Antigen detection method and antigen detection device

Abstract

Provided is a novel antigen detection method capable of detecting an antigen, as a substance to be detected, by using a signal transmission line for transmitting a high-frequency signal. This antigen detection method for detecting an antigen in a sample comprises: a step in which a sample, containing an antigen reacted with both a primary antibody for fixing the antigen to a carrier and a secondary antibody labelled with magnetic particles, is produced; a step in which the sample is placed close to or in contact with a probe in which a signal transmission line is formed; a step in which a DC magnetic field in a direction along the signal transmission line is applied to the sample by a magnetic field application means; a step in which a high-frequency signal is supplied to the signal transmission line by a signal measurement instrument electrically connected to the signal transmission line; a step in which a signal being transmitted along the signal transmission line in a state in which the DC magnetic field is being applied is measured by the signal measurement instrument over a predetermined frequency band; and a step in which the antigen is detected by an arithmetic processing means on the basis of a signal corresponding to the resonance frequency of ferromagnetic resonance observed in the predetermined frequency band.

Description

Antigen detection method and antigen detection device

[0001] The present invention relates to an antigen detection method and an antigen detection device for detecting an antigen, which is a substance to be detected, by magnetic immunoassay using a magnetic marker.

[0002] Immunoassays are used in medical diagnostics to detect biological substances such as disease-related proteins and pathogenic bacteria. Immunoassays utilize an antigen-antibody reaction in which an antigen, the substance to be detected, specifically binds to an antibody. The antibody is labeled with a substance called a marker, and the amount of the antigen can be measured by detecting the signal from the marker on the antibody that has bound to the antigen.

[0003] One type of immunoassay is optical immunoassay, in which an optical marker such as a fluorescent enzyme is added to an antibody whose binding ability to a target substance is known to label it, and the degree of binding with the target substance is optically detected. However, most optical immunoassays require a washing and removal process to separate the optical marker that has bound to the target substance from the optical marker that has not bound, which makes the testing process complicated and time-consuming.

[0004] On the other hand, unlike optical immunoassays, a technology that uses magnetic techniques to detect target substances is known as magnetic immunoassays. Magnetic immunoassays are a method of detecting antigen-antibody reactions using magnetic particles and a magnetic sensor. Magnetic particles (hereinafter referred to as magnetic markers) are attached to antibodies to label them, and the magnetic signal from the magnetic marker is detected by the magnetic sensor, utilizing the differences in the Brownian relaxation properties of the magnetic markers, to determine the degree of binding with the target substance, the antigen.

[0005] Patent Documents 1 to 5 disclose configurations in which an antigen is detected based on a magnetic signal generated by Brownian relaxation of a magnetic marker using a SQUID (Superconducting Quantum Interference Device) as a magnetic sensor.

[0006] Furthermore, Patent Document 6 discloses a magnetic field measurement device that uses a magnetoresistive element (MR sensor) to measure the Brownian relaxation characteristics of a magnetic marker as a difference in AC magnetic susceptibility. That is, a bound marker with a larger volume has lower tracking ability to a high-frequency AC magnetic field than an unbound marker with a smaller volume, and the AC magnetic susceptibility depends on the frequency and the Brownian relaxation time. Therefore, the amount of bound marker can be measured by measuring the AC magnetic susceptibility using a magnetoresistive element (MR sensor).

[0007] Furthermore, Patent Document 7 discloses a magnetic foreign matter inspection device that uses a thin-film magnetic sensor (magnetoresistive sensor, magnetic impedance sensor) that has directivity in the magnetic field detection direction to detect the presence or absence of magnetic foreign matter within an object to be inspected.

[0008] Furthermore, Patent Documents 8 and 9 disclose a magnetic field measuring device that detects antigens, which are substances to be detected, by utilizing changes in magnetic responsiveness, by rotating a sample containing nano-sized magnetic particles as magnetic markers using a rotation mechanism and switching the magnetic field at each rotation period, based on a method proposed by the inventor of the present application.

[0009] Japanese Patent Application Laid-Open No. 2015-163846 Japanese Patent Application Laid-Open No. 2007-240349 Japanese Patent Application Laid-Open No. 2009-115529 Japanese Patent Application Laid-Open No. 1-112161 Japanese Patent Application Laid-Open No. 2001-033455 Japanese Patent No. 5560334 Japanese Patent Application Laid-Open No. 2014-159984 Japanese Patent Application Laid-Open No. 2018-194305 Japanese Patent Application Laid-Open No. 2020-159871

[0010] In the magnetic methods using magnetic particles as exemplified above, the device configuration is relatively large, and a highly sensitive magnetic sensor is required to improve the S / N ratio. The present inventors have now developed a novel antigen detection method that has a relatively simple configuration, does not use a magnetic sensor, and uses a signal transmission line that transmits an ultra-wideband high-frequency signal exceeding 60 GHz as a sensor probe.

[0011] SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a novel antigen detection method and antigen detection device that can detect an antigen, which is a substance to be detected, using a signal transmission line that transmits a high-frequency signal.

[0012] The antigen detection method of the present invention for achieving the above-mentioned object is an antigen detection method for detecting an antigen contained in a sample in which magnetic particles and an antigen bound to the magnetic particles are carried on a carrier, and is characterized by comprising the steps of: generating the sample containing the antigen that has reacted with both a primary antibody immobilized on the carrier and a secondary antibody labeled with the magnetic particles, which specifically binds to the antigen; placing the sample in close proximity to or in contact with a probe on which a signal transmission line is formed; applying a DC magnetic field to the sample in a direction along the signal transmission line by a magnetic field application means; supplying a high-frequency signal to the signal transmission line by a signal measuring instrument electrically connected to the signal transmission line; measuring, by the signal measuring instrument, the signal transmitted through the signal transmission line while the DC magnetic field is applied, over a predetermined frequency band; and detecting, by an arithmetic processing means, the antigen based on the signal corresponding to the resonance frequency of ferromagnetic resonance observed in the predetermined frequency band.

[0013] The antigen detection device of the present invention is an antigen detection device that detects an antigen contained in a sample in which magnetic particles and antigens bound to the magnetic particles are carried on a carrier, and is characterized by comprising: a probe on which a signal transmission line is formed and which is placed in close proximity to or in contact with the sample; a magnetic field application means that applies a DC magnetic field to the sample in a direction along the signal transmission line; a signal measuring device that supplies a high-frequency signal to the signal transmission line and measures the signal transmitted through the signal transmission line over a predetermined frequency band while the DC magnetic field is applied; and an arithmetic processing means that detects the antigen based on the signal corresponding to the resonance frequency of ferromagnetic resonance observed in the predetermined frequency band.

[0014] According to the present invention, in a method for conducting a magnetic immunoassay using magnetic particles, a DC magnetic field is applied to a sample in which magnetic particles and antigens are bound, and antigens can be detected based on ferromagnetic resonance observed by measuring signals over a wide frequency band using a probe using a signal transmission line. By using the signal transmission line, ferromagnetic resonance of magnetic particles can be measured in a high-frequency band, making it possible to evaluate the magnetic susceptibility of magnetic particles over an ultra-wide band up to about 60 GHz, thereby enabling rapid, simple, low-cost, and highly accurate antigen detection using magnetic particles.

[0015] 3(b) is a diagram showing a schematic configuration example of an antigen detection device in an embodiment of the present invention; FIG. 3(c) is a diagram showing a configuration example of a probe 10; FIG. 3(d) is a diagram showing a schematic component structure of a sample 1 containing an antigen to be detected; FIG. 3(e) is a flowchart showing a preparation process of the sample 1 shown in FIG. 3(b); FIG. 3(f) is a flowchart showing a first procedure of an antigen detection method in an embodiment of the present invention; 21 FIG. 1 is a diagram showing an example of measurement results of magnetic susceptibility κ; FIG. 2 is a diagram showing the relationship between magnetic susceptibility and the amount of antigen (GDF15); FIG. 3 is a flowchart showing a second procedure of the antigen detection method according to the embodiment of the present invention; 21 This figure shows an example of the measurement results of the transmission coefficient S 21 FIG. 10 is a graph showing the relationship between the difference value and the amount of antigen (GDF15).

[0016] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. However, the technical scope of the present invention is not limited to these preferred embodiments.

[0017] 1 is a diagram showing a schematic configuration example of an antigen detection device according to an embodiment of the present invention. The antigen detection device according to the embodiment of the present invention includes a probe 10 placed in contact with or in close proximity to a sample 1 containing an antigen, which is a protein to be detected (e.g., GDF15), and magnetic particles bound to the antigen; a network analyzer 20, which is a signal measuring device that supplies a high-frequency signal to the probe 10; an arithmetic processing device (e.g., a computer such as a personal computer) 30 that executes predetermined arithmetic processing such as numerical analysis; and an electromagnetic coil 50, which is, for example, a double-yoke electromagnet, that applies a DC magnetic field to the sample 1 held by the probe 10. The process of preparing the sample 1 will be described later.

[0018] The probe 10 forms a signal transmission line that is, for example, a microstrip line, and is connected to a network analyzer 20 via a signal cable (for example, a coaxial cable) 3. A high-frequency current signal is supplied by the network analyzer 20, which is a current supply source, to measure the transmission coefficient S 21 The signal data is input to a processing unit (computer device) 30, and the magnetic susceptibility of the sample 1 is calculated by a predetermined numerical analysis process. An example of the configuration of the probe 10 will be described later.

[0019] The arithmetic processing device 30 functions as an arithmetic processing means for calculating the magnetic susceptibility of the sample 1, and executes a predetermined arithmetic processing program for calculating the magnetic susceptibility. Based on the calculated magnetic susceptibility, the amount of antigen contained in the sample 1 can be detected.

[0020] The electromagnetic coil 50 is a magnetic field applying means that applies a DC magnetic field to the sample 1 placed in contact with or close to the probe 10 in the direction in which the signal transmission line of the probe 10 extends (in a direction parallel to the signal transmission line).

[0021] 2A and 2B are diagrams showing examples of the configuration of the probe 10, where FIG. 2A shows the form of the probe 10 in a first configuration example, FIG. 2B shows the form of the probe 10 in a second configuration example, and FIG. 2C shows the form of the probe 10 in a third configuration example.

[0022] The first configuration example of the probe 10 shown in Figure 2(a) is configured with a microstrip conductor 11, a flexible substrate 12, a fluororesin substrate 13, a ground conductor 14, and a pair of connectors 15 connected to both ends of the microstrip conductor 11. The microstrip conductor 11 is processed into a straight microstrip line by etching. The microstrip conductor 11 and the flexible substrate 12 are integrally fixed by chemical or thermal treatment. The ground conductor 14 is formed of, for example, copper foil. The configuration in which the flexible substrate 12 and the fluororesin substrate 13, which are dielectrics, are sandwiched between the microstrip conductor 11 and the ground conductor 14 forms a microstrip line.

[0023] A second configuration example of the probe 10 shown in FIG. 2( b) includes a microstrip conductor 11, a flexible substrate (sheet) 12, a ground conductor 14, and a pair of connectors 15 connected to both ends of the microstrip conductor 11. The connector 15 connects to the signal cable 3 ( FIG. 1 ). The microstrip conductor 11 and the flexible substrate 12 are integrally fixed by chemical or thermal treatment. In this second configuration example, the fluororesin substrate 13 of the first configuration example shown in FIG. 2( a) is omitted, and the flexible substrate 12 is pressed against a ground conductor 14 having a planar structure and a curved structure. The ground conductor 14 is made of a metal material such as copper. The microstrip conductor 11 is composed of a central linear portion 11a and curved portions 11b on both sides. The microstrip conductor 11 extends into the ground conductor 14 through an opening 14a provided in the ground conductor 14 and is connected to the connector 15 on the opposite side. The characteristic impedance of both the linear portion 11a and the curved portion 11b of the microstrip conductor 11 is matched to 50 Ω. As in the first configuration example, the configuration in which the flexible substrate 12, which is a dielectric, is sandwiched between the microstrip conductor 11 and the ground conductor 14 forms a microstrip line.

[0024] In a third configuration example of the probe 10 shown in FIG. 2( c), the front shape of the metal body portion including the ground conductor 14 is approximately triangular, with a convexly curved portion formed at the apex. The microstrip conductor 11 and the flexible substrate 12, which is a dielectric, are curved along the convexly curved portion so as to straddle the apex. The connectors 15 are attached along both slopes extending downward from the apex of the ground conductor 14. Preferably, the connectors 15 hold the curved microstrip line 11 at an angle of approximately 90 degrees. Therefore, the hypotenuses sandwiching the apex where the microstrip conductor 11 straddles are formed at an angle of approximately 90 degrees, and the connectors 15 on both sides are positioned at that angle. By setting the angle between the two connectors 15 at approximately 90 degrees, the probe 10 can be placed close to the sample regardless of the size of the sample to be measured. Furthermore, by forming the microstrip line 11 in a curved shape without any bends, degradation of the characteristic impedance is minimized, enabling wide-band measurements with a high S / N ratio.

[0025] 3A and 3B are diagrams showing the component structure of sample 1 containing the antigen to be detected. Fig. 3A shows the component structure of sample 1 prepared by the so-called direct method, and Fig. 3B shows the component structure of sample 1 prepared by the so-called ABC method. In either case, sample 1 has a structure in which the antigen substance to be detected is immobilized on a substrate by a primary antibody, and further, magnetic particles are bound to the antigen by a secondary antibody.

[0026] In this embodiment, the antigen to be detected is growth differentiation factor 15 (GDF15), a type of protein. Detection of GDF15 can be applied to the diagnosis of mitochondrial disease. It is thought that GDF15 may be increased in the blood of patients with mitochondrial disease, and there is a need to establish a method for detecting GDF15.

[0027] In Figure 3(a), antigen (GDF15) 1a contained in sample 1 binds to primary antibody 1c immobilized on a gold (Au) thin film (carrier) 1b on a glass substrate, and antigen (GDF15) 1a further binds to magnetic particles 1e via secondary antibody 1d. In sample 1 of Figure 3(a), primary antibody 1c is human GDF15 antibody (monoclonal mouse IgG2B, R&D Systems) that specifically binds to antigen (GDF15), secondary antibody 1d is also human GDF15 antibody (monoclonal mouse IgG2B, R&D Systems), and magnetic particles 1e are, for example, Micromer-M. Secondary antibody 1d is labeled with magnetic particles 1e. A self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (COOH-SAM) is formed on the surface of a thin gold (Au) film covering a glass substrate, and primary antibody 1c is immobilized thereon. A solution of GDF15, which is antigen 1a, is then exposed to the SAM. Subsequently, magnetic particles 1e that have been pre-reacted with secondary antibody 1d are bound to the SAM to produce sample 1.

[0028] In addition, in the sample 1 shown in Figure 3(b), the antigen (GDF15) 1a binds to a primary antibody 1c immobilized on a gold (Au) thin film (carrier) 1b on a glass substrate. Furthermore, the antigen (GDF15) 1a binds to magnetic particles 1e via a secondary antibody 1d. Furthermore, in the sample 1 shown in Figure 3(b), multiple magnetic particles 1e are linked to the antigen (GDF15) via biotin-coated fluorescent particles 1f. In the sample 1 shown in Figure 3(b), the primary antibody 1c is a human GDF15 antibody (monoclonal mouse IgG2B, R&D Systems), the secondary antibody 1d is a human / primate GDF15 biotinylated antibody (goat poly, R&D Systems), and the magnetic particles 1e are streptavidin-coated magnetic nanoparticles (SPHERO-Coated Superparamagnetic Particles).

[0029] In the sample 1 shown in Figure 3(b), the magnetic particles 1e are coated with avidin (streptavidin), the secondary antibody 1d is coated with biotin, and biotin-coated fluorescent particles are added to bond the avidins together. This utilizes the avidin-biotin interaction to create a component structure in which multiple magnetic particles 1e bind to one antigen 1a. This improves the detection sensitivity and S / N ratio in signal measurement during the antigen detection process described below.

[0030] Fig. 4 is a flowchart showing the fabrication process of Sample 1 shown in Fig. 3(b). A self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (COOH-SAM) is formed on the surface of a gold (Au) thin film covering a glass substrate (S100).

[0031] Next, a solution of the primary antibody, Human GDF15 Antibody (Monoclonal Mouse IgG2B, R&D Systems), is reacted with the COOH-SAM, and the primary antibody 1c is immobilized by covalent bonding to the COOH-SAM on the glass substrate (S102).

[0032] Next, the surface on which the primary antibody 1c is immobilized is exposed to a solution containing an antigen (GDF15) (S104) to allow the antigen-antibody reaction to occur. Note that in the measurement experiment, samples containing different concentrations of the antigen (GDF15 concentrations: 50, 5, 0.5, 0 μg / ml) are prepared.

[0033] Next, avidin-coated magnetic particles (SPHERO streptoavidin-coated Superparamagnetic Particles) that have been pre-reacted with the secondary antibody, Human / Primate GDF15 Biotinylated Antibody (Antibody Polyclonal Goat IgG, R&D Systems), are added (S106).

[0034] Furthermore, biotin-coated fluorescent particles are applied, and then avidin-coated magnetic particles are added (S108). The above steps S106 and S108 are repeated three times to prepare Sample 1.

[0035] 5 is a flowchart showing the first procedure of the antigen detection method according to the embodiment of the present invention. The sample 1 prepared by the above steps is placed in contact with the probe 10 via an insulating PET film (50 μm thick) (S200).

[0036] The electromagnetic coil 50 applies a DC magnetic field to the sample 1 in the direction in which the microstrip conductor 11 of the probe 10 extends (parallel to the microstrip conductor 11), and the signal measuring device (network analyzer) 20 is calibrated (S202). Specifically, a relatively strong DC magnetic field (e.g., 1.8 T) is applied by the electromagnetic coil 50 to magnetically saturate the sample 1, and the network analyzer 20 is calibrated. This eliminates the electrical length of the probe 10 and the coaxial cable 3, the DC impedance of the sample 1, non-magnetic signals, etc. This calibration enables measurements based on the state in which a predetermined magnetic field is applied to the sample.

[0037] After the calibration, the transmission coefficient S of the high frequency current signal when a DC magnetic field is applied by the electromagnetic coil 50 is 21 is measured over a predetermined frequency band (S204). The DC magnetic field applied by the electromagnetic coil 50 is in the direction in which the microstrip conductor 11 extends, which is perpendicular to the AC magnetic field generated by the high-frequency signal transmitted through the microstrip conductor 11.

[0038] Permeability coefficient S 21 In the measurement (S204), the strength of the DC magnetic field is changed, and the transmission coefficient S in each state is measured by applying multiple DC magnetic fields with different strengths (for example, 1.47, 1.1, 0.73, 0.37, 0.18, 0 T). 21 The frequency dependence of the transmission coefficient S corresponding to the change in magnetization in the direction of the DC magnetic field applied by the electromagnetic coil 50 is measured.21 The amount of the antigen GDF15 to be detected in the measurement results of FIG. 6 is 5 μg / ml.

[0039] Figure 6 shows the transmission coefficient S 21 This figure shows an example of the measurement results, and the permeability coefficient S 21 The measurement results in Figure 6 show that the transmission coefficient S 21 The real part of the equation is shown. By applying a relatively large DC magnetic field (greater than 1 T), ferromagnetic resonance is observed in the frequency band above 10 GHz. The absorption of magnetic particles in ferromagnetic resonance was observed around 10-20 GHz. At relatively low DC magnetic fields below 0.37 T, the magnetic particles are randomly oriented, so no clear ferromagnetic resonance was observed. However, by increasing the magnitude of the DC magnetic field, the ferromagnetic resonance becomes sharper and the resonance frequency is shifted higher.

[0040] The measured transmission coefficient S 21 The magnetic susceptibility is calculated by calculation based on the above. In the calculation process to calculate the magnetic susceptibility, first, the transmission coefficient S is calculated by the following formula (1): 21 is converted into the impedance Z of the sample 1 (S206).

[0041] (Number 1) Z=100(1-S 21 ) / S 21 ...(1)

[0042] In step S202, a strong magnetic field is applied by the electromagnetic coil 50 to saturate the sample 1, and the measured transmission coefficient S 21 is used as the background, and the transmission coefficient S 21 Next, the strength of the magnetic field applied by the electromagnetic coil 50 is changed, and the transmission coefficient S 21 The transmission coefficient S 21reflects the magnetic properties due to the magnetic field applied to the sample 1, and it is possible to obtain the impedance Z that reflects the rotation of the magnetization of the magnetic particles in the sample 1. Furthermore, the magnetic susceptibility κ is calculated based on the obtained impedance Z (S208).

[0043] In impedance Z, the real part is the loss (resistance component) R of sample 1, and the imaginary part is the product ωL of the inductance component of sample 1 and the angular frequency ω. The inductance component L corresponds to the real part κ' of the magnetic susceptibility κ of sample 1, and the resistance component R corresponds to the imaginary part κ'' of the magnetic susceptibility κ of sample 1. The magnetic susceptibility (complex number) κ of sample 1 is expressed by the following equation (2):

[0044] (Math. 2) κ=κ'-jκ''...(2)

[0045] FIG. 7 shows an example of measurement results of magnetic susceptibility κ, showing the frequency dependence of magnetic susceptibility κ at a DC magnetic field value of 1.47 T, with FIG. 7(a) showing the real part κ' of magnetic susceptibility κ and FIG. 7(b) showing the imaginary part κ''. The measurement results in FIG. 7 show the magnetic susceptibility κ when the amount of GDF15, the antigen to be detected, is varied to 50, 5, 0.5, and 0 μg / ml.

[0046] In the example of measurement results shown in Figure 7, the value of magnetic susceptibility κ on the vertical axis is the relative value (au) of magnetic susceptibility κ, which is simply calculated by dividing impedance Z by frequency f. The absolute value of magnetic susceptibility κ can be calculated by performing a known electromagnetic field analysis process (e.g., finite element analysis) to obtain table data in advance showing the relationship between magnetic susceptibility and inductance. The table data of magnetic susceptibility and inductance can be obtained by calculating the inductance L when magnetic susceptibility κ is changed using Maxwell's equations through finite element analysis. The computer device 30 calculates the table data showing the relationship between magnetic susceptibility and inductance and stores it as data.

[0047] In Figure 7(a), the real part of the magnetic susceptibility κ varied depending on the amount of GDF15 across the entire frequency range. In particular, in the frequency range below the ferromagnetic resonance (FMR) frequency (10-20 GHz), the real part of the magnetic susceptibility κ increased with increasing GDF15 content. As the antigen (GDF15) density increased, the magnetic particles reacted more with the antigen, resulting in an increase in the real part of the magnetic susceptibility.

[0048] In addition, in FIG. 7(b), the value of the imaginary part of the magnetic susceptibility κ is the loss peak value at the ferromagnetic resonance (FMR) resonance frequency (around 10-20 GHz) that corresponds to the amount of GDF15, and the value of the imaginary part of the magnetic susceptibility κ increases as the amount of GDF15 increases.

[0049] Figure 8 shows the relationship between magnetic susceptibility and the amount of antigen (GDF15), showing the average real part of magnetic susceptibility below 10 GHz and the imaginary part of magnetic susceptibility (loss peak value) at the ferromagnetic resonance frequency as a function of the amount of antigen (antigen density). As is clear from Figure 8, the real part of magnetic susceptibility (average real part of magnetic susceptibility in the frequency band below the resonance frequency (approximately below 10 GHz)) and the imaginary part (loss peak value at the resonance frequency) increased with increasing amount of antigen (GDF15). Therefore, it was confirmed that by measuring the frequency dependence of magnetic susceptibility over a wide frequency range in this embodiment, it is possible to detect antigens and determine the amount of antigen (antigen density).

[0050] In the above-described embodiment of the present invention, a method for measuring the magnetic susceptibility of magnetic particles has been described. However, since the magnetic susceptibility has a relationship with the magnetic permeability of the magnetic particles shown in the following equation (3), the magnetic susceptibility κ can be converted to the relative magnetic permeability μ, which can be considered to be essentially the same as measuring the relative magnetic permeability μ.

[0051] (Math. 3) κ=μ−1 ...(3)

[0052] The signal transmission line configured in the probe 10 is not limited to the microstrip line shown in the above configuration example, but may be configured using, for example, a coplanar line or a coaxial line.

[0053] 9 is a flowchart showing the second procedure of the antigen detection method according to the embodiment of the present invention. In the first procedure shown in FIG. 5, the transmission coefficient S corresponding to the resonance frequency of ferromagnetic resonance is calculated. 21 In the first procedure, the magnetic susceptibility was calculated from the magnetic susceptibility, and the amount of antigen was determined based on the magnetic susceptibility. In the second procedure shown in FIG. 9, two transmission coefficients S 21 A method is proposed to calculate the amount of antigen based on the difference between the values ​​of

[0054] In FIG. 9, similarly to the first procedure, the fabricated sample 1 is placed in direct contact with the probe 10 or via an insulating PET film (50 μm thick) (S200).

[0055] The electromagnetic coil 50 applies a DC magnetic field to the sample 1 in the direction in which the microstrip conductor 11 of the probe 10 extends (parallel to the microstrip conductor 11), and the signal measuring device (network analyzer) 20 is calibrated (S202). Specifically, a relatively strong DC magnetic field (e.g., 2 T) is applied by the electromagnetic coil 50 to magnetically saturate the sample 1, and the network analyzer 20 is calibrated. This eliminates the electrical length of the probe 10 and the coaxial cable 3, the DC impedance of the sample 1, non-magnetic signals, etc. This calibration enables measurements based on the state in which a predetermined magnetic field is applied to the sample.

[0056] After the calibration, the transmission coefficient S of the high frequency current signal when a DC magnetic field is applied by the electromagnetic coil 50 is 21 is measured over a predetermined frequency band (S204). The DC magnetic field applied by the electromagnetic coil 50 is in the direction in which the microstrip conductor 11 extends, which is perpendicular to the AC magnetic field generated by the high-frequency signal transmitted through the microstrip conductor 11.

[0057] Permeability coefficient S 21 In the measurement (S204), the transmission coefficient S is measured when a DC magnetic field with a different strength from that applied by the calibration in S202 is applied, in particular, when a DC magnetic field with a smaller strength (for example, 1.5 T) is applied. 21The frequency dependence of the transmission coefficient S corresponding to the change in magnetization in the direction of the DC magnetic field applied by the electromagnetic coil 50 is measured. 21 The amount of the antigen GDF15 to be detected in the measurement results of FIG. 6 is 5 μg / ml.

[0058] Figure 10 shows the transmission coefficient S 21 10(a), (b), (c), and (d) show examples of measurement results, and the permeability coefficient S 21 9 plots the frequency dependence of the real part of the magnetic field. In each measurement result, two ferromagnetic resonances (FMRs) with different resonance frequencies can be observed. One is the ferromagnetic resonance (indicated as FMR(2T) in the figure) corresponding to the DC magnetic field (e.g., 2T) for calibration applied in step S202 of the second procedure shown in Fig. 9 . The other is the ferromagnetic resonance (indicated as FMR(1.5T) in the figure) corresponding to the DC magnetic field (e.g., 1.5T) applied in step S204 of the second procedure shown in Fig. 9 .

[0059] In FIG. 9, the transmission coefficient S corresponding to the resonance frequencies of these two ferromagnetic resonances 21 The difference value dS of the real part of 21 is calculated, and the amount of antigen (GDF15) is obtained based on the difference value (S210).

[0060] Figure 11 shows the transmission coefficient S 21 The difference value dS 21 11 is a graph showing the relationship between the permeability coefficient S and the amount of antigen (GDF15). As shown in FIG. 11, as the amount (concentration) of antigen (GDF15) increases, the permeability coefficient S 21 The difference value dS 21 Therefore, it was confirmed that the antigen can be detected and the amount of the antigen (antigen density) can be calculated by measuring the frequency dependence of the transmission coefficient over a wide frequency range and calculating the difference in the transmission coefficient corresponding to the resonant frequency of the observed ferromagnetic resonance.

[0061] The present invention is not limited to the above-described embodiments, and it goes without saying that the present invention also includes design changes within the scope of the gist, including various modifications and alterations that would be conceivable to a person with ordinary knowledge in the field of the present invention.

[0062] 1: Sample, 3: Cable, 10: Probe, 11: Microstrip conductor, 12: Flexible substrate, 13: Fluorine resin substrate, 14: Ground conductor, 15: Connector, 20: Signal measuring instrument, 30: Computer device, 50: Electromagnetic coil

Claims

1. A method for detecting an antigen contained in a sample in which magnetic particles and an antigen bound to the magnetic particles are carried on a carrier, the method comprising the steps of: generating the sample containing the antigen that has reacted with both a primary antibody immobilized on the carrier, which specifically binds to the antigen, and a secondary antibody labeled with the magnetic particles; placing the sample in close proximity to or in contact with a probe on which a signal transmission line is formed; applying a DC magnetic field to the sample in a direction along the signal transmission line by a magnetic field application means; supplying a high-frequency signal to the signal transmission line by a signal measurement device electrically connected to the signal transmission line; measuring, over a predetermined frequency band, by the signal measurement device, the signal transmitted through the signal transmission line while the DC magnetic field is applied; and detecting, by a calculation processing means, the antigen based on the signal corresponding to the resonant frequency of ferromagnetic resonance observed in the predetermined frequency band.

2. The antigen detection method according to claim 1, wherein said arithmetic processing means determines the magnetic susceptibility of said sample from said signal, and determines the amount of said antigen based on said magnetic susceptibility.

3. The antigen detection method according to claim 2, wherein the calculation processing means determines the amount of the antigen using the value of the real part or the value of the imaginary part of the magnetic susceptibility.

4. The antigen detection method according to claim 2, characterized in that the calculation processing means detects the amount of the antigen using a peak value of magnetic susceptibility corresponding to the resonance frequency of ferromagnetic resonance observed in the specified frequency band.

5. The antigen detection method according to claim 2, characterized in that the calculation processing means detects the amount of the antigen using an average value of magnetic susceptibility corresponding to a frequency lower than the resonant frequency of ferromagnetic resonance observed in the specified frequency band.

6. The antigen detection method according to claim 1, wherein the signal transmission line is a microstrip line.

7. The antigen detection method according to claim 1, wherein the strength of the DC magnetic field is changed by the magnetic field application means, and the signal transmitted through the signal transmission line is measured.

8. The antigen detection method described in claim 2, characterized in that in the step of determining the magnetic susceptibility, the antigen bound to the magnetic particles contained in the sample is detected based on the frequency at which the magnetic susceptibility peaks.

9. The antigen detection method according to claim 1, wherein the calculation processing means determines the amount of the antigen based on the difference value of each of the signals corresponding to the resonance frequencies of two different ferromagnetic resonances.

10. The antigen detection method according to claim 9, wherein the two signals correspond to the resonant frequencies of ferromagnetic resonance observed when DC magnetic fields of different strengths are applied.

11. The antigen detection method according to claim 9, wherein the signal corresponds to the transmission coefficient of the signal transmission line.

12. An antigen detection device for detecting an antigen contained in a sample in which magnetic particles and antigens bound to the magnetic particles are carried on a carrier, comprising: a probe on which a signal transmission line is formed and which is placed in close proximity to or in contact with the sample; magnetic field application means for applying a DC magnetic field to the sample in a direction along the signal transmission line; a signal measuring device for supplying a high-frequency signal to the signal transmission line and measuring, over a predetermined frequency band, the signal transmitted through the signal transmission line while the DC magnetic field is applied; and arithmetic processing means for detecting the antigen based on the signal corresponding to the resonance frequency of ferromagnetic resonance observed in the predetermined frequency band.

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