Method for detecting analyte or estimating concentration thereof

Abstract

The present invention eliminates the problems of conventional techniques caused by impurities or aggregates, and provides a new method for detecting an analyte or estimating the concentration thereof. The method for detecting an analyte or estimating the concentration thereof is characterized by comprising estimating the concentration of an analyte in a test sample using: a pore sensor having one pore, two chambers connected to both surfaces of the pore, and a pair of electrodes each installed in each of the two chambers; and a reaction reagent including adhesion-type magnetic beads having a magnetic bead surface to which a first adherent that specifically binds to the analyte is adhered, and adhesion-type detection beads having a non-magnetic bead surface to which a second adherent that specifically binds to the analyte is adhered. When the test sample and the reaction reagent are mixed to form a reaction liquid, bonding occurs between the adhesion-type magnetic beads and the adhesion-type detection beads via the analyte in accordance with the concentration of the analyte in the reaction liquid. A magnetic field is applied to a container accommodating the test sample including sandwich complexes, generated by the bonding, of an adhesion-type magnetic bead, the analyte, and an adhesion-type detection bead, to thereby fix the sandwich complexes including the adhesion-type magnetic beads in the container, and in this state, the reaction liquid is replaced with a cleaning liquid. Then, the cleaning liquid is removed, and further an eluate is introduced into the container to cause the adhesion-type detection beads to be released from the sandwich complexes into the eluate, and at the same time, the adhesion-type magnetic beads are fixed in the container by means of the magnetic field, thereby separating the adhesion-type detection beads from the adhesion-type magnetic beads. The eluate containing the separated adhesion-type detection beads is introduced into one of the two chambers of the pore sensor, and an electrolyte is introduced into the other of the two chambers, thereby providing electrical communication between the pair of electrodes. A voltage is applied to the pair of electrodes, and the number of pulse signals due to a current transient change caused in the pair of electrodes each time an adhesion-type detection bead passes through the pore is measured, thereby measuring the presence or absence or the concentration of the analyte in the test sample.

Description

Method for detecting or estimating the concentration of a test substance

[0001] The present invention relates to a method for detecting or estimating the concentration of an analyte based on an electric sensing zone method, and more particularly to a method for detecting or estimating the concentration of an antigen or antibody based on an electric sensing zone method.

[0002] Antigen quantification is a clinical testing technique essential for diagnosing various diseases and is widely used. Conventional techniques for antigen quantification include chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), and fluorescent enzyme immunoassay (FEIA), which use antibody labels to detect immune reactions by fluorescence or chemiluminescence. Other techniques include immunoturbidimetry (latex agglutination), which uses antigen- or antibody-modified beads as reagents and measures the amount of transmitted light obtained by irradiating light onto the beads when immune complexes formed in the presence of the analyte cause the beads to aggregate, and immunonephelometry, which measures scattered light.

[0003] Although CLEIA, CLIA, FEIA, and the like offer high accuracy and sensitivity, they require precise optical systems, resulting in large and expensive instruments. While latex agglutination can be performed using a simple optical system that measures absorbance, it requires the generation of agglutinates at a level that can be optically detected as turbidity, resulting in low detection sensitivity. In recent years, the demand for rapid antigen quantification in clinical settings, and the growing expectations for so-called point-of-care testing, have led to a demand for highly sensitive, highly accurate, rapid, and low-cost testing methods. However, as described above, conventional technologies are unable to combine the high sensitivity and high accuracy of immunoluminescence / fluorescence with the compact size and low cost of latex agglutination. In response to this, a technology has been proposed that uses antibody-modified beads, similar to latex agglutination, to measure the binding or agglutination state of antibody-modified beads by antigen molecules using an electrical detection zone method rather than optical means (Patent Document 1, Patent Document 2).

[0004] The electrical sensing zone method is a technique in which one chamber is filled with a sample and the other with an electrolyte, and a voltage is applied while the upper and lower flow paths are electrically connected through the pore, and the transient change in current that occurs each time a particle passes through the pore. The degree of agglutination can be measured with high precision for each antibody-modified particle or its aggregate generated by an immune reaction as it passes through the pore. This technique allows for the realization of a small, inexpensive measurement system that does not require an optical system for detection. Furthermore, since the binding state of antibody-modified beads with antigen molecules can be observed one by one, high sensitivity can also be achieved.

[0005] JP 2024-002662 A JP 2024-002665 A

[0006] However, the techniques using the electrical sensing zone method disclosed in Patent Documents 1 and 2 have the following two problems.

[0007] The first issue is the increase in noise and deterioration of measurement stability due to impurities. When quantifying antigens in clinical samples using these technologies, not only the antibody-modified particles to be measured but also the impurities in the sample pass through the pores, generating a pulse signal. The pulse signal caused by these impurities is often unstable, causing noise in the analysis and reducing accuracy. In addition, in samples with a high content of impurities, this can cause the pores to become clogged, adversely affecting measurement stability.

[0008] The second problem is that measurement becomes impossible when the agglutinates become larger than the pores. For example, if an attempt is made to increase the concentration of antibody-modified beads in the reagent in order to detect low concentrations of analyte, i.e., to increase detection sensitivity, the agglutinates will become larger than the pores for high concentrations of analyte, making it impossible to measure the high concentrations. In other words, when measuring the degree of agglutination of antibody-modified particles by antigens using the electrical detection zone method, it is generally difficult to expand the measurable concentration range.

[0009] The present invention was completed in consideration of the above problems, and in one embodiment, it is an object of the present invention to solve the above two problems while taking advantage of the advantages of antigen quantification using the electrical detection zone method, which are high sensitivity and high precision, as well as small size and low cost.

[0010] As a result of intensive research, the present inventors have discovered a method for detecting or estimating the concentration of an analyte by using magnetic beads and detection beads to which a specific substance has been attached, processing an eluate, etc., and measuring the concentration of the detection beads rather than the analyte itself. The present invention has been completed based on the above findings, and is exemplified below.

[0011] [1] A method for detecting or estimating the concentration of an analyte, comprising: a pore sensor having one pore, two chambers connected to both sides of the pore, and a set of electrode pairs installed in each of the two chambers; and estimating the concentration of an analyte in a test sample using a reaction reagent including attached magnetic beads having a first attachment attached to the surface of magnetic beads that specifically bind to the analyte, and attached detection beads having a second attachment attached to the surface of non-magnetic beads that specifically bind to the analyte; when the test sample and the reaction reagent are mixed to prepare a reaction solution, binding between the attached magnetic beads and the attached detection beads via the analyte occurs depending on the concentration of the analyte in the reaction solution; applying a magnetic field to a container containing the test sample containing a sandwich complex of the attached magnetic beads, the analyte, and the attached detection beads formed by the binding, thereby fixing the sandwich complex containing the attached magnetic beads in the container; replacing the reaction solution with a washing solution, and then removing the washing solution; the first attachment is a first type of antibody, and the second attachment is a second type of antibody different from the first type of antibody, thereby measuring the presence or concentration of the analyte in the test sample. [3] The method for detecting or estimating the concentration of an analyte according to [1], wherein the analyte is an antigen, and the first and second deposits are antibodies of the same type.[4] The method for detecting or estimating the concentration of a specimen according to [1], wherein the specimen is an antibody, and the first and second deposits are antigens.

[0012] According to one embodiment of the present invention, it is possible to provide a new method for detecting or estimating the concentration of a test substance, while taking advantage of the advantages of antigen quantification using the electrical detection zone method, namely, high sensitivity and high accuracy, as well as small size and low cost, and resolving the problems caused by impurities or aggregates in conventional technology.

[0013] FIG. 1(a) is a diagram showing an example of the structure of a pore sensor that can be used in the analyte detection or concentration estimation method of the present invention. FIG. 1(b) is a diagram showing an electron microscope image of a pore sensor pore 140 used to measure a pulse waveform. FIG. 1(b) is a diagram showing pulse-like transient changes in the ionic current flowing between the electrodes of the pore sensor shown in FIG. 1. FIG. 4(a) is a flowchart showing the flow of a analyte detection or concentration estimation method according to one embodiment of the present invention. FIG. 4(a) is a diagram showing a schematic diagram of a state in which a test sample and a reaction reagent are mixed in a reaction vessel 417. FIG. 4(b) is a diagram showing a schematic diagram of a state in which magnetic beads and latex beads are bound via a target antigen in the test sample to form sandwich complexes 421 and 422. FIG. 4(c) is a diagram showing a state in which the sandwich complex is attracted and fixed near the wall surface of the reaction vessel to which a magnetic field is applied. FIG. 5(a) is a diagram showing a state in which impurity particles have been removed from the reaction vessel. 5(b) is a schematic diagram showing a state in which the bonds between the antigen and magnetic beads and between the antigen and latex beads are broken, and latex beads 522 and antigen 520 are released into the eluate. FIG. 5(c) is an example of the state in which latex beads 501, target antigen 502, latex beads 503, target antigen 504, and latex beads 505 pass through pore 530 in this order.

[0034] FIG. 5(b) is a diagram showing the measurement results for each sample of the calibrator concentration used to create a calibration curve in an example of the present invention.

[0035] FIG. 5(c) is a diagram showing the calibration curve obtained in an example of the present invention.

[0036] FIG. 5(c) is a diagram showing the correlation between antigen concentration and the number of observed pulses in an example of the present invention.

[0014] Next, embodiments of the present invention will be described in detail with reference to the drawings. It should be understood that the present invention is not limited to the following embodiments, and that appropriate design changes and improvements may be made based on the ordinary knowledge of those skilled in the art without departing from the spirit of the present invention.

[0015] The method for estimating the concentration of an analyte of the present invention can be applied to the detection or concentration estimation of an analyte such as an antigen, antibody, sugar chain, or gene having a specific base sequence in a biological sample. In this specification, concentration estimation may be simply referred to as "quantification." In the present invention, a molecule, gene, or the like to be detected or quantified is referred to as an analyte. Furthermore, a sample to be used for the detection or quantification of an analyte is referred to as a test sample.

[0016] In the following, an example will be described in which the analyte is an antigen; however, in the present invention, the analyte in the test sample may also be a sugar chain, a gene having a specific base sequence, a receptor, a ligand, or the like. For example, when the analyte is an antibody, the antigen explained below will be read as antibody, and the antibody as antigen. Furthermore, when the analyte is a sugar chain, the antigen explained below will be read as sugar chain, and the antibody as lectin. Furthermore, when the analyte is a gene having a specific base sequence, the antigen explained below will be read as the gene to be detected, and the antibody as a gene complementary to the gene to be detected. Furthermore, when the analyte is a ligand, the antigen will be read as ligand, and the antibody as receptor.

[0017] FIG. 1( a) shows an example of the structure of a pore sensor that can be used in the analyte detection or concentration estimation method of the present invention. The pore sensor 100 has a cross-sectional structure in which two chambers 110 and 120 are separated by a partition wall 141 and connected via a pore 140 provided in the partition wall 141. Electrodes 112 and 122 are installed in the two chambers, respectively. A sample containing particles suspended in an electrolyte solution is introduced into chamber 110 through inlet 111, and electrolyte solution is introduced into chamber 120 through inlet 121. A voltage is applied to the two electrodes by a voltage source 152. These electrodes are connected to an amplifier 150, an ammeter 151, and the voltage source 152. For example, when a voltage is applied between electrode 112 and electrode 122, an ionic current flows through pore 140.

[0018] FIG. 1(b) shows an electron microscope image of the pore 140 used to measure pulse waveforms in the experiments described below. The pore diameter is selected to be larger than the diameter of the antibody-modified particles to be measured. Note that FIG. 1 is merely one example of a sensor used for measurements in the present invention. The sensor used in the present invention is not particularly limited in terms of the shape or material of the chamber, pore, and inlet, as long as it has one pore, two chambers connected to both sides of the pore, and one pair of electrodes installed in each of the two chambers, and these may be selected appropriately as needed.

[0019] As shown in FIG. 1A , when particles of the test object 101 present in the chamber 110 pass through the aperture 140, the ionic current is temporarily interrupted, and then returns to normal after the test object 101 passes through the chamber 120. Therefore, each time one test object 101 passes through the aperture 140, the ionic current flowing between the electrodes in FIG. 1 exhibits a pulse-like transient change as illustrated in FIG. 2 . In the example of FIG. 1 , this is measured by an ammeter 151. In FIG. 2 , the pulse signal 210 represents a current value 202 at each time 201. The vertical axis may also represent a voltage value. The baseline 200 represents the current value when there is no pulse (or the average over a certain period if there is noise), and the peak current 208 represents the difference between the baseline 200 and the current value at which the largest current phenomenon occurs in the pulse waveform (or the average over a certain period if there is noise).

[0020] The reagents used in one embodiment of the present invention are a reaction reagent containing antibody-solid-phase latex beads, which are antibody-attached magnetic beads having a first attachment (antibody) attached to the surface of magnetic beads that specifically binds to the analyte (antigen), and antibody-attached detection beads, which are antibody-attached detection beads having a second attachment (antibody) attached to the surface of non-magnetic beads that specifically binds to the analyte (antigen), a washing solution, and an elution solution.

[0021] Antibody-immobilized latex beads (hereinafter referred to as "latex beads") are beads in which the surface of a latex substrate is modified with an antibody. The substrate does not have to be latex; any material can be used as long as it does not generate a force that moves the reaction reagent through the liquid when a magnetic field is applied.

[0022] Immobilized antibody magnetic beads (hereinafter referred to as "magnetic beads") may be made of any material, such as iron oxide, that generates a force that moves the reaction reagent through a liquid when a magnetic field is applied. Magnetic beads may also have a magnetic substrate surface coated with polystyrene or silica. The antibody modifying the surface of the latex beads and the antibody modifying the surface of the magnetic beads may be the same or different. Furthermore, antibody modification to these beads may be by covalent or physical bonding. The reaction reagent may be a liquid of any composition as long as it does not inhibit the function of the magnetic beads and latex beads. For example, phosphate buffer, Tris buffer, Good's buffer, etc. can be used, and additives such as protein components such as BSA or skim milk, or surfactants such as Tween 20, may be added.

[0023] The washing solution may have any composition as long as it has the property of breaking the bond between the antigen and antibody but does not have the property of breaking the antibody or antigen from the beads.

[0024] The eluent may be any substance, such as an acid, an alkali, a non-polar solvent, or a reducing agent, as long as it has the property of separating the bond between the antigen and the antibody or separating the antibody or antigen from the beads.

[0025] An outline of a method for detecting or estimating the concentration of an analyte according to one embodiment of the present invention will be described using Figures 3, 4, and 5. First, a test sample containing or potentially containing an antigen to be detected or quantified (hereinafter referred to as "target antigen") is mixed with a reaction reagent containing latex beads and magnetic beads, and the mixture is incubated in a reaction vessel (step S301). Prior to step S301, the test sample may be diluted with a sample dilution solution. In this case, the sample dilution solution may contain a reaction promoter that promotes the reaction between the antigen and the antibody, such as polyethylene glycol (PEG) or polyethylene oxide (PEO), or a blocking agent that suppresses nonspecific binding between the antigen and the antibody, such as BSA, skim milk, or a surfactant.

[0026] 4(a) is a schematic diagram showing a state in which a test sample and a reaction reagent are mixed in a reaction vessel 417. A mixture 418 of the test sample and the reaction reagent contains latex beads 411 modified with an antibody 412, magnetic beads 413 modified with an antibody 414, a target antigen 410, and various impurity particles 419. As an immune reaction progresses in the mixture, the magnetic beads and latex beads bind to each other via the target antigen in the test sample, forming sandwich complexes 421 and 422, as shown in FIG. 4(b) (step S302).

[0027] Next, a magnetic field is applied to the reaction vessel. In this embodiment, a magnetic field is applied to a portion of the reaction vessel by, for example, bringing a permanent magnet 430 close or passing a current through a coil installed near the reaction vessel. Then, as shown in FIG. 4(c), the sandwich complex formed in step S302 is attracted and fixed near the wall of the reaction vessel to which the magnetic field is applied (step S303). Next, while the magnetic field is still applied, the supernatant, i.e., the liquid of the test sample and reaction reagent mixed in step S301, is removed from the reaction vessel (step S304). Next, a cleaning solution is introduced into the reaction vessel and then removed (step S405). These steps S304 and S305 remove impurity particles from the reaction vessel, as shown in FIG. 5(a). These steps S304 and S305 leave sandwich aggregates 431 and 432 in the reaction vessel, while removing impurities contained in the test sample.

[0028] Next, when the eluate is injected into the reaction vessel with a magnetic field applied, the bonds between the antigen and magnetic beads 521 and between the antigen and latex beads 522 are released, leaving the latex beads 522 and antigens 520 free in the eluate 523 (step S306), as shown in Figure 5(b). Meanwhile, the magnetic beads that have been released from the bonds with the latex beads via the antigen remain fixed to the wall of the reaction vessel to which the magnetic field is applied. In the present invention, the concentration of latex beads per unit volume of matrix can be increased, i.e., concentrated, by reducing the amount of eluate 523 compared to the mixture 418 of the test sample and reaction reagent.

[0029] In the present invention, the eluate used in step S306 may contain an acid, an alkali, an organic solvent, a reducing agent, or the like, as long as it has the property of separating the bond between an antigen and an antibody, or the property of separating the antibody from the beads (the base material of the magnetic beads or latex beads in this specification).

[0030] Next, the eluate containing the liberated latex beads is introduced into one chamber of a pore sensor as shown in FIG. 1 , and the electrolyte is introduced into the other chamber, establishing electrical continuity through the pores. A voltage is applied between the electrodes in each chamber, creating a state in which an ionic current flows through the pores (step S307), causing the latex beads and antigens in the eluate to pass through the pores. For example, if the pore diameter is larger than the particle size of the latex beads but not larger than about five times the diameter, a pulse-like transient change occurs in the ionic current between the electrodes each time a latex bead passes through the pore (step S308). Figure 5(c) shows an example of latex bead 501, target antigen 502, latex bead 503, target antigen 504, and latex bead 505 passing through pore 530 in this order. Pulse signals 591, 593, and 595 are measured as latex beads 501, 503, and 505 pass through the pore, respectively. On the other hand, the target antigen is small enough compared to the diameter of the pores, so no pulse signal is measured.

[0031] In Figure 4(b) (step S302), there is a strong positive correlation between the number of antigen molecules in the test sample and the number of latex beads bound to the magnetic beads. Therefore, the number of latex beads released in Figure 5(b) (step S307) also has a strong positive correlation with the number of antigen molecules in the test sample. In the present invention, the antigen in the test sample can be detected and its concentration can be estimated from the number of latex beads passing through the pore in Figure 5(c), i.e., the number of observed pulses (step S309).

[0032] 4 and 5, the antibody 412 modifying the surface of the latex beads and the antibody 414 modifying the surface of the magnetic beads are described as different antibodies. For example, using monoclonal antibodies that bind to target antigens with different epitopes can improve the specificity of target antigen measurement. The antibodies modifying the latex beads and magnetic beads may be the same, or the ratio of the respective antibodies may be different.

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0034] An example of the results of a demonstration experiment of the present invention will be described below. Two types of anti-PSA mouse monoclonal antibodies manufactured by Mikuri Immuno Research Institute, Inc. were used as antibodies. Magnetic beads with a particle size of 180 nm manufactured by Tamagawa Seiki Co., Ltd. and latex beads with a particle size of 300 nm manufactured by Fujikura Kasei Co., Ltd. were used as substrates. One type of antibody was covalently immobilized on each of the magnetic beads and latex beads using a carbodiimide crosslinker. These antibody-immobilized beads were suspended in a matrix consisting of 50 mM Tris-HCl buffer, 150 mM NaCl, 0.5% BSA, and a surfactant. The sample diluent and wash solution consisted of 50 mM Tris-HCl buffer, 150 mM NaCl, 0.5% BSA, and a surfactant. The eluent consisted of 100 mM glycine-HCl, 150 mM NaCl, 0.5% BSA, and a surfactant. In addition, a PSA calibrator manufactured by Fujirebio was used as a standard for the target antigen.

[0035] In this demonstration experiment, the specimen was diluted 1:1 with specimen diluent and used as the test sample. In step S301, the specimen was mixed with the reaction reagent and stirred. In step S301, the incubation was performed at 37°C for 15 minutes after stirring. The magnetic collection in steps S303 to S304 was performed by leaving the tube stationary for 5 minutes. In step S304, the tube was pipetted three times in a tube inside the reaction vessel, then placed in a magnetic collection stand and left stationary for 5 minutes for magnetic collection, after which the supernatant was removed. In step S306, the eluate was added to the tube and stirred for 5 minutes, after which the pore measurement in steps S307 and S308 was performed.

[0036] For pore measurement, an Asahi Rubber pore sensor with a diameter of 1.2 μm and a partition thickness of 50 nm and an Advantest microcurrent measurement device were used, and pulse measurement was performed on the eluate in which the target antigen and latex beads were eluted in step S306 according to the procedures of steps S307 and S308. 18 μL of the eluate in which the target antigen and latex beads were eluted was introduced into the sample-side chamber of the pore sensor, and the voltage applied between the chambers was 100 mV.

[0037] Next, to create a calibration curve, samples with PSA calibrator concentrations of 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, and 100.0 ng / mL, which are the target antigen, were prepared and measured using the method of the present invention. The measurement results for each sample with different calibrator concentrations are shown in Figure 6. In Figure 6, the horizontal axis represents pulse height 601 (peak current value, unit: nA) and the vertical axis represents pulse width 602 (duration, unit: μs), with each plot representing one pulse. As shown in Figure 6, the number of pulses increases as the antigen concentration increases. From this data, a calibration curve 700, as shown in Figure 7, was created. The number of pulses 702 measured correlated well with the target antigen concentration 701, demonstrating that antigen quantification is possible.

[0038] Next, another example of a demonstration experiment of the present invention will be described. In the above demonstration experiment, the elution solution used was 100 mM glycine HCl (pH 2.7), 150 mM NaCl, 0.5% BSA, and a surfactant. In this example, 100 mM Tris HCl (pH 8.5), 150 mM NaCl, and 100 mM dithiothreitol (DTT) and a surfactant were used. DTT is a strong reducing agent and has the ability to reduce disulfide bonds. While it was used to cleave disulfide bonds in the crosslinked structure between the deposit and the beads, it may also be used to directly cleave the deposit structure. Similar reagents include 2-mercaptoethanol and tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). These reagents can also be used under denaturing conditions with the addition of high concentrations of guanidine hydrochloride, urea, and sodium dodecyl sulfate (SDS). Other than that, the process is the same as that described with reference to FIG.

[0039] The verification results of the present invention using the above protocol are shown in Fig. 8. Similar to the results in Fig. 7, the number of pulses 802 observed correlates with the antigen concentration 801, demonstrating that quantification of the antigen is possible.

[0040] Although each embodiment of the present invention has been described above, various inventions can be formed by appropriately combining the multiple components disclosed in each embodiment. For example, some components may be omitted from all the components shown in the embodiments. Furthermore, components of different embodiments may be appropriately combined.

[0041] 100 Pore sensor 101 Test object 110 Chamber 111 Inlet 112 Electrode 120 Chamber 121 Inlet 122 Electrode 140 Pore 141 Diaphragm 150 Amplifier 151 Ammeter 152 Voltage source 200 Baseline 201 Time (horizontal axis) 202 Current value (vertical axis) 208 Peak current 210 Pulse signal 410 Antigen 411 Latex beads 412 Antibody 413 Magnetic beads 414 Antibody 417 Reaction vessel 418 Mixed solution 419 Contaminant particles 421 Sandwich complex 422 Sandwich complex 430 Permanent magnet 431 Sandwich aggregate 432 Sandwich aggregate 501 Latex beads 502 Antigen 503 Latex beads 504 Antigen 505 Latex beads 520 Antigen 521 Magnetic beads 522 Latex beads 523 Eluate 530 Pore 591 Pulse signal 593 Pulse signal 595 Pulse signal 601 Pulse height 602 Pulse width 700 Calibration curve 701 Antigen concentration 702 Number of pulses 801 Antigen concentration 802 Number of pulses

Claims

1. A method for detecting or estimating the concentration of an analyte, comprising: a pore sensor having one pore, two chambers connected to both sides of the pore, and a pair of electrodes installed in each of the two chambers; and estimating the concentration of an analyte in a test sample using a reaction reagent containing attached magnetic beads having a first attachment attached to the magnetic bead surface that specifically binds to the analyte, and attached detection beads having a non-magnetic bead surface that specifically binds to the analyte; when the test sample and the reaction reagent are mixed to prepare a reaction solution, binding occurs between the attached magnetic beads and the attached detection beads via the analyte depending on the concentration of the analyte in the reaction solution; by applying a magnetic field to a container containing the test sample containing a sandwich complex of the attached magnetic beads, the analyte, and the attached detection beads formed by the binding, the sandwich complex containing the attached magnetic beads is fixed in the container; and the reaction solution is replaced with a washing solution, and the washing solution is then removed; Furthermore, the method for detecting or estimating the concentration of an analyte comprises: introducing an elution solution into the container to release the attached detection beads from the sandwich complex into the elution solution, and simultaneously fixing the attached magnetic beads in the container using the magnetic field to separate the attached detection beads from the attached magnetic beads; introducing the elution solution containing the separated attached detection beads into one of the two chambers of the pore sensor; introducing an electrolyte into the other of the two chambers of the pore sensor to electrically connect the electrode pair; applying a voltage to the electrode pair and measuring the number of pulse signals caused by transient current changes in the electrode pair each time the attached detection beads pass through the pore, thereby measuring the presence or concentration of the analyte in the test sample.

2. A method for detecting or estimating the concentration of a test substance as described in claim 1, characterized in that the test substance is an antigen, the first attachment is a first type of antibody, and the second attachment is a second type of antibody different from the first type of antibody.

3. A method for detecting or estimating the concentration of a specimen according to claim 1, characterized in that the specimen is an antigen, and the first and second deposits are antibodies of the same species.

4. A method for detecting or estimating the concentration of a specimen according to claim 1, characterized in that the specimen is an antibody, and the first and second deposits are antigens.

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