Calibration curve creation method, fluorescence polarization immunoassay method, fluorescence polarization immunoassay device, and calibration curve creation kit

Abstract

Disclosed are a calibration curve creation method, a fluorescence polarization immunoassay method, a fluorescence polarization immunoassay device, and a calibration curve creation kit. The calibration curve creation method uses an antibody that binds to a target substance and a fluorescently labeled substance that labels the target substance with a fluorescent dye. The degree of fluorescence polarization Pconc of the sample conc is created on the basis of the degree of fluorescence polarization P0 of a first reference sample free of the target substance and a calibration curve in which the concentration of the target substance added exceeds the concentration CS alpha of the target substance, creating the calibration curve.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Japanese Patent Application No. 2024-68957, filed April 22, 2024, and Japanese Patent Application No. 2024-229081, filed December 25, 2024, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure generally relates to a calibration curve creation method, a fluorescence polarization immunoassay method, a fluorescence polarization immunoassay device, and a calibration curve creation kit. Background Art

[0004] One immunoassay that utilizes the antigen-antibody reaction is an assay called "fluorescence polarization immunoassay" (FPIA). In this method, the concentration of the target substance is estimated by fluorescence polarization measurement. There are two types of FPIA: competitive and non-competitive. In competitive FPIA, the target substance and a fluorescently labeled target substance (tracer) compete for the reaction with the antibody. The tracer that is not bound to the antibody moves violently in the solution and even emits fluorescence randomly when irradiated with polarized excitation light. At the same time, the tracer bound to the antibody is less likely to move and, therefore, emits fluorescence that is biased toward the polarization direction of the excitation light.

[0005] In competitive FPIA, fluorescence intensities parallel to and perpendicular to the polarization direction of the excitation light are measured, and the degree of deviation between the fluorescence intensities in these two directions is measured as the degree of fluorescence polarization. This degree of fluorescence polarization depends on the amount of tracer-antibody conjugate, and thus, the concentration of the target substance can be measured using the degree of fluorescence polarization as an index.

[0006] In FPIA, a calibration curve representing the relationship between the measured fluorescence polarization degree and the concentration of the target substance is created, and the concentration of the target substance contained in the sample being tested is measured from the obtained calibration curve (for example, see the specifications of Unexamined Japanese Patent Application Publication No. 2011-47802 and U.S. Patent Application Publication No. 2009 / 0023595).

[0007] Pure water is an example of a diluent used when creating a calibration curve. Since pure water is readily available, a calibration curve can be created efficiently by using pure water as a diluent.

[0008] However, when a test sample contains contaminants that affect the reaction between the target substance and the antibody, it is difficult to create a calibration curve that takes the influence of the contaminants into account when pure water is used as a diluent.

[0009] One example of a method of creating a calibration curve that takes into account the influence of contaminants in a measured sample is a method that uses the measured sample itself, rather than pure water, as a diluent. That is, it is thought that a calibration curve that takes into account the influence of contaminants can be created by creating calibration curve creation samples by adding a known amount of target substance to a plurality of measured samples.

[0010] However, the fluorescence polarization degree of the low-concentration region of the calibration curve created using this method is susceptible to the influence of the target substance initially contained in the measured sample, and thus, there is a need for further improvement of this method in order to create a calibration curve that is accurate over a wide range from the low-concentration region to the high-concentration region.

[0011] Specifically, when the amount of target substance contained in the measured sample is small, a calibration curve can be created using all of the calibration curve creation samples prepared by adjusting the amount of tracer and the amount of antibody (in other words, in the calibration curve creation method of the present disclosure, the fluorescence polarization degree P dil is used instead of the fluorescence polarization degree P0 to create a calibration curve) in the case where the value of the fluorescence polarization degree P0 is equal to the fluorescence polarization degree P dil However, as described above, this method can only be used when the amount of target substance contained in the measured sample is small.

[0012] The present disclosure was made in view of the above circumstances, and an object of the present disclosure is to provide a calibration curve creation method, a fluorescence polarization immunoassay method, a fluorescence polarization immunoassay device, and a calibration curve creation kit that are more accurate over a wide range from the low-concentration region to the high-concentration region. SUMMARY

[0013] To achieve the above object, the method for creating a calibration curve according to the first aspect of the present application is a method for creating a calibration curve for a fluorescence polarization immunoassay that uses an antibody having a binding ability to a target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, the method comprising:

[0014] Step 1a, preparing a first reference sample that does not contain the target substance;

[0015] Step 1b, after Step 1a, adding the antibody and the fluorescent labeling substance to the first reference sample, and measuring the fluorescence polarization degree P0 of the first reference sample;

[0016] Step 1c, from the first reference sample, preparing a plurality of calibration curve creation samples that contain the target substance at concentrations C Sαseparating a sample number of p, which is an integer number of 3 or more, of the measured sample a from the measured sample a of the target substance, adding different amounts of the target substance to each of the measured samples a1 to ap, and preparing p calibration curve creation samples 1 to p including the concentration of the target substance added later exceeding the initial concentration C of the target substance Sα calibration curve creation samples conc ;

[0017] Step 1d, after Step 1c, adding the antibody and the fluorescent labeling substance to each of the calibration curve creation samples 1 to p in an amount equal to the amount added in Step 1b, and measuring the fluorescence polarization degrees P1 to P P of each of the calibration curve creation samples 1 to p; and

[0018] Step 1e, after Step 1b and Step 1d, creating a calibration curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample based on the fluorescence polarization degree P0 and the fluorescence polarization degrees P conc of the calibration curve creation samples 1 to p of the calibration curve creation samples conc .

[0019] The fluorescence polarization immunoassay method according to the second aspect of the present application is a fluorescence polarization immunoassay method for measuring the concentration C Sβ of a target substance in a measured sample β using an antibody having a binding ability with the target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, the method comprising:

[0020] Step 4a, adding the antibody and the fluorescent labeling substance to the measured sample β in an amount equal to the amount added in Step 1b, and measuring the fluorescence polarization degree P Xβ of the measured sample β; and

[0021] Step 4b, after Step 4a, deriving the concentration C Xβ of the target substance from the calibration curve created according to the method of the first aspect based on the fluorescence polarization degree P Sβ .

[0022] The program according to the third aspect of the present application is a program used in the fluorescence polarization immunoassay method according to the second aspect, which causes a computer to execute the following steps:

[0023] deriving the concentration C Xβ of the target substance from the calibration curve created according to the method of the first aspect based on the fluorescence polarization degree P Sβ of the measured sample β.

[0024] The fluorescence polarization immunoassay device according to the fourth aspect of the present application is a fluorescence polarization immunoassay device that measures the concentration C of a target substance in a sample β to be measured using an antibody having a binding ability with the target substance and a fluorescently labeled substance that labels the target substance with a fluorescent dye Sβ The fluorescence polarization immunoassay device according to the fourth aspect of the present application is a fluorescence polarization immunoassay device that measures the concentration C of a target substance in a sample β to be measured using an antibody having a binding ability with the target substance and a fluorescently labeled substance that labels the target substance with a fluorescent dye

[0025] An emission optical system that emits linearly polarized excitation light on the sample;

[0026] A polarized light adjusting element that selectively allows passage of a linearly polarized component corresponding to the drive signal from the fluorescence emitted from the sample;

[0027] A receiver that detects the intensity of the fluorescence that has passed through the polarized light adjusting element; and

[0028] A controller that outputs the drive signal to the polarized light adjusting element, measures the degree of polarization of the sample in accordance with the drive signal and based on the intensity of the fluorescence detected by the receiver,

[0029] wherein

[0030] The controller includes a storage medium that stores a calibration curve created according to the method of the first aspect and a program used in the fluorescence polarization immunoassay method according to the second aspect, the program causing a computer to execute the steps of:

[0031] Based on the degree of polarization P of the sample β to be measured Xβ deriving the concentration C of the target substance from the calibration curve Sβ .

[0032] The calibration curve creation kit according to the fifth aspect of the present application is a calibration curve creation kit that includes:

[0033] An antibody having a binding ability with the target substance; and a fluorescently labeled substance that labels the target substance with a fluorescent dye;

[0034] and further includes:

[0035] At least one of a means for removing the target substance from a solution containing the target substance and a liquid not containing the target substance.

[0036] It should be understood that the general description above and the following detailed description are exemplary and illustrative, and do not limit the present disclosure.

[0037] In the present disclosure, "calibration curve" sometimes means "function representing the calibration curve", and "creating a calibration curve" includes not only creating a graph that allows visual recognition of the relationship between the amount of the target substance and the degree of fluorescence polarization of the sample, but also deriving a function representing the calibration curve.

[0038] In the present disclosure, the fluorescence polarization degree such as the fluorescence polarization degree P0, the fluorescence polarization degree P conc is calculated by the following equation (I) P A or by the following equation (II) P CB .

[0039]

[0040] In equation (I), A(I II ) is the fluorescence intensity of fluorescence having a polarized light component parallel to the excitation polarization direction when the fluorescent labeling substance and the antibody are added to the sample and measured. A(I ⊥ ) is the fluorescence intensity of fluorescence having a polarized light component perpendicular to the excitation polarization direction when the fluorescent labeling substance and the antibody are added to the sample and measured.

[0041]

[0042] In equation (II), B(I II ) is the fluorescence intensity of fluorescence having a polarized light component parallel to the excitation polarization direction when the sample is measured without modification. B(I ⊥ ) is the fluorescence intensity of fluorescence having a polarized light component perpendicular to the excitation polarization direction when the sample is measured without modification.

[0043] C(I II ) is the fluorescence intensity of fluorescence having a polarized light component parallel to the excitation polarization direction when the fluorescent labeling substance and the antibody are added to the sample and measured. C(I ⊥ ) is the fluorescence intensity of fluorescence having a polarized light component perpendicular to the excitation polarization direction when the fluorescent labeling substance and the antibody are added to the sample and measured.

[0044] When the sample contains a spontaneous fluorescent substance, P A calculated from equation (I) is affected by fluorescence emitted from the spontaneous fluorescent substance, and therefore, P A is not suitable as calibration curve creation data.

[0045] Meanwhile, as in equation (II), in the corrected fluorescence polarization degree (fluorescence polarization degree P CB ) calculated using measurement data of a sample containing a fluorescent labeling substance and an antibody and measurement data of a sample not containing these components, the influence of fluorescence emitted from a spontaneous fluorescent substance is eliminated, and therefore, the corrected fluorescence polarization degree is suitable as calibration curve creation data.

[0046] Examples of cases in which it is preferable to use the case of the corrected fluorescence polarization degree calculated by Equation (II) include, for example, cases in which the sample to be measured is a beverage, food, or the like containing a self-fluorescing substance (such as certain types of vitamins or the like), and cases in which a specific enzyme treatment is performed on the sample to be measured to remove a target substance from the sample to be measured, as described below.

[0047] Therefore, in the calibration curve creation method of the present disclosure, it is preferable to appropriately use P A calculated by Equation (I) and P CB .

[0048] According to the present disclosure, a more accurate calibration curve creation method, a fluorescence polarization immunoassay method, a fluorescence polarization immunoassay device, and a calibration curve creation kit are provided over a wide range from a low concentration region to a high concentration region. BRIEF DESCRIPTION OF DRAWINGS

[0049] A more complete understanding of the present application can be obtained by considering the following detailed description in conjunction with the accompanying drawings, in which:

[0050] Figure 1 is a schematic view showing the configuration of a fluorescence polarization immunoassay device 1;

[0051] Figure 2 is a schematic view of a micro device 23;

[0052] Figure 3 is a graph explaining the preferable concentration of a calibration curve creation sample;

[0053] Figure 4 is a graph showing the creation process (steps 1a to 1b) of a calibration curve;

[0054] Figure 5 is a graph showing the creation process (steps 1c to 1d) of a calibration curve;

[0055] Figure 6 is a graph showing the creation process (step 1e) of a calibration curve;

[0056] Figure 7 is a graph showing the process (steps 2a to 2c) of additional measurement 1;

[0057] Figure 8 is a graph showing the process of additional measurement 1 (step 2d);

[0058] Figure 9 is a graph showing the process (steps 2e to 2f) of additional measurement 1;

[0059] Figure 10is a diagram showing the process (steps 3a to 3d) of additional measurement 2;

[0060] Figure 11 is a diagram showing the process (steps 3e to 3g) of additional measurement 2;

[0061] Figure 12 is a diagram showing the process of additional measurement 2 (step 3h);

[0062] Figure 13 is a diagram showing the process of creation of a calibration curve (steps la to lb);

[0063] Figure 14 is a diagram showing the process of creation of a calibration curve (steps lc to ld);

[0064] Figure 15 is a diagram showing the process of creation of a calibration curve (step le);

[0065] Figure 16 is a calibration curve created in Example 1;

[0066] Figure 17 is a calibration curve created in Example 2;

[0067] Figure 18 is a calibration curve created in Example 6;

[0068] Figure 19 is a calibration curve created in Example 7; and

[0069] Figure 20 is a calibration curve created in Example 8. DETAILED DESCRIPTION

[0070] 1. Calibration curve creation method

[0071] Sample to be measured

[0072] When measuring the concentration C of a target substance contained in a sample to be measured S (hereinafter, can be abbreviated as "the concentration C of a target substance S ") of a sample to be measured, a calibration curve created by the present disclosure is used. Examples of the sample to be measured include beverages, foods, washing liquids, tissue extracts, cell extracts, cell culture supernatants, blood, saliva, urine, lymph, and the like.

[0073] Target substance

[0074] The target substance is a substance of which the concentration is to be measured by the calibration curve created by the present disclosure. The target substance is a compound of which at least a part can be used as an epitope for the preparation of an antibody. Examples of the target substance include antigens, haptens, and the like. Specific examples thereof include proteins, glycoproteins, peptides, polypeptides, oligonucleotides, polynucleotides, antibodies, hormones, drugs, enzymes, receptors, and the like.

[0075] Note that, although haptens bind to antibodies, they have a small molecular weight and thus are a substance that does not exhibit immunogenicity by itself, which is an activity to induce the production of antibodies. In this way, when the target substance is a hapten, the hapten is bound to an immunogenic substance such as a protein or the like to form an antigen that is completely immunogenic, as described later, and thus the concentration in a sample to be measured can be determined.

[0076] Examples of the hapten include histamine, γ-aminobutyric acid (GABA), dopamine, thyroid hormone, steroid hormone, and the like.

[0077] Examples of the immunogenic substance include immunogenic proteins, polypeptides, carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, and the like. Among them, polypeptides or proteins such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), thyroglobulin, and the like are preferred.

[0078] Antibody

[0079] The antibody used in the present disclosure is an antibody capable of binding to the target substance. Specifically, the antibody used in the present disclosure has the ability to recognize and bind to at least a part of the target substance as an epitope. Examples of the antibody include monoclonal antibodies, multispecific antibodies, bifunctional antibodies, human antibodies, humanized antibodies, antibodies derived from birds such as chickens, mammals such as cows and camels, and other animals, recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv ("sdFv"), anti-idiotype ("anti-Id") antibodies, two-domain antibodies, two-variable domain antibodies, and the like.

[0080] When the target substance is a hapten, an antibody using a hapten derivative in which an immunogenic substance is bound to the hapten via a linker is generally used. The linker is a group of atoms introduced between the immunogenic substance and the hapten. Examples of the linker include groups having an amide bond and the like.

[0081] Fluorescently labeled substance

[0082] The fluorescently labeled substance is a compound in which the target substance is labeled with a fluorescent dye.

[0083] A fluorescent dye is a dye that emits fluorescence. Each fluorescent dye has its own fluorescence lifetime. In the present disclosure, a fluorescent dye having a fluorescence lifetime of 1 to 10 nanoseconds, a fluorescent dye having a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, and a fluorescent dye having a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds can be appropriately selected and used depending on the molecular weight and the like of the target substance. Examples of the fluorescent dye having a fluorescence lifetime of 1 to 10 nanoseconds include indolenines; fluorescein compounds such as chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5-amino fluorescein, 6-amino fluorescein, thiourea fluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and, as registered trademarks or product names, Alexa Fluor 488 and other Alexa Fluor series, BODIPY series, DY series, ATTO series, Dy Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, Yakima Yellow, VIC, HEX, R6G, Cy3, TAMRA, Rhodamine Red-X, Redmond Red, ROX, Cal Red, Texas Red, LC Red 640, Cy5, Cy5.5, and LC Red 705. Examples of the fluorescent dye having a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds include naphthalene derivatives such as dialkylaminonaphthalenesulfonyl; and pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrene butyric acid, and alkynylpyrene. Examples of the fluorescent dye having a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds include metal complexes containing platinum, rhenium, ruthenium, osmium, europium, and other metals.

[0084] Examples of the method for labeling the target substance with the fluorescent dye include a method in which the fluorescent dye is directly bound to the target substance, and a method in which the fluorescent dye is bound to the target substance through a suitable linker such as an oligoethylene glycol, an alkyl chain, or the like. In these methods, substituents such as a carboxyl group, an amino group, a hydroxyl group, a thiol group, and a phenyl group contained in the fluorescent dye and / or the target substance can be utilized.

[0085] Fluorescence polarization immunoassay method

[0086] Fluorescence polarization immunoassay is a method for measuring the change in fluorescence polarization caused by the competitive reaction of a substance and the molecular weight change of a competing substance. In the case of irradiating a fluorescent molecule in a liquid with plane polarized light as the excitation light, when the fluorescent molecule does not move much, the fluorescent molecule emits polarized fluorescence in the same plane as the excitation plane. At the same time, when the fluorescent molecule rotates due to Brownian motion in an excited state, the fluorescent molecule emits polarized fluorescence in a plane different from the excitation plane, and therefore the fluorescence polarization decreases as a whole. Therefore, fluorescence polarization is a physical property related to the degree of rotation of the fluorescent molecule from when it is excited to when it emits fluorescence. In addition, the size (molecular weight) of the fluorescent molecule affects the degree of rotation of the fluorescent molecule. For example, low molecular weight molecules (such as free fluorescent markers) rotate violently due to Brownian motion in the solution, while high molecular weight molecules (such as fluorescent markers bound to antibodies) do not show violent motion. Therefore, by measuring fluorescence polarization, information related to the change in the molecular weight of the fluorescent marker in the solution can be obtained. In addition, the concentration C of the target substance in the sample to be measured can be measured by the following method S .

[0087] In a solution containing a mixture of a target substance A, an antibody B capable of binding to target substance A, and a fluorescently labeled substance C labeled with a fluorescent dye, the target substance A, antibody B, and fluorescently labeled substance C compete for reaction in the solution. Therefore, when the concentration of target substance A is high, the amount of the conjugate of target substance A and antibody B increases, resulting in an increase in the amount of free fluorescently labeled substance C. Therefore, the concentration of target substance A affects the amount of free fluorescently labeled substance C (or fluorescently labeled substance C bound to antibody B), and the concentration of target substance A can be measured by creating a calibration curve representing the relationship between the concentration of target substance A and the degree of fluorescence polarization.

[0088] Fluorescence polarization immunoassay device

[0089] In the calibration curve creation method of the present invention, the fluorescence polarization degree of various samples is measured. The measuring device or measuring apparatus for measuring the fluorescence polarization degree is not particularly limited, but the fluorescence polarization degree can be effectively measured by using a micro device.

[0090] Figure 1 An example of a fluorescence polarization immunoassay apparatus using a microdevice is shown.

[0091] The fluorescence polarization immunoassay device 1 includes a light source 10, a focusing lens 11, an aperture 12, a collimator 13, a polarizing element 14, an excitation filter 15, and a dichroic mirror 20. The fluorescence polarization immunoassay device 1 also includes an objective lens 21, a microdevice 23 on which a sample 22 is placed, a stage 24, an absorption filter 25, a polarization adjustment element 26, an imaging lens 27, an imaging element 28, and a controller 30.

[0092] In one example, the light source 10 is implemented as a light emitting diode, and emits excitation light of a wavelength that excites fluorescence of the sample (e.g., blue light having a center wavelength of 470 nm). The excitation light from the light source 10 is focused by the condenser lens 11, and passes through the diaphragm 12. The diaphragm 12 reduces intrusion of external light other than the excitation light.

[0093] The excitation light that has passed through the diaphragm 12 is converted into parallel light by the collimator 13, and enters the polarization element 14. The polarization element 14 is, for example, a polarizing plate, a polarization beam splitter, or a liquid crystal cell, and is a polarizing plate in this example. The polarization element 14 allows passage of light linearly polarized in a specific direction. The linearly polarized excitation light from the polarization element 14 passes through the excitation light filter 15. The excitation light filter 15 is a filter that selects a wavelength range including the wavelength of the excitation light, and reduces light having a wavelength different from the wavelength of the excitation light from the polarization element 14. The dichroic mirror 20 reflects the excitation light that has passed through the excitation light filter 15 toward the objective lens 21.

[0094] The objective lens 21 focuses the linearly polarized excitation light reflected by the dichroic mirror 20 on the sample 22 contained in the micro device 23 on the stage 24. The sample 22 generates fluorescence of a specific wavelength (e.g., green light) from the linearly polarized excitation light from the objective lens 21. The fluorescence becomes parallel light at the objective lens 21, and passes through the dichroic mirror 20 and the absorption filter 25. The dichroic mirror 20 selectively allows passage of light in a specific wavelength region including the fluorescence from the sample 22, and reflects other light. The absorption filter 25 is a filter that selects a wavelength range including the wavelength of the fluorescence from the sample 22, and reduces light other than the fluorescence.

[0095] The fluorescence that has passed through the absorption filter 25 enters the polarization light adjusting element 26. In one example, the polarization light adjusting element 26 is implemented as a polarizing plate, a polarization beam splitter, or a liquid crystal cell. The polarization light adjusting element 26 can be implemented as a polarization filter in a polarization camera. A polarization camera is an imaging device in which a polarization filter is mounted on a sensor, thereby acquiring polarization information of an object. In the following description, the polarization light adjusting element 26 is a liquid crystal cell controlled by a drive signal (applied voltage). The polarization light adjusting element 26 can adjust the transmission light intensity of linearly polarized components. Specifically, the polarization light adjusting element 26 can adjust the transmission light intensity of linearly polarized light parallel to the polarization direction of the excitation light and the transmission light intensity of linearly polarized light perpendicular to the polarization direction of the excitation light. Furthermore, the polarization light adjusting element 26 can adjust the transmission light intensity of light polarized in a direction corresponding to the drive signal, described later.

[0096] The fluorescent light of the linearly polarized light that has passed through the polarization light adjusting element 26 enters the imaging plane of the imaging element 28 via the imaging lens 27. The surface of the sample 22 and the imaging plane of the imaging element 28 are in an imaging relationship. The imaging element 28 includes, for example, a charge-coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) image sensor having multiple pixels. The imaging element 28 generates image data corresponding to the intensity of the fluorescent light generated by the sample 22 and transmits the generated image data to the controller 30.

[0097] The controller 30 performs overall control of the entire fluorescent polarization immunoassay apparatus 1. Specifically, the controller 30 controls the light source 10, the polarization light adjusting element 26, and the imaging element 28. The controller 30 acquires the fluorescent image imaged by the imaging element 28.

[0098] For example, during the measurement operation, the controller 30 causes the light source 10 to emit the excitation light onto the sample 22. The controller 30 outputs a drive signal to the polarization light adjusting element 26 using a DA converter (not shown). By outputting the drive signal to the polarization light adjusting element 26, the controller 30 can control the polarization light component of the fluorescent light that passes through the polarization light adjusting element 26.

[0099] In one example, the polarization light adjusting element 26 includes two transparent substrates facing each other, transparent electrodes provided on the opposite surfaces of the substrates, a liquid crystal material sealed between the substrates, and a polarizing plate provided on the outer surface of the imaging apparatus side (the output side or the downstream side) of the polarization light adjusting element 26. The configuration of the polarization light adjusting element 26 can be determined as needed as long as the polarization light component of the fluorescent light that passes through the polarization light adjusting element 26 can be adjusted.

[0100] The composition of the above-described fluorescent polarization immunoassay apparatus 1 is summarized as follows.

[0101] (A) An emission optical system 10A including the light source 10, the condenser lens 11, the aperture 12, the collimator 13, the polarization element 14, and the excitation light filter 15, which emits linearly polarized excitation light onto the sample 22;

[0102] (B) A micro device 23 that houses the sample 22, a stage 24 on which the micro device 23 is placed;

[0103] (C) An observation optical system 10B including the polarization light adjusting element 26, which adjusts the transmitted linearly polarized component of the fluorescent light emitted from the sample 22 according to a drive signal from the controller 30, and the imaging element 28, which images the fluorescent light that has passed through the polarization light adjusting element 26;

[0104] (D) The controller 30 functions as a driver that outputs a drive signal to the polarized light adjusting element 26 , detects fluorescence intensity based on the fluorescence image imaged by the imaging element 28 , and measures the polarization degree of the sample 22 according to the drive signal to analyze the sample.

[0105] like Figure 2 As shown, the microdevice 23 includes a plurality of channels 23c, each of which includes one end connected to an injection port 23a and the other end connected to an outlet port 23b. Measurement liquids, including calibration curve creation samples or test samples, can be supplied individually as samples 22 to the plurality of channels 23c. The channels 23c and the imaging plane of the imaging element 28 are in an imaging relationship. Thus, if the plurality of channels 23c are imaged by the imaging element 28, a fluorescence image of the plurality of samples 22 supplied to the plurality of channels 23c can be immediately obtained, and the degree of polarization of each sample 22 can be immediately measured. This measurement is performed based on the fluorescence intensity of a region of interest (ROI) of the image data corresponding to the channel 23c in the fluorescence image.

[0106] Calibration curve creation method

[0107] The calibration curve creation method disclosed herein is a method for creating a calibration curve for a fluorescence polarization immunoassay method, which uses an antibody capable of binding to a target substance and a fluorescent labeling substance for labeling the target substance with a fluorescent dye, and comprises:

[0108] Step 1a, preparing a first reference sample that does not contain the target substance;

[0109] Step 1b, after step 1a, adding the antibody and the fluorescent labeling substance to the first reference sample, and measuring the fluorescence polarization degree P0 of the first reference sample;

[0110] Step 1c, starting from a mixture containing a concentration of C Sα Separate the test samples α1 to αp, the number of which is an integer p greater than or equal to 3, from the test sample α containing the target substance, add different amounts of the target substance to each of the test samples α1 to αp, and prepare p calibration curves to create samples 1 to p, including the concentration of the target substance added later exceeding the initial concentration C of the target substance Sα Calibration curve creation samples conc ;

[0111] Step 1d, after step 1c, adding the antibody and the fluorescent labeling substance in an amount equal to that added in step 1b to each of the calibration curve creation samples 1 to p, and measuring the fluorescence polarization degree P1 to P of each calibration curve creation sample 1 to p P ;and

[0112] Step 1e, after Step 1b and Step 1d, creating a calibration curve of the calibration curve creating samples 1 to p based on the fluorescence polarization degree P0 and the calibration curve conc conc The fluorescence polarization degree P of the calibration curve creating samples 1 to p is calculated by the following equation (I).

[0113] Step 1a is a step of preparing a first reference sample containing no target substance.

[0114] The low concentration region of the calibration curve created in the present disclosure is mainly created based on the fluorescence polarization degree P0 of the first reference sample.

[0115] Examples of the first reference sample include pure water, a solution containing no target substance from the beginning, such as physiological saline, and a solution obtained by removing a target substance from a measured sample a.

[0116] Examples of the method for removing a target substance from a measured sample a include a method of adding an enzyme using a target substance as a substrate to a measured sample a, a method of contacting an adsorbent adsorbing a target substance with a measured sample a, and the like.

[0117] When the target substance is histamine, histamine N-methyltransferase, diamine oxidase, or the like can be used as the enzyme. When the target substance is chloramphenicol, chloramphenicol acetyltransferase, hydrolase, nitroreductase, or the like can be used as the enzyme. When the target substance is deoxynivalenol, a mycotoxin-degrading enzyme can be used as the enzyme.

[0118] Note that when a solution obtained by removing a target substance by adding an enzyme to a measured sample a is used as a first reference sample, the first reference sample can include a self-fluorescent substance. Therefore, in this case, it is preferable to use the corrected fluorescence polarization degree calculated by the above equation (II) as the fluorescence polarization degree.

[0119] Bentonite, activated carbon, diatomaceous earth, an ion exchange adsorbent, an affinity adsorbent, or the like can be used as the adsorbent.

[0120] The ion exchange adsorbent is an adsorbent containing an ion exchange resin or the like as an adsorption component. Examples of the ion exchange adsorbent include a cation exchange adsorbent adsorbing a cationic compound, and an anion exchange adsorbent adsorbing an anionic compound. When an ion exchange adsorbent is used as the adsorbent, by selecting a suitable ion exchange adsorbent according to the properties of a target substance, and further adjusting the pH of a measured sample a and the pH of a washing liquid, a target substance can be effectively removed from a measured sample a.

[0121] ​Affinity adsorbents are adsorbents that utilize enzyme / substrate, antibody / antigen, hormone / receptor protein, and similar biospecific interactions. Affinity adsorbents are particularly preferred as a method for removing target substances from a test sample α because they selectively remove target substances and are less likely to adversely affect the test sample α than when using enzymes.

[0122] Note that the method of using the adsorbent is not particularly limited. For example, the target substance can be removed from the sample α by filling a column with the adsorbent and passing the sample α through the column, or by introducing the adsorbent into the sample α and then filtering out the adsorbent.

[0123] Step 1b is a step of adding an antibody and a fluorescent labeling substance to the first reference sample after step 1a, and measuring the fluorescence polarization degree P0 of the first reference sample. For example, the fluorescence polarization immunoassay device 1 can be used to measure the fluorescence polarization degree.

[0124] As described above, the first reference sample does not contain the target substance. However, if it does not affect the results, the fluorescence polarization degree can be measured using a solution obtained by adding a small amount of the target substance to the first reference sample instead of the first reference sample. That is, the "fluorescence polarization degree P0 of the first reference sample" in the present disclosure includes the "fluorescence polarization degree of the solution obtained by adding a small amount of the target substance to the first reference sample."

[0125] As described below, for example, a calibration curve for an antigen-antibody reaction in the present disclosure is created as follows Figure 3 The inverted S-shaped curve shown in FIG. Thus, when the antigen concentration (target substance concentration) is below a certain concentration, the fluorescence polarization degree remains almost unchanged. In the present disclosure, if the concentration is within the range where the fluorescence polarization degree does not change significantly, a "solution obtained by adding a small amount of target substance to the first reference sample" or a "second reference sample" containing a small amount of target substance in a solution described later may be used instead of the "first reference sample."

[0126] Similarly, when the first reference sample is a solution obtained by removing the target substance from the sample α, the first reference sample can be a solution containing a small amount of the target substance that cannot be completely removed as long as the concentration is within the range where the fluorescence polarization degree does not change significantly.

[0127] Step 1c is a step of preparing p calibration curve creation samples 1 to p. The calibration curve creation samples 1 to p are obtained by separating a number p of test samples α1 to αp from the test sample α and adding a different amount of the target substance to each of the test samples α1 to αp.

[0128] The number (p) of the calibration curve creation samples 1 to p is an integer of 3 or more, for example, an integer of 3 to 20, an integer of 3 to 15, or an integer of 3 to 10.

[0129] The calibration curve creation samples 1 to p include the calibration curve creation sample conc in which the concentration of the target substance added later exceeds the initial concentration C of the target substance Sα . The p calibration curve creation samples 1 to p can or can not include the calibration curve creation sample dil in which the concentration of the target substance added later is less than or equal to the initial concentration C of the target substance Sα .

[0130] The middle concentration region to the high concentration region of the calibration curve created in the present disclosure is mainly based on the fluorescence polarization P conc of the calibration curve creation sample conc created.

[0131] Specifically, the calibration curve creation samples 1 to p are prepared based on the measured sample α, so that by using the calibration curve creation samples 1 to p, a calibration curve considering the influence of the contaminant in the measured sample α can be created.

[0132] However, the measured sample α initially contains the target substance at a concentration C Sα , and thus, among the calibration curve creation samples 1 to p, the calibration curve creation sample in which the low concentration target substance is added later (i.e., the calibration curve creation sample dil ) is not suitable for deriving the relationship between the amount of the target substance added later and the fluorescence polarization of the sample.

[0133] Meanwhile, when deriving the relationship between the amount of the target substance added later and the fluorescence polarization of the sample, when the amount of the target substance added later is greater than the amount of the target substance initially contained in the measured sample α, the target substance initially contained in the measured sample α hardly produces any influence.

[0134] Therefore, in the present disclosure, among the fluorescence polarizations P1 to P P of the calibration curve creation samples 1 to p, only the fluorescence polarization P conc of the calibration curve creation sample conc is used.

[0135] As described above, in the calibration curve creation method of the present disclosure, the calibration curve creation sample conc is a necessary sample, and the calibration curve creation sample dil is an optional sample.

[0136] The number of the calibration curve creation samples conc is, for example, an integer of 2 to 20, an integer of 2 to 15, or an integer of 2 to 10.

[0137] Calibration curve creation sample dil The number is, for example, an integer of 0 to 18, an integer of 0 to 13, or an integer of 0 to 8.

[0138] When there is no information related to the concentration C Sα of the target substance at the time of performing step 1c, in step 1c, it is preferable to prepare calibration curve creation samples 1 to p in a wide range from a low concentration to a high concentration. As a result, in step 1c, calibration curve creation samples dil are prepared together with calibration curve creation samples conc .

[0139] In this case, the concentration of the calibration curve creation sample with the highest concentration is, for example, 10 6 to 10 10 times the concentration of the calibration curve creation sample with the lowest concentration.

[0140] As described below, by regressing experimental data with a four-parameter logistic model, a calibration curve is usually created as an inverse s-shaped curve. In order to create a more accurate calibration curve, for example, in the calibration curve shown in FIG. 1, it is preferable to use, at the time of creation, a sample with a concentration close to the polarization degree d, a sample with a concentration close to the polarization degree (a+b) / 2, and a sample with a concentration close to the polarization degree a. Figure 3

[0141] Examples of the “polarization degree close to d”, the “polarization degree close to (a+b) / 2”, and the “polarization degree close to a” are, respectively, d±[(a-d) / 5], [(a+b) / 2]±[(a-d)×3 / 5], and a±[(a-d) / 5], preferably d±[(a-d) / 10], [(a+b) / 2]±[(a-d) / 5], and a±[(a-d) / 10].

[0142] Meanwhile, when there is information related to the concentration C Sα of the target substance at the time of performing step 1c, it is possible to prepare only calibration curve creation samples conc based on the information. Examples of the case where there is information related to the concentration C Sα of the target substance include the case where there is known information on the measured sample a, like when analyzing to confirm the ingredients of a food, a beverage, or the like manufactured by a conventional method, and the case where a tentative concentration C Pα of the target substance in the measured sample a is calculated by an additional measurement described later.

[0143] ​Step 1d is to add the antibody and the fluorescent labeling substance to each of the calibration curve creation samples 1 to p after step 1c, and measure the fluorescence polarization degree P1 to P of each calibration curve creation sample 1 to p. P steps.

[0144] The amounts of the antibody and fluorescent labeling substance added are the same as those added to the first reference sample in step 1b.

[0145] Step 1e is to create a calibration curve for samples 1 to p based on the fluorescence polarization degree P0 of the first reference sample and the calibration curve after step 1b and step 1d. conc The fluorescence polarization degree P conc , a step of creating a calibration curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample.

[0146] Step 1e can be performed, for example, by the following method.

[0147] First, a number of calibration curve creation samples are selected from calibration curve creation samples 1 to p, starting with the highest concentration sample, and a calibration curve is created based on their fluorescence polarization degree and the fluorescence polarization degree P0 of the first reference sample. Next, the fluorescence polarization degree of the test sample α is measured, and the concentration C of the target substance is derived from the calibration curve. Sα By comparing the concentration of target substance C Sα and the concentration of each calibration curve creation sample, the calibration curve creation sample can be determined dil Whether to include the samples used in the calibration curve creation.

[0148] When the calibration curve is created for the sample dil When included in the calibration curve creation samples used, a desired calibration curve can be created by repeatedly performing the process of creating a calibration curve while excluding low-concentration samples from the samples used to create the calibration curve.

[0149] At the same time, when the calibration curve is created, the sample dil When not included in the calibration curve creation samples used, some of the calibration curve creation samples are added in sequence from the high concentration sample among the remaining calibration curve creation samples, and the calibration curve is recreated.

[0150] As described below, if the concentration C of the target substance can be obtained before or during the creation of the calibration curve, Sα If relevant information is available, calibration curves can be created more efficiently.

[0151] For example, in step 1c, the calibration curve was created for the sample dil Create samples with calibration curve conctogether with the fluorescence polarization degree thereof measured in step Id, the calibration curve can be created based on the concentration C Sα The information related to the concentration C conc of the target substance used at this time is an example of information related to the concentration C conc of the target substance, and includes known information about the measured sample α described above, and a tentative concentration C conc of the target substance in the measured sample α calculated through additional measurement described later.

[0152] The information related to the concentration C Sα of the target substance used at this time is an example of information related to the concentration C Pα of the target substance, and includes known information about the measured sample α described above, and a tentative concentration C conc of the target substance in the measured sample α calculated through additional measurement described later.

[0153] Meanwhile, in the case where only the calibration curve creation samples p are prepared in step 1c, the calibration curve can be created based on the fluorescence polarization degree P0obtained in step 1b and the fluorescence polarization degrees P1to P conc (i.e., the fluorescence polarization degrees P conc ) obtained in step Id.

[0154] The "amount of target substance" in the "relationship between the amount of target substance and the fluorescence polarization degree of sample" of the calibration curve can be appropriately selected depending on the purpose of use of the calibration curve. Typically, the concentration of the target substance added later in the calibration curve creation samples 1 to p is used as the "amount of target substance".

[0155] The calibration curve is typically created as an inverse s-shaped curve by regressing the experimental data with a four-parameter logistic model.

[0156] Note that when the first approximation curve and the second approximation curve described later are derived, the aforementioned matters related to the "relationship between the amount of target substance and the fluorescence polarization degree of sample" and the calibration curve creation method (e.g., the preferred concentration of the sample shown in Figure 3 , the method of creating the calibration curve as an inverse s-shaped curve by regressing the experimental data with a four-parameter logistic model, etc.) can be used.

[0157] Embodiment 1

[0158] Figures 4 to 6 The process of creating the calibration curve through steps la to le is shown. However, the calibration curve creation method of the present disclosure is not limited to the order of steps shown in Figures 4 to 6 , and the order of steps can be changed as long as execution is possible.

[0159] Figures 4 to 6 The calibration curve creation method shown in concfluorescence polarization P of the sample conc to create a calibration curve.

[0160] Figure 4 The state after steps 1a and 1b is shown. The fluorescence polarization P0 of the first reference sample is plotted on the y-axis.

[0161] Figure 5 The state after steps 1c and 1d is shown. The fluorescence polarization P1 to P9 of the calibration curve creation samples 1 to 9 prepared in step 1c in a wide range from a low concentration to a high concentration of the target substance are plotted. Sα The state under unknown conditions, the fluorescence polarization P1 to P9 of the calibration curve creation samples 1 to 9 prepared in step 1c in a wide range from a low concentration to a high concentration of the target substance are plotted.

[0162] Figure 6 The state after step 1e is shown. The calibration curve is created based on the fluorescence polarization P0 of the first reference sample and the information related to the concentration C Sα of the target substance selected from the calibration curve. conc The fluorescence polarization P of the sample conc , and the calibration curve representing the relationship between the concentration of the target substance in the sample and the fluorescence polarization of the sample is created.

[0163] Examples of the information related to the concentration C Sα of the target substance used in step 1e include a provisional concentration C Pα(1) of the target substance in the measured sample α derived by the following additional measurement 1 (hereinafter, can be abbreviated as "provisional concentration C Pα(1) of the target substance") and a provisional concentration C Pα(2) of the target substance in the measured sample α derived by the following additional measurement 2 (hereinafter, can be abbreviated as "provisional concentration C Pα(2) of the target substance").

[0164] Note that, for ease of explanation, the tasks repeated with steps 1a to 1e are described again, but in actual measurement, the tasks already performed can be omitted in some cases.

[0165] Additional measurement 1

[0166] Step 2a, prepare a second reference sample containing no target substance;

[0167] Step 2b, after step 2a, separate the second reference sample into a number of second reference samples 1 to q that is an integer greater than or equal to 3 from the second reference sample, add a different amount of the target substance to each of the second reference samples 1 to q, and prepare q additional measurement samples A1 to Aq;

[0168] Step 2c, after step 2b, add an antibody and a fluorescent labeling substance to each of the additional measurement samples A1 to Aq, and measure the fluorescence polarization P of each of the additional measurement samples A1 to Aq.A1 to P Aq ;

[0169] Step 2d, after Step 2c, based on the fluorescence polarization degree P A1 to P Aq , a first approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample is derived;

[0170] Step 2e, the antibody and the fluorescent labeling substance are added to the measured sample a in an amount equal to the amount added in Step 2c, and the fluorescence polarization degree P Xα ; and

[0171] Step 2f, after Step 2d and Step 2e, based on the fluorescence polarization degree P Xα , the provisional concentration C Pα(1) of the target substance in the measured sample a is derived from the first approximation curve.

[0172] The process of deriving the first approximation curve and the provisional concentration C Pα(1) of the target substance by Steps 2a to 2f will be described below with reference to Figures 7 to 9 However, the order of the steps shown in Figures 7 to 9 The order of the steps can be changed as long as execution is possible.

[0173] Step 2a is a step of preparing a second reference sample that does not contain the target substance, and is a step similar to Step la. However, the second reference sample is used to prepare additional measurement samples Al to Aq, and therefore, in the case where the target substance is removed by adding an enzyme, the enzyme activity of the enzyme must be inactivated. Therefore, it is preferable to use a solution that does not contain the target substance from the beginning, such as pure water or the like, or a solution obtained by removing the target substance from the measured sample a by using an adsorbent, as the second reference sample. Note that, as with the first reference sample, when the second reference sample is a solution obtained by removing the target substance from the measured sample a, if the concentration is within a range in which the fluorescence polarization degree does not change significantly, the second reference sample can be a solution containing a small amount of the target substance that could not be completely removed.

[0174] Step 2b is a step of preparing q additional measurement samples Al to Aq. By separating the second reference samples 1 to q of the sample number q from the second reference sample, and adding different amounts of the target substance to these second reference samples 1 to q, the additional measurement samples Al to Aq are obtained.

[0175] The number (q) of the additional measurement samples Al to Aq is an integer of 3 or more, for example, an integer of 3 to 20, an integer of 3 to 15, or an integer of 3 to 10. In step 2b, it is preferable to prepare a wide range of additional measurement samples Al to Aq from a low concentration to a high concentration. The preferable sample concentration range is as described for the calibration curve creation samples 1 to p.

[0176] Step 2c is a step of adding an antibody and a fluorescent labeling substance to each of the additional measurement samples Al to Aq, and measuring the fluorescence polarization degree P A1 to P Aq of each of the additional measurement samples Al to Aq. The amount of the antibody and the fluorescent labeling substance to be added can be the same as or different from the amount of the antibody and the fluorescent labeling substance added to the first reference sample in step lb.

[0177] When the amount of the antibody and the fluorescent labeling substance to be added in step 2c is the same as the amount added in step lb, repeated measurement can be omitted in some cases. When the amount of the antibody and the fluorescent labeling substance to be added in step 2c is different from the amount added in step lb, a suitable first approximation curve can be derived in some cases, which is used to derive a tentative concentration C Pα(1) of the target substance.

[0178] Figure 7 A state after steps 2a to 2c is shown. The fluorescence polarization degrees P A1 to P A9 of the additional measurement samples Al to A9 are plotted.

[0179] Step 2d is a step of deriving a first approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample, based on the fluorescence polarization degrees P A1 to P Aq after step 2c.

[0180] Figure 8 A state after step 2d is shown. The fluorescence polarization degrees P A1 to P A9 of the additional measurement samples Al to A9 and the first approximation curve derived based on the fluorescence polarization degrees P A1 to P A9 are plotted.

[0181] Step 2e is a step of adding an antibody and a fluorescent labeling substance to the measured sample a and measuring the fluorescence polarization degree P Xα of the measured sample a. The amount of the antibody and the fluorescent labeling substance to be added is the same as the amount of the antibody and the fluorescent labeling substance added to the additional measurement samples Al to Aq in step 2c.

[0182] Step 2f is a step of deriving a tentative concentration C Xαderiving a provisional concentration C of the target substance from the first approximation curve Pα(1) .

[0183] Figure 9 The state after steps 2e and 2f is shown. The fluorescence polarization P of the measured sample a Xα is plotted on the y-axis, and the provisional concentration C of the target substance corresponding thereto Pα(1) is plotted on the x-axis.

[0184] Additional measurement 2

[0185] Step 3a, preparing a second reference sample not containing the target substance;

[0186] Step 3b, after step 3a, separating a second reference sample 1 to q of an integer q of 3 or more from the second reference sample, adding a different amount of the target substance to each of the second reference samples 1 to q, and preparing q additional measurement samples Al to Aq;

[0187] Step 3c, after step 3b, adding an antibody and a fluorescent labeling substance to each of the additional measurement samples Al to Aq, and measuring the fluorescence polarization P A1 to P Aq of each of the additional measurement samples Al to Aq;

[0188] Step 3d, after step 3c, deriving a first approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization of the sample, based on the fluorescence polarizations P A1 to P Aq ;

[0189] Step 3e, separating a measured sample a1 to ar of an integer r of 3 or more from the measured sample a, adding a different amount of the target substance to each of the measured samples a1 to ar, and preparing r additional measurement samples Bl to Br;

[0190] Step 3f, after step 3e, adding an antibody and a fluorescent labeling substance to each of the additional measurement samples Bl to Br in an amount equal to the amount added in step 3c, and measuring the fluorescence polarization P B1 to P Br of each of the additional measurement samples Bl to Br;

[0191] Step 3g, after step 3f, deriving a second approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization of the sample, based on the fluorescence polarizations P B1 to P Br ; and

[0192] Step 3h, after Step 3d and Step 3g, deriving a provisional concentration C of the target substance in the measured sample a from the first approximation curve based on the maximum value of the fluorescence polarization degree on the second approximation curve Pα(2) .

[0193] The process of deriving the first approximation curve, the second approximation curve, and the provisional concentration C of the target substance by Steps 3a to 3h is described below Figures 10 to 12 with reference to Pα(2) However, the order of the steps shown in Figures 10 to 12 The order of the steps can be changed as long as execution is possible.

[0194] In Steps 3a to 3d, the same tasks as in Steps 2a to 2d are performed.

[0195] Figure 10 The state after Steps 3a to 3d is shown. The fluorescence polarization degrees P A1 to P A9 of the additional measurement samples A1 to A9 are plotted, and the first approximation curve is derived based on the fluorescence polarization degrees P A1 to P A9 .

[0196] Step 3e is a step of preparing r additional measurement samples B1 to Br. Step 3f is a step of measuring the fluorescence polarization degrees P B1 to P Br of the additional measurement samples B1 to Br, respectively. Like the calibration curve creation samples 1 to p, the additional measurement samples B1 to Br are prepared from the measured sample a. Therefore, Steps 3e and 3f include the same tasks as in Steps 1c and 1d, that is, preparing the calibration curve creation samples 1 to p in a state where there is no information related to the concentration C Sα of the target substance, and measuring the fluorescence polarization degrees P1 to P p of the calibration curve creation samples 1 to p. In this way, in some cases, the measurement result of Step 1d can be used as the measurement result of Step 3f when the amount of the antibody and the fluorescent labeling substance used in Step 3c is equal to the amount added in Step 1b.

[0197] In Step 3g, a second approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization degree is derived based on the fluorescence polarization degrees P B1 to P Br of the additional measurement samples B1 to Br.

[0198] Figure 11 The state after Steps 3e to 3g is shown. The fluorescence polarization degrees P B1 to P B9 of the additional measurement samples B1 to B9 are plotted, and the second approximation curve is derived based on the fluorescence polarization degrees P B1To P B9 The second approximation curve is derived.

[0199] Step 3h is a step of deriving a provisional concentration C Pα(2) of the target substance from the first approximation curve based on the maximum value of the degree of polarization of fluorescence on the second approximation curve.

[0200] The degree of polarization of fluorescence at the concentration of 0 of the target substance added in the second approximation curve indicates the degree of polarization of fluorescence of the measured sample a. Therefore, the maximum value of the degree of polarization of fluorescence on the second approximation curve (the value of the intersection of the second approximation curve with the y-axis) is read, and based on the maximum value, the provisional concentration C Pα(2) of the target substance can be derived from the first approximation curve.

[0201] Figure 12 A state after step 3h is shown. The intersection of the second approximation curve with the y-axis is plotted, and the provisional concentration C Pα(2) of the target substance corresponding thereto is plotted on the x-axis.

[0202] Other Embodiments

[0203] When information related to the concentration C Sα of the target substance is present when step 1c is performed, a calibration curve can be created by a further simplified method shown by Figures 13 to 15 .

[0204] Figures 13 to 15 The calibration curve creation method shown is a method in which only a plurality of calibration curve creation samples conc are prepared in step 1c.

[0205] Figure 13 A state after steps 1a and 1b is shown. The degree of polarization of fluorescence P0 of the first reference sample is plotted on the y-axis. In addition, as known information about the measured sample a, information that the concentration C Sα of the target substance is approximately 1 pg / mL is present and is plotted on the x-axis.

[0206] Figure 14 A state after steps 1c and 1d is shown. The degrees of polarization of fluorescence P Sα of the calibration curve creation samples conc prepared based on information related to the concentration C conc of the target substance are plotted.

[0207] Figure 15 A state after step 1e is shown. Based on the degree of polarization of fluorescence P0 of the first reference sample and the degrees of polarization of fluorescence P conc of the calibration curve creation samples conc , a calibration curve indicating the relationship between the concentration of the target substance and the degree of polarization of fluorescence in the sample is created.

[0208] The calibration curve created by the present disclosure as described above is obtained by combining a low concentration region sample (first reference sample) and a medium to high concentration region sample (calibration curve creation sample). conc ) and is more accurate in a wide range from low-concentration areas to high-concentration areas.

[0209] 2. Fluorescence Polarization Immunoassay

[0210] The fluorescence polarization immunoassay method according to the second aspect of the present disclosure measures the concentration C of the target substance in the test sample β using an antibody having binding ability to the target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye. Sβ , the method comprising:

[0211] Step 4a, adding the antibody and the fluorescent labeling substance to the sample β in an amount equal to the amount added in step 1b, and measuring the fluorescence polarization degree P of the sample β Xβ ;and

[0212] Step 4b, after step 4a, based on the fluorescence polarization degree P Xβ , the concentration C of the target substance was derived from the calibration curve created by this method Sβ .

[0213] That is, the fluorescence polarization immunoassay method disclosed herein includes measuring the fluorescence polarization degree P of the sample β to be tested. Xβ and measure the concentration C of the target substance from the calibration curve created by this method Sβ .

[0214] It is preferable to use the same sample as the sample α or a sample of the same type as the sample α as the sample β.

[0215] Examples of the case where the test sample β is the same as the test sample α include when a calibration curve is created by the above method, and then the remaining test sample α used to create the calibration curve is used to measure the fluorescence polarization degree and derive its concentration. In step 1c and step 1e, when additional measurement 1 is performed, and at this time, the fluorescence polarization degree P of the test sample α is measured using an amount of antibody and fluorescent labeling substance equal to the amount added in step 1b. Xα When the calibration curve is completed, step 4a has been performed, so the concentration C of the target substance is derived at the same time. Sα .

[0216] Examples of the case where the test sample β is the same type as the test sample α include when analyzing the composition of another batch of products manufactured by the same method or a similar method in a factory or the like.

[0217] 3. Procedure

[0218] The program according to the third aspect of the present disclosure is used in the fluorescence polarization immunoassay method according to the second aspect of the present disclosure, and

[0219] causes a computer to execute a step of deriving a concentration C Xβ of a target substance from a calibration curve created by the method. Sβ

[0220] The steps of the program according to the third aspect of the present disclosure are executed by a computer. In one example, the computer includes a processor, a memory, and an input / output device. The processor is constituted by a central processing unit (CPU) or the like, and executes a program stored in the memory. The memory is one example of a storage medium, and stores the program according to the third aspect and the calibration curve obtained by the calibration curve creation method according to the first aspect. The program causes the processor to execute the calculation of the fluorescence polarization immunoassay method according to the second aspect. The input / output device inputs the fluorescence polarization degree P Xβ required for the calculation of the fluorescence polarization immunoassay method according to the second aspect. Further, the input / output device outputs the calculation result, that is, displays the concentration C Sβ or the like. The processor executes the calculation of the fluorescence polarization immunoassay method according to the second aspect on the fluorescence polarization degree P Xβ input by the input / output device according to the program of the third aspect, derives the concentration C Sβ , and outputs the derived concentration C SB from the input / output device.

[0221] The program of the present disclosure is preferably used when the measured sample β is of the same type as the measured sample α.

[0222] Specifically, by initially creating an accurate calibration curve when analyzing the components of a product manufactured in a factory or the like, thereafter, when analyzing the components of another batch of product manufactured by the same method or a similar method, the calibration curve can be used. In this way, by using the program of the present disclosure, the components of a product can be efficiently analyzed.

[0223] 4. Fluorescence polarization immunoassay device

[0224] The fluorescence polarization immunoassay device according to the fourth aspect of the present disclosure measures the concentration C Sβ of a target substance in a measured sample β using an antibody having a binding ability to the target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, and the device includes:

[0225] an emission optical system that emits linearly polarized excitation light on a sample;

[0226] ​a polarized light conditioning element that selectively allows passage of a linearly polarized component corresponding to the driving signal from the fluorescence emitted from the sample;

[0227] a receiver that detects the fluorescence intensity that has passed through the polarized light conditioning element; and

[0228] a controller that outputs the driving signal to the polarized light conditioning element, measures the degree of polarization of the sample in accordance with the driving signal and based on the fluorescence intensity detected by the receiver, wherein

[0229] The controller includes a storage medium that stores the calibration curve created according to the method of the first aspect and a program used in the fluorescence polarization immunoassay method according to the second aspect, which causes a computer to perform the following steps:

[0230] based on the degree of polarization P of the fluorescence of the sample β being measured Xβ derives the concentration C of the target substance from the calibration curve Sβ .

[0231] The emission optical system, the polarized light conditioning element, and the receiver of the fluorescence polarization immunoassay device of the present disclosure are the same as those of the above-described fluorescence polarization immunoassay device 1.

[0232] In one example, the controller of the fluorescence polarization immunoassay device of the present disclosure includes a processor, a memory, and an input / output device. The controller of the fluorescence polarization immunoassay device of the present disclosure includes a storage medium such as a memory and stores the calibration curve created according to the method of the first aspect and the program used in the fluorescence polarization immunoassay method according to the second aspect. The controller of the fluorescence polarization immunoassay device of the present disclosure can perform each step of the program of the present disclosure, and as such can effectively measure the concentration C of the target substance by using the fluorescence polarization immunoassay device of the present disclosure Sβ .

[0233] 5. Calibration curve creation kit

[0234] The calibration curve creation kit according to the fifth aspect of the present disclosure includes:

[0235] an antibody having a binding ability to the target substance, and a fluorescent labeling substance that labels the target substance with a fluorescent dye; and further includes a means for removing the target substance from a solution containing the target substance, and / or a liquid not containing the target substance.

[0236] The antibody having a binding ability to the target substance and the fluorescent labeling substance that labels the target substance with a fluorescent dye are described in the disclosure of the calibration curve creation method.

[0237] These can be in a solution state or a dry state.

[0238] These are usually stored in containers such as bags, bottles, or ampoules, according to their respective states.

[0239] Examples of the means for removing the target substance from the solution containing the target substance include a substance for removing the target substance from the solution containing the target substance, such as an enzyme, an adsorbent, or the like. These are described in the disclosure of the calibration curve creation method.

[0240] Examples of the liquid not containing the target substance include pure water, physiological saline, a buffer solution, or the like.

[0241] Preferably, the calibration curve creation kit of the present disclosure includes exactly the right amount of the antibody and the fluorescent labeling substance according to the intended number of uses.

[0242] The calibration curve creation kit of the present disclosure can further include the micro device of the present disclosure.

[0243] When the calibration curve creation method of the present disclosure described above is performed, the calibration curve creation kit of the present disclosure is preferably used.

[0244] EMBODIMENT

[0245] Reagent

[0246] (1) Antibody solution

[0247] An anti-histamine antibody (manufactured by Progen Biotechnik) was diluted with phosphate buffered saline (PBS(-)) containing 0.01% bovine serum albumin (BSA) to prepare an antibody solution of 1.3 x 10 -7 M.

[0248] (2) Histamine tracer solution

[0249] Histamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was modified with HiLyte Fluor 647 to obtain a histamine tracer. This histamine tracer was dissolved in pure water, and then further diluted with PBS(-) to prepare a histamine tracer solution of 4.56 x 10 -9 M.

[0250] (3) Target substance solution

[0251] Histamine dihydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare a histamine solution of 32 mg / ml. The histamine solution was diluted with pure water to prepare histamine solutions (#1 to #9) having the following concentrations.

[0252] #1: 2.88 x 10-9 M (0.00032 μg / mL)

[0253] #2: 2.88 x 10 -8 M (0.0032 μg / mL)

[0254] #3: 2.88 x 10 -7 M (0.032 μg / mL)

[0255] #4: 2.88 x 10 -6 M (0.32 μg / mL)

[0256] #5: 2.88 x 10 -5 M (3.2 μg / mL)

[0257] #6: 0.000288 M (32 μg / mL)

[0258] #7: 0.002879 M (320 μg / mL)

[0259] #8: 0.02879 M (3,200 μg / mL)

[0260] #9: 0.287899 M (32,000 μg / mL)

[0261] (4) Reference sample (e)

[0262] A histamine-decomposing enzyme was added to the soy sauce to prepare a reference sample (e). As a histamine-decomposing enzyme reagent, reagents of a histamine assay kit (Check Color Histamine (manufactured by Kikkoman Biochemifa)) were used according to the protocol of the kit.

[0263] (5) Reference sample (w)

[0264] Pure water was used as a reference sample (w).

[0265] (6) Reference sample (f)

[0266] A soy sauce diluent was prepared by diluting the soy sauce with a 20 mM phosphate buffer solution having a pH of 6.0. The soy sauce diluent was passed through a column (Sep-Pak Plus Accell CM, manufactured by Waters). Next, a washing liquid (20 mM phosphate buffer solution having a pH of 6.0) was passed through the column. A combination of the soy sauce diluent and the washing liquid that passed through the column was used as a reference sample (f).

[0267] (7) Acylation reagent and acylation buffer

[0268] Reagents of a histamine acylation set (manufactured by Beckman Coulter) were used as the acylation reagent and the acylation buffer.

[0269] Example 1

[0270] The concentration of histamine (target substance) contained in the soy sauce (sample to be measured) was determined by the following method. Example 1 is an example in which there is no known information relating to the concentration of histamine in soy sauce. Preparation of calibration curve creation samples conc and calibration curve creation samples dil The fluorescence polarization degree thereof was measured, and then calibration curve creation samples conc were selected to create a calibration curve.

[0271] Note that the fluorescence polarization degree in Example 1 is a corrected fluorescence polarization degree calculated by the above equation (II).

[0272] To 40 μl of the reference sample (e), 160 μl of PBS(-) was added to obtain a five-fold diluted solution. 100 μl of the obtained five-fold diluted solution, 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain a mixed liquid. 25 μl of the mixed liquid, 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and the mixture was allowed to stand at room temperature in a light-shielded environment for 10 minutes to prepare a measurement liquid containing the reference sample (e), and the fluorescence polarization degree of the measurement liquid containing the reference sample (e) was measured.

[0273] To each of 100 μl of the histamine solutions #1 to #9, 40 μl of the soy sauce and 60 μl of PBS(-) were added to obtain calibration curve creation samples (#1) to (#9). 100 μl of the calibration curve creation samples (#1) to (#9), 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain mixed liquids (#1) to (#9). 25 μl of the mixed liquids (#1) to (#9), 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and these mixtures were allowed to stand at room temperature in a light-shielded environment for 10 minutes to prepare measurement liquids containing the calibration curve creation samples (#1) to (#9), and the fluorescence polarization degrees of these measurement liquids containing the calibration curve creation samples (#1) to (#9) were measured.

[0274] To 100 μl of each of the histamine solutions #1 to #9, 40 μl of the reference sample (w) and 60 μl of PBS (-) were added to obtain additional measurement samples A (#1) to (#9). 100 μl of the additional measurement samples A (#1) to (#9), 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain mixed liquids (#1) to (#9). 25 μl of the mixed liquids (#1) to (#9), 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and the mixtures were allowed to stand at room temperature for 10 minutes in a light-protected environment to prepare measurement liquids (#1) to (#9) containing the additional measurement samples A, and the degrees of fluorescence polarization of the measurement liquids (#1) to (#9) containing the additional measurement samples A were measured. Next, a first approximation curve was derived from the obtained degrees of fluorescence polarization.

[0275] The degrees of fluorescence polarization of the calibration curve creation samples (#1) to (#9) were created as the degrees of fluorescence polarization of additional measurement samples B (#1) to (#9) using the calibration curve, to derive a second approximation curve. The maximum value of the degree of fluorescence polarization on the second approximation curve was read, and a tentative concentration C Pα of histamine in the soy sauce was derived from the first approximation curve based on the maximum value.

[0276] Based on the tentative concentration C Pα , the calibration curve creation samples (#1) to (#9) were divided into calibration curve creation samples conc (#5) to (#9)) and calibration curve creation samples dil (#1) to (#4)), and a calibration curve was created from the degree of fluorescence polarization of the reference sample (e) and the degrees of fluorescence polarization of the calibration curve creation samples (#5) to (#9). The obtained calibration curve is shown in Figure 16 .

[0277] Example 2

[0278] In Example 2, a calibration curve was created in the same manner as in Example 1, except that the reference sample (w) was used instead of the reference sample (e) in Example 1, and the degree of fluorescence polarization calculated by the above equation (I) was used. The obtained calibration curve is shown in Figure 17 .

[0279] Example 3

[0280] The concentration of histamine (target substance) contained in the soy sauce (measured sample) was determined by the following method. Example 3 is an example based on known information relating to the concentration of histamine in soy sauce to prepare only calibration curve creation samples conc .

[0281] Note that the degree of fluorescence polarization in Example 3 is a corrected degree of fluorescence polarization calculated by the above equation (II).

[0282] To 40 μl of the reference sample (e), 160 μl of PBS(-) was added to obtain a five-fold diluted solution. To 100 μl of the obtained five-fold diluted solution, 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain a mixed liquid. To 25 μl of the mixed liquid, 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and the mixture was allowed to stand at room temperature for 10 minutes in a light-shielded environment to prepare a measurement liquid containing the reference sample (e), and the fluorescence polarization degree of the measurement liquid containing the reference sample (e) was measured.

[0283] To each of 100 μl of the histamine solutions #5 to #9, 40 μl of the soy sauce and 60 μl of PBS(-) were added to obtain calibration curve creation samples conc #5 to #9. To 100 μl of the calibration curve creation samples conc (#5) to (#9), 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain mixed liquids (#5) to (#9). To 25 μl of the mixed liquids (#5) to (#9), 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and these mixtures were allowed to stand at room temperature for 10 minutes in a light-shielded environment to prepare measurement liquids (#5) to (#9) containing the calibration curve creation samples conc , and the fluorescence polarization degrees of these measurement liquids (#1) to (#9) containing the calibration curve creation samples conc were measured.

[0284] A calibration curve similar to the calibration curve of Example 1 was created from the fluorescence polarization degrees of the reference sample (e) and the fluorescence polarization degrees of the calibration curve creation samples conc (#5) to (#9).

[0285] Example 4

[0286] In Example 4, a calibration curve similar to the calibration curve of Example 2 was created in the same manner as in Example 3, except that the reference sample (w) was used instead of the reference sample (e) in Example 3, and the fluorescence polarization degree calculated by the above equation (I) was used.

[0287] Example 5

[0288] To 40 μl of the soy sauce, 160 μl of PBS(-) was added to obtain a five-fold diluted solution. 100 μl of the obtained five-fold diluted solution, 25 μl of the acylation reagent, and 200 μl of the acylation buffer were mixed to obtain a mixed liquid. 25 μl of the mixed liquid, 25 μl of the histamine tracer solution, and 25 μl of the antibody solution were mixed, and the mixture was allowed to stand at room temperature for 10 minutes in a light-shielded environment to prepare a measurement liquid containing the soy sauce, and the degree of fluorescence polarization of the measurement liquid containing the soy sauce was measured.

[0289] When the concentration of histamine in the soy sauce was calculated from the calibration curve created in Example 2, the concentration of histamine was 1.02 ppm.

[0290] Note that when the concentration of histamine in the soy sauce was calculated from the first approximate curve created in the process of Example 1, the concentration of histamine was 0.69 ppm.

[0291] Example 6

[0292] In Example 6, a calibration curve was created in the same manner as in Example 1, except that a different soy sauce from that used in Examples 1 to 5 was used as the sample to be measured. The obtained calibration curve is shown in Figure 18 .

[0293] Example 7

[0294] In Example 7, a calibration curve was created in the same manner as in Example 1, except that fish sauce was used as the sample to be measured. The obtained calibration curve is shown in Figure 19 .

[0295] Example 8

[0296] In Example 8, a calibration curve was created in the same manner as in Example 1, except that the reference sample (f) was used instead of the reference sample (e) in Example 1, and the degree of fluorescence polarization calculated by the above equation (I) was used. The obtained calibration curve is shown in Figure 20 .

[0297] The foregoing description of some example embodiments has been presented for purposes of illustration. While the above discussion has given specifics, it is to be understood that modifications and variations can be made in form and detail without departing from the broader spirit and scope of the application. Accordingly, the specification and drawings are to be regarded as illustrative in nature and not as restrictive. Thus, the specific embodiments are not to be taken advantage of, and the scope of the application is to be defined solely by the appended claims and their equivalents.

Claims

1. A method for creating a calibration curve of a fluorescence polarization immunoassay, which uses an antibody having a binding ability to a target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, the method comprising: Step la, preparing a first reference sample that does not contain the target substance; Step lb, after Step la, adding the antibody and the fluorescent labeling substance to the first reference sample, and measuring a fluorescence polarization degree P0 of the first reference sample; Step 1c, from the measured sample α containing the target substance at a concentration C Sα , separate measured samples α1 to αp of which the number is an integer p of 3 or more, add different amounts of the target substance to each of the measured samples α1 to αp, and prepare p calibration curve creation samples 1 to p including a calibration curve creation sample in which the concentration of the target substance added later exceeds the initial concentration C Sα of the target substance conc ; Step 1d, after Step 1c, the antibody and the fluorescent labeling substance are added to each of the calibration curve creation samples 1 to p in an amount equivalent to the added amount in Step 1b, and the fluorescence polarization degree P1 to Pp of each calibration curve creation sample 1 to p is measured. P ; and Step 1e, after Step 1b and Step 1d, creating a calibration curve in the sample 1 to p based on the fluorescence polarization degree P0 and the calibration curve conc conc , creating a calibration curve representing the relationship between the amount of target substance and the fluorescence polarization degree of the sample.​ 2. The method for creating a calibration curve according to claim 1, wherein In step 1c, the concentration C of the target substance is determined based on the information related to the target substance Sα The information related to the target substance is only prepared for creating a plurality of calibration curves conc , or In step le, the concentration C of the target substance is determined based on the information relating to the fluorescence intensity I of the sample Sα The information relating to the fluorescence intensity I of the sample is determined based on the information relating to the fluorescence intensity I of the sample conc The information relating to the fluorescence intensity I of the sample is determined based on the information relating to the fluorescence intensity I of the sample conc The information relating to the fluorescence intensity I of the sample is determined based on the information relating to the fluorescence intensity I of 3. The method for creating a calibration curve according to claim 2, wherein information on a concentration C of the target substance Sα related to the target substance is a tentative concentration C of the target substance obtained by an additional measurement 1 including: Pα(1) an additional measurement 1 including: Step 2a, preparing a second reference sample that does not contain the target substance; Step 2b, after Step 2a, separating a second reference sample 1 to q of a sample number of an integer q of 3 or more from the second reference sample, preparing q additional measurement samples Al to Aq by adding different amounts of the target substance to the second reference sample 1 to q, respectively; Step 2c, after step 2b, adding the antibody and the fluorescently labeled substance to each of the additional measurement samples A1 to Aq and measuring the degree of polarization P of the respective additional measurement samples A1 to Aq A1 to P Aq ; Step 2d, after step 2c, deriving a first approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample based on said fluorescence polarization degree P A1 to P Aq , deriving a first approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample based on said fluorescence polarization degree P Step 2e, the antibody and the fluorescent labeling substance are added to the sample α to be measured in an amount equivalent to the amount added in Step 2c, and the fluorescence polarization P of the sample α to be measured is measured Xα ; and Step 2f, after Step 2d and Step 2e, the fluorescence polarization degree P is calculated based on the following formula: Xα The provisional concentration C of the target substance in the measured sample a is derived from the first approximation curve Pα(1) .

4. The method for creating a calibration curve according to claim 2, wherein information on the concentration C of the target substance Sα related to the concentration C of the target substance is a tentative concentration C of the target substance obtained by an additional measurement 2, which includes: Pα(2) an additional measurement 2 Step 3a, preparing a second reference sample that does not contain the target substance; Step 3b, after Step 3a, separating a second reference sample 1 to q of a sample number of an integer q of 3 or more from the second reference sample, preparing q additional measurement samples Al to Aq by adding different amounts of the target substance to the second reference sample 1 to q, respectively; Step 3c, after step 3b, adding the antibody and the fluorescently labeled substance to each of the additional measurement samples Al to Aq and measuring the degree of polarization P of the respective additional measurement samples Al to Aq A1 to P Aq ; Step 3d, after step 3c, deriving a first approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample based on the fluorescence polarization degree P A1 to P Aq , deriving a first approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample Step 3e, separating a measured sample a 1 to ar of a sample number of an integer r of 3 or more from the measured sample a, preparing r additional measurement samples Bl to Br by adding different amounts of the target substance to the measured sample a 1 to ar, respectively; Step 3f, after step 3e, the antibody and the fluorescent marker substance are added to each of the additional measurement samples B1 to Br in an amount equal to the added amount in step 3c, and the fluorescence polarization degree P of the respective additional measurement samples B1 to Br is measured B1 to P Br ; Step 3g, after step 3f, deriving a second approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample based on the fluorescence polarization degree P B1 to P Br , deriving a second approximation curve representing the relationship between the amount of target substance and the fluorescence polarization degree P of the sample and Step 3h, after step 3d and step 3g, deriving a tentative concentration C of the target substance in the measured sample a from the first approximation curve on the basis of the maximum value of the fluorescence polarization degree on the second approximation curve Pα(2) .

5. The method for creating a calibration curve according to claim 2, wherein the information related to the concentration C Sα is known information about the sample α under test.

6. The method for creating a calibration curve according to claim 1, wherein the first reference sample is pure water.

7. The method for creating a calibration curve according to claim 1, wherein the first reference sample is a solution obtained by removing the target substance from a measured sample a.

8. The method for creating a calibration curve according to claim 7, wherein the method for removing the target substance from the measured sample a is a method of adding an enzyme that uses the target substance as a substrate to the measured sample a.

9. The method for creating a calibration curve according to claim 7, wherein the method for removing the target substance from the measured sample a is a method of bringing an adsorbent that adsorbs the target substance into contact with the measured sample a.

10. The method for creating a calibration curve according to claim 3 or 4, wherein the second reference sample is pure water.

11. The method for creating a calibration curve according to claim 3 or 4, wherein the second reference sample is a solution obtained by removing the target substance from a measured sample a.

12. The method for creating a calibration curve according to claim 11, wherein the method for removing the target substance from the measured sample a is a method of adding an enzyme that uses the target substance as a substrate to the measured sample a.

13. The method for creating a calibration curve according to claim 11, wherein the method for removing the target substance in the measured sample a is a method of bringing an adsorbent that adsorbs the target substance into contact with the measured sample a.

14. The method for creating a calibration curve according to claim 1, wherein the calibration curve is created by regressing experimental data with a four-parameter logistic model.

15. The method for creating a calibration curve according to claim 3, wherein the first approximation curve is created by regressing experimental data with a four-parameter logistic model.

16. The method for creating a calibration curve according to claim 4, wherein the first approximation curve and the second approximation curve are each created by regressing experimental data with a four-parameter logistic model.

17. A fluorescence polarization immunoassay method, which uses an antibody capable of binding to a target substance and a fluorescent labeling substance in which the target substance is labeled with a fluorescent dye to measure the concentration C of the target substance in a sample β. Sβ , the method comprising: Step 4a, the antibody and the fluorescent labeling substance are added to the sample β to be measured in an amount equivalent to the amount added in Step 1b, and the fluorescence polarization P of the sample β to be measured is measured Xβ ; and Step 4b, after step 4a, deriving the concentration C of the target substance based on the fluorescence polarization P Xβ deriving the concentration C of the target substance from the calibration curve created by the method according to any one of claims 1 to 4 Sβ .

18. A fluorescence polarization immunoassay device which measures the concentration C of a target substance in a sample β by using an antibody having a binding ability with the target substance and a fluorescent labeling substance which labels the target substance with a fluorescent dye Sβ , the device comprising: an emission optical system that emits linearly polarized excitation light on a sample; a polarized light adjusting element that selectively allows passage of a linearly polarized component corresponding to a drive signal from the fluorescence emitted from the sample; a receiver that detects the intensity of the fluorescence that has passed through the polarized light adjusting element; and a controller that outputs a drive signal to the polarized light adjusting element, measures the degree of polarization of the sample in accordance with the drive signal and based on the intensity of the fluorescence detected by the receiver, wherein the controller includes a storage medium that stores a calibration curve created in accordance with the method of claim 1 and a program used in the fluorescence polarization immunoassay method according to claim 17, which causes a computer to perform the following steps: The fluorescence polarization degree P of the sample β to be measured Xβ The concentration C of the target substance is derived from the calibration curve Sβ .

19. A calibration curve creation kit comprising: an antibody having a binding ability to a target substance; and a fluorescent labeling substance that labels the target substance with a fluorescent dye; and further comprising: at least one of a tool for removing the target substance from a solution containing the target substance and a liquid that does not contain the target substance.

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