Fluorescence polarization immunity measuring method and fluorescence polarization immunity measuring apparatus

Abstract

To provide a fluorescence polarization immunity measuring method capable of obtaining polarization degree inherent to competitive reaction.SOLUTION: The fluorescence polarization immunity measuring method includes: measuring polarization degrees of a first reference sample containing no target substance and a plurality of second reference samples produced by adding antibody, fluorescent label substance and target substance to the first reference sample and containing target substances respectively in different concentrations; measuring polarization degrees of a first measurement object sample and a second measurement object sample produced by adding to the first measurement object sample the antibody and the fluorescent label substance in same quantity as those added to the second reference sample; correcting the polarization degree of the second reference sample with the polarization degree of the first reference sample to calculate a first corrected polarization degree; creating a first calibration curve representing the relation between concentration of target substance contained in the second reference sample and the first corrected polarization degree; correcting the polarization degree of the second measurement object sample with the polarization degree of the first measurement object sample to calculate second corrected polarization degree; and acquiring on the first calibration curve, concentration corresponding to the second corrected polarization degree as concentration of target substance contained in the first measurement object sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a fluorescence polarization immunoassay method and a fluorescence polarization immunoassay device. [Background technology]

[0002] One of the immunoassays 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 of competitive or non-competitive type. Of these, competitive FPIA uses a competition between the target substance and a fluorescently labeled target substance (tracer) to react with the antibody. Tracers that are not bound to antibodies move violently 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 biased toward the polarization direction of the excitation light.

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

[0004] In FPIA, a calibration curve showing the relationship between the measured degree of polarization and the concentration of the target substance is created, and the concentration of the target substance contained in the sample to be measured is quantified based on the obtained calibration curve (see, for example, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

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

[0006] The measurement sample contains autofluorescent substances in addition to the tracer. The degree of polarization of the fluorescence is affected by the fluorescence emitted from the autofluorescent substances contained in the measurement sample. Due to the influence of the fluorescence from the autofluorescent substances, the degree of polarization that should be obtained from the competitive reaction cannot be obtained.

[0007] The present disclosure has been made under the above circumstances, and has an object to provide a fluorescence polarization immunoassay method and a fluorescence polarization immunoassay device that can obtain the inherent polarization degree of a competitive reaction. [Means for solving the problem]

[0008] In order to achieve the above object, a fluorescence polarization immunoassay according to a first aspect of the present disclosure comprises: A fluorescence polarization immunoassay for measuring a concentration of a target substance contained in a first measurement sample using an antibody capable of binding to the target substance and a fluorescent labeling substance obtained by labeling the target substance with a fluorescent dye, comprising: Measure the degree of polarization of a first reference sample that does not contain the target substance; measuring the degree of polarization of a plurality of second reference samples, each of which is generated by adding the antibody, the fluorescent labeling substance, and the target substance to the first reference sample and has a different concentration of the target substance; measuring the degree of polarization of the first measurement sample; measuring the degree of polarization of a second measurement sample generated by adding the antibody and the fluorescent labeling substance to the first measurement sample in the same amounts as those added to the second reference sample; correcting the degree of polarization of the second reference sample with the degree of polarization of the first reference sample to calculate a first corrected degree of polarization, and generating a first calibration curve showing the relationship between the concentration of a target substance contained in the second reference sample and the first corrected degree of polarization; calculating a second corrected degree of polarization by correcting the degree of polarization of the second measurement sample with the degree of polarization of the first measurement sample; In the first calibration curve, a concentration corresponding to the second corrected degree of polarization is determined as a concentration of the target substance contained in the first measurement sample.

[0009] A fluorescence polarization immunoassay device according to a second aspect of the present disclosure, 1. A fluorescence polarization immunoassay device for measuring a concentration of a target substance contained in a first measurement sample using an antibody capable of binding to the target substance and a fluorescent labeling substance obtained by labeling the target substance with a fluorescent dye, comprising: an illumination optical system that illuminates the sample with linearly polarized excitation light; a polarization adjustment element that selectively passes a linearly polarized component of the fluorescence emitted from the sample in response to a drive signal; a light receiving unit that detects the intensity of the fluorescence that has passed through the polarization adjustment element; a control unit that outputs the drive signal to the polarization adjustment element and measures the degree of polarization of a sample based on the fluorescence intensity detected by the light receiving unit; Equipped with The control unit is A first reference sample not containing the target substance is used as a sample, and the polarization degree of the first reference sample is measured; a plurality of second reference samples each having a different concentration of the target substance are prepared by adding the antibody, the fluorescent labeling substance, and the target substance to the first reference sample, and the second reference samples are used as samples, measuring the degree of polarization of each of the second reference samples; The first measurement target sample is used as a sample to measure the degree of polarization of the first measurement target sample; a second measurement sample is prepared by adding the antibody and the fluorescent labeling substance to the first measurement sample in the same amounts as those added to the second reference sample, and the second measurement sample is used as a sample to measure the degree of polarization of the second measurement sample; correcting the degree of polarization of the second reference sample with the degree of polarization of the first reference sample to calculate a first corrected degree of polarization, and generating a first calibration curve showing the relationship between the concentration of a target substance contained in the second reference sample and the first corrected degree of polarization; calculating a second corrected degree of polarization by correcting the degree of polarization of the second measurement sample with the degree of polarization of the first measurement sample; In the first calibration curve, a concentration corresponding to the second corrected degree of polarization is determined as a concentration of the target substance contained in the first measurement sample. Effect of the Invention

[0010] According to the present disclosure, it is possible to obtain the degree of polarization inherent to the competitive reaction. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing a fluorescence polarization immunoassay according to a first embodiment of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram showing the configuration of a fluorescence polarization immunoassay device. [Diagram 3] FIG. 2 is a block diagram showing the hardware configuration of a control unit. [Figure 4] FIG. 1 is a schematic diagram of a microdevice. [Diagram 5] FIG. 2 is a schematic diagram showing the principle of detecting the concentration of a target substance using a first calibration curve. [Figure 6] FIG. 2 is a schematic diagram showing a first example of a driving method for a polarization adjusting element. [Figure 7] 13 is a flowchart showing a measurement process of a control unit. [Figure 8] FIG. 11 is a schematic diagram showing a second example of a driving method for the polarization adjusting element. [Figure 9] FIG. 11 is a schematic diagram showing a fluorescence polarization immunoassay according to a second embodiment of the present disclosure. [Figure 10] FIG. 11 is a schematic diagram showing a fluorescence polarization immunoassay according to a third embodiment of the present disclosure. [Figure 11] 1A is a diagram showing a calibration curve of histamine contained in fish sauce and pure water before and after correction, and FIG. 1B is a diagram showing a calibration curve of histamine contained in fish sauce and pure water after correction. [Figure 12] 1A is a diagram showing a calibration curve of histamine contained in soy sauce and pure water before and after correction, and FIG. 1B is a diagram showing a calibration curve of histamine contained in soy sauce and pure water after correction. [Figure 13] (A) shows the calibration curves of histamine contained in wine and pure water before and after correction, and (B) shows the calibration curves of histamine contained in wine and pure water after correction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In each drawing, the same or equivalent parts are denoted by the same reference numerals.

[0013] [Embodiment 1] First, a first embodiment of the present disclosure will be described. In the fluorescence polarization immunoassay according to this embodiment, the concentration of a target substance contained in a measurement target sample is measured. In this fluorescence polarization immunoassay, which is shown in FIG. 1, the target substance of the measurement is a target substance OB contained in a first measurement target sample MS1. This fluorescence polarization immunoassay measures the concentration of the target substance OB contained in the first measurement target sample MS1 using an antibody A having a binding ability to the target substance OB and a tracer T as a fluorescent labeling substance obtained by labeling the target substance OB with a fluorescent dye. Examples of fluorescent dyes include fluorescein, rhodamine, and HiLyte Fluor647.

[0014] [sample] In the fluorescence polarization immunoassay according to this embodiment, four types of samples are prepared. (1) First reference sample RS1 The first reference sample RS1 is a solution that does not contain the target substance OB according to this embodiment. Usually, a different type of solution is used from the first measurement target sample MS1. For example, pure water is prepared as the first reference sample RS1. Pure water is also called a water matrix. The first reference sample RS1 may contain an autofluorescent substance SF. (2) Second reference sample RS2 The second reference sample RS2 is a solution produced by adding antibody A, tracer T and target substance OB to the first reference sample RS1. A plurality of second reference samples RS2 having different target substance OB concentrations C1 to CN are prepared. That is, the second reference sample RS2 includes a total of N samples having different concentrations of target substance OB, including a sample with target substance OB concentration C1, a sample with target substance OB concentration C2, ... and a sample with target substance OB concentration CN. (3) First measurement sample MS1 The first measurement target sample MS1 is a measurement target of the fluorescence polarization immunoassay according to this embodiment. The first measurement target sample MS1 contains an autofluorescent substance SF in addition to the target substance OB. This autofluorescent substance SF may be the same as or different from that contained in the first reference sample RS1. (4) Second measurement sample MS2 The second measurement target sample MS2 is a measurement target of the fluorescence polarization immunoassay according to the present embodiment. The second measurement target sample MS2 is generated by adding the antibody A and the tracer T to the first measurement target sample MS1 in the amounts equal to those added to the second reference sample RS2.

[0015] [Fluorescence polarization immunoassay device] Fluorescence polarization immunoassay is performed by a fluorescence polarization immunoassay device 1 shown in Fig. 2. 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.

[0016] The light source 10 is, for example, a light emitting diode, and emits excitation light of a wavelength that excites the fluorescence of the sample (for example, blue light with a central wavelength of 470 nm). The excitation light from the light source 10 is collected by a collecting lens 11 and passes through an iris 12. The iris 12 reduces the intrusion of external light other than the excitation light.

[0017] The excitation light that has passed 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.

[0018] 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 becomes parallel light in 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 including the wavelength of the fluorescence from the sample 22, and reduces light other than the fluorescence.

[0019] 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 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 polarizing filter on a sensor. In the following description, the polarization adjustment element 26 is a liquid crystal cell to which a drive signal (applied voltage) is controlled. The polarization adjustment element 26 can adjust the transmitted light intensity of a linearly polarized component. 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 described later.

[0020] 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 CCD (Charge-Coupled Device) or CMOS (Complementary Metal-Oxide-Semiconductor) 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.

[0021] The fluorescence polarization immunoassay device 1 may have a configuration different from that shown in Fig. 2. For example, the fluorescence polarization immunoassay device 1 may not include the dichroic mirror 20 by making the excitation light from the light source 10 obliquely incident on the sample 22 and separating the optical axis of the optical system that irradiates the excitation light from the optical axis of the optical system that observes the fluorescence. In addition, the iris 12, the excitation light filter 15, and the absorption filter 25 may not be installed if they are not necessary. The fluorescence polarization immunoassay device 1 may include other lenses in addition to or instead of the condenser lens 11, the objective lens 21, and the imaging lens 27 shown in Fig. 2.

[0022] 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.

[0023] For example, during a 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 adjustment element 26. By outputting the drive signal to the polarization adjustment element 26, the control unit 30 can control the polarization component of the fluorescence passing through the polarization adjustment element 26.

[0024] 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 (output 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 therethrough.

[0025] The fluorescence polarization immunoassay device 1 controls the exposure time (imaging time) of the image sensor 28, i.e., the start time and duration of imaging, along with a drive signal to the polarization adjustment element (liquid crystal cell) 26, to acquire image data of a desired polarization component of fluorescence. The fluorescence polarization immunoassay device 1 analyzes the acquired image data. As described below, the fluorescence polarization immunoassay device 1 captures fluorescent images of the first reference sample RS1, the second reference sample RS2, the first measurement target sample MS1, and the second measurement target sample MS2 of the sample 22 using the image sensor 28, and measures the concentration of the target substance OB contained in the first measurement target sample MS1 based on the image data of the fluorescent images. This makes it possible to reduce measurement errors in the concentration of the target substance OB caused by the autofluorescent substance SF contained in the first measurement target sample MS1.

[0026] The control unit 30 of the fluorescence polarization immunoassay device 1 shown in Fig. 2 is realized, for example, by a software program executed by a computer having the hardware configuration shown in Fig. 3. Specifically, the fluorescence polarization immunoassay device 1 is made up of a CPU (Central Processing Unit) 31 that controls the entire device, a main memory unit 32 that operates as a working area for the CPU 31, an external memory unit 33 that stores the operating program of the CPU 31, an operation unit 34, a display unit 35, an input / output unit 36, and an internal bus 38 that connects these units.

[0027] The main memory unit 32 is composed of a RAM (Random Access Memory). A program 39 to be executed by the CPU 31 is loaded into the main memory unit 32 from the external memory unit 33. The main memory unit 32 is also used as a working area (temporary data storage area) for the CPU 31.

[0028] The external storage unit 33 is composed of a non-volatile memory such as a flash memory and a hard disk. The external storage unit 33 stores in advance a program 39 to be executed by the CPU 31.

[0029] The operation unit 34 is composed of a keyboard and a mouse device, and an interface device that connects these devices to an internal bus 38 .

[0030] The display unit 35 is composed of display devices such as a CRT (Cathode Ray Tube) and a liquid crystal monitor.

[0031] The input / output unit 36 ​​is an interface for transmitting and receiving data to and from external devices. In accordance with a command from the CPU 31, the input / output unit 36 ​​outputs a drive signal to the polarization adjustment element 26 and inputs image data from the imaging element 28. The input image data is stored in the main storage unit 32 or the external storage unit 33 and displayed on the display unit 35.

[0032] The functions of the control unit 30 can be implemented in a computer system consisting of one or more computers including one or more processors and one or more storage devices including a non-transitory storage medium. The multiple computers realize the functions of the control unit 30 while communicating with each other via a communication network. For example, some of the functions of the control unit 30 may be implemented in one computer, and the other parts may be implemented in another computer.

[0033] The components of the above-mentioned fluorescence polarization immunoassay device 1 can be summarized as follows. (A) An illumination optical system 10A that includes a light source 10, a condenser lens 11, an iris 12, a collimator 13, a polarizing element 14, and an excitation light filter 15 and illuminates a sample 22 with linearly polarized excitation light. (B) A microdevice 23 that accommodates a sample 22, and a stage 24 on which the microdevice 23 is mounted. (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 in response to a drive signal from the control unit 30 and an image pickup element 28 that captures a fluorescent image 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 fluorescent image captured by the image capture element 28, measures the degree of polarization of the sample 22 in accordance with the drive signal, and analyzes the sample.

[0034] The fluorescence polarization immunoassay method according to this embodiment performs the following measurement using the fluorescence polarization immunoassay device 1 having the above-described configuration. (1) The degree of polarization P when the first reference sample RS1 is the sample 22 A (2) The polarization degree P of each of the second reference samples RS2 having different concentrations C1 to CN of the target substance OB is set as the sample 22. B (3) The degree of polarization P when the first measurement sample MS1 is the sample 22 C (4) Degree of polarization P when the second measurement sample MS2 is the sample 22 D

[0035] 4, the microdevice 23 has a plurality of flow paths 23c, one end of which is connected to the inlet 23a and the other end of which is connected to the outlet 23b. A first reference sample RS1, a plurality of second reference samples RS2, a first measurement sample MS1, or a second measurement sample MS2 can be individually supplied to the plurality of flow paths 23c as the samples 22. Since there is an imaging relationship between the flow paths 23c and the imaging surface of the image sensor 28, by capturing an image of the plurality of flow paths 23c with the image sensor 28, fluorescent images of the plurality of samples 22 supplied to the plurality of flow paths 23c can be obtained at once, and the polarization degree P of each sample 22 can be obtained. A , P B , P C , P DThis measurement is performed based on the fluorescence intensity of a region of interest (ROI) of image data corresponding to the flow channel 23c in the fluorescence image.

[0036] The control unit 30 controls the polarization degree P of the second reference sample RS2. B The polarization degree P of the first reference sample RS1 A The first correction degree of polarization P BA The concentrations C1 to CN of the target substance OB contained in the first reference sample RS1 and the first corrected polarization degree P BA A first calibration curve DL1 (see FIG. 5) is generated, which shows the relationship between the concentration (logarithm) and the polarization degree. In the graph of FIG. 5, the horizontal axis shows the concentration (logarithm) and the vertical axis shows the polarization degree. The control unit 30 also generates a first calibration curve DL1 (see FIG. 5) which shows the relationship between the concentration (logarithm) and the polarization degree P D The polarization degree P of the first measurement target sample MS1 C The second correction degree of polarization P DC Furthermore, the control unit 30 calculates the second corrected polarization degree P DC The concentration Cx corresponding to this is determined as the concentration of the target substance OB contained in the first measurement sample MS1.

[0037] Calculated second corrected polarization degree P DC is the polarization degree P of the second measurement target sample MS2 D The polarization degree P of the first measurement target sample MS1 C Since the amount of tracer T is fixed, the second correction degree of polarization P DC is determined by the amount of the target substance OB contained in the first measurement sample MS1. On the other hand, the first calibration curve DL1 is determined by the polarization degree P B The polarization degree P of the first reference sample RS1 A The first corrected degree of polarization P BA The first correction degree of polarization P BA is determined by the amount of the target substance OB added to the second reference sample RS2. As shown in FIG. 5, the second corrected polarization degree P DC and the first corrected polarization degree P at point B BAWhen the second corrected polarization degree P DC Then, the second correction degree of polarization P DC If the concentration of the target substance OB at point B corresponding to the first measurement sample MS1 is obtained, the obtained concentration corresponds to the concentration of the target substance OB contained in the first measurement sample MS1.

[0038] As shown in Fig. 6, the control unit 30 applies a rectangular pulse wave that oscillates periodically between specific positive and negative voltage values ​​as a drive signal to turn on the polarization adjustment element 26. This rectangular pulse wave is a first signal 40. The polarization adjustment element 26 is turned on at both positive and negative voltage values ​​of the first signal 40. A detection period K1 is defined within a period (first period) T1 during which the polarization adjustment element 26 is in the on state.

[0039] During the detection period K1, the control unit 30 causes the image sensor 28 to receive the fluorescent light and capture a fluorescent image. When the polarization adjustment element 26 is in the on state, a polarized component perpendicular to the polarization direction of the excitation light of the fluorescent light from the sample 22 is received. The fluorescence intensity I ⊥ is represented by the total amount of light received in the ROI corresponding to the flow path 23c in the fluorescence image during the detection period K1 or its time average.

[0040] The control unit 30 inputs a drive signal with a voltage level of 0 to the polarization adjustment element 26 in order to turn the polarization adjustment element 26 off. This drive signal is the second signal 41. The second signal 41 has a signal level different from that of the first signal 40. This drive signal turns the polarization adjustment element 26 into the off state. A detection period K2 is defined within the second period T2 in which the polarization adjustment element 26 is in the off state. The control unit 30 causes the image sensor 28 to receive the fluorescence from the sample 22 and capture a fluorescence image during the detection period K2. When the polarization adjustment element 26 is in the off state, a polarized component parallel to the polarization direction of the excitation light of the fluorescence from the sample 22 is received. The fluorescence intensity I II is represented by the amount of light received in the ROI corresponding to the flow path 23c in the fluorescence image during the detection period K2 or its time average.

[0041] That is, in this embodiment, the control unit 30 inputs to the polarization adjustment element 26 a drive signal that alternates between a first signal 40 that passes only linearly polarized light whose polarization direction is orthogonal to the excitation light incident on the sample 22 and a second signal 41 that passes only linearly polarized light whose polarization direction is parallel to the excitation light incident on the sample 22. The fluorescence intensity detected by the image sensor 28 in the first period T1 when the first signal 40 is input is defined as the first fluorescence intensity, and the fluorescence intensity detected by the image sensor 28 in the second period T2 when the second signal 41 is input is defined as the second fluorescence intensity.

[0042] The control unit 30 determines the fluorescence intensity A (I ⊥ ) and the fluorescence intensity A (I II ) and the polarization degree P of the first reference sample RS1. A Furthermore, when the sample 22 is used as the second reference sample RS2, the control unit 30 measures the fluorescence intensity B (I ⊥ ) and the fluorescence intensity B (I II ) and the polarization degree P of the second reference sample RS2. B Furthermore, the control unit 30 measures the following formula:

number

[0043] Furthermore, when the sample 22 is the first measurement target sample MS1, the control unit 30 calculates the fluorescence intensity C (I ⊥ ) and the fluorescence intensity C(I II ) is the degree of polarization P CFurthermore, when the sample 22 is the second measurement target sample MS2, the control unit 30 measures the fluorescence intensity D(I II ) and the fluorescence intensity D(I ⊥ ) is the polarization degree P of the second measurement sample MS2. D Furthermore, the control unit 30 measures the following equation.

number

[0044] Next, a description will be given of the measurement process, that is, the fluorescence polarization immunoassay method, executed by the control unit 30 of the fluorescence polarization immunoassay device 1. When the setting of the microdevice 23 is completed, the measurement process is started.

[0045] 7, first, the control unit 30 causes the light source 10 to start emitting excitation light (step S1). Then, the control unit 30 starts outputting a drive signal to the polarization adjusting element 26 (step S2).

[0046] Next, the control unit 30 measures the fluorescence intensity (step S3). In this measurement, the control unit 30, in synchronization with the drive signal, determines the fluorescence intensity A (I ⊥ ), the fluorescence intensity B (I ⊥ ), the fluorescence intensity C(I ⊥ ), the fluorescence intensity D(I ⊥ Furthermore, the control unit 30 measures the fluorescence intensity A (I II ), the fluorescence intensity B (I II ), the fluorescence intensity C(III ), the fluorescence intensity D(I II ) is measured.

[0047] Furthermore, the control unit 30 calculates the above formulas (1) and (2) to obtain the second corrected polarization degree P DC and the first corrected polarization degree P BA (Step S4). Next, the control unit 30 calculates the calculated first corrected polarization degree P BA (Step S5). Then, the control unit 30 creates a first calibration curve DL1 based on the second corrected polarization degree P DC The concentration Cx corresponding to is determined as the concentration of the target substance OB contained in the first measurement sample MS1 (step S6). After step S6 is completed, the control unit 30 ends the measurement process.

[0048] In this embodiment, the polarization adjustment element 26 is controlled by a drive signal that is repeatedly turned on and off, but this is not limited to this. The control unit 30 may input, as a drive signal, a signal that sinusoidally changes the first transmitted light intensity of linearly polarized light perpendicular to the polarization direction of the excitation light and the second transmitted light intensity of linearly polarized light parallel to the polarization direction of the excitation light. In this case, as shown in FIG. 8, for example, the drive signal changes over time so as to invert between positive and negative values ​​at a high frequency. The positive and negative envelopes of this drive signal are linearly symmetrical with respect to the horizontal axis (time axis). The envelope changes periodically with a period longer than the inversion period.

[0049] When such a drive signal is input to the polarization adjustment element 26, the first transmitted light intensity changes in a form close to a sine wave, as shown in FIG. 8. The change in the second transmitted light intensity is in opposite phase to the change in the first transmitted light intensity. The first transmitted light intensity and the second transmitted light intensity decrease as the absolute value of the drive signal increases, and increase as the drive signal decreases. As long as the changes in the first transmitted light intensity and the second transmitted light intensity can be close to a sine wave, the waveform of the voltage applied to the polarization adjustment element 26 is not particularly limited.

[0050] The control unit 30 calculates the fluorescence intensity of the ROI in the image of the fluorescent sample in each of the first period D1, the second period D2, the third period D3, and the fourth period D4, for example. The fluorescence intensity in each period is the integral value of the fluorescence intensity of the ROI in each period over time, and is the total amount of light received by the pixels of the ROI in each period (exposure period). The control unit 30 evaluates the fluorescence polarization of the sample based on the light intensity in each of the first period D1 to the fourth period D4. In each of the first period D1 to the fourth period D4, the image sensor 28 receives light of a specific polarization component of the fluorescence. The polarization component in each period has a width of the polarization orientation.

[0051] That is, the control unit 30 inputs, as a drive signal, a signal for sinusoidally changing the first transmitted light intensity of linearly polarized light perpendicular to the polarization direction of the excitation light and the second transmitted light intensity of linearly polarized light parallel to the polarization direction of the excitation light to the polarization adjustment element 26. The polarization adjustment element 26 changes the first transmitted light intensity and the second transmitted light intensity sinusoidally and in opposite phases to each other.

[0052] One period of the transmitted light intensity, which changes sinusoidally, is divided equally into four periods, which are designated as a first period D1, a second period D2, a third period D3, and a fourth period D4. When the sample 22 is designated as a first reference sample RS1, the control unit 30 calculates the average value p 1A , p 2A , p 3A , p 4A The polarization degree P of the first reference sample RS1 A Furthermore, when the sample 22 is used as a second reference sample RS2, the control unit 30 calculates the average value p of the fluorescence intensity detected by the image sensor 28 in the first period, the second period, the third period, and the fourth period. 1B , p 2B , p 3B , p 4B The polarization degree P of the second reference sample RS2 B Then, the control unit 30 measures p xBA =p xB -p xA Calculate (x=1,2,3,4) and then calculate the following equation:

number

[0053] Furthermore, when the sample 22 is the first measurement target sample MS1, the control unit 30 calculates the average value p of the fluorescence intensity detected by the image sensor 28 in the first period D1, the second period D2, the third period D3, and the fourth period D4. 1C , p 2C , p 3C , p 4C The polarization degree P of the first measurement target sample MS1 C Furthermore, when the sample 22 is the second measurement target sample MS2, the control unit 30 measures the average value p 1D , p 2D , p 3D , p 4D The polarization degree P of the second measurement sample MS2 D Measured as:

[0054] Then, the control unit 30 xDC =p xD -p xC (x=1,2,3,4). Furthermore, the control unit 30 calculates the following equation:

number

[0055] [Embodiment 2] Next, a second embodiment of the present disclosure will be described. In the fluorescence polarization immunoassay according to the first embodiment, a first reference sample RS1, a plurality of second reference samples RS2, a first measurement target sample MS1, and a second measurement target sample MS2 are used as samples 22 to measure the concentration of the target substance OB contained in the first measurement target sample MS1. As shown in Fig. 9, in this embodiment, a third measurement target sample MS3 is further prepared.

[0056] The third measurement target sample MS3 is generated by adding to the first measurement target sample MS1 the antibody A, the tracer T, and the high concentration target substance HOB in the same amounts as those added to the second reference sample RS2. The concentration of the high concentration target substance HOB is the concentration at which the degree of polarization is minimized in the first calibration curve DL. The third measurement target sample MS3 is contained in the microdevice 23, like the other samples 22. The control unit 30 controls the degree of polarization P of the third measurement target sample MS3 contained in the microdevice 23. E Measure.

[0057] As shown in FIG. 9, the control unit 30 controls the polarization degree P E The polarization degree P of the first measurement target sample MS1 A The third correction degree of polarization P EA The control unit 30 calculates the first calibration curve DL1 and the third corrected polarization degree P EA and are displayed on the display unit 35 (see FIG. 3) in a comparative manner. This display allows the level of the minimum value of the first calibration curve DL and the third corrected degree of polarization P EA It can be confirmed whether the difference between is within a tolerance. This makes it possible to confirm that the influence of the autofluorescent substance SF in the first measurement sample MS1 is cancelled out from the measurement result.

[0058] The control unit 30 determines the minimum level of the first calibration curve DL1 and the third corrected polarization degree P EA The first calibration curve DL1 may be shifted in the vertical axis direction so that the first calibration curve DL1 coincides with the first calibration curve DL2.

[0059] [Embodiment 3] Next, a third embodiment of the present disclosure will be described. In the fluorescence polarization immunoassay according to the first embodiment, a first reference sample RS1, a plurality of second reference samples RS2, a first measurement target sample MS1, and a second measurement target sample MS2 are used as samples 22 to measure the concentration of the target substance OB contained in the first measurement target sample MS1. In this embodiment, as shown in Fig. 10, in addition to these samples 22, a plurality of fourth measurement target samples MS4 are prepared, which are generated by adding an antibody A, a tracer T, and a target substance to the first measurement target sample MS1 and have different concentrations of the target substance OB.

[0060] The fluorescence polarization immunoassay device 1 measures the polarization degree P of a plurality of fourth measurement samples MS4 having different concentrations of the target substance OB. F The control unit 30 measures the degree of polarization P F The polarization degree P of the first measurement target sample MS1 C The fourth correction degree of polarization P FC The control unit 30 calculates the concentration of the target substance OB contained in the fourth measurement sample MS4 and the fourth corrected polarization degree P FC A second calibration curve DL2 is generated, which shows the relationship between the polarization degree and the added target substance OB. By looking at the second calibration curve DL2, it is possible to confirm the change in the polarization degree in the second measurement sample MS2 according to the added target substance OB.

[0061] The control unit 30 compares and displays the first calibration curve DL1 and the second calibration curve DL2 on the display unit 35 (see FIG. 3). Depending on whether the difference between the two curves in the high concentration region is within an allowable range, it can be confirmed that the error due to the autofluorescent substance SF has been reduced.

[0062] The control unit 30 may shift the first calibration curve DL1 in the vertical axis direction so that the minimum value level of the first calibration curve DL1 coincides with the minimum value level of the second calibration curve DL2.

[0063] The control unit 30 measures both the third measurement sample MS3 and the fourth measurement sample MS4, and confirms that the error due to the autofluorescent substance SF has been reduced. Then, the control unit 30 calculates the first calibration curve DL1, the second calibration curve DL2, and the third corrected degree of polarization P EA The first calibration curve DL1 may be corrected by comparing it with the above.

[0064] [Example] The measurement targets (first measurement target samples) were fish sauce, soy sauce, and wine, and the histamine contained therein was used as the target substance OB, and the histamine concentrations were measured. The measurement procedure was as follows. [Measurement of the first reference sample] A.: Pure water was used as the first reference sample RS1 and was placed in the nine flow paths 23c of the microdevice 23, and the degree of polarization of each was measured.

[0065] [Measurement of the second reference sample] B.: Antibody A, tracer T and target substance OB were added to pure water to prepare nine second reference samples (aqueous solutions with nine concentration levels) containing different concentrations of target substance OB, and the degree of polarization of each was measured. 1) As the tracer T, the target substance OB, histamine, was modified with HiLyte Fluor 647. This was dissolved in pure water, and a 4.56 nM solution was prepared in phosphate buffered saline (PBS(-)). 2) Anti-histamine antibody was diluted with PBS(-) containing 0.01% bovine serum albumin (BSA) to a concentration of 1.3 × 10 -7 A solution of M was prepared. 3) The target substance OB, histamine, was dissolved in pure water to prepare a 32 mg / ml solution. 4) The obtained histamine solution was adjusted with pure water to nine different concentrations as shown in the table below. [Table 1] 5) Each of the nine levels of histamine solution was mixed with an acylation reagent and an acylation buffer according to the following table. [Table 2] 6) 25 μL of each of the above mixed solutions, 25 μL of the histamine tracer in 1) above, and 25 μL of the antibody solution in 2) above were mixed and left at room temperature for 10 minutes in the dark, after which the polarization degree of each mixed solution was measured.

[0066] [Measurement of the first measurement sample] C. Only the first measurement target sample MS1 was placed into the nine flow paths 23c of the microdevice 23 for measurement. Three types of measurement targets, soy sauce, fish sauce, and wine, were each diluted five-fold with PBS(-) and then placed into the nine flow paths 23c of the microdevice 23, and the degree of polarization was measured.

[0067] [Measurement of the second and fourth measurement samples] D. Nine concentration levels of the target substance OB were added to the first measurement sample MS1 to generate a second measurement sample MS2 and a fourth measurement sample MS4, and the degree of polarization was measured. 1) Histamine was modified with HiLyte Fluor 647 to generate tracer T. This was dissolved in pure water and a 4.56 nM solution was prepared in PBS(-). 2) Anti-histamine antibody was diluted with PBS(-) containing 0.01% BSA to a concentration of 1.3 × 10 -7 A solution of M was prepared. 3) Histamine was dissolved in pure water to prepare a 32 mg / mL solution. 4) The resulting histamine solution was adjusted with pure water to nine different concentration levels as shown in the table below. [Table 3] 5) Each histamine solution was added to nine concentration levels so that the samples were diluted 5-fold in the solution, as shown in the table below. [Table 4] [Table 5] 6) Each of the sample solutions containing nine different concentrations of histamine was mixed with an acylation reagent and an acylation buffer as shown in the table below. [Table 6] 7) 25 μL of each of the above mixed solutions, 25 μL of the histamine tracer solution in 1) above, and 25 μL of the antibody solution in 2) above were mixed and allowed to stand at room temperature for 10 minutes in the dark, after which the fluorescence polarization degree of the mixed solutions was measured.

[0068] The measurement target is fish sauce, and the histamine contained therein is the target substance OB. The results of measuring the concentration of the target substance OB are shown in Figs. 11(A) and 11(B). As shown in Fig. 11(A), when the first calibration curve DL1 and the second calibration curve DL2 are generated without correction using a 5-fold diluted fish sauce as the first measurement target sample MS1 and pure water as the first reference sample RS1, deviations occur even in the high concentration range. However, as shown in Fig. 11(B), the first calibration curve DL1 and the second calibration curve DL2 generated while performing correction using the fluorescence polarization immunoassay according to the present embodiment have smaller deviations between them in the high concentration range. This indicates that the effect of the autofluorescent substance SF is canceled in the first calibration curve DL1 and the second calibration curve DL2 by the correction.

[0069] The measurement results of the concentration of the target substance OB, which is soy sauce and the histamine contained therein, are shown in Fig. 12(A) and Fig. 12(B). As shown in Fig. 12(A), when the first calibration curve DL1 and the second calibration curve DL2 are generated without correction using a 5-fold dilution of soy sauce as the first measurement target sample MS1 and pure water as the first reference sample RS1, deviations occur even in the high concentration region. However, as shown in Fig. 12(B), the first calibration curve DL1 and the second calibration curve DL2 generated while performing correction using the fluorescence polarization immunoassay according to the present embodiment have smaller deviations between them in the high concentration region. This indicates that the influence of the autofluorescent substance SF is canceled in the first calibration curve DL1 and the second calibration curve DL2 by the correction.

[0070] The measurement results of the concentration of the target substance OB, which is wine and histamine contained therein, are shown in Figs. 13(A) and 13(B). As shown in Fig. 13(A), when the first calibration curve DL1 and the second calibration curve DL2 are generated without correction using a 5-fold diluted wine as the first measurement sample MS1 and pure water as the first reference sample RS1, deviations occur even in the high concentration region. However, as shown in Fig. 13(B), the first calibration curve DL1 and the second calibration curve DL2 generated while performing correction using the fluorescence polarization immunoassay according to the present embodiment have smaller deviations between them in the high concentration region. This indicates that the influence of the autofluorescent substance SF is canceled in the first calibration curve DL1 and the second calibration curve DL2 by the correction. As shown in Fig. 13(B), for wine, the second calibration curve DL2 and the first calibration curve DL1 are within the allowable range over the entire region.

[0071] The second reference sample RS2 for the calibration curve may have a known concentration of the target substance OB and may have a plurality of different concentration levels. In the above embodiment, pure water is used as the first reference sample RS1, but a buffer solution such as phosphate buffered saline (PBS) or other solvents may be used. Any solvent may be selected as long as it is capable of dissolving the target substance OB and does not inhibit the antigen-antibody reaction. For example, it is difficult to use organic solvents or solvents with extremely high or low pH that denature the antibody (antibody).

[0072] In the above embodiment, the light receiving unit is the image sensor 28, but is not limited to this. When detecting the degree of polarization of each sample 22, such as the first reference sample RS1, the second reference sample RS2, the first measurement sample MS1, and the second measurement sample MS2, individually, the light receiving unit may be a device that detects only the fluorescence intensity, such as a photodiode.

[0073] The measurement subject and the target substance are not limited to those mentioned above. Basically, any substance capable of competing with an antigen-antibody reaction, or a measurement subject containing such a substance, can be used.

[0074] The hardware and software configurations of the control unit 30 are merely examples and can be changed or modified as desired.

[0075] The core part of the processing of the control unit 30, which is composed of the CPU 31, the main memory unit 32, the external memory unit 33, the operation unit 34, the display unit 35, the input / output unit 36, and the internal bus 38, can be realized by using a normal computer system, not a dedicated system. For example, the computer program for executing the above-mentioned operations may be stored in a computer-readable recording medium (flexible disk, CD-ROM, DVD-ROM) and distributed, and the computer program may be installed on a computer to configure the control unit 30 for executing the above-mentioned processing. Also, the computer program may be stored in a storage device of a server device on a communication network of the Internet, and the control unit 30 may be configured by downloading the computer program into a normal computer system.

[0076] When the functions of the control unit 30 are realized by sharing between an OS (operating system) and an application program, or by cooperation between the OS and the application program, only the application program portion may be stored in a recording medium or storage device.

[0077] It is also possible to superimpose a computer program on a carrier wave and distribute it via a communication network. For example, the computer program may be posted on a bulletin board system (BBS) on the communication network and distributed via the network. Then, the computer program may be started and executed under the control of the OS in the same way as other application programs, thereby enabling the above-mentioned processing to be performed.

[0078] Various embodiments and modifications of the present invention are possible without departing from the broad spirit and scope of the present invention. The above-described embodiments are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not the embodiments. Various modifications made within the scope of the claims and the scope of the invention equivalent thereto are considered to be within the scope of the present invention. [Explanation of symbols]

[0079] 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 section), 30 control section (drive section), 31 CPU, 32 main memory section, 33 external memory section, 34 operation section, 35 display section, 36 input / output section, 38 internal bus, 39 program, 40 first signal, 41 second signal, A antibody, DL1 first calibration curve, DL2 second calibration curve, MS1 first measurement target sample, MS2 Second measurement sample, MS3 Third measurement sample, MS4 Fourth measurement sample, OB Target substance, RS1 First reference sample, RS2 Second reference sample, SF Autofluorescent substance, T Tracer (fluorescent labeling substance)

Claims

1. A fluorescence polarization immunoassay method for measuring the concentration of a target substance contained in a first measurement target sample, using an antibody having a binding ability to the target substance and a fluorescently labeled substance obtained by labeling the target substance with a fluorescent dye, comprising: measuring the polarization degree of a first reference sample that does not contain the target substance; measuring the polarization degrees of a plurality of second reference samples generated by adding the antibody, the fluorescently labeled substance, and the target substance to the first reference sample, wherein the concentrations of the target substance are different from each other; measuring the polarization degree of the first measurement target sample; measuring the polarization degree of a second measurement target sample generated by adding the antibody and the fluorescently labeled substance to the first measurement target sample in the same amounts as those added to the second reference samples; calculating a first corrected polarization degree by correcting the polarization degree of the second reference sample with the polarization degree of the first reference sample, and generating a first calibration curve showing the relationship between the concentration of the target substance contained in the second reference sample and the first corrected polarization degree; calculating a second corrected polarization degree by correcting the polarization degree of the second measurement target sample with the polarization degree of the first measurement target sample; obtaining, as the concentration of the target substance contained in the first measurement target sample, the concentration corresponding to the second corrected polarization degree in the first calibration curve; A fluorescence polarization immunoassay method.

2. measuring the polarization degree of a third measurement target sample generated by adding the antibody, the fluorescently labeled substance, and the target substance to the first measurement target sample, wherein the concentration of the target substance is the concentration at which the polarization degree becomes minimum in the first calibration curve; calculating a third corrected polarization degree by correcting the polarization degree of the third measurement target sample with the polarization degree of the first measurement target sample; The fluorescence polarization immunoassay method according to Claim 1.

3. measuring the polarization degrees of a plurality of fourth measurement target samples generated by adding the antibody, the fluorescently labeled substance, and the target substance to the first measurement target sample, wherein the concentrations of the target substance are different from each other; calculating a fourth corrected polarization degree by correcting the polarization degree of the fourth measurement target sample with the polarization degree of the first measurement target sample, and generating a second calibration curve showing the relationship between the concentration of the target substance contained in the fourth measurement target sample and the fourth corrected polarization degree; The fluorescence polarization immunoassay method according to Claim 1.

4. A fluorescence polarization immunoassay device for measuring the concentration of a target substance contained in a first measurement target sample, using an antibody having a binding ability to the target substance and a fluorescence-labeled substance obtained by labeling the target substance with a fluorescent dye, comprising: An irradiation optical system for irradiating the sample with linearly polarized excitation light; A polarization adjustment element that selectively passes a linearly polarized light component corresponding to a drive signal among the fluorescence emitted from the sample; A light receiving unit that detects the fluorescence intensity that has passed through the polarization adjustment element; A control unit that outputs the drive signal to the polarization adjustment element and measures the polarization degree of the sample according to the drive signal based on the fluorescence intensity detected by the light receiving unit; Comprising: The control unit: Measures the polarization degree of a first reference sample that does not contain the target substance as a sample; Uses a plurality of second reference samples generated by adding the antibody, the fluorescence-labeled substance, and the target substance to the first reference sample, and having different concentrations of the target substance as samples, and measures the polarization degree of each of the second reference samples; Uses the first measurement target sample as a sample and measures the polarization degree of the first measurement target sample; Uses a second measurement target sample generated by adding the same amount of the antibody and the fluorescence-labeled substance as added to the second reference sample to the first measurement target sample as a sample, and measures the polarization degree of the second measurement target sample; Corrects the polarization degree of the second reference sample with the polarization degree of the first reference sample to calculate a first corrected polarization degree, and generates a first calibration curve showing the relationship between the concentration of the target substance contained in the second reference sample and the first corrected polarization degree; Corrects the polarization degree of the second measurement target sample with the polarization degree of the first measurement target sample to calculate a second corrected polarization degree; Obtains, as the concentration of the target substance contained in the first measurement target sample, the concentration corresponding to the second corrected polarization degree in the first calibration curve; Fluorescence polarization immunoassay device.

5. The control unit inputs the drive signal that alternately repeats a first signal and a second signal having a different signal level from the first signal to the polarization adjustment element; The polarization adjustment element passes only a linearly polarized light component having a polarization direction orthogonal to the linearly polarized excitation light incident on the sample during a first period when the first signal is input, and passes only a linearly polarized light component having a polarization direction perpendicular to the linearly polarized excitation light incident on the sample during a second period when the second signal is input; The control unit: When the sample is used as the first reference sample, the fluorescence intensity A(I ⊥ ) detected by the light receiving unit in the first period and the fluorescence intensity A(I II ) detected by the light receiving unit in the second period are measured as the polarization degrees of the first reference sample, When the sample is used as the second reference sample, the fluorescence intensity B(I ⊥ ) detected by the light receiving unit in the first period and the fluorescence intensity B(I II ) detected by the light receiving unit in the second period are measured as the polarization degrees of the second reference sample, 【Number 1】 Calculate to obtain the first corrected polarization degree P ＢＡ and obtain The control unit: When the sample is used as the first measurement target sample, the fluorescence intensity C(I ⊥ ) detected by the light receiving unit in the first period and the fluorescence intensity C(I II ) detected by the light receiving unit in the second period are measured as the polarization degree of the first measurement target sample, When the sample is used as the second measurement target sample, the fluorescence intensity D(I ⊥ ) detected by the light receiving unit in the first period and the fluorescence intensity D(I II ) detected by the light receiving unit in the second period are measured as the polarization degree of the second measurement target sample, 【Number 2】 Calculate to obtain the second corrected polarization degree P ＤＣ thereof. The fluorescence polarization immunoassay device according to claim 4.

6. The control unit inputs, as the drive signal, a signal that sinusoidally varies the first transmitted light intensity of linearly polarized light perpendicular to the polarization direction of the excitation light and the second transmitted light intensity of linearly polarized light parallel to the polarization direction of the excitation light to the polarization adjustment element, The polarization adjustment element varies the first transmitted light intensity and the second transmitted light intensity in antiphase, The control unit, When one cycle of the transmitted light intensity that varies sinusoidally is equally divided into four periods, and each period is defined as a first period, a second period, a third period, and a fourth period, When the sample is used as the first reference sample, the average values p of the fluorescence intensities detected by the light receiving unit in the first period, the second period, the third period, and the fourth period １Ａ , p ２Ａ , p ３Ａ , p ４Ａ are measured as the polarization degrees of the first reference sample, When the sample is used as the second reference sample, the average values p of the fluorescence intensities detected by the light receiving unit in the first period, the second period, the third period, and the fourth period １Ｂ , p ２Ｂ , p ３Ｂ , p ４Ｂ are measured as the polarization degrees of the second reference sample, p ｘＢＡ = p ｘＢ - p ｘＡ (x = 1, 2, 3, 4) is calculated, [Number 3] Calculate to obtain the first corrected polarization degree P ＢＡ and obtain The control unit, When the sample is the first measurement target sample, the average values p of the fluorescence intensities detected by the light receiving unit in the first period, the second period, the third period, and the fourth period １Ｃ , p ２Ｃ , p ３Ｃ , p ４Ｃ are measured as the polarization degrees of the first measurement target sample, When the sample is used as the second measurement target sample, the average values p of the fluorescence intensities detected by the light receiving unit in the first period, the second period, the third period, and the fourth period １Ｄ , p ２Ｄ , p ３Ｄ , p ４Ｄ are measured as the polarization degrees of the second measurement target sample, p ｘＤＣ = p ｘＤ - p ｘＣ (x = 1, 2, 3, 4) is calculated, 【Number 4】 Calculate to obtain the second corrected polarization degree P ＤＣ thereof. The fluorescence polarization immunoassay apparatus according to claim 4.

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