Protein detection method

Abstract

Provided is a protein detection method for detecting a protein contained in a biological tissue collected from a living body. The protein detection method comprises: a step for preparing a specimen sample by adding, to a sample containing a protein collected from a living body, antibody-carrying magnetic particles that bind to the protein, and mixing the antibody-carrying magnetic particles and the sample; a measurement step for measuring a magnetic signal corresponding to a magnetic field generated by the specimen sample by applying, to the specimen sample, a switching magnetic field in which the magnetic field direction reverses; and a detection step for detecting the protein contained in the specimen sample on the basis of the magnetic signal.

Description

Protein detection methods

[0001] The present invention relates to a protein detection method for detecting proteins contained in biological tissue collected from a living organism, and more particularly to a protein detection method applicable to rapid intraoperative diagnosis.

[0002] Sentinel lymph node biopsy is a surgical procedure used to assess whether cancer has metastasized to the lymph nodes in breast cancer treatment. The sentinel lymph node is the lymph node that cancer cells are most likely to reach first, and sentinel lymph node biopsy is usually performed as part of breast cancer surgery.

[0003] In sentinel lymph node biopsy, for example, sections of lymph nodes stained with HE are excised, a specimen is prepared, and a histopathological diagnosis is made by microscopic examination. The specimen is prepared by fixing the excised tissue with formalin, solidifying it with paraffin to create a tissue block, then cutting it into sections and staining them. Although histopathological diagnosis is highly accurate, there has been a problem in that it takes a long time to obtain the diagnosis.

[0004] In addition, intraoperative rapid diagnosis may be performed. In intraoperative rapid diagnosis, the excised tissue is quickly frozen (frozen specimen) during surgery, thinly sliced, and stained to create a specimen for pathological examination to check for the presence of cancerous tissue and metastasis to lymph nodes. Intraoperative rapid diagnosis of lymph node metastasis allows for the determination of whether or not metastasis has occurred during surgery, and if the result is negative for metastasis, lymph node dissection, which was previously required, can be omitted.

[0005] Various diagnostic methods have been proposed for rapid intraoperative diagnosis, such as methods for detecting proteins that are biomarkers of lymph nodes, like cytokeratin (Non-patent documents 1-3). However, there are still challenges in making accurate diagnoses in a short amount of time.

[0006] The inventors of this application have been conducting research and development on magnetic field measurement technology for magnetic immunoassays utilizing antigen-antibody reactions (Patent Documents 1-4). Magnetic immunoassays are methods for detecting a substance to be measured that has undergone an antigen-antibody reaction using magnetic particles and a magnetic sensor. Magnetic particles (hereinafter referred to as magnetic markers) are attached to the antibody to label it, and the degree of binding to the antigen, which is the substance to be measured, is detected by the magnetic signal from the magnetic marker using a magnetic sensor. Now, using this magnetic field measurement technology, we have developed a protein detection method applicable to rapid intraoperative diagnosis.

[0007] C. Favrot, M. Linek, J. Fontaine, L. Beco, A. Rostaher, N. Fischer, N. Couturier, S. Jacquenet, and BE Bihain” Western blot analysis of sera from dogs with suspectedfood allergy”, Veterinary Dermatology, 28, pp.180 (2017).DM Rissin, CW Kan, TG Campbell, SC Howes, DR Fournier, L. Song, T. Piech, PP Patel, L. Chang, AJ Rivnak, EP Ferrell, JD Randall, GK Provuncher, RR Walt, and DC F\Duffy, “Single -molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations”, Nature Biotechnology, 28, pp. 595-599 (2010).K. Tanaka, H. Waki, Y Ido, S. Akita, Y. Yoshida, T. Yoshida, T. Matsuo, “Protein and polymer analyzes up to m / z 100 000 by laser ionization time-of-flight mass spectrometry”, Rapid Communications in Mass Spectrometry, 2, pp. 151-153 (1988).

[0008] Japanese Patent Publication No. 2018-194305, Japanese Patent Publication No. 2020-159871, Japanese Patent Publication No. 2023-134959, Japanese Patent Publication No. 2024-125105

[0009] The object of the present invention is to provide a protein detection method that can detect proteins more easily and in a shorter amount of time.

[0010] The present invention provides a protein detection method for achieving the above objective, comprising the steps of: generating a sample by adding and mixing antibody-supported magnetic particles that bind to proteins to a sample containing proteins collected from a living organism; a measurement step of applying a switching magnetic field that reverses the direction of the magnetic field to the sample and measuring a magnetic signal corresponding to the magnetic field emitted from the sample; and a detection step of detecting proteins contained in the sample based on the magnetic signal.

[0011] According to the present invention, magnetic particles are bound to a protein to be detected using an antigen-antibody reaction, and the presence or absence of the protein is detected by detecting the magnetic signal of the magnetic particles bound to the protein. The process is relatively simple and can be measured in a short time, making it applicable to rapid intraoperative diagnosis.

[0012] This is a flowchart showing the processing steps of the protein detection method in this embodiment. This is a diagram showing a schematic configuration example of a magnetic field measuring device for measuring the magnetic response of a sample. This is a diagram showing a schematic arrangement example of the magnetic field sensor 40. This is a diagram showing the processing steps of the magnetic field measurement method using the magnetic field measuring device in this embodiment. This is a diagram showing an example of the first measurement result. This is a diagram showing an example of the first measurement result. This is a diagram showing an example of the second measurement result. This is a diagram showing an example of the second measurement result. This is a diagram showing the gradient of the change in the measured value of the first measurement result shown in Figure 7. This is a diagram showing a schematic configuration example of a magnetic susceptibility measuring device for measuring the magnetic response of a sample. This is a diagram showing the magnetic susceptibility measurement step using the magnetic susceptibility measuring device in the protein detection method. This schematically shows a method of evaluating proteins by performing antigen-antibody reactions and biotin-avidin reactions with magnetic particles and resin particles, respectively. The concentration of GDF15 as the protein (antigen) is 0, 0.1 μ g / ml, 1 μ g / ml, 10 μ This shows the change in the real part of the magnetic susceptibility when the concentration is varied in g / ml.

[0013] 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.

[0014] Figure 1 is a flowchart showing the processing steps of a protein detection method according to an embodiment of the present invention. First, biological tissue to be examined is collected from a living organism (S100). In sentinel lymph node biopsy, lymph node tissue to be examined is collected from the patient. Next, the collected biological tissue is prepared (S102). In sample preparation, for example, the sample is homogenized using a homogenizer in a protein measurement solution containing the sample, the solution is diluted to a predetermined number of times, and the supernatant is collected. Furthermore, a specimen sample is prepared from the prepared biological tissue (S104). The specimen sample is prepared by diluting the extracted supernatant with physiological saline (PBS-T), adding and mixing an equal amount of antibody-carrying magnetic particles, and allowing the reaction to occur. The antibody-carrying magnetic particles are magnetic particles (FG beads, 180 nmφ) coated with protein G via a primary antibody (anti-cytokeratin AE1 / AE3 antibody (monoclonal mouse IgG1)), and cytokeratin, a biomarker for lymph node metastasis, reacts with and binds to these antibody-carrying magnetic particles. Magnetic particles have a nano-sized particle size and are sometimes referred to as magnetic nanoparticles.

[0015] The sample to which these antibody-supported magnetic particles have been added is placed in a magnetic field measuring device, a switching magnetic field that reverses the direction of the magnetic field is applied, and the magnetic response is measured (S106). The means for measuring the magnetic response of the sample is described below.

[0016] Figure 2 shows a schematic example of a magnetic field measuring device for measuring the magnetic response of a sample. Figure 2(a) shows the overall configuration of the magnetic field measuring device, and Figure 2(b) shows the state in the dotted-line-enclosed area P of Figure 2(a) where the permanent magnet 38 has been removed, as will be described later, and the tip of the yoke 36 surrounded by the excitation coil 34 is positioned close to the bottom of the container 12.

[0017] The magnetic field measuring device comprises a rotation mechanism 20 that rotates a container 12 containing a sample 10 around a rotation axis, a magnetic field application unit 30 that applies a switching magnetic field to the sample 10 contained in the container 12 to reverse and switch the direction of the magnetic field, and a magnetic field sensor 40 that is positioned at a distance such that it is not substantially affected by the magnetic field from the magnetic field application unit 30 and detects the leakage magnetic field emitted from the sample 10 contained in the rotating container 12.

[0018] The rotating mechanism 20 has a motor-driven rotating shaft 24 attached to the base 22, and an arm portion 26 attached to the rotating shaft 24 in the radial direction, with the container 12 held at the tip of the arm portion 26. By rotating the rotating shaft 24 with the motor, the container 12 held by the arm portion 26 revolves around the rotating shaft 24. The rotating mechanism 20 is not limited to the configuration of a rotating shaft 24 and an arm portion 26, but may also have a configuration with a disc plate that revolves around the rotating shaft 24. Furthermore, the mechanism for moving the container 12 containing the sample 10 is not limited to a rotating mechanism, but may employ other forms of movement, such as reciprocating linear motion.

[0019] The magnetic field application unit 30 is configured to include an oscillator 32, an excitation coil 34 that generates a magnetic field under its control, and a yoke 36 that is arranged concentrically with its central axis 24. The oscillator 32 reverses the direction of the magnetic field generated by the excitation coil 34 with a rotation period, and the container 12 revolves directly above the excitation coil 34, and the direction of the magnetic field is switched with each rotation of the container 12. In other words, for each rotation of the container 12, the direction of the magnetic field applied to the sample 10 contained in the container 12 is reversed for odd-numbered and even-numbered rotations.

[0020] The yoke 36 is a magnetic material made of a material with high magnetic permeability, such as NiFe (permalloy), and one of its ends is formed into a thin, sharp, pointed shape, for example, a cone shape. The top of the cone-shaped yoke 36 is the pointed tip, and the yoke 36 is positioned in the cavity of the excitation coil 34 with a small gap between its pointed top and the bottom surface of the container 12. Preferably, the top of the yoke 36 protrudes from the excitation coil 34 and faces in close proximity to the bottom surface of the container 12.

[0021] By directing the pointed tip of the yoke 36 toward the bottom surface of the container 12, the magnetic flux generated by the excitation coil 34 can be focused to a narrower area, causing the sample 10 inside the container 12 to aggregate into smaller clumps (aggregates 10a), thereby improving sensor sensitivity.

[0022] The magnetic field sensor 40 is a magnetic impedance sensor (MI sensor) that detects a magnetic field using the magnetic impedance effect. The magnetic impedance effect is a phenomenon in which the impedance changes due to a change in the skin depth caused by a change in the permeability in the circumferential direction when a high-frequency current is passed through a high-frequency alloy magnetic material such as amorphous alloy wire, due to the application of an external magnetic field. This makes the magnetic sensor 40 a sensor that can be miniaturized, highly sensitive, and consume less power. While it is preferable to use an MI sensor for the magnetic field sensor 40 in terms of miniaturization and high sensitivity of the device, it is not limited to this, and other sensors that have the function of detecting a magnetic field, such as a magnetoresistive sensor (MR sensor), may also be used.

[0023] The signal processing unit 50 is a means of processing the sensor voltage value corresponding to the output signal (magnetic signal) from the magnetic field sensor 40. It converts the analog output signal into a digital signal, processes the digital signal using a predetermined arithmetic processing unit, and determines the sensor voltage value corresponding to the intensity of the magnetic signal. The signal processing unit 50 is implemented using a general-purpose computer or a specific digital arithmetic circuit.

[0024] Figure 3 shows a schematic example of the arrangement of the magnetic field sensor 40. The magnetic field sensor 40 has two sensor elements 40a and 40b arranged in parallel in a direction perpendicular to the rotational movement direction of the container 12, and operates as a differential sensor. As will be described later, in the configuration of the differential sensor, the container 12 (a small mass (aggregate 10a) that has accumulated at the bottom of the container 12) is allowed to pass directly over one of the two sensor elements 40a and 40b, while the container 12 is not allowed to pass over the other element, thereby canceling out background noise and improving sensitivity.

[0025] Figure 4 shows the processing steps of the magnetic field measurement method using the magnetic field measuring device in this embodiment. The sample 10 is placed in the container 12 and stirred, and then set in a predetermined position on the arm portion 26 of the rotating mechanism 20 (S200). The sample 10 is a mixture of the magnetic particles described above and the substance to be measured (protein) that can bind to them.

[0026] The initial position of the container 12 is directly above the coil 34 of the magnetic field application unit 30. Before rotating the sample 10, a permanent magnet (e.g., an NdFeB magnet) 38 is placed close to the container 12 and magnetized for approximately 5 minutes to collect the sample 10 at the bottom of the container 12 (S202). The permanent magnet 38 is inserted, for example, manually into the gap between the excitation coil 34 and the bottom of the container 12. Magnetization by the permanent magnet 38 causes the sample 10 in the container 12 to condense and collect in an area approximately the same size as one of the sensor elements 40a and 40b, so that it passes directly over one of the elements during rotation.

[0027] After magnetization by the permanent magnet 38, the permanent magnet 38 is removed from the vicinity of the container 12. Subsequently, the oscillator 32 energizes the excitation coil 34 to generate a magnetic field, which is then applied to the sample 10 (S204). The placement and removal of the permanent magnet 38 may be done manually or mechanically. As an example, the magnetic field strength (or magnetic flux density) of the magnetic field applied by the excitation coil 34 is approximately 78 mT, and the application time is approximately 5 minutes. The direction of the magnetic field applied initially is the same as the magnetic field direction used for magnetization by the permanent magnet 38, for example, the positive direction. The generated magnetic field is focused to a single point by the sharp tip shape of the yoke 36 inside the excitation coil 34, and the sample 10 in the container 12 is aggregated into small clumps (aggregates 10a) as schematically shown in Figure 2(b).

[0028] In this way, before rotation begins, magnetization is performed by the permanent magnet 38 and the excitation coil 34 to collect the sample 10 containing antibody-supported magnetic particles bound to the substance to be measured at the bottom of the container 12. Furthermore, the magnetic field is focused by the pointed yoke 36 to aggregate it into the smallest possible clumps (aggregates 10a), and by ensuring that aggregates that have aggregated into sufficiently small clumps pass near the magnetic field sensor, the detection sensitivity of the magnetic field sensor 40 is increased and the signal-to-noise ratio is improved.

[0029] Next, the container 12 (and the sample 10 inside it) directly above the excitation coil 34 (yoke 36) is rotated while gradually increasing the magnetic field strength applied by the excitation coil 34 by a predetermined increment in dB. Specifically, with the sample 10 stopped on the excitation coil 34, the increased magnetic field (the initial predetermined increment in dB is 0) is applied with the magnetic field direction set to the positive direction (S206). The applied magnetic field strength is increased from the previous rotation. Magnetic field strength B applied in odd-numbered rotations. 2n+1 (2n+1: number of rotations, initial value of n is 0) is the magnetic field strength B applied to the previous rotation 2n. 2n The value is obtained by increasing the initial value by a predetermined increment in dB. The predetermined increment in dB is, for example, 6 mT, and the magnetic field strength B0 = 0 before the first rotation. Therefore, the magnetic field strength B applied before the first rotation is the value obtained by adding the predetermined increment in dB to the initial value B0 = 0, so the magnetic field strength applied before the first rotation is 6 mT. The direction of the magnetic field applied in the first rotation can be either the positive direction (forward direction) or the negative direction (negative direction), but the direction of the magnetic field is reversed with each rotation and the magnetic field direction is switched alternately.

[0030] After applying a magnetic field increased by a predetermined dB intensity in the positive direction for approximately 30 seconds, the magnetic field application is stopped, and the sample 10 is rotated once, passing directly over one element of the magnetic field sensor 40 during the rotation, and the leakage magnetic field of the sample 10 is measured (S208). For example, after 3 / 4 of a cycle, the sample 10 passes directly over one element of the magnetic field sensor 40, and the leakage magnetic field of the sample 10 is measured for each rotation.

[0031] Since the magnetic field sensor 40 has a differential sensor configuration, a highly accurate output signal (sensor voltage value) with background noise canceled out is obtained by obtaining the difference in output between one element over which the sample 10 passes directly and the other element over which the sample 10 does not pass directly. The rotation speed is, for example, about 200 degrees / s. The rotation speed should be such that the acceleration due to centrifugal force is sufficiently small compared to the acceleration due to gravity, as a high rotation speed would destabilize the liquid phase. The gap between one element of the magnetic field sensor 40 and the bottom of the container 12 containing the sample 10 is preferably about 200 μm to 300 μm. The narrower the gap, the higher the sensitivity to detecting leakage magnetic fields from small amounts of sample 10, enabling more accurate protein detection.

[0032] After applying a positive magnetic field in the direction of the previous rotation and causing one full rotation, the sample 10 is stopped on the excitation coil 34, and then the magnetic field is applied by the excitation coil 34, switching to a negative magnetic field direction with the same magnetic field strength as the previous rotation (S210). That is, for the even-numbered 2n+2th period rotation, the magnetic field strength B 2n+2 = B 2n+1 = B 2n The magnetic field is +dB and applied in the opposite direction to the magnetic field applied during the 2n+1th period of movement. Therefore, the negative magnetic field applied before the second rotation is 6mT, the same as the previous rotation, and the application time is about 30 seconds, similar to the application in the positive direction. After applying the magnetic field in the negative direction for 30 seconds, the application of the magnetic field is stopped, and the sample 10 is rotated once, passing directly over one element of the magnetic field sensor 40 during the rotation, as in the previous rotation, and the leakage magnetic field of the magnetic bead is measured (S212). A magnetic field measurement with a positive magnetic field application and a magnetic field measurement with a negative magnetic field application are considered as one set, and for each set of magnetic field measurements, the applied magnetic field is increased by a predetermined increment of dB (S220), and the magnetic field measurement is repeated.

[0033] The process described in steps S206 to S212, which is carried out by gradually increasing the applied magnetic field by a predetermined increment in dB (S220), is repeated until the number of rotations reaches a predetermined number (for example, about 20 to 50 rotations) (S214). That is, for rotations from the first rotation onward, at odd-numbered rotations, the magnetic field strength is increased by a predetermined increment in dB, a positive magnetic field is applied, and the sample 10 is rotated. At even-numbered rotations, a negative magnetic field is applied with the same magnetic field strength as the previous positive rotation, but with the direction of the magnetic field reversed, and the sample 10 is rotated, and measurements are taken by the magnetic field sensor 40 after each rotation.

[0034] In this invention, a magnetic field with reversed polarity is alternately applied to the sample 10 to cause it to rotate. As a result, the magnetic particles contained in the sample 10 attempt to rotate in the direction of the magnetic field. At this time, the aggregates 10a of magnetic particles and proteins bound to them that are formed in the sample 10 become more aggregated due to the rotation, and come together into smaller aggregates 10a. The leakage magnetic field from the aggregates 10a, which are more aggregated due to the rotation, becomes stronger than the leakage magnetic field from the relatively spread aggregates, enabling highly sensitive measurement. The time for applying the reversing magnetic field is relatively long, about 30 seconds each time, but the magnetic field is applied for at least 10 seconds to ensure that the magnetic particles rotate reliably.

[0035] Once a predetermined number of rotations have been performed and measurements have been taken for each rotation, the obtained sensor voltage values ​​are processed by the signal processing unit 50 (S216). The sensor voltage values ​​correspond to the magnetic signal strength, and the signal processing unit 50 obtains the absolute values ​​of the positive and negative voltage values, which are reversed with each rotation.

[0036] The time required to obtain a diagnostic result from the processing used in the magnetic field measuring device in this embodiment and the calculation of the sensor voltage value of the magnetic field measuring device is approximately 30 minutes. Therefore, a rapid intraoperative diagnosis can be performed at the site of surgery to determine whether or not a patient with a specimen has cancer.

[0037] Figures 5 and 6 are diagrams showing a first measurement result example. Figure 5 shows measurement values of sensor voltage values corresponding to the number of rotations of the magnetic field measurement device at concentrations of cytokeratin as a protein of 0 μg / ml, 10 μg / ml, and 100 μg / ml, and Figure 6 is a photograph taken with an optical microscope showing the state of aggregates of the specimen sample for each concentration of cytokeratin.

[0038] In the graph of Figure 5, the magnetic signal intensity (sensor voltage value) in the measurement where the direction of the magnetic field applied for each rotation is reversed and switched is shown. The higher the concentration of cytokeratin, the greater the sensor voltage value, that is, the magnetic field strength, and the greater the number of rotations, the stronger the magnetic field strength. Since cytokeratin aggregates magnetic particles, the magnetic signal becomes stronger as the amount of cytokeratin increases. Also, due to the rotational motion of the aggregates according to the reversing switching magnetic field, the aggregates are further aggregated, and the magnetic signal increases as the number of rotations increases. It is shown that the concentration of cytokeratin can be measured according to the strength of the magnetic signal.

[0039] As shown in Figure 6, it is confirmed that as the cytokeratin concentration increases, the dark-colored region of the aggregates expands, and in the region where it is estimated that there is little cytokeratin, it can be confirmed that the color of the aggregates is light and they are weakly aggregated. As the antigen cytokeratin increases, the magnetic binding between cytokeratin and magnetic particles increases. By applying a switching magnetic field with the reversed magnetic field direction, it is presumed that the magnetic particles are further aggregated by the rotational motion of the magnetic particles in the container, and the magnetic signal also increases. By applying a switching magnetic field with the reversed magnetic field direction, it can be evaluated that there is a statistically significant difference in the intensity of the detected magnetic signal according to the concentration of cytokeratin, suggesting the applicability of the protein detection method of this embodiment to intraoperative rapid diagnosis.

[0040] Figures 7 and 8 are diagrams showing a second measurement result example. Figure 7 shows measurement values of sensor voltage values corresponding to the number of rotations of the magnetic field measurement device in two cancer patients (cancer 1, cancer 2) and a non-cancer patient (non-cancer), and Figure 8 is a photograph taken with an optical microscope showing the state of aggregates of the specimen sample of each patient.

[0041] The graph in FIG. 7 shows the magnetic signal intensity (sensor voltage value) in the measurement where the direction of the magnetic field applied for each rotation is switched. For the specimen sample of a cancer patient containing cytokeratin, compared with the specimen sample of a non-cancer patient not containing cytokeratin, the magnetic field intensity (sensor voltage value) becomes larger, and also, the more the number of rotations, the stronger the magnetic field intensity. Also in the second measurement result example, as in the first measurement result example, since cytokeratin aggregates magnetic particles, the magnetic signal becomes stronger as the amount of cytokeratin increases, and also, as the aggregates are more aggregated for each rotation, the magnetic signal becomes larger for each rotation. It is shown that the concentration of cytokeratin can be measured according to the strength of the magnetic signal.

[0042] As shown in FIG. 8, it is confirmed that in the aggregates corresponding to the specimen sample of a cancer patient, compared with the aggregates corresponding to the specimen sample of a non-cancer patient, the region with a darker color of the aggregates is more expanded. By the rotational movement of the magnetic particles by the switching magnetic field, the magnetic particles become more aggregated, and the aggregated region (the region with a darker color) expands. It is presumed that as the antigen, side cytokeratin, increases, the magnetic binding between cytokeratin and the magnetic particles increases, so that the magnetic particles become more aggregated and the magnetic signal also increases. In the above measurement result example, it can be evaluated that there is a statistically significant difference between cancer patients and non-cancer patients, suggesting the applicability of the protein detection method of the present embodiment to intraoperative rapid diagnosis.

[0043] FIG. 9 is a diagram showing the gradient of the change in the measured value of the first measurement result shown in FIG. 7. The gradient value of a cancer patient is relatively larger than the gradient value of a non-cancer patient, suggesting the applicability to the evaluation of the presence or absence of cytokeratin. Thus, for the determination of the presence or absence of cytokeratin, as shown in FIG. 9, the gradient of the magnetic field intensity that changes (increases) for each rotation, that is, the change in the magnetic field intensity can be used, or the magnitude of the magnetic field intensity itself after rotating a predetermined number of rotations shown in FIG. 7 can also be used.

[0044] Next, another embodiment of the means for measuring the magnetic response of the specimen sample (step S106 in FIG. 2) will be described.

[0045] Figure 10 shows a schematic example of a magnetic susceptibility measuring device for measuring the magnetic response of a sample. The magnetic field measuring device described in Figure 2 above measures the leakage magnetic field of the sample while applying a reversing switching magnetic field to the sample 10, and detects proteins based on the magnetic field strength (sensor voltage). However, protein detection can also be performed using the magnetic susceptibility measuring device shown in Figure 10.

[0046] Using the magnetic field measuring device shown in Figure 2 or another means, a switching magnetic field that reverses the direction of the magnetic field is applied to the sample 10 to further aggregate the aggregates formed in the sample 10. Then, the sample 10 is set in the magnetic susceptibility measuring device shown in Figure 10, and the magnetic susceptibility of the sample 10 is determined by the apparatus configuration described below, thereby enabling protein detection.

[0047] The magnetic susceptibility measuring device shown in Figure 10 comprises a detection coil 61 that detects a signal corresponding to the magnetic field emitted from a sample 10 contained in a container 12, an excitation coil 62 that applies an alternating magnetic field to the sample 10, and a support part 63 that brings the container 12 close to the detection coil 61 and positions it concentrically with its central axis.

[0048] The detection coil 61 detects a signal corresponding to the magnetic field emitted from aggregates formed by agglomerating antibodies bound to antibody-supported magnetic particles contained in the sample 10 within a container 12, which is concentrically positioned with the detection coil 61, while an alternating magnetic field is applied to the sample 10 by the excitation coil 62.

[0049] After performing magnetization treatment using a permanent magnet (not shown) and aggregate formation treatment by applying a switching magnetic field, the container 12 containing the aggregated sample 10 is set in the support unit 63.

[0050] The support portion 63 is a container fixing device that supports the container 12 such that the central axis of the container 12 and the central axis of the detection coil 61 are concentric, and the bottom of the container 12 is positioned directly above the detection coil 61. By attaching the container 12 to the support portion 63, the container 12 is positioned upright directly above the central axis of the detection coil 61. The shape and support method of the support portion 63 are designed appropriately according to the shape and size of the container 12.

[0051] The detection coil 61 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 10(b), preferably, the outer diameter of the detection coil 61 is designed to be approximately the same as or smaller than the diameter of the aggregate at the bottom of the container 12. By making the size of the detection coil 61 (outer diameter of the coil) the same as or smaller than the size of the aggregate, the response to magnetic field components other than the aggregate is suppressed, and the detection signal of the detection coil 61 becomes a response to the magnetic field component from the aggregate as much as possible, thereby improving the signal-to-noise ratio of the detection signal.

[0052] The excitation coil 62 is positioned around the detection coil 61 and is preferably concentric with the central axis of the detection coil 61, applying an alternating magnetic field to the container 12 which is concentric with it. The oscillator 70 energizes the excitation coil 62 to generate an alternating magnetic field of a predetermined frequency, and applies the alternating magnetic field to the sample in the container 12.

[0053] The signal detected by the detection coil 61 is amplified by an amplifier (not shown) and input to the measuring device 72. The measuring device 72 is, for example, a so-called lock-in amplifier, and by using the frequency signal of the excitation coil 62 as a reference signal, the detection signal of the detection coil 61 can be measured with high sensitivity.

[0054] The signal processing device 73 is a means for processing the output signal of the measuring device 72 based on the detection signal of the detection coil 61. It calculates the magnetic susceptibility from the output signal of the measuring device 72 and calculates calculation results related to the presence or absence and quantity of the object to be detected in the container 12 based on its magnitude. The signal processing device 73 can be implemented using a general-purpose computer or a specific digital processing unit.

[0055] Figure 11 shows the magnetic susceptibility measurement process using a magnetic susceptibility measuring device in the protein detection method of this embodiment. In this process, the magnetic field measuring device shown in Figure 2 is used as a switching magnetic field application means. In Figure 11, a switching magnetic field is applied to the sample 10 in advance using the magnetic field measuring device shown in Figure 2, in which the direction of the magnetic field reverses and switches with each rotation (S301). That is, the processes of S202-S212 and S220 in Figure 4 are performed. In this case, the leakage magnetic field detection process (S208, S212) does not need to be performed. Instead of the leakage magnetic field detection process, the magnetic susceptibility is determined using the magnetic susceptibility measuring device in Figure 10.

[0056] The container containing the sample 10 to which the switching magnetic field has been applied is set on the support unit 13 (S302). As a result, the container 50 is positioned close to the detection coil 61 and concentric with its central axis.

[0057] The excitation coil 12 is driven to apply an alternating magnetic field (S303), and the output signal (voltage signal) Vm of the detection coil 61 for the sample 10 in which aggregates have formed at the bottom of the container 50 is measured (S304). The output signal Vm of the detection coil 61 is the response signal of the aggregated magnetic particles to the alternating magnetic field, and 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.

[0058] After the signal measurement in the aggregated state in step S304 is completed, the application of the alternating magnetic field by the excitation coil 12 is stopped, and then the container 50 is replaced, and the container 50 containing a reference sample that does not contain magnetic particles or collected biological tissue is set on the support unit 13 (S305). 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 collected biological tissue are added to the container 50. The container 50 containing the reference sample is prepared in advance.

[0059] With the container 50 containing a reference sample that does not contain magnetic particles or collected biological tissue set on the support unit 13, the excitation coil 12 is driven to apply an alternating magnetic field (S306), and the output signal (voltage signal) Vref of the detection coil 11 is measured (S307). 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 collected biological tissue, and the background signal can be measured. In the measurement of the reference sample, 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 sweep and the output signal may be measured.

[0060] After the signal measurement for the reference sample in step S307 is completed, the application of the alternating magnetic field by the excitation coil 12 is stopped. Subsequently, the signal processing device 73 uses the signals measured in steps S304 and S307 to perform the following calculation using equation (1) to calculate the magnetic susceptibility κ (S308). Magnetic susceptibility κ = (Vm - Vref) / Vh (1)

[0061] Vm is the output signal of the detection coil 61 in the aggregated state measured in S304, Vref is the output signal of the detection coil 61 for the reference sample measured in S307, and Vh is the output signal of the magnetic field detection coil 64 when the container 50 containing the sample is not placed on the support part 13 (when no sample is placed), and these are measured in advance. If the detection coils 61 are differentially connected, the output signal from one of the coils is taken as Vh. 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 (concentration) of protein (cytokeratin) contained in the sample 10, and the amount (concentration) of protein can be measured based on the value of the magnetic susceptibility κ.

[0062] Figure 12 schematically shows a method for evaluating a protein by subjecting it to antigen-antibody reactions and biotin-avidin reactions with magnetic particles (e.g., Micromer-M_proteinA) and resin particles (Polystyrene Microsphere), respectively. The protein binds to magnetic particles to which a primary antibody is bound, and further binds to resin particles to which a secondary antibody is bound. The resin particles are, for example, polystyrene particles (diameter 7 μm).

[0063] Specifically, the method for producing a sample by binding resin particles to a protein carrying a secondary antibody is as follows: First, magnetic particles and a primary antibody are mixed and reacted in a solution to immobilize the primary antibody on the magnetic particles. Next, the magnetic particles to which the primary antibody is immobilized and the protein, which is the antigen to be detected, are mixed and reacted in a solution to bind the protein and the magnetic particles via the primary antibody. Further, resin particles and a secondary antibody are mixed and reacted in a solution containing the bound protein and magnetic particles to bind the protein and the resin particles via the secondary antibody. For example, avidin (streptavidin) -coated resin particles are used, and biotin-coated secondary antibodies are used, and the resin particles and the secondary antibody are bound using the avidin-biotin interaction.

[0064] Since the protein has a microscopic size on the order of nm, after the antigen-antibody reaction of the magnetic particles, the primary antibody, and the protein (antigen), the secondary antibody and the resin particles are bound by the biotin-avidin reaction to increase the aggregate size. Thus, when the protein (antigen) is at a low concentration, the amount of the secondary antibody and the resin particles bound is small, so the density of the magnetic particles in the aggregate is high, resulting in a high magnetic susceptibility. On the other hand, when the protein is at a high concentration, the amount of the resin particles bound to the secondary side increases, and the density of the magnetic particles in the aggregate decreases, so the magnetic susceptibility decreases. When the protein alone is subjected to an antigen-antibody reaction, the density change in the aggregate of the magnetic particles is small, so there is an effect of amplifying the change in magnetic susceptibility by mixing resin particles between the magnetic particles in proportion to the amount of antigen.

[0065] Figure 13 shows the concentration of GDF15 as the protein (antigen) being 0, 0.1 μ g / ml, 1 μg / ml, 10 μ This shows the change in the real part of the magnetic susceptibility when the concentration in g / ml is varied. As the protein concentration increases, the real part of the magnetic susceptibility decreases. It is thought that this decrease in the real part of the magnetic susceptibility is due to the polymer beads, which are bound via the protein as intended, reducing the magnetic coupling between the magnetic nanoparticles. This principle can be used to evaluate the protein concentration.

[0066] 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.

[0067] 10: Sample, 10a: Aggregate, 12: Container, 20: Rotation mechanism, 22: Stand, 24: Rotation shaft, 26: Arm section, 30: Magnetic field application section, 32: Oscillator, 34: Excitation coil, 36: Yoke, 40: Magnetic field sensor, 50: Signal processing section, 61: Detection coil, 62: Excitation coil, 63: Support section, 64: Magnetic field detection coil, 72: Measuring device, 73: Signal processing device

Claims

1. A protein detection method characterized by comprising the steps of: preparing a sample by adding and mixing antibody-supported magnetic particles that bind to proteins to a sample containing proteins collected from a living organism; applying a switching magnetic field in which the direction of the magnetic field is reversed to the sample and measuring a magnetic signal corresponding to the magnetic field emitted from the sample; and detecting proteins contained in the sample based on the magnetic signal.

2. The protein detection method according to claim 1, characterized in that a protein is detected in the detection step based on the intensity of the magnetic signal or a change in the intensity of the magnetic signal.

3. The protein detection method according to claim 1, characterized in that, in the detection step, the magnetic susceptibility corresponding to the magnetic signal is calculated, and the protein is detected based on the value of the magnetic susceptibility.

4. The protein detection method according to claim 1, characterized in that the protein is cytokeratin.

5. The protein detection method according to claim 1, further comprising the steps of: binding resin particles having a particle size larger than the protein with the protein; and mixing the protein bound to the resin particles with the magnetic particles to produce the sample.

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