Production method and storage method for immunological measurement reagent containing insoluble carrier, measurement method for substance to be measured, and immunological measurement reagent

Abstract

To provide a production method for an immunological measurement reagent containing an insoluble carrier, which is able to prevent a change in sensitivity during the storage of the immunological measurement reagent containing the insoluble carrier, a storage method for improving the storage stability of the immunological measurement reagent containing the insoluble carrier, an immunological measurement reagent containing the insoluble carrier, which has improved storage stability, and a measurement method for a substance to be measured using the immunological measurement reagent.SOLUTION: A production method for an immunological measurement reagent containing an insoluble carrier includes a step 1 of storing, at 11°C to 60°C, an insoluble carrier carrying an antigen or an antibody against a substance to be measured; and a step 2 of storing the insoluble carrier at 2°C to 10°C after the step 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an immunoassay reagent containing an insoluble carrier, and a storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier.The present invention further relates to a method for measuring a substance to be measured, and an immunoassay reagent. [Background technology]

[0002] Immunoassay reagents using insoluble carriers are widely used as a method for measuring trace components in biological samples using antigen-antibody reactions. Maintaining the stability of performance of immunoassay reagents using insoluble carriers is important in the manufacture of the reagents. Attempts to improve the storage stability of immunoassay reagents have been investigated, and methods for evaluating and assessing the storage stability of immunoassay reagents have been reported. Patent Document 1 describes a method for evaluating the storage stability of immunoassay reagents in which an antibody or antigen specific to a substance to be measured is supported on an insoluble carrier. The immunoassay reagent is stored at 30 to 45°C for 3 to 5 days, and the storage stability is evaluated based on the change in absorbance measured before and after storage. Patent Document 2 describes a measurement reagent for a substance to be measured, which includes a carrier on which a substance specifically binding to the substance to be measured is immobilized, and which contains 100 mM or more cations and anions, respectively. The difference in absorbance before and after 50 days of storage at 30°C is evaluated. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-108679 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-227027 Summary of the Invention [Problem to be solved by the invention]

[0004] When measuring immunoreaction reagents or biochemical reagents used as in vitro diagnostics using an automated analyzer, creating a calibration curve for each measurement and calculating the measured value is inefficient in terms of the amount of reagent used and the measurement work involved. In reality, measurement values ​​are calculated using a calibration curve created once over several days. Therefore, it is desirable that the sensitivity of these reagents does not fluctuate over that period. However, immunoassay reagents prepared by binding an antibody or antigen to an insoluble carrier may experience changes in reagent sensitivity over time due to structural changes in the antibody or antigen that accompany the binding and the influence of substances contained in the solvent.

[0005] An object of the present invention is to provide a method for producing an immunoassay reagent containing an insoluble carrier that can suppress changes in sensitivity during storage of the immunoassay reagent containing an insoluble carrier.A further object of the present invention is to provide a storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier.A further object of the present invention is to provide an immunoassay reagent containing an insoluble carrier with improved storage stability, and a method for measuring a substance to be measured using the immunoassay reagent. [Means for solving the problem]

[0006] As a result of extensive research to solve the above problems, the present inventors have found that the rate of change in reagent sensitivity over time can be reduced by exposing an immunoassay reagent containing an insoluble carrier to a temperature condition above a predetermined temperature for a certain period of time. The present invention was completed based on this finding.

[0007] According to the present invention, the following inventions are provided. <1> Step 1: storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C; Step 2 includes storing the insoluble carrier at 2°C to 10°C after step 1. A method for producing an immunoassay reagent containing an insoluble carrier. <2> The temperature in step 1 is 20 to 45°C. <1> The method described below. <3> The insoluble carrier is a latex particle. <1> or <2> The method described below. <4> The immunoassay reagent is a reagent used in latex turbidimetry; <1> from <3> 1. The method according to claim 1 , <5> Step 1: storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C; Step 2 includes storing the insoluble carrier at 2°C to 10°C after step 1. A storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier. <6> The temperature in step 1 is 20 to 45°C. <5> The method described below. <7> The insoluble carrier is a latex particle. <5> or <6> The method described below. <8> The immunoassay reagent is a reagent used in latex turbidimetry; <5> from <7> 1. The method according to claim 1 , <9> <1> from <4> 2. Producing an immunoassay reagent containing an insoluble carrier by any one of the methods described above; and Bringing the manufactured immunoassay reagent into contact with the substance to be measured. A method for measuring a substance to be measured, comprising: <10> <1> from <4> 1. An immunoassay reagent comprising an insoluble carrier, which is produced by any one of the methods described above. <11> <10> A method for measuring a substance to be measured, comprising contacting the substance to be measured with the immunoassay reagent according to claim 1. [Effects of the Invention]

[0008] According to the present invention, the storage stability of an immunoassay reagent can be improved, and changes in sensitivity of the immunoassay reagent during storage can be suppressed. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0010] <Method for producing an immunoassay reagent containing an insoluble carrier, and storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier> The method for producing an immunoassay reagent containing an insoluble carrier according to the present invention includes step 1 of storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C, and step 2 of storing the insoluble carrier at 2°C to 10°C after step 1.

[0011] The storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier according to the present invention includes step 1 of storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C, and step 2 of storing the insoluble carrier at 2°C to 10°C after step 1.

[0012] Step 1 is a step of storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C. Storage treatment at 11°C to 60°C for the purpose of improving the storage stability of immunoassay reagents has not been reported previously, and the effect of the present invention is unexpected. In particular, Patent Document 1 indicates that immunoassay reagents deteriorate when treated at high temperatures, so the improvement in storage stability by storage treatment at 11°C to 60°C is an unexpected effect.

[0013] In the present invention, the substances to be measured are not particularly limited, and include all substances that are generally considered to be measurable by complement immunoassay, such as biologically and clinically important drugs, metabolites, vitamins, pesticides, steroids, peptides, hormones, hepatitis markers, cancer markers, antibodies, and serum proteins. Specific examples of endocrine function-related substances include thyroid-stimulating hormone (TSH), parathyroid hormone (iPTH), growth hormone (GH), somatomedin C (IGF-1), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, enkephalin, β-endorphin, thyroxine, triiodothyronine, thyroglobulin, and antithyroglobulin anti- Body, anti-T3 antibody, anti-T4 antibody, anti-TSH antibody, calcitonin, catecholamine, dopamine, serotonin, aldosterone, renin, angiotensin, cortisol, deoxycortisol, cortisone, corticosterone, deoxycorticosterone, androsterone, progesterone, pregnenolone, estrogen, estrone, estriol, estradiol, testosterone, gonadotropins, insulin, anti-insulin antibody, C-peptide, glutamic acid ... Examples of tumor-related substances include CEA, ferritin, β2-microglobulin, elastase, α-fetoprotein, nerve-specific enolase, prostate-specific antigen, CA19-9, etc. Examples of drugs and vitamin-related substances include phenobarbital, phenytoin, carbamazepine, primidone, ethoxybenzone, benzocaine, benzodiazepine ... Succimide, valproic acid, acetazolamide, sulthiame, glutethimide, clonazepam, nitrazepam, diazepam, pentobarbital, secobarbital, bupivacaine, mepivacaine, lidocaine, procainamide, quinidine, digoxin, digitoxin, theophylline, amitriptyline, imipramine, amikacin, gentamicin, tobramycin, cephalexin, sulfamethoxazole, methotrexate, cyclosporine, methylprednisolone,Examples of serum or plasma protein-related substances include albumin, α1-microglobulin, α1-antitrypsin, α2-macroglobulin, haptoglobulin, hemopexin, transferrin, myoglobin, CK-MB (creatine kinase B1), and the like. Examples of substances to be measured include CK-MB, troponin, BNP, NTpro-BNP, IgG, IgM, IgA, IgD, IgE, fibrinogen, antithrombin, plasminogen, antiplasmin, protein C, rheumatoid factor, anti-DNA antibody, anti-CCP antibody, anti-gastric parietal cell antibody, C-reactive protein, etc., and examples of substances related to viruses and infectious diseases include HBs antigen, HBs antibody, HBc antibody, HTLV-I antibody, HTLV-III antibody, TPHA, various viral antigens, various viral antibodies, etc. Specific examples of substances to be measured include, but are not limited to, ferritin and CK-MB, which were measured in the experiments described below.

[0014] In the present invention, when the substance to be measured is an antibody, an antigen for the antibody is carried on an insoluble carrier. In the present invention, when the substance to be measured is an antigen, an antibody against the antigen is carried on an insoluble carrier.

[0015] The insoluble carrier is not particularly limited, but examples thereof include plates (e.g., plastic microtiter plates), latex particles, and magnetic particles. The insoluble carrier is preferably latex particles. Examples of latex particles that can be used include polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and polyvinyl acetate acrylate. Examples of magnetic particles that can be used include magnetic silica particles.

[0016] The temperature in step 1 is 11 to 60°C, preferably 20 to 45°C, and more preferably 35 to 45°C.

[0017] The storage period in step 1 is 12 hours to 30 days, preferably 1 day to 7 days, and more preferably 1 day to 3 days.

[0018] The immunoassay reagent may be any insoluble carrier carrying an antigen or antibody against the substance to be measured, but is preferably a reagent used in latex turbidimetry.

[0019] In step 2, after step 1, the insoluble carrier is stored at 2°C to 10°C.

[0020] <Immunoassay reagents containing insoluble carriers> According to the present invention, there is provided an immunoassay reagent containing an insoluble carrier, which is produced by the method for producing an immunoassay reagent containing an insoluble carrier according to the present invention.

[0021] Details of the insoluble carrier and the immunoassay reagent are as described above. The immunoassay reagent containing the insoluble carrier of the present invention can be used in a method for measuring a substance to be measured.

[0022] <Measuring method for target substance> According to the present invention, an immunoassay reagent containing an insoluble carrier is produced by the method for producing an immunoassay reagent containing an insoluble carrier according to the present invention; and Bringing the manufactured immunoassay reagent into contact with the substance to be measured. There is provided a method for measuring a substance to be measured (measurement method 1), which includes the steps of: The present invention further provides a method for measuring a substance to be measured (measurement method 2), which comprises contacting the substance to be measured with the immunoassay reagent of the present invention. Hereinafter, the above-mentioned measurement method 1 and measurement method 2 will be collectively referred to as the measurement method of the present invention.

[0023] The measurement method of the present invention preferably includes contacting a sample with an antigen or antibody against the substance to be measured, which may have a label.

[0024] The sample is not particularly limited, but is preferably a biological sample, more preferably a human-derived sample. Examples of the sample include, but are not limited to, blood-derived samples (blood, plasma, serum, etc.), urine, saliva, lymph, cerebrospinal fluid, pleural effusion, ascites, tears, semen, bladder washings, tissue extracts, tissue sections, tissue biopsy samples, and samples prepared therefrom. The sample is preferably a blood-derived sample, more preferably serum or plasma, and particularly preferably serum.

[0025] <Labeling method> In one example, the analyte in the sample can be measured based on the label. The substance to be measured may be measured according to the sandwich method or competitive method described in the literature [e.g., Enzyme Immunoassay Method, 2nd Edition (edited by Ishikawa Eiji et al., Igaku-Shoin), 1982], which are commonly used in the field, and the measurement method described in Japanese Patent Laid-Open No. 6-130063.

[0026] In the sandwich method, for example, an antigen or antibody specific to the analyte is immobilized on the surface of a solid support such as magnetic silica particles. The sample, the solid support, and a labeled "substance that binds to the analyte" are mixed together, and the immobilized "antigen or antibody specific to the analyte" is brought into contact with the analyte in the sample and the labeled "substance that binds to the analyte." This allows the formation of a labeled complex, which is a complex of the immobilized "antigen or antibody specific to the analyte," the analyte in the sample, and the labeled "substance that binds to the analyte." The solid support carrying the labeled complex is then subjected to B / F separation to measure the amount of label in the labeled complex, and the amount of the analyte in the sample is determined based on the amount of label in the labeled complex.

[0027] Any solid-phase carrier (especially an insoluble support) commonly used in immunoassays can be used, including organic materials such as polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, and ethylene-maleic anhydride copolymer; inorganic materials such as glass, silicon oxide, diatomaceous earth, porous glass, ground glass, alumina, silica gel, and metal oxides; magnetic materials such as iron, cobalt, nickel, magnetite, and chromite; and alloys of these magnetic materials. Immobilization can be performed on any solid phase (e.g., beads, magnetic beads, membranes, plates, and other surfaces). A variety of commercially available magnetic beads can be used. As magnetic beads, for example, beads disclosed in WO2012 / 173002A may be used.

[0028] Bound / Free (B / F) separation in the sandwich method refers to the separation of substances supported on a solid phase carrier from other substances.

[0029] The sandwich method can also be performed using a microfluidic chip (micro-total analysis system; μTAS). The microfluidic chip has a flow channel with a sample inlet. For example, at the sample inlet, an immunoassay reagent bound to a polycation or polyanion (e.g., DNA) is contacted with the analyte in the sample to form a complex between the immunoassay reagent and the analyte. An electric field is applied to move the complex. When the complex comes into contact with a labeled "substance that binds to the analyte" present in the flow channel, a complex is formed between the immunoassay reagent, the analyte, and the labeled "substance that binds to the analyte." The electric field further moves the complex through the flow channel, and the labeled complex undergoes B / F separation until it reaches a detection unit on the flow channel, where the amount of label in the labeled complex is measured. The amount of the analyte in the sample can be measured based on the amount of label in the labeled complex.

[0030] A competitive method is a method for measuring the amount of a substance to be measured in a sample by, for example, making the substance to be measured in the sample compete with a substance identical to the substance to be measured, or by making the substance to be measured in the sample compete with a substance similar to the substance to be measured, against an immunological assay reagent that specifically binds to the substance to be measured.

[0031] In one example of a competitive method, an antigen or antibody against the analyte is immobilized on the surface of a solid support, and the sample, the solid support, and a labeled "analyte or its analog" are mixed together. This allows the analyte in the sample to compete with the labeled "analyte or its analog" and contact the immobilized "antigen or antibody against the analyte," forming a labeled complex containing the labeled "analyte or its analog" and the immobilized "antigen or antibody against the analyte." The solid support carrying the labeled complex is then subjected to B / F separation to measure the amount of label in the labeled complex, and the amount of the analyte in the sample can be determined based on the amount of label in the labeled complex.

[0032] In this method, the analyte in the sample, the immobilized "antigen or antibody against the analyte," and the labeled "analyte or its analogue" are simultaneously subjected to a competitive reaction; however, the analyte in the sample may be added to the immobilized "antigen or antibody against the analyte," and then the labeled "analyte or its analogue" may be added to cause a competitive reaction.

[0033] In the competitive assay, B / F separation refers to the separation of a substance supported on a solid support from other substances. Specifically, it refers to the separation of a labeled complex containing a labeled "substance to be measured or its analog" and an immobilized "antigen or antibody against the substance to be measured" from other components (components in the sample other than the substance to be measured).

[0034] The method for contacting the analyte in the sample, "antigen or antibody against the analyte," labeled "substance that binds to the analyte," the analyte or its analogue, etc., may be by conventional treatments such as stirring and mixing. The reaction time may be set appropriately depending on the sandwich method, competitive method, etc., but is usually 1 minute to 24 hours, preferably 1 minute to 1 hour, more preferably 1 to 10 minutes, and particularly preferably 1 to 5 minutes.

[0035] B / F separation can be performed, for example, by utilizing the magnetic properties of the solid carriers. The solid carriers are collected from the outside of the reaction vessel using a magnet or the like, the reaction solution is discharged, a washing solution is added, the magnet is removed, the solid carriers are mixed and dispersed, and then washed. This procedure may be repeated 1 to 3 times. The washing solution is not particularly limited as long as it is one commonly used in this field.

[0036] The label is preferably an enzyme used in enzyme immunoassay (EIA). Examples of the enzyme include alkaline phosphatase, β-galactosidase, horseradish peroxidase (HRP), peroxidases such as microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase. Among these, alkaline phosphatase, peroxidase, and glucose oxidase are more preferred, and peroxidase is particularly preferred.

[0037] In the present invention, it is preferable to use an enzyme as a label and carry out an enzymatic reaction, as described above. A preferred example of the enzymatic reaction is a reaction with peroxidase carried out in the presence of hydrogen peroxide.

[0038] The above-mentioned labels can be bound to a substance that binds to the substance to be measured, or to a substance similar to the substance to be measured, by using methods commonly used in this field (e.g., Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Ishikawa Eiji, Kawai Tadashi, and Miyai Kiyoshi, 2nd Edition, Igaku Shoin, 1982, etc.).

[0039] The amount of label used can be appropriately determined depending on the type of label used. For example, when peroxidase is used as the label, the substance that binds to the analyte and the label are preferably used in a molar ratio of, for example, usually 1:1 to 20, preferably 1:1 to 10, and more preferably 1:2 to 6. Furthermore, the "substance that binds to the analyte" labeled with peroxidase may be used by being contained in a buffer solution commonly used in this field, such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, or Good's buffer (e.g., MES (2-morpholinoethanesulfonic acid) buffer).

[0040] The pH of the buffer solution may be within a range that does not inhibit the antigen-antibody reaction, and is usually 5 to 9. Such a buffer solution may contain stabilizers such as albumin, globulin, water-soluble gelatin, and polyethylene glycol, surfactants, sugars, etc., as long as they do not inhibit the antigen-antibody reaction. In this specification, pH refers to the value measured in accordance with JIS K0400-12-10:2000 (measurement temperature: 25°C).

[0041] Methods for measuring the label include colorimetric or chemiluminescent immunoassays (ECLIA, CLIA and CLEIA).

[0042] Examples of the chromogenic substrate include 3',3',5',5'-tetramethylbenzidine (TMB), 4-chloro-1-naphthol (4-CN), and 3,3'-diaminobenzidine (DAB).

[0043] The luminescent substrate is preferably a nitrogen-containing heterocyclic compound having an amino group or a salt thereof, such as luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (such as alkali metal salts), 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione or a salt thereof.

[0044] <Non-labeling method> Measurement methods using antigen-antibody reactions include label-free methods, i.e., label-free methods, such as immunodiffusion using precipitation reactions, immunoturbidimetry, immunonephelometry, and latex agglutination. Label-free methods, such as latex agglutination, do not require labeling and can quantify the target component by photometry at specific wavelengths in the ultraviolet, visible, and near-infrared regions. Therefore, all that is required is a general-purpose, compact spectroscopic device, making them easy to implement in small-scale medical facilities and POCT settings. In particular, latex agglutination is a method that is relatively simple to prepare during reagent manufacturing. Furthermore, users simply add a sample to the latex reagent to initiate the measurement, and then measure at specific wavelengths in the ultraviolet, visible, and near-infrared regions for a set period of time. Measurement is typically completed within a few minutes to several tens of minutes, making it a rapid, simple, and versatile method. Preferably, in the present invention, the target substance in a sample can be measured by an agglutination method, and more preferably, the target substance in a sample can be measured by latex agglutination.

[0045] The insoluble carrier used in the agglutination method can be any known carrier material used in the field of immune agglutination reactions, without particular limitation. Examples of such materials include synthetic polymer powders such as polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and polyvinyl acetate acrylate, with latex, a homogeneously suspended form of these, being preferred. Other examples include other organic polymer powders, inorganic powders, microorganisms, blood cells, cell membrane fragments, and plastic microtiter plates. Examples of inorganic powders include metal pieces such as gold, titanium, and nickel, as well as silica and alumina.

[0046] The particle size of the insoluble carrier is usually 0.01 to 1.0 μm, more preferably 0.05 to 0.7 μm.

[0047] The antigen or antibody against the substance to be measured can be supported on an insoluble carrier by either physical adsorption or covalent chemical bonding. After the antigen or antibody against the substance to be measured is supported on the carrier, a blocking agent can be used to cover the surface of the carrier that is not coated with the antigen or antibody against the substance to be measured. Known substances such as BSA (bovine serum albumin), Block Ace, skim milk, and casein can be used. The blocking agent may be subjected to pretreatment such as partial denaturation using heat, acid, alkali, etc., as needed.

[0048] In agglutination measurements, a dispersion of a carrier carrying an antigen or antibody against the substance to be measured is brought into contact with a sample containing the substance to be measured to initiate a reaction, and the formation of aggregates accompanying the antigen-antibody reaction is quantified by measuring at specific wavelengths in the ultraviolet, visible, or near-infrared range for a set period of time. Measurements are usually completed within a few minutes to a few hours. The reaction may be initiated using two liquids: the carrier dispersion and the sample. Alternatively, a solution containing a stabilizer or agglutination promoter may be prepared separately and the reaction may be carried out using three liquids. The stabilizer or agglutination promoter may be contained in the carrier dispersion or the sample; these substances can be added as needed.

[0049] The stabilizer is not particularly limited as long as it stabilizes the various reagents and components during the reaction, or the progress of the reaction itself, and may include, for example, buffer solutions such as Good's buffer, synthetic or natural polymers such as polyethylene glycol and polysaccharides, surfactants, etc. The same applies when these substances act not only as stabilizers but also as promoters of the agglutination reaction. The various reagents and components during the reaction are, for example, "antigens or antibodies against the substance to be measured" bound to an insoluble carrier, or the substance to be measured, and sugars or amino acids may be added to stabilize proteins such as antibodies and enzymes.

[0050] Examples of the aggregation promoter include synthetic or natural polymers such as polyethylene glycol, polyglycosyl methacrylate, polyvinylpyrrolidone, carboxymethylcellulose, dextran, pullulan, etc. Examples of surfactants having a similar effect include polyethylene glycol fatty acid monoester derivatives.

[0051] The buffer solution that can be used has only to have buffering capacity under the pH conditions under which a typical antigen-antibody reaction is carried out, and such pH conditions are preferably 5 to 11, more preferably 6 to 10. Examples of buffer solutions that have buffering capacity under these pH conditions include phosphate buffer, Tris buffer, MES buffer, HEPES buffer, glycine buffer, citrate buffer, and Good's buffer, and any buffer solution that can be used in typical biochemical experiments can be used. Furthermore, for liquids involved in agglutination reactions, such as dispersions of insoluble carriers carrying antigens or antibodies against the substance to be measured or samples containing the substance to be measured, surfactants, synthetic or natural polymers, organic and inorganic reagents, etc. can be added as needed. For example, surfactants known for immune agglutination reactions, such as nonionic, anionic, and cationic surfactants, can be used.

[0052] When carrying out an agglutination reaction accompanying an antigen-antibody reaction, the degree of agglutination can be measured by visual inspection, videography, etc., as well as spectroscopic methods. Spectroscopic measurement can be carried out by a known method using a reaction solution in which a dispersion of an insoluble carrier carrying an antigen or antibody against the substance to be measured is brought into contact with a sample containing the substance to be measured, and for example, changes (increases or decreases) in scattered light intensity, absorbance, or transmitted light over the particle size and concentration of the carrier and the reaction time are measured.

[0053] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0054] Example 1: Preparation of anti-human ferritin rabbit polyclonal antibody sensitized latex test solution (1) Sensitization (immobilization) of anti-human ferritin rabbit polyclonal antibody to latex 0.5 mL of 50 mmol / L borate buffer (pH 7) containing 0.5 mg of anti-human ferritin rabbit polyclonal antibody (Agilent Technologies) was mixed with 0.5 mL of 50 mmol / L borate buffer (pH 7) containing 1% (w / v) polystyrene latex (average particle size 0.3 μm) and incubated for 2 hours at 25°C. The latex was then separated by centrifugation, washed with 50 mmol / L borate buffer (pH 7), and suspended in 50 mmol / L borate buffer (pH 7) containing 0.5% (w / v) BSA to a concentration of 0.1% (w / v). This was used as the anti-human ferritin rabbit polyclonal antibody-sensitized latex test solution [1].

[0055] (2) Heat treatment The anti-human ferritin rabbit polyclonal antibody-sensitized latex test solution [1] was stored at 40°C for 1 day to obtain anti-human ferritin rabbit polyclonal antibody-sensitized latex test solution [2].

[0056] (3) Refrigerated storage The above anti-human ferritin rabbit polyclonal antibody-sensitized latex test solutions [1] and [2] were stored under refrigerated conditions (7°C) for 7 days, and the test solutions after storage were designated as anti-human ferritin rabbit polyclonal antibody-sensitized latex test solutions [1'] and [2'].

[0057] Example 2: Measurement of ferritin by latex immunoturbidimetry (1) Preparation of ferritin samples A ferritin standard (manufactured by HDM Labs) was diluted with 10 mmol / L phosphate buffer (containing 0.85% NaCl) containing 1% BSA to a concentration of 100 to 500 ng / mL.

[0058] (2) Test solution Reagent [1] was a 20 mmol / L HEPES buffer solution (pH 7) containing 0.1% (W / V) BSA, and this reagent [1], along with the anti-human ferritin rabbit polyclonal antibody-sensitized latex reagent solutions [1] and [2], and the anti-human ferritin rabbit polyclonal antibody-sensitized latex reagent solutions [1'] and [2'] prepared in Example 1 above, were used.

[0059] (3) Ferritin measurement (two-point end method) The following measurements were performed using a Hitachi 7170 automatic analyzer (Hitachi High-Technologies Corporation). 10 μL of the ferritin sample prepared in (1) above was mixed with 150 μL of the reagent [1] in (2) above, and the mixture was incubated at 37°C for 5 minutes. The change in absorbance at 660 nm was measured from 30 seconds to 5 minutes after the addition of the latex test solution [1']. In addition, ferritin in the same sample was measured using anti-human ferritin rabbit polyclonal antibody-sensitized latex test solution [2'] using the same procedure. As a control, ferritin in the same sample was measured using the same reagents, measuring equipment, and method, except for using the anti-human ferritin rabbit polyclonal antibody-sensitized latex test solutions [1] and [2] described above (2).

[0060] (4) Results (Comparison of changes in measurement sensitivity of latex test solution) The measurement results are shown in Table 1. The change in measurement sensitivity of latex test solution [2'], which was refrigerated after heating treatment, was 98% to 100% compared to the measurement sensitivity of latex test solution [2] (at the start of refrigerated storage), while the change in measurement sensitivity of latex test solution [1'], which was not heated, was 90% to 94% compared to the measurement sensitivity of latex test solution [1] (at the start of refrigerated storage). These results demonstrate that heating treatment stabilizes the sensitivity of the reagent during refrigerated storage.

[0061] [Table 1]

[0062] Example 3: Preparation of anti-human CK-MB mouse monoclonal antibody sensitized latex test solution (1) Sensitization (immobilization) of anti-human CK-MB mouse monoclonal antibody to latex 0.5 mL of 50 mmol / L borate buffer (pH 7) containing 0.5 mg of anti-human CK-MB mouse monoclonal antibody (Roche Diagnostics) was mixed with 0.5 mL of 50 mmol / L borate buffer (pH 7) containing 1% (w / v) polystyrene latex (average particle size 0.3 μm) and incubated for 2 hours at 25°C. The latex was then separated by centrifugation, washed with 50 mmol / L borate buffer (pH 7), and suspended in 50 mmol / L borate buffer (pH 7) containing 0.5% (w / v) BSA to a concentration of 0.1% (w / v). This was used as the anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [3].

[0063] A 0.5 mL solution of 0.5 mg of a different anti-human CK-MB mouse monoclonal antibody (Roche Diagnostics) from that used in the anti-human CK-MB mouse monoclonal antibody-sensitized latex reagent [3] was mixed with 0.5 mL of 50 mmol / L borate buffer (pH 7) containing 1% (w / v) polystyrene latex (average particle size 0.3 μm) and incubated at 25°C for 2 hours. The latex was then separated by centrifugation, washed with 50 mmol / L borate buffer (pH 7), and resuspended in 50 mmol / L borate buffer (pH 7) containing 0.5% (w / v) BSA to a concentration of 0.1% (w / v). This was used as the anti-human CK-MB mouse monoclonal antibody-sensitized latex reagent [4].

[0064] (2) Heat treatment The above anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [3] was stored at 37°C for 2 days to prepare anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [5]. The above anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [4] was stored at 37°C for 2 days to prepare anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [6]. Anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [7] was prepared by mixing equal amounts of anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [3] and [4]. Anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [8] was prepared by mixing equal amounts of anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [5] and [6].

[0065] (3) Refrigerated storage The anti-human CK-MB mouse monoclonal antibody-sensitized latex test solutions [7] and [8] were stored under refrigerated conditions (7°C) for 7 days, and the stored test solutions were designated as anti-human CK-MB mouse monoclonal antibody-sensitized latex test solutions [7'] and [8'].

[0066] Example 4: Measurement of CK-MB by latex immunoturbidimetry (1) Preparation of CK-MB samples CK-MB (manufactured by Veritas Corporation) was diluted with Good's buffer solution (50 mmol / L, pH 7) containing 3% BSA to a concentration of 6 to 78 ng / mL.

[0067] (2) Test solution The reagent [9] was a 20 mmol / L HEPES buffer solution (pH 7) containing 0.1% (W / V) BSA, and the anti-human CK-MB mouse monoclonal antibody-sensitized latex reagent solutions [7] and [8], and the anti-human CK-MB mouse monoclonal antibody-sensitized latex reagent solutions [7'] and [8'] prepared in Example 3 above were used.

[0068] (3) Measurement of CK-MB (two-point end method) The following measurements were carried out using a Hitachi 7170 automatic analyzer (Hitachi High-Technologies Corporation). Specifically, 10 μL of the CK-MB sample prepared in (1) above was mixed with 150 μL of the reagent [9] prepared in (2) above, and the mixture was incubated at 37°C for 5 minutes. The change in absorbance at 660 nm was measured from 30 seconds to 5 minutes after the addition of the latex test solution [7']. In addition, CK-MB in the same samples was measured using the same procedure with anti-human CK-MB mouse monoclonal antibody-sensitized latex test solution [8']. As a control, CK-MB was measured in the same sample using the same reagents, measuring equipment, and method, except for the anti-human CK-MB mouse monoclonal antibody-sensitized latex test solutions [7] and [8] described above in (2).

[0069] (4) Results (Comparison of changes in measurement sensitivity of latex test solution) The measurement results are shown in Table 2. The change in measurement sensitivity of latex test solution [8'] that was refrigerated after heating treatment was 102% to 109% compared to the measurement sensitivity of latex test solution [8] (at the start of refrigerated storage), while the change in measurement sensitivity of latex test solution [7'] that was not heated was 126% to 144% compared to the measurement sensitivity of latex test solution [7] (at the start of refrigerated storage). These results demonstrate that heating treatment stabilizes the sensitivity of the reagent during refrigerated storage.

[0070] [Table 2]

Claims

1. Step 1: storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C; Step 2 includes storing the insoluble carrier at 2°C to 10°C after step 1. A method for producing an immunoassay reagent containing an insoluble carrier.

2. The method according to claim 1, wherein the temperature in step 1 is 20 to 45°C.

3. The method of claim 1 , wherein the insoluble carrier is a latex particle.

4. The method according to claim 1, wherein the immunoassay reagent is a reagent used in latex turbidimetry.

5. Step 1: storing an insoluble carrier carrying an antigen or antibody against a substance to be measured at 11°C to 60°C; Step 2 includes storing the insoluble carrier at 2°C to 10°C after step 1. A storage method for improving the storage stability of an immunoassay reagent containing an insoluble carrier.

6. The method according to claim 5, wherein the temperature in step 1 is 20 to 45°C.

7. The method according to claim 5, wherein the insoluble carrier is a latex particle.

8. The method according to claim 5, wherein the immunoassay reagent is a reagent used in latex turbidimetry.

9. Producing an immunoassay reagent containing an insoluble carrier by the method according to any one of claims 1 to 4; and Bringing the manufactured immunoassay reagent into contact with the substance to be measured. A method for measuring a substance to be measured, comprising:

10. An immunoassay reagent comprising an insoluble carrier, produced by the method according to any one of claims 1 to 4.

11. A method for measuring a substance to be measured, comprising contacting the substance to be measured with the immunoassay reagent according to claim 10.

Images