.75 g, DVB used was 1.30 g, ion-exchanged water used was 1190.7 g, and V-50 used was 3.11 g, and the stirring speed was changed from 100 rpm to 140 rpm. A dispersion of core particles B was obtained by the same experimental procedure as in Example 1. Next, base particles B were prepared by the same experimental procedures as in steps 2 and 3 of Example 1, except that core particles B were used, and then a dispersion of latex particles 4 was obtained. The physical properties of latex particles 4 were evaluated and the results are shown in Table 1.

[0063] [Synthesis of latex particles 5] (Preparation of mother particle C) The following materials were weighed into a 2 L four-neck separable flask to prepare a mixed solution. 12.0 g of styrene (St: Kishida Chemical Co., Ltd.) 18.0 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) 0.45g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.) 1200g of ion-exchanged water The mixture was stirred at 200 rpm and maintained at 70°C. A nitrogen flow at a flow rate of 200 ml / min was used to deoxygenate the four-neck separable flask. Next, a solution of 0.68 g of V-50 (Fujifilm Wako Pure Chemical Corporation) dissolved in 30 g of ion-exchanged water, which had been prepared separately, was added to the mixture to initiate soap-free emulsion polymerization. Two hours after the start of polymerization, 3.0 g of GMA was added, and the reaction was continued for 20 hours, yielding a dispersion of base particles C having a copolymer of St, GMA, and DVB. After slowly cooling the dispersion to room temperature, a portion was sampled, and the polymerization conversion was evaluated using gas chromatography, confirming that it was essentially 100%.

[0064] (Preparation of latex particles 5) In step 3 of Example 1, mother particles C were used instead of mother particles A, the molar fraction of MSA:3MPD was changed from 7:3 to 2:8, and the total number of moles of MSA and 3MPD was changed to the same amount as the number of moles of GMA used in producing mother particles C. Otherwise, a dispersion of particles 5 was obtained by the same experimental procedure as in step 3 of Example 1. The physical properties of particles 5 were evaluated and the results are shown in Table 1.

[0065] [Synthesis of comparative particle 1] A dispersion of core particles D was obtained by the same experimental procedure as in Example 1, except that in step 1 of Example 1, the amount of St used was 99.40 g, the amount of DVB was 1.80 g, the amount of ion-exchanged water was 1150.15 g, and the amount of V-50 was 4.31 g, and the stirring speed was changed from 100 rpm to 200 rpm. Thereafter, mother particles D and comparative particles 1 were obtained by the same experimental procedures as steps 2 and 3 of Example 1, except that core particles D were used. The physical properties of comparative particles 1 were evaluated and the results are shown in Table 1.

[0066] [Table 1]

[0067] [Example 1 (Test Reagent 1)] The test reagent 1 is composed of two liquids: a liquid (first reagent liquid) 1 in which affinity particles are dispersed, and a buffer solution (second reagent liquid) A. (Preparation of liquid 1 in which affinity particles are dispersed) An HCV antigen solution (TRINA BIOREACTIVES) was subjected to solvent exchange using an ultrafiltration device (Amicon Ultra 10K, Merck). Subsequently, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the antigen was a protein with a molecular weight of 10,000 or more. The protein concentration in the antigen solution was then measured using a Protein Assay BCA Kit (Wako Pure Chemical Industries) and BSA standard protein, as described above in "Method for calculating the amount of protein supported on the surface of latex particles." The solution was diluted to a protein concentration of 0.14 mg / mL to obtain "HCV antigen solution R." The diluent used was 10 mM HEPES buffer supplemented with 20 mM (±) dithiothreitol (DTT: Fujifilm Wako Pure Chemical Industries, Ltd.). 60 μL of a 1.7% by mass water suspension of latex particles 1 was placed in a microtube. 30 μL of a 0.1% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 30 μL of a 0.1% aqueous solution of sodium N-hydroxysulfosuccinimide were added. The mixture was stirred at room temperature for 30 minutes to obtain a dispersion of particles with activated carboxy groups (activated particle dispersion). After centrifugation and washing, 50 μL of 10 mM HEPES buffer was added, and the particles with activated carboxyl groups were dispersed by ultrasonication. 50 μL of "HCV antigen solution R" (0.14 mg / mL solution) was added (7.0 μg of HCV antigen contacted per mg of particles), and the mixture was stirred at room temperature for 1 hour to bind the antigen to the carboxyl groups of the particles. After centrifugation and washing, 240 μL of masking buffer (0.3 M glycine solution containing 0.1% Tween 20, pH 8.0) was added, and the mixture was stirred at room temperature for 1 hour, then left to stand overnight at 4°C to bind glycine to the remaining activated carboxyl groups, forming affinity particles. After centrifugation and washing, 0.5 mL of storage buffer (10 mM HEPES containing 0.01% Tween 20, pH 7.9) was added and the particles were dispersed by ultrasonic waves to obtain Liquid 1 in which affinity particles were dispersed. The volume average particle size of the affinity particles dispersed in Liquid 1 was evaluated to be 318 nm. The amount of protein supported on the surface of the latex particles in Liquid 1 was evaluated to be 6.4 μg per 1 mg of affinity particles.

[0068] (Buffer A) The following materials were mixed and dissolved in ultrapure water to the concentrations shown below, and the resulting solution was adjusted to pH 7.5 to prepare buffer solution A. 100mM HEPES 500mM sodium chloride ·0.1 mass% Tween20 0.5% by mass CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) 0.2% by mass polyethylene glycol (molecular weight approximately 500,000) This resulted in a test reagent 1 consisting of two liquids: liquid 1 in which affinity particles were dispersed and buffer solution A. The physical properties of test reagent 1 were evaluated and the results are shown in Table 2.

[0069] [Examples 2 and 3 (Test Reagents 2 and 3)] The protein concentration of the "HCV antigen solution R" used in Example 1 (preparation of liquid 1 in which affinity particles are dispersed) was changed from 0.14 mg / mL to 0.04 mg / mL and 0.40 mg / mL, respectively (2.0 μg and 20.0 μg of HCV antigen were contacted with 1 mg of particles, respectively). Affinity particle-dispersed liquid 2 and affinity particle-dispersed liquid 3 were otherwise prepared in the same manner as in Example 1. This resulted in test reagent 2, consisting of two liquids: affinity particle-dispersed liquid 2 and buffer solution A. Test reagent 3, consisting of affinity particle-dispersed liquid 3 and buffer solution A, was also obtained. The physical properties of test reagent 2 and test reagent 3 were evaluated and the results are shown in Table 2.

[0070] [Examples 4 to 7 (Test Reagents 4 to 7)] Affinity particle dispersions 4 to 7 were prepared in the same manner as in Example 1, except that the latex particles 1 used in Example 1 (preparation of affinity particle dispersion 1) were replaced with latex particles 2 to 5, respectively. As a result, test reagent 4 was obtained, which consisted of two liquids: affinity particle dispersion 4 and buffer solution A; test reagent 5, which consisted of affinity particle dispersion 5 and buffer solution A; test reagent 6, which consisted of affinity particle dispersion 6 and buffer solution A; and test reagent 7, which consisted of affinity particle dispersion 7 and buffer solution A. The physical properties of test reagents 4 to 7 were evaluated and the results are shown in Table 2.

[0071] [Examples 8 and 9 (Test Reagents 8 and 9)] The following buffer solution B and buffer solution C were prepared. (Buffer B) Buffer solution B was prepared by mixing the following materials with ultrapure water to the concentrations listed below. PBS(-) (pH 7.4, contains sodium chloride at a concentration of 137 mM based on buffer solution B) ·0.1 mass% Tween20 ·0.5 mass% CHAPS 0.2% by mass polyethylene glycol (molecular weight approximately 500,000)

[0072] (Buffer C) Buffer solution C was prepared in the same manner as buffer solution A, except that the 0.5% by mass of CHAPS in buffer solution A was changed to 0.02% by mass of sodium dodecyl sulfate (SDS). As a result, test reagent 8 consisting of two liquids, liquid 1 in which affinity particles are dispersed and buffer solution B, and test reagent 9 consisting of two liquids, liquid 1 in which affinity particles are dispersed and buffer solution C, were obtained.

[0073] [Comparative Examples 1 and 2 (Comparative Reagents 1 and 2)] Affinity particle-dispersed solutions 8 and 9 were prepared in the same manner as in Example 1, except that the protein concentration of the "HCV antigen solution R" used in Example 1 (preparation of affinity particle-dispersed solution 1) was changed from 0.14 mg / mL to 0.60 mg / mL and 0.018 mg / mL, respectively (30.0 μg and 0.9 μg of antigen were contacted per mg of particles). Comparative reagent 1, consisting of two solutions, affinity particle-dispersed solution 8 and buffer solution A, and comparative reagent 2, consisting of affinity particle-dispersed solution 9 and buffer solution A, were thus obtained. The physical properties of comparative reagents 1 and 2 were evaluated and the results are shown in Table 2.

[0074] [Comparative Example 3 (Comparative Reagent 3)] Affinity particle-dispersed liquid 10 was prepared in the same manner as in Example 1, except that the latex particles 1 used in Example 1 (preparation of affinity particle-dispersed liquid 1) were replaced with comparative particles 1. This yielded comparative reagent 3, which consisted of two liquids: affinity particle-dispersed liquid 10 and buffer A. The physical properties of comparative reagent 3 were evaluated and the results are shown in Table 2.

[0075] [Comparative Example 4 (Comparative Reagent 4)] (Preparation of liquid 11 in which affinity particles are dispersed) A 0.14 mg / mL "HCV antigen solution R" was obtained by the same experimental procedure as in (Preparation of solution 1 in which affinity particles are dispersed). The latex particles 1 used in the affinity particle dispersion liquid 1 were changed to carboxy group-containing polystyrene particles (product name "Fujikura Latex FK-C400D" manufactured by Fujikura Chemical Co., Ltd.). Except for this, the same experimental procedures as in (preparation of affinity particle dispersion liquid 1) were used to bind the antigen to the carboxy group of the particles, and then glycine was bound to the remaining activated carboxy group. After repeated centrifugation washing with storage buffer (50 mM HEPES, pH 7.6 containing 3% BSA and 0.05% Tween 20), 0.5 mL of storage buffer was added to disperse the particles, yielding a liquid 11 containing dispersed affinity particles. As a result, a comparative reagent 4 consisting of two liquids, namely, a liquid 11 in which affinity particles are dispersed and buffer solution A, was obtained.

[0076] [Table 2] *1: The "NaCl content relative to the test reagent" in Table 2 indicates the NaCl content in the test reagent (total of the buffer solution and the liquid in which the affinity particles are dispersed) at the time of the measurement below. *2: The "NaCl content relative to the reaction solution" in Table 2 indicates the NaCl content not derived from human samples in the reaction solution below (total of serum, buffer solution, and solution in which affinity particles are dispersed).

[0077] [Evaluation 1: Measurement of serum from healthy individuals] Using test reagents 1 to 9 and comparative reagents 1 to 4, serum from healthy subjects was measured. Five types of healthy human sera A to E (personal serum samples, purchased from Tennessee Blood Service) and a negative control serum (ACCURUN810 multimarker negative control: Minaris Medical Co., Ltd., hereafter referred to as "Serum N") were used as healthy human sera. Measurements were performed on each of the six types of healthy human sera as follows. 10 μL of healthy human serum and 65 μL of buffer solution (either buffer solutions A to C depending on the test reagent being evaluated) were mixed in a cell and incubated at 37°C for 5 minutes. Next, 25 μL of a solution containing dispersed affinity particles (either affinity particle dispersion solutions 1 to 11 depending on the test reagent being evaluated) was added to the cell and mixed and stirred. Immediately after stirring, the absorbance of the mixed solution (volume 100 μL) was measured at the wavelengths listed in Table 3. The absorbance was measured using an Eppendorf Biospectrometer. After allowing the mixed solution to stand at 37°C for 5 minutes, the absorbance at the same wavelength was measured again, and the change in absorbance, ΔABS × 10,000, was calculated.

[0078] [Evaluation 2: Measurement of HCV-positive serum] Test Reagents 1 to 9 and Comparative Reagents 1 to 4 were used to measure HCV-positive serum.

[0079] (Sample preparation) The following test was carried out using an HCV positive control serum (ACCURUN series Infectrol D: Minaris Medical Co., Ltd.) and the negative control serum used in Evaluation 1 ("Serum N"). The HCV positive control serum was diluted 2-fold, 4-fold, 8-fold, and 16-fold with the negative control serum ("Serum N"). Hereafter, the 16-fold dilution will be referred to as "Serum P1," the 8-fold dilution as "Serum P2," the 4-fold dilution as "Serum P3," the 2-fold dilution as "Serum P4," and the HCV positive control serum as "Serum P5." The HCV positive control serum used had a COI value of 34.9 as measured by the Lumipulse Presto Ortho HCV (Presto II / L2400) reference value.

[0080] (measurement) Measurement was carried out in the same manner as in Evaluation 1, except that the six types of sera from healthy subjects used in Evaluation 1 were changed to serum P1 to P5.

[0081] [Evaluation results] The results of Evaluation 1 and Evaluation 2 are shown in Table 3. FIG. 1 also shows the results of six serogroups from healthy individuals in Evaluation 1 and the results of serum P1 (low positive value) in Evaluation 2. 2 shows the results of serum N (negative control) in Evaluation 1 and positive sera P1 to P5 in Evaluation 2. For Comparative Reagent 1, the detected amounts of serum P1 and P2 in Evaluation 2 could not be distinguished from the negative serum group, so evaluation of serum P3 to P5 in Evaluation 2 was not performed.

[0082] [Table 3]

[0083] As shown in Figures 1-1 to 1-9, Test Reagents 1 to 9 of Examples 1 to 9, in which the affinity particles have a volume-average particle diameter of 400 nm or less, the protein amount supported on the surface of the latex particles is 1.0 to 20.0 μg / mg, and the latex particles before antigen loading contain a polymer having a structural unit represented by formula (1), exhibited high detection levels of low-positive serum P1 relative to the detection levels of serum from healthy subjects, demonstrating excellent detection sensitivity. In particular, Test Reagents 1 to 7, in which the buffer solution of the test reagent contains a nonionic surfactant, a zwitterionic surfactant, and a sufficient amount of sodium chloride (not derived from serum, resulting in 325 mM in the reaction solution and 361 mM in the test reagent during measurement), exhibited small variations in serum from healthy subjects and exhibited excellent detection sensitivity.

[0084] On the other hand, as shown in Figure 1-10, Comparative Reagent 1, which has a protein amount of more than 20.0 μg / mg supported on the latex particle surface, was unable to distinguish between the healthy subject group and the low positive serum P1 due to the large variation in the healthy subject serum group and the small amount of detected P1 in the low positive serum. It is thought that with Comparative Reagent 1, the antigen molecules were not properly spaced apart due to the excessive antigen on the particles, resulting in a decrease in sensitivity in positive samples due to the antigen-antibody reaction being completed within a single particle, and in some healthy subject samples, agglutination worsened due to nonspecific adsorption.

[0085] As shown in Figure 1-11, Comparative Reagent 2, which has a protein load of less than 1.0 μg / mg on the particles, detected very little positive serum P1, making it difficult to distinguish it from the healthy control group. This is thought to be due to an insufficient amount of antigen to detect the target immunoglobulin G.

[0086] As shown in Figures 1-12 and 13, comparative reagents 3 and 4, which have affinity particles with a volume average particle size of more than 400 nm, showed excellent results in terms of detection sensitivity in evaluation 1, similar to test reagents 1 to 9.

[0087] As shown in Figures 2-1 and 2-2, test reagents 1 to 9, in which the volume average particle diameter of affinity particles was 400 nm or less and the amount of protein supported on the latex particle surface of the affinity particles was 1.0 to 20.0 μg / mg, showed an increase in the proportion of HCV-positive serum P5 in the specimen. In other words, the detection amount increased as the amount of target immunoglobulin G in the specimen increased.

[0088] On the other hand, as shown in Figure 2-3, in Comparative Reagent 2, in which the amount of protein supported on the surface of the latex particles in the affinity particles is less than 1.0 μg / mg, and in Comparative Reagents 3 and 4, in which the volume average particle size of the affinity particles exceeds 400 nm, the detection amount does not increase in the range where the amount of target immunoglobulin G in the sample is above a certain level, and it is found that a sufficient measurement range cannot be secured.

[0089] The disclosure of this embodiment includes the following configurations and methods. (Configuration 1) Affinity particles for detecting immunoglobulin G in a human specimen by latex agglutination, comprising: The volume average particle size is 400 nm or less, A method for producing a latex particle-supported protein-containing polymer, comprising: The protein comprises an antigen having a molecular weight of 10,000 or more, Affinity particles characterized in that the amount of protein supported on the surface of the latex particles per 1 mg of the affinity particles is 1.0 μg or more and 20.0 μg or less. (Configuration 2) 2. The affinity particle according to claim 1, wherein the antigen is an HCV antigen or an HBc antigen. (Configuration 3) the latex particles have a skeleton structure composed of structural units derived from a polymerizable monomer, The skeletal structure is composed of structural units represented by the following formula (1): 2 containing a structure corresponding to the structure except for The antigen is represented by the formula (1) 2 3. The affinity particle according to claim 1 or 2, wherein the affinity particle is supported on the surface of the latex particle by binding to a group represented by the formula: [ka] (In formula (1), R 1 represents a hydrogen atom or a methyl group. R 2 represents a group having an epoxy group, a group having a hydroxy group, or a group having a carboxy group. R 1 and R 2 may be different for each structural unit.) (Configuration 4) In the skeletal structure, the structural unit represented by the formula (1) is 2 4. The affinity particle according to configuration 3, wherein the content of the structure corresponding to the structure excluding the structure is 5% by mass or more and less than 50% by mass. (Configuration 5) 5. The affinity particle according to configuration 3 or 4, wherein the structural unit represented by formula (1) contains a structural unit represented by formula (1-A): [ka] (In formula (1-A), R 3 represents a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C): [ka] (In formula (1-B), R 4 represents a single bond or a methylene group. R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and the group represented by formula (1-B) has at least one hydroxy group. Y 1 represents a sulfur atom or an imino group. *1 indicates the bond position.) [ka] (In formula (1-C), R 8 represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y 2 represents a sulfur atom or an imino group. Y 3 represents a single bond or a methylene group. *2 indicates the bond position.) (Configuration 6) 6. The affinity particles according to any one of aspects 1 to 5, wherein the proportion of the antigen in the protein supported on the surface of the latex particles is 70% by mass or more. (Configuration 7) A test reagent comprising a first reagent solution in which the affinity particles according to any one of configurations 1 to 6 are dispersed. (Configuration 8) 8. The test reagent according to claim 7, comprising a nonionic surfactant and a zwitterionic surfactant, and further comprising at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride. (Configuration 9) a test reagent comprising a first reagent solution in which the affinity particles are dispersed and a second reagent solution which is a buffer solution; The test reagent according to configuration 8, wherein the second reagent solution does not contain the affinity particles, but contains the nonionic surfactant and the zwitterionic surfactant, and further contains at least one metal salt selected from the group consisting of sodium chloride, potassium chloride, and magnesium chloride. (Configuration 10) 10. The test reagent according to claim 8 or 9, wherein the nonionic surfactant comprises at least one selected from polyoxyethylene sorbitan monolaurate and n-octyl-β-D-glucopyranoside. (Configuration 11) 11. The test reagent according to claim 8, wherein the zwitterionic surfactant comprises at least one selected from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxysulfonic acid (CHAPSO). (Method 12) A method for detecting immunoglobulin G in a human specimen by a latex agglutination method using a test reagent comprising a first reagent solution in which affinity particles are dispersed and a second reagent solution which is a buffer solution, comprising: a first step of obtaining a mixture containing the human specimen and the second reagent solution; a second step of mixing the mixed solution with the first reagent solution to cause a reaction and obtain a reaction solution; and a third step of measuring the concentration of the immunoglobulin G in the reaction solution, The affinity particles have a volume average particle size of 400 nm or less, the affinity particles include latex particles and proteins supported on the surfaces of the latex particles; The protein comprises an antigen having a molecular weight of 10,000 or more, A detection method characterized in that the amount of protein supported on the surface of the latex particles is 1.0 μg or more and 20.0 μg or less per 1 mg of the affinity particles. (Method 13) the second reagent solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride; 13. The detection method according to Method 12, wherein the total content of at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride in the second reagent solution is 120 mM or more and 500 mM or less. (Method 14) at least one of the first reagent solution and the second reagent solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride; 14. The detection method according to Method 12 or 13, wherein the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride relative to the total content of the first reagent solution and the second reagent solution is 90 mM or more and 400 mM or less. (Method 15) the reaction solution in the third step contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride that is not derived from the human specimen; 15. The detection method according to any one of Methods 12 to 14, wherein the total content of at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride not derived from the human sample in the reaction solution in the third step is 80 mM or more and 360 mM or less.

Claims

1. 1. Affinity particles for detecting immunoglobulin G in a human specimen by latex agglutination, comprising: The volume average particle size is 400 nm or less, A method for producing a latex particle-supported protein-containing polymer, comprising: The protein comprises an antigen having a molecular weight of 10,000 or more, Affinity particles characterized in that the amount of protein supported on the surface of the latex particles per 1 mg of the affinity particles is 1.0 μg or more and 20.0 μg or less.

2. The affinity particle according to claim 1, wherein the antigen is an HCV antigen or an HBc antigen.

3. the latex particles have a skeleton structure composed of structural units derived from a polymerizable monomer, The skeletal structure is composed of structural units represented by the following formula (1) ２ containing a structure corresponding to the structure except for The antigen is represented by the formula (1) ２ The affinity particle according to claim 1, wherein the affinity particle is supported on the surface of the latex particle by binding to a group represented by the formula: 【Chemical Formula 1】 (In formula (1), R １ represents a hydrogen atom or a methyl group. R ２ represents a group having an epoxy group, a group having a hydroxy group, or a group having a carboxy group. R １ and R ２ may be different for each structural unit.)

4. In the skeletal structure, the structural unit represented by the formula (1) is ２ The affinity particle according to claim 3, wherein the content of the structure corresponding to the structure excluding is 5% by mass or more and less than 50% by mass.

5. 4. The affinity particle according to claim 3, wherein the structural unit represented by formula (1) includes a structural unit represented by formula (1-A): 【Chemistry 2】 (In formula (1-A), R ３ represents a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C): 【Chemistry 3】 (In formula (1-B), R ４ represents a single bond or a methylene group. R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and the group represented by formula (1-B) has at least one hydroxy group. Y １ represents a sulfur atom or an imino group. *1 indicates the bond position.) 【Chemistry 4】 (In formula (1-C), R ８ represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y ２ represents a sulfur atom or an imino group. Y ３ represents a single bond or a methylene group. *2 indicates the bond position.)

6. 2. The affinity particle according to claim 1, wherein the proportion of the antigen in the protein carried on the surface of the latex particle is 70% by mass or more.

7. A test reagent comprising a first reagent solution in which the affinity particles according to claim 1 are dispersed.

8. The test reagent according to claim 7 , comprising a nonionic surfactant and a zwitterionic surfactant, and further comprising at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride.

9. a test reagent comprising a first reagent solution in which the affinity particles are dispersed and a second reagent solution which is a buffer solution; The test reagent of claim 8, wherein the second reagent solution does not contain the affinity particles, but contains the nonionic surfactant and the zwitterionic surfactant, and further contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride.

10. 9. The test reagent according to claim 8, wherein the nonionic surfactant comprises at least one selected from polyoxyethylene sorbitan monolaurate and n-octyl-β-D-glucopyranoside.

11. 9. The test reagent according to claim 8, wherein the zwitterionic surfactant comprises at least one selected from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxysulfonic acid (CHAPSO).

12. 1. A method for detecting immunoglobulin G in a human specimen by a latex agglutination method using a test reagent comprising a first reagent solution in which affinity particles are dispersed and a second reagent solution which is a buffer solution, a first step of obtaining a mixture containing the human specimen and the second reagent solution; a second step of mixing the mixed solution with the first reagent solution to cause a reaction and obtain a reaction solution; and a third step of measuring the concentration of the immunoglobulin G in the reaction solution, the volume average particle size of the affinity particles is 400 nm or less; the affinity particles include latex particles and proteins supported on the surfaces of the latex particles; The protein comprises an antigen having a molecular weight of 10,000 or more, A detection method characterized in that the amount of protein supported on the surface of the latex particles is 1.0 μg or more and 20.0 μg or less per 1 mg of the affinity particles.

13. the second reagent solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride; 13. The detection method according to claim 12, wherein the total content of at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride in the second reagent solution is 120 mM or more and 500 mM or less.

14. at least one of the first reagent solution and the second reagent solution contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride; 13. The detection method according to claim 12, wherein the total content of the at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride relative to the total content of the first reagent solution and the second reagent solution is 90 mM or more and 400 mM or less.

15. the reaction solution in the third step contains at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride that is not derived from the human specimen; 13. The detection method according to claim 12, wherein the total content of at least one metal salt selected from sodium chloride, potassium chloride, and magnesium chloride not derived from the human specimen in the reaction solution in the third step is 80 mM or more and 360 mM or less.

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