Particles for immunoturbidimetry, test reagents for in vitro diagnostics

Abstract

To provide particles for immunoturbidimetric methods that can improve the detection sensitivity of target biomolecules by using an immunoassay-based agglutination reaction. [Solution] Particles for immunoturbidiometry comprising diamond particles and a ligand that specifically binds to a biomolecule, wherein the ligand is bound to the diamond particles and the particle size of the immunoturbidimetric particles is 50 nm or more and 1000 nm or less.

Description

Technical Field

[0001] The present invention relates to particles for immunoturbidimetry and an in vitro diagnostic test reagent.

Background Art

[0002] As a method for detecting a substance to be detected in a biological sample (hereinafter referred to as a specimen) in the clinical examination field, a method using an immunological agglutination reaction (hereinafter sometimes referred to as immunoturbidimetry) is known. In this method, a dispersion of particles conjugated with a ligand such as an antibody or an antigen is mixed with a specimen that may contain a biological substance (antigen, antibody, etc.) to be detected. If the biological substance to be detected is contained in the specimen, the particles conjugated with the ligand cause an agglutination reaction. Therefore, by measuring this agglutination reaction as the amount of change in light intensity such as scattered light intensity, transmitted light intensity, or absorbance, the presence or absence and quantification of the substance to be detected can be specified.

[0003] Patent Document 1 discloses particles containing a copolymer of styrene and glycidyl (meth) acrylate as particles used in immunological agglutination reactions.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Conventional particles containing organic polymers have room for improvement in the detection sensitivity of biological substances to be detected. Therefore, an object of the present invention is to provide particles for immunoturbidimetry that can improve the detection sensitivity of biological molecules to be detected by a method using an immunological agglutination reaction.

Means for Solving the Problems

[0006] The particles for immunoturbidimetry according to the present invention are particles for immunoaggregation, comprising diamond particles and a ligand that specifically binds to a biomolecule, wherein the ligand is bound to the diamond particles, and the particle size of the particles for immunoturbidimetry is 50 nm or more and 1000 nm or less. [Effects of the Invention]

[0007] The particles for immunoturbidimetry according to the present invention increase the amount of change in light intensity caused by immunoassay and improve the detection sensitivity of the target biological substance. [Brief explanation of the drawing]

[0008] [Figure 1] A graph showing the detection results of anti-ferritin antibodies using a reagent containing particles for immunoturbidimetry according to an example of the present invention. [Modes for carrying out the invention]

[0009] The embodiments of the present invention will be described in detail below, but the technical scope of the present invention is not limited to these embodiments.

[0010] (Particles for immunoturbidimetry) The particles for immunoturbidimetric analysis according to this embodiment include diamond particles and ligands that specifically bind to biomolecules, with the ligands bound to the diamond particles and the particle size of the immunoturbidimetric particles being between 50 nm and 1000 nm. In immunoturbidimetric analysis, scattered light that increases and transmitted light that decreases due to particle aggregation caused by an immunoaggregation reaction are detected. Transmitted light decreases with increasing particle concentration, optical path length, and scattering efficiency. In other words, one way to improve the detection sensitivity of immunoturbidimetric analysis is to increase the scattering efficiency of the particles. One way to improve scattering efficiency is to increase the refractive index of the particles. The particles for immunoturbidimetric analysis in this embodiment include diamond particles (nanodiamond particles) with nano-order particle size, and the refractive index of the nanodiamond particles is 2.4 or higher. This refractive index is very high compared to particles containing organic polymers that are generally used in immunoturbidimetric analysis. Therefore, the scattering efficiency of light incident on the immunoturbidimetric particles is high, and the change in light intensity is large when the target biomolecule is detected. Therefore, the particles for immunoturbidimetric analysis according to this embodiment can detect target biological substances with high sensitivity in immunoturbidimetric analysis. Immunoturbidimetric analysis can also be called immunoaggregation or latex immunoaggregation.

[0011] Furthermore, if the diamond particles in this embodiment are non-fluorescent, there is an advantage in that the light incident on the diamond particles is not used for fluorescence emission but is instead used to reduce the transmitted light due to particle aggregation. Here, non-fluorescent diamond particles mean that when the diamond particles are excited, they do not emit fluorescence at wavelengths between 400 nm and 1000 nm. The excitation light used here is light with a wavelength between 400 nm and 1000 nm. Not emitting light with wavelengths between 400 nm and 1000 nm means that they emit a small amount of fluorescence that does not significantly affect the improvement of sensitivity when detecting the target biological substance in immunoturbidimetric methods, that is, they substantially do not emit fluorescence. Therefore, if the diamond particles are non-fluorescent, the amount of transmitted light (transmitted light) as a result of incident light on the particles for immunoturbidimetric methods is large when the particles are aggregated and when they are not aggregated. This is because if the energy of the incident light is used for fluorescence emission, the energy of the transmitted light will be small regardless of whether or not aggregation is present. For this reason, it is preferable that the diamond particles in this embodiment are non-fluorescent. In particular, it is preferable that the light used in immunoturbidimetry is non-fluorescent when the wavelength of the light used as the excitation light is used.

[0012] (Binding of ligand and diamond particles) In this embodiment, the ligand and the diamond particles are bonded by at least one of the following: covalent bonds, ionic bonds, hydrogen bonds, and hydrophobic interactions. Ionic bonds, hydrogen bonds, and hydrophobic interactions can be rephrased as non-covalent bonds. The diamond particles in this embodiment may be modified with a compound having at least one of the functional groups of a carboxyl group, a hydroxyl group, and an amino group. In this case, the ligand and the diamond particles can be bonded by a reaction between the ligand and these functional groups. Compounds having at least one of the functional groups of a hydroxyl group, a carboxyl group, and an amino group can be modified mainly on the particle surface of the diamond particles by known chemical reactions.

[0013] (particle size) In this embodiment, the particle size for immunoturbidimetry is the number-average particle size in water, and is between 50 nm and 1000 nm. More preferably, the particle size for immunoturbidimetry in this embodiment is between 100 nm and 500 nm.

[0014] When the particle size of the particles used for immunoturbidimetry is between 100 nm and 500 nm, it offers excellent handling during centrifugation and has a large non-surface area. In this embodiment, the number-average particle size can be measured by dynamic light scattering. Specifically, the dispersion of the target particles is measured by dynamic light scattering, the obtained light intensity is converted into a number distribution, and the average value is taken to obtain the number-average particle size.

[0015] (Ligang) In this embodiment, the ligand is a binding substance that specifically binds to the biomolecule to be detected.

[0016] The ligand in this embodiment can be any of the following: an antibody, an antigen, a fragmented antibody, or a VHH (Variable domain of heavy chain of heavy chain antibody) antibody.

[0017] Furthermore, if the diamond particles according to this embodiment are modified with a compound having a carboxyl group, a ligand can be attached to the carboxyl group. Particles to which a ligand has been attached according to this embodiment can be called ligand-sensitized particles. Also, the process of attaching a ligand to diamond particles in this embodiment can be called ligand sensitization.

[0018] In this embodiment, a ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is determined and has selective or specifically high affinity. Examples include an antigen and an antibody, an enzyme protein and its substrate, a signal substance such as a hormone or a neurotransmitter and its receptor, nucleic acids, etc., but the ligand of this embodiment is not limited to these. The particles for immunoturbidimetry in this embodiment can also be referred to as ligand-sensitized particles having selective or specifically high affinity (affinity) for the target substance.

[0019] A method of chemically reacting the carboxy group possessed by the particles for immunoprecipitation according to this embodiment with the ligand can apply a conventionally known method within the range capable of achieving the object of the present invention. For example, a carbodiimide-mediated reaction and an NHS ester activation reaction are commonly used chemical reactions. However, the method of chemically reacting the carboxy group with the ligand in this embodiment is not limited to these.

[0020] In the ligand-sensitized particles in this embodiment, carboxyl groups remaining without binding of the ligand may be activated esterified to bind a hydrophilic molecule. This is generally referred to as active ester inactivation, or carboxyl group blocking treatment, or masking treatment, etc., and is performed to reduce non-specific adsorption of proteins to the carboxyl group and improve the dispersion stability of the ligand-sensitized particles. In this embodiment, the hydrophilic molecule is preferably polyethylene glycol (PEG) or tris(hydroxymethyl)aminomethane (Tris). Binding PEG to the particles for immunoprecipitation can greatly reduce the adsorption of proteins to the particles, which is particularly preferable. As will be described later in the examples, when performing active ester inactivation using PEG, the molecular weight of PEG is important. If the molecular weight is large, there is a possibility of inhibiting the antigen-antibody reaction. Therefore, the molecular weight of PEG is preferably 350 or more and 5000 or less, and particularly preferably 1000 or more and 2000 or less. As the PEG in this embodiment, those having a functional group reactive with a carboxyl group or an active ester, for example, PEG having an amino group, are preferable, and PEG having a primary amine is particularly preferable. PEG may be a linear polymer or a branched polymer. Tris is represented by the following formula (1), and an example of PEG is represented by the following formulas (2) and (3). Note that n in the following formulas (2) and (3) is an integer of 1 or more and 50 or less indicating the number of oxyethylene units.

[0021] [Chemical formula]

[0022] CH3O-(CH2CH2O) n -CH2CH2NH2 Formula (2) CH3O-(CH2CH2O) n -CH2CH2CH2NH2 Formula (3) That is, the particles for immunoturbidimetry according to this embodiment can have a structure in which the above functional group is a carboxyl group, and a compound represented by any of the structural formulas of the esterified carboxyl group and the above formulas (1), (2), and (3) is bound.

[0023] The amount of ligand bound is also an important factor; if the amount of ligand bound (which can also be called the immobilized amount) is too low, the reactivity of the antigen-antibody will decrease, which is undesirable. Conversely, if the amount of ligand bound is too high, it will worsen the dispersibility of the ligand-sensitized particles. Although it depends on the particle size, if the average particle size is around 200 nm, it is preferable to react 1 μg to 500 μg of ligand with 1 mg of diamond particles, and particularly preferable to react 10 μg to 200 μg of ligand.

[0024] The particles for immunoaggregation in this embodiment use antibodies or antigens as ligands and are preferably applicable to immunoturbidimetric methods (latex immunoaggregation), which are widely used in fields such as clinical testing and biochemical research. When general particles are applied to latex immunoaggregation, target substances such as antigens (antibodies) or foreign substances in serum are nonspecifically adsorbed to the particle surface, which can lead to the detection of unintended particle agglutination and hinder accurate measurement. Therefore, to reduce deceptive noise, biological substances such as albumin are usually coated onto the particles as a blocking agent to reduce nonspecific adsorption to the particle surface. For example, bovine serum albumin (BSA) may be present (coated) on the surface of the particles for immunoturbidimetric methods in this embodiment.

[0025] (Test reagents) The test reagent according to this embodiment comprises particles for immunoturbidimetry (ligand-sensitized particles) according to this embodiment and a dispersion medium for dispersing the particles for immunoturbidimetry. The amount of ligand-sensitized particles contained in the test reagent in this embodiment is preferably 0.001% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass. The test reagent according to this embodiment may further contain third substances such as solvents and blocking agents in addition to the ligand-sensitized particles according to this embodiment, to the extent that the objective of the present invention can be achieved. Two or more types of third substances such as solvents and blocking agents may be included in combination. Examples of solvents used in the present invention include at least one of phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvent contained in the test reagent according to this embodiment is not limited to these.

[0026] (Test kit) The in vitro diagnostic test kit in this embodiment comprises a test reagent according to this embodiment and a housing that encloses the test reagent. Preferably, the test kit according to this embodiment further comprises a reaction buffer containing albumin (hereinafter referred to as Reagent 2) in addition to the test reagent according to this embodiment (hereinafter referred to as Reagent 1). Examples of the albumin include serum albumin, which may be protease-treated. The amount of albumin contained in Reagent 2 is approximately 0.001% to 5% by mass, but the test kit in this embodiment is not limited to this. Reagent 1 and Reagent 2, or either one, may contain a sensitizer for latex immunoaggregation. Examples of sensitizers for latex immunoaggregation include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid. Reagent 1 and Reagent 2, or either one, may contain a surfactant. Since surfactants have the effect of stabilizing particles and proteins, for example, polyoxyethylene sorbitan monolaurate and poly(oxyethylene) octylphenyl ether are preferably used. Furthermore, the test kit in this embodiment may include, in addition to reagent 1 and reagent 2, a positive control, a negative control, a serum diluent, etc. As the medium for the positive control and negative control, in addition to serum and physiological saline that do not contain the target substance that can be measured, a solvent may be used.

[0027] (Detection method) The latex immunoaggregation method for detecting target substances in a sample according to this embodiment is characterized by mixing ligand-sensitized particles for latex immunoaggregation according to this embodiment with a sample that may contain a target substance. Furthermore, the mixing of the ligand-sensitized particles for latex immunoaggregation according to this embodiment with the sample is preferably performed in the pH range of 3.0 to 11.0. The mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 10 seconds to 30 minutes. In the detection method of this embodiment, the concentration of the ligand-sensitized particles for latex immunoaggregation according to this embodiment is preferably 0.001% to 5% by mass, more preferably 0.01% to 1% by mass, in the reaction system. The detection method of this embodiment optically detects the agglutination reaction that occurs as a result of mixing the ligand-sensitized particles for latex immunoaggregation according to this embodiment with the sample. Specifically, by optically detecting the agglutination reaction, the target substance in the sample can be detected, and the concentration of the target substance can also be measured. To optically detect agglutination reactions, one can use optical instruments capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc., and measure the changes in these values. [Examples]

[0028] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0029] (Example 1) Diamond powder MD200 (manufactured by Tomei Diamond) was dissolved in 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer pH 7.9 to obtain a dispersion of 10 mg / mL diamond particles. Next, anti-ferritin antibody (manufactured by Mikli Immunotherapy Co., Ltd.) was dissolved in 10 mM HEPES pH 7.9 to a concentration of 5.6 mg / mL. 200 μL of each solution was mixed and shaken overnight at 4°C. 200 μL of the shaken mixture was dispensed into two aliquots. 120 μL of 10 mg / mL BSA solution (manufactured by Sigma-Aldrich Japan, dissolved in 10 mM HEPES pH 7.9) was added to one of the aliquots, and the mixture was shaken overnight at 4°C. After that, the unadded solution and the BSA-added solution were centrifuged to collect ligand-sensitized particles (particles for immunoturbidimetric analysis), and the supernatant was discarded. The recovered ligand-sensitized particles were redispersed in 10 mM HEPES pH 7.9 and then centrifuged again. The recovery of ligand-sensitized particles by centrifugation and redispersion in 10 mM HEPES pH 7.9 was repeated twice.

[0030] The resulting particle suspensions were referred to as MD200-Ab (particles without BSA) and MD200-Ab-BSA (particles with BSA). DLS (Digital Light Saturation) was used to evaluate the average particle size of the obtained immunoturbidimetric particles, which were found to be 234 nm and 387 nm, respectively.

[0031] Next, the obtained MD200-Ab was diluted to 0.01%, and its absorbance was measured. A UV-Vis spectrophotometer (Biospectrometer Kinetic, Eppendorf) was used to measure the absorbance; the sample was injected into a plastic cell and measured with a path length of 10 mm. For comparison, synthetic polystyrene particles (particle size approximately 300 nm) were similarly diluted to 0.01%, and their absorbance was also measured. The results showed that the absorbance of MD200-Ab was 1.5, while the absorbance of the synthetic polystyrene particles was 0.5. This indicates that the particles used for immunoturbidimetric analysis in this example have a higher scattering efficiency compared to synthetic polystyrene particles. In other words, highly sensitive detection of target biomolecules can be expected in immunoturbidimetric analysis.

[0032] Next, protein quantification was used to confirm that the anti-ferritin antibody had sensitized (immobilized) the diamond particles. Specifically, this involved reacting ligand-sensitized particles (particles for immunoturbidimetry) with the BCA reagent. First, 25 μL (particle volume 25 μg) of the ligand-sensitized particle dispersion (0.1% solution) was taken. 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (Wako Pure Chemical Industries) were mixed to make solution AB. 200 μL of solution AB was added to the particle solution (25 μL) and incubated at 60°C for 30 minutes.

[0033] The solution was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and 200 μL of the supernatant was collected by pipette. Absorbance at 562 nm was measured using a multimode microplate reader (SynergyMX, BioTek) along with standard samples (several points of antibody in the range of 0 to 200 μg / mL using 10 mM HEPES). The amount of antibody was calculated from the standard curve. The amount of antibody sensitization to the particles (amount of antibody bound per particle weight (antibody immobilization amount) (μg / mg)) was determined by dividing the calculated amount of antibody by the particle weight (0.025 mg in this case). As a result, the amount of antibody sensitization for MD200-Ab was 84 μg / mg, confirming that the anti-ferritin antibody sensitized the particles.

[0034] The sensitivity of ligand-sensitized particles (particles for immunoturbidimetric analysis) was evaluated using latex immunoaggregation (immunoturbidimetric analysis). Specifically, antibody-sensitized particles were reacted with an antigen to form aggregates of immune complexes, and these aggregates were irradiated with light. The attenuation of the irradiated light due to scattering (absorbance) was measured using a spectrophotometer. The proportion of aggregates and the absorbance increased depending on the amount of antigen contained in the sample. In evaluating sensitivity, it is desirable that the increase in absorbance at a predetermined antigen concentration (expressed as ΔODx10000) be large. The aforementioned UV-Vis spectrophotometer was used to measure absorbance, and the sample was injected into a plastic cell and measured with a path length of 10 mm. The measurement method is described below in detail.

[0035] Specifically, 15 μL of ferritin solution (ferritin concentration 0 ng / mL or 1000 ng / mL) and 15 μL of 10 mM HEPES pH 7.9 were mixed in a plastic cell and heated at 37°C for 5 minutes (hereinafter referred to as the sample). 30 μL of a dispersion solution of ligand-sensitized particles (MD200-Ab, MD200-Ab-BSA) (particle concentration 0.01 wt%, 10 mM HEPES, pH 7.9) was added to the above sample and quickly pipetted while taking care not to introduce air bubbles to obtain the sample-particle mixture. The absorbance of the sample-particle mixture at 572 nm was read and designated as Abs1. After heating the sample-particle mixture at 37°C for 5 minutes, the absorbance at 572 nm was read and designated as Abs2. The value obtained by subtracting Abs1 from Abs2 was multiplied by 10,000 to obtain the ΔODx10000 value.

[0036] The results are shown in Figure 1. In this example, the ligand-sensitized particles (particles for immunoturbidimetry) showed an increase in ΔODx 10000 in the presence of ferritin. This is a result of the ligand-sensitized particles binding to the antigen, ferritin, and forming particle aggregates, indicating that they function as particles for use in latex immunoaggregation (immunoturbidimetry).

[0037] This embodiment includes the following configurations and methods.

[0038] (Composition 1) Diamond particles and Ligands that specifically bind to biomolecules, Particles for immunoaggregation having, The ligand is bound to the diamond particles, Particles for immunoturbidimetry having a particle size of 50 nm or more and 1000 nm or less.

[0039] (Configuration 2) The particle for immunoturbidimetry according to configuration 1, wherein the ligand and the diamond particle are bound together by at least one of covalent bonds, ionic bonds, hydrogen bonds, and hydrophobic interactions.

[0040] (Composition 3) The diamond particles are particles for immunoturbidimetry according to configuration 1 or 2, wherein when the diamond particles are excited with light of a wavelength of 400 nm to 1000 nm, they do not emit fluorescence of a wavelength of 400 nm to 1000 nm.

[0041] (Composition 4) The diamond particles are modified with a compound having at least one of the functional groups of a carboxyl group, a hydroxyl group, and an amino group, and the ligand and the diamond particles are bound by a reaction between the ligand and the functional group, as described in any one of the particles for immunoturbidimetry according to configuration 1 to 3.

[0042] (Composition 5) The particle for immunoturbidimetry according to configuration 4, wherein the functional group is a carboxyl group, and the esterified carboxyl group is bonded to a compound represented by any of the following structural formulas (1), (2), and (3).

[0043] [ka]

[0044] CH3O-(CH2CH2O) n -CH2CH2NH2 formula (2) CH3O-(CH2CH2O) n -CH2CH2CH2NH2 formula (3) In equations (2) and (3) above, n is an integer between 1 and 50.

[0045] (Composition 6) The immunoturbidimetric particle according to any one of the configurations 1 to 5, wherein the particle size of the immunoturbidimetric particle is 100 nm or more and 500 nm or less.

[0046] (Composition 7) Particles for immunoturbidimetry according to any one of claims 1 to 6, wherein the ligand is one of an antibody, an antigen, a fragmented antibody, and a VHH (Variable domain of heavy chain of heavy chain antibody) antibody.

[0047] (Composition 8) Particles for immunoturbidimetry according to any one of constructs 1 to 7, wherein the ligand is an anti-ferritin antibody.

[0048] (Composition 9) The immunoturbidimetric particle according to any one of claims 1 to 8, wherein bovine serum albumin is present on the surface of the immunoturbidimetric particle.

[0049] (Composition 10) An in vitro diagnostic reagent comprising particles for immunoturbidimetry as described in any one of items 1 to 9, and a dispersion medium for dispersing the particles for immunoturbidimetry.

[0050] (Composition 11) The in vitro diagnostic reagent according to configuration 10, wherein the amount of the particles for immunoturbidimetry contained in the test reagent is 0.001% by mass or more and 20% by mass or less.

[0051] (Composition 12) The test reagent further comprises at least one of phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, according to configuration 10 or 11.

[0052] (Composition 13) An in vitro diagnostic test kit comprising a test reagent described in any one of items 10 to 12 and a housing containing the test reagent.

Claims

1. Diamond particles and Ligands that specifically bind to biomolecules, Particles for immunoaggregation having, The ligand is bound to the diamond particles, Particles for immunoturbidimetry having a particle size of 50 nm or more and 1000 nm or less.

2. The particle for immunoturbidimetry according to claim 1, wherein the ligand and the diamond particle are bound together by at least one of covalent bonds, ionic bonds, hydrogen bonds, and hydrophobic interactions.

3. The diamond particles, as described in claim 1, do not emit fluorescence at wavelengths of 400 nm to 1000 nm when excited with light of a wavelength of 400 nm to 1000 nm.

4. The particles for immunoturbidimetry according to claim 1, wherein the diamond particles are modified with a compound having at least one of a carboxyl group, a hydroxyl group, and an amino group, and the ligand and the diamond particles are bound by a reaction between the ligand and the functional group.

5. The particle for immunoturbidimetry according to claim 4, wherein the functional group is a carboxyl group, and the esterified carboxyl group is bonded to a compound represented by any of the following structural formulas (1), (2), and (3). 【Chemistry 1】 CH ３ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ NH ２ Formula (2) CH ３ O-(CH ２ CH ２ O) ｎ -CH ２ CH ２ CH ２ NH ２ Formula (3) In equations (2) and (3) above, n is an integer between 1 and 50.

6. The particle for immunoturbidimetry according to claim 1, wherein the particle size of the particle for immunoturbidimetry is 100 nm or more and 500 nm or less.

7. The particle for immunoturbidimetry according to claim 1, wherein the ligand is one of an antibody, an antigen, a fragmented antibody, and a VHH (Variable domain of Heavy chain of Heavy chain antibody) antibody.

8. The particle for immunoturbidimetry according to claim 1, wherein the ligand is an anti-ferritin antibody.

9. The immunoturbidimetric particle according to claim 1, wherein bovine serum albumin is present on the surface of the aforementioned immunoturbidimetric particle.

10. An in vitro diagnostic reagent comprising particles for immunoturbidimetry as described in claim 1, and a dispersion medium for dispersing the particles for immunoturbidimetry.

11. The in vitro diagnostic reagent according to claim 10, wherein the amount of the particles for immunoturbidimetry contained in the test reagent is 0.001% by mass or more and 20% by mass or less.

12. The in vitro diagnostic reagent according to claim 10, further comprising at least one of phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer.

13. An in vitro diagnostic test kit comprising a test reagent according to claim 10 and a housing containing the test reagent.

Images