JP2007120976A - Immunoassay, its kit, its device and reader used in immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide immunoassay capable of simply evaluating the modification ratio of modified protein. <P>SOLUTION: In the immunoassay for calculating the modification ratio of modified protein in a reaction system, the reaction system has the antibody specifically bonded to the modified protein and a fluorescent specific substance for specifically recognizing the modifier of the modified protein to sensitize a fluorescent substance. The concentration of the protein in the reaction system is measured on the basis of an immunoagglutination assay, and the concentration of the modified protein in the reaction system on the basis of a fluorescent polarization measuring theory, and the modification ratio of the modified protein is calculated from both measuring results. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immunoassay for analyzing a modified protein in a sample.

  An immunoassay (hereinafter simply referred to as an immunoassay) is a measurement method using an antigen-antibody reaction. So far, immunoassays based on various measurement principles have been reported. For example, measurement methods that require binding / free separation (hereinafter simply referred to as BF separation), such as enzyme immunoassay, fluorescence measurement, or luminescence measurement, to separate immune complexes from unreacted substances, and turbidimetry There are measurement methods that do not require BF separation, such as a method, a ratio method, and a latex agglutination method. These are methods for optically detecting changes in the size of the detection object. By utilizing the fact that the scattered light intensity increases and the transmitted light intensity decreases as the size of the detection object changes greatly. Yes.

  On the other hand, as another method for detecting a change in the size of an object to be detected, an immunoassay using fluorescence polarization (hereinafter referred to as a fluorescence polarization immunoassay) has attracted attention. Even in the fluorescence polarization immunoassay, since no BF separation is required, no washing operation is required. In addition, since fluorescence is basically measured, highly sensitive measurement is possible.

  The phenomenon called fluorescence polarization or fluorescence depolarization began to be applied to biopolymers after 1950. Since the latter half of the 1970s, a wide range of applied research has been conducted in fields such as biochemistry and biology. Has been done in earnest.

  In short, a dedicated measurement device for measuring fluorescence polarization (hereinafter referred to as fluorescence polarization measurement device) includes two polarizing plates in addition to the same configuration as a conventional fluorometer. (For example, refer to Patent Document 1).

  The fluorescence polarization measuring device obtains a monochrome wave (monochromatic light) from a light beam emitted from a light source by a filter or a grating prism, and polarizes it by a polarizer to obtain excitation light. When the excitation light that has passed through the polarizer is incident on the cell, it is absorbed only by molecules having a fluorescent dye in the solution. Thereafter, the vertical and parallel components of the fluorescence emitted within the characteristic fluorescence relaxation time of the fluorescent dye are detected. The detected fluorescence intensity of the vertical component and the parallel component is applied to the Perrin equation, and as a result, the degree of fluorescence polarization is calculated.

The value of the degree of fluorescence polarization depends in principle on the difference in the speed of the rotational movement of the molecule depending on the size of the molecule. For this reason, even if there is an impurity that inhibits the detection of fluorescence, the calculated degree of fluorescence polarization is not directly affected. Therefore, except for the situation where no fluorescence is detected, the degree of fluorescence polarization can be measured without pre-processing the sample, and there is no need for BF separation.
JP-A-11-813332

  In the concept of proteome, a phosphorylated, glycated or acetylated modified protein plays an important role in vivo, and its identification and function elucidation are important. However, in order to perform identification and functional elucidation, an expensive apparatus such as two-dimensional electrophoresis, HPLC (High Performance Liquid Chromatography), mass spectrometry, NMR (Nuclear Magnetic Resonance), or SPR (Surface Plasma Resonance) is used. Had to be evaluated. In addition, a high degree of expertise was often required.

  The present invention has been made to solve the conventional problems, and an object thereof is to provide an immunoassay capable of easily performing qualitative measurement and quantitative measurement.

  The immunoassay of the present invention is an immunoassay for calculating the modification rate of a modified protein in a reaction system, wherein the reaction system specifically recognizes an antibody that specifically binds to the protein of the modified protein and a modified product of the modified protein. A fluorescent specific substance to which the fluorescent substance is sensitized, and the concentration of the protein in the reaction system is measured based on the principle of immunoagglutination measurement, and the reaction system is measured based on the principle of fluorescence polarization measurement. The concentration of the modified protein is measured, and the modification rate of the modified protein is calculated from the measurement results of both.

  With this configuration, the immunoassay of the present invention is a modification that includes a basic protein and a modified product modified to the protein by simultaneously performing a simple non-BF separation-type immunoagglutination measurement principle and a fluorescence polarization immunoassay principle. Protein concentration can be measured simultaneously.

  The immunoassay of the present invention includes (1) a step of causing a test substance containing the modified protein to act on the fluorescent specific substance to form a specific binding complex, and (2) a reaction solution containing the specific binding complex. A step of measuring the degree of fluorescence polarization and the intensity of transmitted light or scattered light, (3) a step of causing an antibody to act on the reaction solution containing the specific binding complex to form a specific binding aggregate complex, (4) ) A step of measuring the fluorescence polarization degree and the transmitted light intensity or scattered light intensity of the specific binding aggregation complex, (5) the difference in fluorescence polarization degree obtained from (2) and (4), and the transmitted light intensity (6) calculating the concentration of the modified protein from the difference in the degree of fluorescence polarization, and calculating the concentration of the protein from the difference in transmitted light intensity or the difference in scattering intensity Obtained by (7) and (6) And it has a structure in which to include the steps of calculating the modification ratio than the concentration and the concentration of the protein in the modified protein.

  With this configuration, the immunoassay of the present invention is a modification that includes a basic protein and a modified product modified to the protein by simultaneously performing a simple non-BF separation-type immunoagglutination measurement principle and a fluorescence polarization immunoassay principle. The protein concentration can be measured simultaneously, and the modification rate can be easily measured.

  Furthermore, the immunoassay of the present invention has a configuration in which the transmitted light intensity measurement or the scattered light intensity measurement and the fluorescence polarization degree measurement are performed using monochromatic light.

  With this configuration, the immunoassay of the present invention does not need to have a diffraction grating or the like, so that it is possible to measure transmitted light intensity or scattered light intensity and measure fluorescence polarization with a simple optical system.

  Further, in the immunoassay of the present invention, the modified protein is composed of a glycated protein, the concentration of all the proteins in the reaction system is measured based on the principle of immunoagglutination measurement, and the concentration of the glycated protein is determined based on the principle of fluorescence polarization measurement. The ratio of the glycated protein with respect to all the proteins is calculated by measuring in this manner.

  With this configuration, the immunoassay of the present invention can be applied to the examination field because it targets a glycated protein that can be a marker substance for diabetes.

  Furthermore, in the immunoassay of the present invention, the specific substance possessed by the fluorescent specific substance is composed of an antibody that recognizes a sugar-modified amino acid region.

  Furthermore, in the immunoassay of the present invention, the specific substance possessed by the fluorescent specific substance is composed of a boronic acid derivative that forms an affinity with a diol of a sugar.

  Furthermore, in the immunoassay of the present invention, the glycated protein is composed of HbA1c.

  Furthermore, the immunoassay of the present invention has a configuration in which an antibody that specifically binds to the protein of the modified protein can form an aggregate composed of the protein.

  With this configuration, in the immunoassay of the present invention, since the scattering intensity of light incident on the reaction system changes in proportion to the square of the volume of the aggregate, the protein in the reaction system is changed based on the change in the scattering intensity. The concentration can be measured.

  Furthermore, the immunoassay of the present invention has a configuration in which the antibody is sensitized to microparticles.

  Furthermore, the immunoassay kit of the present invention is an immunoassay kit that includes a reaction system and is used in an immunoassay for measuring the modification rate of the modified protein in the reaction system, and the reaction system of the immunoassay kit includes the protein of the modified protein and An antibody that specifically binds, and a fluorescent specific substance that specifically recognizes a modified product of the modified protein and is sensitized with the fluorescent substance, and the protein in the reaction system based on an immunoagglutination measurement principle The concentration of the modified protein is measured, the concentration of the modified protein in the reaction system is measured based on the principle of fluorescence polarization measurement, and the modification rate of the modified protein is calculated from the measurement results of both.

  With this configuration, the immunoassay kit of the present invention includes a basic protein and a modified product modified to the protein by simultaneously performing a simple non-BF separation type immunoagglutination measurement principle and a fluorescence polarization immunoassay principle. The concentration of the modified protein can be measured simultaneously, and the modification rate can be easily measured.

  Furthermore, the immunoassay device of the present invention is an immunoassay device that includes a reaction system and is used in an immunoassay for measuring the modification rate of the modified protein in the reaction system, and the reaction system of the immunoassay device includes the protein of the modified protein and An antibody that specifically binds, and a fluorescent specific substance that specifically recognizes a modified product of the modified protein and is sensitized with the fluorescent substance, and the protein in the reaction system based on an immunoagglutination measurement principle The concentration of the modified protein is measured, the concentration of the modified protein in the reaction system is measured based on the principle of fluorescence polarization measurement, and the modification rate of the modified protein is calculated from the measurement results of both.

  With this configuration, the immunoassay device of the present invention includes a basic protein and a modified product modified to the protein by simultaneously performing a simple non-BF separation type immunoagglutination measurement principle and a fluorescence polarization immunoassay principle. The concentration of the modified protein can be measured simultaneously, and the modification rate can be easily measured.

  Furthermore, the reading apparatus of the present invention is used in the above-described immunoassay and has a configuration for simultaneously measuring the transmitted light intensity and the fluorescence polarization degree.

  Furthermore, the reading apparatus of the present invention is used for the above-described immunoassay and has a configuration for simultaneously measuring the scattered light intensity and the fluorescence polarization degree.

  The present invention simultaneously measures the concentration of a base protein and a modified protein containing a modified product modified by the above protein by simultaneously performing a simple non-BF separation type immunoagglutination measurement principle and a fluorescence polarization immunoassay principle. It is possible to provide an immunoassay that can easily measure the modification rate. Further, among the modified proteins, an immunoassay that can be applied to the examination of glycated proteins particularly important as a marker for diabetic patients can be provided.

  Hereinafter, an immunoassay according to an embodiment of the present invention will be described with reference to FIGS.

  The immunoassay of one embodiment of the present invention is a method for measuring the modification rate of the modified protein 13 in the reaction system 1, and the reaction system 1 includes an antibody 16 that specifically binds to the protein 11 of the modified protein 13, and the modified protein. A fluorescent specific substance that specifically recognizes the 13 modified products 12 and the fluorescent substance 15 is sensitized, and measures the concentration of the protein 11 in the reaction system 1 based on the principle of immunoagglutination measurement, The concentration of the modified protein 13 in the reaction system 1 is measured based on the principle of fluorescence polarization measurement, and the modification rate of the modified protein 13 is measured from the measurement results of both.

  A schematic diagram showing the state of the reaction system 1 is shown in FIG. As shown in FIG. 1 (a), the immunoassay of the present embodiment uses a modified protein 13 in which the protein 11 is modified with a modified product 12 among all the proteins 11 contained in the specimen 10, and the modified protein The modification rate for all 13 proteins 11 is simply measured.

  In measuring the modification rate, what is necessary is a fluorescent specific substance such as the fluorescent sensitized antibody 14, and as shown in FIG. 1 (b), the fluorescent sensitized antibody 14 is shown in FIG. 1 (a). It is introduced into the reaction system 1 in the state shown. The fluorescent sensitizing antibody 14 is sensitized with a fluorescent substance 15. In addition to this, the fluorescent specific substance may be, for example, a boronic acid derivative that exhibits a specific bond with a diol group in the glycated product of the glycation-modified protein. In this case, the fluorescent substance 15 is sensitized to boronic acid. In any case, any substance that specifically binds to the modified product 12 may be used.

  Furthermore, as a necessary requirement, there is an antibody 16 that specifically binds to the common region of the modified protein 13 and the unmodified protein 11, that is, the parent protein 11, and as shown in FIG. 16 is introduced into the reaction system 1 in the state shown in FIG.

  The antibody 16 forms an aggregated complex 17 through the protein 11 serving as a matrix. Therefore, the antibody 16 is composed of a polyclonal antibody, an antibody sensitized to fine particles such as latex or gold colloid, or a mixture of monoclonal antibodies recognizing different antigenic determinants (epitopes). If the parent protein 11 is a multimer, the antibody 16 may be composed of a monoclonal antibody that recognizes only one epitope.

  As described above, by causing the fluorescent sensitized antibody 14 and the antibody 16 to act on the specimen 10 containing the modified protein 13, the final reaction system 1 shown in FIG. Fluorescent sensitized antibody 14 and antibody 16 will be present.

  When light having an arbitrary wavelength is incident on the reaction system 1 from a light source (not shown in FIG. 1), the scattering intensity of the incident light changes before and after the formation of the aggregated complex 17, and the fluorescence polarization degree also changes. Therefore, the concentration of the entire protein 11 is calculated based on the measurement of the change in scattering intensity, the concentration of the modified protein 13 is calculated based on the measurement of the change in the degree of fluorescence polarization, and the modification of the protein is performed based on these results. Measure the rate.

  In the immunoassay of the present invention, important factors are the difference between the size of each substance before the reaction and the amount of change in the size of the aggregated complex 17, and the fluorescence lifetime of the fluorescent substance 15.

  The change in the scattering intensity is proportional to the square of the change in the volume of the substance to be detected. The fluorescence polarization degree is maintained when the rotational speed due to the size of the substance is slowed down, so that the difference in fluorescence polarization degree before and after the reaction becomes large. The fluorescence lifetime of the fluorescent substance 15 is not able to catch the difference in rotational movement if the time until the fluorescence disappears is early, and conversely, if the time until the fluorescence disappears is long, the rotational movement before and after the reaction is affected by the Brownian motion. There is no change. Therefore, the fluorescence lifetime is preferably several tens of nsec to several hundreds of nsec. A typical fluorescent substance 15 having a fluorescence lifetime of several tens of nsec has a dansyl skeleton, and a representative one of several hundred nsec of fluorescent substance 15 has a pyrene skeleton.

  In designing the immunoassay reaction system 1 according to the present invention, it is more important than designing the conventional immunoassay reaction system for all the size distributions of the aggregate complex 17 resulting from the change in scattering intensity. Thus, the degree of fluorescence polarization is kept constant. If the degree of fluorescence polarization changes within the range of the change in the size of the molecule indicating the change in scattering intensity, the correct concentration of the modified protein 13 cannot be calculated. Therefore, the reaction system 1 is designed in consideration of the important factors listed above, the difference between the size of each substance before the reaction and the size of the aggregated complex 17, the fluorescence lifetime of the fluorescent substance 15, and the like. There must be.

  The change in the degree of fluorescence polarization is statistically determined by the abundance ratio of the fluorescent sensitizing antibody 14 and the aggregated complex 17 in the reaction system 1. That is, it depends on the amount of the modified product 12 of the modified protein 13 that is the measurement object. Therefore, the concentration of the fluorescent sensitizing antibody 14 in the reaction system 1 should be set strictly. If more than necessary fluorescent sensitizing antibody 14 is introduced into reaction system 1, it becomes noise and the degree of fluorescence polarization decreases, resulting in a smaller dynamic range to be detected.

  In the present embodiment, as the reaction conditions for forming the aggregated complex 17, the factors that determine the reaction conditions when constructed by the conventional immunonephelometry and immunoturbidimetry are applied as they are. Here, the factor means reaction system pH, salt concentration, polymer additive, isoelectric point, purity, etc. of antibody or measurement object. It is also important not to cause non-specific aggregation in the reaction system 1.

  The measurement operation of the immunoassay of the present invention includes (1) a step of causing a test substance containing a modified protein to act on a fluorescent specific substance to form a specific binding complex, and (2) a reaction solution containing a specific binding complex. A step of measuring the degree of fluorescence polarization and the intensity of transmitted light or scattered light; (3) a step of causing an antibody to act on a reaction solution containing a specific binding complex to form a specific binding aggregation complex; and (4) specific binding. A step of measuring the fluorescence polarization degree and the transmitted light intensity or scattered light intensity of the aggregated composite, (5) the difference in fluorescence polarization degree obtained from (2) and (4), and the difference or scattered light intensity A step of calculating the difference in intensity; (6) a step of calculating the concentration of the modified protein from the difference in the degree of fluorescence polarization; and the concentration of the protein from the difference in transmitted light intensity or the difference in scattering intensity; (7) (6) Modified protein concentration and protein concentration obtained in Comprising the step of calculating a more modification ratio.

  The measurement operation of the immunoassay of the present embodiment can be performed using a general-purpose absorption spectrophotometer or fluorescence spectrophotometer. However, when the optical system shown in FIG. 2 and FIG. An immunoassay can be performed.

  FIG. 2 is a schematic diagram showing an example of the configuration of an optical system for performing the immunoassay of the present embodiment. One light source 21 and a scattering intensity detector 22 at a position of 90 ° with respect to the light source 21 are shown. And a detector 23 for measuring fluorescence. In order to measure the degree of fluorescence polarization, a fixed polarizer 24 is installed between the cell 20 constituting the reaction system 1 and the light source 21, while between the cell 20 and the detector 23 for fluorescence measurement. A rotatable polarizer 25 is installed. This is an optical system that can simultaneously perform immuno-ratio measurement and fluorescence polarization measurement.

  FIG. 3 is a configuration diagram showing another example of the optical system for carrying out the immunoassay of the present embodiment. The basic configuration is the same as that of the optical system shown in FIG. Instead of the detector 22, a detector 26 of transmitted light intensity is provided.

  Here, when monochromatic light is used for the measurement, it is sufficient for the optical system to include one light source, and it is not necessary to include a diffraction grating or the like for separating light waves of a plurality of wavelengths. ,preferable. In this case, it is preferable to use a light source on the long wavelength side, particularly in the vicinity of 500 to 700 nm because it is not affected by impurities or the like when measuring the scattering intensity. Accordingly, it is preferable that the fluorescent material 15 is also excited by the wavelength of light in this region.

  Further, the scattering intensity detector 22 or the transmitted light intensity detector 26, the fluorescence measurement detector 23, and the light source 21 constitute a part of the reader. Therefore, the reader can simultaneously measure the scattering intensity or transmitted light intensity of the light wave incident on the cell 20 from the light source 21 and the fluorescence polarization degree.

  The method for producing the fluorescent sensitized antibody 14 is that the primary to tertiary amino group, carboxyl group, thiol group, phenyl group, phenol group or hydroxyl group contained in the antibody for producing the fluorescent sensitized antibody 14 and the fluorescent substance 15 are used. Sensitize by reacting with. For this reason, as the fluorescent substance 15, a substance into which a functional group such as an isothiocyano group, a succinimidyl group, a sulfonyl chloride group, an azide group, a thiol group, or a maleimide group is introduced according to a known method may be used. Further, as the fluorescent substance 15, a metal complex or the like may be used.

  The modified protein 13 expresses an important function in vivo, and is, for example, a protein such as acetylation, phosphorylation, sulfation, methylation, hydroxylation, amidation, saccharification and the like. Here, for example, the function is estimated to be a protein degradation inhibitory function in the case of acetylation, an intracellular signal transduction function in the case of phosphorylation, and a hormone activation function or a protein transport function in the case of sulfation.

  In addition, glycated protein is important because it can be a marker substance for diabetes, and is preferable as a measurement object of the immunoassay of the present invention, and it can be expected that the immunoassay is applied to the examination field. Here, typical glycated proteins include glycated albumin and HbA1c. HbA1c has been conventionally measured based on the immunoassay principle and the boronic acid affinity principle. However, application to the immunoassay according to the present invention is no exception.

  As another embodiment of the present invention, there is an immunoassay kit that includes a reaction system and is used in an immunoassay for measuring the modification rate of the modified protein in the reaction system. The reaction system specifically binds to the protein of the modified protein. It has an antibody and a fluorescent specific substance in which the modified substance of the modified protein is specifically recognized and the fluorescent substance is sensitized, and the concentration of the protein in the reaction system is measured based on the principle of immunoagglutination measurement, It can be provided as an immunoassay kit characterized by measuring the concentration of the modified protein in the reaction system based on the principle of fluorescence polarization measurement and measuring the modification rate of the modified protein from the measurement results of both. The immunoassay kit has a fluorescent specific substance and an antibody, for example, in a dry state, as a necessary minimum configuration. In addition to this, for example, when the measurement object is HbA1c, it has a diluent, a denaturant, and a control reagent.

  Furthermore, as another embodiment of the present invention, there is an immunoassay device that includes a reaction system and is used in an immunoassay for measuring the modification rate of the modified protein in the reaction system. The reaction system specifically binds to the protein of the modified protein. It has an antibody and a fluorescent specific substance in which the modified substance of the modified protein is specifically recognized and the fluorescent substance is sensitized, and the concentration of the protein in the reaction system is measured based on the principle of immunoagglutination measurement, The immunoassay device may be characterized in that the concentration of the modified protein in the reaction system is measured based on the principle of fluorescence polarization measurement, and the modification rate of the modified protein is measured from the measurement results of both. In this case, for example, a device 40 having a disk shape as shown in FIG. 4 is conceivable. In the device 40, first to third chambers 41 to 43 are formed along a radial direction, and the first chamber 41 and the second chamber 42, and the second chamber 42 and the third chamber 43 include The flow paths 44 communicate with each other. The fluorescent specific substance is held in a dry state in the second chamber 42, and the antibody is held in a dry state in the third chamber 43.

  The device 40 is rotated by a rotating device 70 as shown in FIG. The rotating device 70 includes a clamper 71 that fixes the device 40, a turntable 73 that supports the device 40, a spindle motor 74 that rotates the turntable 73, and a control device 75 that controls the rotation of the spindle motor 74. Yes. Therefore, when the rotation of the device 40 is controlled by the rotating device 70, the liquid containing the measurement object introduced into the first chamber 41 is transferred through the flow path 44 by centrifugal force. The immunoassay of the invention can be performed.

  Preliminary examination results as to whether or not the immunoassay of the present invention can be carried out are shown in FIGS. Preliminary examination measured the degree of fluorescence polarization using a monoclonal antibody and a polyclonal antibody as CRP (C-reactive-protein) as a measuring object.

  In FIG. 6, black dots indicate the relationship between the concentration of CRP and the degree of fluorescence polarization when a fluorescent antibody specific for a monoclonal antibody that recognizes one epitope is added. The white dots indicate the relationship between the concentration of CRP and the degree of fluorescence polarization when a polyclonal antibody is added for comparison.

  As shown in FIG. 6, when the monoclonal antibody is added, the fluorescence polarization degree response is not observed. However, when a polyclonal antibody is further added in the presence of the monoclonal antibody, the monoclonal antibody exhibits fluorescence polarization degree responsiveness as shown in FIG. Here, the fluorescent monoclonal antibody corresponds to the antibody against the modified product 12 in the reaction system 1 according to one embodiment of the present invention, and the polyclonal antibody is a matrix protein in the reaction system 1 according to one embodiment of the present invention. This corresponds to an antibody against 11. Therefore, it was confirmed that the immunoassay of the present invention was feasible.

  As described above, the immunoassay according to the present invention is useful as an immunoassay having an effect that it is possible to easily evaluate a modified protein that provides an important function in a living body and can be used for a preliminary evaluation of proteome analysis. .

The schematic diagram which shows the state of the reaction system of the immunoassay of one embodiment of this invention 1 is a schematic diagram showing an example of the configuration of an optical system according to an embodiment of the present invention. 1 is a schematic diagram showing an example of the configuration of an optical system according to an embodiment of the present invention. Schematic diagram showing the structure of an immunoassay device according to another embodiment of the present invention. The schematic diagram which shows the structure of the rotation apparatus in other embodiment of this invention. A graph showing the relationship between the concentration of CRP and the degree of fluorescence polarization when a monoclonal antibody is added Graph showing the relationship between the concentration of CRP and the degree of fluorescence polarization when a monoclonal antibody and a polyclonal antibody are added

Explanation of symbols

1 Reaction System 10 Specimen 11 Protein 12 Modified Product 13 Modified Protein 14 Fluorescent Sensitized Antibody 15 Fluorescent Substance 16 Antibody 17 Aggregation Complex 20 Cell 21 Light Source 22 Scattering Intensity Detector 23 Fluorescence Measurement Detector 24 Fixed Polarizer 25 Rotating Polarizer 26 Detector of Transmitted Light Intensity 40 Device 41 First Chamber 42 Second Chamber 43 Third Chamber 44 Channel 70 Rotating Device 71 Clamper 73 Turntable 74 Spindle Motor 75 Control Device

Claims (13)

An immunoassay for calculating a modification rate of a modified protein in a reaction system,
The reaction system includes an antibody that specifically binds to the protein of the modified protein, and a fluorescent specific substance that specifically recognizes the modified product of the modified protein and sensitizes the fluorescent substance,
Measure the concentration of the protein in the reaction system based on the principle of immunoagglutination measurement,
Measure the concentration of the modified protein in the reaction system based on the principle of fluorescence polarization measurement,
An immunoassay, wherein the modification rate of the modified protein is calculated from the measurement results of both. The immunoassay is:
(1) a step of causing a test substance containing the modified protein to act on the fluorescent specific substance to form a specific binding complex; (2) a fluorescence polarization degree of a reaction solution containing the specific binding complex and transmitted light; A step of measuring intensity or scattered light intensity, (3) a step of causing an antibody to act on the reaction solution containing the specific binding complex to form a specific binding aggregation complex, and (4) the specific binding aggregation complex. A step of measuring the degree of fluorescence polarization and the intensity of transmitted light or scattered light, (5) the difference in fluorescence polarization obtained from (2) and (4), the difference in transmitted light intensity or the difference in scattered intensity (6) calculating the concentration of the modified protein from the difference in the degree of fluorescence polarization, and calculating the concentration of the protein from the difference in transmitted light intensity or the difference in scattering intensity; (7) (6 The concentration of the modified protein obtained in Step of calculating the modification ratio than the concentration of white matter,
The immunoassay according to claim 1, comprising: The immunoassay according to claim 2, wherein the measurement of the transmitted light intensity or the measurement of the scattered light intensity and the measurement of the fluorescence polarization degree are performed using monochromatic light. The modified protein is composed of a glycated protein, the concentration of all proteins in the reaction system is measured based on the principle of immunoaggregation measurement, and the concentration of the glycated protein is measured based on the principle of fluorescence polarization measurement. The immunoassay according to claim 1 or 2, wherein the ratio of the glycated protein to the protein is calculated. The immunoassay according to claim 4, wherein the specific substance possessed by the fluorescent specific substance comprises an antibody that recognizes a sugar-modified amino acid region. 5. The immunoassay according to claim 4, wherein the specific substance possessed by the fluorescent specific substance is composed of a boronic acid derivative that forms an affinity with a sugar diol. The immunoassay according to claim 6, wherein the glycated protein is composed of HbA1c. The immunoassay according to claim 1 or 2, wherein the antibody that specifically binds to the protein of the modified protein can form an aggregate composed of the protein. The immunoassay according to claim 8, wherein the antibody is sensitized to microparticles. An immunoassay kit for use in an immunoassay comprising a reaction system and measuring the modification rate of the modified protein in the reaction system,
The reaction system includes an antibody that specifically binds to the protein of the modified protein, and a fluorescent specific substance that specifically recognizes the modified product of the modified protein and sensitizes the fluorescent substance,
Measure the concentration of the protein in the reaction system based on the principle of immunoagglutination measurement,
Measure the concentration of the modified protein in the reaction system based on the principle of fluorescence polarization measurement,
An immunoassay kit, wherein the modification rate of the modified protein is calculated from the measurement results of both. An immunoassay device for use in an immunoassay comprising a reaction system and measuring a modification rate of a modified protein in the reaction system,
The reaction system includes an antibody that specifically binds to the protein of the modified protein, and a fluorescent specific substance that specifically recognizes the modified product of the modified protein and sensitizes the fluorescent substance,
Measure the concentration of the protein in the reaction system based on the principle of immunoagglutination measurement,
Measure the concentration of the modified protein in the reaction system based on the principle of fluorescence polarization measurement,
An immunoassay device, wherein the modification rate of the modified protein is calculated from the measurement results of both. 12. A reader which is used in the immunoassay according to any one of claims 1 to 11 and simultaneously measures the transmitted light intensity and the fluorescence polarization degree. 12. A reader which is used in the immunoassay according to any one of claims 1 to 11 and simultaneously measures the scattered light intensity and the fluorescence polarization degree.

Images