Antigen detection device, antigen detection method, and detection kit

Abstract

Provided is an improved, highly convenient antigen detection device enabling more accurate and efficient measurement. This antigen detection device detects a detection-target antigen in a vessel accommodating a sample containing magnetic particles carrying antibodies and a specimen having the detection-target antigen which can bind to the antibodies of the magnetic particles. The antigen detection device comprises: an excitation coil for applying an alternating current magnetic field to the sample; a sensing coil for sensing a signal of the sample corresponding to the alternating current magnetic field; and a support section that brings the vessel close to the sensing coil and arranges the vessel concentrically with a central axis of the sensing coil. The sensing coil senses the signal, corresponding to the alternating current magnetic field, of the sample to which the alternating current magnetic field is being applied by the excitation coil, the sample being in an agglutinated state in which the detection-target antigen that is bound to the magnetic particles is agglutinated in the vessel, which is arranged concentrically with the sensing coil. In addition, the sensing coil senses a signal, corresponding to the alternating current magnetic field, of a reference sample to which the alternating current magnetic field is applied by the excitation coil, the reference sample not containing magnetic particles or the detection-target antigen. There is provided a signal processing unit for finding the amount of the detection-target antigen on the basis of the signal corresponding to the magnetic field from the sample in the agglutinated state and the signal corresponding to the magnetic field from the reference sample.

Description

Antigen detection device, antigen detection method, and detection kit

[0001] The present invention relates to an antigen detection device and an antigen detection method for detecting a target antigen by magnetic immunoassay using a magnetic marker.

[0002] Immunoassays for detecting biological substances such as proteins and bacteria (pathogens) derived from diseases are used in medical diagnosis. Immunoassays utilize an antigen-antibody reaction in which an antigen, which is the target substance to be detected, specifically binds to an antibody. This antibody is labeled with a substance called a marker, and by detecting the signal from the marker of the antibody bound to the antigen, it becomes possible to measure the amount of the antigen.

[0003] As one type of immunoassay, an optical immunoassay is performed in which an optical marker such as a fluorescent enzyme is added to an antibody whose binding ability to the target substance is known and labeled, and the degree of binding to the target substance is optically detected. Here, in many optical immunoassays, there is an aspect that a washing and removing step for separating the optical marker bound to the target substance from the optical marker that did not bind is required, and the inspection process is complicated and time-consuming.

[0004] On the other hand, different from optical immunoassays, a technique for detecting a target substance by a magnetic method is known as magnetic immunoassay (Patent Documents 1 and 2). Magnetic immunoassay is a method for detecting an antigen-antibody reaction using magnetic particles and a magnetic sensor. An antibody is labeled by adding magnetic particles (hereinafter referred to as magnetic markers), and the degree of binding to the antigen, which is the target substance, is detected using a magnetic sensor as a magnetic signal from the magnetic marker. Specifically, a sample is prepared by binding the target substance and an antibody to which magnetic markers are added in a solution, and a direct current magnetic field is externally applied to the sample to magnetize the magnetic markers. After blocking the application of the direct current magnetic field, the magnetic marker-added antibody bound to the target substance (hereinafter referred to as the bound marker) forms aggregates and has a larger volume than the magnetic marker-added antibody not bound to the target substance (unbound marker). Therefore, the Brownian rotational motion slows down and the Brownian relaxation time becomes relatively long. As a result, the bound marker has a long residual magnetization time.

[0005] On the other hand, antibodies with magnetic markers that did not bind to the substance to be detected (unbound markers) also exist in the solution. Because unbound markers exist individually, they have a small volume and undergo rapid Brownian rotation. Consequently, the direction of the magnetic moment of unbound marker antibodies tends to be random, their Brownian relaxation time is short, and unbound markers have a short residual magnetism period. By utilizing the difference in Brownian times between bound and unbound markers, it is possible to selectively detect the magnetic signal of only the bound marker.

[0006] Thus, by utilizing the differences in the Brownian relaxation properties of magnetic markers, magnetic immunoassays can measure the degree of binding to the substance to be detected without the need for a washing and removal process of the magnetic marker-added antibody.

[0007] Patent documents 1-5 disclose a configuration for detecting an antigen based on a magnetic signal derived from Brownian relaxation of a magnetic marker, using a SQUID (Superconducting Quantum Interference Device) as a magnetic sensor.

[0008] Patent Document 6 discloses a magnetic field measuring device that uses a magnetoresistive effect element (MR sensor) to measure the Brownian relaxation characteristics of a magnetic marker as a difference in AC magnetic susceptibility. That is, a coupled marker with a larger volume has lower tracking ability to high-frequency AC magnetic fields than an uncoupled marker with a smaller volume, and the AC magnetic susceptibility depends on the frequency and Brownian relaxation time. From this, the amount of coupled markers can be measured by measuring the AC magnetic susceptibility using a magnetoresistive effect element (MR sensor).

[0009] Patent Document 7 discloses a magnetic foreign matter inspection device that uses a thin-film magnetic sensor (magnetoresistive sensor, magnetic impedance sensor) having directionality in the magnetic field detection direction to detect the presence or absence of magnetic foreign matter in an object to be inspected.

[0010] Patent documents 8 and 9 disclose a magnetic field measuring device that detects an antigen, which is a substance to be detected, by utilizing Brownian relaxation, which is achieved by rotating a sample containing a magnetic marker (magnetic particle) using a rotation mechanism and switching the magnetic field at each rotation period, as proposed by the inventor of the present invention.

[0011] Patent Document 10 discloses another antigen detection method proposed by the present inventor, which discloses a configuration in which a liquid sample containing magnetic particles and an antigen to be detected that can bind thereto is placed in close proximity to a detection coil, and the amount of the antigen to be detected is measured based on the output signal of the detection coil.

[0012] Japanese Unexamined Patent Publication No. 2015-163846, Japanese Unexamined Patent Publication No. 2007-240349, Japanese Unexamined Patent Publication No. 2009-115529, Japanese Unexamined Patent Publication No. Hei 1-112161, Japanese Unexamined Patent Publication No. 2001-033455, Japanese Patent No. 5560334, Japanese Unexamined Patent Publication No. 2014-159984, Japanese Unexamined Patent Publication No. 2018-194305, Japanese Unexamined Patent Publication No. 2020-159871, Japanese Unexamined Patent Publication No. 2023-134959

[0013] The inventors of this application have diligently conducted research and development to further improve upon the antigen detection method disclosed in Patent Document 10, and have now developed an improved antigen detection device and antigen detection method that enable more accurate measurements and offer greater convenience.

[0014] The object of the present invention is to provide an improved antigen detection device and antigen detection method that can measure with higher accuracy and efficiency, and is also convenient.

[0015] The antigen detection device of the present invention for achieving the above objective is an antigen detection device for detecting a detectable antigen in a container containing a sample comprising magnetic particles carrying antibodies and a sample having a detectable antigen capable of binding to the magnetic particles, comprising an excitation coil for applying an alternating magnetic field to the sample, a detection coil for detecting a signal corresponding to the alternating magnetic field of the sample, and a support portion that brings the container close to the detection coil and is positioned concentrically with its central axis, wherein the detection coil detects a signal corresponding to the alternating magnetic field of the sample in an aggregated state, where the detectable antigen bound to the magnetic particles has been aggregated in the container concentrically positioned with the detection coil, and further comprises a signal processing unit that determines the amount of the detectable antigen based on the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample.

[0016] The present invention provides an antigen detection method for detecting a detectable antigen in a container containing a solution-like sample having an antibody-supported magnetic particle and a detectable antigen capable of binding to the antibody on the magnetic particle, comprising the steps of: positioning the container close to a detection coil and concentrically with its central axis; applying an alternating magnetic field to the aggregated sample, in which the detectable antigen bound to the magnetic particle has been aggregated in the container concentrically with the detection coil, and detecting a signal corresponding to the alternating magnetic field of the aggregated sample; applying an alternating magnetic field to a reference sample that does not contain the magnetic particle or the detectable antigen capable of binding to the magnetic particle, and detecting a signal corresponding to the alternating magnetic field of the reference sample, wherein the method further comprises determining the amount of the detectable antigen based on the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample.

[0017] The detection kit of the present invention is characterized by comprising an antigen detection device and magnetic particles carrying antibodies.

[0018] According to the present invention, magnetic immunoassay can be performed using an antigen detection device with a relatively simple configuration. The antigen detection device is small, lightweight, portable, and can be constructed at low cost due to its simple configuration without moving parts.

[0019] This figure shows an example configuration of an antigen detection device in an embodiment of the present invention. This is a flowchart of the antigen detection method using the antigen detection device in this embodiment. This figure shows the magnetization state using a yoke. This figure shows an example of measurement using the antigen detection method of this embodiment. This figure shows another example of measurement using the antigen detection method of this embodiment. This figure shows a schematic example of the external configuration of the antigen detection device 10. This figure shows an example of the configuration of the excitation coil 12 for enhancing the cooling effect. This figure schematically shows aggregates 30 when the antigen to be detected is bacteria and aggregates 30 when the antigen to be detected is protein. This figure schematically shows a state in which resin particles are bound to protein, and further, magnetic particles are bound to it. This is a flowchart of the method for preparing a sample in which resin particles are bound to protein. This figure shows an example of measurement using the antigen detection method of this embodiment when the antigen to be detected is protein.

[0020] Embodiments of the present invention will be described below with reference to the drawings. However, these embodiments do not limit the technical scope of the present invention.

[0021] Figure 1 is a diagram showing an example configuration of an antigen detection device in an embodiment of the present invention. Figure 1(a) is a diagram showing an example configuration of an antigen detection device, and Figure 1(b) is an enlarged schematic view of the dotted circled area P in Figure 1(a) as seen from the bottom side of the container. In Figure 1(a), the antigen detection device 10 is an antigen detection device that detects a detectable antigen in a container 50 containing a solution-like sample containing magnetic particles on which an antibody, which is a magnetic marker, is supported, and a detectable antigen that can specifically bind to the antibody on the magnetic particles. The device comprises a detection coil 11 that detects a signal corresponding to a magnetic field emitted from magnetic microparticles bound to the sample contained in the container 50, an excitation coil 12 that applies an alternating magnetic field to the sample, and a support part 13 that brings the container 50 close to the detection coil 11 and is positioned concentrically with its central axis.

[0022] The detection coil 11 detects a signal corresponding to the magnetic field emitted from an aggregate 30, which is formed by agglomerating the antigen to be detected, bound to magnetic particles, in a container 50 concentrically arranged with the detection coil 11, while an alternating magnetic field is applied to the sample by the excitation coil 12.

[0023] Container 50 is a cylindrical tube, such as a microtube. A sample containing magnetic particles (sometimes called magnetic beads) carrying antibodies, which act as magnetic markers, and a target antigen that specifically binds to the antibodies, is placed in the water (physiological saline) inside container 50. In other words, the sample is a suspension mixture of physiological saline (PBS) containing antibody-carrying magnetic particles and a target antigen capable of binding to them. The target antigen is an antigen of the target protein, bacteria, or microorganism. The magnetic particles, acting as magnetic markers, carry a predetermined antibody, and within container 50, the target antigen binds to the magnetic particles through an antigen-antibody reaction. Mixing may be performed to promote the antigen-antibody reaction. The magnetic particles are nano-sized magnetic nanoparticles; for example, Micromer-M can be used.

[0024] By bringing a magnet close to the bottom of the container 50 containing the sample, the magnetic particles dispersed in the container 50 and the antigen to be detected bound to them aggregate at the bottom of the container 50, forming small aggregates 30 of magnetic particles and the antigen to be detected bound to them. Figure 1 shows the aggregated state in which aggregates 30 are formed inside the container 50. Before placing the container 50 on the support unit 13, a magnet (not shown) is brought close to the bottom of the container 50 to form aggregates 30 at the bottom of the container 50, and then the container 50 is placed on the support unit 13.

[0025] The support portion 13 is a container fixing device that supports the container 50 such that the central axis of the container 50 and the central axis of the detection coil 11 are concentric, and the bottom of the container 50 is positioned directly above the detection coil 11. By attaching the container 50 to the support portion 13, the container 50 is positioned upright directly above the central axis of the detection coil 11. The shape and support method of the support portion 13 are designed appropriately according to the shape and size of the container 50.

[0026] The detection coil 11 is a magnetic field sensor that outputs a voltage signal corresponding to the magnitude of the magnetic field to be detected, and is composed of, for example, two differentially connected coils. These two coils constituting the detection coil 11 are positioned so that their central axes are concentric and overlap in the axial direction, and are differentially connected. As shown in Figure 1(b), the outer diameter of the detection coil 11 is designed to be approximately the same as, or smaller than, the diameter of the aggregate 30 at the bottom of the container 50. By making the size of the detection coil 11 (outer diameter of the coil) the same as or smaller than the size of the aggregate 30, the response to magnetic field components other than the aggregate 30 is suppressed, and the detection signal of the detection coil 11 is, as far as possible, a response only to the magnetic field component from the aggregate 30, thereby improving the signal-to-noise ratio of the detection signal. The diameter of the aggregate 30 formed by measurement with this antigen detection device is about 1 to 2 mm, and therefore, it is preferable that the outer diameter of the detection coil 11 is 2 mm or less.

[0027] The excitation coil 12 is positioned around the detection coil 11, preferably concentric with the central axis of the detection coil 11, and applies an alternating magnetic field to the container 50 which is concentric with it. The oscillator 60 energizes the excitation coil 12 to generate an alternating magnetic field of a predetermined frequency, and applies the alternating magnetic field to the sample inside the container 50.

[0028] The signal detected by the detection coil 11 is amplified by an amplifier (not shown) and input to the measuring device 62. The measuring device 62 is, for example, a so-called lock-in amplifier, and by using the frequency signal of the excitation coil 12 as a reference signal, the detection signal of the detection coil 11 can be measured with high sensitivity.

[0029] The signal processing device 63 is a means for processing the output signal of the measuring device 62 based on the detection signal of the detection coil 11. It calculates the magnetic field (specifically, the magnetic susceptibility) from the output signal of the measuring device 62 and calculates calculation results related to the presence or absence and quantity of the object to be detected in the container 50 based on its magnitude. The signal processing device 63 can be implemented using a general-purpose computer or a specific digital processing unit.

[0030] The magnitude of the magnetic field around the sample in container 50 varies depending on the amount of antigen to be detected bound to the magnetic particles. The antigen detection device of the present invention determines the difference in the magnitude of the magnetic field based on the difference in the state of the sample, and determines the presence or absence of the antigen to be detected and its amount (number, concentration). The measurement procedure using the antigen detection device according to the first configuration example is described below.

[0031] Figure 2 is a flowchart of the antigen detection method using the antigen detection device of this configuration example. First, the solution-like sample containing magnetic particles and the antigen to be detected is magnetized (S101). Specifically, the bottom of a container 50 containing a solution-like sample containing the antigen to be measured and magnetic microparticles on which antibodies that specifically bind to the antigen to be measured are supported and dispersed in physiological saline solution are brought close to a predetermined magnet, and the magnetic particles bound to the antigen to be measured are magnetized. Due to magnetization, the antigen to be measured that is bound to the antibodies supported on the magnetic particles is attracted to the bottom of the container 50 and aggregates, and the antigen to be measured that is bound to the antibodies supported on the magnetic particles forms aggregates 30, which are like small clumps that have aggregated in the solution, resulting in an aggregated state.

[0032] Magnetization using magnets involves bringing the bottom of the container 50 close to the surface of a magnet, such as a samarium cobalt (SmCo) magnet or a neodymium iron boron (NdFeB) magnet, and magnetizing it for several minutes. Subsequently, the bottom of the container 50 is brought close to the tip of a probe-shaped yoke that has been magnetized by another magnet, and magnetized thereafter.

[0033] Figure 3 shows the magnetization state using a yoke. By bringing the probe-shaped yoke 41, which is in contact with the magnet 40, close to the bottom of the container 50 and magnetizing it, magnetic particles and the antigen to be detected bound to them can be precisely aggregated at the bottom of the container 50, forming smaller aggregates 30.

[0034] In Figure 2, the container 50 containing the aggregated sample is set on the support unit 13 (S102). As a result, the container 50 is positioned close to the detection coil 11 and concentric with its central axis.

[0035] When the sample is in an aggregated state, the excitation coil 12 is driven to apply an alternating magnetic field (S103), and the output signal (voltage signal) Vm of the detection coil 11 is measured in the aggregated state in which the aggregate 30 is formed at the bottom of the container 50 (S104). The output signal Vm of the detection coil 11 is the response signal of the magnetic particles bound to the antigen to be detected in the aggregated state to the alternating magnetic field. The frequency of the alternating magnetic field from the excitation coil 12 may be fixed to a single frequency, or the frequency may be changed by frequency sweeping to measure the output signal.

[0036] After the signal measurement in the aggregated state in step S104 is completed, the application of the alternating magnetic field by the excitation coil 12 is stopped, and then the container 50 is replaced, and a container 50 containing a reference sample that does not contain magnetic particles or the antigen to be detected is set on the support unit 13 for the sample to be measured. The reference sample is a sample in which only physiological saline, especially phosphate-buffered saline (PBS), is placed in the container 50, and no magnetic particles or the antigen to be detected are added to the container 50. The container 50 containing the reference sample is prepared in advance.

[0037] With the container 50 containing a reference sample that does not contain magnetic particles or the antigen to be detected set on the support unit 13, the excitation coil 12 is driven to apply an alternating magnetic field (S106), and the output signal (voltage signal) Vref of the detection coil 11 is measured (S107). The output signal Vref of the detection coil 11 is the response signal to the alternating magnetic field in a state that does not contain magnetic particles or the antigen to be detected, and it is possible to measure a background signal similar to the response signal when the aggregate 30 is stirred and dispersed. The reference sample is a non-magnetic sample that does not contain magnetic particles and does not generate a magnetic field, and even when the aggregate 30 is stirred and the magnetic particles and the antigen to be detected bound to them are dispersed in the container, the magnetic field is substantially canceled out, and by measuring the reference sample, it is possible to measure a signal that is substantially the same as when the magnetic particles and the antigen to be detected bound to them are dispersed in the container. By preparing the reference sample in a separate container, measurement is possible by changing the container without performing the stirring work for dispersion, and the measurement procedure is simplified. In the measurement of the reference sample, the frequency of the AC magnetic field generated by the excitation coil 12 may be fixed to a single frequency, or the frequency may be varied by frequency sweeping and the output signal may be measured.

[0038] After the signal measurement for the reference sample in step S107 is completed, the application of the alternating magnetic field by the excitation coil 12 is stopped, and then the signal processing device 63 performs the following calculation using the signals measured in steps S104 and S107, according to equation (1), to calculate the magnetic susceptibility κ (S108).

[0039] Magnetic susceptibility κ=(Vm-Vref) / Vh (1)

[0040] Vm is the output signal of the detection coil 11 in the aggregated state measured in S104, Vref is the output signal of the detection coil 11 for the reference sample measured in S107, and Vh is the output signal of the detection coil 11 when the container 50 containing the sample is not placed in the support part 13 (when no sample is placed), and these are measured in advance. If the detection coils 11 are differentially connected, Vh is the output signal from one of the coils. Vh is also determined for frequencies matched to Vm and Vref, or for frequencies changed by frequency sweep. The magnetic susceptibility κ calculated has a correlation with the amount of sample, i.e., the amount (number) of antigen, and the amount of antigen can be measured with high sensitivity based on the value of the magnetic susceptibility κ.

[0041] Figure 4 shows an example of measurement using the antigen detection method of this embodiment. The measurement example shown in Figure 4 shows the measurement results using Fusobacterium nucleatum, a bacterium known to be associated with periodontal disease, as the antigen to be detected, and is a graph showing the value of the magnetic susceptibility κ (real part) when the amount of Fusobacterium nucleatum contained in the sample is changed. Specifically, the amount of Fusobacterium nucleatum is changed to 0, 10, and so on. 6 , 10 8 , 10 9 In the measurement example shown in Figure 4, for a sample with a concentration of [CFU / ml], the output signal of the detection coil 11 is a frequency domain signal obtained by frequency sweeping the alternating magnetic field applied by the excitation coil 12 over a predetermined frequency band. Figure 4 shows a graph illustrating the relationship between the frequency of the alternating magnetic field and the magnetic susceptibility κ (real part). From Figure 4, it becomes clear that in the frequency range below 10 kHz, there is a correlation between the amount of antigen and the calculated magnetic susceptibility κ, and that the magnetic susceptibility κ tends to increase as the amount of antigen increases. Thus, the amount of antigen can be determined based on the magnetic susceptibility κ. By creating the calibration curve shown in Figure 4, the amount of antigen corresponding to the magnetic susceptibility κ can be determined by measuring the magnetic susceptibility κ of a sample containing an unknown amount of antigen.

[0042] FIG. 5 is a diagram showing another measurement example by the antigen detection method of the present embodiment example. In the measurement example of FIG. 5, the same antigen to be detected as in FIG. 4 is used as a sample. Different from the measurement in FIG. 4, the output signal of the detection coil 11 is a time-domain signal obtained by fixing the alternating magnetic field applied by the excitation coil 12 at a predetermined frequency. For example, the excitation frequency of the alternating magnetic field is 310 Hz, and the excitation current is 8 A. FIG. 5(a) shows the measurement results of repeatedly measuring the signal of the aggregated sample and the signal of the reference sample every 20 seconds in a measurement for about 100 seconds. The output signal Vm corresponding to the aggregated sample has a significantly increased magnetic signal compared to the output signal Vref corresponding to the reference sample. In the measurement, temperature drift occurs within the measurement time, and the change due to the temperature drift can be approximated by a quadratic curve. FIG. 5(b) shows the measurement results of the output signals Vm and Vref calibrated with the approximated quadratic curve. According to the process of step S108 in FIG. 2, by evaluating the difference between this output signal Vm and Vref, the influence of temperature drift can be reduced and the amount of antigen can be measured.

[0043] The antigen detection device of this configuration example can measure a sample containing an antigen in a stationary state, has a relatively simple configuration, does not require a movable part, and can be miniaturized and lightened. The component elements necessary for the output of the detection signal can be housed in the housing, and it can be made into a portable device. Also, the cost of the antigen detection device can be reduced.

[0044] Further, the user can mix the specimen and the magnetic particles carrying the antibody to be measured in the container and set the container in the antigen detection device for measurement. Thus, even without specialized knowledge, the amount of bacteria or the like contained in the specimen can be specified quickly.

[0045] FIG. 6 is a diagram showing a schematic external configuration example of the antigen detection device 10. The antigen detection device 10 is configured to be built in a small and portable box-shaped housing 45 having a display 43 for displaying measurement results, and preferably includes a fan 42 for cooling the excitation coil 12. Temperature drift due to heat generation of the excitation coil 12 is suppressed, and measurement accuracy is improved. Further, in order to enhance the cooling effect by the fan 42, the excitation coil 12 may be configured to allow the air flow from the fan 42 to pass through the inside of the excitation coil 12.

[0046] FIG. 7 is a diagram showing a configuration example of the excitation coil 12 for enhancing the cooling effect. The excitation coil 12 is configured to be wound with a spacer 46 at intervals for each single winding or multiple windings. By sandwiching the spacer 46, the air blown from the fan 42 can be passed through the intervals formed by the spacer 46, and the cooling effect of the excitation coil 12 can be enhanced.

[0047] FIG. 8 is a diagram schematically showing the aggregate 30 when the detected antigen is a bacterium and the aggregate 30 when the detected antigen is a protein. Generally, bacteria are on the order of 1 μm to 10 μm in size, whereas proteins are on the order of several nm to several tens of nm, which is approximately 1 / 1000 of the size of bacteria. Also, the magnetic particles are approximately several hundred nm to several μm.

[0048] As described above, in the aggregate 30 formed by binding the antigen to be detected with magnetic particles by an antigen-antibody reaction and agglomerating it with a magnet, when the antigen to be detected is a protein, as shown in the figure, the number of magnetic particles per unit volume increases compared to when the antigen to be detected is a bacterium, and the change in the density of magnetic particles in response to the amount of protein becomes relatively smaller. For this reason, when the antigen to be detected is a protein, the change in magnetic susceptibility κ corresponding to the amount of protein becomes smaller compared to when the antigen to be detected is a bacterium, and it may not be possible to measure with sufficiently high accuracy. Therefore, in this embodiment, when the antigen to be detected is a protein, a secondary antibody is supported on resin particles (sometimes called polymer beads) with a particle size of approximately the same size as bacteria, and these are bound to the protein. By using the protein bound to these resin particles as the antigen to be detected, it becomes possible to measure with almost the same detection sensitivity as when the antigen to be detected is a bacterium. The resin particles (polymer beads) can also be used as pseudobacteria when used in experiments. The method for preparing a sample in which resin particles are bound to protein will be described below.

[0049] Figure 9 schematically shows a state in which resin particles are bound to a protein, and then magnetic particles are further bound to it. Figure 10 is a flowchart of a method for preparing a sample in which resin particles are bound to a secondary antibody-carrying protein. The protein binds to the magnetic particles to which the primary antibody is bound, and then to the resin particles to which the secondary antibody is bound. The resin particles are, for example, polystyrene particles (7 μm in diameter).

[0050] In Figure 10, first, magnetic particles and a primary antibody are mixed in solution and reacted to immobilize the primary antibody on the magnetic particles (S201). Next, the magnetic particles with the immobilized primary antibody are mixed in solution with the protein that is the antigen to be detected and reacted to bind the protein to the magnetic particles via the primary antibody (S202). Furthermore, resin particles and a secondary antibody are mixed in the solution containing the bound protein and magnetic particles and reacted to bind the protein to the resin particles via the secondary antibody (S203). For example, avidin (streptavidin) coated resin particles are used, and for example, biotin coated secondary antibodies are used, and the resin particles and secondary antibodies are bound using the avidin-biotin interaction.

[0051] Figure 11 shows an example of measurement using the antigen detection method of this embodiment when the antigen to be detected is a protein. Figure 11(a) shows the measurement result of magnetic susceptibility κ when resin particles are bound to the protein, and Figure 11(b) shows the measurement result of magnetic susceptibility κ when resin particles are not bound to the protein. Note that the protein in the measurement result of Figure 11(a) is GDF15, and the protein in the measurement result of Figure 11(b) is cytokeratin. Although these are measurements of different proteins, the size order of both is almost the same, and it can be inferred that the difference in measurement results is due to the presence or absence of resin particles as a measurement condition.

[0052] The measurement results in Figure 11(a) clearly show a tendency for the magnetic susceptibility κ to decrease as the amount of antigen (protein (GDF15)) increases from 0, 0.1, and 1 [μg / ml] in the frequency range of 1 kHz to less than 10 kHz. By capturing the change in magnetic susceptibility κ according to the amount of antigen, the amount of antigen can be determined. Note that there is virtually no distinction in magnetic susceptibility κ for amounts of GDF15 of 1 and 10 μg / ml. It is presumed that by adjusting (increasing) the amount of resin particles bound to the protein, the magnetic susceptibility κ can be changed even when the amount of antigen is relatively large.

[0053] On the other hand, the measurement results in Figure 11(b) show that in all frequency bands, there is no change in magnetic susceptibility κ in response to the amount of antigen, which is a protein (cytokeratin), and the error bars overlap with each other, indicating that the amount of antigen cannot be separated by magnetic susceptibility κ.

[0054] Thus, when the antigen to be detected is a protein that is much smaller than bacteria, it has been found that measuring by attaching it to resin particles is effective, and this antigen detection method also makes it possible to measure the amount of protein with high accuracy.

[0055] In the antigen detection device according to the embodiment of the present invention, by detecting the signal corresponding to the alternating magnetic field of an aggregated sample in which the antigen to be detected is bound to magnetic particles and aggregated, and the signal corresponding to the alternating magnetic field of a reference sample that does not contain magnetic particles or the antigen to be detected (eliminating the need to detect the signal corresponding to the alternating magnetic field of a sample in which the antigen to be detected is dispersed and bound to magnetic particles), magnetic immunoassay becomes possible with a simpler process. Furthermore, by providing a cooling means (fan) to cool the detection coil and excitation coil, temperature drift is suppressed, enabling more accurate measurements. In addition, by making the outer diameter of the detection coil the same as or smaller than the diameter of the aggregate, the signal-to-noise ratio of the detection signal is improved. Furthermore, even when the antigen to be detected is a protein, by binding resin particles to the protein and measuring, it is possible to measure with the same level of accuracy as when the antigen to be detected is a bacterium. With the above-described configurations, the antigen detection device can be made small and portable, and a highly sensitive magnetic immunoassay can be performed using a relatively simple measurement process.

[0056] The present invention is not limited to the embodiments described above, and of course, design changes that do not depart from the spirit of the invention, including various modifications and alterations that can be conceived by a person with ordinary skill in the art of the present invention, are also included in the present invention.

[0057] 10: Antigen detection device, 11: Detection coil, 12: Excitation coil, 13: Support part, 20: Antigen detection device, 30: Aggregate, 40: Magnet, 41: Yoke, 42: Fan, 43: Display, 45: Box-shaped housing, 50: Container, 60: Oscillator, 62: Measuring device, 63: Signal processing device

Claims

1. An antigen detection device for detecting a detectable antigen in a container containing a sample having magnetic particles carrying antibodies and a detectable antigen capable of binding to the antibodies on the magnetic particles, comprising: an excitation coil for applying an alternating magnetic field to the sample; a detection coil for detecting a signal corresponding to the alternating magnetic field of the sample; and a support portion that brings the container close to the detection coil and positions it concentrically with its central axis, wherein the detection coil detects a signal corresponding to the alternating magnetic field of the sample in an aggregated state, where the detectable antigen bound to the magnetic particles has been aggregated in the container concentrically positioned with the detection coil, and further, the detection coil detects a signal corresponding to the alternating magnetic field of a reference sample, where an alternating magnetic field has been applied by the excitation coil and the reference sample does not contain the magnetic particles or the detectable antigen, and the device further comprises a signal processing unit that determines the amount of the detectable antigen based on the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample.

2. The antigen detection device according to claim 1, characterized in that the support portion supports the container such that the bottom of the container is positioned near the upper end of the central axis of the detection coil.

3. The antigen detection device according to claim 1 or 2, further comprising a cooling means for cooling the excitation coil.

4. The antigen detection device according to claim 3, characterized in that the cooling means has a fan that blows air onto the excitation coil.

5. The antigen detection device according to claim 4, wherein the cooling means includes spacers for forming gaps between the layers of the plurality of winding coils of the excitation coil, and the air blown from the fan is used to ventilate the gaps.

6. The antigen detection device according to claim 1 or 2, characterized in that the diameter of the detection coil is substantially the same as or smaller than the diameter of the aggregate formed by agglutinating the antigen to be detected bound to the magnetic particles.

7. An antigen detection method comprising: detecting a detectable antigen in a container containing a solution-like sample having an antibody-supported magnetic particle and a detectable antigen capable of binding to the antibody on the magnetic particle, the method comprising: placing the container close to a detection coil and positioning it concentrically with its central axis; applying an alternating magnetic field to the aggregated sample, in which the detectable antigen bound to the magnetic particle has been aggregated in the container positioned concentrically with the detection coil, and detecting a signal corresponding to the alternating magnetic field of the aggregated sample; applying an alternating magnetic field to a reference sample that does not contain the magnetic particle and the detectable antigen capable of binding to the magnetic particle, and detecting a signal corresponding to the alternating magnetic field of the reference sample; and determining the amount of the detectable antigen based on the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample.

8. The antigen detection method according to claim 7, further comprising the steps of: the antigen to be detected is a protein, and the step of binding resin particles having a larger particle size than the antigen to be detected to the antigen to be detected; and mixing the antigen to be detected bound to the resin particles with magnetic particles to produce the sample.

9. The antigen detection method according to claim 7, characterized in that the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample are frequency domain signals obtained by sweeping the frequency of the applied AC magnetic field.

10. The antigen detection method according to claim 7, characterized in that the signal corresponding to the magnetic field of the aggregated sample and the signal corresponding to the magnetic field of the reference sample are time-domain signals obtained by fixing the frequency of the applied alternating magnetic field.

11. A detection kit comprising the antigen detection device described in claim 1 and magnetic particles carrying antibodies.

12. The detection kit according to claim 11, further comprising resin particles having a particle size larger than the antigen to be detected and on which a secondary antibody that binds to the antigen to be detected is supported.

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