Method for measuring immunosuppressant in whole blood

Abstract

The present invention provides a method for measuring an immunosuppressant in whole blood by immunoassay, the method making it possible to suppress a non-specific reaction. More specifically, the present invention provides a method for measuring an immunosuppressant in whole blood, the method comprising (a) a step for mixing a treatment liquid and whole blood that could contain an immunosuppressant to prepare a mixed liquid, and (b) a step for measuring the concentration of the immunosuppressant in the mixed liquid by immunoassay. The treatment liquid contains an extractant. The treatment liquid furthermore contains a non-ionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5-5 mass% and / or has a pH of 4.8-6.4.

Description

Method for measuring immunosuppressants in whole blood

[0001] The present invention relates to a method for measuring an immunosuppressant in whole blood.

[0002] Immunosuppressants such as cyclosporine and tacrolimus are mostly present in the red blood cell fraction, so whole blood containing the red blood cell fraction is used as the sample for TDM.

[0003] When measuring such immunosuppressants, a pretreatment step is required to extract the immunosuppressants from the red blood cell fraction. Known pretreatment solutions used for extracting immunosuppressants from whole blood include organic solvent-based pretreatment solutions (Patent Document 1) and buffer-based pretreatment solutions (Patent Document 2). Patent Document 3 also describes treating a blood sample with an acid or alkali to improve the efficiency of immunosuppressant extraction.

[0004] JP 2011-508209 A International Publication No. 2009 / 020468 International Publication No. 2019 / 131380

[0005] Organic solvent-based pretreatment solutions require careful handling, etc. Furthermore, when performing measurements using buffer-based pretreatment solutions, especially when measuring fresh whole blood samples, sudden nonspecific reactions (nonspecific reactions sometimes occur and sometimes do not occur with each measurement, even with the same sample) can occur.

[0006] An object of the present invention is to provide a method for measuring immunosuppressants in whole blood by immunoassay, which can prevent non-specific reactions.

[0007] As a result of extensive research, the present inventors have found that by mixing whole blood with a specific treatment liquid containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.4, nonspecific reactions can be suppressed in measuring the concentration of an immunosuppressant in whole blood by immunoassay, and have completed the present invention.

[0008] That is, the present invention provides the following [1] to

[12] . [1] A method for measuring an immunosuppressant in whole blood, comprising the following steps: (a) mixing whole blood that may contain an immunosuppressant with a treatment solution to prepare a mixture, and (b) measuring the concentration of the immunosuppressant in the mixture by immunoassay, wherein the treatment solution contains an extractant, and the treatment solution further contains a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or has a pH of 4.8 to 6.4. [2] The method of [1], wherein the nonionic surfactant having a polyoxyethylene alcohol structure is at least one selected from the group consisting of alcohol ethoxylates and polyoxyethylene-polyoxypropylene block copolymers. [3] The method of [1] or [2], wherein the treatment solution contains the nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.9 to 1.4% by mass. [4] The method according to any one of [1] to [3], wherein the treatment solution further contains a pH buffer. [5] The method according to [4], wherein the pH buffer is at least one selected from the group consisting of 2-morpholinoethanesulfonic acid, phosphoric acid, tartaric acid, citric acid, acetic acid, trifluoroacetic acid, phthalic acid, glycine, and carbonic acid. [6] The method according to any one of [1] to [5], wherein the immunosuppressant is a cyclic polypeptide immunosuppressant or a macrolide immunosuppressant. [7] The method according to any one of [1] to [6], wherein the immunosuppressant is at least one selected from the group consisting of cyclosporine, tacrolimus, everolimus, sirolimus, temsirolimus, zotarolimus, biolimus, novolimus, pimecrolimus, myolimus, and deforolimus. [8] The method according to any one of [1] to [7], wherein the extractant contains a chaotropic denaturant and / or a surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure.[9] The method according to any one of [1] to [8], wherein the non-specific reaction of the immunoassay is suppressed by containing a non-ionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.4.

[10] The method according to any one of [1] to [9], wherein the non-ionic surfactant having a polyoxyethylene alcohol structure is contained at a concentration of 0.5 to 5% by mass and has a pH of 4.8 to 6.4.

[11] A kit for measuring immunosuppressants in whole blood by immunoassay, comprising a treatment liquid, the treatment liquid containing an extractant, and the treatment liquid further containing a non-ionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.4.

[12] A method for inhibiting non-specific reactions in an immunoassay for measuring an immunosuppressant in whole blood, comprising the steps of: (a) mixing whole blood that may contain an immunosuppressant with a treatment liquid to prepare a mixed liquid, wherein the treatment liquid contains an extractant, and the treatment liquid further contains a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass, and / or has a pH of 4.8 to 6.4.

[0009] According to the present invention, non-specific reactions such as spontaneous non-specific reactions can be suppressed when measuring the concentration of an immunosuppressant in whole blood by immunoassay.

[0010] [1. Method for measuring immunosuppressants in whole blood] The present invention provides a method for measuring immunosuppressants in whole blood by immunoassay, comprising the following steps: (a) mixing whole blood that may contain an immunosuppressant with a treatment liquid to prepare a mixture, and (b) measuring the concentration of the immunosuppressant in the mixture by immunoassay.

[0011] [1-1. Step (a)] [Whole Blood] In the present invention, the blood sample serving as the specimen is whole blood that may contain an immunosuppressant. Such whole blood can be collected from an animal to which an immunosuppressant has been administered. The animal from which such whole blood is derived is preferably a mammal (e.g., primates such as humans, monkeys, and chimpanzees; rodents such as mice, rats, and rabbits; and livestock and working animals such as cows, pigs, horses, goats, and sheep), more preferably a primate, and particularly preferably a human. The majority of the immunosuppressant is present in the blood fraction (red blood cell fraction) of whole blood. The whole blood may also be collected from a mammal suffering from a disease for which immunosuppression is desired (e.g., autoimmune disease, rheumatoid arthritis, myasthenia gravis, Crohn's disease, lupus nephritis, active ulcerative colitis, polymyositis, graft-versus-host disease (GVDH), interstitial pneumonia associated with dermatomyositis), or a mammal that has undergone organ, tissue, or cell transplantation (e.g., heart transplant, kidney transplant, bone marrow transplant, lung transplant, liver transplant, pancreas transplant, small intestine transplant). Whole blood collected from such mammals may be subjected to preliminary treatment (e.g., filtration, heating, hemolysis).

[0012] The whole blood may preferably be fresh whole blood. Fresh whole blood refers to a sample collected within 7 days, preferably within 3 days, and more preferably within 1 day. The storage temperature after collection is usually 2 to 30°C, preferably 2 to 10°C.

[0013] Furthermore, the whole blood may be a specimen that has been frozen (frozen specimen) or a specimen that has never been frozen (non-frozen specimen), but preferably a non-frozen specimen. A frozen specimen is a specimen obtained by freezing whole blood and then thawing it. The freezing temperature and freezing time are not particularly limited as long as the specimen is frozen, but the freezing temperature can be, for example, −100 to −10°C, and preferably −90 to −50°C. The freezing time is, for example, 30 minutes or more, 1 hour or more, or 1.5 hours or more. The thawing temperature and thawing time are not particularly limited as long as the frozen specimen is thawed, but the thawing temperature is, for example, room temperature (e.g., 1 to 30°C, 15 to 25°C).

[0014] [Treatment Solution] The treatment solution contains an extractant, and further contains a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5 mass %, and / or has a pH of 4.8 to 6.4. The treatment solution may further contain a pH buffering agent described below.

[0015] In the present invention, the extractant is a substance that can lyse cells and dissociate immunosuppressants from their binding proteins in whole blood to extract them. Note that the extractant in the present invention does not include nonionic surfactants having a polyoxyethylene alcohol structure, which will be described later.

[0016] The extractant preferably contains a chaotropic denaturant and / or a surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure.

[0017] Examples of chaotropic denaturants include urea, thiourea, guanidine hydrochloride, guanidine thiocyanate, sodium salicylate, sodium thiocyanate, sodium perchlorate, acetamide, and formamide, with urea being preferred. As the chaotropic denaturant, one of the above-mentioned components may be used alone, or a mixture of two or more (e.g., two, three, four, or five) may be used.

[0018] The concentration of the extractant in the treatment solution is usually 3% by mass or more, preferably 6% by mass or more, and more preferably 12% by mass or more. The upper limit is usually 63% by mass or less, preferably 46% by mass or less, and more preferably 37% by mass or less. Therefore, the concentration of the extractant in the treatment solution is usually 3 to 63% by mass, preferably 6 to 46% by mass, and more preferably 12 to 37% by mass.

[0019] The concentration of the extractant in the mixture of the treatment liquid and whole blood (hereinafter simply referred to as the "mixture") is typically 2.5% by mass or more, preferably 5% by mass or more, and more preferably 10% by mass or more. The upper limit is typically 52.5% by mass or less, preferably 38.4% by mass or less, and more preferably 30.9% by mass or less. Therefore, the concentration of the extractant in the mixture is typically 2.5 to 52.5% by mass, preferably 5 to 38.4% by mass, and more preferably 10 to 30.9% by mass.

[0020] When the treatment solution contains a chaotropic denaturant, the concentration of the chaotropic denaturant in the treatment solution is usually 0.5 M (3% by mass) or more, preferably 1 M (6% by mass) or more, and more preferably 2 M (12% by mass) or more. The upper limit is usually 8 M (48% by mass) or less, preferably 6 M (36% by mass) or less, and more preferably 5 M (30% by mass) or less. Therefore, the concentration of the chaotropic denaturant in the treatment solution is usually 0.5 to 8 M (3 to 48% by mass), preferably 1 to 6 M (6 to 36% by mass), and more preferably 2 to 5 M (12 to 30% by mass). When the concentration of the chaotropic denaturant satisfies the above numerical range, the effects of the present invention can be more suitably exhibited.

[0021] When the treatment solution contains a chaotropic denaturant, the concentration of the chaotropic denaturant in the mixture is usually 0.4 M (2.5% by mass) or more, preferably 0.8 M (5% by mass) or more, and more preferably 1.6 M (10% by mass) or more. The upper limit is usually 6.7 M (40% by mass) or less, preferably 5 M (30% by mass) or less, and more preferably 4.2 M (25% by mass) or less. Therefore, the concentration of the chaotropic denaturant in the mixture is usually 0.4 to 6.7 M (2.5 to 40% by mass), preferably 0.8 to 5 M (5 to 30% by mass), and more preferably 1.6 to 4.2 M (10 to 25% by mass). When the concentration of the chaotropic denaturant satisfies the above numerical range, the effects of the present invention can be more suitably exhibited.

[0022] Examples of surfactants other than nonionic surfactants having a polyoxyethylene alcohol structure include nonionic surfactants other than nonionic surfactants having a polyoxyethylene alcohol structure, cationic surfactants, anionic surfactants, and zwitterionic surfactants.

[0023] The nonionic surfactant other than the nonionic surfactant having a polyoxyethylene alcohol structure may be any nonionic surfactant other than the components exemplified in the section below titled "Nonionic surfactant having a polyoxyethylene alcohol structure," and examples thereof include N-D-gluco-N-methylalkanamides (e.g., the MEGA series (e.g., octanoyl-N-methylglucamide (MEGA 8) and decanoyl-N-methylglucamide (MEGA 10))).

[0024] Examples of cationic surfactants include quaternary ammonium salts. Examples of salts include salts with halogens (e.g., fluorine, chlorine, bromine, iodine). Examples of quaternary ammonium salts include Cn alkyl trimethyl ammonium bromide (CnTAB) and Cn alkyl trimethyl ammonium chloride (CnTAC). Examples of CnTAB include octyl trimethyl ammonium bromide (C8TAB), nonyl trimethyl ammonium bromide (C9TAB), dodecyl trimethyl ammonium bromide (C12TAB), tetradecyl trimethyl ammonium bromide (C14TAB), and hexadecyl trimethyl ammonium bromide (C16TAB). Examples of CnTAC include dodecyltrimethylammonium chloride (C12TAC), tetradecyltrimethylammonium chloride (C14TAC), hexadecyltrimethylammonium chloride (C16TAC), and octadecyltrimethylammonium chloride (C18TAC).

[0025] Examples of anionic surfactants include carboxylic acid surfactants (e.g., N-decanoyl sarcosinate sodium (NDS), N-lauroyl sarcosinate sodium hydrate (NLS)), sulfonic acid surfactants (e.g., 1-sodium nonanesulfonate (NSS), sodium dodecylbenzenesulfonate (SDBS)), carboxylic acid-sulfonic acid surfactants (e.g., sodium chondroitin sulfate (CSSS)), and sulfate ester surfactants (e.g., sodium dodecyl sulfate (SDS)).

[0026] Examples of the zwitterionic surfactant include quaternary ammonium-sulfonic acid surfactants. Examples of the quaternary ammonium-sulfonic acid surfactant include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), 3-(N,N-dimethyloctylammonio)propanesulfonate (C8APS), 3-(decyldimethylammonio)propanesulfonate (C10APS), and N-dodecyl-N,N-dimethyl-3-ammonio. ammonium propanesulfonate (NDSB-195), 3-[dimethyl-(2-hydroxyethyl)ammonio]-1-propanesulfonate (NDSB-211), and 3-(benzenedimethylammonio)propanesulfonate (NDSB-256).

[0027] The surfactant other than the nonionic surfactant having a polyoxyethylene alcohol structure is preferably a nonionic surfactant other than the nonionic surfactant having a polyoxyethylene alcohol structure, or an amphoteric surfactant, more preferably N-D-gluco-N-methylalkanamide, a quaternary ammonium-sulfonic acid surfactant, and even more preferably octanoyl-N-methylglucamide (MEGA8), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). As the surfactant other than the nonionic surfactant having a polyoxyethylene alcohol structure, one of the above-mentioned components may be used alone, or a mixture of two or more (e.g., two, three, four, or five) may be used in combination.

[0028] When the treatment liquid contains a surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure, the concentration of the surfactant in the treatment liquid is typically 0.01% by mass or more, preferably 0.1% by mass or more, and more preferably 1% by mass or more. The upper limit is typically 15% by mass or less, preferably 10% by mass or less, and more preferably 7% by mass or less. Therefore, the concentration of the surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure in the treatment liquid is typically 0.01 to 15% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 7% by mass. By ensuring that the concentration of the surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure in the treatment liquid satisfies the above numerical range, the effects of the present invention can be more suitably exhibited.

[0029] When the treatment liquid contains a surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure, the concentration of the surfactant in the mixture is typically 0.008% by mass or more, preferably 0.08% by mass or more, and more preferably 0.8% by mass or more. The upper limit is typically 12.5% ​​by mass or less, preferably 8.4% by mass or less, and more preferably 5.9% by mass or less. Therefore, the concentration of the surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure in the mixture is typically 0.008 to 12.5% ​​by mass, preferably 0.08 to 8.4% by mass, and more preferably 0.8 to 5.9% by mass. When the concentration of the surfactant other than a nonionic surfactant having a polyoxyethylene alcohol structure in the mixture satisfies the above numerical range, the effects of the present invention can be more suitably exhibited.

[0030] -Nonionic surfactant having a polyoxyethylene alcohol structure- The nonionic surfactant having a polyoxyethylene alcohol structure is preferably a nonionic surfactant having a -O-(-CH ２ -CH ２ -O-) ｘ The compound is a linear or branched-chain compound (a compound not containing a cyclic structure) containing a structure represented by -H (a polyoxyethylene alcohol structure). x may be an integer of 1 or greater, preferably an integer of 5 or greater, more preferably an integer of 15 or greater, and even more preferably an integer of 20 or greater. x may also be an integer of 400 or less, preferably an integer of 350 or less, more preferably an integer of 300 or less, even more preferably an integer of 280 or less, and particularly preferably an integer of 270 or less. More specifically, x may be an integer of 1 to 400, preferably an integer of 5 to 350, more preferably an integer of 15 to 300, even more preferably an integer of 20 to 280, and particularly preferably an integer of 20 to 270. In this specification, the value of x represents an average value.

[0031] The nonionic surfactant having a polyoxyethylene alcohol structure may be composed of only a single bond or may contain an unsaturated bond (double / triple bond), but a compound composed of only a single bond is preferred. Such a compound may contain, as a structure other than the polyoxyethylene alcohol structure, for example, an alkyl structure (linear or branched chain) and / or a polyoxyalkylene structure (linear or branched chain). The alkyl structure may be, for example, C ６～３１ It may be an alkyl structure, ６～３１ A linear alkyl structure is preferred. The alkylene in the polyoxyalkylene structure is, for example, C １～６ It may be alkylene. １～６ Examples of alkylene include C １ Alkylene (methylene), C ２ Alkylene (ethylene group, ethylidene group), C ３ Alkylene (propylidene, propylene, trimethylene, isopropylidene), C ４ Alkylene (e.g., tetramethylene), C ５ Alkylene (e.g., pentamethylene), C ６ Alkylene (e.g., hexamethylene) is an example. Such compounds may consist of carbon, hydrogen, and oxygen atoms.

[0032] More specifically, examples of nonionic surfactants having a polyoxyethylene alcohol structure include alcohol ethoxylates and polyoxyethylene-polyoxyalkylene block copolymers (e.g., polyoxyethylene-polyoxypropylene block copolymers).

[0033] Alcohol ethoxylate, also known as poly(oxyethylene) alkyl ether, is a compound in which a hydrophilic polyoxyethylene (POE) chain and a hydrophobic alkyl group are bonded via an ether bond. Alcohol ethoxylate can be represented by the following formula (1):

[0034] (wherein x1 is an integer of 1 or more; y1 is an integer of 0 or more; z1 is an integer of 0 or more; and y1≧z1.) In this specification, the values ​​of x1, y1, and z1 represent average values.

[0035] x1 may be an integer of 1 or greater, preferably an integer of 5 or greater, more preferably an integer of 10 or greater, even more preferably an integer of 15 or greater, and particularly preferably an integer of 20 or greater. x1 may also be an integer of 300 or less, preferably an integer of 250 or less, more preferably an integer of 200 or less, even more preferably an integer of 150 or less, and particularly preferably an integer of 100 or less, 80 or less, 60 or less, or 50 or less. More specifically, x1 may be an integer of 1 to 300, preferably an integer of 5 to 250, more preferably an integer of 10 to 200, even more preferably an integer of 15 to 150, and particularly preferably an integer of 20 to 100, 20 to 80, 20 to 60, or 20 to 50.

[0036] y1 is an integer of 0 or greater, preferably an integer of 1 or greater, and more preferably an integer of 5 or greater. y1 may also be an integer of 30 or less, preferably an integer of 20 or less, and even more preferably an integer of 13 or less. More specifically, y1 may be an integer of 0 to 30, preferably an integer of 1 to 20, and even more preferably an integer of 5 to 15.

[0037] z1 is an integer of 0 or greater, and may be an integer of 1 or greater, but is preferably 0. z1 may also be an integer of 15 or less, preferably an integer of 10 or less, or 7 or less. More specifically, z1 is an integer from 0 to 15, preferably an integer from 1 to 10, more preferably an integer from 0 to 7, and even more preferably 0.

[0038] Preferably, y1 and z1 may satisfy the relationship y1≧z1. Also, y1 and z1 may satisfy the relationship that y1+z1 is an integer of 5 to 30. Preferably, y1+z1 may satisfy the relationship that y1+z1 is an integer of 10 to 14.

[0039] The alcohol ethoxylate may be either a branched chain alcohol ethoxylate or a straight chain alcohol ethoxylate. Preferably, the alcohol ethoxylate may be a straight chain alcohol ethoxylate.

[0040] Branched-chain alcohol ethoxylates correspond to compounds represented by the above formula (1) in which y1 is an integer of 1 or greater and z1 is an integer of 1 or greater. Examples of such branched-chain alcohol ethoxylates include polyoxyethylene (40) sec-tridecyl ether. Specific examples of such branched-chain alcohol ethoxylates include compounds in the TERGITOL (registered trademark) series, such as Tergitol 15-S-40.

[0041] A linear alcohol ethoxylate corresponds to a compound represented by the above formula (1) in which z1 is 0. Specific examples of linear alcohol ethoxylate include compounds in the BRIJ (registered trademark) series such as Brij 58 and Brij S 100. A linear alcohol ethoxylate can also be represented by the following formula (1'):

[0042] (In the formula, x1 is an integer of 1 or more; and y1 is an integer of 1 or more.)

[0043] Preferred examples of x1 and y1 in formula (1') are the same as the preferred examples of x1 and y1 in formula (1), respectively.

[0044] Preferably, the compound represented by formula (1) is as follows: x1 is an integer of 40 to 100; y1 is an integer of 0 or greater; z1 is an integer of 0 or greater; y1≧z1; and y1+z1 is an integer of 5 to 30. (The preferred ranges for x1, y1, and z1 are the same as those described above.)

[0045] More specifically, examples of alcohol ethoxylates corresponding to the compound represented by formula (1) include Brij 58, Tergitol 15-S-40, and Brij S 100. Preferably, the alcohol ethoxylate is Brij 58.

[0046] A "polyoxyethylene-polyoxyalkylene block copolymer" is a block copolymer containing a polyoxyethylene block and a polyoxyalkylene block. The alkylene in the polyoxyalkylene block is, for example, C １～６ It may be alkylene, for example, C １ Alkylene (methylene), C ２ Alkylene (ethylene, ethylidene), C ３ Alkylene (propylidene, propylene, trimethylene, isopropylidene), C ４ Alkylene (e.g., tetramethylene), C ５ Alkylene (e.g., pentamethylene), C ６ Examples of the polyoxyethylene-polyoxyalkylene block copolymer include a block copolymer having a structure of HO-[polyoxyethylene block]-[polyoxyalkylene block]-[polyoxyethylene block]-OH. The polyoxyethylene-polyoxyalkylene block copolymer may preferably be a polyoxyethylene-polyoxypropylene block copolymer.

[0047] The "polyoxyethylene-polyoxypropylene block copolymer" is a compound represented by the following formula (2).

[0048] (wherein x2, y2, and z2 are integers of 1 or more) In this specification, the values ​​of x2, y2, and z2 represent average values.

[0049] x2 and z2 are each an integer of 1 or greater. The sum of x2 and z2 may be an integer of 2 or greater, preferably an integer of 20 or greater, more preferably an integer of 80 or greater, and particularly preferably an integer of 150 or greater. The sum of x2 and z2 may also be an integer of 400 or less, preferably an integer of 350 or less, more preferably an integer of 300 or less, and particularly preferably an integer of 270 or less. More specifically, the sum of x2 and z2 may be an integer of 2 to 400, preferably an integer of 20 to 350, more preferably an integer of 80 to 300, and particularly preferably an integer of 150 to 270.

[0050] y2 may be an integer of 1 or greater, preferably an integer of 5 or greater, more preferably an integer of 10 or greater, even more preferably an integer of 15 or greater, and particularly preferably an integer of 20 or greater. y2 may also be an integer of 200 or less, preferably an integer of 150 or less, more preferably an integer of 100 or less, even more preferably an integer of 80 or less, and particularly preferably an integer of 70 or less. More specifically, x1 may be an integer of 1 to 200, preferably an integer of 5 to 150, more preferably an integer of 10 to 100, even more preferably an integer of 15 to 80 or less, and particularly preferably an integer of 20 to 70.

[0051] Examples of polyoxyethylene-polyoxypropylene block copolymers include poloxamer 188, poloxamer 388, and poloxamer 407. Specific examples of polyoxyethylene-polyoxypropylene block copolymers include compounds in the PLURONIC (registered trademark) series, such as Pluronic F68, Pluronic F108, and Pluronic F127.

[0052] Preferably, the compound represented by formula (2) is as follows: x2 is an integer of 1 or more; y2 is an integer of 15 to 80; z2 is an integer of 1 or more; and x2+z2 is an integer of 80 to 280. (The preferred ranges of x2, y2, and z2 are the same as those described above.)

[0053] More specifically, examples of polyoxyethylene-polyoxypropylene block copolymers corresponding to the compound represented by formula (2) include poloxamer 188 (e.g., Pluronic F68), poloxamer 108 (e.g., Pluronic F38), poloxamer 217 (e.g., Pluronic F77), poloxamer 237 (e.g., Pluronic F87), poloxamer 238 (e.g., Pluronic F88), poloxamer 288 (e.g., Pluronic F98), poloxamer 388 (e.g., Pluronic F108), and poloxamer 407 (e.g., Pluronic F127). Preferably, the polyoxyethylene-polyoxypropylene block copolymer is poloxamer 188 (eg, Pluronic F68) or poloxamer 388 (eg, Pluronic F108).

[0054] For reference, the relationship between the nonionic surfactant (alcohol ethoxylate / polyoxyethylene-polyoxypropylene block copolymer) and the compound represented by the above formula (1) or (2) is shown below.

[0055]

[0056]

[0057] The average molecular weight of the nonionic surfactant having a polyoxyethylene alcohol structure is preferably 900 or more, more preferably 1000 or more, and even more preferably 1100 or more. The upper limit is preferably 18000 or less, more preferably 15000 or less, and even more preferably 14800 or less. Therefore, the average molecular weight of the nonionic surfactant having a polyoxyethylene alcohol structure is preferably 900 to 18000, more preferably 1000 to 15000, and even more preferably 1100 to 14800. By having the average molecular weight satisfy the above numerical range, the effects of the present invention can be more suitably exhibited. Note that, unless otherwise specified, "average molecular weight" means weight average molecular weight.

[0058] The nonionic surfactant having a polyoxyethylene alcohol structure is preferably an alcohol ethoxylate or a polyoxyethylene-polyoxypropylene block copolymer, more preferably polyoxyethylene (20) cetyl ether, poloxamer 188, or poloxamer 388. Specific examples of such nonionic surfactants include compounds of the PLURONIC (registered trademark) series such as Pluronic F-68 and Pluronic F-108, and compounds of the BRIJ (registered trademark) series such as Brij 58.

[0059] As the nonionic surfactant having a polyoxyethylene alcohol structure, one type of nonionic surfactant may be used, or multiple types (e.g., two, three, or four) of nonionic surfactants may be used in combination.

[0060] The concentration of the nonionic surfactant having a polyoxyethylene alcohol structure in the treatment solution is preferably 0.5% by mass or more, more preferably 0.8% by mass or more, even more preferably 0.9% by mass or more, and even more preferably 1.0% by mass or more (it may exceed 1.0% by mass if necessary). This allows for suppression of nonspecific reactions in immunoassays. The upper limit is preferably 5.0% by mass or less, more preferably 4.0% by mass or less, even more preferably 3.0% by mass or less, even more preferably 2.0% by mass or less, and most preferably 1.4% by mass or less. This allows for prevention of a decrease in detection sensitivity in immunoassays. Therefore, the concentration of the nonionic surfactant having a polyoxyethylene alcohol structure in the treatment solution is preferably 0.5 to 5.0% by mass, more preferably 0.8 to 4.0% by mass, even more preferably 0.9 to 3.0% by mass, even more preferably 1.0 to 2.0% by mass, and even more preferably 1.0 to 1.4% by mass.

[0061] The concentration of the nonionic surfactant having a polyoxyethylene alcohol structure in the mixed solution is preferably 0.4% by mass or more, more preferably 0.6% by mass or more, even more preferably 0.75% by mass or more, even more preferably 0.8% by mass or more, and most preferably 0.9% by mass or more. This allows for suppression of nonspecific reactions in immunoassays. The upper limit is preferably 4.2% by mass or less, more preferably 3.4% by mass or less, even more preferably 2.5% by mass or less, even more preferably 1.7% by mass or less, and most preferably 1.2% by mass or less. This allows for prevention of a decrease in detection sensitivity in immunoassays. Therefore, the concentration of the nonionic surfactant having a polyoxyethylene alcohol structure in the mixed solution is preferably 0.4 to 4.2% by mass, more preferably 0.6 to 3.4% by mass, even more preferably 0.75 to 3.4% by mass, even more preferably 0.8 to 2.5% by mass, even more preferably 0.8 to 1.7% by mass, and most preferably 0.9 to 1.2% by mass.

[0062] - pH - The pH of the treatment solution is preferably 4.8 or higher, more preferably 5.5 or higher, even more preferably 6.0 or higher, and most preferably 6.1 or higher. By ensuring that the lower limit of the pH satisfies the above-mentioned numerical range, non-specific reactions can be suppressed. Furthermore, the upper limit is preferably 6.4 or lower, more preferably 6.3 or lower, and most preferably 6.2 or lower. By ensuring that the upper limit of the pH satisfies the above-mentioned numerical range, a decrease in sensitivity in immunoassays can be prevented. Therefore, the pH of the treatment solution is preferably 4.8 to 6.4, more preferably 5.5 to 6.3, even more preferably 6.0 to 6.3, and most preferably 6.1 to 6.2.

[0063] The pH can be measured by a method known in the art. Preferably, the pH is measured at 25° C. using a pH meter with a glass electrode.

[0064] - pH Buffer - The pH of the treatment solution can be adjusted using a pH buffer. That is, the treatment solution may further contain a pH buffer. Examples of pH buffers include 2-morpholinoethanesulfonic acid (MES), phosphoric acid, tartaric acid, citric acid, acetic acid, trifluoroacetic acid, phthalic acid, glycine, and carbonic acid. Of these, 2-morpholinoethanesulfonic acid is preferred.

[0065] [Immunosuppressants] Immunosuppressants are not particularly limited as long as they are compounds that have the effect of reducing or inhibiting the activity of the immune system, but cyclic polypeptide immunosuppressants or macrolide immunosuppressants are preferred. Macrolide immunosuppressants contain a macrolide structure (a cyclic lactone structure consisting of 12 or more atoms).

[0066] Examples of cyclic polypeptide immunosuppressants include cyclosporine (cyclosporine A). Examples of macrolide immunosuppressants include tacrolimus (FK506), sirolimus (rapamycin), and derivatives thereof (e.g., everolimus, temsirolimus), zotarolimus, biolimus, novolimus, pimecrolimus, myolimus, myolimus, and deforolimus.

[0067] Of the above immunosuppressants, cyclosporine, tacrolimus, sirolimus, everolimus, temsirolimus, zotarolimus, biolimus, novolimus, pimecrolimus, myolimus, myolimus, and deforolimus are preferred, and cyclosporine is more preferred.

[0068] -Mixing Treatment- In step (a), the whole blood is mixed with the treatment solution. This allows the immunosuppressant that may be contained in the whole blood to be extracted from the whole blood (red blood cell fraction). Mixing can be performed in any manner. For example, mixing can be performed by inversion and / or stirring, with stirring being preferred. The order in which the whole blood and treatment solution are mixed is not particularly limited. The mixing time is not particularly limited as long as it is a time sufficient for mixing the whole blood and treatment solution, and may be, for example, 1 second to 5 minutes, or 10 seconds to 1 minute. The temperature during mixing may be, for example, 4 to 37°C, or 15 to 30°C. After mixing the whole blood and treatment solution, the mixture may be incubated. The incubation time may be, for example, 1 second to 60 minutes, preferably 5 seconds to 20 minutes. The temperature during incubation is not particularly limited, but is 20 to 45°C, preferably 30 to 39°C.

[0069] [1-2. Step (b)] [Immunoassay] In immunoassay, the concentration of the immunosuppressant in the mixture is measured using an antibody.

[0070] Immunoassays include, for example, enzyme immunoassays (EIA) (e.g., chemiluminescence EIA (CLEIA), enzyme-adsorbed EIA (ELISA)), fluorescent immunoassays, chemiluminescence immunoassays, electrochemiluminescence immunoassays, agglutination assays, immunostaining, flowmetry, biolayer interferometry, in situ PLA, chemically amplified luminescence proximity homogeneous assays, line blotting, and Western blotting. Of these, sandwich-type immunoassays (enzyme-adsorbed EIA (ELISA)) are preferred.

[0071] The antibody used in the present invention may be any antibody against the immunosuppressant to be measured, and is not particularly limited. Preferably, the antibody is an antibody against a cyclic polypeptide immunosuppressant or a macrolide immunosuppressant. Such an antibody may be either a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody. The antibody may be of any isotype of immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE, IgY, HCAb). The antibody may also be a full-length antibody. A full-length antibody refers to an antibody comprising a heavy chain and a light chain, each of which comprises a variable region and a constant region (e.g., an antibody comprising two Fab portions and an Fc portion). The antibody may also be an antibody fragment derived from such a full-length antibody. An antibody fragment is a portion of a full-length antibody, and may be, for example, a constant region-deleted antibody (e.g., F(ab') ２ , Fab', Fab, Fv, VHH). The antibody may also be a modified antibody such as a single-chain antibody. Preferably, the antibody may be IgG, IgM, or an antibody fragment thereof. In the present invention, not only one type of antibody but also two or more types (e.g., two or three types) of antibodies may be used.

[0072] Antibodies against the immunosuppressant of interest can be produced using any method known in the art. For example, antibodies can be produced using the immunosuppressant of interest as an antigen. In addition, if antibodies against the immunosuppressant of interest are commercially available, such commercially available products can also be used.

[0073] In the present invention, one or more (e.g., one or two) antibodies against the immunosuppressant of interest can be used. In the present invention, a secondary antibody against the antibody can also be used.

[0074] The antibody may be immobilized on a solid phase. Herein, an antibody immobilized on a solid phase may be simply referred to as a solid-phase antibody. Examples of solid phases include solid phases that can accommodate or carry a liquid phase (e.g., containers such as well plates, microchannels, glass capillaries, nanopillars, monolith columns, and tubes; and supports such as plates, membranes, and filter paper), as well as solid phases that can be suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles). Examples of solid-phase materials include glass, plastic, metal, and carbon. Nonmagnetic or magnetic materials can also be used as solid-phase materials, but magnetic materials are preferred for solid-phase carriers from the standpoint of ease of operation. Any method known in the art can be used to immobilize an antibody on a solid phase. Examples of such methods include physical adsorption, covalent bonding, methods using affinity substances (e.g., biotin, streptavidin), and ionic bonding.

[0075] The antibody may be labeled with a labeling substance. In this specification, an antibody labeled with a labeling substance may be simply referred to as a labeled antibody. Examples of the labeling substance include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin), fluorescent substances or proteins (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), light-emitting or light-absorbing substances (e.g., luciferin, aequorin, acridinium), radioactive substances (e.g., ３ H. １４ C. ３２ P. ３５ S. １２５ I) can be mentioned.

[0076] Steps (a) and (b) may be performed in parallel or separately. For example, when an antibody against the immunosuppressant described above is used in mixing whole blood and a treatment solution, extraction of the immunosuppressant from the red blood cell fraction with the treatment solution and measurement of the immunosuppressant concentration by antibody-based immunoassay can be performed in parallel. For example, whole blood, a treatment solution, and an antibody against the immunosuppressant described above may be mixed simultaneously. A second antibody (e.g., a labeled antibody) against the immunosuppressant different from the first antibody (e.g., a solid-phase antibody) against the immunosuppressant may also be used. On the other hand, for example, when whole blood, a treatment solution, and an antibody against the immunosuppressant described above are not mixed simultaneously, extraction of the immunosuppressant with the treatment solution and measurement of the immunosuppressant concentration can be performed separately. In this case, steps (a) and (b) can be performed continuously or discontinuously. When steps (a) and (b) are performed discontinuously, step (a) is usually performed first, followed by step (b). In step (a), the whole blood mixed with the treatment solution may be subjected to further treatment, such as pretreatment (e.g., filtration, heating, hemolysis), pH change, or treatment with a specific chemical.

[0077] In a specific embodiment, the method of the present invention may include the following steps: (a) mixing whole blood containing an immunosuppressant with a treatment solution to prepare a mixture, and (b) measuring the concentration of the immunosuppressant in the mixture by immunoassay. Such a method is useful for measuring the count of the immunosuppressant in whole blood containing the immunosuppressant in a method for measuring an immunosuppressant in whole blood.

[0078] In another specific embodiment, the method of the present invention may comprise the steps of: (a) mixing immunosuppressant-free whole blood with a treatment solution to prepare a mixture, and (b) measuring the concentration of the immunosuppressant in the mixture by immunoassay. Such a method is useful for measuring background counts in immunosuppressant-free whole blood in a method for measuring an immunosuppressant in whole blood.

[0079] [2. Kit for Measuring Immunosuppressants] The present invention also provides a kit for measuring immunosuppressants in whole blood by immunoassay, which includes a treatment solution that contains an extractant and further satisfies the following requirements: a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.1 to 5% by mass; and / or a pH of 4.8 to 6.4.

[0080] The nonionic surfactant having a polyoxyethylene alcohol structure, the extractant, and the pH conditions of the treatment solution are as described above. The kit of the present invention may also include a pH buffer. The pH buffer is as described above. The kit of the present invention may further include an antibody against an immunosuppressant. The antibody against an immunosuppressant is as described above.

[0081] The kit of the present invention may also contain an immunosuppressant (sample) to be measured. The immunosuppressant can be used, for example, as a positive control and / or to prepare a calibration curve for quantification.

[0082] The reagent of the present invention may further contain components necessary for immunoassay, such as the above-mentioned labeling substance and enzyme, their substrates, diluents, secondary antibodies and antibody stabilizers, as well as instruments capable of collecting samples from mammals (e.g., syringes, biopsy needles).

[0083] 3. Method for inhibiting non-specific reactions in immunoassays for measuring immunosuppressants in whole blood The present invention also provides a method for inhibiting non-specific reactions in immunoassays for measuring immunosuppressants in whole blood, comprising the steps of: (a) mixing whole blood that may contain an immunosuppressant with a treatment liquid to prepare a mixture;

[0084] The above method can be carried out in the same manner as step (a) in the method for measuring an immunosuppressant in whole blood. Therefore, whole blood that may contain an immunosuppressant may be whole blood containing an immunosuppressant or whole blood not containing an immunosuppressant. The above method is useful for measuring counts for each type of whole blood. Therefore, the expression "method for measuring an immunosuppressant in whole blood" used in the present invention includes not only determining the concentration of an immunosuppressant contained in whole blood, but also determining the presence or absence of an immunosuppressant in whole blood.

[0085] The present invention will be described below with reference to examples, which are not intended to limit the scope of the present invention.

[0086] <Materials used in Test Examples 1 to 4> Measuring instrument: Lumipulse (registered trademark) L2400 (Fujirebio Inc.) Measuring reagents: Buffer solution containing 0.04% ferrite particles bound to anti-cyclosporine monoclonal antibody (mouse) (particle solution) Buffer solution containing 1 μg / mL of ALP-labeled anti-cyclosporine immune complex monoclonal antibody (alpaca) (labeled body fluid) Lumipulse Presto reagents (substrate solution, washing solution)

[0087] (1) Test Example 1 (1-1) Treatment Solution and Measurement Object Used in Test Example 1 The treatment solution used in Test Example 1 is as follows. Treatment solution 1-A: 100 mM MES (component (C)), 4 M urea (component (B-1)), 6.0% CHAPS (component (B-2)), 0.75% MEGA8 (component (B-2)), 0.1% ProClin 300, 0.005% Antifoam SI, pH 6.5 Treatment solution 1-B: Treatment solution 1-A adjusted to pH 6.0 Treatment solution 1-C: Treatment solution 1-A to which 2.2% Brij (registered trademark) 58 (component (A)) was added Treatment solution 1-D: Treatment solution 1-A to which 0.338% sodium taurocholate was added Treatment solution 1-E: Treatment solution 1-B to which 2.2% Brij 58 and 0.338% sodium taurocholate were added

[0088] The measurement objects used in Test Example 1 are as follows. Measurement objects: phosphate buffer solution containing cyclosporine (standard solution), fresh whole blood sample (EDTA2Na) not containing cyclosporine (sample not containing cyclosporine, 4 days after blood collection, storage temperature 2-8°C).

[0089] (1-2) Experimental Method The following reactions and measurements were performed using a Lumipulse L2400. Specifically, the samples to be measured were treated with a treatment solution, followed by immunoassay (n = 2 for each dilution series of labeled body fluids, and n = 2 for each cyclosporine-free sample (samples 1 to 9)). (1) 20 μL of the samples to be measured (standard solution and cyclosporine-free sample) were dispensed into 100 μL of treatment solution, stirred, and then incubated at 37°C for 6.5 minutes. For the standard solution, phosphate buffer was added to dilute the cyclosporine to concentrations of 30, 100, 300, 800, and 2000 ng / mL, and 20 μL of each was used as the samples to prepare a dilution series (Cal 0 to 2000). (2) 50 μL of the particle solution was dispensed into the reaction solution in (1), stirred, and then incubated at 37°C for 8 minutes. (3) B / F separation was performed using a magnet, the reaction solution was removed, and the antibody-bound particles were washed with washing solution. (4) 50 μL of labeled body fluid was mixed with the antibody-bound particles washed in (3) and incubated at 37°C for 8 minutes. (5) B / F separation was performed using a magnet, the reaction solution was removed, and the antibody-bound particles were washed with washing solution. (6) 200 μL of substrate solution was added to the antibody-bound particles washed in (5), and the reaction was allowed to proceed at 37°C for 4 minutes. (7) The amount of luminescence (counts) at a wavelength of 463 nm was measured. A Lumipulse L2400 was used to measure the amount of luminescence. The results of the luminescence measurement are shown in Table 1.

[0090] The method for calculating the measured values ​​(ng / mL) of cyclosporine in whole blood samples is as follows. A calibration curve was drawn using the standard solution, and the measured values ​​of samples containing no cyclosporine were calculated. More specifically, a linear approximation formula for the calibration curve was obtained from the counts obtained when measuring the luminescence intensity of the standard solutions obtained by steps (1) to (7) above and the cyclosporine concentrations set in the dilution series in step (1). Using the linear approximation formula for the calibration curve, the measured values ​​(ng / mL) of cyclosporine were calculated based on the count values ​​obtained when measuring samples containing no cyclosporine. The results are shown in Table 2.

[0091] Non-specific reactions were evaluated as follows. A measurement value of 30 ng / mL or higher in the whole blood sample was defined as a non-specific reaction occurring, and the occurrence of non-specific reactions was evaluated. More specifically, the proportion of samples in which the measurement value of the whole blood sample was 30 ng / mL or higher was calculated as the frequency of non-specific reactions. The results are shown in Table 2.

[0092]

[0093]

[0094] [Notes for Table 2] *: "Sensitivity (30 / 0)" is the ratio of the average luminescence amount at Cal 30 in Table 1 to the average luminescence amount at Cal 0. The same applies to Tables 5, 6 and 9 below.

[0095] (1-3) Results Non-specific reactions occurred with treatment solution 1-A, whereas the occurrence of non-specific reactions was suppressed with treatment solutions 1-B, 1-C, and 1-E. On the other hand, treatment solution 1-D, which used sodium taurocholate instead of Brij58, was unable to suppress non-specific reactions.

[0096] (2) Test Example 2 (2-1) Treatment Solution and Measurement Object Used in Test Example 2 The treatment solution used in Test Example 2 is as follows. Treatment solution 2-A: 100 mM MES (component (C)), 4 M urea (component (B-1)), 6.0% CHAPS (component (B-2)), 0.75% MEGA8 (component (B-2)), 0.1% ProClin 300, 0.005% Antifoam SI, pH 6.5 Treatment solution 2-B: Treatment solution 2-A with the pH adjusted to 5.8 Treatment solution 2-C: Treatment solution 2-A with the pH adjusted to 6.0 Treatment solution 2-D: Treatment solution 2-A with the pH adjusted to 6.2 Treatment solution 2-E: Treatment solution 2-A with 1.0% Pluronic (registered trademark) F-68 (component (A)) added Treatment solution 2-F: Treatment solution 2-A with 1.0% Pluronic F-108 (component (A)) added

[0097] The measurement objects used in Test Example 2 are as follows. Measurement objects: phosphate buffer solution containing cyclosporine (standard solution), fresh whole blood sample (EDTA2Na) not containing cyclosporine (sample not containing cyclosporine, 5 days after blood collection, storage temperature 2-8°C), sample obtained by freezing the whole blood sample in a -80°C freezer for 2 hours and then thawing it in a water bath at room temperature (frozen sample not containing cyclosporine).

[0098] (2-2) Experimental Method The same procedures as in Test Example 1 were carried out to measure the amount of luminescence, except that the above-mentioned treatment solutions 2-A to 2-F were used as treatment solutions, and that the above-mentioned standard solutions, cyclosporine-free samples, and also cyclosporine-free frozen samples were used as the measurement objects (each dilution series of the standard solution: n=3, each cyclosporine-free sample (samples 10 to 19): n=2, each cyclosporine-free frozen sample (frozen samples 10 to 19): n=1). The results are shown in Tables 3 and 4.

[0099] Furthermore, the measured cyclosporine values ​​(ng / mL) were calculated based on the count values ​​obtained from the measurement of the cyclosporine-free sample and the cyclosporine-free frozen sample using the linear approximation of the calibration curve obtained from the count values ​​obtained from the measurement of the standard solution, in the same manner as in Test Example 1. The results are shown in Table 5.

[0100] The sensitivity (30 / 0) and the frequency of non-specific reactions were calculated for the cyclosporine-free samples and the cyclosporine-free frozen samples in the same manner as in Test Example 1. The results are shown in Table 5.

[0101]

[0102]

[0103]

[0104] (2-3) Results Non-specific reactions occurred in treatment solution 2-A, which had a pH of 6.5 and did not contain a non-ionic surfactant having a polyoxyethylene alcohol structure, whereas non-specific reactions did not occur in treatment solutions 2-B to 2-D, which had pHs of 5.8, 6.0, and 6.2, respectively. Furthermore, non-specific reactions did not occur in treatment solutions 2-E and 2-F, which contained Pluronic F-68 or F-108, a non-ionic surfactant having a polyoxyethylene alcohol structure. Furthermore, no non-specific reactions occurred under any of the conditions for the 10 frozen specimens that did not contain cyclosporine.

[0105] (3) Test Example 3 (3-1) Treatment Solution and Measurement Object Used in Test Example 3 The treatment solution used in Test Example 3 is as follows. Treatment solution 3-A: 100 mM MES (component (C)), 4 M urea (component (B-1)), 6.0% CHAPS (component (B-2)), 0.75% MEGA8 (component (B-2)), 0.1% ProClin 300, 0.005% Antifoam SI, pH 6.5 Treatment solution 3-B: Treatment solution 3-A adjusted to pH 6.0 Treatment solution 3-C: Treatment solution 3-A to which 1% Pluronic F-68 (component (A)) was added Treatment solution 3-D: Treatment solution 3-B to which 1% Pluronic F-68 (component (A)) was added

[0106] The measurement objects used in Test Example 3 are as follows: Measurement objects: phosphate buffer solution containing cyclosporine (standard solution), fresh whole blood sample (EDTA2Na) not containing cyclosporine (sample not containing cyclosporine, within 24 hours after blood collection, storage temperature 2-8°C).

[0107] (3-2) Experimental Method The same procedure as in Test Example 1 was carried out, except that the above-mentioned treatment solutions 3-A to 3-D were used as the treatment solutions, and the luminescence intensity was measured (each dilution series of the standard solution: n=2, each cyclosporine-free specimen (samples 20 to 58): n=1). The results are shown in 6 and 7.

[0108] Furthermore, the measured values ​​of cyclosporine (ng / mL) were calculated based on the counts of the cyclosporine-free samples using the linear approximation of the calibration curve obtained from the counts during measurement of the standard solutions, in the same manner as in Test Example 1. The results are shown in Table 8.

[0109] The sensitivity (30 / 0) and the frequency of non-specific reactions in cyclosporine-free samples were calculated in the same manner as in Test Example 1. The results of sensitivity (30 / 0) are shown in Table 6, and the results of the frequency of non-specific reactions are shown in Table 8.

[0110]

[0111]

[0112]

[0113] (3-3) Results Non-specific reactions occurred in treatment solution 3-A, which had a pH of 6.5 and did not contain a surfactant having a polyoxyethylene alcohol structure, whereas the occurrence of non-specific reactions was suppressed in treatment solutions 3-B to 3-D.

[0114] (4) Test Example 4 (4-1) Treatment solutions and measurement objects used in Test Example 4 The materials used in Test Example 4 are as follows. Treatment solution 4-A: 100 mM MES (component (C)), 4 M urea (component (B-1)), 6.0% CHAPS (component (B-2)), 0.75% MEGA8 (component (B-2)), 0.1% ProClin 300, 0.005% Antifoam SI, pH 6.5 Treatment solutions 4-B to 4-F: Treatment solution 4-A to which 1%, 0.5%, 1.4%, 2%, and 3% of Pluronic F-68 (component (A)) were added, respectively. Treatment solutions 4-G and 4-H: Treatment solution 4-A with the pH adjusted to 5.0 and 5.5, respectively.

[0115] The measurement objects used in Test Example 4 are as follows: Measurement objects: phosphate buffer solution containing cyclosporine (standard solution), fresh whole blood sample (EDTA2Na) not containing cyclosporine (sample not containing cyclosporine, 3 days after blood collection, storage temperature 2-8°C).

[0116] (4-2) Experimental Method The same procedure as in Test Example 1 was carried out, except that the above-mentioned treatment solutions 4-A to 4-H were used as the treatment solutions, and the luminescence intensity was measured (each dilution series of the standard solution: n=2, each specimen (samples 59 to 75): n=1). The results are shown in Tables 9 and 10.

[0117] Furthermore, the measured cyclosporine values ​​(ng / mL) were calculated based on the count values ​​measured in the cyclosporine-free sample using the linear approximation of the calibration curve obtained from the count values ​​measured in the standard solution in the same manner as in Test Example 1. The results are shown in Table 11.

[0118] The sensitivity (30 / 0) and the frequency of non-specific reactions in samples not containing cyclosporine were calculated in the same manner as in Test Example 1. The results of sensitivity (30 / 0) are shown in Table 9, and the results of the frequency of non-specific reactions are shown in Table 11.

[0119]

[0120]

[0121]

[0122] (4-3) Results Non-specific reactions occurred in treatment solution 4-A, which had a pH of 6.5 and did not contain a POE surfactant, whereas the occurrence of non-specific reactions was suppressed in treatment solutions 4-B to 4-H.

[0123] (5) Test Example 5 (5-1) Treatment Solutions and Measurement Objects Used in Test Example 5 The treatment solutions used in Test Example 5 are as follows. Treatment solution 5-A: 100 mM MES (component (C)), 4 M urea (component (B-1)), 6.0% CHAPS (component (B-2)), 0.75% MEGA8 (component (B-2)), 0.1% ProClin 300, 0.005% Antifoam SI, pH 6.5 Treatment solution 5-B: Treatment solution 5-A to which 1% Pluronic F-68 (component (A)) was added Treatment solution 5-C: Treatment solution 4-A with the pH adjusted to 6.0

[0124] The measurement objects used in Test Example 5 are as follows. Measurement objects: phosphate buffer solution containing cyclosporine (standard solution), a sample in which cyclosporine was added to a fresh whole blood sample (EDTA2Na) not containing cyclosporine (cyclosporine-containing sample, cyclosporine concentration 50 ng / mL, within 48 hours after blood collection, storage temperature 2-8°C), and a sample in which cyclosporine was added to a purchased whole blood sample (stored in a -80°C freezer) thawed (thawed in a water bath at room temperature) (cyclosporine-containing frozen sample, cyclosporine concentration 50 ng / mL).

[0125] (5-2) Experimental Method The same procedures as in Test Example 1 were repeated to measure the luminescence intensity, except that the treatment solutions 5-A to 5-C described above were used as the treatment solutions, and that cyclosporine-containing samples and cyclosporine-containing frozen samples were used as measurement targets instead of cyclosporine-free samples (each dilution series of the standard solution: n=2, each cyclosporine-containing sample (samples 76 to 85): n=2, each cyclosporine-containing frozen sample (sample 86): n=2). The results for each dilution series of the standard solution are shown in Table 12, and the results for the cyclosporine-containing samples and cyclosporine-containing frozen samples are shown in Table 13.

[0126] Furthermore, the measured values ​​of cyclosporine (ng / mL) were calculated based on the count values ​​of the cyclosporine-containing specimen and the cyclosporine-containing frozen specimen using the linear approximation of the calibration curve obtained from the count values ​​of the standard solution in the same manner as in Test Example 1. The results are shown in Table 14.

[0127] The sensitivity (30 / 0) and the frequency of non-specific reactions in the cyclosporine-containing samples were calculated in the same manner as in Test Example 1. The results are shown in Table 12.

[0128] In Test Example 5, the occurrence of a nonspecific reaction was defined as a measurement value of a fresh whole blood sample to which cyclosporine had been added (a cyclosporine-containing sample) that was 120% or more of the measurement value of a purchased whole blood sample to which cyclosporine had been added (a cyclosporine-containing frozen sample), and the occurrence of a nonspecific reaction was evaluated. The results of the frequency of nonspecific reactions are shown in Table 14.

[0129]

[0130]

[0131]

[0132] [Notes for Table 14] *1: "N.D." means no data. *2: "Frozen whole blood avg." is the average of the measured values ​​(n=2) of cyclosporine-containing frozen specimens (86 samples) treated with each treatment solution. *3: "Frozen whole blood avg. x 1.2" is the value obtained by multiplying "frozen whole blood avg." by 1.2. In other words, in Test Example 5, it was defined that a nonspecific reaction occurred when the measured value of the cyclosporine-containing specimen exceeded the value of "frozen whole blood avg. x 1.2."

[0133] (5-3) Results Non-specific reactions occurred in treatment solution 5-A, which had a pH of 6.5 and did not contain a surfactant having a polyoxyethylene alcohol structure, whereas the occurrence of non-specific reactions was suppressed in treatment solutions B to C.

Claims

1. The following steps: (a) mixing whole blood that may contain an immunosuppressant and a treatment solution to prepare a mixed solution; and (b) measuring the concentration of the immunosuppressant in the mixed solution by immunoassay, wherein the treatment solution contains an extractant, and the treatment solution further satisfies the condition of containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.

4. A method for measuring an immunosuppressant in whole blood.

2. The method according to claim 1, wherein the nonionic surfactant having a polyoxyethylene alcohol structure is at least one selected from the group consisting of alcohol ethoxylates and polyoxyethylene-polyoxypropylene block copolymers.

3. The method according to claim 1 or 2, wherein the treatment solution contains the nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.9 to 1.4% by mass.

4. The method according to claim 1 or 2, wherein the treatment solution further contains a pH buffer.

5. The method according to claim 4, wherein the pH buffer is at least one selected from the group consisting of 2-morpholinoethanesulfonic acid, phosphoric acid, tartaric acid, citric acid, acetic acid, trifluoroacetic acid, phthalic acid, glycine, and carbonic acid.

6. The method according to claim 1 or 2, wherein the immunosuppressant is a cyclic polypeptide immunosuppressant or a macrolide immunosuppressant.

7. The method according to claim 1 or 2, wherein the immunosuppressant is at least one selected from the group consisting of cyclosporine, tacrolimus, everolimus, sirolimus, temsirolimus, zotarolimus, biolimus, novolimus, pimecrolimus, myolimus, myocimus, and deforolimus.

8. The method according to claim 1 or 2, wherein the extractant contains a chaotropic denaturant and / or a surfactant other than the nonionic surfactant having a polyoxyethylene alcohol structure.

9. The method according to claim 1 or 2, wherein the non-specific reaction of the immunoassay is suppressed by containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.

4.

10. The method according to claim 1 or 2, which satisfies both containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and having a pH of 4.8 to 6.

4.

11. A kit for measuring an immunosuppressant in whole blood by immunoassay, which comprises a treatment liquid, wherein the treatment liquid contains an extractant, and the treatment liquid further satisfies containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.

4.

12. A method for suppressing non-specific reaction in an immunoassay for measuring an immunosuppressant in whole blood, which comprises the following step: (a) a step of mixing whole blood that may contain an immunosuppressant and a treatment liquid to prepare a mixed liquid, wherein the treatment liquid contains an extractant, and the treatment liquid further satisfies containing a nonionic surfactant having a polyoxyethylene alcohol structure at a concentration of 0.5 to 5% by mass and / or having a pH of 4.8 to 6.4.

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