Highly sensitive immunoassay reagent and measurement method

Abstract

The present invention provides a highly sensitive measurement reagent and a measurement method which make it possible to easily and accurately measure the sCLEC-2 concentration in blood even at levels found in healthy individuals. The measurement reagent contains a carrier on which a first immunological partner that binds to a bioactive substance contained in a biological sample is fixed, and a second immunological partner that binds to the bioactive substance. The measurement reagent further contains: an amphiphilic substance that contains a linear, branched, or annular saturated or unsaturated hydrocarbon group in which a hydrophobic part is constituted by 6-12 carbons, an aromatic hydrocarbon group constituted by 6-12 carbons, or a steroid skeleton; or a chaotropic substance.

Description

Highly sensitive immunoassay reagent and assay method

[0001] The present invention relates to a reagent and a method for measuring a substance in a biological sample with high sensitivity.

[0002] Currently, clinical diagnostic testing is required to measure a wide range of substances that serve as indicators for disease diagnosis in a large number of samples in a short period of time with high accuracy, and to provide the results promptly and accurately as feedback to the treatment site. For example, accurate quantification of trace amounts of target substances is often carried out using measurement systems in which physiologically active substances (antibodies, enzymes, proteins such as receptors, antigens, nucleic acid substances such as DNA and RNA, or sugar chains) are bound to the surface of particles, or immunological measurements using antigen-antibody reactions.

[0003] In general, the performance of in vitro diagnostic reagents used in clinical diagnostic tests depends heavily on the performance of their main raw materials. There are two main types of main raw materials: one is a physiologically active substance, such as an antibody, that recognizes the target substance, and the other is particles that support the physiologically active substance. However, a sufficient measurement method cannot be constructed with only physiologically active substances or particles that have the desired reactivity and properties.

[0004] In vitro diagnostic reagents that utilize an antigen-antibody reaction using an antibody contain, in addition to physiologically active substances such as antibodies and particles that support the physiologically active substances, various additives such as surfactants, salts, human antibodies, preservatives, and sensitizers, and these additives are also factors that affect the performance of in vitro diagnostic reagents.

[0005] As an example of a reagent that requires such highly accurate measurements, a reagent for measuring sCLEC-2 will be described. C-type lectin-like receptor 2 (CLEC-2) was identified on platelets as a receptor for the platelet-activating snake venom rhodocytin. In humans, CLEC-2 is expressed almost exclusively in platelets and megakaryocytes, and can be said to be a platelet-specific molecule. It has been reported that this CLEC-2 is released into the blood as soluble CLEC-2 (hereinafter abbreviated as sCLEC-2) upon platelet activation (Patent Document 1, Non-Patent Document 1).

[0006] High blood sCLEC-2 concentrations may indicate some pathological condition, and a highly sensitive and specific measurement method is essential to quickly identify such conditions.

[0007] Although blood sCLEC-2 has been measured by ELISA (Patent Document 1) (Non-Patent Documents 1 to 3), this method is not sufficiently sensitive and has presented practical problems in clinical practice. For example, in Patent Document 1, the sample is diluted five-fold before measurement to take into account the influence of inhibitors present in the blood.

[0008] Japanese Patent Application No. 2021-003671

[0009] F. Kazama et al., Platelets 2015; 26(8):711-719Y. Yamashita et al., Thrombosis Research 178 (2019) 54-58X. Zhang et al., Stroke.2019;50:45-52

[0010] Measurement of sCLEC-2 in blood has been shown to be useful for diagnosing cerebral infarction, myocardial infarction, deep vein thrombosis, pulmonary thromboembolism, thrombotic microangiopathy, disseminated intravascular coagulation, and other conditions. These diseases are extremely serious, with many cases requiring intensive care and a high mortality rate, making early diagnosis crucial. To accurately diagnose these diseases, it is essential to be able to clearly distinguish between healthy individuals and patients with thrombosis. Therefore, it is extremely important to be able to more accurately measure sCLEC-2 levels in the blood of healthy individuals.

[0011] Measurement of blood sCLEC-2 levels has been suggested to be useful not only for disease diagnosis but also for monitoring therapeutic effects and determining prognosis (Patent Documents 1 and 2). Administration of antiplatelet drugs to patients with thrombosis is expected to reduce blood sCLEC-2 levels to the sCLEC-2 concentration range of healthy individuals. Therefore, even when using this method to monitor therapeutic effects in patients with thrombosis, it is extremely important to be able to accurately measure sCLEC-2 levels within the blood sCLEC-2 concentration range of healthy individuals.

[0012] The above-mentioned diagnostic applications, therapeutic effect monitoring, and prognostic assessment applications are all made possible by more accurate measurement of blood sCLEC-2 concentrations in healthy individuals. Therefore, an object of the present invention is to provide a highly sensitive assay reagent and assay method that enable accurate and simple measurement of blood sCLEC-2 concentrations at the same level as healthy individuals.

[0013] To accurately measure the blood concentration of sCLEC-2 in healthy individuals, it is first necessary to increase the measurement sensitivity so that lower concentrations of sCLEC-2 can be measured, and it is also necessary to eliminate the influence of interfering substances present in blood that affect sCLEC-2 so that accurate measurement of sCLEC-2 concentrations can be achieved.

[0014] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they discovered a method for measuring blood sCLEC-2 with high sensitivity by adding an amphipathic substance containing a linear, branched, or cyclic, saturated or unsaturated hydrocarbon group having 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or a steroid skeleton in the hydrophobic moiety to a blood sCLEC-2 measurement system, and completed the present invention. In particular, it was surprising that amphipathic substances containing a linear, branched, or cyclic, saturated or unsaturated hydrocarbon group having a relatively short hydrophobic moiety of 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or a steroid skeleton have the above-mentioned effects, while amphipathic substances containing a hydrophobic moiety of 13 or more carbon atoms (excluding steroid skeletons) do not have the above-mentioned effects.

[0015] It has also been found that a significant effect can be obtained when these amphiphilic substances are added at relatively high concentrations of 0.05% to 5.33%, preferably 0.27% to 3.73%, and more preferably 1.07% to 2.67%.

[0016] That is, the present invention provides the following: [1] A measurement reagent for a physiologically active substance, comprising a support on which a first immunological partner that binds to a physiologically active substance contained in a biological sample is immobilized, and a second immunological partner that binds to the physiologically active substance, the measurement reagent further comprising an amphipathic substance or a chaotropic substance, the hydrophobic portion of which comprises a linear, branched, or cyclic, saturated or unsaturated hydrocarbon group having 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or a steroid skeleton. [2] The measurement reagent of [1], wherein the amphipathic substance the hydrophobic portion of which comprises a linear, branched, or cyclic hydrocarbon group having 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or a steroid skeleton is a compound having an anionic functional group, a cationic functional group, an amphoteric functional group, a pyranoside group, a glucamine group, an oxyethylene group, or an aromatic ring that may be substituted. [3] The measurement reagent of [1] or [2], wherein two or more types of amphipathic substances are used, or the amphipathic substance is used in combination with a chaotropic compound. [4] The measuring reagent of any of [1] to [3], wherein the concentration of the amphipathic substance in the reaction solution is 0.05% to 5.33%, and the concentration of the chaotropic compound in the reaction solution is 0.27 mol / L to 3.2 mol / L. [5] The measuring reagent of any of [1] to [4], wherein the measurement target item is soluble CLEC-2. [6] A method for measuring a physiologically active substance contained in a biological sample using the measuring reagent of any of [1] to [5], comprising the following steps: (1) reacting the physiologically active substance in the sample with a carrier having a first immunological partner that binds to the physiologically active substance immobilized thereon, and a second immunological partner that binds to the physiologically active substance, in the presence of an amphipathic substance; (2) separating the carrier from substances other than the carrier in the reaction solution after step (1); and (3) measuring the second immunological partner on the carrier separated in step (2).

[0017] The measuring reagent and measuring method of the present invention enable highly sensitive measurement without being affected by measurement-interfering substances in the blood, making it possible to accurately measure the concentration of a substance to be measured in the blood of a healthy person, thereby enabling accurate diagnosis, monitoring of treatment effects, and prognosis prediction.

[0018] 1 shows a standard curve prepared using human sCLEC-2 protein as a standard. 2 shows the dilution linearity obtained using the CLEIA method. 3 shows the distribution of sCLEC-2 from healthy individuals obtained using the CLEIA method.

[0019] The substances to be measured in the present invention are not particularly limited as long as they can be measured by immunoassay. Examples include soluble CLEC-2, soluble interleukin-2 receptor, prostate-specific antigen, cardiac troponin, presepsin, brain natriuretic peptide, human brain natriuretic peptide precursor N-terminal fragment, myoglobin, CK-MB, D-dimer, thrombin-antithrombin complex, estrogen, progesterone, human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, prolactin, testosterone, procalcitonin, CRP, thrombomodulin, thyroid-stimulating hormone, FT4, and FT3. However, any substance that can be measured by immunological heterogeneous assay is not particularly limited. Hereinafter, an embodiment of the present invention will be described in detail using sCLEC-2 measurement as an example, but the use method is not limited thereto.

[0020] As used herein, "CLEC-2" refers to a platelet-activating receptor belonging to the C-type lectin family, which is normally present on the platelet membrane and is released into the blood upon platelet activation. As used herein, the term "soluble CLEC-2 (sCLEC-2)" refers to sCLEC-2 or a molecule derived from sCLEC-2 that is released from platelets and detected in the blood (or in the buffer when incubated in a buffer).

[0021] sCLEC-2 is believed to include proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Patent Document 1). The proteins with molecular weights of approximately 40 kDa and approximately 32 kDa are presumed to be present on the platelet membrane surface and released in a state contained in microparticles produced upon platelet activation. These proteins are thought to have glycosylated chains. On the other hand, the protein with molecular weight of approximately 25 kDa is thought to be cleaved by proteases upon platelet activation and released from the platelets. In the present invention, the amount of sCLEC-2 as described above is measured. Regarding sCLEC-2, the proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa may be detected together, or only the protein with molecular weight of approximately 25 kDa may be detected.

[0022] CLEC-2 that can be used in the present invention has been confirmed in a wide range of animal species, including humans, cows, pigs, guinea pigs, rats, and mice, with human CLEC-2 being particularly preferred. The amino acid sequence may contain one to several substitutions depending on race, etc., and the CLEC-2 to be detected may contain one to several (e.g., five, preferably two) substitutions.

[0023] The samples (specimens) used in the present invention are preferably blood, plasma containing an anticoagulant, or whole blood containing an anticoagulant, but any biological fluid can also be measured, including body fluids such as urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, nasal discharge, and tears, as well as culture media for cultured cells, pharyngeal or nasal swabs, and plasma with suspended platelets.

[0024] The reagent of the present invention is not particularly limited as long as it is used in the method for detecting the substance to be measured, but preferably the immunological method using the antibody that recognizes the substance to be measured.As the immunological method for detecting protein, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunochromatography and other immunoassays using labeled antibodies, or Western blotting under non-denaturing conditions, latex agglutination, immunoturbidimetry, chemiluminescence-enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA) and other commonly used methods can be used, among which, from the viewpoint of high sensitivity measurement, chemiluminescence-enzyme immunoassay (CLEIA), electrochemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA) and fluorescent immunoassay are preferably used, and those skilled in the art can appropriately select and use them after considering the measurement equipment required to realize measurement.

[0025] The immunological partner that can be used in the present invention may be a partner that immunologically binds specifically to the substance to be measured, for example, an immunological substance (i.e., an antigen or antibody) that can specifically bind to the various proteins, polysaccharides, lipids, nucleic acids, haptens, and complexes or fragments thereof. When the immunological partner is an antibody, the antibody used may be a monoclonal antibody or a polyclonal antibody, or a fragment thereof treated with an enzyme or the like. The antibody fragment is preferably a functional fragment containing the antigen-binding region of the antibody or its variable region, for example, F(ab') ２ , Fab', Fab, etc. F(ab') ２ Fab' is an antibody fragment produced by treating an immunoglobulin with a proteolytic enzyme (e.g., pepsin or papain) across the disulfide bond present between the two H chains in the hinge region. Furthermore, multiple types of immunological partners may be used in combination.

[0026] The antibody that reacts with the analyte is characterized by recognizing and reacting with an antigenic determinant contained in the analyte. When the analyte is sCLEC-2, such an antibody may be one that recognizes a protein having a molecular weight of approximately 40 kDa, a protein having a molecular weight of approximately 32 kDa, and a protein having a molecular weight of approximately 25 kDa. Alternatively, an antibody that recognizes only a protein having a molecular weight of approximately 25 kDa may be selected from antibodies produced by a hybridoma and used.

[0027] Anti-CLEC-2 antibodies can be produced, for example, using a polypeptide comprising all or part of the amino acid sequence of sCLEC-2 as an immunogen. The antigen polypeptide may be a synthetic polypeptide chemically synthesized according to known methods, or may be one produced by genetic recombination or the like. Samples used for measurement are preferably derived from humans, but samples derived from animals other than humans may also be used to understand the pathology of experimental animals. Experimental animals are not particularly limited, and examples include guinea pigs, rats, mice, and dogs.

[0028] Furthermore, the antibody used in the present invention can be used as an immobilized antibody supported on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance.

[0029] An immobilized antibody refers to an antibody that is supported on an insoluble carrier by physical adsorption, chemical bonding, or the like. These immobilized antibodies can be used to detect or quantify a substance to be measured contained in a sample. Examples of insoluble carriers that can be used to support antibodies include polymeric materials such as latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), and silicone; agarose; gelatin; red blood cells; and inorganic materials such as silica gel, glass, inert alumina, and magnetic materials. These may be used alone or in combination.

[0030] The labeling substance is not particularly limited as long as it can be used in a conventional immunoassay, and examples thereof include enzymes, fluorescent substances, radioisotopes, insoluble granular substances, etc. Examples of the labeling enzyme include alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, acid phosphatase, etc. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), luciferin, etc. Examples of the radioisotope include １２５ I, １４ C. ３２ Examples include P.

[0031] Furthermore, when the labeling substance is an enzyme, the labeling substance can be measured by carrying out a luminescence, fluorescence, or color reaction using a substrate for the enzyme. For example, when the enzyme is alkaline phosphatase, the substrate can be CDP-star (registered trademark) (2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium), CSPD (registered trademark) (3-(4-methoxyspiro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.1 ３，７ ]decane}-4-yl)phenyl phosphate disodium), AMPPD (registered trademark) (adamantyl methoxyphenyl phosphoryl dioxycetane), APS-5 or other chemiluminescent substrates; 4-methylumbelliferyl phosphate or other fluorescent substrates; p-nitrophenyl phosphate, BCIP (5-bromo-4-chloro-3-indolyl phosphate), NBT (4-nitroblue tetrazolium chloride), INT (iodonitrotetrazolium) or other chromogenic substrates can be used.

[0032] The immunoassay method of the present invention can be carried out using an immunoassay reagent composed of one liquid or two or more liquids.

[0033] When the reagent of the present invention is composed of one liquid, it can be, for example, a reaction reagent containing at least an insoluble carrier carrying an immunological partner for forming an immunological complex. A sample is reacted with the reagent according to a known method, and a signal is detected. The amphiphilic substance used in the present invention may be added to the reagent before the sample reacts with the reagent, but is preferably supplied in a state where it has been added to the reagent.

[0034] When the reagent of the present invention is composed of two or more liquids, it can be composed of, for example, a stabilizing reagent and a reaction reagent containing an insoluble carrier carrying at least an antibody or antigen for forming an immunological complex. The stabilizing reagent is a reagent used to dilute the sample to an appropriate concentration or perform pretreatment, and can be prepared according to known methods. According to known methods, the sample is reacted with the stabilizing reagent, and then reacted with the reaction reagent, and a signal is detected. The amphiphilic substance used in the present invention is preferably added to the stabilizing reagent and / or the reaction reagent before the sample reacts with the reaction reagent, but preferably can be supplied in a state where it has been added to the stabilizing reagent and / or the reaction reagent.

[0035] The amphiphilic substance used in the present invention may be in any form that is ultimately mixed with the sample, and may be contained in a stabilizing reagent or in a reagent containing an insoluble carrier. In the present invention, we have found that adding a certain concentration range of amphiphilic substance to the reagent is essential to achieve high sensitivity in the measurement system. This is thought to be a phenomenon derived from the physicochemical properties of the substance to be measured, but if there is a small amount of amphiphilic substance, a sufficient signal cannot be obtained in a high-sensitivity measurement system.

[0036] The amphipathic substance may be contained in a reaction solution in which an immune reaction with the substance to be measured takes place, and may be added to, for example, a dilution solution for pre-diluting the measurement sample, a reagent for mixing the measurement sample with a solid-phased antibody bound to an insoluble carrier and causing a reaction between the substance to be measured and the solid-phased antibody, or a reagent for mixing and reacting a labeled antibody with the measurement sample and causing a reaction between the labeled antibody and the substance to be measured, or to each of these reagents.

[0037] Amphiphiles are generally molecules that have both hydrophobic and hydrophilic portions. In the present invention, the hydrophobic portion can be one with a relatively short carbon chain of C6 to C12. Amphiphiles with long carbon chains, which are generally used in reagents that utilize immune reactions to measure analyte substances contained in biological samples, are unsuitable. Examples of the hydrophobic portion include linear, branched, or cyclic saturated or unsaturated hydrocarbon groups with carbon chains of C6 to C12, aromatic hydrocarbon groups with carbon chains of C6 to C12, and steroid skeletons.

[0038] The hydrophilic portion of the amphiphilic substance may be anionic, cationic, nonionic, or amphoteric, and may be a compound having an anionic functional group, a pyranoside group, a glucamine group, an oxyethylene group, or an aromatic ring which may be substituted (particularly an aromatic ring substituted with a hydroxyl group).

[0039] Examples of the salts having an anionic functional group include, but are not limited to, sodium octanoate, sodium n-nonanoate, sodium 1-pentanesulfonate, sodium 1-hexanesulfonate, sodium 1-heptanesulfonate, and sodium 1-octanesulfonate.

[0040] Examples of substances having a cationic functional group include, but are not limited to, n-octylamine hydrochloride, n-octyltrimethylammonium chloride, and decyltrimethylammonium chloride. Alternatively, the substance may be a zwitterionic amphiphilic substance. Examples of zwitterionic amphiphilic substances include, but are not limited to, dimethyl(n-octyl)(3-sulfopropyl)ammonium hydroxide inner salt, decyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt, and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate). Alternatively, an amphiphilic substance whose hydrophobic portion has a branched structure can also be used. For example, an amphiphilic substance having a branched structure includes, but is not limited to, sodium 2-ethylhexyl sulfate.

[0041] Those having a pyranoside group may be glycosides of pyranose, and examples of pyranose include ribopyranose, arabinopyranose, xylopyranose, lyxopyranose, allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. Specific examples of compounds having a pyranoside group include, but are not limited to, N-octyl-β-D-glucopyranoside, n-heptyl-β-D-thioglucopyranoside, N-octyl-β-D-maltopyranoside, n-nonyl-β-D-thiomaltopyranoside, N-decyl-β-D-maltopyranoside, n-octanoyl-N-methyl-D-glucamine, α-D-glucopyranosyl-α-D-glucopyranoside monooctanoate, α-D-glucopyranosyl-α-D-glucopyranoside monodecanoate, and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate.

[0042] Those having a glucamine group may be any as long as they have the structure of N-methyl-D(-)-glucamine, and examples thereof include, but are not limited to, n-nonanoyl-N-methyl-D-glucamine and n-octanoyl-N-methyl-D-glucamine.

[0043] The substance having an oxyethylene group may be any substance having an oxyethylene group with ethylene glycol as the basic structure, and examples thereof include tetraethylene glycol monooctyl ether and poly(ethylene glycol) octyl ether, but are not limited thereto.

[0044] Examples of compounds having a cyclic skeleton include, but are not limited to, aromatic rings which may be substituted, for example, aromatic rings having a sodium salicylate structure, and compounds having a steroid group such as cholic acid and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate).

[0045] The amphiphilic substance that can be used in the present invention is a general term for compounds that have both a hydrophilic portion and a hydrophobic portion, and includes surfactants (hereinafter, may be referred to as surfactants). Surfactants are not limited to those described above, and a person skilled in the art can appropriately select and use a substance that has appropriate reactivity.

[0046] Amphiphiles aggregate in water to form micelles or bilayer membranes, typically lipid bilayers. They also function as surfactants by adsorbing to the interface between the aqueous and organic phases, lowering the surface tension and forming micelles that mix uniformly to form emulsions. Taking advantage of this property, amphiphiles are sometimes added to reagents to prevent antigen adsorption to insoluble carriers or to exert a stronger cleaning effect, thereby suppressing nonspecific reactions.

[0047] Generally, the properties of an amphipathic substance are determined by the balance between the hydrophilic and hydrophobic portions, the chain length, and the bulkiness. Amphipathic substances are classified according to the properties of the hydrophilic portion, and the selection criteria for which amphipathic substance to select and use are, for example, focused on the ability to solubilize only the target substance to be measured and not to solubilize other substances that may be inhibitors. In other words, considering the effect of dispersing a water-insoluble substance in an aqueous solution, it is common knowledge for those skilled in the art that the effect can be achieved by selecting an amphipathic substance with a long hydrophobic portion, i.e., a large number of carbon atoms. Therefore, it is expected that the desired effect will not be obtained even if an amphiphilic substance with a short hydrophobic group is added to a reagent, for reasons such as its weak hydrophobicity making it difficult to form micelles of sufficient size. Therefore, a person skilled in the art would not normally select such a substance. However, it was an unexpected discovery that the addition of an amphiphilic substance whose hydrophobic moiety is a short, linear, branched, or cyclic, saturated or unsaturated hydrocarbon group with a carbon number of 6 to 12, an aromatic hydrocarbon group with a carbon number of 6 to 12, or a steroid skeleton contributes to increasing the sensitivity of the reagent.

[0048] In addition to the above-mentioned amphipathic substances, chaotropic substances can also be added and used. Chaotropic substances that can be used in the present invention include, but are not limited to, urea. Amphipathic substances are sometimes used for the purpose of denaturation, such as protein solubilization, but in the present invention, as shown in the examples described below, chaotropic substances used as denaturing agents also have the same effect as amphipathic substances, so it is presumed that the denaturing effect contributes to the promotion of immune responses.

[0049] The amphipathic substances used in the present invention may be used in combination with multiple types. It was surprising that when two or more types of amphipathic substances were used in combination, the reactivity was equal to or greater than that observed when a single type of amphipathic substance was used alone, even when the concentration added to the reagent was low. Since too high a concentration of an amphipathic substance added to a reagent can inhibit the main reactions, such as immune reactions and luminescence measurement, it is common knowledge for those skilled in the art to keep the addition concentration as low as possible, and therefore, it is preferable to use a lower addition concentration than when a single substance is used, since it can be used in combination with multiple types of amphipathic substances.

[0050] The amphiphilic substance added to the reagent is preferably present in a concentration of 0.05% to 5.33%, more preferably 0.27% to 3.73%, and even more preferably 1.07% to 2.67% in the reaction solution. This concentration is higher than that typically added to reagents used in immune reactions. As previously mentioned, when using amphiphilic substances whose hydrophobic moiety is a linear, branched, or cyclic saturated or unsaturated hydrocarbon group having a short carbon number of 6 to 12, an aromatic hydrocarbon group having a carbon number of 6 to 12, or a steroid skeleton, the expected effect is small, and therefore it is expected that the amphiphilic substance must be added at a high concentration. However, when used at a high concentration, foaming is likely to occur, making it difficult to use as a reagent. However, the amphiphilic substance was surprisingly effective in increasing sensitivity and could be used without foaming, which would affect the immune reaction. Here, the reaction solution may be any reaction space in which a reaction between the analyte and the immunological partner to form an immune complex takes place, and those skilled in the art can select and use either a homogeneous assay, which is distinguished by whether or not a step (B / F separation) is included to remove unreacted immunological partner and analyte by washing, or a heterogeneous assay. Furthermore, the heterogeneous assay may be either a one-step method or a two-step method, as described below.

[0051] The most suitable one is selected from these to prepare a reaction solution for the antigen-antibody reaction. By combining the reaction solution, reaction procedure, and reaction format using the amphiphilic substance of the present invention, a measurement method suited to the equipment used for the immunoassay can be performed.

[0052] Specific measurement methods using the assay reagent of the present invention include, for example, reacting a sample with a solid-phase antibody and simultaneously reacting with a labeled antibody (one-step method), or reacting with a labeled antibody after washing (two-step method) to form a solid-phase antibody-antigen-labeled antibody complex. The unbound labeled antibody is then washed away, and the amount of antigen in the sample can be measured based on the amount of bound labeled antibody. Specifically, in the case of enzyme immunoassay, a substrate is reacted with the labeled enzyme under optimal conditions, and the amount of the reaction product is measured by optical methods or the like. In the case of fluorescence immunoassay, the fluorescence intensity of the fluorescent substance label is measured, and in the case of radioimmunoassay, the radiation dose of the radioactive substance label is measured. In the case of chemiluminescent immunoassay, the amount of luminescence produced by a chemiluminescent reaction system is measured. The presence of the analyte in the test sample is detected by forming an immune complex between the analyte in the reaction solution and an antibody specific to the analyte (antigen in the case of an antibody detection system), and the signal resulting from this formation is appropriately detected. This method includes a step of performing measurement after removing unreacted sample and excess labeled substance by washing. The antibodies to be used can be determined appropriately depending on the measurement system, but for highly sensitive quantitative measurement, a sandwich immunoassay using two or more antibodies can be selected. The sandwich immunoassay method may be carried out in one or more stages (two stages, three stages, etc.).

[0053] Furthermore, the reagent of the present invention may include an instruction manual that describes the procedure for carrying out an immunoassay using the reagent of the present invention, and notes on the storage and handling of the reagent itself.

[0054] In general, the treatment of thrombosis involves administering antiplatelet drugs such as aspirin in the case of arterial thrombosis, such as myocardial infarction, cerebral infarction, and arteriosclerosis obliterans, where the pathology progresses due to platelet activation and aggregation. In the case of venous thrombosis, such as deep vein thrombosis, atrial fibrillation, cardiogenic cerebral embolism, and pulmonary embolism, or thrombosis where the pathology progresses due to activation of the coagulation system in the atrium, anticoagulants such as warfarin and DOAC are administered. While the use of the present invention is intended to test and measure platelet-related thrombosis, platelets also play an important role in thrombosis where the coagulation system is the primary factor, and this measurement may be useful. Accurate risk assessment of platelet-based thrombosis using the present invention may also enable the selection of appropriate antiplatelet drugs.

[0055] Based on these findings, measuring the blood sCLEC-2 concentration in patients with or suspected of having thrombosis using the present invention will provide more reliable measurement values ​​not only for diagnosing thrombosis but also for monitoring the course of treatment, making it possible to provide extremely useful information for the diagnosis and treatment of thrombosis.

[0056] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0057] Example 1: Preparation of sCLEC-2 Assay Reagent and Measurement (Preparation of Assay Reagent and Test Sample) - Sample diluent: 0.1 mol / L HEPES buffer solution (pH 7.5) containing a preservative was used. The antibody contained in the reagent was the antibody described in the Examples of Japanese Patent No. 6078845 and was prepared as follows. - First antibody solution: A mouse monoclonal antibody (11D5) that recognizes sCLEC-2 was bound to magnetic latex particles (JSR Corporation) and dispersed in 0.01 mol / L MES buffer solution (pH 6.0) containing a preservative. - Second antibody solution: Another mouse monoclonal antibody (11E6) that recognizes sCLEC-2 was labeled with alkaline phosphatase (ALP) using the maleimide method and dispersed in 0.01 mol / L MES buffer solution (pH 6.0) containing a preservative. Luminescent substrate solution: 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium (CDP-Star®: Applied Biosystems) was used. B / F washing solution: A buffer containing 0.1 mol / L citric acid (pH 6.5), 0.15 mol / L NaCl, and 0.1% Triton X-100 was used. Test sample: Recombinant hCLEC-2 protein diluted with a buffer (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA) was used as test sample 1, and diluted with citrated plasma was used as test sample 2.

[0058] (Measurement using measurement reagents) A fully automated clinical testing system, STACIA (registered trademark, manufactured by LSI Medience Corporation), was used for the measurement. The prepared specimen dilution solution, first antibody solution (magnetic latex reagent), and second antibody solution (enzyme-labeled antibody reagent) were each filled into STACIA-specific bottles and placed in the instrument. Measurement was performed according to the operating method of the instrument. Specifically, 40 μL of specimen dilution solution was added to 10 μL of sample, and the mixture was heated at 37°C for several minutes. Then, 25 μL of first antibody solution (magnetic latex reagent) was added and heated at 37°C for several minutes. Next, B / F separation was performed, 50 μL of second antibody solution (enzyme-labeled antibody reagent) was added, and the mixture was heated at 37°C for several minutes. B / F separation was then performed again, and 100 μL of luminescent substrate solution was added. After a few minutes of reaction at 37°C, the signal intensity (counts) was measured.

[0059] Example 2: Effect of adding surfactants to reaction solution on sCLEC-2 reactivity The procedure of Example 1 was repeated, except that surfactants with different hydrophobic and hydrophilic moieties were added to the specimen dilution solution to the following concentrations in the reaction solution (75 μL) after addition of the first antibody solution. Surfactants with long carbon chains in the hydrophobic moiety (C12 or longer) were added to the specimen dilution solution at concentrations of 0.05%, 0.27%, and 1.07%, respectively. As a sample with a short carbon chain in the hydrophobic portion (C8), sodium octanoate was added to the specimen dilution solution at concentrations of 0.05%, 0.27%, 1.07%, 2.67%, 3.73%, and 5.33%, and n-octyl-β-D-glucoside was added to the specimen dilution solution at concentrations of 0.05%, 0.27%, and 1.07%. The measurement results for Test Sample 1 are shown in Table 1-1, and the measurement results for Test Sample 2 are shown in Table 1-2.

[0060]

[0061]

[0062] Compared to the results without surfactant addition, an increase in the measured values ​​(counts) was observed when sodium octanoate was added at 0.27% to 3.73% and when n-octyl-β-D-glucoside was added at 0.27% and 1.07%. On the other hand, no increase in the measured values ​​(counts) was observed when other surfactants were added. This indicates that surfactants with a small molecular weight in the hydrophobic part are effective in improving reactivity in sCLEC-2 measurements.

[0063] Example 3: Effect of adding various surfactants with hydrophobic carbon chains shorter than C12 to the reaction solution on the reactivity of sCLEC-2 The procedure of Example 2 was followed, except as noted below. Surfactants with different hydrophobic and hydrophilic structure shown in Table 2 were added to the specimen dilution solution to give concentrations of 0.27% and 1.07% in the reaction solution (75 μL) after addition of the first antibody solution. The measurement results for Test Sample 1 are shown in Table 3-1, and the measurement results for Test Sample 2 are shown in Table 3-2.

[0064]

[0065]

[0066]

[0067] Compared to the results without surfactant addition, an increase in the measured values ​​(counts) was observed under all conditions. The effect was particularly large (more than five times) with HTG, sodium octanoate, MEGA-8, OG, and sodium pelargonate. Hydrophilic types with small molecular weights, such as fatty acids, glucose types, and glucamine types, tended to be more effective.

[0068] Example 4: Effect of adding sulfonates (C5-C8) with different hydrophobic fatty acid carbon chain lengths to the reaction solution on the reactivity of sCLEC-2 The procedure of Example 1 was followed, except as noted below. Sodium hexanesulfonate, sodium heptanesulfonate, and sodium octanesulfonate were added to the specimen diluent so that the concentrations in the reaction solution (75 μL) after addition of the first antibody solution were 0.27%, 1.07%, and 2.67%. The measurement results for Test Sample 1 are shown in Table 4-1, and the measurement results for Test Sample 2 are shown in Table 4-2.

[0069]

[0070]

[0071] Compared to the results without surfactant addition, increases in the measured values ​​(counts) were observed for sodium hexanesulfonate of 1.07% or more, sodium heptanesulfonate of 0.27% to 2.67%, and sodium octanesulfonate of 0.27% and 1.07%. No increase in the measured values ​​(counts) was observed for sodium octanesulfonate of 2.67%.

[0072] Furthermore, sodium octanesulfonate was added to the specimen dilution solution to concentrations of 0.05%, 0.27%, 1.07%, and 2.67%. The measurement results for Test Sample 1 are shown in Table 4-3, and the measurement results for Test Sample 2 are shown in Table 4-4. It was confirmed that the measurement value (count) also increased when 0.05% sodium octanesulfonate was added.

[0073]

[0074]

[0075] Example 5: Effect of Addition of a Chaotropic Substance and a Surfactant with a Non-Linear Hydrophobic Moiety on sCLEC-2 Reactivity to the Reaction Solution The procedure of Example 2 was repeated, except that the chaotropic substance and surfactant were added to the sample dilution solution to the following concentrations, calculated as the concentration in the reaction solution (75 μL) after addition of the first antibody solution. Urea was added to the sample dilution solution as a chaotropic substance to give concentrations of 0.27 mol / L, 1.1 mol / L, and 3.2 mol / L. Sodium salicylate, a surfactant with a cyclic hydrophobic moiety, was added to the sample dilution solution to give concentrations of 0.05%, 0.27%, 1.07%, and 2.67%. Sodium cholate, an anionic surfactant with a steroidal hydrophobic moiety, and CHAPS, a zwitterionic surfactant, were added to the sample dilution solution to give concentrations of 0.05%, 0.27%, 1.07%, and 2.67%, respectively. The measurement results of test sample 1 are shown in Table 5-1, and the measurement results of test sample 2 are shown in Table 5-2.

[0076]

[0077]

[0078] Compared with the results without the addition of surfactant, increases in the measured values ​​(counts) were observed at 0.27% or more for sodium cholate and CHAPS, 2.67% for sodium salicylate, and 3.2 mol / L for urea.

[0079] Example 6: Reduction of sample matrix differences in sCLEC-2 measurement by adding various surfactants in combination. The procedure of Example 2 was followed, with the exceptions noted below. After addition of the first antibody solution, 1.07% sodium octanoate or a combination of 1.07% sodium octanoate and the following surfactants was added to the sample diluent to a concentration of 0.27% or 1.07% in the reaction solution (75 μL): n-heptyl-β-D-thioglucoside (HTG), n-octyl-β-D-glucoside (OG), MEGA-9. The measurement results for Test Sample 1 are shown in Table 6-1, and the measurement results for Test Sample 2 are shown in Table 6-2.

[0080]

[0081] Based on the results in Tables 6-1 and 6-2, the measured values ​​(counts) in Test Sample 1 with the addition of sCLEC-2 are summarized in Table 6-3, and the measured values ​​(counts) in Test Sample 2 with the addition of sCLEC-2 are summarized in Table 6-4. The difference between the measured values ​​in each test sample without the addition of sCLEC-2 (0 pg / mL) and the samples with the addition of sCLEC-2 (500 pg / mL or 1,000 pg / mL) was calculated (Δ counts).

[0082]

[0083]

[0084] The results in Table 6-4 under each measurement condition were divided by the results in Table 6-3 to compare the reactivity of sCLEC-2 measurement in different sample matrices (Table 6-5).

[0085]

[0086] Compared to the condition where sodium octanoate was added alone, the difference in reactivity due to the difference in sample matrix was reduced by combining with other surfactants. The effect of the difference in matrix was even smaller when combined with OG. This demonstrated the feasibility of using multiple surfactants in combination.

[0087] Example 7: Sensitivity Test This test was performed according to the procedure of Example 2, with the exceptions noted below. Sample diluent: Sodium octanoate and n-octyl-β-D-glucoside (OG) were combined and added to the sample diluent so that the concentration in the reaction solution (75 μL) after addition of the first antibody solution was 1.07% and 0.27%, respectively. Test sample: Recombinant hCLEC-2 protein was diluted to 0, 10, 25, 50, or 100 pg / mL using a buffer solution (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA). Test samples at each concentration were measured at a multiplicity of n = 10, and the mean and standard deviation (SD) of the measured values ​​(counts) were calculated and evaluated. The results are shown in Table 7 and Figure 1. The +2SD of 0 pg / mL and the -2SD of 10 pg / mL did not overlap, indicating that 10 pg / mL or more was measurable.

[0088]

[0089] Example 8: Dilution Linearity Test (CLEIA Method) This test was performed according to the procedure of Example 2, with the exceptions noted below. Sample diluent: Sodium octanoate 1.07% and n-octyl-β-D-glucoside (OG) 0.27% were combined and added to the sample diluent. Reference standard: Recombinant hCLEC-2 protein was diluted to 0 and 500 pg / mL using a buffer solution (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA). The sCLEC-2 concentration of the test sample was calculated by comparing the measured value (counts) with that of the reference standard. Test sample: Two types of pooled plasma (Plasma-1: citrated plasma, Plasma-2: EDTA-added plasma) were diluted 2-fold, 4-fold, and 8-fold with physiological saline (Otsuka Pharmaceutical Co., Ltd.). Each test sample was measured in duplicate. The results are shown in Table 8 and Figure 2. Good dilution linearity was observed from the measurement of the undiluted stock solution to an 8-fold dilution.

[0090]

[0091] Example 9: Distribution in Healthy Volunteers (CLEIA Method) Test Sample: 5 mL of citrated plasma collected from a healthy volunteer was centrifuged at 2,000 g for 15 minutes to prepare a test sample. The procedure in Example 2 was repeated, with the exceptions noted below. Sample Diluent: Sodium octanoate 1.07% and n-octyl-β-D-glucoside (OG) 0.27% were combined and added to the sample diluent. Reference Standard: Recombinant hCLEC-2 protein was diluted to 0.300 pg / mL using a buffer solution (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA). The sCLEC-2 concentration of the test sample was calculated by comparing the measured values ​​(counts) with those of the reference standard. The results are shown as STACIA sCLEC-2.

[0092] The results are shown in Table 9. All of the obtained sCLEC-2 measurement results were above the analytical sensitivity shown in Example 7. The mean, standard deviation, and median were 59.1, 16.7, and 54.6 pg / mL, respectively, and the reference range, expressed as the 2.5th to 97.5th percentile values, was 34.0 to 101.7 pg / mL.

[0093]

[0094] When using a measurement method or reagent in a clinical test, it is necessary to set a cutoff value to identify the pathological condition. This involves determining a range that is considered normal based on a reference range. However, in the case of a measurement method or reagent with a large variance, the confidence interval is wide, and the reference range derived from such a sample must be said to be unreliable, and as a result, the set cutoff value also becomes unreliable. Therefore, not only is the sensitivity of measurement by the ELISA method insufficient, but it cannot be said that reliable results can be obtained in terms of measurement accuracy itself.

[0095] Example 10: Effect of adding surfactants with cationic functional groups, zwitterionic functional groups, and branched structures in the hydrophobic moiety to the reaction solution on the reactivity of sCLEC-2 The procedure of Example 2 was repeated, with the exceptions noted below. The following surfactants with different hydrophobic and hydrophilic moieties were added to the sample dilution solution so as to achieve the following concentrations in the reaction solution after the addition of the first antibody solution. n-Octyltrimethylammonium chloride, a surfactant with a cationic functional group, was added to the sample dilution solution so as to achieve concentrations of 0.05%, 0.27%, 1.07%, and 2.67%. Dimethyl(n-octyl)(3-sulfopropyl)ammonium hydroxide inner salt and decyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt, a surfactant with a zwitterionic functional group, were added to the sample dilution solution so as to achieve concentrations of 0.05%, 0.27%, 1.07%, and 2.67%. Sodium 2-ethylhexyl sulfate, a surfactant with a branched hydrophobic structure, was added to the specimen dilution solution at concentrations of 0.05%, 0.27%, 1.07%, and 2.67%. The measurement results for Test Sample 1 are shown in Table 10-1, and the measurement results for Test Sample 2 are shown in Table 10-2.

[0096]

[0097]

[0098] Compared to the results without surfactant addition, an increase in the measured value (count) was observed for n-octyltrimethylammonium chloride from 0.27% to 2.67%. An increase in the measured value (count) was observed for dimethyl(n-octyl)(3-sulfopropyl)ammonium hydroxide inner salt from 1.07% to 2.67%. An increase in the measured value (count) was observed for decyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt from 0.05% to 1.07%, but no increase in the measured value (count) was observed for test sample 1 at 2.67%.

[0099] By using the measurement reagent and measurement method of the present invention, highly sensitive measurements are possible without being affected by measurement-interfering substances. Therefore, it is possible to accurately measure the concentration of a substance to be measured in the blood of a healthy person. Therefore, by using concentration measurement not only for accurate diagnosis but also for selecting an appropriate treatment, monitoring the effectiveness of treatment, and predicting prognosis, highly reliable measurements can be provided, providing extremely useful information for medical treatment.

Claims

1. A measurement reagent for a physiologically active substance, comprising a carrier on which a first immunological partner that binds to a physiologically active substance contained in a biological sample is immobilized, and a second immunological partner that binds to the physiologically active substance, and further comprising a hydrophobic portion that is a linear, branched, or cyclic, saturated or unsaturated hydrocarbon group having 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or an amphipathic substance containing a steroid skeleton, or a chaotropic substance.

2. The measurement reagent according to claim 1, wherein the amphiphilic substance, the hydrophobic portion of which is a linear, branched, or cyclic hydrocarbon group having 6 to 12 carbon atoms, an aromatic hydrocarbon group having 6 to 12 carbon atoms, or a steroid skeleton, is a compound having any one of an anionic functional group, a cationic functional group, an amphoteric functional group, a pyranoside group, a glucamine group, an oxyethylene group, and an aromatic ring which may be substituted.

3. The measuring reagent according to claim 1 or 2, wherein two or more types of the amphipathic substance are used in combination, or the amphipathic substance is used in combination with a chaotropic compound.

4. The measurement reagent according to any one of claims 1 to 3, wherein the concentration of the amphipathic substance in the reaction solution is 0.05% to 5.33%, and the concentration of the chaotropic compound in the reaction solution is 0.27 mol / L to 3.2 mol / L.

5. The measuring reagent according to any one of claims 1 to 4, wherein the item to be measured is soluble CLEC-2.

6. A method for measuring a physiologically active substance contained in a biological sample using the measuring reagent according to any one of claims 1 to 5, comprising the following steps: (1) a step of reacting the physiologically active substance in the sample with a carrier having a first immunological partner that binds to the physiologically active substance immobilized thereon, and a second immunological partner that binds to the physiologically active substance, in the presence of an amphipathic substance, (2) a step of separating the carrier from substances other than the carrier in the reaction solution after step (1), and (3) a step of measuring the second immunological partner on the carrier separated in step (2).