Composition for pretreatment and method for preparing pretreated sample

Abstract

In order to provide a technology for pretreatment that is suited to measuring forms of vitamin D by homogeneous assay, one embodiment of the present disclosure provides a composition for pretreatment for measuring forms of vitamin D in a sample by homogeneous assay, said composition being characterized in that the pH of the composition is in the range 6.0-9.0 and in that the composition includes at least one selected from the group consisting of unsubstituted or substituted benzoic acid represented by formula (1) (where X is selected from the group consisting of H, OH, COCH3, SH, and NH2, m instances of Y are each independently chosen from the group consisting of alkyl groups, halogens, and alkoxy groups, and m is an integer in the range 0-4), warfarin, azapropazone, phenylbutazone, acenocoumarol, indomethacin, tolbutamide, chlorpropamide, iophenoxate, iodipamide, sulfadimethoxine, and the like.

Description

Pretreatment composition and method for preparing pretreated samples

[0001] This disclosure relates to a pretreatment composition and a method for preparing a pretreated sample.

[0002] Vitamin D is primarily produced in the body in response to ultraviolet light exposure. ３ Vitamin D obtained from food ２ Vitamin D is classified into two types, but they have equivalent physiological functions. Vitamin D is stored in the liver as stable 25-hydroxyvitamin D, and is metabolized in the kidneys to produce the active form 1,25-dihydroxyvitamin D, which exerts various physiological effects such as regulating calcium metabolism. Vitamin D deficiency has a significant impact on bone metabolism. Furthermore, vitamin D deficiency has been reported to be involved in decreased physical function due to muscle mass reduction, the development of sarcopenia, and cancer and diabetes. Therefore, it is becoming increasingly important to detect vitamin D deficiency early, monitor its progress, provide guidance on improving lifestyle habits, and provide appropriate treatment.

[0003] 25-hydroxyvitamin D exists in the blood as a complex with vitamin D-binding protein (DBP) and other substances. Blood levels of 25-hydroxyvitamin D are used as an indicator of the amount of vitamin D in the body. Generally, a serum 25-hydroxyvitamin D concentration of 20 ng / mL or higher but less than 50 ng / mL is considered normal; levels below this indicate a deficiency and are considered to have adverse health effects. In particular, serum 25-hydroxyvitamin D concentrations below 12 ng / mL are suspected to be associated with rickets in children and osteomalacia in adults (see Non-Patent Literature 1). Recent studies have published numerous reports suggesting that many people, regardless of race or nationality, are deficient in 25-hydroxyvitamin D, with blood concentrations below 30 ng / mL. For example, a study by Miyamoto et al. on Japanese subjects revealed, using mass spectrometry, that 98% of Japanese people, regardless of sex or age, had levels below 30 ng / mL (see Non-Patent Literature 2).

[0004] In clinical testing, electrochemiluminescence immunoassay (ECLIA) and chemiluminescence enzyme immunoassay (CLEIA) are currently the most common methods used to test for 25-hydroxyvitamin D in the blood. These methods can be automated using specialized equipment, and because the specialized equipment is equipped with a washing mechanism, it is less susceptible to the influence of DBP and other contaminants. However, the reaction time is several tens of minutes, which is not particularly short. Because these methods require specialized equipment, it is difficult to perform rapid measurements in all laboratories.

[0005] Other testing methods, such as mass spectrometry (LC-MS / MS), can accurately quantify vitamin D without cross-referencing similar structures, but this method is virtually impossible to implement in a typical laboratory. While lateral flow-based rapid testing reagents are commercially available, they are impractical in clinical settings due to their unsuitability for high-volume testing and the inability to guarantee accuracy through visual inspection.

[0006] In light of these circumstances, a method for measuring vitamin D and reagents for its use have been developed that are applicable to general-purpose instruments such as automated biochemical analyzers. It should be noted that, since automated biochemical analyzers do not usually have a washing mechanism, the measurement is a homogeneous assay performed with DBP present in the system. To accurately measure 25-hydroxyvitamin D, it is necessary to release 25-hydroxyvitamin D into a free state. The binding rate between DBP and 25-hydroxyvitamin D is lowest in the acidic range, especially around pH 4-5, and it is known that a large amount of free 25-hydroxyvitamin D is present (see Non-Patent Literature 3). However, since antibodies, which are common capture substances for vitamin D, perform best under neutral conditions, it becomes difficult to measure vitamin D in a sufficiently free state when DBP is present in the system.

[0007] Various methods have been reported and put into practical use for measuring 25-hydroxyvitamin D while avoiding the effects of DBP. For example, Patent Document 1 discloses a method for measuring vitamin D by latex immunoaggregation using a homogeneous assay with antibodies and anti-metatype antibodies. Patent Documents 2 and 3 disclose the liberation of vitamin D using sodium salicylate. In addition, Patent Documents 4 to 6 disclose methods using fluorinated surfactants, organic solvents such as alcohols, and strong bases such as sodium hydroxide as liberants.

[0008] International Publication No. 2018 / 023066, International Publication No. 2015 / 200182, International Publication No. 2002 / 057797, International Publication No. 2015 / 040319, Japanese Patent Publication No. 2015-137891, International Publication No. 2007 / 039194

[0009] Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: National Academy Press, 2010.H. Miyamoto, et al., Determination of a Serum 25-Hydroxyvitamin D Reference Ranges in Japanese Adults Using Fully Automated Liquid Chromatography-Tandem Mass Spectrometry. The Journal of Nutrition, Volume 153 (2023) 1253-1264R. Bouillon, et al., Comparative study of the affinity of the serum vitamin d-binding protein, Journal of Steroid Biochemistry, Volume 13, 1029-1034, September 1980

[0010] The technology described in Patent Document 1 specifies acidic conditions, particularly preferably pH 4.5 to 5.5, as the conditions for releasing vitamin D from DBP. However, the pH suitable for the immunoassay to measure the released vitamin D is generally neutral. Therefore, if the reagents for the immunoassay are under neutral to basic conditions, the final reaction solution will deviate from the conditions suitable for vitamin D release, which can lead to recombination of vitamin D and DBP, potentially hindering accurate measurement.

[0011] The technologies described in Patent Documents 2 and 3 are all heterogeneous assays that involve a washing step and are unsuitable for rapid measurement. The technologies described in Patent Documents 4 to 6 have room for improvement when considering environmental impact, effects on captured substances in the system, and suitability for automated biochemical analyzers.

[0012] One aspect of this disclosure aims to provide a technique for pretreatment suitable for the measurement of vitamin D compounds by homogeneous assay.

[0013] To solve the above problems, a pretreatment composition according to one aspect of the present disclosure is a pretreatment composition for measuring vitamin D compounds in a sample by homogeneous assay, wherein the pH of the pretreatment composition is 6.0 or higher and 9.0 or lower, and the pretreatment composition contains a ligand for the vitamin D compounds, wherein the ligand is an unsubstituted or substituted benzoic acid represented by the following formula (1) (wherein X is H, OH, COCH ３ SH and NH ２each m Y is independently selected from the group consisting of alkyl groups, halogens, and alkoxy groups, and m is an integer of 0 to 4 inclusive), warfarin, azapropazone, phenylbutazone, acenocoumarol, indomethacin, tolbutamide, chlorpropamide, iophenoxate, iodipamide, sulfadimethoxine, sulfathiazole, furosemide, tenoxicam, sodium valproate, piretanide, spironolactone, potassium canrenoate, quercetin, benzylthiouracil, oxyphenbutazone, S-carbenicillin, cosalane, bilirubin, eicosanoid, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, and is at least one selected from the group consisting of ions thereof, wherein the ions are anions.

[0014] According to one aspect of the present disclosure, there is provided a technique relating to a pretreatment suitable for measuring vitamin D by a homogeneous assay.

[0015] It is a graph showing the calibration curve prepared in Example 1. It is a graph showing the calibration curve prepared in Example 1. It is a graph showing the calibration curve prepared in Example 3.

[0016] <Vitamin D> In the present disclosure, vitamin D refers to a compound having a basic skeleton common to the calciferol compound group. Examples of vitamin D include vitamin D ２ (ergocalciferol), vitamin D ３ (cholecalciferol), vitamin D ４ , vitamin D ５ , vitamin D ６ , and vitamin D ７ ; vitamin D ２ to D ７ any precursor or metabolite thereof; and vitamin D ２ to D ７ any one thereof and any analog of the aforementioned precursors or metabolites; examples thereof are given. Vitamin D ２ to D ７ As an example of any metabolite thereof, 25-hydroxyvitamin D ２, 25-hydroxyvitamin D ３ , 1,25-dihydroxyvitamin D ２ , 1,25-dihydroxyvitamin D ３ , 24,25-dihydroxyvitamin D ２ , and 24,25-dihydroxyvitamin D ３ Examples include dihydrotachisterol, an example of an analog.

[0017] In particular, the vitamin D compounds to be measured are preferably 25-hydroxyvitamin D, as they are useful as indicators of the amount of vitamin D in the body. In this disclosure, "25-hydroxyvitamin D" means 25-hydroxyvitamin D ２ and 25-hydroxyvitamin D ３ It refers to at least one of the following. Hereafter, "25-hydroxyvitamin D" may be abbreviated as "25OHVD". Also, among 25-hydroxyvitamin D, "25-hydroxyvitamin D ３ This is sometimes abbreviated as "25OHVD3".

[0018] Furthermore, the vitamin D to be measured may be 24,25-dihydroxyvitamin D, as it is useful as an indicator of hypercalcemia. In this disclosure, "24,25-dihydroxyvitamin D" means 24,25-dihydroxyvitamin D ２ and 24,25-dihydroxyvitamin D ３ It refers to at least one of the following.

[0019] Multiple vitamin D derivatives may be measured. For example, the 24,25-dihydroxyvitamin D / 25-hydroxyvitamin D ratio (VMR), calculated by measuring both 24,25-dihydroxyvitamin D and 25-hydroxyvitamin D, is useful in the differential diagnosis of conditions such as CYP24A1 deficiency, vitamin D overdose, and granulomatous disease.

[0020] <Sample> In this disclosure, the sample may be any liquid and is not particularly limited. Examples of samples include: biological samples such as blood (serum, plasma, or whole blood), urine, lymph, cerebrospinal fluid, nasal fluid, and saliva, as well as dilutions or purified products thereof; medical liquid compositions such as hemodialysis fluid, oral rehydration solution, and intravenous infusion solution; beverages such as sports drinks, fruit juice, and coffee; and environmental liquids such as tap water, river water, and industrial wastewater. For example, serum, plasma, or whole blood may be used as the sample, and 25OHVD in these samples may be used as the vitamin D compounds to be measured.

[0021] In this disclosure, "measurement sample" refers to a sample that has been processed and prepared for the purpose of being used as a measurement sample in its original form. For example, if blood collected as a sample is subjected to various processes so that it can be used for measurement by immunological methods, the liquid obtained after such processing can be called a measurement sample.

[0022] <Pretreatment Composition (First Composition)> One aspect of the present disclosure relates to a pretreatment composition. The pretreatment composition according to one aspect of the present disclosure is a pretreatment composition for measuring vitamin D compounds in a sample by homogeneous assay, wherein the pH of the pretreatment composition is 6.0 or higher and 9.0 or lower, and the pretreatment composition contains a ligand for the vitamin D compounds, the ligand being an unsubstituted or substituted benzoic acid represented by the following formula (1) (wherein X is H, OH, COCH ３ SH and NH ２The composition is characterized by being selected from the group consisting of, where m Y groups are independently selected from the group consisting of alkyl groups, halogens, and alkoxy groups, and m is an integer between 0 and 4), warfarin, azapropazone, phenylbutazone, asenocumarol, indomethacin, tolbutamide, chlorpropamide, iofenoxate, iodipamide, sulfadimethoxine, sulfathiazole, furosemide, tenoxicam, sodium valproate, pyretanide, spironolactone, potassium canrenoate, quercetin, benzylthiouracil, oxyfenbutazone, S-carbenicillin, cosaran, bilirubin, eicosanoids, and 3-carboxy-4-methyl-5-propyl-2-furanopanoic acid, and ions thereof, wherein the ion is an anion. Furthermore, in the following description, the "pretreatment composition" may be referred to as the "first composition".

[0023]

[0024] The first composition is used to measure vitamin D compounds in a sample by a homogeneous assay, and is particularly used for sample pretreatment. Pretreatment using the first composition involves releasing vitamin D compounds from the complex of vitamin D-binding protein (DBP) and vitamin D compounds present in the sample, either before or simultaneously with the immunoassay, in order to facilitate the measurement of vitamin D compounds by the homogeneous assay. Here, the measurement by the homogeneous assay may utilize a specific reaction involving vitamin D compounds, i.e., an immunoassay. Typically, pretreatment is achieved by adding the first composition to the sample during the preparation process of the sample to be used for vitamin D measurement.

[0025] (pH of First Composition) In one aspect of the present disclosure, the first composition is an aqueous composition containing water as a solvent. The pH of the first composition is in the range from neutral to weakly basic. The first composition enables release of vitamin D from a complex of DBP and vitamin D within this pH range suitable for immune reactions. When pretreatment is performed using the first composition, a subsequent immune reaction can be carried out at the same or similar pH as that of the pretreatment, and recombination of vitamin D with DBP and denaturation of various components caused by pH change are reduced. Therefore, the first composition can be used for pretreatment suitable for measuring vitamin D by homogeneous assay.

[0026] In the prior art, release of vitamin D has been performed under acidic conditions. In contrast, in the present disclosure, release of vitamin D is enabled by a combination of selecting the pH of the first composition within the range from neutral to weakly basic, and selecting components such as a ligand contained in the first composition within a specific range. Details of the components contained in the first composition will be described later.

[0027] The pH of the first composition is not less than 6.0 and not more than 9.0, preferably not less than 6.5 and not more than 8.5, more preferably not less than 6.8 and not more than 8.2, and still more preferably not less than 7.0 and not more than 8.0. The higher the pH of the first composition is within these ranges, the greater the effect that the subsequent immune reaction can be performed at a pH closer to that of the pretreatment, and the lower the pH of the first composition is within these ranges, the greater the effect that release of vitamin D in the pretreatment is further promoted.

[0028] In order to amplify the above effect, the pH of the first composition may be selected depending on the type of ligand for vitamin D contained in the first composition. For example, when the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and ions thereof, the pH of the first composition is 6.0 or higher, preferably 6.5 or higher, more preferably 6.8 or higher, and most preferably 7.0 or higher. Further, when the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and ions thereof, the pH of the first composition is preferably 9.0 or lower, more preferably 8.5 or lower, still more preferably 8.2 or lower, and most preferably 8.0 or lower.

[0029] (Ligand) The first composition contains a ligand for vitamin D. Said ligand is a compound having an effect of releasing vitamin D from DBP, for example, a compound that competes with vitamin D and binds to DBP.

[0030] The ligand may be selected from compounds that specifically bind to Site I of human serum albumin, which belongs to the albumin family same as DBP, under neutral to weakly basic conditions (pH 7.4). Such a ligand is at least one selected from the group consisting of unsubstituted or substituted benzoic acid represented by the following formula (1), warfarin, azapropazone, phenylbutazone, acenocoumarol, indomethacin, tolbutamide, chlorpropamide, iophenoxate, iodipamide, sulfadimethoxine, sulfathiazole, furosemide, tenoxicam, sodium valproate, piretanide, spironolactone, potassium canrenoate, quercetin, benzylthiouracil, oxyphenbutazone, S-carbenicillin, cosalane, bilirubin, eicosanoid, 3-carboxy-4-methyl-5-propyl-2-furanopropanoic acid, and ions thereof, wherein said ion is an anion.

[0031]

[0032] In formula (1), X is H, OH, COCH ３ , SH and NH ２It is selected from the group consisting of the following, and is preferably OH. Furthermore, X is preferably bonded to the carboxyl group at the ortho, meta, or para position, and more preferably at the ortho position.

[0033] In formula (1), each m Y is independently selected from the group consisting of alkyl groups, halogens, and alkoxy groups.

[0034] The alkyl group preferably has 1 to 2 carbon atoms, and more preferably 1 carbon atom. The alkyl group is preferably selected from the group consisting of a methyl group and an ethyl group.

[0035] The halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0036] The number of carbon atoms in the alkoxy group is preferably 1 or more and 2 or less, and more preferably 1. The alkoxy group is preferably selected from the group consisting of a methoxy group and an ethoxy group.

[0037] In formula (1), m is an integer between 0 and 4, preferably between 0 and 2, and more preferably between 0 and 1.

[0038] The benzoic acid ion represented by formula (1) is formed when the salt of benzoic acid dissociates, and is represented by the following formula (2).

[0039]

[0040] A salt of benzoic acid is a salt of the carboxylate ion of benzoic acid represented by formula (2) and an inorganic or organic cation. Examples of inorganic cations include: alkali metal cations such as sodium, potassium, lithium, and cesium; alkaline earth metal cations such as magnesium, calcium, and barium. Examples of organic cations include: quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, choline, imidazolium, and pyridinium; quaternary phosphonium cations such as phosphonium, tetramethylphosphonium, tetraethylphosphonium, tetrapropylphosphonium, and tetrabutylphosphonium. The cation is preferably at least one selected from the group consisting of sodium ions and quaternary ammonium cations, and is preferably a sodium ion. There may be one type of cation or multiple types.

[0041] From the viewpoint of further promoting the release of vitamin D compounds under neutral to basic conditions, the ligand is preferably at least one selected from the group consisting of benzoic acid represented by formula (1) and its ions. From the same viewpoint, the ligand is preferably at least one selected from the group consisting of 2-hydroxybenzoic acid (salicylic acid), 3-hydroxybenzoic acid, 4-hydroxybenzoic acid and their ions, and more preferably at least one selected from the group consisting of salicylic acid and its ions. The salicylate ion is preferably at least one selected from the group consisting of sodium salicylate, potassium salicylate, magnesium salicylate, calcium salicylate, tetrabutylammonium salicylate, and choline salicylate; more preferably sodium salicylate; and is included in the first composition as an ionized form derived from these.

[0042] The ligand concentration in the first composition may be, for example, 10 mM or more and 1000 mM or less, but can be appropriately selected depending on the type of ligand. For example, if the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and its ions, the concentration of benzoic acid ions represented by formula (1) in the first composition is preferably 50 mM or more and 980 mM or less, more preferably 100 mM or more and 950 mM or less, even more preferably 120 mM or more and 900 mM or less, and even more preferably 150 mM or more and 800 mM or less. The higher the concentration of benzoate ions within these ranges, the more the release of vitamin D compounds is promoted, and the lower the concentration of benzoate ions within these ranges, the better the sensitizing effect per unit amount of water-soluble polymer added as an immunoaggregation reaction promoter in the first composition, and the more possible it is to reduce the amount of water-soluble polymer added.

[0043] (Water-soluble polymer) The first composition may further contain a water-soluble polymer. The role of the water-soluble polymer in the first composition may include, but is not limited to, an immunoaggregation reaction promoter, a solvent, a specific gravity modifier, a viscosity modifier, etc. The type of water-soluble polymer can be appropriately selected depending on the mechanism of action used by the vitamin D measurement method using the first composition. The first composition containing a water-soluble polymer is preferred because it is endowed with a function due to the water-soluble polymer. The water-soluble polymer may be one type or multiple types.

[0044] Water-soluble polymers may be those known in the art. Examples of water-soluble polymers include: polyoxyalkylene glycols such as polyethylene glycol, polypropylene glycol, and polyoxyethylene-polyoxypropylene copolymer; oxygen-containing polyvinyls such as polyvinylpyrrolidone and polyvinyl alcohol; amine cationic polymers such as polyethyleneimine, polyallylamine, polyvinylamine, dimethylaminoethyl dextran, aminoethanesulfonic acid derivatives, and aminopropanesulfonic acid derivatives; nonionic polysaccharides such as dextran, alginic acid, starch, pullulan, and gelatin; anionic polysaccharides such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate; anionic polymers such as xanthan gum, carrageenan, sodium alginate, gum arabic, pectin, and carboxyvinyl polymer; cellulose lower alkyl ether compounds such as carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, and hydroxypropylcellulose; and polyacrylates such as polyglucosyloxyethyl methacrylate.

[0045] When a water-soluble polymer is used as an immunoaggregation reaction promoter, that is, a sensitizer that promotes the aggregation of insoluble particles by an immune reaction, it is preferable that the water-soluble polymer is at least one selected from the group consisting of polyoxyalkylene glycol and oxygen-containing polyvinyl.

[0046] The concentration of the water-soluble polymer in the first composition can be appropriately selected, for example, 0% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass or less. When a water-soluble polymer is used as an immunoaggregation reaction promoter, the concentration of the water-soluble polymer is preferably 0% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass or less.

[0047] (Buffering agent of the first composition) The first composition may further contain a buffering agent that exhibits buffering capacity in the neutral to basic range of pH 7.0 to 9.0. The buffering agent may be one known in the art. Examples of buffering agents include: acid salts such as phosphates, citrates, and amino acid salts; Good's buffers such as bicine, glycinamide, tricine, acetamidoglycine, HEPES, TES, BES, coramine hydrochloride, ACES, PIPES, ADA, and MES; and other buffering agents such as Tris. There may be one type of buffering agent or multiple types.

[0048] The concentration of the buffer in the first composition can be selected as appropriate, for example, 1 mM or more and 1000 mM or less.

[0049] (Ions and salts of the first composition) The first composition may further contain ions or salts to bring the first composition closer to physiological conditions. The ions or salts may be known to be present in living organisms. Examples of ions include: alkali metal cations such as sodium ions, potassium ions, and lithium ions; alkaline earth metal cations such as magnesium ions and calcium ions; inorganic base cations such as ammonium; halide anions such as fluoride ions, bromide ions, chloride ions, and iodide ions; and inorganic acid anions such as phosphate ions, nitrate ions, and sulfate ions. Examples of salts include salts of the aforementioned cations and anions, as well as salts containing organic bases such as trimethylamine, triethylamine, and pyridine. The ions or salts may be one type or multiple types.

[0050] The concentration of ions or salts in the first composition can be appropriately selected depending on the type of ions or salt and the type of sample. For example, if the sample is blood, the concentration of potassium ions in the first composition may be adjusted to approximate the physiological concentration of potassium ions in blood.

[0051] (Proteins in the First Composition) The first composition may further contain proteins. The role of the proteins in the first composition may include, but are not limited to, nonspecific aggregation reaction inhibitors, reagent stabilizers, reaction stabilizers, etc. The type of protein may be appropriately selected depending on the mechanism of action used by the vitamin D measurement method using the first composition.

[0052] Examples of proteins include bovine serum albumin (BSA), globulin, casein, and collagen. The protein may be one type or multiple types.

[0053] (Surfactants of the First Composition) The first composition may further contain a surfactant. The surfactant may be one known in the art and is selected from the group consisting of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. There may be one surfactant or multiple surfactants.

[0054] Examples of cationic surfactants include lauryltrimethylammonium chloride and trimethyloctadecylammonium chloride. Examples of anionic surfactants include sodium dodecylbenzenesulfonate, sodium dialkylsulfosuccinate, sodium lauryl sulfate, sodium polyoxyethylene alkyl ether sulfate, sodium polyoxyethylene alkylphenyl ether sulfate, ammonium polyoxyethylene alkyl ether sulfate, ammonium polyoxyethylene alkylphenyl ether sulfate, polyoxyethylene alkyl ether phosphate, and polyoxyethylene alkylphenyl ether phosphate. Examples of nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, polyoxyethylene oxypropylene block polymer, polyethylene glycol fatty acid ester, and polyoxyethylene sorbitan fatty acid ester. Examples of amphoteric surfactants include lauryl betaine and lauryldimethylamine oxide.

[0055] The concentration of the surfactant in the first composition can be adjusted as appropriate, and may be, for example, 0% by mass or more and 10% by mass or less.

[0056] (Preservative in the first composition) The first composition may further contain a preservative to improve its long-term shelf life. The preservative may be one known in the art. Examples of preservatives include: azides such as sodium azide; isothiazolinones such as chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT); and as a preservative, ProClin ＴＭ Commercially available products such as those listed above may be used. The preservative may be one type or multiple types.

[0057] The concentration of the preservative in the first composition can be adjusted as appropriate, and may be, for example, 0% by mass or more and 10% by mass or less.

[0058] (Vitamin D-scavenging component of the first composition) The first composition may further contain a vitamin D-scavenging component. The vitamin D-scavenging component of the first composition specifically captures vitamin D released from the complex. Typically, the scavenging component is not bound to a carrier such as an insoluble carrier and is included in the first composition in a free state, but the disclosure is not limited to this configuration.

[0059] The capture component plays a role in returning vitamin D compounds released from the complex from their free state to their bound state, thereby inhibiting their participation in the immune response after pretreatment. The first composition containing the capture component is useful when measuring vitamin D compounds by a competitive method.

[0060] The capture component specifically captures vitamin D derivatives and is at least one selected from the group consisting of antibodies, their antigen-binding fragments, and aptamers. The antibody may be either a polyclonal antibody or a monoclonal antibody. In this disclosure, the antibody includes proteins and amino acid polymers (peptides) with a molecular weight smaller than that of proteins.

[0061] (Other components of the first composition) The components included in the first composition are not limited to those described above, and other known components used in homogeneous assays may be further included in the first composition as other components. Examples of other components include: anticoagulants such as ethylenediaminetetraacetic acid (EDTA) salt, heparin, sodium citrate, and quaternary ammonium salts for the purpose of improving reactivity, etc.; sugars such as sucrose, trehalose, and mannitol; chaotropic agents such as guanidine and urea; and reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol (2ME).

[0062] As described above, the first composition releases vitamin D compounds from the DBP-vitamin D complex through the action of a ligand on vitamin D compounds. Therefore, the first composition can sufficiently induce the release of vitamin D compounds even without containing a protease that digests DBP, and is therefore advantageous for use in pretreatment. In other words, the first composition does not need to contain a protease that digests DBP. However, the disclosure is not limited to this configuration, and the first composition may contain a protease that digests DBP.

[0063] In embodiments in which the first composition is used in a step of a homogeneous assay, it is preferable that the first composition does not contain any components that require washing, separation, or extraction. Such components may be selected from the group consisting of, for example, proteases.

[0064] <Method for preparing pre-treated samples> The pre-treatment composition (first composition) can be used to prepare pre-treated samples for measuring vitamin D compounds in a sample by homogeneous assay. Accordingly, one aspect of the present disclosure relates to a method for preparing pre-treated samples using the first composition. This method is a method for preparing pre-treated samples for measuring vitamin D compounds in a sample by homogeneous assay, and is characterized by including a step of contacting the sample with the pre-treatment composition (first composition). Hereinafter, the step of contacting the sample with the first composition will be referred to as the "first contact step".

[0065] The first contact step yields a pre-treated sample containing the sample and the first composition. In the pre-treated sample, vitamin D compounds are well released from the DBP-vitamin D complex, and subsequent processing can be successfully applied to the released vitamin D compounds. Therefore, the pre-treated sample is suitable for use in measuring vitamin D compounds by homogeneous assay.

[0066] The operation of the first contact step is not particularly limited as long as the sample and the first composition come into contact. For example, after adding the first composition to the sample, the mixture may be mixed by inversion, shaking, pipetting or tapping, or by rotational stirring using a stirrer or rotator.

[0067] The amount of the first composition used in the first contact step can be adjusted as appropriate. Without limiting the scope of this disclosure, the amount of the first composition used per 1 volume of sample may be between 1 volume and 1000 volumes.

[0068] The amount of the first composition used may be determined so that the amount of ligand in the mixture of the sample and the first composition is within a predetermined range. The amount of ligand in the mixture may be, for example, 1 μmol or more and 100 μmol or less, but it may be appropriately selected depending on the type of ligand. The higher the amount of ligand within these ranges, the more the release of vitamin D compounds is promoted. Also, the lower the amount of ligand within these ranges, the better the sensitizing effect per amount of water-soluble polymer added as an immunoaggregation reaction promoter, and the less water-soluble polymer is added. When the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and its ions, the amount of benzoic acid ions represented by formula (1) contained in the first composition used per 1 μL of sample is preferably 3 μmol or more and 25 μmol or less.

[0069] In the first contact step, the contact time between the sample and the first composition may be long enough to allow the release of vitamin D compounds, while at the same time shortening the time required for the preparation of the pre-treated sample, it may be, for example, 1 second to 1000 seconds, or 30 seconds to 500 seconds. The temperature in the first contact step may be, for example, 0°C to 50°C, and 37°C as an example.

[0070] In the first contact step, compositions other than the first composition may be added to the sample. The compositions other than the first composition may be known components used in homogeneous assays, and examples include, but are not limited to, diluents.

[0071] The preparation method for pre-treated samples may be carried out as a step in a homogeneous assay. In such embodiments, the preparation method does not include a washing step, and therefore, it is preferable that each composition used in the preparation method does not contain components that require washing, separation, or extraction, such as proteases, acids, bases, or salts at concentrations that inhibit a series of reactions. For example, when latex particles are used as an insoluble carrier in the reaction composition described later, the above components should be selected so as to prevent nonspecific aggregation of the latex particles by salting out or coagulation in the homogeneous assay.

[0072] <Reaction Composition (Second Composition)> The above-described pretreatment composition (first composition) may be used in combination with the reaction composition in the measurement of vitamin D compounds in a sample by homogeneous assay. As the reaction composition, a known composition for inducing a desired immunoreaction involving free vitamin D compounds can be used. The pH and components of a reaction composition preferred for use in combination with the first composition are described below. In the following description, the "reaction composition" may be referred to as the "second composition".

[0073] The second composition is used to measure vitamin D compounds in a sample by homogeneous assay, and in particular to induce a specific reaction involving vitamin D compounds. Typically, in the preparation process of a sample to be measured for vitamin D compound measurement, the first composition is added to the sample, followed by the second composition.

[0074] The immune response may be a known reaction, as long as it is a specific reaction regulated by the presence of vitamin D compounds. The immune response may be an agglutination reaction, a color reaction, a luminescence reaction, a fluorescence reaction, etc., of the components in the second composition. Among these, in homogeneous assays, an agglutination reaction is preferred from the viewpoint of ease of application to general-purpose equipment such as automated biochemical analyzers. The regulation of the specific reaction by the presence of vitamin D compounds may be either inhibition or promotion of the reaction, and therefore the immune response may be a reaction utilizing either a competitive or non-competitive method for vitamin D compounds. The form of involvement of vitamin D compounds in the immune response may also be a known method, such as a direct method, an indirect method, a sandwich method, or a method utilizing anti-metatype antibodies. The method for observing the immune response may appropriately employ a method known in the field of homogeneous assays, and examples include the turbidimetric method, the metric wax method, the AlphaScreen® method, the LOCI® method, etc. The second composition may contain a label to be observed, depending on a predetermined method for observing the immune response.

[0075] (pH of the second composition) In one embodiment of the present disclosure, the second composition is an aqueous composition containing water as a solvent. The pH of the second composition is preferably in the range of neutral to weakly basic. This pH range is suitable for immunoreactions and reduces the recombination of vitamin D derivatives released from the complex with DBP and the denaturation of various components.

[0076] The pH of the second composition is preferably 7.0 or higher. The higher the pH of the second composition within this range, the more the immune response is promoted. The pH of the second composition is preferably 8.0 or lower. The lower the pH of the second composition within this range, the less the recombination of free vitamin D compounds is reduced.

[0077] To amplify the above effects, the pH of the second composition may be selected according to the pH of the first composition. For example, the difference in pH (absolute value) between the first composition and the second composition is preferably 2.0 or less, and more preferably 1.0 or less.

[0078] (Insoluble carrier) The second composition contains an insoluble carrier. The insoluble carrier carries the supported reaction component described later and plays a role in making the immunoreaction observable as an agglutination reaction through the aggregation of the insoluble carrier. This aggregation can be observed using known methods such as turbidimetric methods and waxy metering methods.

[0079] The insoluble carrier may be one known in the art and is typically a particle insoluble in water. Examples of insoluble carriers include: latex particles composed of latex such as polystyrene, polymethyl methacrylate, polymethylnaphthalene, poly(divinylbenzene), polyvinylnaphthalene, styrene copolymer, divinylbenzene acrylate, and naphthalene; metal colloids composed of metals or metal oxides such as gold, silver, chromium dioxide, and titanium dioxide; silica particles; and the like. From the viewpoint of particle size uniformity and dispersibility in aqueous solvents, the insoluble carrier is preferably latex particles. The insoluble carrier may be one type or multiple types.

[0080] The average particle size of the insoluble carrier can be appropriately selected within a range in which aggregation of the insoluble carrier can be observed. For example, the average particle size of the insoluble carrier is 10 nm to 1 μm, another example is 30 nm to 600 nm, and yet another example is 50 nm to 500 nm. The average particle size may be selected according to the type of insoluble carrier. When the insoluble carrier is latex particles, the average particle size of the latex particles is preferably 100 nm to 500 nm, from the viewpoint of increasing the change in transmitted or scattered light intensity due to the aggregation reaction and making it easier to observe the aggregation reaction.

[0081] (Reaction Components) The second composition contains at least one reaction component involved in a specific reaction regulated by the presence of vitamin D derivatives. The reaction component may be known in the art. The reaction component can be appropriately selected depending on whether a competitive or non-competitive immunoassay is used.

[0082] The insoluble carrier supports at least one supported reactive component as at least one reactive component. The method of support may be adhesion due to intermolecular interactions or by utilizing chemical bonding, and a known linker or spacer component may be interposed between the insoluble carrier and the supported reactive component; it is not particularly limited. The at least one supported reactive component may be one type or multiple types. When multiple types of supported reactive components are used, the multiple types of supported reactive components may be supported on the same type of insoluble carrier, or each may be supported on a different type of insoluble carrier.

[0083] At least one of the supported reaction components may specifically capture vitamin D derivatives. Multiple such supported reaction components may be used to induce a sandwich immunoassay-type immune response. Alternatively, at least one of the supported reaction components may be captured by a reaction component that specifically captures vitamin D derivatives, i.e., vitamin D derivatives themselves. Such supported reaction components may be used in combination with a supported reaction component that specifically captures vitamin D derivatives to induce a competitive immunoassay-type immune response. In both the sandwich immunoassay type and the competitive immunoassay type, it is preferable that at least one of the supported reaction components specifically captures vitamin D derivatives.

[0084] At least one supported reaction component can be selected from the group consisting of antibodies, their antigen-binding fragments, and aptamers. Various types of such supported reaction components have been developed, which can provide the second composition with design flexibility according to the desired immunoassay.

[0085] Without limiting the scope of this disclosure, we will describe the reaction components that may be used to induce an anti-metatype immunoassay type immune response. In one example, the insoluble carrier carries at least one supported reaction component, which is a first supported reaction component that specifically captures vitamin D derivatives and a second supported reaction component that specifically captures a complex of vitamin D derivatives and the first supported reaction component.

[0086] As another example not limiting the scope of this disclosure, we describe reaction components that may be used to induce a competitive immunoassay-type immune response. In one example, an insoluble carrier supports vitamin D directly or indirectly via a known linker or spacer component as at least one supported reaction component, and the second composition further comprises a capture component (vitamin D capture component) that specifically captures vitamin D. In this case, vitamin D is used as an antigen and is usually introduced to the insoluble carrier by chemical bonding. An example of a capture component is an anti-vitamin D antibody.

[0087] (Buffering agent of the second composition) The second composition may further contain a buffering agent that exhibits buffering capacity in the neutral to basic range of pH 7.0 to 9.0. The buffering agent may be one known in the art. Examples of buffering agents include: acid salts such as phosphates, citrates, and amino acid salts; Good buffers such as bicine, glycinamide, tricine, acetamidoglycine, HEPES, TES, BES, coramine hydrochloride, ACES, PIPES, ADA, MES; and other buffering agents such as Tris. There may be one type of buffering agent or multiple types.

[0088] The concentration of the buffer in the second composition can be selected as appropriate, for example, 1 mM or more and 1000 mM or less.

[0089] (Ions and salts of the second composition) The second composition may further contain ions or salts to bring the second composition closer to physiological conditions. The ions or salts may be known to be present in living organisms. Examples of ions include: alkali metal cations such as sodium ions, potassium ions, and lithium ions; alkaline earth metal cations such as magnesium ions and calcium ions; inorganic base cations such as ammonium; halide anions such as fluoride ions, bromide ions, chloride ions, and iodide ions; and inorganic acid anions such as phosphate ions, nitrate ions, and sulfate ions. Examples of salts include salts of the aforementioned cations and anions, as well as salts containing organic bases such as trimethylamine, triethylamine, and pyridine. There may be one type of ion or salt, or there may be multiple types.

[0090] The concentration of ions or salts in the second composition can be appropriately selected depending on the type of ions or salt and the type of sample. For example, if the sample is blood, the concentration of potassium ions in the second composition may be adjusted to approximate the physiological concentration of potassium ions in blood.

[0091] (Proteins in the second composition) The second composition may further contain proteins. The role of the proteins in the second composition may include, but are not limited to, nonspecific aggregation inhibitors, reagent stabilizers, reaction stabilizers, etc. The type of protein may be appropriately selected depending on the mechanism of action used by the vitamin D measurement method using the second composition.

[0092] Examples of proteins include bovine serum albumin (BSA), globulin, casein, and collagen. The protein may be one type or multiple types.

[0093] (Surfactants of the second composition) The second composition may further contain a surfactant. The surfactant may be one known in the art and is selected from the group consisting of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. There may be one surfactant or more surfactants.

[0094] Examples of cationic surfactants include lauryltrimethylammonium chloride and trimethyloctadecylammonium chloride. Examples of anionic surfactants include sodium dodecylbenzenesulfonate, sodium dialkylsulfosuccinate, sodium lauryl sulfate, sodium polyoxyethylene alkyl ether sulfate, sodium polyoxyethylene alkylphenyl ether sulfate, ammonium polyoxyethylene alkyl ether sulfate, ammonium polyoxyethylene alkylphenyl ether sulfate, polyoxyethylene alkyl ether phosphate, and polyoxyethylene alkylphenyl ether phosphate. Examples of nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, polyoxyethylene oxypropylene block polymer, polyethylene glycol fatty acid ester, and polyoxyethylene sorbitan fatty acid ester. Examples of amphoteric surfactants include lauryl betaine and lauryldimethylamine oxide.

[0095] The concentration of the surfactant in the second composition can be adjusted as appropriate, and may be, for example, 0% by mass or more and 10% by mass or less.

[0096] (Preservative in the second composition) The second composition may further contain a preservative to improve its long-term shelf life. The preservative may be one known in the art. Examples of preservatives include: azides such as sodium azide; isothiazolinones such as chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT); and as a preservative, ProClin ＴＭ Commercially available products such as those listed above may be used. The preservative may be one type or multiple types.

[0097] The concentration of the preservative in the second composition can be adjusted as appropriate, and may be, for example, 0% by mass or more and 10% by mass or less.

[0098] (Other components of the second composition) The components included in the second composition are not limited to those described above, and other known components used in homogeneous assays may be included in the second composition as other components. Examples of other components include: anticoagulants such as ethylenediaminetetraacetic acid (EDTA) salt, heparin, sodium citrate, and quaternary ammonium salts for the purpose of improving reactivity; sugars such as sucrose, trehalose, and mannitol; chaotropic agents such as guanidine and urea; and reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol (2ME).

[0099] In embodiments in which the second composition is used in a step of a homogeneous assay, it is preferable that the second composition does not contain components that require washing, separation, or extraction. Such components may be selected from the group consisting of, for example, proteases.

[0100] <Method for Measuring Vitamin D Substances> The first and second compositions can be used to measure vitamin D substances in a sample by homogeneous assay. Accordingly, one aspect of the present disclosure relates to a method for measuring vitamin D substances using the first and second compositions. This method is a method for measuring vitamin D substances in a sample by homogeneous assay, and is characterized by comprising the steps of contacting the sample with a pretreatment composition (first composition) and contacting the pretreatment sample with a reaction composition (second composition). Hereinafter, the step of contacting the sample with the first composition will be referred to as the "first contact step," and the step of contacting the pretreatment sample with the reaction composition (second composition) will be referred to as the "second contact step."

[0101] (First Contact Step) The first contact step in the method for measuring vitamin D is the same as the first contact step in the method for preparing a pre-treated sample, so a detailed explanation is omitted. According to the first contact step in the method for measuring vitamin D, a mixture containing the sample and the first composition is obtained as a pre-treated sample.

[0102] (Second Contact Step) In the second contact step, a mixture containing the pre-treated sample and the second composition is obtained as the measurement sample, and a specific reaction regulated by the presence of vitamin D proceeds in the measurement sample. In the measurement sample, vitamin D is readily released from the DBP-vitamin D complex, and the specific reaction is strongly regulated by the released vitamin D, making it possible to observe the specific reaction with high sensitivity. For this reason, the measurement sample is suitable for use in measuring vitamin D by homogeneous assay.

[0103] The operation of the second contact step is not particularly limited, as long as the pre-treated sample and the second composition come into contact. For example, after adding the second composition to the pre-treated sample, the mixture may be mixed by inversion, shaking, pipetting or tapping, or by rotational stirring using a stirrer or rotator. After stirring the mixture by these operations, the mixture may be allowed to stand.

[0104] The amount of the second composition used in the second contact step can be adjusted as appropriate within a range in which the progress of the specific reaction can be observed. Without limiting the scope of this disclosure, the amount of the second composition used per 1 volume of pre-treated sample may be between 0.1 and 10 volumes.

[0105] The amount of the second composition used may be determined so that the content of the insoluble carrier in the measurement sample is within a predetermined range. The content of the insoluble carrier in the measurement sample may be, for example, 0.001 w / v% to 10 w / v%, but it may be appropriately selected depending on the type of insoluble carrier. When the insoluble carrier is latex particles, the content of latex particles in the measurement sample is preferably 0.005 w / v% to 5 w / v%.

[0106] In the second contact step, the contact time between the pre-treated sample and the second composition should be sufficient for the specific reaction to be completed. However, from the viewpoint of shortening the time required for the second contact step, the contact time should be, for example, 1 second to 3600 seconds, more preferably 10 seconds to 900 seconds, and even more preferably 60 seconds to 600 seconds. When the specific reaction is an agglutination reaction, from the viewpoint of performing rapid measurement of vitamin D compounds with sufficient sensitivity, the contact time should be 900 seconds or less, more preferably 600 seconds or less, and even more preferably 300 seconds or less. In other words, the agglutination reaction should preferably be completed within 15 minutes from the time of contact, i.e., from the start of the reaction, more preferably within 10 minutes, and even more preferably within 5 minutes. The temperature in the second contact step may be, for example, 0°C to 50°C, and 37°C as an example.

[0107] In the second contact step, compositions other than the second composition may be added to the pre-treated sample. The compositions other than the second composition may be known components used in homogeneous assays, and examples include, but are not limited to, diluents.

[0108] The method for measuring vitamin D compounds may be carried out as a homogeneous assay. In such an embodiment, the measurement method does not include a washing step, and therefore, it is preferable that each composition used in the measurement method does not contain components that require washing, separation, or extraction, such as proteases or acids, bases, salts, or surfactants at concentrations that can inhibit the immune response. For example, when latex particles are used as the insoluble carrier in the second composition, the above components should be selected so as to prevent nonspecific aggregation of the latex particles by salting out or coagulation in the homogeneous assay.

[0109] (Observation step) The method for measuring vitamin D compounds preferably further includes a step of observing a specific reaction. Hereinafter, the step of observing a specific reaction will be referred to as the "observation step".

[0110] The method used in the observation step is selected from methods known in the art, depending on the type of specific reaction. Examples of methods used in the observation step include turbidimetric methods, metric efflux methods, luminescence methods, and fluorescence methods. For example, if the specific reaction is an agglutination reaction of an insoluble carrier, the observation step typically includes measuring the transmitted light intensity or scattered light intensity of a mixture of the pre-treated sample and the second composition (measurement sample). In other words, the observation step may be a step using a turbidimetric method that involves measuring transmitted light intensity, or a metric efflux method that involves measuring scattered light intensity. The measurement of transmitted light intensity or scattered light intensity may be an endpoint assay performed at only one point in time, but from the viewpoint of further improving measurement accuracy, it is preferable to perform it over time.

[0111] The measurement accuracy of the vitamin D measurement method can be evaluated using the coefficient of variation (CV value) of the transmitted light intensity or scattered light intensity measured in the observation step for samples with a vitamin D concentration of 5 ng / mL to 100 ng / mL. For example, the CV value is preferably less than 10%. In this disclosure, the CV value of the transmitted light intensity or scattered light intensity is determined based on the method described in the examples below.

[0112] <Kit> One aspect of the present disclosure relates to a kit for a method of measuring vitamin D compounds in a sample by homogeneous assay. The kit is for a method of measuring vitamin D compounds and comprises a pretreatment composition (first composition) and a reaction composition (second composition).

[0113] The kit may further comprise any components such as: diluents or other compositions other than the first and second compositions; instruments used in a method for measuring vitamin D, particularly for observing immune responses, such as containers; and instructions for using the kit.

[0114] The user manual may be a paper document with instructions printed on it, or it may be an electronic document that stores the instructions for the user. Alternatively, instead of explaining how to use the kit, the user manual may be a paper or electronic document that explains how to access the instructions for the user (for example, a URL for a document that can be viewed on the internet).

[0115] <Summary> As can be understood from the above explanation, this disclosure encompasses the following aspects:

[0116] Embodiment 1: A pretreatment composition (first composition) for measuring vitamin D compounds in a sample by homogeneous assay, wherein the pH of the pretreatment composition is 6.0 or higher and 9.0 or lower, and the pretreatment composition contains a ligand for the vitamin D compounds, the ligand being an unsubstituted or substituted benzoic acid represented by the following formula (1) (wherein X is H, OH, COCH ３ SH and NH ２ A pretreatment composition characterized by being selected from the group consisting of, where m Y are each independently selected from the group consisting of alkyl groups, halogens and alkoxy groups, and m is an integer between 0 and 4), warfarin, azapropazone, phenylbutazone, asenocumarol, indomethacin, tolbutamide, chlorpropamide, iofenoxate, iodipamide, sulfadimethoxine, sulfathiazole, furosemide, tenoxicam, sodium valproate, pyretanide, spironolactone, potassium canrenoate, quercetin, benzylthiouracil, oxyfenbutazone, S-carbenicillin, cosaran, bilirubin, eicosanoids, and 3-carboxy-4-methyl-5-propyl-2-furanopanoic acid, and ions thereof, wherein the ion is an anion. According to this embodiment, a composition is provided that can be used for pretreatment suitable for measurement of vitamin D compounds by homogeneous assay.

[0117]

[0118] Embodiment 2: The pretreatment composition according to Embodiment 1, wherein the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and its ions, and the concentration of the benzoic acid ion represented by formula (1) is 100 mM or more and 800 mM or less. According to this embodiment, the release of vitamin D compounds is further promoted under neutral to basic conditions.

[0119] Embodiment 3: A pretreatment composition according to Embodiment 2, wherein the pH of the pretreatment composition is 7.0 or higher and 9.0 or lower. According to this embodiment, the subsequent immunoassay can be carried out at a pH closer to that of the pretreatment, and the release of vitamin D compounds is further promoted during the pretreatment.

[0120] Embodiment 4: A pretreatment composition according to any one of Embodiments 1 to 3, further comprising a water-soluble polymer. According to this embodiment, the first composition is provided with a function due to the water-soluble polymer.

[0121] Embodiment 5: A method for preparing a pre-treated sample for measuring vitamin D compounds in a sample by a homogeneous assay, characterized by comprising the step of contacting the sample with a pre-treatment composition according to any one of Embodiments 1 to 4. According to this embodiment, a pre-treated sample suitable for measuring vitamin D compounds by a homogeneous assay is prepared.

[0122] Embodiment 6: A method for preparing a pre-treated sample according to Embodiment 5, wherein 1 μL of the sample is brought into contact with the pre-treatment composition containing 3 μmol to 25 μmol of benzoic acid ions represented by formula (1). According to this embodiment, the release of vitamin D compounds is further promoted under neutral to basic conditions.

[0123] Embodiment 7: A method for measuring vitamin D compounds in a sample by homogeneous assay, comprising the steps of: contacting the sample with a pretreatment composition according to any one of Embodiments 1 to 4; and measuring the vitamin D compounds in the pretreatment sample by an immunological method. This embodiment provides a method for measuring vitamin D compounds with good accuracy by homogeneous assay.

[0124] <Additional Notes> This disclosure is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of this disclosure.

[0125] An embodiment of this disclosure is described below, but each embodiment is not limiting to this disclosure.

[0126] <Example 1: Preparation of Calibration Curve> Pretreatment compositions containing salicylate ions at different concentrations were prepared, and calibration curves for 25-hydroxyvitamin D (25OHVD) concentration were created using each pretreatment composition and reaction composition.

[0127] (Preparation of the composition) The following composition was prepared.

[0128] - Pretreatment composition C1 (Comparative example) Water (as solvent) Sodium salicylate 0 mM pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 0.3 w / v% - Pretreatment compositions 2 to 8 (Examples) These were the same as pretreatment composition C1, except for the changes shown in Table 1 below.

[0129]

[0130] • Reaction composition 1: Water (as solvent), latex particles sensitized with anti-25-hydroxyvitamin D antibody, bovine serum albumin 1 w / v%, trehalose dihydrate 5 w / v%

[0131] • Sample 1: Mass Spect Gold Vitamin D Free Human Serum (Golden West Biologicals) (as solvent), 25OHVD3 (calcifediol synthesis) 0, 5, 10, 20, 50, or 100 ng / mL • Sample 2: Water (as solvent), HEPES 25 mM, Bovine serum albumin 1 w / v%, Sodium chloride 150 mM, 25OHVD3 (calcifediol synthesis) 0, 5, 10, 20, 50, or 100 ng / mL

[0132] (Preparation of Calibration Curve) In a 96-well plate, 54 μL of pretreatment composition C1 was added to 2 μL of sample 1 and mixed. 18 μL of reaction composition 1 was added to the resulting mixture (pretreatment sample), and the absorbance at a wavelength of 700 nm was measured every 30 seconds using a plate reader while incubating at 37°C for 5 minutes. The difference between the average absorbance at 30 and 60 seconds from the start of incubation and the average absorbance at 270 and 300 seconds was taken and plotted as a calibration curve. The same measurements were performed for sample 2 and pretreatment compositions 2-8. The obtained calibration curves are shown in Figures 1 and 2.

[0133] (Results) As shown in Figures 1 and 2, in pretreatment composition C1 which does not contain salicylate ions, the absorbance of serum-based sample 1 at the same 25OHVD3 concentration was significantly lower than that of buffer-based sample 2. This lower deviation is thought to be because 25OHVD3 binds to DBP contained in serum and forms a complex. On the other hand, this lower deviation was reduced in pretreatment compositions 2 to 8 which contain salicylate ions. From this, it was found that the pretreatment compositions according to one aspect of the present disclosure can be used for pretreatment suitable for the measurement of vitamin D compounds by homogeneous assay.

[0134] Furthermore, since pretreatment compositions 2 to 7 showed a high absorbance response to an increase in 25OHVD3 concentration, it was found that salicylate ions in a concentration range of 125 mM to less than 937 mM are particularly useful.

[0135] <Example 2: Determination of CV Value> The CV value was determined using pretreatment composition 4. As the sample, serum that had been valued by mass spectrometry was diluted with vitamin D-free serum (Golden West Bio.) to a calcifediol (25OHVD3) concentration of 5 ng / mL or 20 ng / mL. Absorbance based on transmitted light intensity was measured for 10 separate samples (No. 1 to 10) prepared under the same conditions using the biochemical automated analyzer Bioliss 30i (Tokyo Boeki Medisys Co., Ltd.). The results are shown in Table 2.

[0136]

[0137] As shown in Table 2, we confirmed that the CV value was less than 5% at a 25OHVD3 concentration of 5 ng / mL and at 20 ng / mL, which is the diagnostic criterion for vitamin D deficiency in clinical testing.

[0138] <Example 3: Examination of Benzoic Acid Species> As alternative species of benzoic acid, we investigated using isomers and organic salts of salicylic acid.

[0139] (Preparation of the composition) The following composition was prepared.

[0140] • Pretreatment composition C11 (Comparative Example) Water (as solvent) Sodium salicylate 0 w / v% (0 mM) pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 1.0 w / v% • Pretreatment composition 12 (Example) Water (as solvent) Sodium salicylate (2-hydroxybenzoate sodium) 10 w / v% (625 mM) pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 1.7 w / v% • Pretreatment composition 13 (Example) Water (as solvent) Sodium 3-hydroxybenzoate 10 w / v% (625 mM) pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 1.5 w / v% • Pretreatment composition 14 (Example) Water (as solvent) Sodium 4-hydroxybenzoate 10 w / v% (625 mM) pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 1.5 w / v% • Pretreatment composition 15 (Example) Water (as solvent) Tetrabutylammonium salicylate 10 w / v% (263 mM) pH 7.3 Sodium chloride 150 mM Bovine serum albumin 1 w / v% Polyvinylpyrrolidone (PVP) 1.0 w / v%

[0141] A calibration curve was prepared using the pretreatment composition in the same manner as in Example 1. The results are shown in Figure 3.

[0142] As shown in Figure 3, the additive effect was confirmed with all isomers: o-isomer (sodium salicylate), m-isomer (sodium 3-hydroxybenzoate), and p-isomer (sodium 4-hydroxybenzoate), but the o-isomer showed the best effect.

[0143] Because the molecular weight of the cation portion of the organic salt tetrabutylammonium salicylate differs from that of the sodium salt, when added in the same weight percent as the sodium hydroxybenzoate isomer, the ion concentration is lower (263 mM) (see comparison between compositions 12-14 and composition 15). As a result, although pretreatment composition 15 showed an effect from the addition of tetrabutylammonium salicylate, its effect was not as good as that of pretreatment compositions 12-14 containing the same weight percent of sodium hydroxybenzoate.

Claims

1. A pretreatment composition for measuring vitamin D compounds in a sample by homogeneous assay, wherein the pH of the pretreatment composition is 6.0 or higher and 9.0 or lower, and the pretreatment composition contains a ligand for the vitamin D compounds, wherein the ligand is an unsubstituted or substituted benzoic acid represented by the following formula (1) (wherein X is H, OH, COCH ３ SH and NH ２ At least one selected from the group consisting of warfarin, azapropazone, phenylbutazone, asenocumarol, indomethacin, tolbutamide, chlorpropamide, iofenoxate, iodipamide, sulfadimethoxine, sulfathiazole, furosemide, tenoxicam, sodium valproate, pyretanide, spironolactone, potassium canrenoate, quercetin, benzylthiouracil, oxyfenbutazone, S-carbenicillin, kosaran, bilirubin, eicosanoids, and 3-carboxy-4-methyl-5-propyl-2-furanopanoic acid, and their ions, wherein the ion is an anion. A pretreatment composition characterized by the following features.

2. The pretreatment composition according to claim 1, wherein the ligand is at least one selected from the group consisting of benzoic acid represented by formula (1) and its ions, and the concentration of the benzoic acid ions represented by formula (1) is 100 mM or more and 800 mM or less.

3. The pretreatment composition according to claim 2, wherein the pH of the pretreatment composition is 7.0 or higher and 9.0 or lower.

4. A pretreatment composition according to any one of claims 1 to 3, further comprising a water-soluble polymer.

5. A method for preparing a pre-treated sample for measuring vitamin D compounds in a sample by homogeneous assay, comprising the step of contacting the sample with a pre-treatment composition according to any one of claims 1 to 4.

6. A method for preparing a pre-treated sample according to claim 5, wherein the pre-treatment composition containing 3 μmol to 25 μmol of benzoic acid ions represented by formula (1) is brought into contact with 1 μL of the sample.

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