Non-specific reaction inhibitor, method for using non-specific reaction inhibitor, non-specific reaction inhibition method, reagent for biochemical measurement, sample pretreatment liquid, and kit for biochemical measurement

Abstract

The present invention relates to a non-specific reaction inhibitor which has a constituent unit (A) derived from 2-methacryloyloxyethyl phosphorylcholine and exhibits a copolymer having a specific physical property (X) as measured by Fourier transform infrared spectroscopy by attenuated total reflection measurement (ATR method). And a non-specific reaction inhibitor for use in inhibiting a non-specific reaction when measuring an antibody or antigen contained in a biological sample by mixing the copolymer with the biological sample.

Description

Technical Field

[0001] The present invention relates to a nonspecific reaction inhibitor, a method for using a nonspecific reaction inhibitor, a nonspecific reaction inhibition method, a biochemical assay reagent, a specimen pretreatment solution, and a biochemical assay kit. This application claims priority based on Japanese Patent Application No. 2022-161609 filed in Japan on October 6, 2022, and its contents are incorporated herein by reference. Background Art

[0002] Biochemical measurement methods are conventionally known as means for detecting various components contained in human biological samples. Among biochemical measurement methods, immunological measurement methods utilize specific binding between proteins such as antigen-antibody reaction and are therefore very specific measurement methods.

[0003] On the other hand, due to the presence of various substances in the sample, a combination not based on the original specific reaction is sometimes produced, or a specific reaction is hindered. These are referred to as non-specific reactions, and the cause substances are sometimes referred to as non-specific factors. When non-specific reactions occur in biochemical assays, measurement errors are produced. Therefore, various non-specific factors have been elucidated so far, and the inhibition method of non-specific reactions has been studied.

[0004] For example, as a non-specific factor, heterophilic antibodies are known (non-patent document 1). Heterophilic antibodies refer to the general term for human antibodies that show reactivity to animal-derived antibodies as the main component of immunological assays, and HAMA (human anti-mouse immunoglobulin antibody) is known as a representative antibody. In addition, as a non-specific factor, rheumatoid factor (RF) is known. Rheumatoid factor is an antibody of the main IgM class that recognizes the Fc region of IgG, which is not only present in patients with rheumatism, but also in the blood of healthy people. As a method for suppressing non-specific reactions caused by heterophilic antibodies and rheumatoid factors, it is known that anti-human IgM antibodies, anti-human IgA antibodies, or anti-human IgG antibodies are mixed with samples in advance (patent document 1). In this way, the component added to the reagent in order to suppress non-specific reactions is called a non-specific reaction inhibitor.

[0005] However, high molecular polymers having segments derived from 2-methacryloyloxyethyl phosphorylcholine are commonly used components in immunological assay reagents. For example, in patent document 2, in latex immunoturbidimetry, it is added to the assay reagent for the purpose of stabilizing the dispersion of latex particles. In addition, it is known that there are effects of suppressing the prozone phenomenon in latex immunoturbidimetry (patent document 3), effects of enabling high-sensitivity measurements in antigen-antibody reactions, enzyme-substrate reactions, etc. (patent document 4), effects of improving reproducibility in agglutination immunoassays (patent document 5), and protein adsorption inhibition effects on polystyrene plates used in ELISA methods (patent document 6). In addition, patent document 7 discloses that in a reagent based on the latex immunoturbidimetry method for detecting the presence or absence of anti-syphilis Treponema pallidum antibodies in a serum specimen, a copolymer containing a segment derived from 2-methacryloyloxyethyl phosphorylcholine and a segment derived from a hydrophilic monomer is used as a sensitizer to prevent the occurrence of serum interference. In physiological saline and serum, the types of proteins, lipids, and other various components contained are very different. Serum interference described in Patent Document 7 refers to a phenomenon in which measured values ​​differ from each other when measuring physiological saline to which an anti-Treponema pallidum antibody standard solution is added and syphilis-positive serum to which the same amount of the standard solution is added. The addition of the above component reduces the difference in measured values.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Biophysical Chemistry 2007, 51, 223-226

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Laid-Open No. 07-012818

[0011] Patent Document 2: Japanese Patent Application Publication No. 2001-228149

[0012] Patent Document 3: Japanese Patent Application Publication No. 2001-318099

[0013] Patent Document 4: Japanese Patent Application Publication No. 2002-022740

[0014] Patent Document 5: International Publication No. 2002 / 018953

[0015] Patent Document 6: Japanese Patent Application Publication No. 2015-148558

[0016] Patent Document 7: Japanese Patent No. 4629564 Summary of the invention

[0017] Technical problem solved by the invention

[0018] The method of suppressing non-specific reactions by anti-human IgM antibodies etc. described in Patent Document 1 is a method currently put into practical use in various reagents. However, it is known from the test of the present inventors that there are non-specific reactions that cannot be suppressed by these antibodies. These non-specific reactions cannot be suppressed even if the components described in Patent Document 7 are used. The problem of the present invention is to suppress such non-specific reactions.

[0019] One of the objects of the present invention is to provide a nonspecific reaction inhibitor, a method for using a nonspecific reaction inhibitor, a method for inhibiting a nonspecific reaction, a biochemical assay reagent, a specimen pretreatment solution, and a biochemical assay kit that can inhibit nonspecific reactions and improve measurement accuracy.

[0020] Technical means of solving problems

[0021] [1] A non-specific reaction inhibitor, which is a copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X:

[0022] The non-specific reaction inhibitor is used for suppressing a non-specific reaction when measuring a substance to be measured contained in a biological sample by mixing the copolymer with the biological sample.

[0023] [2] A method for using a non-specific reaction inhibitor, which is a method for using a copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X as a non-specific reaction inhibitor, wherein:

[0024] By mixing the copolymer with a biological sample, a non-specific reaction is suppressed when measuring a substance to be measured contained in the biological sample.

[0025] [3] A method for inhibiting a non-specific reaction, wherein:

[0026] A copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X is mixed with a biological sample to suppress a nonspecific reaction when measuring a substance to be measured contained in the biological sample.

[0027] [4] A biochemical assay reagent, which is a reagent used in a biochemical assay, wherein:

[0028] The reagent includes a copolymer having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X.

[0029] [5] The biochemical assay reagent according to [4], which is a reagent used in biochemical assays, wherein

[0030] The reagent comprises a copolymer having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X,

[0031] The copolymer is contained in the reagent as a nonspecific reaction inhibitor for suppressing nonspecific reactions in a biological sample.

[0032] [6] The biochemical assay reagent according to any one of [4] to [5], wherein

[0033] The biochemical assay is a homogeneous method.

[0034] [7] The biochemical assay reagent according to any one of [4] to [6], wherein

[0035] The biochemical assay is an assay based on antigen-antibody reaction.

[0036] [8] The biochemical assay reagent according to any one of [4] to [7], wherein

[0037] The biochemical assay was latex immunoturbidimetry.

[0038] [9] The biochemical assay reagent according to any one of [4] to [8], wherein

[0039] The biological sample to be measured in the above-mentioned biochemical measurement is a blood sample.

[0040]

[10] The biochemical assay reagent according to any one of [4] to [9], wherein

[0041] The biochemical assay is an assay for detecting an antigen-antibody reaction between one or more selected from soluble interleukin-2 receptor and pulmonary surfactant protein D contained in a biological sample as an assay object and an antibody added to the biological sample.

[0042]

[11] A sample pretreatment solution, which is a reagent used in biochemical assays, wherein:

[0043] The reagents include:

[0044] A copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X; and

[0045] A liquid medium which dissolves or disperses the copolymer.

[0046]

[12] A biochemical assay kit comprising:

[0047] The biochemical assay reagent according to any one of [4] to

[10] or the sample pretreatment solution according to

[11] .

[0048]

[13] A method for using a biochemical assay reagent, wherein:

[0049] The biochemical assay is performed by mixing the biochemical assay reagent described in any one of [4] to

[10] or the sample pretreatment solution described in

[11] with a biological sample.

[0050]

[14] A method for inhibiting nonspecific reactions of biochemical assay reagents, wherein:

[0051] When the biochemical assay reagent according to any one of [4] to

[10] or the sample pretreatment solution according to

[11] is mixed with a biological sample to perform a biochemical assay, the copolymer suppresses a nonspecific reaction in the biological sample.

[0052] <Physical Properties X>

[0053] In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

[0054] Effects of the Invention

[0055] According to the present invention, a nonspecific reaction inhibitor, a method for using the nonspecific reaction inhibitor, a method for inhibiting nonspecific reactions, a biochemical assay reagent, a sample pretreatment solution, and a biochemical assay kit are provided, which can inhibit nonspecific reactions and improve measurement accuracy.

[0056] Furthermore, when the nonspecific reaction inhibitor of the present invention is used, it is possible to suppress only the nonspecific reaction without reducing the measurement sensitivity.

[0057] " suppressing non-specific reaction " recorded in this specification refers to alleviating the non-specific reaction of the sample source produced by some reasons, and becomes the state that the measured value is closer to the original measured value (true value). With regard to the true value, if there is the measured object component of the benchmark determination method, the true value is the measured value based on the benchmark determination method. If there is no measured object component of the benchmark determination method, then the determination based on the heterogeneous method (the determination method of washing process and B / F separation process is carried out when measuring) is usually not easy to have non-specific reaction relative to the homogeneous method (the determination method of washing process and B / F separation process is not carried out when measuring). Even if it is the heterogeneous method, it is also affected by the non-specific reaction, and sometimes the true value cannot be obtained. In this case, the sample can be diluted and measured again by the component that does not include the measured object component such as physiological saline, by judging whether it is the measured value corresponding to the dilution ratio, so as to grasp the degree of the influence of the non-specific reaction, as the true value.

[0058] In the case of suppressing nonspecific adsorption, it is considered to use a method of coating all the components involved in the reaction with a certain component. For example, the case of using a nonspecific adsorption inhibitor is equivalent to this. The nonspecific adsorption inhibitor can be fixed to the inner wall of the reagent container, the inner wall of the reaction container (for example, the inner wall of the microplate used in the ELISA method, the inner wall of the reaction unit of the automatic analyzer, etc.), the membrane in the immunochromatography, the pad, etc., and the surface is coated. In addition, the nonspecific adsorption inhibitor can seal the solid phase (microplate, latex particles, magnetic particles, fluorescent particles, membrane in immunochromatography, etc.) bound to antigens and antibodies. When such a nonspecific adsorption inhibitor is added to a biochemical assay reagent, there is a situation where the biochemical reaction that was originally intended to occur is not easy to occur, and the measurement sensitivity is reduced. In this specification, it is intended not to cause such a reduction in measurement sensitivity and to suppress nonspecific reactions that are difficult to suppress in nonspecific adsorbents. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] [ Figure 1A ] is a graph showing infrared absorption spectra of various products having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine, obtained by Fourier transform infrared spectroscopy (FT-IR) analysis based on attenuated total reflectance measurement (ATR method), and is the spectrum of Lipidure used in Examples 1 to 3.

[0060] [ Figure 1B ] is a diagram showing a part of the spectrum of the copolymer Lipidure used in Examples 1 to 3, which is enlarged.

[0061] [ Figure 1C ] is a diagram showing a part of the spectrum of the copolymer Lipidure used in Examples 1 to 3, which is enlarged.

[0062] [ Figure 1D] is a diagram showing a part of the spectrum of the copolymer Lipidure used in Examples 1 to 3, which is enlarged.

[0063] [ Figure 1E ] is the spectrum of the copolymer Lipidure used in Comparative Examples 3 to 4 and Comparative Examples 6 to 10.

[0064] [ Figure 1F ] is a diagram showing a part of the spectrum of the copolymer Lipidure used in Comparative Examples 3 to 4 and Comparative Examples 6 to 10, which is enlarged.

[0065] [ Figure 1G ] is the spectrum of the copolymer Lipidure used in Comparative Examples 11 to 16.

[0066] [ Figure 1H ] is a diagram showing a part of the spectrum of the copolymer Lipidure used in Comparative Examples 11 to 16, which is enlarged.

[0067] [ Figure 2 ] is a scatter plot showing the measurement results of soluble interleukin 2 receptor (sIL-2R) in human serum, comparing the results of the LTIA method and the CLEIA method.

[0068] [ Figure 3 ] is a scatter plot showing the measurement results of lung surfactant protein D (SP-D) in human serum, comparing the results of the LTIA method and the CLEIA method. DETAILED DESCRIPTION

[0069] <<Non-specific reaction inhibitors>>

[0070] The first aspect of the present invention is a copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X. Here, the structural unit may be referred to as a segment or a repeating unit.

[0071] <Physical Properties X>

[0072] In the infrared absorption spectrum obtained by Fourier transform infrared spectroscopy (FT-IR) analysis based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

[0073] [Infrared absorption spectrum]

[0074] The infrared absorption spectrum is an absorption spectrum that summarizes the vibration frequencies (hereinafter sometimes referred to as "wave numbers") unique to the functional groups generated by the functional groups in the molecule when the molecule is irradiated with infrared light. In the present invention, the infrared absorption spectrum is a value measured by the attenuated total reflection measurement method (ATR method) using the Fourier transform infrared spectroscopy (FT-IR) analysis method. The infrared absorption spectrum has transmittance (%) on the vertical axis and wave number (unit: cm) on the horizontal axis. -1 ), is shown as a graph of curves connecting consecutive measured values. When there is a peak with maximum absorption in a specific wave number range, the graph changes from decrease to increase in the peak.

[0075] The numerical values ​​of the infrared absorption spectra described in this specification are based on the values ​​measured using the following apparatus and measurement conditions. If a measurement apparatus having performance equal to or better than the following is used and the same measurement conditions are applied, the same measurement results should be obtained.

[0076] <Measurement Device>

[0077] Thermo Scientific Nicolet TM iS5 FT-IR

[0078] <Measurement Conditions>

[0079] Crystal: Diamond

[0080] Angle of incidence: 45°

[0081] Correction: None

[0082] In the first embodiment of the present invention, the infrared absorption spectrum of the copolymer is 3000 to 2800 cm -1 The range has at least two peaks of maximum absorption. Among the peaks in the range, the wave number ranges of the peak on the high wave number side (hereinafter sometimes referred to as P1) and the peak on the low wave number side (hereinafter sometimes referred to as P2) are as follows.

[0083] In one embodiment of the method, P1 appears at 3000-2880 cm -1 The maximum absorption of P1 is 2946~2906cm -1 , preferably 2936~2916cm -1 P2 appears in the range of 2879 to 2800 cm -1 The maximum absorption of P2 is 2870~2833cm -1 , preferably 2865~2833cm -1 , more preferably 2863 to 2843 cm -1The range. The number of peaks with the maximum absorption in the range of P1 and the range of P2 is also considered to be affected by the resolution of the infrared absorption spectroscopic device, and is not particularly limited. It is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and most preferably 1. The transmittance (%) of the maximum absorption can be P1 > P2, can be P1 = P2 or P1 ≈ P2, and can be P1 < P2. Preferably, P1 < P2.

[0084] Generally, in the range of 3000 - 2800 cm -1 The region is the region where peaks of CH stretching, CH2 symmetric stretching, CH3 symmetric stretching, CH2 antisymmetric stretching, and CH3 antisymmetric stretching appear, and it is presumed that P1 and P2 belong to at least any one or more of these. In particular, CH2 symmetric stretching has a peak around 2853 cm -1 and CH2 antisymmetric stretching has a peak around 2926 cm -1 (Identification Method of Spectra of Organic Compounds, 8th Edition, published by Tokyo Kagaku Dojin, 2016, p84). Generally, the peaks of the infrared absorption spectrum are shifted by the influence of the surrounding molecular structure. In the region of 3000 - 2800 cm -1 , the variation of the peaks generally falls within the range of ±10 cm -1 .

[0085] In the first aspect of the present invention, the infrared absorption spectrum of the copolymer further shows a peak with the maximum absorption in the range of 1000 - 920 cm -1 (hereinafter, sometimes referred to as P3).

[0086] In one embodiment of the above aspect, the maximum absorption of P3 is in the range of 975 - 940 cm -1 . The ratio (P1 / P3) of the intensity of the maximum absorption of P1 to the intensity of the maximum absorption of P3 (100%) is 16% or more, preferably 20% or more, more preferably 26.6% or more, further preferably 27% or more, and most preferably 28% or more. The ratio (P2 / P3) of the intensity of the maximum absorption of P2 to the intensity of the maximum absorption of P3 (100%) is 8% or more, preferably 10% or more, more preferably 12% or more, more preferably 15% or more, and further preferably 18% or more. It should be noted that P3 may be a peak attributed to the -P - O - C - structure contained in the constituent unit A (Japanese Patent Laid-Open No. 2002 - 143294).

[0087] In the first aspect of the present invention, the infrared absorption spectrum of the copolymer further shows a peak in the range of 1300 - 1200 cm -1 (hereinafter, sometimes referred to as P4).

[0088] In one embodiment of the method, the maximum absorption of P4 is 1252-1215 cm -1 The ratio (P1 / P4) of the intensity of the maximum absorption of P1 to the intensity of the maximum absorption of P4 (100%) is 18% or more, preferably 25% or more, more preferably 28.4% or more, further preferably 29% or more, and most preferably 30% or more. The ratio (P2 / P4) of the intensity of the maximum absorption of P2 to the intensity of the maximum absorption of P4 (100%) is 9% or more, preferably 12% or more, more preferably 15% or more, and further preferably 20% or more. It should be noted that P4 may be a peak attributable to the P=O structure contained in the constituent unit A (Japanese Patent Application Laid-Open No. 3-39309).

[0089] In the first embodiment of the present invention, the infrared absorption spectrum of the copolymer further shows that the infrared absorption spectrum is between 1100 and 1000 cm -1 In one embodiment of the method, the maximum absorption of P5 is 1090-1040 cm -1 P5 may be a peak attributed to the PO-O-structure contained in the structural unit A (Japanese Patent Application Laid-Open No. 10-324634).

[0090] The intensity of the infrared absorption spectrum is expressed as formula (1), and is represented by the absorbance (%) obtained by subtracting the transmittance (%) at each wave number obtained by measurement from 100 (%).

[0091] Intensity of infrared absorption spectrum = 100 (%) - transmittance of the wave number (%)... (1)

[0092] The ratio R of the intensity K2 of the maximum absorption at the second wave number to the intensity K1 of the maximum absorption at the first wave number is calculated by equation (2).

[0093] Ratio R = Strength K2 / Strength K1 × 100 (%) (2)

[0094] [Kinematic viscosity]

[0095] In the first embodiment of the present invention, the kinematic viscosity (unit: m 2 / s), it is preferably in the following range at 25°C. That is, it is preferably 0.2 to 4.0 m / s. 2 / s, more preferably in the range of 0.4 to 3.8, and further preferably in the range of 0.6 to 3.6. In addition, within the preferred range, the lower limit value may be greater than 0.8, greater than 1.0, greater than 1.2, or greater than 1.4. In addition, the upper limit value may also be less than 3.0, less than 2.7, or less than 2.4. These lower limits and upper limits may be arbitrarily combined, for example, may be 1.4 to 2.4.

[0096] Kinematic viscosity refers to the value obtained by dividing the viscosity by the density of the liquid under the same conditions (temperature, pressure). The method for measuring viscosity is specified by Japanese Industrial Standard JIS Z8803. Kinematic viscosity can be measured using a capillary viscometer. Those skilled in the art can purchase any measuring equipment to measure the kinematic viscosity. Measuring equipment is sold, for example, by Shibata Science Co., Ltd., A&D Co., Ltd., etc.

[0097] [Surface tension]

[0098] The surface tension of the aqueous solution obtained by dissolving the copolymer in water at a concentration of 0.1 wt% (unit: ×10 -3 N / m), it is preferably in the following range at 25°C. That is, it is preferably 45 to 75 × 10 -3 In the preferred range, the lower limit may be 47 or more or 49 or more. In addition, the upper limit may be 73 or less, 71 or less, or 69 or less. These lower limits and upper limits may be arbitrarily combined, for example, 49 to 69.

[0099] Surface tension is represented by surface energy per unit area. Surface tension can be measured by the Wilhelmy plate method. Measuring equipment is sold by, for example, Kyowa Interface Science Co., Ltd., Sanyo Trading Co., Ltd., and the like.

[0100] [Constitutional units of the copolymer]

[0101] The structural unit A of the copolymer is the same as that of the copolymer described in Patent Document 7.

[0102] The copolymer preferably has one or more constituent units B that do not have a functional group that is ionized in water. The constituent unit B has a structure that is the source of at least the peak of P2 in the infrared absorption spectrum. If the copolymer has the constituent unit B, the copolymer described in Patent Document 7 is different from the copolymer of this embodiment in terms of the infrared absorption spectrum and the constituent unit B. The copolymer of this embodiment can show the infrared absorption spectrum (peak P1, peak P2, peak intensity ratio P1 / P3, P2 / P3, P1 / P4, P2 / P4) by having the constituent unit B, and present a specific range of kinematic viscosity and surface tension, further suppressing non-specific reactions.

[0103] The molar fraction of the constituent unit A relative to the total constituent units constituting the copolymer of this embodiment is preferably 2 to 98%, more preferably 10 to 95%, more preferably 30 to 95%, and most preferably 50 to 90%. In addition, it can be 60 to 90%, 65 to 90%, 70 to 90%, 70 to 85%, or 75 to 85%.

[0104] The molar fraction of the total of the constituent units A and B relative to the total constituent units constituting the copolymer of this embodiment is preferably 60 to 100%, more preferably 80 to 100%, further preferably 85 to 100%, and most preferably 90 to 100%. When it is 100%, the copolymer is composed of only the constituent units A and B.

[0105] When the molar fraction of each structural unit is within the above range, non-specific reactions can be further suppressed.

[0106] The ratio of the constituent unit A to the constituent unit B in the copolymer of this embodiment may be, for example, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, or 1:6 to 6:1 in terms of molar ratio. In addition, it may be 1:5 to 5:1, 1:4 to 4:1, or 7:3 to 3:7. In addition, it may be 1:1 to 6:1, 2:1 to 6:1, 3:1 to 6:1, or 4:1 to 6:1. When it is within the above range, non-specific reactions can be further suppressed.

[0107] The molar fraction of the constituent unit B relative to the remaining part after removing the constituent unit A from all the constituent units constituting the copolymer of this embodiment can be 10 to 100%, 20 to 100%, 40 to 100%, or 50 to 100%. In addition, it is preferably 60 to 100%, and more preferably 70 to 100%. It can be 80 to 100%, 90 to 100%, or 95 to 100%. In addition, it is preferably 55 to 95%, 60 to 95%, 65 to 95%, 70 to 95%, 55 to 90%, 60 to 90%, 65 to 90%, 70 to 90%, 55 to 85%, 60 to 85%, 65 to 85%, or 70 to 85%. The remaining part may include constituent units C other than the constituent unit B.

[0108] When the molar fraction of the constituent unit B is within the above range, non-specific reactions can be further suppressed. It should be noted that the ratio of the constituent unit A to the constituent unit B in the copolymer of the present embodiment and the molar fraction of the constituent unit B relative to the remaining portion after removing the constituent unit A from all the constituent units can be measured, for example, using thermal decomposition gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy (1H-NMR, 13C-NMR), etc.

[0109] The monomer forming the structural unit A has a functional group having a structure represented by formula (3). A , R B and R C Each is independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group having 1 to 6 carbon atoms, and n is an integer of 1 to 4. Among them, R A , R B and R C A structure in which the group is methyl and n=2.

[0110] [Chemical formula 1]

[0111]

[0112] As a specific example of a monomer having a structure represented by formula (3), 2-(meth)acryloyloxyethyl phosphorylcholine (preferably 2-methacryloyloxyethyl phosphorylcholine) represented by formula (4) can be cited. In the formula, X represents a hydrogen atom or a methyl group, preferably a methyl group. Formula (4) has a (meth)acryloyl group and can therefore be copolymerized with the polymerizable monomer that forms constituent unit B. It should be noted that in this specification, the expression of (meth)acrylic acid is an abbreviation of acrylic acid or methacrylic acid, and the same applies to other similar expressions.

[0113] The copolymer of the structural unit A and the structural unit B, or the copolymer of the structural unit A, the structural unit B, and the structural unit C has a structure showing the infrared absorption spectra P3 and P4.

[0114] [Chemical formula 2]

[0115]

[0116] The structural unit B has an infrared absorption spectrum at 2870-2833 cm -1 It has maximum absorption and does not have a functional group that is ionized in water. Here, "functional group that is ionized in water" refers to a functional group that can have a positive charge or a negative charge by donating or accepting a proton in water. For example, acid groups such as carboxyl and sulfonic acid groups release protons in water and ionize. In addition, amino groups introduce protons in water and ionize. It should be noted that the hydroxyl group of alcohol does not ionize, but the phenolic hydroxyl group bonded to the aromatic ring ionizes in water.

[0117] As the functional group that the constituent unit B may have that is not ionized in water, for example, an alcoholic hydroxyl group, a straight-chain or branched hydrocarbon group that may have a cyclic portion, etc. may be mentioned. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group, preferably an aliphatic hydrocarbon group. In addition, a carbonyl group and / or an ether bond may be included. As the number of carbon atoms possessed by the hydrocarbon group, for example, 1 to 24 may be mentioned. In this specification, a functional group that is not ionized in water is sometimes referred to as a hydrophobic functional group.

[0118] Examples of the polymerizable monomer that forms the structural unit B include compounds represented by formula (5). The structural unit B may be composed of one or more compounds represented by formula (5).

[0119] [Chemical formula 3]

[0120]

[0121] In the formula, X represents a hydrogen atom or a methyl group, and is preferably a methyl group.

[0122] In the formula, R1 represents a compound selected from -C6H4-, -C6H 10 -, -(C=O)O-, -O-, -(C=O)NH-, -O-(C=O)- and -O-(C=O)-O-.

[0123] In the formula, R2 represents a hydrogen atom, -(CH2) g -R3 and -((CH2) p -O) h -Hydrophobic functional group in R3. Here, g represents an integer of 7 to 24, h represents an integer of 1 to 24, p represents an integer of 3 to 5, and R3 represents a functional group selected from a hydrogen atom, a methyl group, -C6H5, and -O-C6H5.

[0124] Among them, the structure represented by formula (6) in which R1 is -(C=O)O- is preferred. In formula (6), X is preferably a methyl group.

[0125] [Chemical formula 4]

[0126]

[0127] R2 is preferably a structure without a hydrogen-bonding functional group. "Without a hydrogen-bonding functional group" means that it does not have a functional group that can form a hydrogen bond with a polar molecule such as water or alcohol, and / or a functional group that can form a hydrogen bond between the side chains R2 in the copolymer. Examples of hydrogen-bonding functional groups include hydroxyl (-OH), amino (-NH2), carboxyl (-C(=O)OH), and phosphate (-P(=O)(OH)2).

[0128] Examples of R2 having no hydrogen-bonding side chain include straight-chain or branched aliphatic hydrocarbon groups, cyclic alkyl groups, aromatic groups, etc. Among them, R2 shows CH2 symmetric stretching and CH2 antisymmetric stretching in the infrared absorption spectrum, i.e., 2870 to 2833 cm -1 has the maximum absorption, so R2 is preferably -(CH2) g -R3, g is preferably 7 or more, 8 or more, 10 or more, or 12 or more. In order to make the copolymer of this embodiment easily soluble in the buffer solution constituting the biochemical assay reagent, g may be 24 or less, 22 or less, 20 or less, or 18 or less. g may be 8-24, 10-24, 12-24. In addition, g may be 8-22, 10-22, 12-22. In addition, g may be 8-20, 10-20, 12-20. In addition, g may be 8-18, 10-18, 12-18. Among them, g is preferably 12-22, 12-20, 12-18, and most preferably 12-18. R3 is preferably a hydrogen atom.

[0129] As specific polymerizable monomers (hydrophobic monomers) forming the constituent unit B, for example, straight or branched alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aromatic (meth)acrylates, etc. having an aliphatic hydrocarbon group can be cited. More specifically, for example, straight or branched alkyl (meth)acrylates having an aliphatic hydrocarbon group such as octyl (meth)acrylate, decyl (meth)acrylate, lauryl (meth)acrylate, tetradecyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, oleyl (meth)acrylate, eicosyl (meth)acrylate, behenyl (meth)acrylate, etc.; cyclic alkyl (meth)acrylates such as cyclohexyl (meth)acrylate; aromatic (meth)acrylates such as benzyl (meth)acrylate and phenoxyethyl (meth)acrylate; hydrophobic polyalkylene glycol (meth)acrylates such as polypropylene glycol (meth)acrylate, etc. can be cited. It should be noted that the expression of (meth)acrylic acid is an abbreviation of acrylic acid or methacrylic acid. Among these hydrophobic monomers, linear or branched alkyl (meth)acrylates are preferred, and octyl (meth)acrylate, decyl (meth)acrylate, lauryl (meth)acrylate, myristyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, oleyl (meth)acrylate, eicosyl (meth)acrylate, and behenyl (meth)acrylate are preferred. Lauryl (meth)acrylate, myristyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, and oleyl (meth)acrylate are most preferred.

[0130] If the constituent unit B is in the range of 2870 to 2833 cm in the infrared absorption spectrum, -1 The monomer showing the maximum absorption may be used in combination of two or more.

[0131] Constituent unit C can be used as a constituent component of the copolymer of this embodiment as needed. Constituent unit C is a constituent unit derived from any polymerizable monomer that can be polymerized with constituent unit A and constituent unit B. Constituent unit C is not constituent unit A and B. Constituent unit C can be a structure having a functional group that is ionized in water. In addition, constituent unit C can be a structure that does not have a functional group that is ionized in water. As a specific polymerizable monomer (hydrophobic monomer) forming constituent unit C, for example, methyl (meth) acrylate, ethyl (meth) acrylate, and butyl (meth) acrylate can be cited.

[0132] Constituent unit C can be copolymerized within the range that the polymer composed of constituent units A and constituent unit B exhibits practical non-specific reaction inhibition ability. Constituent unit C can be copolymerized for the purpose of improving the production stability, handling ease, storage stability, etc. of the polymer composed of constituent units A and constituent unit B.

[0133] The weight average molecular weight of the copolymer of this embodiment is not particularly limited, and can be, for example, 5,000 to 5,000,000, 10,000 to 4,000,000, 20,000 to 3,000,000, or 20,000 to 2,000,000. In addition, the weight average molecular weight is preferably 20,000 to 1,000,000, 20,000 to 500,000, 20,000 to 200,000, or 20,000 to 100,000. The weight average molecular weight is most preferably 20,000 to 50,000.

[0134] When the molecular weight is within the above range, non-specific reactions can be further suppressed.

[0135] When the weight average molecular weight is 5000 or more, the affinity with non-specific reaction substances becomes high, and the effect of the copolymer as a non-specific reaction inhibitor is easily obtained. In addition, when it is 5000000 or less, the viscosity of the reagent of the present invention containing the copolymer is suppressed from becoming too high, and the target measurement is easily and smoothly performed.

[0136] In addition, the method for measuring the weight average molecular weight of the copolymer of this embodiment can refer to WO2022 / 124288.

[0137] The copolymer of this mode can be synthesized according to the method of recording in patent document 2 (Japanese Patent Publication No. 2001-228149). In addition, the copolymer of this mode can be purchased as a commercially available product. For example, BL1002, BL1003, and BL1103 of the BL series in the various series of Lipidure (registered trademark) sold by NOF Corporation can be cited as preferred series. These can consider other components contained in the biochemical assay reagent, such as the type, pH, concentration of the buffer solution constituting the reagent, the type and concentration of the polymer substance other than this copolymer, and are appropriately combined within the range of not affecting the stirring, dispersion, etc. of the reagent.

[0138] Among various series of Lipidure (registered trademark) sold by NOF Corporation, the series shown below do not exhibit a non-specific reaction inhibitory effect and do not conform to the inherent properties of the copolymer of the present embodiment.

[0139] BL400 series does not exhibit the physical property X regarding infrared absorption spectrum. According to the NOF Corporation website, the side chain characteristics of the structural unit B in BL400 series are anionic, which is not preferable in that structural units ionized in water are included in addition to structural unit A.

[0140] The BL500 series does not exhibit the physical property X regarding infrared absorption spectrum. According to the website, the side chain of the structural unit B is cationic, which is not preferable in that a structural unit that is ionized in water is included in addition to the structural unit A.

[0141] The BL800 series does not exhibit the physical property X regarding infrared absorption spectrum. In addition, according to the homepage, the side chain characteristics of the constituent unit B are "hydrophilic / hydrophobic," which is not preferred in terms of containing a constituent unit that is ionized in water in addition to the constituent unit A. In addition, when the kinematic viscosity exceeds 4.0 m 2 / s and surface tension exceeding 70×10 -3 It is not preferred in terms of N / m.

[0142] In addition, the BL205, BL206, BL1201, BL1202, and BL1301 series do not exhibit the physical property X regarding the infrared absorption spectrum.

[0143] The various series of Lipidure (registered trademark) described above can be used in combination with the copolymer of the present embodiment for the purpose of exerting effects different from the nonspecific reaction inhibition effect, such as sensitization effect, blocking effect, and nonspecific adsorption inhibition effect.

[0144] <Uses of non-specific reaction inhibitors>

[0145] The copolymer of this mode is mixed with a biological sample (specimen) (hereinafter, sometimes only recorded as a sample), and is used to suppress the non-specific reaction caused by the sample when the measurement object substance contained in the sample is measured. By "suppressing non-specific reactions", the measurement error can be reduced when the measurement object substance contained in the sample is measured, and the true measurement value is approached. As the non-specific reaction, for example, the reaction of reducing or suppressing the interaction between components that should interact with each other, the reaction of causing the interaction between components that should not interact with each other, and the reaction of promoting the interaction between components that should only weakly interact to the extent that cannot be detected, etc. can be cited.

[0146] The biological sample may be of human or animal origin. The sample may be blood, culture supernatant, urine, stool, spinal fluid, saliva, sweat, ascites, or extracts of cells or tissues, etc. The blood may be whole blood, plasma, serum, or a sample subjected to any treatment such as dilution or purification of the blood.

[0147] As the measurement target substance, the measurement target substance of the following immunological measurement method can be mentioned.

[0148] <<How to use non-specific reaction inhibitors>>

[0149] The second embodiment of the present invention is a method for using a non-specific reaction inhibitor, which suppresses non-specific reactions when measuring a substance to be measured contained in the sample by mixing the non-specific reaction inhibitor of the first embodiment with the sample. The description of the first embodiment can also be applied to the second embodiment, so the description repeated with the first embodiment is omitted.

[0150] In the method of use of this embodiment, the method of mixing the non-specific reaction inhibitor with the sample is not particularly limited. For example, a method of preparing a reagent S containing the non-specific reaction inhibitor at a desired concentration in an arbitrary buffer and mixing it with the sample can be cited. The reagent can be a reagent included in a biochemical assay kit, such as the first reagent shown in the examples of this specification, or a sample diluent or pretreatment solution.

[0151] When the reagent S is a reagent included in a biochemical assay kit, the sample is generally added with 10- to 100-fold volume of the reagent S. In this case, the concentration of the non-specific reaction inhibitor is preferably, for example, 0.001 to 5.00% (w / v), more preferably 0.002 to 1.00% (w / v), further preferably 0.003 to 0.60% (w / v), particularly preferably 0.005 to 0.50% (w / v), and most preferably 0.04 to 0.30% (w / v) relative to the total volume of the reagent S.

[0152] When the reagent S is a sample diluent or pretreatment solution, the sample is generally added with 0.1 to 10 times the volume of the reagent S. The concentration of the non-specific reaction inhibitor relative to the total volume of the reagent S in this case is preferably, for example, 0.001 to 5.00% (w / v), more preferably 0.01 to 4.50% (w / v), and further preferably 0.10 to 3.00% (w / v), 0.20 to 2.50% (w / v), 0.5 to 2.00% (w / v). When it is within these appropriate ranges, the dispersibility of the non-specific reaction inhibitor in the reagent is improved, and the sample is easily mixed evenly.

[0153] In the mixed solution obtained by mixing the non-specific reaction inhibitor with the sample, it is preferred to place the mixed solution at room temperature, for example, 37° C. for more than 5 minutes, so that the non-specific reaction inhibitor and the components contained in the sample are more likely to contact and react with each other, which is used to exert the non-specific reaction inhibitory effect of the present invention. When such a mixing time is set, non-specific reactions can be fully suppressed when measuring the analyte contained in the sample.

[0154] The concentration of the non-specific reaction inhibitor relative to the total volume of the mixed solution is preferably, for example, 0.001 to 5.00% (w / v), more preferably 0.002 to 1.00% (w / v), further preferably 0.003 to 0.60% (w / v), particularly preferably 0.02 to 0.40% (w / v), and most preferably 0.04 to 0.30% (w / v). When it is within these appropriate ranges, the dispersibility of the non-specific reaction inhibitor in the mixed solution is improved, and the interaction can fully occur.

[0155] <<Non-specific reaction inhibition method>>

[0156] The third embodiment of the present invention is a non-specific reaction inhibition method, which suppresses non-specific reactions when measuring a substance to be measured contained in the sample by mixing the non-specific reaction inhibitor of the first embodiment with the sample. The description of the first to second embodiments can also be applied to the third embodiment, so repeated descriptions are omitted.

[0157] By sufficiently dispersing the nonspecific reaction inhibitor in a mixed solution obtained by mixing the nonspecific reaction inhibitor with a sample, a nonspecific reaction can be sufficiently suppressed when measuring the analyte contained in the sample.

[0158] As an example of the present embodiment, there can be mentioned an analysis method in which at least one of a biochemical reaction and a measurement is performed in the presence of the non-specific reaction inhibitor of the first embodiment.

[0159] As another example of the present method, there is a biochemical measurement method for biochemically measuring a substance to be measured in a sample: in a liquid phase containing the sample, a reaction product based on a reaction between the substance to be measured and a specific affinity substance is measured in the presence of a non-specific reaction inhibitor of the first method.

[0160] Here, relative to the total volume of the liquid phase in which the generated reaction product is measured, the content of the non-specific reaction inhibitor of the first embodiment is, for example, preferably 0.0005 to 5.00% (w / v), more preferably 0.001 to 1.00% (w / v), further preferably 0.003 to 0.60% (w / v), particularly preferably 0.004 to 0.40% (w / v), and most preferably 0.03 to 0.30% (w / v). When it is these suitable ranges, the non-specific reaction in the liquid phase measured can be fully suppressed. These suitable concentration ranges are equally suitable in other examples of this embodiment and other embodiments.

[0161] As another example of the present method, there is the following biochemical assay method for measuring a substance to be measured in a sample by immunoturbidimetric methods such as LTIA, which comprises: in a liquid phase, in the presence of a non-specific reaction inhibitor of the first method, bringing the sample into contact with insoluble carrier particles carrying a substance with specific affinity for the substance to be measured.

[0162] As another example of the present embodiment, there is a biochemical measurement method for measuring a substance to be measured in a sample by an immunoturbidimetric method such as LTIA, which includes the following steps 1 to 3.

[0163] (Step 1) a step of contacting the sample with the non-specific reaction inhibitor of the first embodiment in a liquid phase;

[0164] (Step 2) After step 1, a step of adding insoluble carrier particles carrying a substance having a specific affinity for the substance to be measured to the liquid phase;

[0165] (Step 3) After step 2, a step of measuring the agglutination reaction between the substance to be measured and the insoluble carrier particles.

[0166] In biochemical assays, specific affinity substances for the substance to be assayed are used. If the substance to be assayed is an antibody, the specific affinity substance of step 2 can use an antigen corresponding thereto. If the substance to be assayed is an antigen, the specific affinity substance can use an antibody against it. In addition, when the substance to be assayed is labeled with avidin or biotin, the specific affinity substance can use biotin or avidin. In addition, if the substance to be assayed is a sugar or a sugar chain, the specific affinity substance of step 2 can use a lectin corresponding thereto. In addition, as a specific affinity substance, aptamers, oligonucleotides, and peptides can also be used. Among them, the assay utilizing antigen-antibody reaction is referred to as immunological assay. The "non-specific reaction" described in this specification is a reaction that hinders the normal reaction between the substance to be assayed and the specific affinity substance. The sample-derived component that is the cause of the non-specific reaction interacts with the substance to be assayed, interacts with the specific affinity substance, or interacts with both, whereby the measured value of the substance to be assayed deviates from the true value.

[0167] As an example of a more specific measurement method of the present embodiment, there can be mentioned a method for measuring sIL-2R in a blood sample by optically measuring or visually observing the degree of agglutination caused by the antigen-antibody reaction between an insoluble carrier carrying an anti-sIL-2R antibody and sIL-2R in the blood sample, using a sIL-2R measurement reagent comprising an insoluble carrier carrying an anti-sIL-2R antibody and the non-specific reaction inhibitor of the first embodiment. Here, sIL-2R is a soluble interleukin-2 receptor.

[0168] In addition, as an example of a more specific measuring method of the present embodiment, there can be mentioned a measuring method for measuring SP-D in a sample by optically measuring or visually observing the degree of agglutination produced by the antigen-antibody reaction between an insoluble carrier carrying an anti-SP-D antibody and SP-D in a blood sample, using an SP-D measuring reagent comprising an insoluble carrier carrying an anti-SP-D antibody and the non-specific reaction inhibitor of the first embodiment. Here, SP-D is pulmonary surfactant protein D.

[0169] <<Biochemical Assay Reagents>>

[0170] The fourth aspect of the present invention is a biochemical assay reagent for detecting the binding of a biochemical assay target substance occurring in a sample with a substance having a specific affinity for the assay target substance, wherein the reagent comprises the non-specific reaction inhibitor of the first aspect, the non-specific reaction inhibitor being included as an effective component for inhibiting a non-specific reaction in the sample. The description of the first to third aspects is also applicable to the fourth aspect, and therefore repeated descriptions are omitted.

[0171] As a representative example of the binding occurring in the sample, an antigen-antibody reaction can be cited. In the antigen-antibody reaction, the substance to be measured can be an antigen or an antibody. As binding other than the antigen-antibody reaction, for example, the binding of avidin to biotin, the binding of protein A to the Fc region of immunoglobulin, the binding of protein G to the Fc region of immunoglobulin, the binding of protein L to the κ chain of immunoglobulin, etc. can be cited.

[0172] Hereinafter, as an example of the present embodiment, an immunological assay reagent used in an immunological assay for detecting an antigen-antibody reaction will be described in detail.

[0173] The type of immunological assay is not particularly limited and various known methods can be applied, for example, turbidimetric immunoassay (TIA), latex turbidimetric immunoassay (LTIA), immunochromatography (lateral flow, flow-through), electrochemiluminescence assay (ECLIA), chemiluminescence assay (CLIA), enzyme-linked immunosorbent assay (ELISA), etc.

[0174] Generally, immunological assays are roughly divided into homogeneous methods and heterogeneous methods. The homogeneous method is a method in which no washing treatment (so-called binding (B) / non-binding (F) separation treatment) is performed, and the washing treatment separates the reaction product generated by the participation of the sample and the assay object substance produced in the mixed solution with the reagent solution, and other substances (inclusions, etc.) contained in the sample. According to the reagent of this method, non-specific reactions in the sample can be suppressed, and the detection accuracy of the antigen-antibody reaction can be improved. Therefore, the reagent of this method is preferably used in the homogeneous method. In addition, the suppression of non-specific reactions shown by the reagent of this method is as significant in the presence of latex particles as in the absence of latex particles, so the reagent of this method is more preferably used in LTIA.

[0175] As an example of this embodiment, there is mentioned an immunological assay reagent comprising: the non-specific reaction suppressor of the first embodiment and a reagent for LTIA.

[0176] <ltia>

[0177] Hereinafter, LTIA will be described in detail as an example of the immunological assay method of the present embodiment.

[0178] LTIA (latex immunoturbidimetry) is a method for measuring a substance to be measured using latex particles on which a substance having a specific affinity for the substance to be measured, such as an antigen or an antibody, is immobilized, and is widely used in the field of clinical examinations.

[0179] Methods for measuring antigens as measurement targets by LTIA are roughly divided into the following two types.

[0180] (a) A method in which latex particles immobilized with antibodies against a substance to be measured are reacted with an antigen as the substance to be measured to form a sandwich-type immune complex, and the substance to be measured (antigen) is measured based on the degree of aggregation of the latex particles accompanying the formation of the immune complex.

[0181] (b) A method of causing latex particles immobilized with antigens and antigens (substance to be measured) in a sample to compete with free antibodies added to a reagent to inhibit the formation of immune complexes between the latex particles and the antibodies, and measuring the substance to be measured (antigen) by the degree of inhibition of agglutination of the latex particles accompanied by the inhibition of the formation of immune complexes. In this method, a combination of free antibodies added to a reagent and latex particles immobilized with antibodies against the substance to be measured can also be used. Both antibodies and antigens can also be immobilized on latex particles.

[0182] In LTIA, due to certain components contained in the sample, agglutination that should not occur (positive measurement error) or agglutination that should not occur (negative measurement error) may occur in the latex particles fixed with a specific affinity substance for the substance to be measured. These are called non-specific reactions and impair the measurement accuracy and reliability.

[0183] By suppressing the non-specific reaction, the measurement error can be reduced, and the positive or negative measurement error of the measurement value can be brought closer to the original measurement value (true value).

[0184] It should be noted that the cause of non-specific reactions is not always clear, but it is sometimes believed to be related to immunoglobulins such as HAMA and rheumatoid factor, lipoproteins such as very-low-density lipoprotein (VL DL), chylomicrons, simple lipids, and lipids such as neutral fat. In particular, the state in which the values ​​of VLDL and chylomicrons are high is called "chyle."

[0185] In LTIA, the test substance can be measured by optically or electrochemically observing the degree of agglutination produced. As a method of optical observation, there can be cited methods of measuring scattered light intensity, absorbance or transmitted light intensity using optical equipment (endpoint method, rate method, etc.). The measured values ​​of absorbance etc. obtained by measuring the sample are compared with the measured values ​​of absorbance etc. obtained by measuring the standard substance (a sample in which the concentration of the measured substance is known), and the concentration (quantitative value) of the measured substance contained in the sample is calculated. The measurement of the absorbance of transmitted light or scattered light etc. can be a single-wavelength measurement or a two-wavelength measurement (the difference or ratio of two wavelengths). The measurement wavelength is usually selected from 500 to 900 nm.

[0186] The determination of LTIA can be performed manually or using a determination device. The determination device can be a general analysis device or a dedicated automatic determination device. LTIA is generally performed using multiple operation steps such as a two-step method (two-reagent method).

[0187] The measurement object substance in the immunological measurement of this method is not particularly limited as long as it is a substance that can be measured by a known immunological measurement method. For example, proteins (antigens, haptens, antibodies), carbohydrates, lipids, nucleic acids, chemical substances (hormones, drugs), etc. can be mentioned. Among them, antigens or antibodies are preferred, and antigens containing proteins are more preferred. As specific examples, soluble interleukin-2 receptor (sIL-2R), CRP, fibrin and fibrinogen degradation products, D dimer, soluble fibrin (SF), lipoprotein (a) (Lp (a)), matrix metalloproteinase-3 (MMP-3), prostate specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α 2-macroglobulin, heme-binding protein, antithrombin-III, alpha-fetoprotein, CEA (carcinoembryonic antigen), ferritin, HBs-Ag (hepatitis B surface antigen), Anti-HBs (anti-hepatitis B surface antibody), HBe-Ag (hepatitis B e antigen), Anti-HBe (anti-hepatitis B e antibody), Anti-HBc (anti-hepatitis B core antibody), SARS-Cov-2, human brain natriuretic peptide (BNP), pulmonary surfactant protein D (SP-D), procalcitonin, etc.

[0188] As specific affinity substances for the substance to be measured supported on insoluble carrier particles such as latex, proteins, peptides, amino acids, lipids, sugars, nucleic acids, haptens, etc. used in known immunological assays can be cited. Generally, antibodies or antigens are often used. The antibody can be a polyclonal antibody or a monoclonal antibody.

[0189] The antibody may be a whole antibody molecule or a functional fragment having antigen-antibody reactivity.

[0190] The antibody may be an antibody obtained by immunizing an animal such as a mouse, or an antibody synthesized by protein engineering. These antibodies include, for example, antibodies treated with protease, F(ab')2, Fab', single-chain antibodies (scFv), chimeric antibodies, humanized antibodies, bispecific antibodies (BsAb), etc. The antibody is obtained by a known method.

[0191] The latex particles used in LTIA are not particularly limited, and latex particles commonly used as immunological assay reagents can be applied. The latex particles are formed, for example, by polymerizing one or more of the following monomers. Specific examples of monomers include: polymerizable monomers having a phenyl group such as styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene; polymerizable monomers having a phenyl group and a sulfonate such as styrene sulfonate, divinylbenzenesulfonate, o-methylstyrenesulfonate, and p-methylstyrenesulfonate; polymerizable unsaturated aromatics such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate; polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; and polymerizable unsaturated aromatics such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid. Polymerizable unsaturated carboxylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylate, and tribromophenyl (meth)acrylate; (meth)acrylonitrile, (meth)acrolein, (meth)acrylamide, N-hydroxymethyl-(meth)acrylamide, methylenebis(meth)acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, unsaturated carboxylic acid amides such as vinyl chloride, vinylidene chloride, and vinyl bromide; polymerizable unsaturated nitriles; vinyl halides; and conjugated dienes.

[0192] The average particle size of the latex particles used in LTIA may be, for example, 0.02 to 1.6 μm, preferably 0.1 to 0.4 μm, in consideration of the concentration of the substance to be measured in the sample and the detection sensitivity of the measuring device. The average particle size can be measured using a particle size distribution meter.

[0193] The method for supporting one or more specific affinity substances on the latex particles used in LTIA is not particularly limited, and the latex particles may be supported by a known method such as physical adsorption (hydrophobic bonding) or chemical bonding.

[0194] In order to prevent non-specific adsorption of impurities on latex particles carrying specific affinity substances, the latex particles can be subjected to a known blocking treatment (masking treatment) in which they are brought into contact with proteins such as bovine serum albumin (BSA), casein, gelatin, egg white albumin or its salts, polymer compounds such as polysaccharides, surfactants or skimmed milk powder.

[0195] <Reagent Composition>

[0196] The composition of the immunological assay reagent of this mode is as long as it is a composition containing the non-specific reaction inhibitor of the first mode, for example, a double reagent type composition containing the non-specific reaction inhibitor in the first reagent and latex particles in the second reagent can be cited. In addition, it can also be a multi-reagent type, which contains the nth reagent (n represents an integer greater than 3) after the third reagent containing other components. The non-specific reaction inhibitor can also be included in the second reagent and the nth reagent other than the first reagent together with other components. The non-specific reaction inhibitor is preferably included in the first reagent.

[0197] In the immunological assay reagent of this mode, known buffer, protein, peptide, amino acid, nucleic acid, lipid, phospholipid, sugar, inorganic salt, macromolecular compound, surfactant, other non-specific reaction inhibitor, preservative etc. can be included in the scope that does not hinder its non-specific reaction inhibition effect. As the component of buffering, adjusting the pH, ionic strength, osmotic pressure etc. of sample, for example, buffer and Good's buffer such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole, their sodium salt, potassium salt, calcium salt etc. can be included. In addition, as the component that strengthens agglutination formation, known macromolecules such as polyvinyl pyrrolidone, phospholipid polymer can be included in the past. As the phospholipid polymer that strengthens agglutination formation, Lipidure (registered trademark) BL400 series sold by NOF Corporation can be cited.

[0198] As a specific example of the reagent composition, as shown in the examples below, there can be cited a dual-reagent immunological assay reagent in which a reagent having a nonspecific reaction inhibitor added to a buffer for diluting a sample is used as a first reagent, and a reagent having latex particles carrying a specific affinity substance dispersed in the same buffer is used as a second reagent.

[0199] Furthermore, when a diluent, a pretreatment solution, or the like is added to a sample before subjecting the sample to immunoassay, the non-specific reaction inhibitor of the first embodiment may be contained in the diluent or the pretreatment solution.

[0200] <Concentration of non-specific inhibitory substances>

[0201] In the immunoassay reagent of the present embodiment, when the first reagent contains a non-specific reaction inhibitor, the concentration in the first reagent is preferably, for example, 0.001 to 5.00% (w / v), more preferably 0.002 to 1.00% (w / v), further preferably 0.003 to 0.60% (w / v), particularly preferably 0.005 to 0.50% (w / v), and most preferably 0.04 to 0.30% (w / v), 0.02 to 0.20% (w / v). When it is within these appropriate ranges, the dispersibility of the non-specific reaction inhibitor in the reagent is improved, and it is easy to mix uniformly with the sample.

[0202] <How to use the reagent>

[0203] As an example of a method for using the immunological assay reagent of the present embodiment, the following methods (1) to (4) can be cited, which involve contacting a biological sample containing a substance to be assayed with latex particles carrying a substance having a specific affinity for the substance to be assayed in the presence of a nonspecific reaction inhibitor of the first embodiment.

[0204] (1) A method of mixing a biological sample with a first reagent containing a non-specific reaction inhibitor and an optional buffer solution, and then mixing the mixture with a second reagent containing latex particles carrying a substance having a specific affinity for a substance to be measured and an optional buffer solution.

[0205] (2) A method of simultaneously mixing a biological sample, a first reagent containing a non-specific reaction inhibitor and an optional buffer, and a second reagent containing latex particles carrying a substance having a specific affinity for a substance to be measured and an optional buffer.

[0206] (3) A method in which a biological sample is mixed with a second reagent including latex particles carrying a substance having a specific affinity for a substance to be measured and an optional buffer solution, and then a first reagent including a non-specific reaction inhibitor and an optional buffer solution is added to the mixture and mixed.

[0207] (4) A method of mixing a first reagent including a non-specific reaction inhibitor and an optional buffer solution with a second reagent including latex particles carrying a substance with specific affinity for a substance to be measured and an optional buffer solution, and then adding a biological sample to the mixture and mixing the mixture.

[0208] Among the above methods, from the viewpoint of suppressing non-specific reactions, (1) (2) (4) are preferred, (1) (4) are more preferred, and (1) is most preferred.

[0209] As an example of a specific reagent of the present embodiment, there can be cited a sIL-2R assay reagent for measuring sIL-2R in a blood sample by optically measuring or visually observing the degree of agglutination produced by the antigen-antibody reaction between an insoluble carrier carrying an anti-sIL-2R antibody and sIL-2R in the blood sample, which comprises an insoluble carrier carrying an anti-sIL-2R antibody and the non-specific reaction inhibitor of the first embodiment.

[0210] As an example of another specific reagent of the present method, there can be cited an SP-D measuring reagent for measuring the degree of agglutination produced by the antigen-antibody reaction between an insoluble carrier carrying anti-SP-D antibodies and SP-D in the blood sample by optical measurement or visual observation, which comprises an insoluble carrier carrying anti-SP-D antibodies and a non-specific reaction inhibitor of the first method.

[0211] <<Biochemical Assay Kit>>

[0212] The fifth aspect of the present invention is a kit comprising the biochemical assay reagent of the fourth aspect. The description of the first to fourth aspects is also applicable to the fifth aspect, and therefore, duplicate descriptions are omitted.

[0213] Examples of the components other than the biochemical assay reagent of the fourth embodiment that may be included in this embodiment include instructions for use of the reagent, blood sample collection tools (collection pipette, syringe, cotton swab, filter, etc.), detection devices, and analysis devices.

[0214] <<Specimen pretreatment solution>>

[0215] The sixth aspect of the present invention is a specimen pretreatment liquid, which is a reagent used in biochemical assays, wherein the reagent comprises: a copolymer having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the physical property X; and a liquid medium that dissolves or disperses the copolymer.

[0216] The biological sample used as the specimen here may be of human or animal origin. The sample may be blood, culture supernatant, urine, stool, spinal fluid, saliva, sweat, ascites, or cell or tissue extract, etc. The blood may be whole blood, plasma, serum, or a sample subjected to any treatment such as dilution or purification of the blood.

[0217] Here, specimen pretreatment refers to adding a certain component to the specimen before mixing the specimen and the assay reagent. For example, a solution containing the non-specific reaction inhibitor of the present invention may be added to the specimen to complete the reaction between the non-specific factors in the specimen and the non-specific reaction inhibitor before mixing with the assay reagent. Sometimes, a "specimen diluent" is added for the purpose of simply diluting the specimen, and such a "specimen diluent" is also included in the "specimen pretreatment solution" of the present invention.

[0218] The liquid medium is not particularly limited as long as it is a medium that can be mixed with the assay reagent, but is preferably an aqueous medium.

[0219] In the range that does not hinder the non-specific reaction inhibitory effect of the non-specific reaction inhibitor of the present invention, the specimen pretreatment liquid may contain conventionally known buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, inorganic salts, polymer compounds, surfactants, non-specific reaction inhibitors that do not belong to the present invention, preservatives, etc. As components for buffering and adjusting the pH, ionic strength, osmotic pressure, etc. of the sample as the specimen, for example, buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole, and Good's buffer, their sodium salts, potassium salts, calcium salts, etc. may be included. In addition, particularly in the case of LTIA reagents, as components that enhance the aggregation formation of the solute (non-volatile component) mixed with the specimen pretreatment liquid, conventionally known polymers such as polyvinyl pyrrolidone and phospholipid polymers may be included. As phospholipid polymers that enhance aggregation formation, the Lipidure (registered trademark) BL400 series sold by NOF Corporation can be cited.

[0220] The concentration of the copolymer relative to the total volume of the specimen pretreatment solution is preferably 0.001 to 5.00% (w / v), more preferably 0.01 to 4.50% (w / v), and further preferably 0.10 to 3.00% (w / v), 0.20 to 2.50% (w / v), or 0.5 to 2.00% (w / v) relative to the total volume of the specimen pretreatment solution, when the specimen pretreatment solution is added in an amount 0.1 to 10 times the volume of the sample as the specimen. When it is within these appropriate ranges, the dispersibility of the non-specific reaction inhibitor in the specimen pretreatment solution is improved, and the specimen sample is easily mixed uniformly.

[0221] The specimen pretreatment solution can be mixed with the specimen in any ratio. For example, 1 to 10 volumes of the specimen pretreatment solution can be added to 1 volume of the specimen. When the specimen and the specimen pretreatment solution are mixed, it can be done manually using a micropipette or automatically using a specimen dilution device. The specimen and the specimen pretreatment solution can also be mixed and then incubated at 10 to 40°C for 30 seconds to 60 minutes before measurement.

[0222] Example

[0223] [Preliminary Test 1] Measurement of Infrared Absorption Spectrum

[0224] 1. Determination method

[0225] 500 μL of each Lipidure (5 wt% aqueous solution) shown in Table 1 was added to 5000 μL of tetrahydrofuran (THF) to precipitate the components of the Lipidure. Then, centrifuge at 8000 rpm for 3 minutes and remove the supernatant. 5000 μL of THF was added to the obtained precipitate again to precipitate the components of the Lipidure, and centrifuge at 8000 rpm for 3 minutes again. The supernatant was removed and dried at 95°C for 7 hours. The infrared absorption spectrum of the obtained Lipidure powder was measured by Fourier transform infrared spectroscopy.

[0226] The results are as follows Figure 1A to Figure 1H The legend in the figure indicates the model number following "Lipidure-" in Table 1.

[0227] <Measurement Device>

[0228] Thermo Scientific Nicolet TM iS5 FT-IR

[0229] <Measurement Conditions>

[0230] Crystal: Diamond

[0231] Angle of incidence: 45°

[0232] Correction: None

[0233] 2. Measurement results

[0234] Table 1 shows the results of infrared absorption spectrum measurement, physical property data of side chain characteristics described on the NOF Corporation website, and kinematic viscosity and surface tension read from graphs described in the catalog.

[0235] In the table, the column "Constitutional unit B does not have an ionizable functional group" is represented by "○" when it does not have an ionizable functional group, and by "×" when it has an ionizable functional group.

[0236] "P1: 2946~2906cm -1 The columns with "maximum absorption" are indicated by "○" if they are present, and "×" if they are not present. The maximum absorption is present at 2924 cm in Lipidure-BL1002 and Lipidure-BL1003. -1 , which is present at 2926cm in Lipidure-BL1103 -1 .

[0237] "P2: 2870~2833cm -1 The columns with "maximum absorption" are indicated by "○" if they are present, and "×" if they are not present. The maximum absorption is present at 2852 cm in Lipidure-BL1002 and Lipidure-BL1003. -1 , which is present at 2854cm in Lipidure-BL1103 -1 .

[0238] The transmittance (%) of P1 and the transmittance (%) of P2 are both P1 in the case of Lipidure-BL1002, 1003, and 1103. <P2。

[0239] P3 is between 975 and 940 cm -1 The maximum absorption peak is in the range of . All samples measured have maximum absorption at P3. The wave number varies depending on the model of Lipidure, and is 964 to 951 cm -1 range.

[0240] P4 is between 1252 and 1215 cm -1 The maximum absorption peak is in the range of . Except for Lipidure-BL405, the measured samples have the maximum absorption in P4. The wave number varies depending on the model of Lipidure, and is 1242~1225cm -1 range.

[0241] In the table, the columns of ratio A to ratio D are described as follows. In addition, the strength and the ratio were calculated by the method of formula (1) and formula (2) in the specification.

[0242] Ratio A: P1 / P3 in the presence of P1. In the absence of P1, for the models with side chain characteristics of "hydrophobic" in Table 1B, 2925 cm -1 The intensity ratio of P to P3.

[0243] Ratio B: P1 / P4 in the presence of P1. In the absence of P1, for the models with "hydrophobic" side chain characteristics in Table 1B, 2925 cm -1 The intensity ratio of P4.

[0244] Ratio C: P2 / P3 in the presence of P2. In the absence of P2, for the models with side chain characteristics of "hydrophobic" in Table 1B, 2852 cm -1 The intensity ratio of P to P3.

[0245] Ratio D: P2 / P4 in the presence of P2. In the absence of P2, for the models with side chain characteristics of "hydrophobic" in Table 1B, 2852 cm -1 The intensity ratio of P4.

[0246] It should be noted that "ND" in the table means not determined, cannot be calculated because P1 or P2 does not exist, or there is no information on the homepage or directory.

[0247] [Table 1A]

[0248]

[0249] [Table 1B]

[0250]

[0251] [Experimental Example 1] Use of a non-specific reaction inhibitor in a soluble interleukin-2 receptor (sIL-2R) assay reagent - 1

[0252] The following is an experiment on measuring the sIL-2R concentration in a sample using a reagent based on the LTIA method to which Lipidure-BL1002, 1003, 1103, and other components are added as examples of the non-specific reaction inhibitor of the present invention. The materials used are as follows.

[0253] ·HBR-1 manufactured by SCANTIBODIES LABORATORY, INC. was used.

[0254] · Various types of Lipidure were made by NOF Corporation.

[0255] 1. Measurement based on the CLEIA method (Reference Example 1)

[0256] The CLEIA method implements a B / F separation operation and has a washing step. Therefore, it is a measurement method that is not easily affected by non-specific reactions from samples. The measurement value based on this method is used as the control value of the sIL-2R concentration in each sample.

[0257] 1-1. Assay reagents

[0258] LUMIPULSE PRESTO (registered trademark) IL-2R (FUJIREBIO CORPORATION)

[0259] 1-2. Sample

[0260] Human serum samples are sample numbers 1 to 17 (however, the donors of each sample are different).

[0261] 1-3. Measurement steps

[0262] The measurement was performed using LUMIPULSE (registered trademark)-L2400 (Fujire Bio Co., Ltd.) according to the package instructions of the measurement reagent.

[0263] 2. Measurement based on LTIA reagent (Comparative Examples 1 to 16, Examples 1 to 3)

[0264] 2-1. Assay reagents

[0265] The first reagent and the second reagent were prepared according to the methods described in Japanese Patent Application Publication Nos. 2017-181377 and 2018-173429.

[0266] In Comparative Example 1, the first reagent was used as it was.

[0267] In Comparative Example 2, a reagent was used in which HBR-1, a commercially available non-specific reaction inhibitor, was added to the first reagent so as to have a concentration of 100 μg / mL.

[0268] In Comparative Examples 3 to 16 and Examples 1 to 3, reagents obtained by adding various Lipidures shown in Table 2 to the first reagent so as to give a final concentration of 0.1% (w / v) were used.

[0269] [Table 2]

[0270]

[0271] 2-2. Samples

[0272] Same as sample 1-2.

[0273] 2-3. Measurement steps

[0274] The first reagent and the second reagent are combined, and the sIL-2R concentration in the sample is measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent is added to 5.6 μL of the sample, and after incubating at 37°C for 5 minutes, 40 μL of the second reagent is added and stirred. Within the next 5 minutes, the absorbance change accompanying the formation of agglutination is measured at a main wavelength of 570 nm and a secondary wavelength of 800 nm, and the absorbance change is substituted into the calibration curve obtained by measuring a standard substance with a known concentration, and the measured value is calculated. For each specimen, the value obtained by dividing the measured value in Comparative Examples 1 to 16 and Examples 1 to 3 by the measured value in Reference Example 1 is calculated and evaluated as a relative ratio (%). When the relative ratio is greater than 85% and less than 115%, it is judged that there is no deviation from the measured value of Reference Example 1.

[0275] 3. Results and Investigation

[0276] The measurement results are shown in Table 3. It should be noted that "ND" in the table means that the measurement was not performed.

[0277] The measurement sensitivity of the calibration curve obtained by measuring the standard substance with known concentration (the net measurement sensitivity caused by the antigen-antibody reaction, minus the measurement sensitivity of the physiological saline under each condition) was calculated. When the sensitivity of Comparative Example 1 was taken as 100%, the sensitivity decreased to 86% in Comparative Example 3. In addition, the sensitivity increased to 115% in Comparative Example 9. Under the other conditions, it was approximately equivalent to 101 to 111%.

[0278] [Table 3]

[0279]

[0280] (1) Control specimens

[0281] Specimens 1 to 7, in which the ratio (relative ratio) of the measured value of the LTIA method reagent without the addition of additives (Comparative Example 1) to the measured value of the CLEIA method (Reference Example 1) was 102 to 111%, were evaluated as "control specimens" that did not cause non-specific reactions in the LTIA method. Among the control specimens, the measured values ​​of the LTIA method reagents to which Lipidure-BL1002, Lipidure-BL1003, and Lipidure-BL1103 of the present invention were added (Examples 1 to 3) were 89 to 113% relative to Reference Example 1, and the measurement results were substantially the same as those of Reference Example 1. Therefore, it can be seen that the addition of Lipidure-BL1002, Lipidure-BL1003, and Lipidure-BL1103 of the present invention does not affect the measured values ​​of specimens that do not show non-specific reactions.

[0282] In Comparative Example 3, the relative ratio of sample 4 was 116%, which was out of the range of 85% to 115%, and was relatively inferior to the condition (Comparative Example 1) without the addition of the present compound. In addition, the relative ratios of sample 2 in Comparative Example 4, all samples 1 to 7 in Comparative Example 5, and sample 4 in Comparative Example 6 were out of the range of 85% to 115%, and there were many cases in which the relative ratio became inferior even in the control samples by adding Lipidure which did not have the properties of the present invention. Only Comparative Examples 2, 8, 11, 15, Examples 1, 2, and 3 were able to measure all the control samples at the same relative ratio as Reference Example 1 and Comparative Example 1.

[0283] (2) Deviating sample measurement value

[0284] Specimens 8 to 17, for which the ratio (relative ratio) of the measured value of the LTIA method reagent without additives (Comparative Example 1) to the measured value of the CLEIA method (Reference Example 1) was less than 85% or greater than 115%, were evaluated as "deviant specimens" that caused non-specific reactions in the LTIA method.

[0285] In specimen No. 8, the relative ratio of the measured value of the LTIA method reagent to which no nonspecific reaction inhibitor was added (Comparative Example 1) was 124%, which deviated from Reference Example 1, and it was considered that a nonspecific reaction occurred. In contrast, when HBR-1, which is a commercially available nonspecific reaction inhibitor, was added (Comparative Example 2), the relative ratio was 126%, and the deviation in specimen No. 8 was not improved in HBR-1. When 1% of Lipidure, which does not have the properties of the present invention, was added (Comparative Examples 3 to 14, 16), the relative ratio was 117 to 171%, and the measured value sometimes deviated further significantly. On the other hand, the measured values ​​of the LTIA method reagent to which 0.1% (w / v) of Lipidure-BL1002, Lipidure-BL1003, and Lipidure-BL1103 having the properties of the present invention were added (Examples 1 to 3), and the relative ratios were 103%, 106%, and 109%, respectively, which were improved from the relative ratio of Reference Example 1. The same results were obtained in specimens 9 to 17. In Comparative Example 15, the relative comparison was improved in Sample No. 8, but was greatly deviated in Samples 9 to 17.

[0286] As described above, in the LTIA method, which is a homogeneous immunoassay method, non-specific reactions derived from the sample can be suppressed by allowing Lipidure-BL1002, Lipidure-BL1003, and Lipidure-BL1103 having the properties of the present invention to be present in the reaction system. The Lipidure series are all high molecular weight polymers having a segment derived from 2-methacryloyloxyethyl phosphorylcholine, and by adding them to the LTIA reagent, even for the control sample that can be measured at a relative ratio of 85% or more and 115% or less relative to Reference Example 1 when not added, relatively poor samples are more likely to appear. As for the components that do not affect the measured value of the control sample, further suppress the non-specific reaction that has been a problem so far, and reduce the deviation of the measured value, only Lipidure-BL1002, 1003, and 1103 discovered by the present inventors have the effect of reducing the deviation of the measured value.

[0287] It should be noted that Lipidure-NH01 of Comparative Example 3 does not satisfy the physical property X regarding infrared absorption spectrum and does not belong to the present invention. In addition, Lipidure-NH01 is a reactive polymer having an amino group and can react with functional groups (carboxyl groups, etc.) on the surface of the substrate. Therefore, it is considered that Lipidure-NH01 is not preferred in terms of having an amino group in addition to not satisfying the physical property X.

[0288] Lipidure-AC01 of Comparative Example 4 does not satisfy the physical property X regarding infrared absorption spectrum and does not belong to the present invention. In addition, it is a copolymer of the structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and the structural unit having a carboxyl group in the side chain. That is, the structural unit has a functional group that is ionized in water. Therefore, it is considered that Lipidure-AC01 is not preferred in terms of having a structural unit containing a functional group that is ionized in water in addition to not satisfying the physical property X.

[0289] Lipidure-SF08 of Comparative Example 5 is a substance having a phosphorylcholine group and is sold as a low-molecular zwitterionic surfactant. It is not a copolymer and does not belong to the present invention.

[0290] According to the catalog, Lipidure is known to have a high sensitivity effect (Lipidure-BL103, 400 series), a non-specific adsorption inhibition effect (blocking) (Lipidure-BL200, 800, 1200 series), and a stabilization effect (Lipidure-BL800 series, SF08) in the latex immunoturbidimetric system, but the effect of inhibiting the deviation of the measured value for a specimen showing a non-specific reaction is unknown. The present invention has discovered a new effect different from the hitherto unknown non-specific reaction inhibition in the Lipidure-BL1000 series and the Lipidure-BL1100 series.

[0291] It should be noted that it is known that the copolymer of this mode can inhibit the non-specific reaction caused by lipid and immunoglobulin by the method known in the past. By verifying the change of the measured value when adding the component that may be a non-specific factor to the sample or removing these components, it is possible to infer which component is a non-specific factor. It should be noted that the copolymer of this mode may also inhibit the non-specific reaction caused by the non-specific factors other than the above.

[0292] It should be noted that Lipidure-BL405 studied in Comparative Example 9 is "highly hydrophilic" according to the catalog, and is presumed to be a component described in Patent Document 7 that exhibits an inhibitory effect on serum interference. However, this component did not show an effect of reducing the deviation of the measured value, and the measurement accuracy of the control sample was also poor.

[0293] [Experimental Example 2] Use of non-specific reaction inhibitors in soluble interleukin-2 receptor (sIL-2R) assay reagents-2

[0294] 1.2. Determination based on LTIA reagents

[0295] 1-1. Assay reagents

[0296] The first reagent and the second reagent were prepared according to the method described in Japanese Patent Application Laid-Open No. 2017-181377 and Japanese Patent Application Laid-Open No. 2018-173429. For the first reagent, Lipidure-BL1002 found in the present application was added in such a manner that the final concentration was as follows.

[0297] Example 4 0.01% (w / v)

[0298] Example 5 0.05% (w / v)

[0299] Example 6 0.2% (w / v)

[0300] Example 7 0.5% (w / v)

[0301] 1-2. Samples

[0302] Human serum sample Sample Nos. 8, 11, 13, 15 used in Experimental Example 1

[0303] 1-3. Measurement steps

[0304] Same as 2-3 of Experimental Example 1.

[0305] 2. Results and Investigation

[0306] The measurement results are shown in Table 4.

[0307] The measurement sensitivity of the calibration curve obtained by measuring the standard substance with known concentration was calculated (the net measurement sensitivity caused by the antigen-antibody reaction, minus the measurement sensitivity of the physiological saline under each condition). When the sensitivity of Comparative Example 1 is taken as 100%, the measurement sensitivities of Examples 1 and 4 to 6 are 100 to 108%, which is approximately the same as Comparative Example 1. The measurement sensitivity of Example 7 is 92%.

[0308] [Table 4]

[0309]

[0310] The concentration of Lipidure-BL1002 added to the first reagent was varied between 0.005% (w / v) and 0.5% (w / v). In specimens No. 8, 11, 12, and 15, the relative ratio was improved compared to Comparative Example 1. In particular, 0.05% (w / v) to 0.2% (w / v) was a preferred condition in which the relative ratio was 85% or more and 115% or less in all specimens, indicating an effect of suppressing nonspecific reactions.

[0311] [Experimental Example 3] Use of non-specific reaction inhibitors in soluble interleukin-2 receptor (sIL-2R) assay reagents-3

[0312] 1. Measurement based on the CLEIA method (Reference Example 2)

[0313] 1-1. Assay reagents

[0314] Same as Reference Example 1

[0315] 1-2. Sample

[0316] 85 human serum samples (each sample has a different donor).

[0317] 1-3. Measurement steps

[0318] Same as Reference Example 1

[0319] 2. Determination based on LTIA reagents

[0320] 2-1. Assay reagents

[0321] In Comparative Example 16, the same reagents as those in Comparative Example 1 were used.

[0322] In Example 8, the same reagents as in Example 1 were used.

[0323] 2-2. Samples

[0324] Same as Reference Example 2

[0325] 2-3. Measurement steps

[0326] Same as Experimental Example 1.

[0327] 3. Results and Investigation

[0328] A scatter diagram was prepared with the measured values ​​based on Reference Example 2 as the horizontal axis and the measured values ​​based on Comparative Example 16 as the vertical axis ( Figure 2 (a)). In addition, a scatter diagram was prepared with the measured values ​​based on Reference Example 2 as the horizontal axis and the measured values ​​based on Example 8 as the vertical axis ( Figure 2 (b)). In addition, the correlation coefficient and the slope of the regression line calculated by the least square method for each curve are shown in the figure.

[0329] like Figure 2 As shown in (a), in Comparative Example 16, the slope of the regression equation relative to the measured value of Reference Example 2 is 1.11, and the correlation coefficient R is 0.983. Figure 2 As shown in (b), in Example 8, the slope is 0.97 and R=0.992. The LTIA reagent to which the components of the present invention are added has a significantly improved correlation convergence with respect to the measured value of the CLEIA method. In particular, it is significantly improved in the sample distributed between 1000 and 3000 U / mL.

[0330] [Experimental Example 4] Use of non-specific reaction inhibitors in pulmonary surfactant protein D (SP-D) assay reagents - 1

[0331] Hereinafter, an experiment is described in which the SP-D concentration in a sample is measured using a reagent based on the LTIA method to which Lipidure-BL1002 and 1003, which are examples of the non-specific reaction inhibitor of the present invention, and other components are added. The materials used are as follows.

[0332] ·HBR-1 manufactured by SCANTIBODIES LABORATORY, INC. was used.

[0333] · Various types of Lipidure were made by NOF Corporation.

[0334] 1. Measurement based on the CLEIA method (Reference Example 3)

[0335] The CLEIA method implements a B / F separation operation and has a washing step. Therefore, it is a measurement method that is not easily affected by non-specific reactions from samples. The measurement value based on this method is used as a control value for the SP-D concentration in each specimen.

[0336] 1-1. Assay reagents

[0337] CL SP-D "Yamasa" NX (made by Yamasa Soy Sauce Co., Ltd.)

[0338] 1-2. Samples

[0339] Human serum samples: Sample numbers 18 to 22 (however, the donors of each sample are different).

[0340] 1-3. Measurement steps

[0341] The assay was performed using a fully automatic chemiluminescence immunoassay device, CL-JACK NX, according to the package instructions of the assay reagent.

[0342] 2. Determination based on LTIA reagents

[0343] 2-1. Assay reagent (Comparative Example 17)

[0344] (1) First Reagent

[0345] 100 mM MES-NaOH (pH 6.0)

[0346] 500mM NaCl

[0347] 0.5%(w / v)BSA

[0348] Sensitizer (not Lipidure-BL1002, 1003, 1103)

[0349] (2) Second Reagent

[0350] Anti-human SP-D monoclonal antibody sensitized latex

[0351] 5mM MOPS-NaOH (pH 7.0)

[0352] The anti-human SP-D monoclonal antibody sensitized latex was prepared as follows. An anti-human SP-D monoclonal antibody solution diluted to 0.35 mg / mL with an equal amount of 10 mM MOPS buffer was added to a 1% (w / v) polystyrene latex solution (manufactured by SEKISUI MEDICAL Co., Ltd.) (10 mM MOPS buffer) having an average particle size of 317 nm, and stirred at 4°C for 2 hours. Then, an equal amount of 0.5% (w / v) BSA solution (10 mM MOPS buffer) was added, and stirred at 4°C for 1 hour to prepare an anti-human SP-D monoclonal antibody sensitized latex solution.

[0353] The anti-human SP-D monoclonal antibody was obtained by a method known to those skilled in the art using human SP-D (manufactured by GenScript) as an antigen.

[0354] 2-2. LTIA reagent (Comparative Examples 18 to 22, Examples 9 and 10)

[0355] The first reagent and the second reagent described in Comparative Example 17 were used. As the first reagent, a reagent to which various components shown in Table 5 were added to Comparative Example 17 so as to have the final concentrations shown in the table was used.

[0356] [Table 5]

[0357]

[0358] 2-3. Calibrators

[0359] 100 mM citrate buffer (pH 5.0)

[0360] 1% (w / v) BSA

[0361] 10 mM calcium chloride

[0362] SP-D antigen

[0363] 2-4. Samples

[0364] Same as 1-2.

[0365] 3. Determination method

[0366] The first reagent and the second reagent of each embodiment or comparative example were combined, and the samples were measured using Hitachi automatic analyzer 3500. Specifically, 120 μL of the first reagent was added to 5 μL of each sample, and the mixture was kept warm at 37°C for 5 minutes. Then, 40 μL of the second reagent was added each time and stirred. Next, the absorbance change was measured for 5 minutes at a main wavelength of 570 nm and a sub-wavelength of 800 nm.

[0367] 4. Evaluation Method

[0368] First, the calibration samples were measured to create calibration curves for each test using each reagent. Using the obtained calibration curves, each sample was measured to determine the concentration. It should be noted that the measurement sensitivity was approximately the same under each condition, 97 to 106%, with Comparative Example 17 being 100%.

[0369] For each sample, the measured value in Comparative Examples 17 to 22 and Examples 9 and 10 was divided by the measured value in Reference Example 3 and evaluated as a relative ratio (%). When the relative ratio was 90% or more and 110% or less, it was determined that the measured value did not deviate from the measured value in Reference Example 3.

[0370] [Table 6]

[0371]

[0372] 5. Results and Investigation

[0373] (1) Control specimens

[0374] Specimens 18 to 20, in which the relative ratio of the measured value of the LTIA method reagent without additives (Comparative Example 17) to the measured value of the CLEIA method (Reference Example 3) was 92 to 109%, were evaluated as "control specimens" that did not cause non-specific reactions in the LTIA method. Among the control specimens, the relative ratio of the measured value of the LTIA method reagent to which Lipidure-BL1002 and Lipidure-BL1003 of the present invention were added (Examples 9 and 10) to Reference Example 3 was 99 to 109%, and the measurement results were substantially the same as those of Comparative Example 17. Therefore, it can be seen that the addition of Lipidure-BL1002 and Lipidure-BL1003 having the properties of the present invention does not affect the measured value of the specimen that does not show non-specific reactions.

[0375] (2) Deviating sample measurement value

[0376] Specimens 21 and 22, for which the relative ratio of the measurement value of the LTIA method reagent without additives (Comparative Example 17) to the measurement value of the CLEIA method (Reference Example 3) was less than 90%, were evaluated as "deviant specimens" that caused non-specific reactions in the LTIA method.

[0377] In specimen No. 21, the relative ratio of the LTIA reagent without additives (Comparative Example 17) to the measured value of the CLEIA method was 86%. In contrast, when 0.1% (w / v) of Lipidure, which does not have the properties of the present invention, was added (Comparative Examples 19 to 22), the relative ratio was 86 to 88%, and the relative ratio was almost unchanged. On the other hand, the measured values ​​of the LTIA reagent to which 0.1% (w / v) of Lipidure-BL1002 and Lipidure-BL1003 having the properties of the present invention were added (Examples 9 and 10), and the relative ratios were 90% and 95%, respectively, and the relative ratio was improved. Since the relative ratio was 90% when the commercially available non-specific reaction inhibitor HBR-1 was added (Comparative Example 18), Lipidure-BL1002 was at the same level as Comparative Example 18, and Lipidure-BL1003 had an improvement effect of the relative ratio greater than that of Comparative Example 18. The same result was obtained in specimen 22. In sample 22, it was found that both Lipidure-BL1002 and 1003 had an improvement effect greater than that of Comparative Example 18.

[0378] In SP-D, similarly to Experimental Example 1, the LTIA reagent to which the compound discovered in the present application was added could suppress non-specific reactions originating from the sample. That is, the relative ratio of the measured value to the measured value by the CLEIA method, which is a measurement method for B / F separation, was improved.

[0379] [Experimental Example 5] Use of non-specific reaction inhibitors in pulmonary surfactant protein D (SP-D) assay reagents -2

[0380] 1. Measurement based on the CLEIA method (Reference Example 4)

[0381] 1-1. Assay reagents

[0382] Same as Reference Example 3

[0383] 1-2. Sample

[0384] 200 human serum samples (each sample has a different donor).

[0385] 1-3. Measurement steps

[0386] Same as Reference Example 3

[0387] 2. Determination based on LTIA reagents

[0388] 2-1. Measurement reagent (Comparative Examples 23 to 25, Example 11)

[0389] (1) First Reagent

[0390] In Comparative Example 23, the first reagent described in Comparative Example 17 was used.

[0391] In Comparative Example 24, 25 μg / mL of anti-human Ig M antibody was added to the first reagent described in Comparative Example 17.

[0392] As the anti-human IgM antibody, the antibody described in Japanese Patent No. 4625879 (microorganism deposit number: FERM BP-11134) was used.

[0393] In Comparative Example 25, 100 μg / mL of commercially available Heterobloc k (OMEGA Biologicals) was added to the first reagent described in Comparative Example 17.

[0394] In Example 11, Lipidure-BL1002 was added to the first reagent described in Comparative Example 17 so that the final concentration was 0.1% (w / v).

[0395] (2) Second Reagent

[0396] The reagent described in Comparative Example 17 was used.

[0397] 2-2. Calibrators

[0398] Same as Experimental Example 4.

[0399] 2-3. Samples

[0400] Same as 1-2.

[0401] 3. Determination method

[0402] Same as Experimental Example 4.

[0403] 4. Evaluation Method

[0404] First, the calibrator is measured to create a calibration curve for each test using each reagent, and each sample is measured using the obtained calibration curve to determine the concentration.

[0405] Next, a scatter plot was prepared with the control value based on Reference Example 4 as the horizontal axis and the measured value of any one of Comparative Examples 23 to 25 or Example 11 as the vertical axis ( Figure 2 ). Figure 2 The upper row shows all the samples that were measured. Figure 2 The lower part is a graph showing an extract of a specimen of a reference example in which the SP-D measurement value is 100 ng / mL or less. In addition, the graph shows the correlation coefficient and the slope of the regression line calculated by the least square method for each curve.

[0406] 5. Results

[0407] The correlation coefficient of the LTIA measurement value with the measurement value based on Reference Example 4, which is a measurement method for B / F separation, was 0.987 to 0.989 in Comparative Examples 23 to 25, whereas it was improved to 0.992 in Example 11. In particular, the improvement was remarkable in the specimens indicated by the dotted circle on the low value side in the figure.

[0408] 6. Inspection

[0409] In SP-D, in the LTIA reagent to which the components of the present invention are added, the convergence of the measurement method with respect to B / F separation is also improved.

[0410] As described above, it is considered that the non-specific reaction inhibitory effect of Lipidure-BL1002, 1003, and 1103 having the properties of the present invention is not limited to measurement items, but can be widely applied to LTIA reagents.

[0411] [Experimental Example 6] Use of non-specific reaction inhibitors in soluble interleukin-2 receptor (sIL-2R) assay reagents -4

[0412] 1. Measurement based on the CLEIA method (Reference Example 5)

[0413] 1-1. Assay reagents

[0414] Same as Reference Example 1

[0415] 1-2. Samples

[0416] Human serum samples: Sample numbers 23, 24, and 25 (however, the donors of each sample are different).

[0417] 1-3. Measurement steps

[0418] Same as Reference Example 1

[0419] 2. Measurement by ELISA method (Comparative Examples 26 to 29, Example 12)

[0420] 2-1. Methods

[0421] (1) Anti-sIL-2R monoclonal antibody (clone number 92212) was dissolved in 20 mM phosphate buffer (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride to a concentration of 10 μg / mL, and 100 μL of the dissolved solution was dispensed into each well of a 96-well microplate and allowed to stand at 4°C overnight.

[0422] (2) Each well was washed three times with 400 μL of PBS containing 0.05% (w / v) Tween (registered trademark) 20 (hereinafter referred to as PBST), and then 200 μL of PBST containing 1% (w / v) bovine serum albumin (hereinafter referred to as BSA-PBST) was added and blocked at room temperature for 1 hour. This was used as an ELISA plate.

[0423] (3) After each well of the ELISA plate was washed three times with 400 μL of PBST, 100 μL of a sample diluted 40-fold with a sample diluent prepared by adding the components listed in Table 7 to BSA-PBST was added to each well and allowed to stand at room temperature for 1 hour.

[0424] (4) After washing each well three times with 400 μL of PBST, 100 μL of biotin-labeled anti-sIL-2R monoclonal antibody (clone number 92204R) diluted to 0.50 μg / mL with the sample diluent described in Table 7 was dispensed into each well and allowed to stand at room temperature for 1 hour.

[0425] (5) After washing each well three times with 400 μL of PBST, 100 μL of HRP-labeled streptavidin (ThermoFisher Scientific) diluted to 0.20 μg / mL with BSA-PBST was injected into each well and allowed to stand at room temperature for 30 minutes.

[0426] (6) After washing each well three times with 400 μL of PBST, add 50 μL of citric acid buffer (pH 5) containing 0.2% (w / v) o-phenylenediamine and 0.02% (w / v) hydrogen peroxide. After standing at room temperature for 10 minutes, add 50 μL of 4.5 N sulfuric acid to stop the enzyme reaction, and measure the absorbance at a wavelength of 492 nm.

[0427] (7) Using a standard substance of known concentration as a calibrator, calculate the sIL-2R value in the test sample.

[0428] [Table 7]

[0429] additive Concentration (weight %) Comparative Example 26 Not added - Comparative Example 27 Lipidure-BL206 0.10％ Comparative Example 28 Lipidure-BL802 0.10％ Comparative Example 29 Lipidure-BL1301 0.10％ Example 12 Lipidure-BL1002 0.10％

[0430] 2-2. Samples

[0431] Same as 1-2.

[0432] 3. Evaluation Method

[0433] Using the measurement results of the calibrator, a calibration curve for each test using each reagent was prepared. Using the obtained calibration curve, the measurement value of each specimen was obtained. It should be noted that the measurement sensitivity of Comparative Examples 27, 28, and Example 12 was 106 to 108% when Comparative Example 26 was taken as 100%, which was roughly the same as Comparative Example 26. The sensitivity of Comparative Example 29 was 117%.

[0434] For each sample, the value obtained by dividing the measured value in Comparative Examples 26 to 29 and Example 12 by the measured value in Reference Example 5 was calculated and evaluated as a relative ratio (%). When the relative ratio was 90% or more and 110% or less, it was determined that there was no deviation from the measured value in Reference Example 5. The obtained results are shown in Table 8.

[0435] 4. Results

[0436] (1) Control specimens

[0437] Specimens 23 and 24, in which the ratio (relative ratio) of the measurement value of the ELISA method without adding additives (Comparison Example 26) to the measurement value of the CLEIA method (Reference Example 5) was 108-109%, were evaluated as "control specimens" that did not undergo non-specific reactions in the ELISA method. The relative ratio of the control specimen to Reference Example 5 in Example 12 was 106%, and no non-specific reactions occurred.

[0438] (2) Deviation of specimen

[0439] Sample 25, in which the ratio (relative ratio) of the measured value by the ELISA method without the addition of additives (Comparative Example 26) to the measured value by the CLEIA method (Reference Example 5) was 114%, was evaluated as a "deviant sample" that caused a non-specific reaction in the ELISA method.

[0440] When 0.1% (w / v) of Lipidure not having the properties of the present invention was added (Comparative Examples 27 to 29), the relative ratio was 114%, and the deviation was not improved. On the other hand, in the ELISA method (Example 12) in which 0.1% (w / v) of Lipidure-BL1002 having the properties of the present invention was added, the relative ratio was 101%, which was improved compared to the relative ratio of Reference Example 5.

[0441] [Table 8]

[0442]

[0443] 5. Inspection

[0444] When the component of the present invention is added to a sample diluent in an ELISA reagent as a measurement method involving B / F separation, the consistency of the measurement value with respect to the CLEIA method as a measurement method involving B / F separation is improved.

[0445] As described above, it is considered that the non-specific reaction inhibitory effect of Lipidure-BL1002, 1003, and 1103 having the properties of the present invention is not limited to measurement methods without B / F separation, but can be widely applied to biochemical measurement methods.< / ltia>

Claims

1. A non-specific reaction inhibitor, which is a copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X, The non-specific reaction inhibitor is used for the following purpose: by mixing the copolymer with a biological sample, a non-specific reaction is suppressed when a substance to be measured contained in the biological sample is measured. Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

2. A method for using a non-specific reaction inhibitor, which is a method for using a copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X as a non-specific reaction inhibitor, wherein: By mixing the copolymer with a biological sample, a non-specific reaction is suppressed when measuring a substance to be measured contained in the biological sample. Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

3. A method for inhibiting a non-specific reaction, wherein: By mixing a copolymer with a biological sample, a non-specific reaction is suppressed when a substance to be measured contained in the biological sample is measured. The copolymer has a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibits the following physical property X: Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

4. A biochemical assay reagent, which is a reagent used in biochemical assays, wherein: The reagent comprises a copolymer having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X, Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

5. A biochemical assay reagent, which is a reagent used in biochemical assays, wherein: The reagent comprises a copolymer having a constituent unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X, The copolymer is contained in the reagent as a nonspecific reaction inhibitor for suppressing nonspecific reactions in a biological sample. Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

6. The biochemical assay reagent according to claim 4 or 5, wherein The biochemical assay is a homogeneous method.

7. The biochemical assay reagent according to claim 5, wherein The biochemical assay is an assay based on antigen-antibody reaction.

8. The biochemical assay reagent according to claim 4 or 5, wherein The biochemical assay was latex immunoturbidimetry.

9. The biochemical assay reagent according to claim 4 or 5, wherein The biological sample to be measured in the above-mentioned biochemical measurement is a blood sample.

10. The biochemical assay reagent according to claim 4 or 5, wherein The biochemical assay is an assay for detecting an antigen-antibody reaction between one or more selected from soluble interleukin-2 receptor and pulmonary surfactant protein D contained in a biological sample as an assay object and an antibody added to the biological sample.

11. A sample pretreatment liquid, which is a reagent used in biochemical assays, wherein: The reagents include: A copolymer having a structural unit A derived from 2-methacryloyloxyethyl phosphorylcholine and exhibiting the following physical property X; and a liquid medium which dissolves or disperses the copolymer, Physical Properties X: In the spectrum of Fourier transform infrared spectroscopy based on attenuated total reflectance measurement (ATR method), there are: -1 The peak with maximum absorption.

12. A biochemical assay kit comprising: The biochemical assay reagent according to claim 4 or 5.

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