Diagnosis method, diagnosis device, and antibody regeneration method

Abstract

This diagnosis method comprises: a marker complex concentration measurement step for disposing, on a substrate, a photoacid and an anti-marker antibody that specifically binds with a marker and providing a test sample thereon to measure the concentration of a marker complex in which the anti-marker antibody and a marker in the test sample are bound; and a marker dissociation step for irradiating the photoacid with light to dissociate the marker from the marker complex.

Description

Diagnostic method, diagnostic device, and antibody regeneration method

[0001] The present invention relates to a diagnostic method, a diagnostic device, and a method for regenerating an antibody.

[0002] Antigen-antibody reactions are an excellent molecular recognition technology that can be applied to most antigens, such as proteins, as long as the antibody is available, and have become the mainstream analytical method in clinical testing. However, in conventional technologies, in order to regenerate the antibody for repeated use, it is necessary to dissociate the antigen from the antigen-antibody complex using a dissociation solution (see, for example, Patent Document 1), which requires the replenishment of reagents and increases the size of the equipment, making it difficult to create a device that can be attached to a living body to perform time-series monitoring.

[0003] As a prior art related to antibody regeneration using photoacid, for example, Non-Patent Document 1 reports the application of photoacid as a sensor to the regeneration of DNA hybridization. However, Non-Patent Document 1 describes the local control of the DNA hybridization / dehybridization process, and does not disclose the use of photoacid for antibody regeneration.

[0004] Furthermore, Non-Patent Document 2 reports the application of photoacid to a protein concentration sensor. However, Non-Patent Document 2 uses photoacid to selectively separate (collect) target proteins, and does not intend to use photoacid as a concentration sensor for antibody regeneration.

[0005] Japanese Patent Application Publication No. 3-214051

[0006] Hagit Peretz-Soroka et al. , :Manipulating and Monitoring On-Surface Biological Reactions by Light-Triggered Local pH Alterations. Nano Lett. 15(7):4758-68, 2015. doi: 10.1021 / acs. nanolett. 5b01578. Ella Borberg et al. , :Light-Controlled Selective Collection-and-Release of Biomolecules by an On-Chip Nanostructured Device. Nano Lett. 19(9), 5868-5878, 2019. doi: 10.1021 / acs. nanolett. 9b01323.

[0007] The present invention aims to solve the above-mentioned problems of the prior art and to achieve the following objectives: That is, the present invention aims to provide a diagnostic method and diagnostic device that can regenerate an anti-marker antibody in a detection unit that recognizes a marker, thereby enabling repeated measurement of the marker and realizing time-series monitoring of the marker, and an antibody regeneration method that can easily regenerate an antibody for repeated use.

[0008] Means for solving the above problems are as follows. That is, <1> A diagnostic method comprising: a marker complex concentration measurement step of arranging an anti-marker antibody that specifically binds to a marker and a photoacid on a substrate, and measuring the concentration of a marker complex in the test sample, in which the marker and the anti-marker antibody are bound, by providing the test sample; and a marker dissociation step of irradiating the photoacid with light to dissociate the marker from the marker complex. <2> The diagnostic method according to <1>, in which the concentration of the marker complex is repeatedly measured at intervals. <3> The diagnostic method according to <1> or <2>, in which the substrate is a vibrating body. <4> The diagnostic method according to any one of <1> to <3>, in which the light is laser light. <5> The diagnostic method according to any one of <1> to <4>, wherein the marker is a cardiac disease marker, the anti-marker antibody is an anti-cardiac disease marker antibody, and the cardiac disease marker is at least one of brain natriuretic peptide (BNP) and N-terminal fragment of brain natriuretic peptide precursor (NT-proBNP). <6> The diagnostic method according to any one of <1> to <5>, wherein the photoacid is 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). <7> A diagnostic device comprising: an oscillator having an anti-marker antibody that specifically binds to the marker and a photoacid disposed on its surface; a detection unit that detects the resonant frequency of the oscillator; and a light irradiation unit that irradiates the photoacid with light. <8> The diagnostic device according to <7>, wherein the oscillator is placed on a vibrator, and the oscillator and the vibrator are not mechanically coupled, and the oscillator is not mechanically coupled to any member. <9> An antibody regeneration method, comprising: an antigen-antibody complex formation step of arranging an antibody that specifically binds to an antigen to be measured and a photoacid on a substrate, and providing a test sample to form an antigen-antibody complex in which the antigen and the antibody are bound; and an antigen dissociation step of irradiating the photoacid with light to dissociate the antigen from the antigen-antibody complex.

[0009] According to the present invention, the above-mentioned problems in the prior art can be solved, the above-mentioned object can be achieved, and a diagnostic method and a diagnostic device can be provided that can regenerate the anti-marker antibody in the detection unit that recognizes the marker, thereby enabling the marker to be repeatedly measured and realizing time-series monitoring of the marker, as well as an antibody regeneration method that can easily regenerate antibodies for repeated use.

[0010] Figures 1(a) to 1(c) are schematic diagrams illustrating the principle of the antibody regeneration method. Figure 2 is a diagram illustrating the chemical reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited. Figure 3 is a fluorescence spectrum diagram of HPTS. Figure 4 is a diagram illustrating the change in pH over time when an HPTS solution is irradiated with laser light having a wavelength of 405 nm. Figure 5 is a diagram illustrating the relationship between laser irradiation intensity and the rate of pH change when an HPTS solution is irradiated with laser light having a wavelength of 405 nm. Figure 6 is a diagram illustrating the reproducibility of the pH controllability of an HPTS solution by laser light irradiation. Figure 7 is a diagram illustrating an example of HPTS pretreatment. Figure 8 is a schematic diagram illustrating an example of a method for immobilizing anti-BNP antibodies and HPTS on the vibrator surface. Figures 9(a) to 9(d) are schematic diagrams illustrating the principle of the diagnostic method. Figure 10 is a schematic diagram illustrating an example of a diagnostic device. FIG. 11 is a graph showing that if the cardiac disease marker BNP is not repeatedly measured over time, high and low BNP levels may be overlooked and deemed normal, leading to a risk of misdiagnosing cardiac disease. FIGS. 12(a) and 12(b) are schematic diagrams showing a state in which a wearable cardiac disease diagnostic device is worn on the arm and cardiac disease markers are measured over time and displayed. FIGS. 13(a) to 13(f) are schematic diagrams showing the BNP measurement method in the examples. FIG. 14 is a schematic diagram showing the method of irradiating a microplate with laser light. FIG. 15 is a graph showing the relationship between HPTS concentration and absorbance of a TMB solution in Example 1 and Comparative Example 1. FIG. 16A is an image showing an Au mesh substrate in Example 2. FIG. 16B is a schematic diagram showing immobilization of anti-BNP antibodies on an Au mesh substrate and the antigen-antibody-based BNP detection reaction in Example 2. FIG. 17 is a graph showing the relationship between BNP concentration and luminescence intensity in Example 2.

[0011] (Method for Regenerating Antibodies) The method for regenerating antibodies of the present invention comprises an antigen-antibody complex formation step and an antigen dissociation step, and may further comprise other steps as necessary.

[0012] In the present invention, a photoacid is placed near an antibody that specifically binds to the antigen to be measured, and by irradiating the photoacid with light, the antigen can be dissociated from the antigen-antibody complex, making it possible to reuse the antibody simply by physical stimulation, and the antibody can be used repeatedly. By utilizing the principle of this antibody regeneration method, as described below, it is possible to repeatedly measure the marker and realize time-series monitoring of the marker.

[0013] <Antigen-antibody complex formation step> The antigen-antibody complex formation step is a step in which an antibody that specifically binds to the antigen to be measured and a photoacid are placed on a substrate, and a test sample is provided to form an antigen-antibody complex in which the antigen and the antibody are bound to each other.

[0014] -Substrate- The substrate is not particularly limited in shape, size, structure, material, etc., and can be appropriately selected depending on the purpose. Examples of the shape of the substrate include beam, rod, plate, film, sheet, and microplate. The size of the substrate is not particularly limited and can be appropriately selected depending on the application, etc. The structure of the substrate is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, a single-layer structure or a multi-layer structure. The material of the substrate is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include resin, ceramics, crystal, and metal.

[0015] Examples of the resins include polystyrene resin, acrylic resin, polycarbonate resin, polyvinylidene fluoride (PVDF), polylactic acid, polyethylene terephthalate resin, polyvinyl chloride resin, and polystyrene resin. Examples of the ceramics include silicon, barium titanate, lead titanate, lead zirconate titanate (PZT), potassium niobate, lithium niobate, sodium tungstate, and lithium tantalate. Examples of the crystals include quartz. Examples of the metals include iron, silicon steel, ferrite, cobalt, nickel, aluminum, and alnico.

[0016] -Antigen- An antigen refers to a component contained in a test sample that is to be measured through an antigen-antibody reaction, and examples thereof include amino acids, peptides, proteins, nucleic acids, lipids, carbohydrates, electrolytes, viruses, bacteria, pollen, and other low-molecular-weight metabolites.

[0017] -Antibodies- Antibodies include immunoglobulin molecules themselves that specifically bind to the antigen to be measured, as well as Fab and F(ab') produced by proteolytic enzymes such as papain and pepsin, or by chemical degradation. ２ Decomposition products such as fragments are also included. The antibody may be either a polyclonal antibody or a monoclonal antibody. There are no particular limitations on the method for obtaining the antibody, and any commonly used method can be used.

[0018] -Photoacid- The photoacid is not particularly limited as long as it is a compound that generates an acid upon irradiation with light, and can be appropriately selected depending on the purpose. The photoacid preferably has an acid strength (pKa) value in the range of 5 to 10. In the singlet electron state, it becomes a stronger acid. The most common photoacids are oxo salts, but only some of them can have their acid strength enhanced by photoexcitation. Examples of the photoacid include 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), phenol, 7-cyano-2-naphthol, and tryptophan. These may be used alone or in combination of two or more. Among these, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is preferred due to its high acid strength and fluorescent spectrum in the visible light region.

[0019] The method for disposing the photoacid and antibody on the substrate is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a method in which the antibody is immobilized on the substrate by a method using a self-assembled monolayer, and the photoacid is immobilized instead of a blocking treatment for filling the gaps between the immobilized antibodies. Details of the method for disposing the photoacid and antibody on the substrate will be described later.

[0020] Test Sample: The test sample is not particularly limited and can be selected appropriately depending on the purpose. However, a liquid test sample is preferred because the pH is lowered by hydrogen ions released from the photoacid. Examples of liquid test samples include whole blood (before coagulation), plasma, serum, amniotic fluid, secretions from the mucosal epithelium of the upper and lower respiratory tract, saliva, exudate, tissue fluid, secretions, breast milk, sweat, nasal mucus, serous fluid, transudate, cyst fluid, urine, cerebrospinal fluid, tears, gastric juice, bile, pancreatic juice, and mucus. When the test sample is liquid, it can be analyzed directly without pretreatment. This allows for simple and rapid analysis and avoids denaturation such as decomposition and oxidation of the test sample, thereby enabling accurate and precise analysis of the components contained in the test sample. Liquid test samples may also be subjected to pretreatment, such as dilution, deproteinization, lipid elution, and concentration, as needed.

[0021] <Antigen Dissociation Step> The antigen dissociation step is a step in which a photoacid is irradiated with light to dissociate the antigen from the antigen-antibody complex. In the antibody regeneration method of the present invention, after measurement of the concentration of the antigen-antibody complex contained in the test sample is completed, the antibody can be regenerated by irradiating the photoacid with light to dissociate the antigen from the antigen-antibody complex. The method for measuring the concentration of the antigen contained in the test sample is not particularly limited and can be appropriately selected depending on the purpose. Examples include ELISA (Enzyme-Linked Immunosorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead analysis, and a method using a resonant mass sensor that detects the resonant frequency associated with a change in mass when an antigen adheres to an oscillator vibrating at a constant frequency. Among these, a resonant mass sensor is preferred because it does not require replenishment of reagents and dissociation solution and allows for device miniaturization.

[0022] The light irradiated onto the photoacid is preferably laser light. The wavelength of the laser light is not particularly limited and can be selected appropriately depending on the type of photoacid, but is preferably 400 nm or more and 1,000 nm or less, and more preferably 400 nm or more and 800 nm or less. The output of the laser light is not particularly limited and can be selected appropriately depending on the purpose, but is preferably 1 mW or more. Irradiating the photoacid with laser light excites the photoacid, releasing hydrogen ions, and lowering the pH of the test sample, thereby dissociating the antigen from the antigen-antibody complex and regenerating the antibody.

[0023] <Other Steps> The other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include a cleaning step and a control step.

[0024] Here, Figures 1(a) to 1(c) are schematic diagrams illustrating the principle of the antibody regeneration method. As shown in Figure 1(a), photoacid 3 is immobilized near antibody 4, which specifically binds to the antigen to be measured and is immobilized on substrate 1, and test sample 2 is then applied. Then, as shown in Figure 1(b), antigen 5 in the test sample undergoes an antigen-antibody reaction with antibody 4, forming antigen-antibody complex 6. Next, as shown in Figure 1(c), photoacid 3 is irradiated with light of a specific wavelength for several seconds, causing hydrogen ions to be released from the photoacid, changing the pH of test sample 2 and causing antigen 5 to dissociate from antigen-antibody complex 6 in an extremely short time. Then, test sample 2 is discarded, allowing antibody 4 to be regenerated and reused.

[0025] Figure 2 shows the reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited. When HPTS in the ground state is irradiated with laser light having a wavelength of 405 nm, HPTS becomes excited, releases hydrogen ions, and emits fluorescence with a wavelength of 512 nm. To verify the excitation light wavelength effective for efficiently releasing hydrogen ions from HPTS, the fluorescence spectroscopic characteristics of HPTS were measured (FP8600, manufactured by JASCO Corporation). The results are shown in Figure 3. From the fluorescence spectrum of HPTS shown in Figure 3, a fluorescence peak was observed at excitation light wavelengths of 400 nm to 430 nm, indicating that the optimal wavelength for HPTS is 405 nm.

[0026] Next, to verify the pH control ability of the HPTS solution by laser light irradiation, the change in pH of the HPTS solution over time was measured. First, HPTS (30080, manufactured by Cayman Chemical Company) was dissolved in distilled water, and 1.0 × 10 －３A 100 mol / L HPTS aqueous solution was prepared. Next, a pH meter (PAL-pH, manufactured by Atago Co., Ltd.) was fixed onto a plate shaker, and 1 mL of the prepared HPTS aqueous solution was dripped onto the electrode of the pH meter. Next, a laser irradiation device (D405C-300-11-1C-11, manufactured by Kyocera SOC Corporation) and a diffusion lens (AL1210M-A, manufactured by Thorlabs Inc.) were installed, and the dripped HPTS aqueous solution was irradiated with laser light having a wavelength of 405 nm. At this time, the distance between the diffusion lens and the pH meter was set to 100 mm to ensure that the entire HPTS aqueous solution was irradiated with laser light. The plate shaker was set to 300 rpm, and pH measurements were performed while stirring. The results are shown in Figures 4 and 5. From the results of the change in pH of the HPTS aqueous solution over time shown in Fig. 4, it was confirmed that continuous irradiation of the HPTS aqueous solution with a laser beam having a wavelength of 405 nm lowered the pH of the HPTS solution from 6 to around 3. Furthermore, from the results in Fig. 5, it was found that the rate of change in pH of the HPTS aqueous solution increased in proportion to the irradiation intensity of the laser beam, and that irradiation with a laser beam having an irradiation intensity of 50 mW or more resulted in a pH change that was sufficient to dissociate the antigen from the antigen-antibody complex.

[0027] Next, the reproducibility of the pH control ability of the HPTS solution by laser light irradiation was verified. 1 mL of a 1 mmol / L HPTS (30080, manufactured by Cayman Chemical Company) aqueous solution was added to the detection section of a pH meter (PAL-pH, manufactured by Atago Co., Ltd.). The HPTS solution in the detection section was repeatedly irradiated (output 50 mW) with a 405 nm laser (D405C-300-11, manufactured by Kyocera SOC Corporation) and stopped, and the pH change was measured. The results are shown in Figure 6. From Figure 6, it can be seen that laser light irradiation caused the release of H from HPTS. ＋ It was confirmed that the dissociation (deprotonation) of H occurs, causing a decrease in pH, and when the laser light irradiation is stopped, the original molecular structure is quickly restored, causing the pH to increase. ＋ It was shown that the emission / absorption of

[0028] (Diagnostic Method) The diagnostic method of the present invention comprises a marker complex concentration measuring step and a marker dissociation step, and may further comprise other steps as necessary.

[0029] <Marker complex concentration measurement process> The marker complex concentration measurement process is a process in which an anti-marker antibody that specifically binds to a marker and a photoacid are placed on a substrate, and a test sample is provided to measure the concentration of a marker complex in which the marker and the anti-marker antibody are bound in the test sample.

[0030] The marker is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include heart disease markers, cytokines (IL-1β, IL-6, IL-4, INFγ, TNF, etc.), proteins (zonulin, LBP (lipopolysaccharide-binding protein), etc.), etc. Among these, heart disease markers are preferred.

[0031] - Cardiac Disease Markers - Examples of cardiac disease markers include biochemical myocardial markers such as brain natriuretic peptide (BNP), N-terminal fragment of brain natriuretic peptide precursor (NT-proBNP), troponin T (TnT), myoglobin, and CK-MB, as well as heart-type fatty acid-binding protein. These may be used alone or in combination of two or more. Of these, BNP and NT-proBNP are recognized as markers for heart failure, and troponin is recognized as a marker for myocardial infarction. These are items tested in early diagnosis, have high cardiac disease specificity, and can be the most important diagnostic information for cardiac disease.

[0032] The production of the BNP and NT-proBNP originates from the same BNP gene. After transcription and translation, the BNP gene produces a BNP precursor (proBNP [1-108]). This is then cleaved into physiologically inactive NT-proBNP (76 amino acids [1-76] from the N-terminus of proBNP) and physiologically active mature BNP (the remaining 32 amino acids [77-108]). In other words, BNP and NT-proBNP are secreted in equimolar amounts from the myocardium. Gene expression of the BNP and NT-proBNP is enhanced primarily in response to wall stress (progressive stress) in the ventricle, leading to their rapid production and secretion. Therefore, in heart failure with increased wall stress, the blood concentrations of the BNP and NT-proBNP increase depending on the severity of the condition.

[0033] The anti-marker antibody is not particularly limited and can be appropriately selected depending on the purpose. Examples include anti-cardiac disease marker antibodies, antibodies whose antigen is a cytokine (IL-1β, IL-6, IL-4, INFγ, TNF, etc.), and antibodies whose antigen is a protein (zonulin, LBP (lipopolysaccharide-binding protein), etc.). Among these, anti-cardiac disease marker antibodies are preferred.

[0034] Anti-cardiac disease marker antibodies include immunoglobulin molecules themselves that specifically bind to cardiac disease markers, as well as Fab and F(ab') antibodies generated by proteolytic enzymes such as papain and pepsin, or by chemical degradation. ２ Decomposition products such as fragments are also encompassed. Furthermore, the anti-cardiac disease marker antibody may be either a polyclonal antibody or a monoclonal antibody. The anti-cardiac disease marker antibody can be obtained by a commonly used method. For example, when the cardiac disease marker is BNP, an anti-BNP monoclonal antibody is used. Furthermore, when the cardiac disease marker is NT-proBNP, an anti-NT-proBNP monoclonal antibody is used.

[0035] - Substrate - The substrate is not particularly limited and can be appropriately selected depending on the purpose, and the same substrates as those used in the antibody regeneration method described above can be used. Among these, it is preferable to use a vibrating body as the substrate, because the concentration of the marker can be measured by measuring the resonant frequency of the vibrating body.

[0036] Photoacid: The photoacid is not particularly limited and can be appropriately selected depending on the purpose. The same photoacids as those used in the antibody regeneration method described above can be used. Among these, it is preferable to use 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as the photoacid because of its high acid strength and fluorescent spectrum in the visible light region.

[0037] Here, we will explain a method for immobilizing anti-BNP antibodies as anti-cardiac disease marker antibodies and HPTS as photoacid on the surface of a vibrating body as a substrate. A thin metal film is formed on the surface of the vibrating body, and a molecular layer-order molecular film is formed on this metal film by sputtering or the like. The antibody can be immobilized on the vibrating body by modifying this molecular film with an antibody. The metal film can be an adhesion layer and a platinum (Pt) film formed on the adhesion layer. Examples of the adhesion layer include Ti and Cr. The molecular layer-order molecular film can be a self-assembled monolayer (hereinafter sometimes referred to as a "SAM film"). Specifically, a titanium (Ti) and platinum (Pt) film is formed to a thickness of 500 Å on one surface of the vibrating body (quartz plate) by sputtering. To immobilize anti-BNP antibodies on the platinum (Pt) film surface, a SAM film is formed and the SAM film is modified with the anti-BNP antibody. Amine coupling can be used to bond the SAM film to the anti-BNP antibody. As a result of the above, the anti-BNP antibody is immobilized on the surface of the vibrating body 11 as shown in FIG. 8(a).

[0038] Next, as shown in FIG. 7, the four groups (three SO ３ Of the OH groups, OH is protected with an acetyl group, and three SO ３ One of the Na atoms is replaced with Cl, and as shown in FIG. 8C, the NH ２－ Trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate is immobilized on the end.

[0039] Pretreatment (Functional Group Substitution)—The functional groups of trisodium 8-hydroxypyrene-1,3,6-trisulfonate were substituted to form 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride, as shown in the reaction scheme in Figure 7. First, trisodium 8-hydroxypyrene-1,3,6-trisulfonate (20 g, 0.038 mol) was dissolved in 30 mL of water containing NaOH (2.4 g, 0.06 mol) and the solution was cooled to approximately 0°C. Next, acetic anhydride (5 g, 4.8 mL, 0.48 mol) was added dropwise to the solution, and the reaction mixture was stirred for 2 hours. Next, ethanol (20 mL) was added to complete the precipitation. The precipitate was collected by filtration, washed with ethanol (3 x 10 mL), and dried under reduced pressure for 24 hours to obtain a yellow solid, trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate (17 g, 80% yield). Next, a mixture of trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate (5 g, 0.0088 mol) and toluene (150 mL) is placed in a 0.25-liter round-bottom flask equipped with an automatic water separator (Dean-Stark trap) and a condenser. The mixture is then heated to reflux for 2 hours to dry the reaction mixture. The dried reaction mixture is then cooled to 60°C, and oxalyl chloride (6 mL) and N,N-dimethylformamide (DMF) (2 drops) are added. The mixture is then heated. The heated mixture is then refluxed for 8 hours to distill off the mixture of toluene and excess oxalyl chloride (30 mL). The sodium chloride precipitate is then filtered, and the solvent is removed from the filtrate under reduced pressure. The solid residue is then dried in vacuo for 24 hours to give 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride (4 g) (81.5% yield).

[0040] - Immobilization on the vibrating body surface - As shown in Figure 8(a), the vibrating body 11 on which the anti-BNP antibody has been immobilized is incubated for 24 hours in a mixed solution of 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride and pyridine obtained in the pretreatment described above, and then exposed to a saturated sodium bicarbonate solution of phenol functional groups. As a result, the anti-BNP antibody and trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate can be immobilized on the surface of the vibrating body 11, as shown in Figure 8(c).

[0041] - Measuring the Concentration of Marker Complex - The method for measuring the concentration of a marker complex formed by binding a marker and an anti-marker antibody in a test sample is not particularly limited and can be selected appropriately depending on the purpose. Examples include ELISA (Enzyme-Linked Immunosorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead analysis, and a method using a resonant mass sensor that detects the resonant frequency associated with a change in mass when a cardiac disease marker adheres to an oscillator vibrating at a constant frequency. Among these, a method using a resonant mass sensor is preferred because it does not require the replenishment of reagents and dissociation solutions, allows for device miniaturization, enables the regeneration of anti-marker antibodies simply by irradiating the photoacid with light, allows the anti-marker antibodies to be reused, and enables time-series monitoring. In a method using a resonant mass sensor, for example, when an oscillator on which an anti-marker antibody that specifically binds to the marker and a photoacid are immobilized is provided to a test sample, the marker in the test sample is captured by the anti-marker antibody, the mass of the oscillator increases, and the resonant frequency of the oscillator decreases. As a result, the concentration of the marker in the test sample can be measured from the rate of change in the resonant frequency.

[0042] <Marker Dissociation Step> The marker dissociation step is a step in which the photoacid is irradiated with light to dissociate the marker from the marker complex. In the diagnostic method of the present invention, after measurement of the concentration of the marker contained in the test sample is completed, the photoacid is irradiated with light to dissociate the marker from the marker complex in which the marker and the anti-marker antibody are bound, thereby regenerating the anti-marker antibody. The light is preferably laser light. By irradiating the photoacid with laser light, hydrogen ions are released from the photoacid, and the pH of the liquid test sample decreases, thereby dissociating the marker from the marker complex and regenerating the anti-marker antibody.

[0043] In the present invention, time-series monitoring of the marker can be achieved by regenerating the anti-marker antibody and repeatedly measuring the concentration of the marker complex at intervals.

[0044] <Other Steps> The other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include a cleaning step and a control step.

[0045] Here, Figures 9(a) to 9(d) are schematic diagrams illustrating the principle of the diagnostic method. As shown in Figure 9(a), photoacid 3 is immobilized on an oscillator 11 near an anti-marker antibody 14 that specifically binds to the marker to be measured, and a test sample 2 is then provided. Then, as shown in Figure 9(b), marker 15 in the test sample undergoes an antigen-antibody reaction with anti-marker antibody 14, forming a marker complex 16. Next, as shown in Figure 9(c), marker 15 in the test sample is captured by anti-marker antibody 14, and the mass of oscillator 11 increases. As a result, the resonant frequency of oscillator 11 decreases, and the concentration of the marker in the test sample can be measured from the rate of change in resonant frequency. The change in the resonant frequency of oscillator 11 can be measured using a laser displacement meter. Next, as shown in Figure 9(d), the photoacid 3 is irradiated with light of a specific wavelength, changing the pH of the test sample 2 and causing marker 15 to dissociate from marker complex 16. Thereafter, when the test sample 2 is discharged, the anti-marker antibody 14 can be regenerated as shown in FIG. 9(a) and can be used repeatedly.

[0046] As described above, the diagnostic methods of the present invention include methods for quantifying markers contained in a test sample and methods for evaluating markers contained in a test sample.

[0047] (Diagnostic device) The diagnostic device of the present invention comprises an oscillator having an anti-marker antibody that specifically binds to the marker and a photoacid on its surface, a detection unit that detects the resonant frequency of the oscillator, and a light irradiation unit that irradiates the photoacid with light, and further comprises other means as necessary.

[0048] <Vibrator> An anti-marker antibody and photoacid that specifically bind to the marker are disposed on the surface, and a piezoelectric crystal or the like can be used. Piezoelectric crystals are materials that exhibit an electrical response when strained and, conversely, generate strain when a voltage is applied, and examples thereof include quartz, barium titanate, lead titanate, lead zirconate titanate (PZT), potassium niobate, lithium niobate, sodium tungstate, and lithium tantalate. The method for disposing the anti-marker antibody and photoacid on the vibrator surface is not particularly limited and can be selected appropriately depending on the purpose, and can be carried out in the same manner as the above-mentioned diagnostic method.

[0049] It is preferable that the vibrating body is placed on a vibrator, and that the vibrating body and the vibrator are not mechanically coupled, and that the vibrating body is not mechanically coupled to any member, which is preferable in that the resonance phenomenon is not inhibited by power supply or fixing, restrictions on the design of the shape and dimensions of the vibrating body can be eliminated, and mass changes can be measured with high sensitivity.

[0050] <Detection Unit> The detection unit is a means for detecting the resonant frequency of the vibrating body, and an optical detection unit consisting of a light-emitting element and a light-receiving element can be used. The optical detection unit may measure any of the frequency, displacement, velocity, and acceleration of the vibrating body. A laser displacement meter or the like can be used as the detection unit. The laser displacement meter can measure changes in the resonant frequency of the vibrating body in a non-contact manner.

[0051] <Light Irradiation Unit> The light irradiation unit is a means for irradiating the photoacid with light, and is preferably a laser light irradiation unit. The laser light irradiation unit has, for example, a laser light source, a diffusion lens, and a power source.

[0052] <Other Means> Examples of other means include input means, recording means, display means, communication means, and maintenance means.

[0053] Fig. 10 is a schematic diagram showing an example of a diagnostic device. The diagnostic device 10 in Fig. 10 includes an oscillator 11 having an anti-marker antibody 14 that specifically binds to the marker and a photoacid 3 on its surface, a detector 12 that detects the resonance frequency of the oscillator 11, a light irradiator 13 that irradiates the photoacid 3 with light, and a power source 17 that drives the oscillator 11.

[0054] There are no particular limitations on the vibrating body 11, and the vibrating body can be used alone, but it is preferable to use the vibrating body 11 mounted on a vibrator. In this case, the vibrating body 11 and the vibrator are not mechanically coupled, and the vibrating body 11 is not mechanically coupled to any member, that is, the vibrating body 11 has a structure that is not fixed anywhere, which is preferable in terms of not inhibiting the resonance phenomenon due to power supply or fixing, eliminating design constraints on the shape and dimensions of the vibrating body 11, and enabling high-sensitivity measurement of mass changes.

[0055] The vibrating body 11 is processed to have a shape and dimensions that are optimal for the resonance phenomenon. The vibrating body 11 is placed in an unconstrained state on a vibrator such as a piezoelectric element, and is structured so that when the piezoelectric element vibrates at a given frequency, the vibrating body 11 can vibrate at its own resonant frequency. When an AC voltage close to the natural frequency of the beam-shaped vibrating body is applied to the piezoelectric element, the vibrating body resonates at its own natural resonant frequency. In this way, there are no restrictions on the material or shape and dimensions of the vibrating body, and the degree of freedom in design can be dramatically improved.

[0056] The vibration mode of the vibrating body 11 is set so as not to be a vibration mode such as uniaxial vibration that moves in a certain direction, like a standing wave ultrasonic motor. In other words, the vibration mode of the vibrating body 11 is not a vibration mode that moves in a certain direction, and the amplitude is small, on the order of nanometers (nm), so the vibrating body 11 will not detach from the vibrator. For details of the vibrating body and the resonant mass sensor having the vibrator, see, for example, Japanese Patent No. 6086347.

[0057] When the oscillator 11, on which the anti-marker antibody 14 and photoacid 3 that specifically bind to the marker are immobilized, is applied to a test sample, the marker 15 in the test sample is captured by the anti-marker antibody 14, and the mass of the oscillator 11 increases. As a result, the resonant frequency of the oscillator 11 decreases, and the concentration of the marker 15 in the test sample can be measured from the rate of change in the resonant frequency. The change in the resonant frequency of the oscillator 11 is detected by the detection unit 12. The detection unit 12 uses a laser displacement meter as a means for measuring the resonant frequency of the oscillator 11. Next, after measuring the concentration of the marker 15 contained in the test sample, the light irradiation unit 13 irradiates the photoacid 3 with laser light. Then, hydrogen ions released from the photoacid decrease the pH of the test sample, causing the marker 15 to dissociate from the marker complex 16, allowing the anti-marker antibody 14 to be regenerated.

[0058] Commercially available stand-alone cardiac disease diagnostic devices, such as portable and desktop devices, are very useful for initial diagnosis in small medical institutions. However, immediate information acquisition (POCT) is generally performed at facilities with on-site specialist physicians, and the widespread use of portable and desktop cardiac disease diagnostic devices is limited. Furthermore, measurement of BNP concentration as a cardiac disease marker requires repeated time-series measurements at intervals using a wearable cardiac disease diagnostic device, as shown in FIG. 11 . Unless measurements are taken repeatedly over time at intervals, high and low BNP levels may be overlooked and interpreted as normal, resulting in an incorrect diagnosis of cardiac disease. (a) of FIG. 12 is a schematic diagram showing an example of a wearable cardiac disease diagnostic device capable of rapid, on-site, and repeated measurement of cardiac disease markers. This wearable cardiac disease diagnostic device 22 can be worn on the patient's wrist to repeatedly measure BNP concentration at intervals. Furthermore, by transmitting the BNP concentration measured by the body-attached cardiac disease diagnosis device 22 to the smartphone 21 at any time, the BNP concentration can be displayed in chronological order as shown in Figure 12 (b), making it possible to accurately and quickly diagnose cardiac disease.

[0059] According to the diagnostic method and diagnostic device of the present invention, by placing a photoacid near an anti-marker antibody that specifically binds to a marker and irradiating the photoacid with light, the marker can be dissociated from the marker complex. This makes it possible to reuse the anti-marker antibody with just physical stimulation, and since the anti-marker antibody can be used repeatedly, it is possible to realize chronological monitoring of the marker.

[0060] Examples of the present invention will be described below, but the present invention is not limited to these examples in any way.

[0061] Example 1 The dissociation of a cardiac marker (BNP) from a cardiac marker complex by light irradiation of a photoacid (HPTS) was experimentally demonstrated, and the effect of changes in HPTS concentration on the degree of dissociation of BNP was evaluated.

[0062] First, as shown in FIG. 13(a), an anti-BNP antibody (14) (4BNP2cc-50E1cc, manufactured by HyTest Ltd.) was immobilized in the wells of a microplate (SpectraPlate-384 HB, manufactured by PerkinElmer Inc.) serving as a substrate (1) by a method using a self-assembled monolayer. Then, BNP (15) (Nanopia (registered trademark) BNP control for BNP, manufactured by Sekisui Medical Co., Ltd.) was added as a cardiac disease marker under the conditions shown in Table 1, and the mixture was incubated for 1 hour to form a cardiac disease marker complex (16).

[0063] Next, after washing the microplate, as shown in FIG. 13(b), an enzyme-labeled detection antibody (7) (4BNP2cc-24C5cc, manufactured by HyTest Ltd.) was added and incubated for 1 hour, and the anti-BNP antibody (14), BNP (15), and the enzyme-labeled detection antibody (7) were bound to the wells of the microplate in this order (see FIG. 13(c)).

[0064] HPTS (30080, manufactured by Cayman Chemical Company) was dissolved in distilled water to give a 1.0 × 10 －３ mol / L~7.0×10 －３A 1000 mol / L HPTS aqueous solution was prepared (see Table 1). Next, after washing the microplate, the HPTS aqueous solution prepared to the predetermined concentration was added, and the microplate was irradiated with laser light for 30 minutes, as shown in Figure 13(d). This lowered the pH of the HPTS aqueous solution, resulting in dissociation of BNP (15) and the enzyme-labeled detection antibody (7) from the anti-BNP antibody (14). Figure 14 shows the method for irradiating the microplate with laser light. A laser irradiation device (33) (D405C-300-11-1C-11, manufactured by Kyocera SOC Corporation) emitted a 405 nm wavelength laser beam through an f=10 diffusion lens (32) (AL1210M-A, manufactured by Thorlabs Inc.), and the diffused laser beam was irradiated onto the bottom surface of the microplate (31). The number of wells in the microplate (31) irradiated with laser light can be adjusted by changing the distance between the f=10 diffusing lens (32) and the microplate (31). In Figure 14, reference numeral 34 denotes a variable output power supply. In this example, the distance between the f=10 diffusing lens 32 and the microplate 31 was set to 200 mm (irradiation diameter: 40 mm) to irradiate 10 wells with laser light.

[0065] Next, as shown in Figure 13(e), a TMB solution (8a) (E102, manufactured by Bethyl Laboratories, Inc.), which reacts with the enzyme labeled on the detection antibody (7) to develop color, was added to each well after laser light irradiation, and the wells were incubated for 1 hour.

[0066] Next, as shown in Figure 13(f), the absorbance of the colored TMB solution (8b) was measured using a plate reader (Wallac 1420, manufactured by PerkinElmer Inc.) to determine the rate of BNP dissociation (measurement wavelength: 355 nm). The results are shown in Table 2 and Figure 15. Note that a higher absorbance of the TMB solution indicates a higher amount of BNP bound, while a lower absorbance indicates more BNP dissociation and a lower amount of BNP bound.

[0067]

[0068] Comparative Example 1 The rate of BNP dissociation was measured in the same manner as in Example 1, except that laser light irradiation was not performed after the addition of the HPTS aqueous solution. The results are shown in Table 2 and FIG. 15 .

[0069]

[0070] From the results of Table 2 and Figure 15, in Example 1, in which the HPTS aqueous solution was added and then the laser beam was irradiated, the absorbance decreased as the concentration of the HPTS aqueous solution increased. This is because hydrogen ions (H ＋ ) was released, the pH of the HPTS aqueous solution decreased, and as a result, BNP dissociation occurred. In contrast, in Comparative Example 1, in which laser light was not irradiated after the addition of the HPTS aqueous solution, no change in absorbance was observed with an increase in the concentration of the HPTS aqueous solution. This indicates that the HPTS aqueous solution alone does not affect the antigen-antibody reaction.

[0071] Example 2: To demonstrate a method for diagnosing cardiac disease, a BNP analytical sensor was constructed and the effect of changes in BNP concentration on luminescence intensity was evaluated. (1) Preparation of Au Mesh Substrate: A stainless steel mesh (SUS304, wire diameter 160 μm, mesh coarseness 30 mesh, E9107, manufactured by Kyūho Metal Works, Ltd.) was cut into a 6 mm diameter circle, and a 100 nm thick gold thin film was formed using a sputtering device (MSP-20UM, manufactured by Vacuum Device Co., Ltd.) to obtain an Au mesh substrate (see FIG. 16A). Using a substrate with such a mesh shape allows light from irradiation to reach not only the front side of the substrate but also the HPTS immobilized on the back side, thereby improving sensor performance. An optical signal detection unit consisting of a light-emitting element and a light-receiving element can also be installed on the back side. The method for forming the Au thin film is not limited to vapor deposition; plating or other methods may also be used.

[0072] (2) Immobilization of BNP Antibody The prepared Au mesh substrate was washed with piranha solution to remove proteins, and then immersed in 10 mmol / L 6-amino-1-hexaneethioiol (A425, Dojindo Laboratories, Inc.) dissolved in ethanol at 25°C for 16 hours to form a self-assembled monolayer (SAM). The SAM was then activated by immersion for 30 minutes in a 100 mmol / L solution of bis(sulfosuccinimidyl)suberate (BS3, product number B574, Dojindo Laboratories, Inc.) dissolved in a pH 7.8 buffer solution (phosphate buffered saline, PBS). The mesh was then immersed in a 10 μmol / L solution of BNP capture antibody (4BNP2 cc-50E1 cc, HyTest, Ltd.) dissolved in PBS (pH 7.8) for 30 minutes to immobilize the antibody on the mesh surface. For blocking, the mesh was immersed in a solution of ethanolamine (012-12455, Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in PBS (pH 7.8) for 60 minutes, and then in a blocking agent solution of skim milk (UKB80, KAC Corporation) for 120 minutes.

[0073] (3) BNP Concentration Measurement Step: The antibody-immobilized mesh was immersed in 0 pg / mL, 1 pg / mL, and 100 pg / mL BNP standard solutions (Nanopia (registered trademark) BNP Control for BNP, manufactured by Sekisui Medical Co., Ltd.) for 30 minutes, washed with a cleaning solution, and then immersed in an ALP-labeled BNP detection antibody (4BNP2cc-24C5cc, HyTest, Ltd.) for 30 minutes (see FIG. 16B). After washing, the mesh was transferred to a holder, and 50 μL of a luminescent substrate (Chemiluminescent AP Microwell, manufactured by SurModics) was added. The luminescence intensity was measured for 900 seconds using a detector consisting of a photomultiplier tube and a counting unit. The results are shown in FIG. 17.

[0074] The results of FIG. 17 show that with such a sensor configuration and process, the detection signal increases depending on the BMP concentration, and the sensor functions as a BNP sensor.

[0075] This international application claims priority based on Japanese Patent Application No. 2022-180727, filed on November 11, 2022, the entire contents of which are incorporated herein by reference.

[0076] REFERENCE SIGNS LIST 1 Substrate 2 Test sample 3 Photoacid 4 Antibody 5 Antigen 6 Antigen-antibody complex 7 Enzyme-labeled detection antibody 8a TMB 8b Colored TMB 10 Cardiac disease diagnostic device 11 Vibrator 12 Detection unit 13 Light irradiation unit 14 Anti-marker antibody 15 Marker 16 Marker complex 17 Power supply 20 Body-mounted cardiac disease diagnostic device 21 Smartphone 31 Microplate 32 Diffusion lens 33 Laser irradiation device 34 Variable output power supply

Claims

1. An anti-marker antibody that specifically binds to a marker and a photoacid are disposed on both surfaces of a mesh-shaped substrate, and by providing a test sample, a marker complex concentration measurement step of measuring the concentration of a marker complex in which the marker in the test sample and the anti-marker antibody are bound, A marker dissociation step of irradiating the photoacid with light to dissociate the marker from the marker complex; A diagnostic method characterized by including.

2. The diagnostic method according to claim 1, wherein the concentration of the marker complex is repeatedly measured at intervals.

3. The diagnostic method according to claim 1 or 2, wherein the substrate is a vibrating body.

4. The diagnostic method according to any one of claims 1 to 3, wherein the light is laser light.

5. The marker is a heart disease marker, The anti-marker antibody is an anti-heart disease marker antibody, The diagnostic method according to any one of claims 1 to 4, wherein the heart disease marker is at least one of brain natriuretic peptide (BNP) and N-terminal fragment of brain natriuretic peptide precursor (NT-proBNP).

6. The diagnostic method according to any one of claims 1 to 5, wherein the photoacid is 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS).

7. A mesh-shaped vibrating body having an anti-marker antibody that specifically binds to a marker and a photoacid disposed on both surfaces, A detection unit that detects the resonance frequency of the vibrating body, A light irradiation unit that irradiates the photoacid with light; A diagnostic apparatus characterized by having.

8. The diagnostic apparatus according to claim 7, wherein the vibrating body is placed on a vibrator, the vibrating body and the vibrator are not mechanically coupled, and the vibrating body is not mechanically coupled to any member.

9. An antibody and a photoacid that specifically bind to an antigen to be measured are disposed on both surfaces of a mesh-shaped substrate, and an antigen-antibody complex formation step of forming an antigen-antibody complex in which the antigen and the antibody are bound by providing a test sample, An antigen dissociation step of irradiating the photoacid with light to dissociate the antigen from the antigen-antibody complex; An antibody regeneration method characterized by including.