Latex agglutination reaction sensitizer, latex agglutination reagent, and method of measuring target substance in sample by latex agglutination method using the same

Abstract

To provide a latex agglutination reagent which contains polyoxazoline, is suitable for use in latex agglutination reaction, has a high sensitizing effect, and is capable of suppressing non-specific reactions, and to provide a method of measuring a target substance in a sample by the latex agglutination method.SOLUTION: A latex agglutination reagent containing a polyoxazoline having a repeating unit represented by a general formula (1) below is provided, the latex agglutination reagent being designed to be used in a method of measuring a target substance in a sample using the latex agglutination method that utilizes an insoluble carrier carrying an antibody or antigen. (In the formula (1), R represents a hydrogen atom, methyl group, ethyl group, or propyl group, and n is an integer greater than or equal to 100.)SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a latex agglutination reaction sensitizer, a latex agglutination reagent, and a method for measuring a target substance in a specimen by a latex agglutination method using the same.

Background Art

[0002] For detecting and measuring a specific antigen or antibody (hereinafter referred to as "target substance"), immunoassay methods using antigen-antibody reactions are used in clinical tests and the like. Among them, an immunoassay method using an insoluble carrier (such as latex particles) carrying an antibody or antigen is known as a latex agglutination method.

[0003] In an example of the latex agglutination method, a specimen (such as serum) that may contain a target substance is reacted with a latex agglutination reagent containing particles (also referred to as "antibody-sensitized particles") carrying an antibody against the target substance, and for the aggregates generated by this antigen-antibody reaction, by visually or optically detecting this aggregation, the target substance can be measured qualitatively and quantitatively.

[0004] In another example of the latex agglutination method, a specimen that may contain a target substance is mixed with a first reagent (sometimes referred to as "R1") of a latex agglutination reagent. Next, a sensitized particle dispersion liquid containing sensitized particles to which an antibody or antigen against the target substance is fixed (the second reagent of the latex agglutination reagent, sometimes referred to as "R2") is further mixed. When the first reagent and the second reagent are mixed, if the target substance is present, due to the antigen-antibody reaction, the sensitized particles will aggregate, so by visually or optically detecting this aggregation, the target substance can be measured qualitatively and quantitatively.

[0005] In clinical tests, highly sensitive chemiluminescent immunoassay methods are also used, but since BF separation (a process for removing unreacted components) is required, the test time tends to be long. On the other hand, in the latex agglutination method, although the test time is short and the test throughput is high, compared with the chemiluminescent immunoassay method, the detection sensitivity may be insufficient, and further improvement in detection sensitivity is required.

[0006] Polymer compounds are used as sensitizers to improve the sensitivity of the latex agglutination method. More specifically, latex agglutination reagents containing sensitizers are used. As sensitizers, water-soluble polymers such as polyethylene glycol (PEG) and sodium alginate are added to the reaction system to promote the agglutination reaction. Patent documents 1 and 2 propose polyethylene glycol, a nonionic polymer, as a sensitizer. Patent document 3 proposes alginate, an anionic polymer, as a sensitizer.

[0007] However, while these polymers sensitize the antigen, they can also increase the viscosity of the solution and cause polymer salting-out, potentially promoting reactions unrelated to the antigen-antibody reaction, i.e., non-specific agglutination reactions. This can lead to problems such as high blank values, false positives, and reduced measurement accuracy. Therefore, there is a need for a latex agglutination reagent containing a sensitizer that exhibits sensitizing effects while suppressing non-specific reactions and maintaining good measurement accuracy.

[0008] Furthermore, Patent Document 4 proposes polyoxazoline, a nonionic polymer, as an additive to enhance chemiluminescence detection.

[0009] However, the sensitizing effect of polyoxazolines in latex agglutination methods for measuring target substances in samples using insoluble carriers loaded with antibodies or antigens was previously unknown. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Unexamined Patent Publication No. 58-47256 [Patent Document 2] Japanese Patent No. 2682697 [Patent Document 3] Japanese Unexamined Patent Publication No. 2003-294753 [Patent Document 4] Japanese Patent No. 3632856 [Patent Document 1] Japanese Unexamined Patent Publication No. 58-47256 [Patent Document 2] Japanese Patent No. 2682697 [Patent Document 3] Japanese Unexamined Patent Publication No. 2003-294753 [Patent Document 4] Japanese Patent No. 3632856 [Patent Document 1] Japanese Unexamined Patent Publication No. 58-47256 [Patent Document 2] Japanese Patent No. 2682697 [Patent Document 3] Japanese Unexamined Patent Publication No. 2003-294753 [Patent Document 4] Japanese Patent No. 3632856 [Patent Document 1] Japanese Unexamined Patent Publication No. 58-47256 [Patent Document 2] Japanese Patent No. 2682697 [Patent Document 3] Japanese Unexamined Patent Publication No. 2003-294753 [Patent Document 4] Japanese Patent No. 3632856 [Overview of the project] [Problems that the invention aims to solve]

[0011] Therefore, the present invention aims to provide a latex agglutination reagent comprising a novel latex agglutination reaction sensitizer that is suitable for use in latex agglutination methods using an insoluble carrier supporting an antibody or antigen, exhibits a sensitizing effect while suppressing nonspecific reactions and having good measurement accuracy, and a method for measuring a target substance in a sample by latex agglutination using the same. [Means for solving the problem]

[0012] Therefore, the present inventors diligently investigated the matter and found that the above objective can be achieved by using a latex agglutination reagent containing polyoxazoline as a latex agglutination reaction sensitizer.

[0013] In other words, the present invention relates to a latex agglutination reagent comprising a polyoxazoline (latex agglutination reaction sensitizer) having repeating units represented by the following general formula (1), used in a method for measuring target substances in a sample by latex agglutination using an insoluble carrier on which an antibody or antigen is supported. [Chemical Formula 1] (In the formula, R represents a hydrogen atom or a methyl group, ethyl group, or propyl group. n is an integer greater than or equal to 100.)

[0014] The present invention also relates to a latex agglutination reagent kit comprising the above-mentioned latex agglutination reagent as a component. Furthermore, the present invention relates to a method for measuring target substances in a sample by latex agglutination using an insoluble carrier supporting an antibody or antigen, using the above-mentioned latex agglutination reagent or latex agglutination reagent kit. [Effects of the Invention]

[0015] The latex agglutination reagent of the present invention, comprising polyoxazoline, exhibits a higher sensitizing effect compared to conventional latex agglutination reagents comprising sensitizers, while simultaneously suppressing nonspecific reactions. Furthermore, the latex agglutination reagent of the present invention provides a latex agglutination reagent that is easy to handle due to its good measurement accuracy and low viscosity. By using the latex agglutination reagent of the present invention, it becomes possible to measure target substances in a sample with high sensitivity and accuracy using a latex agglutination method employing an insoluble carrier supporting an antibody or antigen. [Modes for carrying out the invention]

[0016] The following describes latex agglutination reaction sensitizers, latex agglutination reagents, and methods for measuring target substances in a sample by latex agglutination using the same, according to embodiments of the present invention (hereinafter referred to as "this embodiment"). However, the present invention is not limited thereto. The latex agglutination reagent of this embodiment is used for measuring target substances in a sample by latex agglutination using an insoluble carrier supporting an antibody or antigen. In this specification, latex agglutination refers to an immunoassay method based on an agglutination reaction, and includes, for example, immunoassay methods called latex agglutination immunoturbidimetry, immunoturbidimetry, and immunobug.

[0017] (Polyoxazoline as a sensitizer) The latex agglutination reagent of this embodiment is characterized by containing at least polyoxazoline, which functions as a sensitizer for the latex agglutination reaction. For example, the latex agglutination reagent can be prepared by adding polyoxazoline to various solutions used in the latex agglutination method.

[0018] In the latex agglutination reagent of this embodiment, the mechanism by which polyoxazoline sensitizes the agglutination reaction is thought to be depletion agglutination. Depletion agglutination is interparticle agglutination caused by osmotic pressure acting between particles as dissolved polymers with a finite extent are expelled from the gaps between particles when particles approach each other. In order to effectively induce depletion agglutination, the polymer must have weak interactions with the medium and carrier in aqueous solution and have an appropriate size in aqueous solution. In this embodiment, as will be described later, polyoxazoline has weak interactions with salts and ions, as well as weak interactions with proteins and insoluble carriers. Furthermore, since polyoxazoline does not significantly increase the viscosity of the solution, it has been found to function effectively as a sensitizer in the latex agglutination reagent.

[0019] In this embodiment, polyoxazoline refers to a polymer having repeating units represented by the following general formula (1). In this embodiment, polyoxazoline is a general term for poly(N-acylethyleneimine). In a preferred embodiment, the polyoxazoline used according to this embodiment is a 2-alkyl-2-oxazoline polymer and may be called poly(2-oxazoline). [Chemical Formula 2]

[0020] In formula (1), R constituting the polymer side chain is selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group, or a combination thereof. Examples of these include poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropyl-2-oxazoline), and poly(2-n-propyl-2-oxazoline). The hydrophobicity of R increases as the alkyl chain length increases. Polyoxazolines with longer alkyl side chains may be undesirable because the increased hydrophobicity of the polymer reduces its solubility in water. In formula (1), R constituting the polymer side chain is preferably a methyl group or an ethyl group, and more preferably an ethyl group. Examples of preferred polymers include poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline), but poly(2-ethyl-2-oxazoline), represented by the following general formula (2), which has a good balance of water solubility and hydrophobicity, is particularly preferred. [Chemical Formula 3]

[0021] In formulas (1) and (2), n, which represents the degree of polymer polymerization (also called the number of repeating units), is an integer of 100 or more, preferably between 100 and 100,000. In the case of a homopolymer of poly(2-ethyl-2-oxazoline) of formula (2), the weight-average molecular weight of the polymer is approximately 10,000 to 1,000,000 within this range of polymer polymerization degrees. If the polymer polymerization degree n is less than 100, the sensitizing effect decreases due to the small molecular weight. On the other hand, if it exceeds 100,000, the molecular weight is large and the viscosity of the solution becomes too high, which is undesirable.

[0022] In this embodiment, the polyoxazoline may be a homopolymer or a copolymer, as long as it has repeating units represented by general formula (1). Specific examples of such copolymers include those of polyoxazoline and polyethylene glycol, polyoxazoline and polylactic acid, and polyoxazoline and polyacrylic acid.

[0023] These polyoxazolines can be used alone or in combination in the latex agglutination reagent and the method for measuring target substances using the same according to this embodiment. Preferably, the latex agglutination reagent contains poly(2-ethyl-2-oxazoline) of formula (2), and the method for measuring target substances using the same.

[0024] Polyoxazolines can be produced, for example, by polymerizing oxazoline monomers using known polymerization reactions. For example, polyoxazolines can be synthesized by cationic ring-opening polymerization of 2-oxazoline under appropriate conditions. As an example of a method for synthesizing polyoxazolines, poly(2-ethyl-2-oxazoline) (hereinafter sometimes abbreviated as "POX") will be explained. Under a dry argon atmosphere, methyl tosylate (initiator) and 2-ethyl-2-oxazoline (monomer) are added in acetonitrile to carry out cationic ring-opening polymerization. After reacting at a temperature of 40°C for about 10 days, sodium hydroxide is added to stop the reaction in order to introduce hydroxyl groups (termination agent) at the ends of the polymer. The reaction solution is purified by dialyzing with water, and the solid is obtained by vacuum drying to synthesize poly(2-ethyl-2-oxazoline) homopolymer.

[0025] The terminal structure of polyoxazolines is not particularly limited, but for example, one terminal may be a functional group derived from a polymerization initiator and the other terminal may be a functional group derived from a polymerization inhibitor. Furthermore, in cationic ring-opening polymerization, it is possible to introduce arbitrary functional groups to both ends of the polymer chain by appropriately selecting an electrophilic polymerization initiator and a nucleophilic polymerization inhibitor. Examples of electrophilic polymerization initiators include alkyl halides, acid halides, polyfunctional tosylates, polyfunctional triflates, and polyfunctional nosylates. Examples of nucleophilic polymerization inhibitors include hydroxides, amines, and carboxylates. Examples of terminal structures of polyoxazolines include methyl groups and hydroxyl groups.

[0026] The range of the weight-average molecular weight of polyoxazoline is not particularly limited, but is preferably 10,000 or more and 1,000,000 or less. Regarding the degree of polymerization of the polymer, as described above, in order to exhibit a sensitizing effect, it is necessary for the polymer to have a certain degree of spread in water, while on the other hand, it is desirable as a latex flocculation reagent that the viscosity of the solution is not too high. Therefore, when polyoxazoline having a weight-average molecular weight of 50,000, more preferably at least 200,000, and most preferably at least 400,000 is used, a high sensitizing effect is recognized. When the weight-average molecular weight is less than 10,000, it is difficult to obtain a sufficient sensitizing effect because the molecular size in water is small. When the weight-average molecular weight exceeds 1,000,000, the viscosity of the solution becomes high, resulting in poor handling and insufficient effect. The weight-average molecular weight of polyoxazoline is determined based on gel permeation chromatography (GPC) or viscosity measurement.

[0027] (Latex flocculation reagent) The latex flocculation reagent of the present embodiment is characterized by comprising at least polyoxazoline that functions as a sensitizer. For example, a latex flocculation reagent can be prepared by adding polyoxazoline to an aqueous medium used in the latex flocculation method.

[0028] In the latex flocculation reagent of the present embodiment, the concentration of polyoxazoline is preferably 0.01 w / v% or more and 2.0 w / v% or less from the viewpoint of solubility. If the concentration of polyoxazoline is within this range, the viscosity of the solution is also low, making it easy to handle the solution. If the concentration of polyoxazoline is too low, it is difficult to obtain a sufficient sensitizing effect. As will be described later, in the measurement method based on the latex flocculation method of the present embodiment, the polyoxazoline according to the present embodiment is added and used so that the final concentration in the reaction solution is usually 0.005 w / v% or more and 1.9 w / v% or less, preferably 0.1 w / v% or more and 1.9 w / v% or less, more preferably 0.15 w / v% or more and 1.9 w / v% or less. Also, the concentration can be changed according to the type of the target substance or the insoluble carrier to be measured.

[0029] The latex agglutination reagent of this embodiment is an aqueous solution of polyoxazoline. Examples of the aqueous medium include various buffers such as purified water, phosphate buffer, glycine buffer, Good buffer, Tris buffer, ammonia buffer, etc. Particularly, phosphate buffer and Good buffer are preferred.

[0030] The latex agglutination reagent of this embodiment comprises polyoxazoline in an aqueous medium, but may also contain additives such as salts, bovine serum albumin (BSA), surfactants, immunoglobulins, etc. Particularly, for adjusting the solution osmotic pressure and stabilizing proteins, addition of sodium chloride is preferred.

[0031] (Insoluble carrier) Any insoluble carrier that can be used in a normal latex agglutination reaction can be used as the insoluble carrier that can be preferably used with the latex agglutination reagent of this embodiment, but a granular carrier (hereinafter may be abbreviated as "particle") is preferred. Examples of the material of the insoluble carrier include polymers such as polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, acrylic acid polymer, agarose, dextran, etc., inorganic substances such as silica, alumina, etc., and metals such as gold colloid. The synthesis and preparation of the insoluble carrier can be carried out according to known methods.

[0032] An example of a preferred carrier is latex particles. Here, latex particles are polymer particles capable of immobilizing an antigen or an antibody against a target substance, and mean particles used in a latex agglutination reaction. Preferred examples of latex particles include polystyrene particles, polystyrene particles having siloxane, polystyrene particles having polyglycidyl (meth)acrylate (hereinafter may be abbreviated as "SG particles"), etc. These particles have advantages such as relatively easily obtaining nano-sized particles and being able to chemically modify the particle surface according to the purpose, and are carriers that can be preferably used with the latex agglutination reagent of this embodiment.

[0033] Among these carriers, as will be described later in the examples, polystyrene particles containing siloxane and SG particles in particular have chemically hydrophilic particle surfaces, which can highly suppress nonspecific adsorption. Therefore, there is almost no interaction between the polyoxazoline and the particles in this embodiment, making it the most ideal combination of particles and sensitizer for promoting aggregation by depletion effect. Specifically, when polystyrene particles containing siloxane and SG particles are used as carriers, the polyoxazoline exhibits low viscosity and does not adsorb to the particles, so increasing the concentration of polyoxazoline can increase sensitivity and further improve measurement accuracy.

[0034] The size of the insoluble carrier is preferably 0.01 μm to 10 μm in terms of its long axis diameter, and more preferably 0.05 μm to 1 μm. By setting the size of the insoluble carrier to 0.05 μm or larger, the absorbance in the visible range observed by latex agglutination measurement is within an appropriate range. By setting the size of the insoluble carrier to 1 μm or smaller, the dispersion stability in solution is improved, and the absorbance in the visible range is within an appropriate range. From the viewpoint of reactivity and dispersion stability, it is preferable to use an insoluble carrier between 0.05 μm and 0.40 μm. As described above, one example of a suitable insoluble carrier is a particle. Therefore, a suitable example of size can be expressed in terms of average particle diameter. The average particle diameter can be the volume-average particle diameter measured by scattering particle size distribution measurement. The preferred average particle diameter for particles as an insoluble carrier is 0.05 μm to 1 μm.

[0035] Furthermore, the amount of insoluble carrier used can be adjusted as appropriate, depending on its size, and is such that, from the viewpoint of reactivity and solution turbidity, the concentration of insoluble carrier in the solution during the agglutination reaction is between 0.01 w / v% and 0.5 w / v%, or between 0.02 w / v% and 0.3 w / v%.

[0036] Insoluble carriers are preferably those on which an antigen or antibody is immobilized. The antigen or antibody may be immobilized on the insoluble carrier by chemical bonding or by physical adsorption, but immobilization by chemical bonding is preferred. Immobilization of the antigen or antibody can be carried out according to conventional methods, but the functional groups of the insoluble carrier can be reacted with primary amines, secondary amines, carboxyl groups, thiol groups, etc., present in the antigen or antibody. Examples of functional groups of the insoluble carrier include carboxyl groups, amino groups, aldehyde groups, epoxy groups, thiol groups, maleimide groups, etc. The insoluble carrier may have only one type of these functional group, or it may have two or more types.

[0037] Alternatively, antigen-binding fragments such as Fab, F(ab'), F(ab'), Fv, and scFv can be used instead of antibodies.

[0038] In addition to configurations in which antigens or antibodies are immobilized on an insoluble carrier, configurations in which appropriate substances are bound to the target substance may also be used. For example, a configuration in which enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin, and biotin may be bound to the insoluble carrier.

[0039] (Target Substance) The latex agglutination reagent of this embodiment is used to measure a target substance, and the target substance can be any substance that can be measured by an immunoassay, such as antigens and antibodies. Examples include, but are not limited to, CRP (C-reactive protein), ferritin, albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin E (IgE), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), KL-6, pepsinogen (PG), rheumatoid factor (RF), lipoprotein, insulin, infection-related antigens and antibodies against them, hormones, vitamins, and antibiotics.

[0040] (Example of preparation of latex agglutination reagent) By incorporating the polyoxazoline according to this embodiment into various reagents used in immunoassays, latex agglutination reagents with high sensitizing activity can be prepared. An example of the latex agglutination reagent of this embodiment is shown.

[0041] (One-solution latex agglutination reagent) Polyoxazoline is dissolved in phosphate-buffered saline (sometimes abbreviated as PBS). By adding polystyrene particles (insoluble carriers) sensitized with antibodies against the target substance to this polyoxazoline solution, the resulting solution can be used as an example of an embodiment of the latex agglutination reagent of the present invention. A sample such as serum or plasma (hereinafter sometimes abbreviated as sample) is added to this one-solution latex agglutination reagent, and the turbidity is measured using a spectrophotometer. If the sample does not contain the target substance, the polystyrene particles remain dispersed, and no change in turbidity over time is observed. On the other hand, if the sample contains the target substance, the polystyrene particles agglutinate due to an antigen-antibody reaction with the target substance. This agglutination causes a change in turbidity over time; that is, the turbidity increases. By obtaining a calibration curve (a graph showing the relationship between turbidity and the concentration of the target substance) by measuring the turbidity using a standard solution of the target substance with a known concentration, it becomes possible to quantify the target substance contained in the sample from the increase in turbidity of the sample. Various additives may be included in the latex agglutination reagent of this embodiment. Generally, pH buffers (e.g., Tris buffer, phosphate buffer, borate buffer, Good's buffer, etc.), proteins such as albumin and globulin, amino acids, surfactants, preservatives, etc. may be included.

[0042] (Latex Agglutination Reagent Kit) One embodiment of the present invention is a latex agglutination reagent kit comprising a latex agglutination reagent containing polyoxazoline as at least one component, and a specific example is a kit in which the latex agglutination reagent is composed of at least two or more solutions. Examples of these solutions include a sample diluent, a dispersion of an insoluble carrier, a diluent of the dispersion of the insoluble carrier, a standard solution for obtaining a calibration curve (a solution containing a known concentration of the target substance), and a control for confirming measurement accuracy. An example of a kit is a latex agglutination reagent kit comprising at least a first reagent consisting of a latex agglutination reagent containing polyoxazoline and a second reagent consisting of an insoluble carrier on which an antibody or antigen is supported. The first reagent consisting of a latex agglutination reagent containing polyoxazoline is sometimes called a sample diluent. This sample diluent may not contain polyoxazoline, while the second reagent containing the insoluble carrier may contain polyoxazoline. Generally, from the viewpoint of storage stability, it is preferable to include polyoxazoline in the first reagent or the sample diluent.

[0043] In the measurement of target substances by latex agglutination, for example, the sample is added to a first reagent consisting of a latex agglutination reagent containing polyoxazoline, and then this solution is mixed with a second reagent containing an insoluble carrier on which an antibody or antigen is supported. At this time, it is preferable to perform the latex agglutination reaction when the polyoxazoline concentration is in the range of 0.005 w / v% to 1.0 w / v%, and the concentration of polyoxazoline in the first reagent should be set after setting the amount of sample, the amount of the first reagent and the amount of the second reagent. During the latex agglutination reaction, within the aforementioned range of polyoxazoline concentrations, the viscosity of the solution is low, the solution is easy to handle, and a sufficient sensitization effect can be obtained. Furthermore, the concentration can be appropriately set depending on the target substance to be measured and the type of insoluble carrier.

[0044] (Method for measuring target substances in a sample) This embodiment is a method for measuring target substances in a sample by latex agglutination using an insoluble carrier on which an antibody or antigen is supported, characterized in that it uses the latex agglutination reagent or latex agglutination reagent kit of this embodiment. Here, measurement includes both qualitative and quantitative measurement. In the method for measuring target substances in a sample, a preferred form is a latex agglutination method using latex particles on which an antibody or antigen is supported.

[0045] In one example of the latex agglutination method, a sample potentially containing the target substance (such as serum) is reacted with a latex agglutination reagent containing latex particles carrying antibodies against the target substance. The agglutinations produced by this antigen-antibody reaction can then be detected visually or optically to qualitatively and quantitatively measure the target substance.

[0046] In another example of the latex agglutination method, a sample potentially containing the target substance is mixed with a sample diluent (the first latex agglutination reagent). Next, a latex particle dispersion containing latex particles immobilized with antibodies or antigens against the target substance (the second latex agglutination reagent) is further mixed. When the first and second reagents are mixed, if the target substance is present, the latex particles agglutinate as a result of the antigen-antibody reaction. By visually or optically detecting this agglutination, the target substance can be measured qualitatively and quantitatively.

[0047] As described above, it is preferable to perform the latex agglutination method when the polyoxazoline concentration during the reaction is in the range of 0.005 w / v% to 1.0 w / v%. When the polyoxazoline concentration is within this range, the measurement of target substances in the sample by the latex agglutination method becomes highly sensitive and enables measurement of target substances with high accuracy.

[0048] The reaction temperature for latex agglutination is generally in the range of 4°C to 50°C. Since low temperatures reduce the reactivity of the antigen-antibody, and high temperatures reduce the stability of the antigen-antibody immune complex, a temperature of 10°C to 40°C, and particularly 30°C to 40°C, is preferred. The reaction time for latex agglutination is generally in the range of 0 minutes to 60 minutes, preferably 1 minute to 30 minutes.

[0049] Aggregates generated by antigen-antibody reactions can be detected visually or optically to measure the target substance. In other words, the target substance can be measured qualitatively and quantitatively. In this detection step, aggregation formation may be observed visually, but from the viewpoint of detection reproducibility and throughput, it is preferable to measure the degree of aggregation using an optical device. As the optical device, a device capable of measuring scattered light or transmitted light is preferred, and a spectrophotometer or the like can be used.

[0050] In the latex agglutination method of this embodiment, the insoluble carrier is preferably polystyrene particles, polystyrene particles containing siloxane, or SG particles. In the latex agglutination method, the polyoxazoline concentration during the reaction can be varied depending on the insoluble carrier used.

[0051] The target substance measured by the latex agglutination method of this embodiment can be any substance that can be measured by an immune reaction, such as antigens and antibodies. Examples include, but are not limited to, CRP (C-reactive protein), ferritin, albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin E (IgE), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), KL-6, pepsinogen (PG), rheumatoid factor (RF), lipoprotein, insulin, infection-related antigens and antibodies against them, hormones, vitamins, and antibiotics.

[0052] In the latex agglutination method, the polyoxazoline concentration during the reaction can be varied depending on the target substance. [Examples]

[0053] The following describes an example of the present invention (hereinafter referred to as "this example"): a latex agglutination reagent kit composed of two liquids, but the present invention is not limited thereto.

[0054] (Example 1: Preparation of the first reagent of a latex agglutination reagent kit) An example of a latex agglutination reagent kit consisting of two solutions is a first reagent for diluting the sample and a second reagent containing antibody-sensitized latex. Here, an example of the preparation of the first reagent is shown.

[0055] <Example 1-1: Preparation of a latex agglutination reagent (first reagent) containing poly(2-ethyl-2-oxazoline)> Poly(2-ethyl-2-oxazoline) (average molecular weight 400,000, Sigma-Aldrich) was dissolved in PBS solution (pH 7.4). Hereafter, poly(2-ethyl-2-oxazoline) may be abbreviated as "POX". The concentration of POX was adjusted to 0.5 w / v% to obtain the latex agglutination reagent (first reagent) of this example. Hereafter, this first reagent will be referred to as R1+POX.

[0056] (Comparative Example 1-1: Preparation of Conventional Latex Agglutination Reagent) For comparison, a first reagent (i.e., PBS solution) without poly(2-ethyl-2-oxazoline) was prepared in the same manner as in Example 1-1. This will be denoted as R1. In addition, PEG (average molecular weight 400,000, Sigma-Aldrich), a nonionic polymer commonly used as a sensitizer, and sodium alginate (80-120, Wako Pure Chemical Industries) (SA), an anionic polymer, were dissolved in PBS to a concentration of 0.5 w / v% to prepare R1+PEG and R1+SA, respectively, as first reagents for comparison.

[0057] <Example 1-2: Viscosity Measurement of Latex Agglutination Reagent (First Reagent)> The viscosity of the latex agglutination reagent (first reagent) R1+POX in this example was measured. Viscosity was measured using an E-type rotational viscometer (RE-80, Toki Sangyo). The results are shown in Table 1.

[0058] (Comparative Examples 1-2) For comparison, viscosity measurements were performed for R1+PEG and R1+SA. The results are shown in Table 1.

[0059] [Table 1]

[0060] As shown in Table 1, in solutions of the same polymer concentration, R1+POX had a higher viscosity than comparative example R1, but a lower viscosity compared to conventional flocculant-containing solutions R1+PEG and R1+SA. The poly(2-ethyl-2-oxazoline) used in this example had similar properties and did not increase the viscosity of the solution compared to PEG, a polymer with a similar molecular weight. SA showed particularly high viscosity, but this is likely due to its molecular weight, which is estimated to be several million.

[0061] <Example 1-3: Viscosity Measurement of Latex Agglutination Reagent (First Reagent)> The viscosity of the latex agglutination reagent (first reagent) R1+POX in this example was measured when the POX concentration was changed. Viscosity was measured using an E-type rotational viscometer (RE-80, Toki Sangyo). The results are shown in Table 2.

[0062] (Comparative Examples 1-3) For comparison, the viscosity of R1+PEG and R1+SA was measured at different polymer concentrations in the same manner as in Examples 1-3. The results are shown in Table 2. "-" indicates that measurement was not performed.

[0063] [Table 2]

[0064] The results in Table 2 show that while the viscosity of R1+POX increases with increasing polymer concentration, this increase is more gradual compared to the comparative examples R1+PEG and R1+SA. The poly(2-ethyl-2-oxazoline) used in this example has the advantage of allowing for a more gradual adjustment of the solution viscosity compared to similar polymers such as PEG and SA.

[0065] (Example 2: Preparation of the second reagent of the latex agglutination reagent kit) An example of a latex agglutination reagent kit consisting of two solutions is a first reagent for diluting the sample and a second reagent containing antibody-sensitized latex. Here, an example of the preparation of the second reagent is shown.

[0066] (Synthesis Example 2-1: Synthesis of SG Particles) 12.0 g of styrene (St: Kishida Chemical Industries Co., Ltd.), 17.9 g of glycidyl methacrylate (GMA: Tokyo Chemical Industries Co., Ltd.), 0.45 g of divinylbenzene (DVB: Kishida Chemical Industries Co., Ltd.), and 2168.6 g of deionized water were weighed into a 2 L four-neck separable flask to form a mixture. This mixture was then maintained at 70°C while stirring at 200 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 1.13 g of V-50 (Fujifilm Wako Pure Chemical Industries, Ltd.) in 30 g of deionized water was added to the mixture to initiate soap-free emulsion polymerization. Two hours after the start of polymerization, 3.1 g of GMA was added to the four-necked separable flask, and the mixture was maintained at 70°C for a further 22 hours while stirring at 200 rpm to obtain an aqueous dispersion containing granular copolymer A. After the dispersion was slowly cooled to room temperature, a portion was taken, and the polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that the polymerization conversion rate was substantially 100%. The dry particle size of granular copolymer A was 151.4 nm, and the particle size in water was 160.2 nm. Granular copolymer A was concentrated by ultrafiltration or diluted with deionized water to obtain a 2.5 w / v% aqueous dispersion, and stored at 4°C under light-shielding conditions. Next, 24 g of a 2.5 w / v% aqueous dispersion of granular copolymer A, 3.3 g of deionized water, 40 mg (0.26 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.) were weighed into a 100 ml round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and then maintained in this state for 18 hours to obtain a dispersion of SG particles (hereinafter abbreviated as "latex A particles").Latex A particles were separated from the dispersion using a centrifuge, and this process of redispersing the latex A particles in ion-exchanged water was repeated eight times to purify the latex A particles. The final aqueous dispersion was adjusted to contain 1.0 w / v% latex A particles and stored at 4°C under light-shielded conditions. The aqueous particle size of the latex A particles was 202.0 nm.

[0067] (Synthesis Example 2-2: Synthesis of Polystyrene Particles Containing Siloxane) 157 g of 50 mM 2-morpholinoethanesulfonic acid (MES, manufactured by Tokyo Chemical Industries, Ltd.) buffer (pH 7.0) and 1.3 g of polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000) were mixed. Next, 4 g of 3-methacrylateoxypropyltrimethoxysilane (Shin-Etsu Chemical Co., Ltd., LS-3380) and 13 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature while blowing nitrogen into it for 10 minutes. After that, the emulsion in the flask was heated to 70°C in an oil bath. 0.5 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in deionized water, and the resulting solution was added to the emulsion heated to 70°C. After stirring at 70°C for 7 hours, heating was stopped and the mixture was stirred overnight to obtain a dispersion of polystyrene particle precursor containing siloxane. Next, the dispersion of polystyrene particle precursor containing siloxane was centrifuged to recover the polystyrene particle precursor containing siloxane, and the supernatant was discarded. The recovered polystyrene particle precursor containing siloxane was washed by redispersing it in ion-exchanged water and repeating the centrifugation process several times. The average particle size of the polystyrene particle precursor containing siloxane obtained in this way was evaluated by dynamic light scattering using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation) and was found to be 203 nm. Next, 0.4 g of polystyrene particle precursor containing siloxane was mixed with Tween® 20 (manufactured by Tokyo Chemical Industry Co., Ltd.) to a final concentration of 1 w / v% and 40 mL of ion-exchanged water. After stirring this mixture at room temperature for 15 minutes, 0.04 mL of X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing a carboxyl group, was added and stirred at room temperature overnight. Subsequently, this dispersion was centrifuged, and the supernatant was discarded. The recovered particles were redispersed in ion-exchanged water and washed by repeating the centrifugation process several times. The resulting particles were designated as siloxane-containing polystyrene latex particles (hereinafter abbreviated as "latex B particles").

[0068] <Example 2-1: Preparation of the second reagent of the latex agglutination reagent kit> The carboxyl groups of latex A particles and latex B particles obtained in Synthesis Examples 2-1 and 2-2 were activated using WSC / NHS (water-soluble carbodiimide / N-hydroxysuccinimide) by a known method. 0.05 mL of a 10 w / v% aqueous solution of the activated latex A particles and latex B particles was mixed with 0.05 mL of 50 mM MES buffer (pH 5.0) containing 1 mg / mL of anti-human CRP polyclonal antibody (manufactured by Oriental Yeast Co., Ltd.), and the mixture was reacted at room temperature for 1 hour. Subsequently, the solution was washed by centrifugation, and the resulting antibody-immobilized particles were called antibody-sensitized latex A particles and antibody-sensitized latex B particles, respectively. The aqueous dispersions thereof were called antibody-sensitized latex A particle dispersion and antibody-sensitized latex B particle dispersion, and these were used as the second reagent. Antibody-sensitized latex A particles and antibody-sensitized latex B particles are referred to as R2-A and R2-B, respectively. In addition, a dispersion of antibody-sensitized latex C particles, i.e., the second reagent, was prepared in the same manner as above, except that commercially available 200 nm particle size polystyrene latex C particles (IMMUTEX® P0113, manufactured by JSR Corporation) were used and a post-coat was performed with bovine serum albumin (BSA). This was referred to as R2-C.

[0069] (Sample) To confirm a non-specific reaction to human serum, CRP-free human serum (Oriental Yeast) was used. To confirm the sensitizing effect, a CRP solution was used as the antigen solution for sensitivity measurement. A CRP solution with a CRP concentration of 0.5 mg / dL was used.

[0070] <Example 2-2: Evaluation of Nonspecific Reactivity and Sensitizing Effect by Latex Agglutination Measurement 1> The nonspecific reactivity of the latex agglutination reagent of this example was confirmed by latex agglutination measurement using human serum. 1 μL of CRP-free human serum from the above sample was added to 50 μL of the first reagent (R1+POX) of this example obtained in Example 1-1. This was placed in a spectrophotometer measurement cell (optical path length 10 mm) and held at 37°C for 5 minutes. Next, 50 μL of the second reagent (R2-A solution) of this example was added to 51 μL of the R1 solution and thoroughly mixed. After this, the absorbance at 572 nm was measured using a spectrophotometer (represented as the absorbance at 0 minutes). After 5 minutes, the absorbance at 572 nm was measured again (represented as the absorbance at 5 minutes). The difference in absorbance between the two obtained points ((absorbance after 5 minutes) - (absorbance at 0 minutes)) is calculated and multiplied by 10,000 to express as the absorbance change. If there is no nonspecific reaction, the value will be close to zero. If a nonspecific reaction occurs, the value will be high. Here, an absorbance change of 100 or more is defined as the occurrence of a nonspecific reaction. The nonspecific reaction of R1+POX was evaluated according to the measurement method described above. The results are shown in Tables 3-4. Here, nonspecific reactivity refers to the degree of the nonspecific reaction described above and is expressed as the absorbance change.

[0071] Furthermore, the sensitizing effect of the latex agglutination reagent in this example was confirmed by measuring the latex agglutination of CRP. 1 μL of a CRP solution with a CRP concentration of 0.5 mg / dL was added to 50 μL of the first reagent (R1+POX) of this example obtained in Example 1-1. This was placed in a spectrophotometer measurement cell (optical path length 10 mm) and held at 37°C for 5 minutes. Next, 50 μL of the second reagent (R2-A solution) of this example was added to 51 μL of the R1 solution and thoroughly mixed. After this, the absorbance at 572 nm was measured using a spectrophotometer (represented as the absorbance at 0 min). After 5 minutes, the absorbance at 572 nm was measured again (represented as the absorbance at 5 min). The difference between the two absorbances obtained ((absorbance at 5 min) - (absorbance at 0 min)) was calculated and multiplied by 10,000 to express the change in absorbance. If particle aggregation occurs due to the reaction between CRP and antibody-antigen, the change in absorbance will increase. A higher change in absorbance indicates higher sensitivity. The CRP measurement sensitivity (CRP concentration 0.5 mg / dL) of R1+POX was evaluated according to the measurement method described above. The results are shown in Table 3. Here, CRP measurement sensitivity refers to the degree of particle aggregation due to the reaction between CRP and antibody-antigen, and is expressed as the change in absorbance.

[0072] (Comparative Example 2-2) For comparison, the non-specific reactivity and sensitizing effect of R1+PEG and R1+SA were evaluated in the same manner as in Example 2-2. The results are shown in Table 3.

[0073] [Table 3]

[0074] Table 3 shows the evaluation results of nonspecific reactivity and sensitization effect by latex aggregation measurement when the second reagent is R2-A solution, i.e., when SG particles are used as the insoluble carrier, and the polymer concentration of the first reagent is changed.

[0075] R1+POX showed higher sensitivity than R1 (comparative example) without a sensitizer, and the sensitivity of CRP measurement improved depending on the POX concentration. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents. Furthermore, R1+POX showed very little nonspecific reactivity to human serum at concentrations up to 0.866%. On the other hand, while the comparative examples R1+PEG and R1+SA showed high sensitivity and functioned as sensitizers, nonspecific reactions occurred. As shown in the table*, in particular, with R1+SA, when the polymer concentration during the reaction exceeded 0.186%, rapid agglutination occurred during the measurement of absorbance at 0 minutes, resulting in an absorbance of 3.0 or higher (range over), which is the upper limit of the spectrophotometer's measurement, making measurement impossible (indicated as "unmeasurable" in the table). With PEG and alginic acid, agglutination due to salt may occur, and the high viscosity of the solution presents challenges such as bubble generation during stirring and reaction reproducibility. Compared to the results of R1+PEG and R1+SA in Comparative Example 2-2, the latex agglutination reagent of this example containing poly(2-ethyl-2-oxazoline) shows a maximum sensitivity of 10,600 when the absorbance change is considered to be 100 or less when no nonspecific reaction occurs, indicating that the absorbance change can be increased. In the comparative examples, the maximum sensitivity when no nonspecific reaction occurred was 3,310 for PEG and 7,030 for SA.

[0076] Based on these results, when SG particles are used as an insoluble carrier, the latex agglutination reagent containing poly(2-ethyl-2-oxazoline) of this example can exhibit a sensitizing effect while suppressing nonspecific reactions, enabling latex agglutination measurement with high sensitivity.

[0077] <Example 2-3: Evaluation of nonspecific reactivity and sensitization effect by latex agglutination measurement 2> In the same manner as in Example 2-2, the second reagent was R2-C solution, i.e., polystyrene latex particles were used as the insoluble carrier, and the nonspecific reactivity and sensitization effect were evaluated by latex agglutination measurement. The results are shown in Table 4.

[0078] (Comparative Example 2-3) For comparison, the non-specific reactivity and sensitizing effect of R1+PEG and R1+SA were evaluated in the same manner as in Example 2-3. The results are shown in Table 4.

[0079] [Table 4]

[0080] Table 4 shows the results of evaluating nonspecific reactivity and sensitization effect by latex aggregation measurement when the second reagent is R2-C solution, i.e., polystyrene latex particles are used as the insoluble carrier, and the polymer concentration of the first reagent is varied.

[0081] Similar to the results of Example 2-2, even when the second reagent was R2-C solution, i.e., when polystyrene latex particles were used as the insoluble carrier, R1+POX showed higher sensitivity than R1 (comparative example) without a sensitizer, and the CRP measurement sensitivity improved depending on the POX concentration. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents. Furthermore, R1+POX did not show non-specific reactivity to human serum at concentrations up to 0.866%. On the other hand, R1+PEG and R1+SA in Comparative Example 2-3 showed high sensitivity and functioned as sensitizers, but non-specific reactions occurred. As shown in the table marked with *, in particular, when the polymer concentration during the reaction was 0.248% or higher for R1+SA, and when the polymer concentration during the reaction was 0.495% or higher for R1+PEG, rapid aggregation occurred during the measurement of absorbance at 0 minutes, resulting in an absorbance of 3.0 or higher (range over), which is the upper limit of the spectrophotometer's measurement, making measurement impossible (indicated as "unmeasurable" in the table). Compared with the results of R1+PEG and R1+SA in Comparative Examples 2-3, the latex agglutination reagent of this example containing poly(2-ethyl-2-oxazoline) shows a maximum sensitivity of 11,410, which is very high, when non-specific reactivity is considered as an absorbance change of 100 or less. The maximum sensitivity of the comparative examples was 5,240 for PEG and 8,250 for SA.

[0082] Based on these results, when polystyrene latex particles are used as an insoluble carrier, the latex agglutination reagent containing poly(2-ethyl-2-oxazoline) of this example can be used to exhibit a sensitizing effect while suppressing nonspecific reactions, enabling latex agglutination measurement with high sensitivity.

[0083] <Example 2-4: Evaluation of Measurement Accuracy> To evaluate the measurement accuracy, the sensitization effect (CRP concentration 0.5 mg / dL) by latex agglutination measurement was repeatedly measured, similar to Examples 2-2 and 2-3. Table 5 shows the results of repeated measurements when the first reagent was R1+POX and the second reagent was R2-A solution, i.e., when SG particles were used as the insoluble carrier. Table 6 shows the results of repeated measurements when the first reagent was R1+POX and the second reagent was R2-C solution, i.e., when polystyrene latex particles were used as the insoluble carrier.

[0084] (Comparative Example 2-4) For comparison, the first reagent was subjected to repeated measurements with R1+PEG and R1+SA in the same manner as in Example 2-4. The results are shown in Tables 5 and 6.

[0085] [Table 5]

[0086] The results in Table 5 show that, compared to the comparative example, the R1+POX of this example had a smaller coefficient of variation and higher repeatability when SG particles were used as the insoluble carrier.

[0087] [Table 6]

[0088] The results in Table 6 show that, compared to the comparative example, the R1+POX of this example had a smaller coefficient of variation and higher repeatability when polystyrene latex particles were used as the insoluble carrier.

[0089] (Example 3: Effect of Molecular Weight of Poly(2-ethyl-2-oxazoline)) Poly(2-ethyl-2-oxazoline) (Sigma-Aldrich), with an average molecular weight of 200,000, was dissolved in PBS in the same manner as in Example 1-1 to prepare the latex agglutination reagent R1+POX-200k of this example. Poly(2-ethyl-2-oxazoline) (Sigma-Aldrich), with an average molecular weight of 50,000, was dissolved in PBS in the same manner as in Example 1-1 to prepare the latex agglutination reagent R1+POX-50k of this example. Nonspecific reactivity and sensitization effect were evaluated by latex agglutination measurement in the same manner as in Example 2-2. To confirm the effect of molecular weight, R1+POX (average molecular weight of POX 400,000) was also evaluated in the same manner. The results are shown in Table 7.

[0090] [Table 7]

[0091] The results in Table 7 show that in the latex agglutination reagent containing poly(2-ethyl-2-oxazoline) in this example, the sensitizing effect increases as the molecular weight of poly(2-ethyl-2-oxazoline) increases.

[0092] (Example 4: Evaluation of nonspecific reactivity and sensitization effect by latex agglutination measurement 3) In the same manner as in Example 2-2, the second reagent was R2-B solution, i.e., polystyrene latex particles containing siloxane were used as the insoluble carrier, and the nonspecific reactivity and sensitization effect were evaluated by latex agglutination measurement. The results are shown in Table 8.

[0093] [Table 8]

[0094] As shown in Table 8, even when the second reagent was R2-B solution, i.e., when polystyrene latex particles containing siloxane were used as the insoluble carrier, R1+POX (with a polymer concentration of 0.5 w / v% in the first reagent) showed a greater change in absorbance than R1 (with a polymer concentration of 0 w / v% in the first reagent) which did not contain a sensitizer. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents containing polystyrene latex particles containing siloxane.

[0095] (Example 5: Preparation of a latex agglutination reagent (Reagent 1B) containing poly(2-ethyl-2-oxazoline)) POX (average molecular weight 400,000, Sigma-Aldrich) was dissolved in HEPES-T solution (pH 7.9, containing 0.01% Tween 20). The concentration of POX was adjusted to 1.5 w / v% to obtain the latex agglutination reagent (Reagent 1B) of this example. Hereafter, this Reagent 1 will be referred to as R1+POX-B.

[0096] (Comparative Example 5: Preparation of a Conventional Latex Agglutination Reagent) For comparison, a POX-free first reagent (i.e., HEPES-T solution) was prepared in the same manner as in Example 5. This will be denoted as R1-B.

[0097] (Example 6) <Example 6-1: Preparation of the second reagent of the latex agglutination reagent kit> Using a known method, commercially available polystyrene latex D particles with a particle size of 153 nm (IMMUTEX® P2118, manufactured by JSR Corporation) were used to prepare an antibody-sensitized latex D particle dispersion, i.e., the second reagent. Antibody sensitization was performed by physically adsorbing the antibody onto the surface of the polystyrene latex D particles and post-coating with BSA. An example of the preparation method is shown below. The polystyrene latex D particles were diluted with HEPES buffer (pH 7.9) to a solid content concentration of 3%. Anti-human CRP polyclonal antibody was diluted with the same buffer to prepare an antibody solution with a concentration of approximately 1.0 mg / ml. 0.1 mL of the antibody solution and 0.1 mL of polystyrene latex D particles were mixed and stirred overnight at 4°C. After centrifugation at 20000 G for 15 minutes, the supernatant was removed, and a 0.2% BSA HEPES solution was added to the precipitate as a blocking agent. After stirring at room temperature for 60 minutes, the mixture was centrifuged at 20,000 G for 15 minutes, and the supernatant was removed. The precipitate was washed twice with HEPES-T solution, and then redispersed by adding HEPES-T solution until the final latex solid content was 0.1%, to obtain the anti-human CRP antibody-conjugated latex reagent. This is called R2-D.

[0098] <Example 6-2: Evaluation of nonspecific reactivity by latex agglutination and sensitization effect of CRP measurement> In the same manner as in Example 2-2, the nonspecific reactivity and sensitization effect by latex agglutination were evaluated using R1+POX-B as the first reagent and R2-D solution as the second reagent, i.e., polystyrene latex particles (with antibodies physically adsorbed) as the insoluble carrier. Nonspecific reactivity was evaluated using CRP-free human serum (CRP concentration 0 mg / dL), and the sensitization effect was evaluated using CRP solutions with CRP concentrations of 0.5, 1, 2, and 4 mg / dL, in the same manner as in Example 2-2. The results are shown in Table 9.

[0099] (Comparative Example 6) For comparison, non-specific reactivity and sensitization effect were evaluated in the same manner as in Example 6-2, except that the first reagent was R1-B. The results are shown in Table 9.

[0100] [Table 9]

[0101] As shown in Table 9, when the target substance (measurement target) was evaluated as CRP, even when the second reagent was R2-D solution, i.e., when polystyrene latex particles (with antibodies physically adsorbed) were used as the insoluble carrier, R1+POX-B showed a larger change in absorbance than R1-B without a sensitizer. Furthermore, non-specific reactivity was also very small. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents containing polystyrene latex particles (with antibodies physically adsorbed).

[0102] (Example 7) <Example 7-1: Preparation of the second reagent of the latex agglutination reagent kit> Except for using anti-human ferritin antibody (manufactured by Mikli Immunology Laboratory Co., Ltd., clone No. 13) as the antibody and setting the final latex solid content concentration to 0.3%, the antibody-sensitized latex D particle dispersion, i.e., the second reagent, was prepared in the same manner as in Example 6-1. This is an anti-human ferritin antibody-conjugated latex reagent, and is referred to as R2-F.

[0103] <Example 7-2: Evaluation of nonspecific reactivity by latex agglutination measurement and sensitization effect of ferritin measurement> In the same manner as in Example 2-2, the nonspecific reactivity and sensitization effect by latex agglutination measurement were evaluated when R1+POX-B was used as the first reagent and R2-F solution was used as the second reagent, i.e., polystyrene latex particles (with antibodies physically adsorbed) were used as the insoluble carrier. For nonspecific reactivity, phosphate-buffered saline (ferritin concentration 0 μg / ml) was used as the sample, and for the sensitization effect, ferritin solutions with ferritin concentrations of 15 and 150 μg / ml were used as the sample. The evaluation was carried out in the same manner as in Example 2-2, except that 10 μL of the above sample was used, 100 μL of the first reagent (R1+POX-B) was used, and the optical path length of the spectrophotometer measurement cell was 2 mm. The results are shown in Table 10.

[0104] (Comparative Example 7) For comparison, non-specific reactivity and sensitization effect were evaluated in the same manner as in Example 7-2, except that the first reagent was R1-B. The results are shown in Table 10.

[0105] [Table 10]

[0106] As shown in Table 10, even when the second reagent was R2-F solution, i.e., polystyrene latex particles (with antibodies physically adsorbed) were used as the insoluble carrier, and the target substance (measurement target) was ferritin, R1+POX-B showed a larger change in absorbance than R1-B without the sensitizer. Furthermore, non-specific reactivity was very small. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents containing polystyrene latex particles (with antibodies physically adsorbed).

[0107] (Example 8) <Example 8-1: Preparation of the second reagent of the latex agglutination reagent kit> Except for using anti-human thyroid-stimulating hormone (TSH) antibody (manufactured by Mikli Immunology Laboratory Co., Ltd., clone 9G11, clone 3F10) as the antibody and setting the final latex solid content concentration to 0.3%, the antibody-sensitized latex D particle dispersion, i.e., the second reagent, was prepared in the same manner as in Example 6-1. This is an anti-human TSH antibody-conjugated latex reagent, and is called R2-T.

[0108] <Example 8-2: Evaluation of nonspecific reactivity by latex agglutination measurement and sensitization effect of TSH measurement> In the same manner as in Example 2-2, the nonspecific reactivity and sensitization effect by latex agglutination measurement were evaluated when R1+POX-B was used as the first reagent and R2-T solution was used as the second reagent, i.e., polystyrene latex particles (with antibodies physically adsorbed) were used as the insoluble carrier. For nonspecific reactivity, phosphate-buffered saline containing 0.1% BSA (TSH concentration 0 μg / ml) was used as the sample, and for the sensitization effect, a TSH solution with a TSH concentration of 10 μg / ml (phosphate-buffered saline containing 0.1% BSA) was used as the sample. The evaluation was carried out in the same manner as in Example 2-2, except that 20 μL of the above sample, 100 μL of the first reagent (R1+POX-B), and the optical path length of the spectrophotometer measurement cell were 2 mm. The results are shown in Table 11.

[0109] (Comparative Example 8) For comparison, non-specific reactivity and sensitization effect were evaluated in the same manner as in Example 8-2, except that the first reagent was R1-B. The results are shown in Table 11.

[0110] [Table 11]

[0111] As shown in Table 11, even when the second reagent was R2-T solution, i.e., polystyrene latex particles (with antibodies physically adsorbed) were used as the insoluble carrier, and the target substance (measurement target) was TSH, R1+POX-B showed a larger change in absorbance than R1-B without the sensitizer. Furthermore, non-specific reactivity was very small. In other words, it was confirmed that POX functions as a sensitizer for latex agglutination reagents containing polystyrene latex particles (with antibodies physically adsorbed).

Claims

1. A method for producing a composition comprising: an aqueous medium; a polyoxazoline dissolved in the aqueous medium; and an insoluble carrier carrying an antibody or an antigen, The polyoxazoline has a repeating unit represented by the following general formula (1): the insoluble carrier is a latex particle, A latex agglutination reagent, wherein the weight average molecular weight of the polyoxazoline is 50,000 or more and 1,000,000 or less. 【Chemistry 1】 (In formula (1), R represents a hydrogen atom, a methyl group, an ethyl group, or a propyl group; and n is an integer of 100 or more.)

2. The latex agglutination reagent described in claim 1, wherein the weight average molecular weight is 200,000 or more.

3. The latex agglutination reagent described in claim 1, wherein the weight average molecular weight is 400,000 or more.

4. A latex agglutination reagent as described in claim 1, wherein the average particle diameter of the latex particles is 0.05 μm or more and 1 μm or less.

5. The latex agglutination reagent described in claim 1, wherein the latex particles are any one of polystyrene particles, polystyrene particles having siloxane, and polystyrene particles having polyglycidyl (meth)acrylate.

6. The latex agglutination reagent of claim 1, wherein the latex particles comprise a copolymer of styrene, glycidyl (meth)acrylate, and divinylbenzene.

7. The latex agglutination reagent according to claim 1, wherein R in the general formula (1) is an ethyl group.

8. 2. The latex agglutination reagent according to claim 1, wherein the concentration of the polyoxazoline is 0.01% w / v or more and 2.0% w / v or less.

9. The latex agglutination reagent of claim 1, wherein the aqueous medium is any one of purified water, phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer.

10. The latex agglutination reagent of claim 1, further comprising any one of salts, bovine serum albumin, surfactants, immunoglobulins, and sodium chloride.

11. The latex agglutination reagent described in claim 1, wherein the insoluble carrier carries an antibody, which is an anti-human CRP polyclonal antibody or an anti-human ferritin antibody.

12. A latex agglutination reagent kit comprising the latex agglutination reagent according to claim 1 as a constituent component.

13. 13. The latex agglutination reagent kit according to claim 12, wherein the latex agglutination reagent is composed of at least two components, and comprises at least a first reagent comprising the latex agglutination reagent according to claim 1, and a second reagent comprising an insoluble carrier carrying an antibody or an antigen.

14. 13. The latex agglutination reagent kit according to claim 12, which is prepared so as to carry out the latex agglutination method at a concentration of the polyoxazoline in the range of 0.005 w / v % or more and 1.9 w / v % or less.

15. 13. A method for measuring a target substance in a specimen by a latex agglutination method using an insoluble carrier carrying an antibody or an antigen, the method comprising using the latex agglutination reagent according to claim 1 or the latex agglutination reagent kit according to claim 12.

16. 16. The method for measuring a target substance in a specimen by the latex agglutination method according to claim 15, wherein the latex agglutination method is performed with a concentration of polyoxazoline in the latex agglutination reagent or the latex agglutination reagent kit in the range of 0.005 w / v % or more and 1.9 w / v % or less.

17. 16. The method for measuring a target substance in a specimen by the latex agglutination method according to claim 15, wherein the insoluble carrier is any one of polystyrene particles, polystyrene particles having siloxane, and polystyrene particles having polyglycidyl (meth)acrylate.

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