Calibration curve creation method, fluorescence polarization immunoassay, program, fluorescence polarization immunity measuring apparatus, and calibration curve creation kit

Abstract

To provide a calibration curve creation method capable of creating a more accurate calibration curve across a wide range from a low-concentration region to a high-concentration region, as well as a fluorescence polarization immunoassay, a program, a fluorescence polarization immunity measuring apparatus, and a calibration curve creation kit that use this calibration curve.SOLUTION: A calibration curve creation method is a method of creating a calibration curve for a fluorescence polarization immunoassay using an antibody having a binding ability to a target substance and a fluorescent-labeled substance obtained by labeling the target substance with a fluorescent dye, and creates the calibration curve on the basis of a fluorescence polarization degree P0 of a first reference sample that does not contain the target substance, and a fluorescence polarization degree Pconc of a calibration curve creation sample conc in which a concentration of the added target substance exceeds a concentration CSα of the target substance.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present disclosure relates to a method for creating a calibration curve, a fluorescence polarization immunoassay, a program, a fluorescence polarization immunoassay device, and a calibration curve creation kit. [Background technology]

[0002] One immunoassay that utilizes antigen-antibody reactions is the fluorescence polarization immunoassay (FPIA), which estimates the concentration of a target substance by measuring fluorescence polarization. FPIA can be divided into competitive and non-competitive types. Of these, competitive FPIA involves pitting the target substance against a fluorescently labeled target substance (tracer) to react with the antibody. Tracers that are not bound to antibodies move vigorously in the liquid, and emit fluorescence randomly even when irradiated with polarized excitation light. On the other hand, tracers that are bound to antibodies are less mobile, and emit fluorescence that is polarized in the direction of the polarization of the excitation light.

[0003] In competitive FPIA, the fluorescence intensity is measured both parallel and perpendicular to the polarization direction of the excitation light, and the degree of polarization between the two directions is measured as the fluorescence polarization index. Because this fluorescence polarization index depends on the amount of tracer-antibody complex, the concentration of the target substance can be measured using the fluorescence polarization index as an index.

[0004] In FPIA, a calibration curve showing 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 to be measured is measured from the resulting calibration curve (see, for example, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-47802 [Patent Document 2] US Patent Application Publication No. 2009 / 0023595 Summary of the Invention [Problem to be solved by the invention]

[0006] Pure water can be used as a diluent when preparing a calibration curve. Pure water is easily available, so using it as a diluent allows for efficient preparation of a calibration curve. However, when the sample to be measured contains impurities that affect the reaction between the target substance and the antibody, it is difficult to prepare a calibration curve that takes into account the influence of the impurities by using pure water as a diluent.

[0007] One possible method for creating a calibration curve that takes into account the influence of impurities in the sample to be measured is to use the sample to be measured itself as a diluent instead of pure water. That is, by adding known amounts of the target substance to each of multiple samples to prepare samples for creating a calibration curve, it is thought that a calibration curve that takes into account the influence of impurities can be created. However, the fluorescence polarization index in the low concentration region of the calibration curve created using this method is easily affected by the target substance originally contained in the sample being measured, and therefore further improvement is required to create an accurate calibration curve over a wide range from low to high concentrations using this method. That is, if the amount of the target substance contained in the sample to be measured is minute, it is possible to create a calibration curve using all of the prepared samples for creating a calibration curve by adjusting the amount of tracer or antibody (in other words, the fluorescence polarization index P dil is a value similar to the fluorescence polarization degree P0, the fluorescence polarization degree P dil However, as mentioned above, this method can only be used when the amount of the target substance contained in the sample to be measured is very small.

[0008] The present disclosure has been made in light of the above-mentioned circumstances, and aims to provide a method for creating a more accurate calibration curve over a wide range from low concentration to high concentration, a fluorescence polarization immunoassay method using this calibration curve, a program, a fluorescence polarization immunoassay device, and a calibration curve creation kit. [Means for solving the problem]

[0009] In order to achieve the above object, a method for creating a calibration curve according to a first aspect of the present disclosure includes: 1. A method for preparing a calibration curve for a fluorescence polarization immunoassay using an antibody capable of binding to a target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, comprising: Step 1a, providing a first reference sample that does not contain the target substance; After step 1a, step 1b is performed by 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. The target substance is added at a concentration of C Sα p measurement target samples α1 to αp, where p is an integer equal to or greater than 3, are taken from a measurement target sample α containing Sα More than 1000 samples for creating calibration curves conc Step 1c of preparing p calibration curve samples, After step 1c, the antibody and the fluorescent labeling substance are added to each of the p calibration curve preparation samples in the same amounts as in step 1b, and the fluorescence polarization indices P1 to P p Step 1d of measuring respectively After step 1b and step 1d, the fluorescence polarization degree P0 and the number of samples for preparing a calibration curve among the p samples for preparing a calibration curve are conc Fluorescence polarization P conc Step 1e., based on the above, creating a calibration curve showing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample; It has.

[0010] A fluorescence polarization immunoassay according to a second aspect of the present disclosure includes: Using an antibody capable of binding to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is measured. SβA fluorescence polarization immunoassay for measuring The antibody and the fluorescent labeling substance are added to the measurement sample β in the same amounts as in step 1b, and the fluorescence polarization index P Xβ Step 4a measuring After step 4a, the fluorescence polarization index P Xβ Based on the above, the concentration C of the target substance is determined from the calibration curve created by the above method. Sβ Step 4b to derive It has.

[0011] A program according to a third aspect of the present disclosure includes: A program used in the fluorescence polarization immunoassay, The fluorescence polarization degree P of the measurement sample β Xβ Based on the above, the concentration C of the target substance is determined from the calibration curve created by the above method. Sβ The method causes the computer to execute a step of deriving

[0012] A fluorescence polarization immunoassay device according to a fourth aspect of the present disclosure, Using an antibody capable of binding to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is measured. Sβ A fluorescence polarization immunoassay device for measuring an illumination optical system that illuminates the sample with linearly polarized excitation light; a polarization adjusting element that selectively passes a linearly polarized component of the fluorescence emitted from the sample in accordance with the drive signal; a light receiving unit that detects the intensity of the fluorescence that has passed through the polarization adjusting element; a control unit that outputs the drive signal to the polarization adjustment element and measures the degree of polarization of the sample in accordance with the drive signal based on the fluorescence intensity detected by the light receiving unit; Equipped with The control unit executes the program and calculates the concentration C of the target substance. Sβ This is to derive the following.

[0013] A calibration curve creation kit according to a fifth aspect of the present disclosure includes: The method comprises: an antibody capable of binding to a target substance; and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye; The apparatus further comprises a means for removing the target substance from the solution containing the target substance and / or a liquid not containing the target substance.

[0014] In the present disclosure, the term "calibration curve" may refer to a "function representing the calibration curve." Furthermore, in the present disclosure, "creating a calibration curve" includes not only creating a graph that visually shows the relationship between the amount of a target substance and the fluorescence polarization degree of a sample, but also deriving a function representing the calibration curve.

[0015] In the present disclosure, the fluorescence polarization degree P0, the fluorescence polarization degree P conc The fluorescence polarization index is calculated by the following formula (I): A or P calculated by formula (II) CB is.

[0016]

number

[0017] In formula (I), A(I II ) is the fluorescence intensity of the fluorescence with a polarization component parallel to the excitation polarization direction when a fluorescent label and antibody are added to the sample and measured, and A(I ⊥ ) is the fluorescence intensity of fluorescence with a polarized component perpendicular to the excitation polarization direction when a fluorescent label and antibody are added to a sample and measured.

[0018]

number

[0019] In formula (II), B(I II ) is the fluorescence intensity of the fluorescence with a polarization component parallel to the excitation polarization direction when the sample is measured as is, and B(I ⊥) is the fluorescence intensity of the fluorescence with a polarization component perpendicular to the excitation polarization direction when the sample is measured as is. C(I II ) is the fluorescence intensity of the fluorescence with a polarization component parallel to the excitation polarization direction when a fluorescent label and antibody are added to the sample and measured, and C(I ⊥ ) is the fluorescence intensity of fluorescence with a polarized component perpendicular to the excitation polarization direction when a fluorescent label and antibody are added to a sample and measured.

[0020] When the sample contains an autofluorescent substance, P calculated by formula (I) A is affected by the fluorescence emitted by autofluorescent substances and is therefore not suitable as data for creating a calibration curve. On the other hand, as shown in formula (II), the corrected fluorescence polarization index (P CB ) is suitable as data for creating a calibration curve because the effects of fluorescence emitted by autofluorescent substances have been eliminated. Examples of cases in which it is preferable to use the corrected fluorescence polarization calculated by formula (II) include when the sample to be measured is a beverage, food, etc. that contains autofluorescent substances such as certain vitamins, or when a specific enzymatic treatment has been performed to remove the target substance from the sample to be measured, as described below. In this way, in the method for creating a calibration curve of the present disclosure, the influence of fluorescence emitted by the autofluorescent substance in the sample is taken into consideration, and P calculated by formula (I) is A and P calculated by formula (II) CB It is preferable to use them appropriately. [Effects of the Invention]

[0021] The present disclosure provides a method for creating a more accurate calibration curve over a wide range from low to high concentration, a fluorescence polarization immunoassay using this calibration curve, a program, a fluorescence polarization immunoassay device, and a calibration curve creation kit. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing the configuration of a fluorescence polarization immunoassay device 1. [Figure 2] FIG. 2 is a schematic diagram of a microdevice 23. [Figure 3] FIG. 1 is a diagram illustrating preferred concentrations of samples for preparing a calibration curve. [Figure 4] FIG. 2 is a diagram showing the process of creating a calibration curve (steps 1a to 1b). [Figure 5] FIG. 1 is a diagram showing the process of creating a calibration curve (steps 1c to 1d). [Figure 6] FIG. 10 is a diagram showing the process of creating a calibration curve (Step 1e). [Figure 7] FIG. 10 is a diagram showing the process of additional measurement 1 (steps 2a to 2c). [Figure 8] FIG. 10 is a diagram showing the process of additional measurement 1 (step 2d). [Figure 9] FIG. 10 is a diagram showing the process of additional measurement 1 (steps 2e to 2f). [Figure 10] FIG. 10 is a diagram showing the process of additional measurement 2 (steps 3a to 3d). [Figure 11] FIG. 10 is a diagram showing the process of additional measurement 2 (steps 3e to 3g). [Figure 12] FIG. 10 is a diagram showing the process of additional measurement 2 (step 3h). [Figure 13] FIG. 2 is a diagram showing the process of creating a calibration curve (steps 1a to 1b). [Figure 14] FIG. 1 is a diagram showing the process of creating a calibration curve (steps 1c to 1d). [Figure 15] FIG. 10 is a diagram showing the process of creating a calibration curve (Step 1e). [Figure 16] 1 shows a calibration curve prepared in Example 1. [Figure 17] 1 is a calibration curve prepared in Example 2. [Figure 18] 1 is a calibration curve prepared in Example 6. [Figure 19] 1 is a calibration curve prepared in Example 7. [Figure 20] 1 is a calibration curve prepared in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0023] 1. How to create a calibration curve [Measurement sample] The calibration curve created by the present disclosure is the concentration C S (Hereinafter, "the concentration of the target substance C S "). Examples of samples to be measured include beverages, foods, cleaning fluids, tissue extracts, cell extracts, cell culture supernatants, blood, saliva, urine, and lymph.

[0024] [Target substance] The target substance is a substance whose concentration is measured using a calibration curve created by the present disclosure. The target substance is a compound from which an antibody can be prepared, with at least a portion of the target substance serving as an epitope. Examples of target substances include antigens and haptens, including proteins, glycoproteins, peptides, polypeptides, oligonucleotides, polynucleotides, antibodies, hormones, drugs, enzymes, and receptors. A hapten is a substance that binds to an antibody but does not exhibit immunogenicity, i.e., the activity of inducing antibody production, by itself due to its small molecular weight. Therefore, when the target substance is a hapten, the hapten can be bound to an immunogenic substance such as a protein to form a complete antigen with immunogenicity, as described below, so that the concentration of the hapten in the sample to be measured can be measured.

[0025] Examples of haptens include histamine, γ-aminobutyric acid (GABA), dopamine, thyroid hormone, and steroid hormone. Immunogenic substances include immunogenic proteins, polypeptides, carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, etc. Among these, proteins or polypeptides such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and thyroglobulin are preferred.

[0026] 〔antibody〕 The antibody used in the present disclosure is an antibody capable of binding to a substance of interest. That is, the antibody used in the present disclosure has the ability to recognize and bind to at least a portion of the substance of interest as an epitope. Examples of antibodies 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 Fvs ("scFvs"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs"), anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, and dual-variable domain antibodies.

[0027] When the target substance is a hapten, an antibody is usually used that uses a hapten derivative as an immunogen, in which an immunogenic substance is bound to the hapten via a linker. The linker is an atomic group introduced between the immunogenic substance and the hapten. Examples of linkers include groups having an acid amide bond, etc.

[0028] [Fluorescent Labeling Substances] A fluorescently labeled substance is a compound in which a target substance is labeled with a fluorescent dye. A fluorescent dye is a dye that emits fluorescence. Each fluorescent dye has its own unique fluorescence lifetime. In the present disclosure, a fluorescent dye with a fluorescence lifetime of 1 to 10 nanoseconds, a fluorescent dye with a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, or a fluorescent dye with a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds can be appropriately selected and used depending on the molecular weight of the target substance, etc. For example, fluorescent dyes having a fluorescence lifetime of 1 to 10 nanoseconds include indolenine; fluorescein compounds such as chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5-aminofluorescein, 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and trademarks such as the Alexa Fluor series (Alexa Fluor 488, etc.), the BODIPY series, the DY series, the ATTO series, the Dy Light series, the Oyster series, the HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, and 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 are examples of fluorescent dyes with a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds. Examples of fluorescent dyes with 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, pyrenebutanoic acid, and alkynylpyrene. Examples of fluorescent dyes with a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds include metal complexes containing platinum, rhenium, ruthenium, osmium, europium, and other metals.

[0029] Methods for labeling a target substance with a fluorescent dye include, for example, a method in which the fluorescent dye and the target substance are directly bonded to each other, and a method in which the fluorescent dye and the target substance are bonded via an appropriate linker such as oligoethylene glycol, an alkyl chain, etc. In these methods, substituents such as carboxyl groups, amino groups, hydroxyl groups, thiol groups, and phenyl groups contained in the fluorescent dye or the target substance can be used.

[0030] [Fluorescence polarization immunoassay] Fluorescence polarization immunoassay utilizes the competitive reaction of substances and the change in fluorescence polarization caused by changes in the molecular weight of the competing substances. When plane-polarized light is irradiated onto fluorescent molecules in a liquid, if the fluorescent molecules are not moving much, they emit polarized fluorescence in the same plane as the excitation plane. On the other hand, if the fluorescent molecules rotate due to Brownian motion while excited, they emit polarized fluorescence in a plane other than the excitation plane, resulting in a decrease in overall fluorescence polarization. Thus, fluorescence polarization is a physical property related to the degree of rotation of a fluorescent molecule between excitation and emission of fluorescence. The size (molecular weight) of the fluorescent molecule affects the degree of rotation. For example, low-molecular-weight molecules such as free fluorescently labeled substances rotate vigorously in solution due to Brownian motion, while high-molecular-weight molecules such as fluorescently labeled substances bound to antibodies do not. Therefore, measuring fluorescence polarization can provide information on changes in the molecular weight of fluorescently labeled substances in solution. Furthermore, the following method can be used to determine the concentration C of a target substance in a sample. S can be measured.

[0031] In a solution containing a mixture of target substance A, antibody B capable of binding to target substance A, and fluorescently labeled substance C, which is target substance A labeled with a fluorescent dye, target substance A, antibody B, and fluorescently labeled substance C compete with each other in the solution, so a high concentration of target substance A increases the amount of complex between target substance A and antibody B, resulting in an increase in the amount of free fluorescently labeled substance C. In this way, the concentration of target substance A affects the amount of free fluorescently labeled substance C (or fluorescently labeled substance C bound to antibody B), so the concentration of target substance A can be measured by creating a calibration curve showing the relationship between the concentration of target substance A and the degree of fluorescence polarization.

[0032] [Fluorescence polarization immunoassay device] In the method for creating a calibration curve of the present disclosure, the degree of fluorescence polarization of various samples is measured. Although there are no particular limitations on the measuring device or measuring instrument used in measuring the degree of fluorescence polarization, the degree of fluorescence polarization can be measured efficiently by using a microdevice.

[0033] An example of a fluorescence polarization immunoassay device using a microdevice is shown in Figure 1. The fluorescence polarization immunoassay device 1 includes a light source 10, a condenser lens 11, an iris 12, a collimator 13, a polarizing element 14, an excitation light 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 image sensor 28, and a control unit 30.

[0034] Light source 10 is, for example, a light-emitting diode, and emits excitation light of a wavelength that excites fluorescence from the sample (for example, blue light with a central wavelength of 470 nm). The excitation light from light source 10 is collected by collecting lens 11 and passes through iris 12. Iris 12 reduces the intrusion of external light other than the excitation light.

[0035] The excitation light that passes through the iris 12 is converted into parallel light by the collimator 13 and enters the polarizing element 14. The polarizing element 14 is, for example, a polarizing plate, a polarizing beam splitter, or a liquid crystal cell, and is a polarizing plate in this example. The polarizing element 14 passes linearly polarized light in a specific direction. The linearly polarized excitation light from the polarizing element 14 passes through the excitation light filter 15. The excitation light filter 15 is a filter that selects a wavelength range that includes the wavelength of the excitation light, and reduces light with wavelengths different from that of the excitation light from the polarizing element 14. The dichroic mirror 20 reflects the excitation light that has passed through the excitation light filter 15 toward the objective lens 21.

[0036] The objective lens 21 focuses the linearly polarized excitation light reflected by the dichroic mirror 20 onto the sample 22 housed in the microdevice 23 on the stage 24. The sample 22 generates fluorescence of a specific wavelength (e.g., green light) in response to the linearly polarized excitation light from the objective lens 21. The fluorescence is collimated by the objective lens 21 and passes through the dichroic mirror 20 and the absorption filter 25. The dichroic mirror 20 selectively passes light in a specific wavelength range, including the fluorescence from the sample 22, and reflects other light. The absorption filter 25 is a filter that selects a wavelength range that includes the wavelength of the fluorescence from the sample 22, and reduces light other than the fluorescence.

[0037] The fluorescence that has passed through the absorption filter 25 is incident on the polarization adjustment element 26. The polarization adjustment element 26 is, for example, a polarizing plate, a polarizing beam splitter, or a liquid crystal cell. The polarization adjustment element 26 may also be a polarizing filter in a polarization camera. A polarization camera is an imaging device that acquires polarization information of a subject by mounting a polarization filter on a sensor. In the following description, the polarization adjustment element 26 is a liquid crystal cell controlled by a drive signal (applied voltage). The polarization adjustment element 26 can adjust the transmitted light intensity of linearly polarized light components. Specifically, the polarization adjustment element 26 can adjust the transmitted light intensity of linearly polarized light parallel to or perpendicular to the polarization direction of the excitation light, as well as polarized light in a direction corresponding to a drive signal, which will be described later.

[0038] The linearly polarized fluorescence that has passed through the polarization adjustment element 26 is incident on the imaging surface of the image sensor 28 via the imaging lens 27. There is an imaging relationship between the surface of the sample 22 and the imaging surface of the image sensor 28. The image sensor 28 includes, for example, a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) image sensor having a plurality of pixels. The image sensor 28 generates image data according to the intensity of the fluorescence generated by the sample 22 and transmits the image data to the control unit 30.

[0039] The control unit 30 comprehensively controls the entire fluorescence polarization immunoassay device 1. Specifically, the control unit 30 controls the light source 10, the polarization adjustment element 26, and the image sensor 28. The control unit 30 acquires a fluorescent image captured by the image sensor 28.

[0040] For example, during measurement operation, the control unit 30 causes the light source 10 to emit excitation light to the sample 22. The control unit 30 uses a DA converter (not shown) to output a drive signal to the polarization adjusting element 26. By outputting the drive signal to the polarization adjusting element 26, the control unit 30 can control the polarization component of the fluorescence passing through the polarization adjusting element 26.

[0041] The polarization adjustment element 26 includes, for example, two opposing transparent substrates, transparent electrodes arranged on the opposing surfaces of the substrates, a liquid crystal material sealed between the substrates, and a polarizing plate arranged on the outer surface on the imaging device side (exit side or downstream side) of the polarization adjustment element 26. The polarization adjustment element 26 may be configured in any way as long as it can adjust the polarization component of the fluorescence passing through it.

[0042] The components of the above-described fluorescence polarization immunoassay device 1 are summarized as follows. (A) An illumination optical system 10A that irradiates a sample 22 with linearly polarized excitation light, the illumination optical system 10A including a light source 10, a condenser lens 11, an iris 12, a collimator 13, a polarizing element 14, and an excitation light filter 15. (B) A microdevice 23 that accommodates the sample 22, and a stage 24 that mounts the microdevice 23. (C) An observation optical system 10B including a polarization adjustment element 26 that adjusts the linearly polarized component of the fluorescence emitted from the sample 22 that is transmitted in response to a drive signal from the control unit 30, and an image pickup element 28 that captures a fluorescence image that has transmitted through the polarization adjustment element 26. (D) A control unit 30 that functions as a drive unit that outputs a drive signal to the polarization adjustment element 26, detects the fluorescence intensity based on the fluorescence image captured by the image capture element 28, measures the polarization degree of the sample 22 in accordance with the drive signal, and analyzes the sample.

[0043] As shown in FIG. 2, the microdevice 23 includes a plurality of flow paths 23c, each connected at one end to an inlet 23a and at the other end to an outlet 23b. Measurement liquids containing a calibration curve preparation sample or a measurement target sample can be individually supplied to the plurality of flow paths 23c. Because the flow paths 23c and the imaging surface of the imaging element 28 are in an imaging relationship, capturing images of the plurality of flow paths 23c with the imaging element 28 simultaneously obtains fluorescence images of the plurality of samples 22 supplied to the plurality of flow paths 23c, allowing the polarization degree of each sample 22 to be simultaneously measured. This measurement is performed based on the fluorescence intensity of a region of interest (ROI) in the image data corresponding to the flow path 23c in the fluorescence image.

[0044] [How to create a calibration curve] The method for creating a calibration curve according to the present disclosure includes: 1. A method for preparing a calibration curve for a fluorescence polarization immunoassay using an antibody capable of binding to a target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, comprising: Step 1a, providing a first reference sample that does not contain the target substance; After step 1a, step 1b is performed by 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. The target substance is added at a concentration of C Sα p measurement target samples α1 to αp, where p is an integer equal to or greater than 3, are taken from a measurement target sample α containing Sα More than 1000 samples for creating calibration curves conc Step 1c of preparing p calibration curve samples, After step 1c, the antibody and the fluorescent labeling substance are added to each of the p calibration curve preparation samples in the same amounts as in step 1b, and the fluorescence polarization indices P1 to P p Step 1d of measuring respectively After step 1b and step 1d, the fluorescence polarization degree P0 and the number of samples for preparing a calibration curve among the p samples for preparing a calibration curve are conc Fluorescence polarization P conc Step 1e., based on the above, creating a calibration curve showing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample; It has.

[0045] Step 1a is a step of preparing a first reference sample that does not contain the target substance. The low concentration region of the calibration curve prepared in the present disclosure is prepared mainly based on the fluorescence polarization degree P0 of the first reference sample. The first reference sample may be a solution that does not contain the target substance from the beginning, such as pure water or physiological saline, or a solution from which the target substance has been removed from the measurement sample α. Methods for removing a target substance from a measurement target sample α include adding an enzyme that uses the target substance as a substrate to the measurement target sample α, or contacting the measurement target sample α with an adsorbent that adsorbs the target substance.

[0046] As the enzyme, if the target substance is histamine, histamine N-methyltransferase, diamine oxidase, etc. can be used; if the target substance is chloramphenicol, chloramphenicol acetyltransferase, hydrolase, nitroreductase, etc. can be used; if the target substance is deoxynivalenol, mycotoxin-degrading enzymes, etc. can be used. When the first reference sample is a solution obtained by adding an enzyme to the measurement sample α to remove the target substance, the first reference sample may contain autofluorescent substances. Therefore, in this case, the corrected fluorescence polarization degree calculated by the above formula (II) is preferable.

[0047] Examples of adsorbents that can be used include bentonite, activated carbon, diatomaceous earth, ion exchange adsorbents, and affinity adsorbents. An ion exchange adsorbent is an adsorbent that uses an ion exchange resin or the like as an adsorbent component. Examples of ion exchange adsorbents include cation exchange adsorbents that adsorb cationic compounds and anion exchange adsorbents that adsorb anionic compounds. When using an ion exchange adsorbent as an adsorbent, the target substance can be efficiently removed from the target sample α by selecting an appropriate ion exchange adsorbent depending on the properties of the target substance and further adjusting the pH of the target sample α and the pH of the cleaning solution. Affinity adsorbents are adsorbents that utilize biospecific interactions such as enzyme / substrate, antibody / antigen, hormone / receptor protein, etc. Affinity adsorbents are particularly preferred as a means of removing target substances from target sample α because they can selectively remove target substances and, compared to enzyme treatment, are less likely to adversely affect target sample α. The method for using the adsorbent is not particularly limited. For example, the target substance in the sample α can be removed by filling a column with the adsorbent and passing the sample α through the column, or by adding the adsorbent to the sample α and then filtering off the adsorbent.

[0048] Step 1b is a step following step 1a in which an antibody and a fluorescent labeling substance are added to the first reference sample and the fluorescence polarization index P0 of the first reference sample is measured. The measurement of the fluorescence polarization index P0 can be performed using, for example, the fluorescence polarization immunoassay device 1.

[0049] As described above, the first reference sample does not contain the target substance, but a sample to which a trace amount of the target substance has been added may be used instead of the first reference sample to measure the degree of fluorescence polarization, as long as this does not affect the results. In other words, the "fluorescence polarization degree P0 of the first reference sample" in this disclosure includes the "fluorescence polarization degree of the first reference sample to which a trace amount of the target substance has been added." As will be described later, the calibration curve for the antigen-antibody reaction in the present disclosure is created as an inverted sigmoid curve, for example, as shown in Figure 3, so that when the antigen concentration (concentration of the target substance) is lower than a certain concentration, the fluorescence polarization index changes very little. Thus, in the present disclosure, the "first reference sample" can be replaced with a "first reference sample to which a trace amount of the target substance has been added," or a "second reference sample" (described later) containing a trace amount of the target substance, as long as the concentration is within a range in which no significant change in the fluorescence polarization index occurs. Similarly, if the first reference sample is a solution from which the target substance in the measurement sample α has been removed, it may contain a trace amount of the target substance that was not completely removed, as long as it is within a concentration range that does not cause a significant change in the degree of fluorescence polarization.

[0050] Step 1c is a step of preparing p samples for creating a calibration curve. The p samples for creating a calibration curve are prepared by taking p measurement target samples α1 to αp from the measurement target sample α, and adding different amounts of the target substance to each of these measurement target samples α1 to αp. The number of samples (p) for preparing a calibration curve is an integer of 3 or more, for example, an integer from 3 to 20, an integer from 3 to 15, or an integer from 3 to 10. The p samples for creating the calibration curve are added at a concentration of C Sα More than 1000 samples for creating calibration curves conc The p samples for preparing the calibration curve are prepared so that the concentration of the added target substance is equal to the concentration C Sα The following samples for creating a calibration curve: dil It may or may not include.

[0051] The medium to high concentration range of the calibration curve prepared in this disclosure is mainly determined by the sample for preparing the calibration curve. conc Fluorescence polarization P conc It is created based on the In other words, since the sample for creating the calibration curve is prepared based on the measurement target sample α, by using the sample for creating the calibration curve, it is possible to create a calibration curve that takes into account the influence of impurities in the measurement target sample α. However, the measurement sample α originally contained the target substance at a concentration of C Sα Therefore, the low-concentration calibration curve preparation sample (i.e., the calibration curve preparation sample dil ) is not suitable for deriving the relationship between the amount of target substance added later and the fluorescence polarization of the sample. On the other hand, when the amount of the target substance added later is in excess of the amount of the original target substance, the influence of the original target substance is small. Therefore, in the present disclosure, among the samples for preparing a calibration curve, the sample for preparing a calibration curve conc Use only.

[0052] As described above, in the method for creating a calibration curve of the present disclosure, conc is an essential sample, and is a sample for creating a calibration curve. dil is an arbitrary sample. Sample for creating a calibration curve conc The number is, for example, an integer from 2 to 20, an integer from 2 to 15, or an integer from 2 to 10. Sample for creating a calibration curve dil The number is, for example, an integer from 0 to 18, an integer from 0 to 13, or an integer from 0 to 8.

[0053] When performing step 1c, the concentration of the target substance, C Sα If there is no information about the concentration of the sample, it is preferable to prepare samples for preparing a calibration curve over a wide range from a low concentration region to a high concentration region in step 1c. conc Along with samples for creating a calibration curve dil is prepared. In this case, the concentration of the most concentrated sample for creating a calibration curve is, for example, 10 times the concentration of the most diluted sample for creating a calibration curve. 6 ~10 10 It's double.

[0054] As will be described later, a calibration curve is usually created as an inverse sigmoid curve by regressing experimental data using a four-parameter logistic model. To create a more accurate calibration curve, for example, in the case of the calibration curve shown in Figure 3, it is preferable to use a sample with a concentration that shows a polarization degree near d, a sample with a concentration that shows a polarization degree near (a+b) / 2, and a sample with a concentration that shows a polarization degree near a. Specific examples of "near a polarization degree d," "near a polarization degree (a+b) / 2," and "near a polarization degree a" include d±[(ad) / 5], [(a+b) / 2]±[(ad)×3 / 5], and a±[(ad) / 5], respectively, with d±[(ad) / 10], [(a+b) / 2]±[(ad) / 5], and a±[(ad) / 10] being preferred.

[0055] On the other hand, when performing step 1c, the concentration of the target substance C Sα If you have information about the sample for creating the calibration curve, conc Only the concentration of the target substance, C Sα For example, when analyzing to confirm the ingredients of manufactured foods, beverages, etc., when there is known information about the measurement target sample α, such as when there is measurement results for the same type of sample, or when there is a provisional concentration C of the target substance in the measurement target sample α by additional measurement, which will be described later. Pα An example is when the following is derived:

[0056] In step 1d, after step 1c, an antibody and a fluorescently labeled substance are added to each of the p samples for preparing a calibration curve, and the fluorescence polarization indices P1 to P2 are measured. p This is a step of measuring each of the above. The amounts of antibody and fluorescent labeling substance added are the same as those added to the first reference sample in step 1b.

[0057] Step 1e is a step of calculating the fluorescence polarization degree P0 of the first reference sample and the fluorescence polarization degree P1 of the calibration curve preparation samples among the p calibration curve preparation samples after Step 1b and Step 1d. conc Fluorescence polarization P concThis is a step of creating a calibration curve that shows the relationship between the amount of the target substance and the degree of fluorescence polarization of the sample based on the above.

[0058] Step 1e can be carried out, for example, by the following method. First, select several samples from the p number of samples for creating a calibration curve in order of concentration, and create a calibration curve based on the fluorescence polarization index of these samples and the fluorescence polarization index P0 of the first reference sample. Next, measure the fluorescence polarization index of the sample α to be measured, and based on the obtained fluorescence polarization index, determine the concentration C of the target substance from the calibration curve. Sα The concentration of the target substance, C Sα By comparing the concentration of each calibration sample with that of the calibration sample, the calibration sample used can be dil It can be determined whether or not it is included. The sample used for creating the calibration curve was dil When the sample contains a low concentration of the compound, the desired calibration curve can be created by repeatedly excluding the low concentration samples from the calibration curve creation samples and creating a new calibration curve. On the other hand, the sample used for creating the calibration curve was dil If the sample does not contain the analyte, a new calibration curve may be prepared by adding some of the remaining samples for preparing the calibration curve in order of increasing concentration.

[0059] In addition, before or during the calibration curve creation, the concentration C of the target substance is calculated as follows: Sα If information on this can be obtained, calibration curves can be created more efficiently. For example, in step 1c, the sample for creating the calibration curve conc Along with samples for creating a calibration curve dil When preparing the target substance, the concentration C Sα Based on this information, the sample for creating the calibration curve conc After selecting only the fluorescence polarization P0 obtained in step 1b, the selected sample for creating the calibration curve was conc Fluorescence polarization P conc A calibration curve can be created based on the above. The concentration of the target substance used at this time is C Sα The information about the target substance in the target sample α is given by the above-mentioned known information and the provisional concentration C of the target substance in the target sample α, which is derived by the additional measurement described later. Pα Examples include: On the other hand, in step 1c, the sample for creating the calibration curve conc If only the fluorescence polarization index P0 obtained in step 1b is prepared, the fluorescence polarization index P0 obtained in step 1d is conc A calibration curve can be created based on the above.

[0060] The "amount of target substance" in the "relationship between the amount of target substance and the fluorescence polarization of the sample" in the calibration curve can be selected appropriately depending on the intended use of the calibration curve. Typically, the "amount of target substance" is the concentration of the target substance added to the sample used to create the calibration curve (the target substance added to create the calibration curve). The calibration curve is usually constructed as an inverse sigmoid curve by regressing the experimental data with a four-parameter logistic model. The above items regarding the "relationship between the amount of target substance and the fluorescence polarization of the sample" and the method for creating a calibration curve (e.g., the preferred concentration of the sample shown in Figure 3, and the method for creating an inverse sigmoid curve by regressing experimental data with a four-parameter logistic model) can be used when deriving the first and second approximation curves described below.

[0061] [First embodiment] The process of creating a calibration curve through steps 1a to 1e is shown in Figures 4 to 6. However, the method of creating a calibration curve of the present disclosure is not limited to the order of steps shown in Figures 4 to 6, and the order of steps may be changed as long as it is feasible. The method for creating the calibration curve shown in Figures 4 to 6 involves the use of multiple calibration curve creation samples in step 1e. conc Only the selected compounds are selected and then a calibration curve is created.

[0062] Figure 4 shows the state after steps 1a-1b. On the y-axis, the fluorescence polarization value P0 of the first reference sample is plotted. FIG. 5 shows the state after steps 1c to 1d. In step 1c, the concentration C Sα The fluorescence polarization indices P1 to P9 of samples for creating a calibration curve over a wide range of concentrations, from low to high, which were prepared under unknown conditions, are shown. Figure 6 shows the state after step 1e. Sα Samples for creating a calibration curve were selected based on information about conc Fluorescence polarization P conc and the fluorescence polarization degree P0 of the first reference sample, a calibration curve is created that shows the relationship between the concentration of the target substance in the sample and the fluorescence polarization degree of the sample.

[0063] The concentration C of the target substance used in step 1e Sα For example, the information about the concentration C of the target substance in the measurement sample α, which is derived in the following additional measurement 1, is Pα(1) (Hereinafter referred to as "provisional concentration C of the target substance" Pα(1) ") and the tentative concentration C of the target substance in the measurement sample α derived in additional measurement 2. Pα(2) (Hereinafter referred to as "provisional concentration C of the target substance" Pα(2) " is sometimes abbreviated as ".) For convenience of explanation, the operations that overlap with steps 1a to 1e will be described again, but in actual measurements, there are cases where operations that have already been performed can be omitted.

[0064] (Additional measurement 1) Step 2a. providing a second reference sample that does not contain the target substance. After step 2a, step 2b is performed in which q second reference samples 1 to q, where q is an integer equal to or greater than 3, are taken from the second reference sample, and different amounts of the target substance are added to the second reference samples 1 to q, respectively, to prepare q additional measurement samples A. After step 2b, the antibody and the fluorescent labeling substance are added to each of the q additional measurement samples A, and the fluorescence polarization index P A1 ~P Aq Measure each of the steps in step 2c. After step 2c, the fluorescence polarization index P A1 ~P Aq and 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 obtained result. The antibody and the fluorescent labeling substance are added to the measurement sample α in the same amounts as in step 2c, and the fluorescence polarization index P Xα Measure step 2e After step 2d and step 2e, the fluorescence polarization index P Xα Based on the above, the tentative concentration C of the target substance in the measurement sample α is calculated from the first approximation curve. Pα(1) Step 2f to derive

[0065] 7 to 9, the first approximation curve and the provisional concentration C of the target substance are calculated by steps 2a to 2f. Pα(1) However, the method for creating a calibration curve of the present disclosure is not limited to the order of steps shown in Figures 7 to 9, and the order of steps may be changed as long as it is feasible.

[0066] Step 2a is a step of preparing a second reference sample that does not contain the target substance, and is similar to step 1a. However, since the second reference sample is used to prepare additional measurement sample A, if the target substance is removed by adding an enzyme, the enzyme activity 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 a solution in which the target substance has been removed from the measurement target sample α using an adsorbent. As with the first reference sample, if the second reference sample is a solution in which the target substance has been removed from the measurement target sample α, it may contain trace amounts of the target substance that were not completely removed, as long as there is no significant change in the fluorescence polarization. Step 2b is a step of preparing q additional measurement samples A. The q additional measurement samples A are prepared by taking q second reference samples 1 to q from the second reference sample and adding different amounts of the target substance to each of these second reference samples 1 to q. The number (q) of additional measurement samples A is an integer of 3 or more, for example, an integer from 3 to 20, an integer from 3 to 15, or an integer from 3 to 10. In step 2b, it is preferable to prepare additional measurement samples A in a wide range of concentrations, from low to high. The preferable sample concentration range is as explained for the samples for preparing a calibration curve. Step 2c is to add an antibody and a fluorescently labeled substance to each of the q additional measurement samples A, and measure their fluorescence polarization indices P A1 ~P Aq The amounts of antibody and fluorescent labeling substance added may be the same as or different from the amounts of antibody and fluorescent labeling substance added to the first reference sample in step 1b. In step 2c, by using the same amount of antibody and fluorescent labeling substance as in step 1b, it may be possible to omit duplicate measurements. In step 2c, by using different amounts of antibody and fluorescent labeling substance than in step 1b, it is possible to reduce the tentative concentration C Pα(1) In some cases, a more suitable first approximation curve can be derived by deriving Figure 7 shows the state after steps 2a to 2c. A1 ~P A9 is written.

[0067] Step 2d is the fluorescence polarization P A1 ~P Aq This is a step of deriving a first approximation curve that represents the relationship between the amount of the target substance and the degree of fluorescence polarization of the sample based on the above. Figure 8 shows the state after step 2d. The fluorescence polarization index P of sample A for additional measurement A1 ~P A9 and the fluorescence polarization P A1 ~P A9 The first approximation curve derived based on the above is shown.

[0068] In step 2e, an antibody and a fluorescent labeling substance are added to the measurement target sample α, and the fluorescence polarization degree P XαThe amounts of antibody and fluorescent labeling substance added are the same as those added to additional measurement sample A in step 2c. Step 2f is the fluorescence polarization degree P Xα Based on this, the tentative concentration C of the target substance is calculated from the first approximation curve. Pα(1) This is the step of deriving 9 shows the state after steps 2e to 2f. Xα is plotted on the y-axis, and the corresponding hypothetical concentration of the target substance, C Pα(1) is marked on the x-axis.

[0069] (Additional measurement 2) Step 3a. providing a second reference sample that does not contain the target substance. After step 3a, step 3b is performed in which q second reference samples 1 to q, where q is an integer equal to or greater than 3, are taken from the second reference sample, and different amounts of the target substance are added to the second reference samples 1 to q, respectively, to prepare q additional measurement samples A. After step 3b, the antibody and the fluorescent labeling substance are added to each of the q additional measurement samples A, and the fluorescence polarization index P A1 ~P Aq Measure each of the following in step 3c After step 3c, the fluorescence polarization index P A1 ~P Aq and 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 obtained result. Step 3e of separating r measurement samples α1 to αr, where r is an integer equal to or greater than 3, from the measurement sample α, and adding different amounts of the target substance to each of the measurement samples α1 to αr to prepare r additional measurement samples B. After step 3e, the same amounts of the antibody and the fluorescent labeling substance as in step 3c are added to each of the r additional measurement samples B, and the fluorescence polarization indices P B1 ~P Br Step 3f: Measure each After step 3f, the fluorescence polarization index P B1 ~P Br and deriving a second approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample based on the above. After steps 3d and 3g, a tentative concentration C of the target substance in the measurement sample α is calculated from the first approximation curve based on the maximum value of the degree of fluorescence polarization in the second approximation curve. Pα(2) Step 3h to derive

[0070] 10 to 12, the first approximation curve, the second approximation curve, and the provisional concentration C of the target substance are calculated by steps 3a to 3h. Pα(2) However, the method for creating a calibration curve of the present disclosure is not limited to the order of steps shown in Figures 10 to 12, and the order of steps may be changed as long as it is feasible.

[0071] Steps 3a to 3d are the same as steps 2a to 2d. 10 shows the state after steps 3a to 3d. A1 ~P A9 and the fluorescence polarization P A1 ~P A9 The first approximation curve derived based on the above is shown.

[0072] Step 3e is a step of preparing r additional measurement samples B, and step 3f is a step of measuring their fluorescence polarization indices P B1 ~P Br The additional measurement sample B is prepared from the measurement target sample α, just like the sample for creating the calibration curve. Therefore, steps 3e to 3f are steps for measuring the concentration C of the target substance in steps 1c to 1d. Sα p samples for creating a calibration curve are prepared without information about the fluorescence polarization indices P1 to P2. pTherefore, if the same amounts of antibody and fluorescent labeling substance are used in step 3c as in step 1b, the measurement results in step 1d may be used as the measurement results in step 3f. In step 3g, the fluorescence polarization P of sample B for additional measurement B1 ~P Br Based on this, a second approximation curve is derived that represents the relationship between the amount of the target substance and the fluorescence polarization degree of the sample. 11 shows the state after steps 3e to 3g. B1 ~P B9 and the fluorescence polarization P B1 ~P B9 A second approximation curve derived based on the

[0073] Step 3h is to calculate the tentative concentration C of the target substance from the first approximation curve based on the maximum value of the fluorescence polarization in the second approximation curve. Pα(2) This is the step of deriving The degree of fluorescence polarization when the concentration of the added target substance is 0 on the second approximation curve represents the degree of fluorescence polarization of the sample α to be measured. Therefore, the maximum value of the degree of fluorescence polarization on the second approximation curve (the value at the intersection of the second approximation curve and the y-axis) is read, and based on this value, the tentative concentration C of the target substance is calculated from the first approximation curve. Pα(2) can be derived. Figure 12 shows the state after step 3h. The intersection of the second approximation curve and the y-axis is plotted, and the corresponding tentative concentration C of the target substance is Pα(2) is marked on the x-axis.

[0074] Other Embodiments When performing step 1c, the concentration of the target substance, C Sα When information on the above is available, a calibration curve can be created by a more simplified method as shown in FIGS. The method for creating the calibration curve shown in FIGS. 13 to 15 involves the steps of: conc Only the above is prepared.

[0075] FIG. 13 shows the state after steps 1a and 1b. The fluorescence polarization degree P0 of the first reference sample is plotted on the y-axis. Also, the concentration C of the target substance is known as known information of the measurement target sample α. Sα is approximately 1 μg / mL, which is shown on the x-axis. Figure 14 shows the state after steps 1c and 1d. Sα Calibration samples prepared based on information on conc Fluorescence polarization P conc is written. 15 shows the state after step 1e. The fluorescence polarization degree P0 of the first reference sample and the sample for preparing the calibration curve conc Fluorescence polarization P conc Based on this, a calibration curve is created that shows the relationship between the concentration of the target substance in the sample and the fluorescence polarization degree of the sample.

[0076] As described above, the calibration curve prepared by the present disclosure is made by dividing the sample for the low concentration range (first reference sample) and the sample for the medium to high concentration range (sample for preparing the calibration curve). conc ) and is more accurate over a wide range from low to high concentration regions.

[0077] 2. Fluorescence Polarization Immunoassay A fluorescence polarization immunoassay according to a second aspect of the present disclosure includes: Using an antibody capable of binding to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is measured. Sβ A fluorescence polarization immunoassay for measuring The antibody and the fluorescent labeling substance are added to the measurement sample β in the same amounts as in step 1b, and the fluorescence polarization index P Xβ Step 4a measuring After step 4a, the fluorescence polarization index P Xβ Based on the above, the concentration C of the target substance is determined from the calibration curve created by the above method. Sβ Step 4b to derive It has.

[0078] That is, in the fluorescence polarization immunoassay method of the present disclosure, the fluorescence polarization degree P Xβ is measured, and the concentration C of the target substance is determined from the calibration curve prepared by the above method. Sβ It measures:

[0079] The measurement sample β is preferably the same as the measurement sample α or the same type as the measurement sample α. In the case where the measurement sample β is the same as the measurement sample α, for example, after preparing a calibration curve by the above-mentioned method, the fluorescence polarization index is measured using the remaining measurement sample α used to prepare the calibration curve, and its concentration is derived. Also, in the additional measurement 1 of step 1c or step 1e when preparing the calibration curve, the same amount of antibody and fluorescent labeling substance as in step 1b are used to measure the fluorescence polarization index P of the measurement sample α. Xα If you are measuring, step 4a has already been performed, so the concentration C of the target substance is calculated at the same time as the calibration curve is completed. Sα is derived.

[0080] An example of a case where the measurement target sample β is of the same type as the measurement target sample α is when analyzing the components of a product from a different lot that was manufactured by the same or a similar method in a factory or the like.

[0081] 3. Program A program according to a third aspect of the present disclosure includes: A program used in a fluorescence polarization immunoassay according to a second aspect of the present disclosure, comprising: The fluorescence polarization degree P of the measurement sample β Xβ Based on the above, the concentration C of the target substance is determined from the calibration curve created by the above method. Sβ The method causes the computer to execute a step of deriving

[0082] A program according to a third aspect of the present disclosure is executed by a computer. The computer includes, for example, a processor, a memory, and an input / output unit. The processor is composed of a CPU (Central Processing Unit) or the like, and executes a program stored in the memory. The memory is an example of a storage medium, and stores the program according to the third aspect and a calibration curve obtained by the calibration curve creation method according to the first aspect. This program causes the processor to execute calculations related to the fluorescence polarization immunoassay according to the second aspect. The input / output unit stores the fluorescence polarization index P required for calculations related to the fluorescence polarization immunoassay according to the second aspect. xβ The input / output section receives the result of the calculation, i.e., the concentration C sβ The processor outputs, for example, by displaying the fluorescence polarization index P xβ Calculation related to the fluorescence polarization immunoassay method according to the second aspect is performed on the sβ is derived and output from the input / output section.

[0083] The program of the present disclosure is preferably used when the measurement target sample β is of the same type as the measurement target sample α. In other words, when analyzing the components of a product manufactured in a factory, etc., by first creating an accurate calibration curve, the calibration curve can be used when analyzing the components of a different lot of product manufactured by the same or a similar method. Therefore, by using the program disclosed herein, product component analysis can be performed efficiently.

[0084] 4. Fluorescence Polarization Immunoassay Device A fluorescence polarization immunoassay device according to a fourth aspect of the present disclosure, Using an antibody capable of binding to a target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is determined. Sβ A fluorescence polarization immunoassay device for measuring an illumination optical system that illuminates the sample with linearly polarized excitation light; a polarization adjusting element that selectively passes a linearly polarized component of the fluorescence emitted from the sample in accordance with the drive signal; a light receiving unit that detects the intensity of the fluorescence that has passed through the polarization adjusting element; a control unit that outputs the drive signal to the polarization adjustment element and measures the degree of polarization of the sample in accordance with the drive signal based on the fluorescence intensity detected by the light receiving unit; Equipped with The control unit executes a program according to the third aspect of the present disclosure to determine the concentration C Sβ This is to derive the following.

[0085] The illumination optical system, polarization adjusting element, and light receiving section of the fluorescence polarization immunoassay device of the present disclosure are the same as those of the fluorescence polarization immunoassay device 1 described above. The control unit of the fluorescence polarization immunoassay device of the present disclosure includes, for example, a processor, a memory, and an input / output unit. The control unit of the fluorescence polarization immunoassay device of the present disclosure can execute the program of the present disclosure, and therefore the concentration C of the target substance can be measured by the fluorescence polarization immunoassay device of the present disclosure. Sβ can be measured efficiently.

[0086] 5. Calibration curve creation kit A calibration curve creation kit according to a fifth aspect of the present disclosure includes: The device comprises an antibody capable of binding to a target substance and a fluorescently labeled substance in which the target substance is labeled with a fluorescent dye, and further comprises a means for removing the target substance from a solution containing the target substance and / or a liquid that does not contain the target substance.

[0087] The antibody capable of binding to the target substance and the fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye are those described in the disclosure of the method for creating a calibration curve. These may be in a solution state or in a dry state. For example, they are stored in bags, bottles, ampoules, etc.

[0088] Examples of means for removing a target substance from a solution containing the target substance include enzymes, adsorbents, etc. These are explained in the disclosure of the method for creating a calibration curve. Examples of liquids that do not contain the target substance include pure water, physiological saline, and buffer solutions.

[0089] The calibration curve preparation kit of the present disclosure preferably contains just the right amount of antibody and fluorescent labeling substance depending on the number of times it is intended to be used. The calibration curve preparation kit of the present disclosure may further include a microdevice. The calibration curve preparation kit of the present disclosure is preferably used when carrying out the calibration curve preparation method of the present disclosure described above. [Example]

[0090] 〔reagent〕 (1) Antibody solution Antihistamine antibody (Progen Biotechnik) was diluted with phosphate-buffered saline (PBS(-)) containing 0.01% bovine serum albumin (BSA) to give a concentration of 1.3 × 10 -7 An antibody solution of M was prepared. (2) Histamine tracer solution Histamine (Fujifilm Wako Pure Chemical Industries, Ltd.) was modified with HiLyte Fluor 647 to obtain a histamine tracer. This was dissolved in pure water and further diluted with PBS(-) to obtain a 4.56 × 10 -9 A histamine tracer solution of M was prepared. (3) Target substance solution Histamine dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare a 32 mg / ml histamine solution, which was then diluted with pure water to prepare histamine solutions with the following concentrations (#1 to #9). #1: 2.88×10 -9 M (0.00032 μg / mL) #2: 2.88×10 -8 M (0.0032 μg / mL) #3: 2.88×10 -7 M (0.032 μg / mL) #4: 2.88×10 -6 M (0.32 μg / mL) #5: 2.88×10 -5 M (3.2 μg / mL) #6: 0.000288M (32μg / mL) #7: 0.002879M (320μg / mL) #8: 0.02879M (3,200μg / mL) #9: 0.287899M (32,000μg / mL) (4) Reference sample (e) A histamine-degrading enzyme was added to soy sauce to prepare a reference sample (e). The histamine-degrading enzyme reagent was a histamine measurement kit (Check Color Histamine (Kikkoman Biochemifa)) according to the kit's protocol. (5) Reference sample (w) Pure water was used as the reference sample (w). (6) Reference sample (f) Soy sauce was diluted with 20 mM phosphate buffer (pH 6.0) to prepare a soy sauce dilution. This soy sauce dilution was passed through a column (Sep-Pak Plus Accell CM, Waters). Next, a wash solution (20 mM phosphate buffer (pH 6.0)) was passed through the column. The soy sauce dilution that had passed through the column and the wash solution were combined and used as the reference sample (f). (7) Acylation reagents, acylation buffers The acylation reagent and acylation buffer used were reagents from Histamine Acylation Set (manufactured by Beckman Coulter).

[0091] Example 1 The concentration of histamine (target substance) contained in soy sauce (measurement sample) was measured by the following method. Example 1 is an experimental example in which there is no known information about the histamine concentration in soy sauce. Samples for creating a calibration curve conc and a sample for creating a calibration curve dil After measuring the fluorescence polarization of these samples, the calibration curve was prepared. concwere selected to create a calibration curve. The fluorescence polarization degree in Example 1 is a corrected fluorescence polarization degree calculated by the above formula (II).

[0092] 160 μl of PBS(-) was added to 40 μl of reference sample (e) to obtain a 5-fold diluted solution. 100 μl of the resulting 5-fold diluted solution was mixed with 25 μl of acylation reagent and 200 μl of acylation buffer to obtain a mixture. 25 μl of this mixture, 25 μl of histamine tracer solution, and 25 μl of antibody solution were mixed and left at room temperature for 10 minutes in the dark to prepare a measurement solution containing reference sample (e), and the fluorescence polarization index was measured.

[0093] To 100 μl of each of the histamine solutions #1 to #9, 40 μl of soy sauce and 60 μl of PBS(-) were added, respectively, to obtain calibration curve preparation samples (#1) to (#9). 100 μl of calibration curve preparation samples (#1) to (#9), 25 μl of acylation reagent, and 200 μl of acylation buffer were mixed to obtain mixtures (#1) to (#9). 25 μl of mixtures (#1) to (#9), 25 μl of histamine tracer solution, and 25 μl of antibody solution were mixed and left at room temperature for 10 minutes in the dark to prepare calibration curve preparation sample-containing measurement solutions (#1) to (#9). The fluorescence polarization index of each was measured.

[0094] 40 μl of the reference sample (w) and 60 μl of PBS (-) were added to 100 μl of each of the histamine solutions #1 to #9, respectively, to obtain additional measurement samples A (#1) to (#9). 100 μl of additional measurement samples A (#1) to (#9), 25 μl of acylation reagent, and 200 μl of acylation buffer were mixed to obtain mixtures (#1) to (#9). 25 μl of mixtures (#1) to (#9), 25 μl of histamine tracer solution, and 25 μl of antibody solution were mixed and left at room temperature for 10 minutes in the dark to prepare additional measurement sample A-containing measurement solutions (#1) to (#9). The fluorescence polarization indices of these samples were then measured. A first fitted curve was then derived from the obtained fluorescence polarization indices.

[0095] The second approximation curve was derived using the fluorescence polarization index of the calibration curve preparation samples (#1) to (#9) as the fluorescence polarization index of the additional measurement samples B (#1) to (#9). The maximum value of the fluorescence polarization index on the second approximation curve was read, and based on that value, the tentative concentration C of histamine in soy sauce was calculated from the first approximation curve. Pα was derived.

[0096] Provisional concentration C Pα Based on the above, the samples (#1) to (#9) for creating the calibration curve were conc [Calibration curve preparation samples (#5) to (#9)] and calibration curve preparation samples dil A calibration curve was created from the fluorescence polarization index of the reference sample (e) and the fluorescence polarization index of the calibration curve samples (#5) to (#9). The resulting calibration curve is shown in Figure 16.

[0097] Example 2 A calibration curve was prepared in the same manner as in Example 1, except that reference sample (w) was used instead of reference sample (e) and that the fluorescence polarization index calculated by the above formula (I) was used. The obtained calibration curve is shown in Figure 17. Example 3 The concentration of histamine (target substance) contained in soy sauce (measurement sample) was measured by the following method. In Example 3, a sample for preparing a calibration curve was prepared based on known information about the histamine concentration in soy sauce. conc This is an experimental example in which only the hydroxybenzoate was prepared. The fluorescence polarization degree in Example 3 is a corrected fluorescence polarization degree calculated by the above formula (II).

[0098] 160 μl of PBS(-) was added to 40 μl of reference sample (e) to obtain a 5-fold diluted solution. 100 μl of the resulting 5-fold diluted solution was mixed with 25 μl of acylation reagent and 200 μl of acylation buffer to obtain a mixture. 25 μl of this mixture, 25 μl of histamine tracer solution, and 25 μl of antibody solution were mixed and left at room temperature for 10 minutes in the dark to prepare a measurement solution containing reference sample (e), and the fluorescence polarization index was measured.

[0099] To 100 μl of each of the histamine solutions #5 to #9, add 40 μl of soy sauce and 60 μl of PBS(-), and use them as samples for creating a calibration curve. conc (#5) to (#9) were obtained. Samples for creating a calibration curve conc Mixtures (#5) to (#9) were obtained by mixing 100 μl of (#5) to (#9), 25 μl of acylation reagent, and 200 μl of acylation buffer. Mixtures (#5) to (#9) were obtained by mixing 25 μl of (#5) to (#9), 25 μl of histamine tracer solution, and 25 μl of antibody solution, and the mixture was left at room temperature for 10 minutes in the dark to prepare samples for preparing a calibration curve. conc The content measurement solutions (#5) to (#9) were prepared and their fluorescence polarization degrees were measured.

[0100] Fluorescence polarization of the reference sample (e) and the sample for creating the calibration curve conc A calibration curve similar to that in Example 1 was created from the fluorescence polarization indices (#5) to (#9).

[0101] Example 4 A calibration curve similar to that of Example 2 was prepared in the same manner as in Example 3, except that reference sample (w) was used instead of reference sample (e) in Example 3 and the fluorescence polarization index calculated by the above formula (I) was used.

[0102] Example 5 160 μl of PBS(-) was added to 40 μl of soy sauce to obtain a 5-fold diluted solution. 100 μl of the resulting 5-fold diluted solution was mixed with 25 μl of acylation reagent and 200 μl of acylation buffer to obtain a mixed solution. 25 μl of this mixed solution, 25 μl of histamine tracer solution, and 25 μl of antibody solution were mixed and left at room temperature for 10 minutes in the dark to prepare a soy sauce-containing measurement solution, and its fluorescence polarization was measured. The histamine concentration in the soy sauce was calculated from the calibration curve prepared in Example 2 to be 1.02 ppm. The histamine concentration in the soy sauce was calculated from the first approximation curve created during Example 1 and was found to be 0.69 ppm.

[0103] Example 6 A calibration curve was prepared in the same manner as in Example 1, except that the sample to be measured was a soy sauce different from the soy sauce used in Examples 1 to 5. The obtained calibration curve is shown in FIG.

[0104] Example 7 Except for using fish sauce as the sample to be measured, a calibration curve was prepared in the same manner as in Example 1. The obtained calibration curve is shown in FIG.

[0105] Example 8 A calibration curve was prepared in the same manner as in Example 1, except that reference sample (f) was used instead of reference sample (e) and that the fluorescence polarization index calculated by the above formula (I) was used. The obtained calibration curve is shown in Figure 20. [Explanation of symbols]

[0106] 1 Fluorescence polarization immunoassay device, 10 light source, 10A illumination optical system, 10B observation optical system, 11 condenser lens, 12 iris, 13 collimator, 14 polarizing element, 15 excitation light filter, 20 dichroic mirror, 21 objective lens, 22 sample, 23 microdevice, 23a inlet, 23b outlet, 23c flow path, 24 stage, 25 absorption filter, 26 polarization adjustment element (liquid crystal cell), 27 imaging lens, 28 image sensor (light receiving unit), 30 control unit

Claims

1. 1. A method for preparing a calibration curve for a fluorescence polarization immunoassay using an antibody capable of binding to a target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, comprising: Step 1a, providing a first reference sample that does not contain the target substance; After step 1a, the antibody and the fluorescent labeling substance are added to the first reference sample, and the fluorescence polarization index P ０ Step 1b, measuring The target substance is added at a concentration of C Ｓα p measurement target samples α1 to αp, where p is an integer of 3 or more, are taken from the measurement target sample α containing Ｓα More than 1000 samples for creating calibration curves ｃｏｎｃ Step 1c of preparing p samples for preparing a calibration curve, After step 1c, the antibody and the fluorescent labeling substance are added to each of the p samples for preparing a calibration curve in the same amounts as in step 1b, and the fluorescence polarization index P １ ~P ｐ and step 1d of measuring respectively After step 1b and step 1d, the fluorescence polarization index P ０ and a sample for preparing a calibration curve among the p samples for preparing a calibration curve ｃｏｎｃ Fluorescence polarization degree P ｃｏｎｃ Step 1e., based on the above, creating a calibration curve showing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample; A method for creating a calibration curve having the following formula:

2. The concentration C of the target substance Ｓα Based on information about In step 1c, a plurality of samples for preparing a calibration curve are ｃｏｎｃ Alternatively, in step 1e, a plurality of samples for preparing a calibration curve are prepared. ｃｏｎｃ and then create the calibration curve. The method for creating a calibration curve according to claim 1.

3. The concentration C of the target substance Ｓα Information about Step 2a. providing a second reference sample that does not contain the target substance; After step 2a, step 2b is performed to prepare q additional measurement samples A by dividing q second reference samples 1 to q, where q is an integer equal to or greater than 3, from the second reference sample and adding different amounts of the target substance to each of the second reference samples 1 to q. After step 2b, the antibody and the fluorescent labeling substance are added to each of the q additional measurement samples A, and the fluorescence polarization index P Ａ１ ~P Ａｑ Step 2c of measuring After step 2c, the fluorescence polarization index P Ａ１ ~P Ａｑ a step 2d 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 above; The antibody and the fluorescent labeling substance are added to the measurement sample α in the same amounts as in step 2c, and the fluorescence polarization index P Ｘα Step 2e measuring After step 2d and step 2e, the fluorescence polarization index P Ｘα Based on the above, a tentative concentration C of the target substance in the measurement sample α is calculated from the first approximation curve. Ｐα（１） Step 2f of deriving The tentative concentration C of the target substance obtained by additional measurement 1 having Ｐα（１） The method for creating a calibration curve according to claim 2, wherein

4. The concentration C of the target substance Ｓα Information about Step 3a: providing a second reference sample that does not contain the target substance; After step 3a, step 3b is performed to prepare q additional measurement samples A by dividing q second reference samples 1 to q, where q is an integer equal to or greater than 3, from the second reference sample and adding different amounts of the target substance to each of the second reference samples 1 to q. After step 3b, the antibody and the fluorescent labeling substance are added to each of the q additional measurement samples A, and the fluorescence polarization index P Ａ１ ~P Ａｑ Step 3c of measuring After step 3c, the fluorescence polarization index P Ａ１ ~P Ａｑ a step 3d 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 above; Step 3e of separating r measurement target samples α1 to αr, where r is an integer equal to or greater than 3, from the measurement target sample α, and adding different amounts of the target substance to each of the measurement target samples α1 to αr to prepare r additional measurement samples B; After step 3e, the antibody and the fluorescent labeling substance are added to each of the r additional measurement samples B in the same amounts as in step 3c, and the fluorescence polarization indices P Ｂ１ ~P Ｂｒ Step 3f of measuring each of After step 3f, the fluorescence polarization index P Ｂ１ ~P Ｂｒ a step 3g of deriving a second approximation curve representing the relationship between the amount of the target substance and the fluorescence polarization degree of the sample based on the above; and After steps 3d and 3g, a tentative concentration C of the target substance in the measurement sample α is calculated from the first approximation curve based on the maximum value of the degree of fluorescence polarization in the second approximation curve. Ｐα（２） Step 3h of deriving The tentative concentration C of the target substance obtained by additional measurement 2 having Ｐα（２） The method for creating a calibration curve according to claim 2, wherein

5. The concentration C of the target substance Ｓα 3. The method for creating a calibration curve according to claim 2, wherein the information about is known information about the measurement target sample α.

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

7. 2. 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 the measurement sample α.

8. 8. The method for creating a calibration curve according to claim 7, wherein the method for removing the target substance from the measurement sample [alpha] comprises adding an enzyme to the measurement sample [alpha], the enzyme using the target substance as a substrate.

9. 8. The method for creating a calibration curve according to claim 7, wherein the method for removing the target substance from the measurement target sample a comprises contacting the measurement target sample a with an adsorbent that adsorbs the target substance.

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

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

12. 12. The method for creating a calibration curve according to claim 11, wherein the method for removing the target substance from the measurement sample [alpha] comprises adding an enzyme that uses the target substance as a substrate to the measurement sample [alpha].

13. 12. The method for creating a calibration curve according to claim 11, wherein the method for removing the target substance from the measurement target sample a comprises contacting the measurement target sample a with an adsorbent that adsorbs the target substance.

14. 2. 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. 4. 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. 5. 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. Using an antibody capable of binding to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is measured. Ｓβ A fluorescence polarization immunoassay for measuring The antibody and the fluorescent labeling substance are added to the measurement sample β in the same amounts as in step 1b, and the fluorescence polarization index P Ｘβ Step 4a measuring After step 4a, the fluorescence polarization index P Ｘβ Based on the above, the concentration C of the target substance can be determined from the calibration curve prepared by the method according to any one of claims 1 to 4. Ｓβ Step 4b of deriving Fluorescence polarization immunoassay with.

18. A program used in the fluorescence polarization immunoassay method according to claim 17, The fluorescence polarization degree P of the measurement sample β Ｘβ Based on the above, the concentration C of the target substance can be determined from the calibration curve prepared by the method according to any one of claims 1 to 4. Ｓβ A program that causes a computer to execute a step of deriving

19. Using an antibody capable of binding to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, the concentration C of the target substance in the measurement sample β is measured. Ｓβ A fluorescence polarization immunoassay device for measuring an illumination optical system that illuminates the sample with linearly polarized excitation light; a polarization adjusting element that selectively passes a linearly polarized component of the fluorescence emitted from the sample in accordance with the drive signal; a light receiving unit that detects the intensity of the fluorescence that has passed through the polarization adjusting element; a control unit that outputs the drive signal to the polarization adjustment element and measures the degree of polarization of the sample in accordance with the drive signal based on the fluorescence intensity detected by the light receiving unit; Equipped with The control unit executes the program according to claim 18 to determine the concentration C of the target substance. Ｓβ A fluorescence polarization immunoassay device that derives the above.

20. The method comprises: an antibody capable of binding to a target substance; and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye; The calibration curve preparation kit further comprises a means for removing the target substance from the solution containing the target substance and / or a liquid not containing the target substance.

Images