Immunological assay reagents, immunoassay methods, and methods for storing immunoassay reagents

Abstract

To provide an immunological assay reagent with improved storage stability, an immunological assay method using the same, and a method for storing the immunological assay reagent. [Solution] An immunoassay reagent comprising HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound. An immunoassay method comprising: confirming the performance of a detection reagent containing a substance that interacts with a substance to be measured using an immunoassay reagent comprising HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound; and measuring the substance to be measured using the detection reagent.

Description

Technical Field

[0001] The present invention relates to an immunological measurement reagent, an immunological measurement method, and a method for storing an immunological measurement reagent.

Background Art

[0002] Biochemical measurement methods are known as means for detecting various components contained in biological samples. Among biochemical measurement methods, immunological measurement methods are highly specific measurement methods because they utilize specific binding between proteins such as antigen-antibody reactions.

[0003] Reagents used in immunological measurements may contain components derived from biological samples. For example, standard reagents for confirming the presence or absence of denaturation of detection reagents for immunological measurements or for creating calibration curves may contain components derived from biological samples.

[0004] For example, Patent Document 1 discloses a reagent for immunological measurement containing a component derived from a biological sample.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Among standard reagents, there is a negative control reagent as a reagent for inspecting whether a reagent that does not contain the measurement target substance correctly determines a negative result for a detection reagent. When a negative control reagent is used, signals caused by the measurement target substance are not detected or only low-level signals are detected in an immunological measurement method.

[0007] However, in latex immunoturbidimetry, when using a negative control reagent containing components derived from biological samples, a phenomenon may occur where a signal originating from the target substance is detected.

[0008] The inventors discovered that the reason a signal originating from the target substance is detected even in a negative control reagent is due to the HDL cholesterol contained in the negative control reagent. After diligent investigation, the inventors found that when HDL cholesterol is included in an immunological measurement reagent, the HDL cholesterol may decrease due to breakdown or other factors during storage of the negative control reagent, and this affects the measurement results of the immunological measurement.

[0009] Furthermore, we found that the storage stability of immunoassay reagents, including not only negative control reagents in latex immunoturbidimetry but also immunoassay reagents containing HDL cholesterol, is improved by coexisting a specific surfactant with an inclusion compound.

[0010] The object of the present invention is to provide an immunological assay reagent with improved storage stability, an immunological assay method using the same, and a method for storing the immunological assay reagent. [Means for solving the problem]

[0011] The present invention encompasses the following embodiments. [1] An immunoassay reagent comprising HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound. [2] The immunoassay reagent according to [1], wherein the surfactant is a nonionic surfactant or an anionic surfactant. [3] The immunoassay reagent according to [1] or [2], wherein the number of phenyl groups contained in one molecule of the surfactant is one. [4] The immunoassay reagent according to any one of [1] to [3], wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin. [5] The immunoassay reagent according to any one of [1] to [3], wherein the inclusion compound has 7 to 8 structural units derived from glucose in its molecular structure. [6] An immunoassay reagent as described in any one of [1] to [5], used as a negative control in an immunoassay method. [7] Using an immunoassay reagent containing HDL cholesterol, a surfactant having optionally substituted phenyl groups, and an inclusion compound, the performance of a detection reagent containing a substance that interacts with the substance to be measured is confirmed. An immunoassay method comprising measuring the target substance using the detection reagent. [8] The immunological measurement method according to [7], wherein the substance to be measured is a non-treponemal lipid antibody. [9] The immunological measurement method according to [7] or [8], wherein the immunological measurement method is latex immunoturbidimetry.

[10] The immunoassay method according to any one of [7] to [9], wherein the surfactant is a nonionic surfactant or an anionic surfactant.

[11] The immunoassay method according to any one of [7] to

[10] , wherein the number of phenyl groups contained in one molecule of the surfactant is one.

[12] The immunoassay method according to any one of [7] to

[11] , wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin.

[13] The immunoassay method according to any one of [7] to

[11] , wherein the inclusion compound has 7 to 8 structural units derived from glucose in its molecular structure.

[14] A method for preserving an immunoassay reagent, comprising adding a surfactant having a phenyl group which may have a substituent and an inclusion compound to an immunoassay reagent containing HDL cholesterol.

[15] The method for storing immunoassay reagents according to

[14] , wherein the surfactant is a nonionic surfactant or an anionic surfactant.

[16] A method for storing an immunoassay reagent according to

[14] or

[15] , wherein the number of phenyl groups contained in one molecule of the surfactant is one.

[17] A method for storing an immunoassay reagent according to any one of

[14] to

[16] , wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin.

[18] The method for storing immunoassay reagents according to

[14] -

[16] , wherein the inclusion compound has 7-8 structural units derived from glucose in its molecular structure. [Effects of the Invention]

[0012] According to the above embodiment, it is possible to provide an immunological assay reagent with improved storage stability, an immunological assay method using the same, and a method for storing the immunological assay reagent. [Modes for carrying out the invention]

[0013] In this invention, the terms "react" and "bind" are used synonymously to describe the reactivity between an antibody and an antigen.

[0014] When a numerical range is written as, for example, "1-10" or "1~10," it means the range from 1 to 10, including the lower limit of 1 and the upper limit of 10. Furthermore, the upper and lower limits of a numerical range can be combined in any way. In addition, the numerical ranges for each physical property, composition, and measurement process can be combined in any way.

[0015] <Immunological assay reagents> An immunoassay reagent in one aspect of the present invention comprises HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound.

[0016] HDL cholesterol is a type of lipoprotein in the blood. HDL cholesterol contains apolipoproteins such as apolipoprotein A-I (hereinafter sometimes referred to as apo A-I) and apolipoprotein A-II (hereinafter sometimes referred to as apo A-II). In the present embodiment, the HDL cholesterol is preferably human-derived HDL cholesterol.

[0017] When an immunological measurement reagent contains a blood-derived component, it may contain HDL cholesterol. For example, for the purpose of accurate measurement, standard reagents such as negative control reagents or positive control reagents for confirming the performance of detection reagents may be used. The detection reagent contains a specific affinity substance for the measurement target substance, while the negative control reagent is a reagent that does not contain the measurement target substance, and the positive control reagent is a reagent that contains the measurement target substance. Also, for the purpose of creating a calibration curve for calculating the concentration of the measurement target substance, a standard reagent containing the measurement target substance at a specified concentration is used.

[0018] The blood-derived component may be any one selected from the group consisting of whole blood, plasma, and serum.

[0019] The proportion of HDL cholesterol contained in the immunological measurement reagent is preferably 10 to 17 mg / dL, more preferably 10 to 13 mg / dL based on the total volume of the immunological measurement reagent.

[0020] The surfactant of the present embodiment is a surfactant having a phenyl group which may have a substituent. The surfactant is preferably a nonionic surfactant or an anionic surfactant.

[0021] In this embodiment, it is preferable that the surfactant contains one phenyl group per molecule. Examples of substituted phenyl groups include phenyl groups, phenyl groups having an alkyl group, and phenyl groups having an anionic group. When the phenyl group has an alkyl group, the alkyl group may be linear or branched. Examples of anionic groups include sulfate groups, carboxyl groups, and sulfo groups.

[0022] Examples of surfactants in this embodiment include polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol, sodium dodecylbenzenesulfonate, and sodium polyoxyethylene nonylphenyl ether sulfate, and may be used individually or in combination of two or more of these.

[0023] When the surfactant in this embodiment is a nonionic surfactant, the HLB value is preferably 10 to 15, and more preferably 11 to 14. When the HLB value is 10 to 15, micelles are more likely to form with factors released from HDL cholesterol during storage of the immunoassay reagent if those factors include lipid components. The HLB value can be calculated from the structure of the surfactant, for example, by raising the sum of the molecular weights of the hydrophilic parts to 20 and dividing by the total molecular weight.

[0024] The proportion of surfactant in the immunoassay reagent is preferably 0.05 to 0.2% by mass relative to the total mass of the immunoassay reagent. When the proportion of surfactant in the immunoassay reagent is 0.05 to 0.2% by mass relative to the total mass of the immunoassay reagent, factors released from HDL cholesterol can be captured.

[0025] The ratio of the mass of surfactant to the mass of HDL cholesterol in the immunoassay reagent is preferably 700 to 1000% by mass. If the ratio of the mass of surfactant to the mass of HDL cholesterol is 700% by mass or more, the surfactant can sufficiently capture the factors released from HDL cholesterol. If the ratio of the mass of surfactant to the mass of HDL cholesterol is 1000% by mass or less, the excess surfactant that is not involved in the factors released from HDL cholesterol is sufficiently encapsulated in the inclusion compound.

[0026] In this specification, an inclusion compound is a compound having a tunnel-like structure, a layered structure, or a network structure, which can incorporate other molecules into its internal pores. Examples of inclusion compounds include compounds having 7 to 8 structural units derived from glucose in their molecular structure. For example, cyclodextrins are examples of inclusion compounds, with β-cyclodextrin, in which 7 glucose units are linked in a ring, and γ-cyclodextrin, in which 8 glucose units are linked in a ring, being preferred. Because the inside of the ring shape of a cyclodextrin is hydrophobic and the outside is hydrophilic, it readily inclusions hydrophobic compounds inside.

[0027] The proportion of inclusion compounds in the immunoassay reagent is preferably 0.1 to 1.5% by mass relative to the total mass of the immunoassay reagent. If the proportion of inclusion compounds in the immunoassay reagent is 0.1% by mass or more relative to the total mass of the immunoassay reagent, excess surfactant that is not involved with the factors released from HDL cholesterol can be sufficiently inclusioned. If the proportion of inclusion compounds in the immunoassay reagent is 1.5% by mass or less relative to the total mass of the immunoassay reagent, it will not adversely affect the measurement system, such as causing fluctuations in measurement values.

[0028] The substance to be measured by the immunological assay reagent of this embodiment is not particularly limited, but is not limited to any substance that can be measured by known immunological assay methods. Examples include proteins (antigens, haptens, antibodies, etc.), carbohydrates, lipids, glycoproteins, glycolipids, nucleic acids, and chemical substances (hormones, drugs). In particular, from the viewpoint of suppressing the influence of factors released from HDL cholesterol, the substance to be measured is preferably a component of the blood. The substance to be measured may be an antigen or antibody contained in the blood. Specific examples of substances to be measured include non-treponemal lipid antibodies, soluble interleukin-2 receptor (sIL-2R), CRP, fibrin and fibrinogen degradation products, D-dimer, soluble fibrin (SF), lipoprotein(a) (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, and α2-macroglobulin. Examples include hemopexin, antithrombin-III, alpha-fetoprotein, tumor embryonic antigen (CEA), ferritin, HBs-Ag (hepatitis B outer antigen), Anti-HBs (anti-hepatitis B outer antibody), HBe-Ag (hepatitis B e antigen), Anti-HBe (anti-hepatitis B e antibody), Anti-HBc (anti-hepatitis B core antibody), SARS-CoV-2, human brain natriuretic peptide (BNP), pulmonary surfactant protein D (SP-D), thymic and activation-regulated chemokines (TARC, CCL-17), and procalcitonin.

[0029] The immunological assay method using the immunological assay reagent of this embodiment is not particularly limited and can be applied to various known methods, such as immunoturbidimetric assay (TIA), latex immunoturbidimetric assay (LTIA), immunochromatography (lateral flow type, flow-through type), electrochemiluminescence assay (ECLIA), chemiluminescence assay (CLIA), chemioenzyme immunoassay (CLEIA), and enzyme-linked immunosorbent assay (ELISA). Since interaction with factors released from HDL cholesterol is likely to occur, LTIA is preferable to obtain the storage stability of the immunological assay reagent, which is the effect of this invention. Furthermore, when the substance to be measured is a non-treponemal lipid antibody, it is preferable that the immunological assay method be LTIA.

[0030] The immunoassay reagent of this embodiment contains a surfactant having a phenyl group, which may have substituents, and an inclusion compound, thereby improving the storage stability of the immunoassay reagent. More specifically, during storage of an immunoassay reagent containing HDL cholesterol, the amount of HDL cholesterol may decrease due to breakdown or other reasons. This is thought to cause factors that affect immunoassay to be performed to be released from HDL cholesterol. These effects on immunoassay include the nonspecific binding of specific affinity substances for the target substance contained in the detection reagent to factors released from HDL cholesterol. As a result, phenomena such as the inability to accurately verify the performance of the detection reagent containing specific affinity substances may occur.

[0031] The immunoassay reagent of this embodiment contains a surfactant having a phenyl group, which may have substituents. This surfactant is thought to capture factors released from HDL cholesterol. Furthermore, while surfactants contained in immunoassay reagents may bind nonspecifically to specific affinity substances and cause elevated measurement values, it is thought that excess surfactant is encapsulated by the inclusion compound. Therefore, even if HDL cholesterol decreases during storage of the immunoassay reagent, it is thought that factors affecting immunoassay measurements can be captured, thereby improving the storage stability of the immunoassay reagent.

[0032] <Immunological measurement method> The immunological measurement method in this embodiment includes confirming the performance of a detection reagent containing a substance that interacts with the substance to be measured, using an immunological measurement reagent containing HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound, and measuring the substance to be measured using the detection reagent.

[0033] In biochemical measurements, a specific affinity substance for the substance being measured is used. If the substance being measured is an antibody, the specific affinity substance can be the corresponding antigen. If the substance being measured is an antigen, the specific affinity substance can be the antibody against it. Furthermore, if the substance being measured is labeled with avidin or biotin, biotin or avidin can be used as the specific affinity substance. Of these, measurements that utilize antigen-antibody reactions are called immunological measurements.

[0034] The type of immunological measurement is not particularly limited, and the measurement methods described in <Immunological Measurement Reagents> are examples, with LTIA being preferred for the same reasons as described in <Immunological Measurement Reagents>.

[0035] Below, LTIA is described in detail as an example of this type of immunological assay. LTIA is a method for measuring target substances, such as antigens or antibodies, using latex particles immobilized with a specific affinity substance for that substance, and is widely used in the field of clinical testing.

[0036] Methods for measuring antigens, which are the target substances, using LTIA can be broadly classified into the following two types. (a) A method for measuring the target substance (antigen) by reacting latex particles immobilized with antibodies against the target substance with an antigen that is the target substance to be measured, forming a sandwich-type immune complex, and measuring the target substance (antigen) from the degree of aggregation of the latex particles accompanying the formation of the immune complex. (b) A method for measuring the target substance (antigen) by having latex particles immobilized with an antigen and the antigen (target substance) in the sample compete with a free antibody added separately to a reagent, thereby inhibiting the formation of an immune complex between the latex particles and the antibody, and measuring the target substance (antigen) from the degree of inhibition of aggregation of the latex particles due to the inhibition of immune complex formation. In this method, a combination of a free antibody added to a reagent and latex particles immobilized with an antibody against the target substance can also be used. Both the antibody and the antigen can also be immobilized on the latex particles.

[0037] In the example above, the substance to be measured is an antigen. However, if the substance to be measured is an antibody, then in (a), the antigen is immobilized on latex particles. In (b), the antibody is immobilized on latex particles, and the free antigen is used.

[0038] In LTIA, certain components in the detection reagent or the sample being measured may cause aggregation that should not occur (positive measurement error) or cause aggregation that should occur to not occur (negative measurement error) in latex particles immobilized with a specific affinity substance for the target substance.

[0039] When an immunoassay reagent contains HDL cholesterol, the amount of HDL cholesterol may decrease during storage due to breakdown or other factors. This is thought to cause factors that affect immunoassays to be released from the HDL cholesterol. These effects on immunoassays include the nonspecific binding of latex particles immobilized with specific affinity substances for the analyte to factors released from HDL cholesterol, resulting in unwanted aggregation. As a result, it may become impossible to accurately confirm the performance of detection reagents containing substances that interact with the analyte using this immunoassay reagent.

[0040] In the immunological assay method of this embodiment, an immunological assay reagent is used that contains HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound. The surfactant having a phenyl group which may have substituents is thought to capture factors released from HDL cholesterol. Furthermore, the surfactant contained in the immunological assay reagent may nonspecifically bind to substances with specific affinity for the target substance, causing an increase in the measured value, but it is thought that the excess surfactant is inclusionated by the inclusion compound. As a result, even if the HDL cholesterol decreases during storage of the immunological assay reagent, it is thought that factors affecting the immunological assay can be captured, and the storage stability of the immunological assay reagent can be improved. Consequently, the performance of a detection reagent containing a substance with specific affinity for the target substance can be accurately confirmed using this immunological assay reagent.

[0041] LTIA allows for the measurement of a target substance by optically or electrochemically observing the degree of aggregation that occurs. Optical observation methods include measuring scattered light intensity, absorbance, and transmitted light intensity using optical instruments (endpoint method, rate method, etc.).

[0042] The absorbance and other measurements obtained from measuring a sample are compared with the absorbance and other measurements obtained from measuring a standard substance (a sample in which the concentration of the target substance is known) to calculate the concentration (quantitative value) of the target substance contained in the sample. The measurement of absorbance and other measurements of transmitted or scattered light may be performed using one wavelength or two wavelengths (difference or ratio between two wavelengths). The measurement wavelength is generally selected from 500 to 900 nm.

[0043] LTIA may be measured using a measuring device. This device may be a general-purpose analytical instrument or a dedicated automated measuring device. LTIA is generally performed using a two-step method (two-reagent method) or other multi-step procedures.

[0044] The immunological assay method in this embodiment includes confirming the performance of a detection reagent containing a substance that interacts with the substance to be measured, using an immunological assay reagent comprising HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound. The immunological assay reagent used here is as described in <Immunological Assay Reagent>, so a detailed explanation is omitted. The immunological assay reagent is, for example, the negative control reagent, positive control reagent, or standard reagent for creating a calibration curve.

[0045] After confirming the performance of a detection reagent containing a substance with specific affinity for the target substance, the target substance is measured using this detection reagent. The target substance is contained in a biological sample.

[0046] The substance to be measured in the immunological measurement method of this embodiment is one of those listed under <Immunological Measurement Reagents>, and is preferably a component of blood. The substance to be measured may be an antigen or antibody contained in the blood.

[0047] Examples of specific affinity substances for the target substance supported on insoluble carrier particles such as latex include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, which are used in known immunoassays. Generally, antibodies or antigens are frequently used.

[0048] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be the entire antibody molecule or a functional fragment having antigen-antibody reaction activity. The antibody may be obtained by immunizing animals such as mice, or it may be synthesized by protein engineering. Examples of these antibodies include antibodies treated with proteolytic enzymes, F(ab')2, Fab', single-chain antibodies (scFv), chimeric antibodies, humanized antibodies, and bispecific antibodies (BsAb). The antibody may be obtained by known methods.

[0049] The latex particles used in LTIA are not particularly limited, and latex particles commonly used as reagents for biochemical measurements can be applied. Latex particles are formed, for example, by polymerizing one or more of the following monomers. Specific examples of monomers include polymerizable monomers having a phenyl group, such as styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene; polymerizable monomers having a phenyl group and sulfonate, such as styrene sulfonate, divinylbenzene sulfonate, o-methylstyrene sulfonate, and p-methylstyrene sulfonate; polymerizable unsaturated aromatics having a naphthyl group, such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate; polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, and (meth)acrylic acid. Examples include polymerizable unsaturated carboxylic acid esters such as 2-hydroxyethyl hydroxylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylate, and tribromophenyl (meth)acrylate; polymerizable unsaturated nitriles such as (meth)acrylonitrile; polymerizable unsaturated aldehydes such as (meth)acrolein; polymerizable unsaturated ketones such as 3-methyl-3-buten-2-one; polymerizable unsaturated carboxylic acid amides such as (meth)acrylamide, N-methylol-(meth)acrylamide, and methylenebis(meth)acrylamide; conjugated dienes such as butadiene and isoprene; vinyl esters such as vinyl acetate; polymerizable unsaturated heterocyclic compounds such as vinylpyridine; vinylamides such as N-vinylpyrrolidone; and vinyl halides such as vinyl chloride, vinylidene chloride, and vinyl bromide.

[0050] The average particle size of the latex particles used in LTIA can be selected from, for example, 0.02 to 1.6 μm, preferably 0.1 μm to 0.4 μm, taking into consideration the concentration of the substance to be measured in the sample or the detection sensitivity of the measuring instrument. Here, the average particle size can be measured using a particle size analyzer.

[0051] The method for supporting one or more of the specific affinity substances on latex particles used in LTIA is not particularly limited, and they may be supported by known methods such as physical adsorption (hydrophobic bonding) or chemical bonding. To prevent nonspecific adsorption of contaminants to the latex particles supporting the specific affinity substances, known blocking treatments (masking treatments) may be performed on the latex particles by contacting them with proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or its salts, high molecular weight compounds such as polysaccharides, surfactants, or skim milk powder.

[0052] In the immunological measurement method of this embodiment, since an immunological measurement reagent with high storage stability is used, the performance of the detection reagent containing a substance with specific affinity for the target substance can be accurately confirmed. As a result, highly accurate immunological measurements can be performed.

[0053] <Storage method for immunoassay reagents> The method for preserving the immunoassay reagent of this embodiment includes adding a surfactant having a phenyl group, which may have substituents, and an inclusion compound to the immunoassay reagent containing HDL cholesterol.

[0054] The immunoassay reagent in this embodiment contains HDL cholesterol. Adding a surfactant and an inclusion compound to this immunoassay reagent improves its storage stability. More specifically, during storage of an immunoassay reagent containing HDL cholesterol, the amount of HDL cholesterol may decrease due to breakdown or other reasons. This is thought to cause factors that affect immunoassay to be released from the HDL cholesterol. These effects on immunoassay include the nonspecific binding of a detection reagent containing a substance with specific affinity for the target substance to factors released from HDL cholesterol. As a result, phenomena such as the inability to accurately verify the performance of the detection reagent containing the substance with specific affinity may occur.

[0055] Surfactants having a phenyl group, which may have substituents, are thought to capture factors released from HDL cholesterol. Furthermore, surfactants contained in immunoassay reagents may bind nonspecifically to specific affinity substances, causing inflated measurement values, but excess surfactant is thought to be encapsulated by inclusion compounds. Therefore, even if HDL cholesterol decreases during storage of immunoassay reagents, it is thought that factors affecting immunoassay measurements can be captured, thereby improving the storage stability of immunoassay reagents. [Examples]

[0056] [Experimental Example 1: Measurement of RPR value and lipid-related parameters of negative control reagent] Lipid-related parameters were measured for multiple negative control reagents used for RPR measurement.

[0057] 1. Detection reagent For measuring non-treponemal lipid antibodies using RPR (Rapid Plasma Reagin), the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo AI, the Apo AI Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo A-II, the Apo A-II Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo B, the Apo B Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo C-II, the Apo C-II Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo C-III, the Apo C-III Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring apo E, the Apo E Auto-N "Daiichi" (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring HDL cholesterol, the Cholestest N-HDL (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring LDL cholesterol, the Cholestest LDL (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring triglycerides, we used Cholestest TG (manufactured by Sekisui Medical Co., Ltd.). For measuring total cholesterol, we used Cholestest CHO (manufactured by Sekisui Medical Co., Ltd.). For measuring phospholipids, we used Pure Auto PL (manufactured by Sekisui Medical Co., Ltd.). For measuring free fatty acids, we used Clinimate NEFA (manufactured by Sekisui Medical Co., Ltd.).

[0058] 2. Sample Ten lots of RPR control (negative) (manufactured by Sekisui Medical Co., Ltd.), which does not contain non-treponemal lipid antibodies, were used as the samples for measurement (samples 1-10).

[0059] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), each sample was measured according to the package insert for each detection reagent mentioned above.

[0060] Table 1 shows the measured values ​​of non-treponemal lipid antibodies (indicated as RPR in the table), apo AI (indicated as ApoA1 in the table), apo A-II (indicated as ApoA2 in the table), apo B, apo C-II (indicated as ApoC2 in the table), apo C-III (indicated as ApoC3 in the table), apo E, HDL cholesterol (indicated as HDL in the table), LDL cholesterol (indicated as LDL in the table), triglycerides (indicated as TG in the table), total cholesterol (indicated as CHO in the table), phospholipids (indicated as PL in the table), and free fatty acids (indicated as NEFA in the table) for samples 1 to 10.

[0061] [Table 1]

[0062] As shown in Table 1, samples 2, 3, and 5, which had high levels of non-treponemal lipid antibodies measured by RPR, tended to have lower levels of HDL cholesterol and apo A1.

[0063] [Experimental Example 2: Increase in non-treponemal lipid antibody levels and decrease in HDL cholesterol levels measured by RPR when the negative control reagent was subjected to vibration treatment] To investigate the mechanism by which non-treponemal lipid antibody levels are elevated by RPR, diluted human serum negative for RPR measurement was vigorously agitated, and non-treponemal lipid antibody levels and lipid-related parameters were measured.

[0064] [Experimental Example 2-1] 1. Detection reagent For measuring non-treponemal lipid antibodies using RPR, the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring HDL cholesterol, the Cholestest N-HDL (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring LDL cholesterol, the Cholestest LDL (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring triglycerides, the Cholestest TG (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring total cholesterol, the Cholestest CHO (manufactured by Sekisui Medical Co., Ltd.) was used. For measuring phospholipids, the Pure Auto PL (manufactured by Sekisui Medical Co., Ltd.) was used.

[0065] 2. Sample After refrigerating serum that did not contain non-treponemal lipid antibodies for one day, an RPR control (negative) sample was prepared by diluting it five-fold with an aqueous solution prepared by disodium hydrogen phosphate dodecahydrate (7.44 g), potassium dihydrogen phosphate (0.58 g), sodium chloride (9 g), bovine serum albumin (4 g), and sodium azide (1 g) in purified water to a total volume of 1000 mL. In this example, refrigeration means storage at 2-8°C.

[0066] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), the samples were measured according to the package inserts for each of the detection reagents mentioned above.

[0067] [Experimental Example 2-2] The sample from Experimental Example 2-1 was prepared by stirring it at 2500 rpm for 1 minute using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 2-1.

[0068] [Experimental Example 2-3] The sample from Experimental Example 2-1 was prepared by stirring it at 2500 rpm for 5 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 2-1.

[0069] [Experimental Example 2-4] The samples were prepared using the same method as in Experimental Example 2-1, except that the refrigeration period was changed to 6 months, and measurements were performed using the same method as in Experimental Example 2-1.

[0070] [Experimental Example 2-5] The samples from Experimental Example 2-4 were prepared by stirring them at 2500 rpm for 1 minute using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 2-1.

[0071] [Experimental Example 2-6] The samples from Experimental Example 2-4 were prepared by stirring them at 2500 rpm for 5 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 2-1.

[0072] Table 2 shows the refrigeration period, stirring time, and measured values ​​of non-treponemal lipid antibodies (indicated as RPR in the table), HDL cholesterol (indicated as HDL in the table), LDL cholesterol (indicated as LDL in the table), triglycerides (indicated as TG in the table), total cholesterol (indicated as CHO in the table), and phospholipids (indicated as PL in the table) for the samples in Experimental Examples 2-1 to 2-4.

[0073] [Table 2]

[0074] As shown in Table 2, comparing Experimental Example 2-4, which was refrigerated for 6 months, with Experimental Example 2-6, it was found that stirring the sample for 5 minutes increased the value of non-treponemal lipid antibodies and decreased HDL cholesterol. Comparing Experimental Example 2-1, which was refrigerated for 1 day, with Experimental Examples 2-2 and 2-3, it was found that stirring the sample did not significantly change the values ​​of non-treponemal lipid antibodies or HDL cholesterol. These results suggest that in samples refrigerated for 6 months, stirring causes the breakdown of HDL cholesterol, and that some substance contained in HDL cholesterol causes the elevated values ​​of non-treponemal lipid antibodies measured by RPR. Subsequently, serum refrigerated for 6 months was used as a model for a degraded RPR control (negative) reagent.

[0075] [Experimental Example 3: Addition of Cyclodextrin to the Sample] Methylated β-cyclodextrin, an inclusion compound, was added to the sample, and non-treponemal lipid antibodies were measured by RPR.

[0076] [Experimental Example 3-1] 1. Detection reagent For the measurement of non-treponemal lipid antibodies using RPR, we used the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.).

[0077] 2. Sample Serum that did not contain non-treponemal lipid antibodies, which had been refrigerated for 6 months, was diluted five-fold with an aqueous solution prepared by disodium hydrogen phosphate dodecahydrate (7.44 g), potassium dihydrogen phosphate (0.58 g), sodium chloride (9 g), bovine serum albumin (4 g), and sodium azide (1 g) dissolved in purified water to a total volume of 1000 mL.

[0078] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), the samples were measured according to the instructions in the package insert for the detection reagents mentioned above.

[0079] [Experimental Example 3-2] Methylated β-cyclodextrin was added to the aqueous solution of Experimental Example 3-1 to prepare an aqueous solution, and a sample was prepared in which the proportion of methylated β-cyclodextrin was 0.2% by mass relative to the total mass of the sample. The sample was then measured using the same method as in Experimental Example 3-1.

[0080] [Experimental Example 3-3] The measurement was performed using the same method as in Experimental Example 3-2, except that a sample was prepared in which the proportion of methylated β-cyclodextrin was 2.0% by mass relative to the total mass of the sample.

[0081] [Experimental Example 3-4] The sample from Experimental Example 3-1 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 3-1.

[0082] [Experimental Example 3-5] The sample from Experimental Example 3-2 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 3-1.

[0083] [Experimental Example 3-6] The sample from Experimental Example 3-3 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 3-1.

[0084] Table 3 shows the stirring time for the samples in Experimental Examples 3-1 to 3-6, the final concentration of methylated β-cyclodextrin in the samples, and the measured values ​​of non-treponemal lipid antibodies by RPR (indicated as RPR measurement values ​​in the table).

[0085] [Table 3]

[0086] As shown in Table 3, adding only methylated β-cyclodextrin, an inclusion compound, to the sample did not suppress the elevated levels of non-treponemal lipid antibodies measured by RPR.

[0087] [Experimental Example 4: Addition of methylated β-cyclodextrin and surfactant to the sample] A nonionic surfactant, a substitute for nonidet P-40, and a methylated β-cyclodextrin, an inclusion compound, were added to the sample and stirred. The levels of non-treponemal lipid antibodies were then measured by RPR. Experimental Examples 4-3 and 4-6 are examples, while Experimental Examples 4-1, 4-2, 4-4, and 4-5 are comparative examples.

[0088] [Experimental Example 4-1] 1. Detection reagent For the measurement of non-treponemal lipid antibodies using RPR, we used the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.).

[0089] 2. Sample Serum that did not contain non-treponemal lipid antibodies, which had been refrigerated for 6 months, was diluted five-fold with an aqueous solution prepared by disodium hydrogen phosphate dodecahydrate (7.44 g), potassium dihydrogen phosphate (0.58 g), sodium chloride (9 g), bovine serum albumin (4 g), and sodium azide (1 g) dissolved in purified water to a total volume of 1000 mL.

[0090] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), the samples were measured according to the instructions in the package insert for the detection reagents mentioned above.

[0091] [Experimental Example 4-2] Nonidet P-40 substitute (a mixture of polyoxyethylene nonylphenyl ether and polyoxyethylene octylphenyl ether, manufactured by Nacalai Tesque) was dissolved in the aqueous solution of Experimental Example 4-1 to a final concentration of 0.1% by mass to obtain the aqueous solution for this experiment. Serum that did not contain non-treponema lipid antibodies, which had been stored under refrigeration for 6 months, was diluted fivefold with this aqueous solution to prepare a sample, and measurements were performed using the same method as in Experimental Example 4-1.

[0092] [Experimental Example 4-3] Serum free of non-treponemal lipid antibodies, stored under refrigeration for 6 months, was diluted 2-fold with the aqueous solution of Experimental Example 4-2. Methylated β-cyclodextrin was dissolved in the aqueous solution of Experimental Example 4-1 to a final concentration of 1.5% to obtain the aqueous solution of this experiment. The above 2-fold diluted solution was further diluted 2.5-fold with the aqueous solution of this experiment to prepare the sample. This sample was measured using the same method as in Experimental Example 4-1.

[0093] [Experimental Example 4-4] The sample from Experimental Example 4-1 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 4-1.

[0094] [Experimental Example 4-5] The sample from Experimental Example 4-2 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 4-1.

[0095] [Experimental Examples 4-6] The sample from Experimental Example 4-3 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 4-1.

[0096] Table 4 shows the stirring times for the samples in Experimental Examples 4-1 to 4-6, the final concentration of the nonidet P-40 substitute in the sample, the final concentration of methylated β-cyclodextrin in the sample, and the measured values ​​of non-treponemal lipid antibodies by RPR (indicated as RPR measurement values ​​in the table).

[0097] [Table 4]

[0098] The results from Experiments 4-2 and 4-4, in which only the nonidet P-40 substitute was added, showed that abnormally high levels of non-treponemal lipid antibodies were observed both when the sample was not stirred and when the sample was stirred. This suggests that nonidet P-40 causes abnormally high levels of non-treponemal lipid antibodies.

[0099] Experimental Example 4-3, in which methylated β-cyclodextrin was added to the nonidette P-40 substitute, showed that the abnormally high RPR values ​​in unmixed samples were suppressed. In other words, it was shown that methylated β-cyclodextrin suppressed the abnormally high measured values ​​of non-treponemal lipid antibodies caused by the nonidette P-40 substitute. In addition, results from Experimental Example 4-6 showed that the phenomenon of elevated measured values ​​of non-treponemal lipid antibodies was not observed after mixing.

[0100] [Experimental Example 5: Evaluation of various surfactants in combination with methylated β-cyclodextrin] Various surfactants and methylated β-cyclodextrin, an inclusion compound, were added to the sample, and non-treponemal lipid antibodies were measured by RPR. Experimental examples 5-16, 5-19, and 5-22 are examples, experimental examples 5-6, 5-10, and 5-15 are control examples, and experimental examples 5-1 to 5-5, 5-7 to 5-9, 5-11 to 5-14, 5-16 to 5-18, 5-20 to 5-22, and 5-24 to 5-27 are comparative examples.

[0101] [Experimental Example 5-1] 1. Detection reagent For RPR measurement, we used the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.).

[0102] 2. Sample Serum that did not contain non-treponemal lipid antibodies, which had been refrigerated for 6 months, was diluted five-fold with an aqueous solution prepared by disodium hydrogen phosphate dodecahydrate (7.44 g), potassium dihydrogen phosphate (0.58 g), sodium chloride (9 g), bovine serum albumin (4 g), and sodium azide (1 g) dissolved in purified water to a total volume of 1000 mL.

[0103] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), the samples were measured according to the instructions in the package insert for the detection reagents mentioned above.

[0104] [Experimental Example 5-2] The sample from Experimental Example 5-1 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE), and measurements were performed using the same method as in Experimental Example 5-1.

[0105] [Experimental Examples 5-3 to 5-12] The surfactants shown in Table 5 were dissolved in the aqueous solution of Experimental Example 5-1 to a final concentration of 0.1% to obtain aqueous solution 1 of Experimental Examples 5-3 to 5-12. Serum that did not contain non-treponemal lipid antibodies, which had been stored in the refrigerator for 6 months, was diluted 2-fold with these aqueous solutions 1 to obtain dilutions. Methylated β-cyclodextrin was dissolved in the aqueous solution of Experimental Example 5-1 to a final concentration of 1.5% to obtain aqueous solution 2. The above 2-fold diluted dilution was further diluted 2.5-fold with aqueous solution 2 to prepare the sample. This sample was measured using the same method as in Experimental Example 5-1.

[0106] [Table 5]

[0107] [Experimental Examples 5-13 to 5-22] The samples from Experimental Examples 5-3 to 5-12 were stirred at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation) to prepare the samples for Experimental Examples 5-13 to 5-22, and measurements were performed using the same method as in Experimental Example 5-1.

[0108] Tables 6 and 7 show the final concentrations of methylated β-cyclodextrin in the samples, the surfactant, the final concentration of the surfactant in the samples, the stirring time, and the measured values ​​of non-treponemal lipid antibodies by RPR (indicated as RPR measurement values ​​in the table) for experimental examples 5-1 to 5-22.

[0109] [Table 6]

[0110] [Table 7]

[0111] In addition to nonidet P-40 substitutes, experimental examples 5-16 and 5-22, which used Neoperex G65 and Triton X-100, which have substituted phenyl groups, as surfactants in combination with methyl β-cyclodextrin, showed a tendency to suppress the increase in measured values ​​of non-treponemal lipid antibodies caused by stirring.

[0112] [Experimental Example 6: Evaluation of γ-cyclodextrin and various surfactants] Various surfactants and γ-cyclodextrin as an inclusion compound were added to the samples, and the levels of non-treponemal lipid antibodies were measured by RPR. Experimental examples 6-17 and 6-19 are examples, experimental examples 6-8 and 6-10 are control examples, and experimental examples 6-1 to 6-7, 6-9, 6-11 to 6-16, 6-18 and 6-20 are comparative examples.

[0113] [Experimental Example 6-1] 1. Detection reagent For RPR measurement, we used the Mediace RPR (manufactured by Sekisui Medical Co., Ltd.).

[0114] 2. Sample Serum that did not contain non-treponemal lipid antibodies, which had been refrigerated for 6 months, was diluted five-fold with an aqueous solution prepared by disodium hydrogen phosphate dodecahydrate (7.44 g), potassium dihydrogen phosphate (0.58 g), sodium chloride (9 g), bovine serum albumin (4 g), and sodium azide (1 g) dissolved in purified water to a total volume of 1000 mL.

[0115] 3. Measurement Procedure Using an automated clinical chemistry analyzer (EV800, manufactured by Hitachi High-Technologies Corporation), the samples were measured according to the instructions in the package insert for the detection reagents mentioned above.

[0116] [Experimental Example 6-2] The sample from Experimental Example 6-1 was prepared by stirring it at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation), and measurements were performed using the same method as in Experimental Example 7-1.

[0117] [Experimental Examples 6-3 to 6-11] The surfactants shown in Table 8 were dissolved in the aqueous solution of Experimental Example 6-1 to a final concentration of 0.1% to obtain aqueous solution 1 of Experimental Examples 6-3 to 6-11. Serum that did not contain non-treponemal lipid antibodies, which had been stored in the refrigerator for 6 months, was diluted 2-fold with these aqueous solutions 1 to obtain dilutions. Methylated β-cyclodextrin was dissolved in the aqueous solution of Experimental Example 6-1 to a final concentration of 1.5% to obtain aqueous solution 2. The above 2-fold diluted dilution was further diluted 2.5-fold with aqueous solution 2 to prepare the sample. This sample was measured using the same method as in Experimental Example 6-1.

[0118] [Table 8]

[0119] [Experimental Examples 6-12 to 6-20] The samples from Experimental Examples 6-3 to 6-11 were stirred at 2500 rpm for 10 minutes using a TRIO test tube mixer (manufactured by AS ONE Corporation) to prepare the samples for Experimental Examples 6-12 to 6-20, and measurements were performed using the same method as in Experimental Example 6-1.

[0120] Tables 9 and 10 show the final concentrations of γ-cyclodextrin sulfated sodium salt hydrate in the samples, the surfactant, the final concentrations of the surfactant in the samples, the stirring time, and the measured values ​​of non-treponemal lipid antibodies by RPR (indicated as RPR measurement values ​​in the table) for experimental examples 6-1 to 6-20.

[0121] [Table 9]

[0122] [Table 10]

[0123] In Experimental Example 6-23, which used Rebenol WZ as the surfactant and γ-cyclodextrin as the inclusion compound, and in Experimental Example 7-26, which used NP-40 as the surfactant and γ-cyclodextrin as the inclusion compound, the suppression of the increase in RPR values ​​due to stirring of the sample was demonstrated.

Claims

1. An immunoassay reagent comprising HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound.

2. The immunoassay reagent according to claim 1, wherein the surfactant is a nonionic surfactant or an anionic surfactant.

3. The immunoassay reagent according to claim 1, wherein the number of phenyl groups contained in one molecule of the surfactant is one.

4. The immunoassay reagent according to claim 1, wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin.

5. The immunoassay reagent according to claim 1, wherein the inclusion compound has 7 to 8 structural units derived from glucose in its molecular structure.

6. An immunoassay reagent according to any one of claims 1 to 5, used as a negative control in an immunoassay method.

7. The performance of a detection reagent containing a substance that interacts with the target substance is confirmed using an immunoassay reagent containing HDL cholesterol, a surfactant having a phenyl group which may have substituents, and an inclusion compound. An immunoassay method comprising measuring the target substance using the detection reagent.

8. The immunological measurement method according to claim 7, wherein the substance to be measured is a non-treponemal lipid antibody.

9. The immunological measurement method according to claim 7 or 8, wherein the immunological measurement method is latex immunoturbidimetry.

10. The immunoassay method according to claim 7 or 8, wherein the surfactant is a nonionic surfactant or an anionic surfactant.

11. The immunoassay method according to claim 7 or 8, wherein the number of phenyl groups contained in one molecule of the surfactant is one.

12. The immunoassay method according to claim 7 or 8, wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin.

13. The immunoassay method according to claim 7 or 8, wherein the inclusion compound has 7 to 8 structural units derived from glucose in its molecular structure.

14. A method for preserving an immunoassay reagent, comprising adding a surfactant having a phenyl group which may have substituents and an inclusion compound to an immunoassay reagent containing HDL cholesterol.

15. The method for storing an immunoassay reagent according to claim 14, wherein the surfactant is a nonionic surfactant or an anionic surfactant.

16. A method for storing an immunoassay reagent according to claim 14 or 15, wherein the number of phenyl groups contained in one molecule of the surfactant is one.

17. The method for storing an immunoassay reagent according to claim 14 or 15, wherein the inclusion compound is at least one of β-cyclodextrin and γ-cyclodextrin.

18. The method for storing an immunoassay reagent according to claim 14 or 15, wherein the inclusion compound has seven to eight structural units derived from glucose in its molecular structure.

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