JP2024044817A - Method of suppressing precipitation of specific binding substance-immobilized carrier particles - Google Patents

Abstract

To provide a method of suppressing precipitation of carrier particles having a substance that specifically binds to a measurement target substance immobilized thereon when the carrier particles are stored in a buffer solution.SOLUTION: A carrier particle precipitation suppression method of the present invention involves storing carrier particles having a substance that specifically binds to a measurement target substance immobilized thereon in a buffer solution containing at least one substance selected from the group consisting of Good's buffers and alkali metal halides.SELECTED DRAWING: None

Description

The present invention relates to a method for suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized, which is used when measuring a substance to be measured.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

There are various known measurement reagents and methods for measuring a substance contained in a sample that utilize reactions between substances with specific affinity, such as antigens and antibodies, sugars and lectins, nucleotide chains and complementary nucleotide chains, and ligands and receptors.

This is done by measuring the presence or absence of binding, or the amount of binding, between the analyte contained in the sample and a substance that specifically binds to the analyte (substance that specifically binds to the analyte). , to measure the presence or absence of a substance to be measured contained in a sample (qualitative measurement), or to measure its content (concentration) (quantitative measurement).

In particular, a method or measurement reagent that uses a carrier on which a substance that specifically binds to the substance to be measured is immobilized is frequently used.

For example, in immunological measurement reagents (immunological measurement methods) that utilize the antigen-antibody reaction (immune reaction, immunological reaction) between an antigen and an antibody, latex immunoturbidimetry using latex particles as a carrier is used. ; Indirect agglutination measurement method using latex particles, organic polymer particles, inorganic particles, metal particles, or red blood cells as carriers; Enzyme immunoassay using microtiter plates, beads, particles, test tubes, containers, etc. as carriers Measurement methods, labeled immunoassay methods such as luminescence immunoassay; or immunochromatography methods using cellulose, nonwoven fabric, etc. as carriers are frequently used.

Among these, the latex immunoturbidimetric method, which uses latex particles as a carrier, is widely used in clinical laboratories of medical institutions such as hospitals and clinics because it is easy to operate and has a short measurement time.

However, in this latex immunoturbidimetric method, the latex particles used as a carrier tend to sediment during storage, so if the latex particles settle, it is necessary to make the latex particles uniform by inverting and mixing during measurement. There are problems in that it is necessary, the operation is complicated, and errors are likely to occur in the measurement of the substance to be measured in the sample.

In order to suppress the settling of latex particles and the like, a method has been proposed in which at least one compound selected from the group consisting of polyanions and their salts, dextran, cyclodextrin, polyethylene glycol, and glycerol is allowed to coexist in a dispersion of latex particles and the like (see, for example, Patent Document 1). However, this method is not necessarily complete.

Japanese Patent Application Publication No. 2007-114129

Therefore, an object of the present invention is to provide a method that can suppress sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is stored in a buffer solution.

As a result of intensive studies aimed at solving the above problems, the present inventor discovered that when storing carrier particles on which a substance that specifically binds to a substance to be measured is stored in a buffer solution, Good buffer is added to the buffer solution. The present inventors have discovered that sedimentation of carrier particles can be suppressed by containing at least one member selected from the group consisting of agents and alkali metal halides, and have completed the present invention.

That is, the present invention provides the following inventions.
(1) A method for inhibiting the sedimentation of carrier particles, characterized in that carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured are stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.
(2) The method for inhibiting precipitation of carrier particles according to (1) above, wherein the substance that specifically binds to the substance to be measured is an anti-HMGB1 antibody.
(3) The method for inhibiting precipitation of carrier particles according to (1) or (2) above, wherein the carrier particles are latex particles.
(4) The method for inhibiting precipitation of carrier particles according to (1) or (2) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.
(5) The method for inhibiting precipitation of carrier particles according to (3) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.
(6) A method for measuring HMGB1 in a sample, characterized by using carrier particles having anti-HMGB1 antibodies immobilized thereon, the carrier particles being stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.
(7) The method for measuring HMGB1 in a sample according to (6) above, wherein the carrier particles are latex particles.
(8) The method for measuring HMGB1 in a sample according to (6) or (7) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.

The present invention is capable of suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized.
As a result, the carrier particles can be stored for a long period of time without sedimentation, so that, for example, by using the carrier particles whose sedimentation has been suppressed according to the present invention in measurements such as latex turbidimetry, errors can be eliminated. It is possible to perform measurements that provide stable performance.

The present invention will be described in detail below, but the following embodiments are illustrative for explaining the present invention, and the present invention is not limited to these embodiments. Moreover, the present invention can be implemented in various forms without departing from the gist thereof.

1. Method for inhibiting sedimentation of carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured The method for inhibiting sedimentation of carrier particles of the present invention comprises storing carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.

(1) Substance that specifically binds to the substance to be measured In the present invention, the substance that specifically binds to the substance to be measured is not particularly limited as long as it is a substance that can specifically bind to the substance to be measured. .

Examples of the substance that specifically binds to the analyte are antibodies that can bind to the analyte (anti-analyte antibody), aptamers (nucleic acid aptamers or peptide aptamers), affibodies, sugars or lectins, Examples include nucleotide chains and receptors.

In addition, examples of antibodies capable of binding to the analyte (anti-analyte antibody) include monoclonal antibodies, polyclonal antibodies, antiserum, antibody fragments [Fab and F( ab')2], single chain antibodies (scFv), and the like.

Furthermore, in the present invention, it is particularly preferable that the substance that specifically binds to the substance to be measured is an anti-HMGB1 antibody.

(2) Carrier Particles In the present invention, the carrier particles are not particularly limited as long as they are capable of immobilizing a substance that specifically binds to the above-mentioned target substance to be measured.

The material of the carrier particles is not particularly limited, and examples include polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid. Copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid polymer, gelatin, silica, alumina, Examples include carbon black, metal compounds, metals, ceramics, and magnetic materials.

Examples of the carrier particles include latex particles, metal colloid particles, liposomes, microcapsules, and particles such as red blood cells.
Further, in the present invention, the carrier particles are preferably latex particles.

In the present invention, a substance that specifically binds to a substance to be measured can be immobilized on carrier particles by a known method such as a physical adsorption method, a chemical bonding method, or a combination thereof.
When using the physical adsorption method, a substance that specifically binds to the substance to be measured and carrier particles are mixed in a solution such as a buffer solution and brought into contact with each other, or dissolved in a buffer solution or the like, according to a known method. This can be carried out by, for example, bringing a substance that specifically binds to the substance to be measured into contact with carrier particles.

In addition, when using a chemical binding method, it can be performed according to known methods described in "Clinical Pathology Special Issue No. 53: Immunoassays for Clinical Tests - Techniques and Applications" edited by the Japanese Society of Clinical Pathology, published by Clinical Pathology Publishing Society, 1983; "New Biochemical Experiment Course 1: Protein IV" edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1991, etc., by mixing and contacting a substance that specifically binds to the substance to be measured and carrier particles with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and reacting the amino groups, carboxyl groups, thiol groups, aldehyde groups, hydroxyl groups, etc. of the substance that specifically binds to the substance to be measured and the carrier particles with the bivalent cross-linking reagent.

Furthermore, if it is necessary to perform treatment to suppress non-specific reactions of carrier particles on which a substance that specifically binds to the substance to be measured is immobilized, the substance that specifically binds to the substance to be measured is immobilized. The surface of the prepared carrier particles is treated by a known method such as contacting and coating a protein such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or a salt thereof, a surfactant, or skim milk powder, etc., The carrier particles may be subjected to blocking treatment (masking treatment).

(3) Good's buffer and alkali metal halide In the present invention, the Good's buffer is particularly limited as long as it has the effect of suppressing sedimentation of carrier particles when the carrier particles are stored in a buffer solution. However, for example, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2- Aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)methane (Bis-Tris), 1 , 3-Bis-Trishydroxymethylaminopropane (Bis-Trispropane), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-hydroxy-3-aminoethanesulfonic acid ( CAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 2-[4-(2- Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]-propanesulfonic acid monohydrate (HEPPSO), 2-morpholinoethane Sulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine- 1,4-bis(2-hydroxy-3-propanesulfonic acid) dehydrate (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl- 2-hydroxy-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), or N-[tris(hydroxymethyl)methyl]glycine (Tricine), or These salts can be mentioned.

In the present invention, the alkali metal halide is not particularly limited as long as it has the effect of suppressing the precipitation of the carrier particles when the carrier particles are stored in a buffer solution, but it is particularly preferable to use sodium chloride, potassium chloride, or sodium bromide.

The Good's buffer and alkali metal halide may be used alone or in combination.

In addition, the concentration of Good's buffer and alkali metal halide to be contained in the buffer solution is particularly limited as long as it has the effect of suppressing sedimentation of the carrier particles when the carrier particles are stored in the buffer solution. It is set appropriately depending on the type.

For example, when using a Good buffer, it is sufficient if it is 0.25 mol/L or more, preferably in the range of 0.25 to 1 mol/L, and preferably in the range of 0.5 to 1 mol/L. More preferably, the range is from 0.75 to 1 mol/L.
In addition, when using an alkali metal halide, it is sufficient if it is 0.5 mol/L or more, preferably in the range of 0.5 to 3 mol/L, and preferably in the range of 0.75 to 2 mol/L. is more preferable, and a range of 1 to 2 mol/L is particularly preferable.

In the present invention, the buffer solution for storing the carrier particles is not particularly limited, but may be, for example, water, physiological saline, or an aqueous solvent such as various buffer solutions such as tris(hydroxymethyl)aminomethane buffer solution [Tris buffer solution], phosphate buffer solution, or phosphate buffered physiological saline solution.

2. Method for measuring HMGB1 in a sample The method for measuring HMGB1 in a sample of the present invention involves contacting "HMGB1 in a sample" with "carrier particles having immobilized anti-HMGB1 antibodies" and measuring the aggregates of "carrier particles having immobilized anti-HMGB1 antibodies" bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample.

That is, "HMGB1 in the sample" and "carrier particles on which anti-HMGB1 antibody is immobilized" are brought into contact, and an immune complex aggregate of "carrier particles on which anti-HMGB1 antibody is immobilized" bound via HMGB1 is generated. The transmitted light and scattered light are measured by an optical method or visually. In other words, it measures the formation of aggregates of "complexes resulting from antigen-antibody reactions" (agglutination reaction method).

The present invention is characterized in that "HMGB1 in a sample" is contacted with "carrier particles having anti-HMGB1 antibodies immobilized thereon, the carrier particles being stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide."

Examples of methods for measuring aggregates of carrier particles on which anti-HMGB1 antibodies are immobilized include latex turbidimetry, latex agglutination method, and particle agglutination method.

Measurement of aggregates of carrier particles on which anti-HMGB1 antibodies are immobilized can be carried out using latex turbidimetry, latex agglutination reaction method, particle agglutination reaction method, etc., and the generation of immune complex aggregates can be performed by optically measuring the transmitted light and scattered light. When the measurement is carried out by a measuring method or a visual measuring method, a phosphate buffer, a glycine buffer, a Tris buffer, a Good's buffer, or the like can be used as a solvent.

In addition, when using latex turbidimetry as the measurement principle, there is no particular restriction on the particle size of the latex particles used as a carrier, but the latex particles may bond through the substance to be measured (HMGB1) to form aggregates. For reasons such as ease of measurement of the produced aggregates and the like, the average particle size of the latex particles is preferably 0.04 to 1 μm.

In addition, when using latex turbidimetry as the measurement principle, the concentration of latex particles immobilized with anti-HMGB1 antibody is determined by the concentration of HMGB1 in the sample, the distribution density of the antibody on the latex particle surface of the present invention, The optimal concentration cannot be generalized because it varies depending on various conditions such as the particle size of the latex particles and the mixing ratio of the sample and the measurement reagent.

However, typically, during the measurement reaction in which the sample and the measurement reagent are mixed and an antigen-antibody reaction takes place between the "anti-HMGB1 antibody" immobilized on the latex particles and the "HMGB1" contained in the sample, the concentration of the "latex particles with immobilized anti-HMGB1 antibody" is adjusted to 0.005 to 1% (w/v) when the "HMGB1 in the sample" comes into contact with the "latex particles with immobilized anti-HMGB1 antibody." In this case, the measurement reagent for HMGB1 in the sample contains a concentration of "latex particles with immobilized anti-HMGB1 antibody" that will result in such a concentration when the "HMGB1 in the sample" comes into contact with the "latex particles with immobilized anti-HMGB1 antibody."

When the measurement principle is an indirect agglutination method such as a latex agglutination method or a particle agglutination method, there are no particular restrictions on the particle size of the particles used as the carrier, but the average particle size is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.3 to 10 μm. The specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.

In addition, when the measuring principle is indirect aggregation reaction method such as latex agglutination reaction method or particle agglutination reaction method, the container used for the measurement is, for example, a test tube made of glass, polystyrene, polyvinyl chloride, or polymethacrylate. , a microplate (microtiter plate), or a tray. The bottom surface of the solution-accommodating portion of these containers (wells of microplates, etc.) is preferably U-shaped, V-shaped, or UV-shaped, with a slope from the center of the bottom surface to the periphery.

The measurement can be performed by known methods, etc., but for example, when measuring by an optical method, the sample is reacted with "carrier particles having immobilized anti-HMGB1 antibodies" and the transmitted light or scattered light is measured by an endpoint method or a rate method.
In addition, in the case of visual measurement, the sample is reacted with "anti-HMGB1 antibody-immobilized carrier particles" in the above-mentioned container such as a plate or microplate, and the state of agglutination is visually determined. Note that instead of this visual measurement, the measurement may be performed using an instrument such as a microplate reader.

In addition, samples in the method for measuring HMGB1 in a sample of the present invention include body fluids such as blood, serum, plasma, urine, spinal fluid, saliva, sweat, tears, ascites or amniotic fluid; stool; organs such as blood vessels or liver; Any biological sample that may contain HMGB1, such as tissue; cells; or extracts of stool, organs, tissues, or cells, is eligible.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of anti-HMGB1 antibody (2H6)
As the anti-HMGB1 antibody (2H6), a monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02843 was used.
The hybridoma identified by the accession number NITE P-02843 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.

[Example 2] (Preparation of anti-HMGB1 antibody (2D4))
As the anti-HMGB1 antibody (2D4), a monoclonal antibody produced by a hybridoma deposited under accession number NITE P-02842 was used.
The hybridoma identified by the accession number NITE P-02842 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.

[Example 3] (Preparation of anti-HMGB1,2 monoclonal antibodies)
As the monoclonal antibody that binds to HMGB1 and HMGB2 (anti-HMGB1,2 monoclonal antibody), a monoclonal antibody produced by a hybridoma deposited under accession number NITE P-02844 was used.
The hybridoma identified by the accession number NITE P-02844 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.

[Example 4] (Confirmation of the effect of Good buffer or alkali metal halide)
The effect of suppressing sedimentation of carrier particles when Good's buffer or an alkali metal halide was contained in the buffer was confirmed.

[1] Reagents for measuring HMGB1 in a sample 1. First reagent A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.

2. Second Reagent (1) Equal amounts of the antibody (2H6) prepared in Example 1 and the antibody (2D4) prepared in Example 2 were mixed to prepare a mixture of anti-HMGB1 monoclonal antibodies. This was named "anti-HMGB1 monoclonal antibody mixture."

(2) Dilute the anti-HMGB1 monoclonal antibody mixture from (1) above with a buffer solution to 1 to 2 mg/mL, and mix 1 mL of this antibody solution with 1 mL of latex particles (Fujikura Kasei Co., Ltd. [Japan]). The anti-HMGB1 monoclonal antibody mixture was immobilized on the latex particles by contacting and standing at 2 to 8°C for 16 hours.

(3) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which the anti-HMGB1 monoclonal antibody mixture was immobilized, and a blocking treatment was performed to obtain latex particles onto which the blocked anti-HMGB1 monoclonal antibody mixture was immobilized.

(4) Dilute the anti-HMGB1,2 monoclonal antibody prepared in Example 3 above with a buffer solution to a concentration of 1 to 2 mg/mL, and add 1 mL of this antibody solution and 1 mL of latex particles (Fujikura Kasei Co., Ltd. [Japan]). The anti-HMGB1,2 monoclonal antibody was immobilized on the latex particles by mixing and contacting and standing at 2 to 8°C for 16 hours.

(5) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles with anti-HMGB1,2 monoclonal antibodies to perform a blocking treatment, thereby obtaining latex particles with anti-HMGB1,2 monoclonal antibodies that had been subjected to a blocking treatment.

(6) Equal amounts of latex particles immobilized with the anti-HMGB1 monoclonal antibody mixture prepared in (3) above and latex particles immobilized with the anti-HMGB1,2 monoclonal antibodies prepared in (4) above were mixed to prepare a mixture of latex particles immobilized with anti-HMGB1 monoclonal antibodies. This was named "anti-HMGB1 monoclonal antibody immobilized latex particle mixture."

(7) The Good's buffer or alkali metal halide shown in Table 1 was added to the "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture" in (6) above to the concentrations shown in Table 1, to prepare 10 types of second reagents each containing (or not containing) a Good's buffer or an alkali metal halide.

[2] Sample Physiological saline (0.9% sodium chloride aqueous solution) was used as the sample.

[3] Storage of second reagents 5 mL of the 10 types of second reagents prepared in 2 of [1] above were dispensed into test tubes and stored under refrigeration (2 to 8°C) for 13 months.

[4] Measurement of sample The sample in [2] was measured using the first reagent in [1] above as the first reagent for measuring HMGB1 in the sample, and the second reagent in [3] above as the second reagent.

The measurements were carried out using a Hitachi 7180 model automatic analyzer (Hitachi High-Tech Corporation, Japan).
The first reagent in item 1 of item 1 of item 1 of item 2 ...
Specifically, the measurement was performed using 24 μL of the sample described in [2] above, 120 μL of the first reagent described in [1] 1 above as the first reagent, and 40 μL of the second reagent described in [3] above as the second reagent.

The reaction temperature was 37°C, and the measurement wavelength was 800 nm. The absorbance was measured at the 16th and 17th points (immediately after the addition of the second reagent) of the measurement time course, and the difference (absorbance difference) was calculated and used as the latex concentration. The absorbance difference (latex concentration) was calculated for the cases where the second reagent in [3] above was not mixed by inversion (no inversion mixing) and where it was mixed by inversion immediately before measurement (mixing by inversion).

[5] Measurement results The measurement results of the samples are shown in Table 1. The values shown in Table 1 indicate the average value of the absorbance difference (latex concentration) when the sample is measured five times. The sedimentation inhibition rate shown in Table 1 indicates the value (unit: %) obtained by dividing the absorbance difference (unit: Abs.) without inversion mixing by the absorbance difference (unit: Abs.) with inversion mixing.

As is clear from Table 1, when the second reagent does not contain a Good's buffer or an alkali metal halide (no addition), the absorbance without mixing by inversion is extremely low and the sedimentation inhibition rate is also 5%. This is presumably due to the fact that the antibody-immobilized latex particles in the second reagent settle during storage, reducing the amount of antibody-immobilized latex particles added and mixed into the first reagent, thereby lowering the absorbance.
In contrast, when the second reagent contained Good's buffer or an alkali metal halide, in either case, there was almost no change in the absorbance difference between the cases with and without inversion mixing, compared to the case where no additive was added, indicating that the sedimentation inhibition rate was improved.

In this way, it was confirmed that when storing antibody-immobilized latex particles in a buffer solution, the sedimentation of the antibody-immobilized latex particles can be suppressed for a long period of time by adding a Good's buffer or an alkali metal halide to the buffer solution. Therefore, it was confirmed that it is not necessary to homogenize the latex particles by inversion mixing or the like during measurement, and that measurements can be performed that are free of errors and provide stable performance.

Example 5 (Confirmation of the effect of Good's buffer or alkali metal halide)
The effect of suppressing the sedimentation of carrier particles when a Good's buffer or an alkali metal halide was contained in a buffer solution was confirmed.

[1] Reagent for measuring HMGB1 in sample 1. First Reagent A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.

2. Second Reagent The operations were performed as described in (1) to (6) of [1] of Example 4 to prepare a mixture of latex particles on which anti-HMGB1 monoclonal antibody was immobilized. This was named "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture."
Good's buffer or alkali metal halide listed in Table 2 was added to this "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture" to the concentrations listed in Table 2, and Good's buffer or alkali metal halide was Twenty-three types of second reagents each containing (or not containing) a compound were prepared.

[2] Sample Physiological saline (0.9% sodium chloride aqueous solution) was used as a sample.

[3] Storage of the second reagent The 23 types of second reagent prepared in [1] 2 above were dispensed into test tubes in 5 mL portions and stored in a refrigerator (2 to 8° C.) for one month.

[4] Measurement of sample The first reagent of 1 above [1] is used as the first reagent for measuring HMGB1 in the sample, and the second reagent of [3] above is used as the second reagent. The sample [2] was measured in the same manner as in Example 4 [4].

[5] Measurement results The measurement results of the samples are shown in Table 2. The values shown in Table 2 indicate the average value of the absorbance difference (latex concentration) when the sample is measured five times. The sedimentation inhibition rate shown in Table 2 indicates the value (unit: %) obtained by dividing the absorbance difference (unit: Abs.) without inversion mixing by the absorbance difference (unit: Abs.) with inversion mixing.

As is clear from Table 2, when the second reagent does not contain Good's buffer or alkali metal halide (no addition), the absorbance is extremely low without mixing by overturning, and the sedimentation inhibition rate is 18%. It has become. This is because the antibody-immobilized latex particles in the reagent precipitated during storage of the second reagent, reducing the amount of antibody-immobilized latex particles added to and mixed with the first reagent, resulting in lower absorbance. It is assumed that this is due to this.
On the other hand, when the second reagent contains Good's buffer or alkali metal halide, no matter the concentration of Good's buffer or alkali metal halide, mixing by overturning is more difficult than when no Good's buffer or alkali metal halide is added. It can be seen that there is little change in the difference in absorbance between without and with overturning mixing, indicating that the sedimentation suppression rate has been improved.

In this way, when storing antibody-immobilized latex particles in a buffer solution, it is possible to suppress sedimentation of antibody-immobilized latex particles for a long period of time by including Good's buffer or an alkali metal halide in the buffer solution. confirmed. Therefore, it was confirmed that it is not necessary to make the latex particles uniform by inverting and mixing during measurement, and that it is possible to perform measurements that do not include errors and provide stable performance.

From these facts, in the method for suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized according to the present invention, the carrier particles can be stored for a long period of time without sedimentation. It was confirmed that by using carrier particles with suppressed sedimentation in measurements, it is possible to perform measurements that do not include errors and provide stable performance.

Claims (8)

A method for inhibiting the sedimentation of carrier particles, characterized in that carrier particles onto which a substance that specifically binds to a substance to be measured is immobilized are stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide. The method for inhibiting the sedimentation of carrier particles according to claim 1, wherein the substance that specifically binds to the substance to be measured is an anti-HMGB1 antibody. The method for inhibiting the settling of carrier particles according to claim 1 or 2, wherein the carrier particles are latex particles. The method for inhibiting the settling of carrier particles according to claim 1 or 2, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide. The method for inhibiting the settling of carrier particles according to claim 3, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide. HMGB1 in a sample is characterized by using carrier particles on which anti-HMGB1 antibodies are immobilized, which are stored in a buffer containing at least one selected from the group consisting of Good's buffer and an alkali metal halide. Measuring method. The method for measuring HMGB1 in a sample according to claim 6, wherein the carrier particles are latex particles. The method for measuring HMGB1 in a sample according to claim 6 or 7, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.