WO2024101447A1 - Diagnosis method, diagnosis device, and antibody regeneration method - Google Patents

Diagnosis method, diagnosis device, and antibody regeneration method Download PDF

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Publication number
WO2024101447A1
WO2024101447A1 PCT/JP2023/040540 JP2023040540W WO2024101447A1 WO 2024101447 A1 WO2024101447 A1 WO 2024101447A1 JP 2023040540 W JP2023040540 W JP 2023040540W WO 2024101447 A1 WO2024101447 A1 WO 2024101447A1
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Prior art keywords
marker
antibody
photoacid
antigen
complex
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PCT/JP2023/040540
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French (fr)
Japanese (ja)
Inventor
昌樹 山口
宏一郎 桑原
匡俊 南澤
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国立大学法人信州大学
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Publication of WO2024101447A1 publication Critical patent/WO2024101447A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a diagnostic method, a diagnostic device, and an antibody regeneration method.
  • Antigen-antibody reactions are an excellent molecular recognition technology that can be applied to most antigens, such as proteins, as long as the antibodies are available, and are the mainstream analytical method in clinical testing.
  • a dissociation liquid see, for example, Patent Document 1
  • Patent Document 1 which necessitates the need to replenish 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 chronological monitoring.
  • Non-Patent Document 1 reports the use of photoacid as a sensor for the regeneration of DNA hybridization.
  • Non-Patent Document 1 describes only the local control of the DNA hybridization/dehybridization process, and does not disclose the use of photoacid for antibody regeneration.
  • Non-Patent Document 2 reports the application of photoacid to a protein concentration sensor.
  • 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.
  • the present invention aims to solve the above-mentioned problems in the past and to achieve the following objectives. That is, the present invention aims to provide a diagnostic method and diagnostic device that can regenerate the anti-marker antibody in the detection unit that recognizes the marker, thereby enabling repeated measurement of the marker and realizing time-series monitoring of the marker, as well as an antibody regeneration method that can easily regenerate antibodies for repeated use.
  • the diagnostic method is characterized by comprising: ⁇ 2> This is a diagnostic method described in ⁇ 1> above, in which the concentration of the marker complex is repeatedly measured at intervals.
  • ⁇ 3> The diagnostic method according to ⁇ 1> or ⁇ 2>, wherein the substrate is a vibrating body.
  • ⁇ 4> The diagnostic method according to any one of ⁇ 1> to ⁇ 3>, wherein the light is laser light.
  • the marker is a cardiac disease marker
  • the anti-marker antibody is an anti-cardiac disease marker antibody
  • BNP brain natriuretic peptide
  • NT-proBNP N-terminal fragment of a precursor of brain natriuretic peptide
  • ⁇ 6> The diagnostic method according to any one of ⁇ 1> to ⁇ 5>, wherein the photoacid is 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS).
  • a vibrator having an anti-marker antibody and a photoacid on its surface that specifically bind to the marker;
  • a detection unit that detects a resonance frequency of the vibrator;
  • a light irradiation unit that irradiates the photoacid with light;
  • the diagnostic device is characterized by having: ⁇ 8> The diagnostic device described in ⁇ 7> above, wherein the vibrating body is placed on a transducer, the vibrating body and the transducer are not mechanically coupled, and the vibrating body is not mechanically coupled to any member.
  • the present invention relates to a method for regenerating an antibody, the method comprising the steps of:
  • the present invention can solve the above-mentioned problems in the prior art and achieve the above-mentioned objective, and can provide a diagnostic method and diagnostic device that can regenerate the anti-marker antibody in the detection section that recognizes the marker, thereby enabling repeated measurement of the marker and realizing time-series monitoring of the marker, as well as an antibody regeneration method that can easily regenerate antibodies for repeated use.
  • FIG. 1(a) to (c) are schematic diagrams illustrating the principle of the antibody regeneration method.
  • FIG. 2 is a diagram showing a chemical reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited.
  • FIG. 3 is a fluorescence spectrum of HPTS.
  • FIG. 4 is a graph showing the change in pH over time when a HPTS solution is irradiated with a laser beam having a wavelength of 405 nm.
  • FIG. 5 is a graph showing the relationship between the laser irradiation intensity and the rate of pH change when a HPTS solution is irradiated with a laser beam having a wavelength of 405 nm.
  • FIG. 1(a) to (c) are schematic diagrams illustrating the principle of the antibody regeneration method.
  • FIG. 2 is a diagram showing a chemical reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photo
  • FIG. 6 is a graph showing the reproducibility of the pH control ability of the HPTS solution by irradiating it with laser light.
  • FIG. 7 is a diagram showing an example of pre-processing of HPTS.
  • FIG. 8 is a schematic diagram showing an example of a method for immobilizing anti-BNP antibody and HPTS on the vibrator surface.
  • 9(a) to (d) are schematic diagrams showing the principle of the diagnostic method.
  • FIG. 10 is a schematic diagram showing an example of a diagnostic device.
  • FIG. 11 is a graph showing that unless BNP, a cardiac disease marker, is repeatedly measured over time, high and low BNP values may be overlooked, leading to a risk of being judged as normal and an erroneous diagnosis of cardiac disease.
  • FIG. 12A and 12B are schematic diagrams showing a state in which a body-worn cardiac diagnosis device is worn on the arm and cardiac disease markers are measured and displayed in chronological order.
  • FIG. 13(a) to (f) are schematic diagrams showing the method for measuring BNP in the examples.
  • FIG. 14 is a schematic diagram showing a method of irradiating a microplate with laser light.
  • FIG. 15 is a graph showing the relationship between the HPTS concentration and the absorbance of the TMB solution in Example 1 and Comparative Example 1.
  • FIG. 16A is an image showing the Au mesh substrate in Example 2.
  • FIG. 16B is a schematic diagram of immobilization of anti-BNP antibodies on an Au mesh substrate and a BNP detection reaction based on an antigen and antibody in Example 2.
  • FIG. 17 is a graph showing the relationship between BNP concentration and luminescence intensity in Example 2.
  • the antibody regeneration method of the present invention includes an antigen-antibody complex formation step and an antigen dissociation step, and may further include other steps as necessary.
  • a photoacid is placed near an antibody that specifically binds to the antigen to be measured, and the photoacid is irradiated with light, thereby dissociating the antigen from the antigen-antibody complex, making it possible to reuse the antibody with just a physical stimulus, and the antibody can be used repeatedly.
  • the antigen-antibody complex formation process is a process 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 antibody are bound to each other.
  • the substrate is not particularly limited in shape, size, structure, material, etc., and can be appropriately selected depending on the purpose.
  • the shape of the substrate include a beam, a rod, a plate, a film, a sheet, and a microplate.
  • the size of the substrate is not particularly limited and can be appropriately selected depending on the application.
  • the structure of the substrate is not particularly limited and may be appropriately selected depending on the purpose.
  • the substrate may have 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. Examples of the material include resins, ceramics, crystals, and metals.
  • the resin examples include polystyrene resin, acrylic resin, polycarbonate resin, polyvinylidene fluoride (PVDF), polylactic acid, polyethylene terephthalate resin, polyvinyl chloride resin, and polystyrene resin.
  • the ceramics include silicon, barium titanate, lead titanate, lead zirconate titanate (PZT), potassium niobate, lithium niobate, sodium tungstate, and lithium tantalate.
  • the crystals include, for example, quartz crystal.
  • the metal include iron, silicon steel, ferrite, cobalt, nickel, aluminum, and alnico.
  • An antigen refers to a component contained in a test sample that is to be measured through an antigen-antibody reaction, and examples of such components include amino acids, peptides, proteins, nucleic acids, lipids, carbohydrates, electrolytes, viruses, bacteria, pollen, or other low molecular weight metabolic products.
  • the antibody includes not only the immunoglobulin molecule itself that specifically binds to the antigen to be measured, but also decomposition products such as Fab and F(ab') 2 fragments generated by proteolytic enzymes such as papain and pepsin, or by chemical decomposition.
  • the antibody may be either a polyclonal antibody or a monoclonal antibody. There is no particular limitation on the method for obtaining the antibody, and any commonly used method may be used.
  • the photoacid is not particularly limited as long as it is a compound that generates an acid by irradiation with light, and can be appropriately selected according to 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 photoacid is an oxo salt, but the acid strength of only some of them can be increased by photoexcitation.
  • Examples of the photoacid include 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), phenol, 7-cyano-2-naphthol, tryptophan, etc. These may be used alone or in combination of two or more. Among these, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is preferred because it has a high acid strength and a fluorescent spectrum in the visible light region.
  • the method for disposing the photoacid and antibody on the substrate is not particularly limited and can be appropriately selected depending on the purpose.
  • 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 gap between the immobilized antibody and the antibody.
  • the method for disposing the photoacid and antibody on the substrate will be described in detail later.
  • test sample is not particularly limited and can be appropriately selected depending on the purpose, but a liquid test sample is preferred because the pH is lowered by hydrogen ions released from the photoacid.
  • 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, exudates, tissue fluids, secretions, breast milk, sweat, nasal mucus, serous fluid, transudate, cyst fluid, urine, cerebrospinal fluid, tears, gastric juice, bile, pancreatic juice, mucus, etc.
  • test sample When the test sample is liquid, it can be directly analyzed without pretreatment, which allows for simple and rapid analysis and prevents the test sample from being decomposed or oxidized, thereby enabling accurate and precise analysis of the components contained in the test sample.
  • Liquid test samples may be subjected to pretreatment such as dilution, deproteinization, lipid elution, and concentration, as necessary.
  • the antigen dissociation step is a step in which the photoacid is irradiated with light to dissociate the antigen from the antigen-antibody complex.
  • 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, and examples thereof include ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead method, and a method using a resonance type mass sensor that detects the resonance frequency associated with the mass change when an antigen adheres to a vibrating body vibrating at a constant frequency.
  • the resonance type mass sensor is preferable because it does not require refilling of reagents and dissociation liquid and can reduce the size of the device.
  • the light irradiated to the photoacid is preferably laser light.
  • the wavelength of the laser light is not particularly limited and can be appropriately selected depending on the type of photoacid, but is preferably from 400 nm to 1,000 nm, and more preferably from 400 nm to 800 nm.
  • the output of the laser light is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1 mW or more.
  • the other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, a cleaning step, a control step, etc. can be mentioned.
  • FIG. 1 are schematic diagrams showing the principle of the antibody regeneration method.
  • 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 provided.
  • antigen 5 in the test sample reacts with antibody 4 to form antigen-antibody complex 6.
  • photoacid 3 is irradiated with light of a specific wavelength for several seconds, causing hydrogen ions to be released from photoacid, changing the pH of test sample 2 and causing antigen 5 to dissociate from antigen-antibody complex 6 in an extremely short time. After that, when test sample 2 is discharged, antibody 4 can be regenerated and the antibody can be used repeatedly.
  • FIG 2 shows the reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited.
  • HPTS 8-hydroxypyrene-1,3,6-trisulfonic acid
  • FIG. 3 shows the reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited.
  • HPTS in the ground state is irradiated with laser light with a wavelength of 405 nm
  • HPTS becomes excited, releases hydrogen ions, and emits fluorescence with a wavelength of 512 nm.
  • 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, and it was found that the optimal wavelength for HPTS is 405 nm.
  • HPTS 30080, manufactured by Cayman Chemical Company
  • HPTS aqueous solution a pH meter (PAL-pH, manufactured by Atago Co., Ltd.) was fixed onto a plate shaker, and 1 mL of the above-prepared HPTS aqueous solution was dropped onto the electrode of the pH meter.
  • PAL-pH manufactured by Atago Co., Ltd.
  • 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 dropped HPTS aqueous solution was irradiated with a laser beam having a wavelength of 405 nm.
  • the distance between the diffusion lens and the pH meter was set to 100 mm.
  • the plate shaker was set to 300 rpm, and the pH was measured while stirring. The results are shown in Figures 4 and 5. From the results of the time-dependent change in pH of the HPTS aqueous solution shown in Fig.
  • 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.
  • 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 in the test sample is bound to the anti-marker antibody.
  • the marker is not particularly limited and can be appropriately selected depending on the purpose. Examples include heart disease markers, cytokines (IL-1 ⁇ , IL-6, IL-4, INF ⁇ , TNF, etc.), proteins (zonulin, LBP (lipopolysaccharide binding protein), etc.). Among these, heart disease markers are preferable.
  • Heart disease markers examples include biochemical cardiac markers such as brain natriuretic peptide (BNP), N-terminal fragment of brain natriuretic peptide precursor (NT-proBNP), troponin T (TnT), myoglobin, and CK-MB, heart-type fatty acid binding protein, etc. These may be used alone or in combination of two or more.
  • BNP and NT-proBNP are recognized as markers for heart failure
  • troponin is recognized as a marker for myocardial infarction. They are items tested for early diagnosis, have high specificity for heart disease, and can be the most important diagnostic information for heart disease.
  • the BNP and NT-proBNP are produced from the same BNP gene. After transcription and translation, the BNP precursor (proBNP [1-108]) is produced from the BNP gene. It 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 cardiac muscle. The gene expression of the BNP and NT-proBNP is enhanced mainly in response to wall stress (progressive stress) in the ventricle, and the BNP and NT-proBNP are rapidly produced and secreted. Therefore, in heart failure in which wall stress increases, the blood concentrations of the BNP and NT-proBNP increase depending on the severity of the condition.
  • the anti-marker antibody is not particularly limited and can be appropriately selected depending on the purpose. Examples include anti-heart disease marker antibodies, antibodies whose antigen is a cytokine (IL-1 ⁇ , IL-6, IL-4, INF ⁇ , TNF, etc.), and antibodies whose antigen is a protein (zonulin, LBP (lipopolysaccharide binding protein), etc.). Among these, anti-heart disease marker antibodies are preferable.
  • the anti-cardiac disease marker antibody includes not only the immunoglobulin molecule itself that specifically binds to the cardiac disease marker, but also decomposition products such as Fab and F(ab') 2 fragments generated by proteolytic enzymes such as papain and pepsin, or by chemical decomposition.
  • the anti-cardiac disease marker antibody may be either a polyclonal antibody or a monoclonal antibody.
  • the anti-cardiac disease marker antibody may be obtained by a commonly used method. For example, when the cardiac disease marker is BNP, an anti-BNP monoclonal antibody is used, and when the cardiac disease marker is NT-proBNP, an anti-NT-proBNP monoclonal antibody is used.
  • the substrate is not particularly limited and can be appropriately selected depending on the purpose, and the same substrate as that used in the antibody regeneration method can be used. Among these, it is preferable to use a vibrator as the substrate, because the concentration of the marker can be measured by measuring the resonance frequency of the vibrator.
  • the photoacid is not particularly limited and may be appropriately selected depending on the purpose, and the same photoacids as those used in the antibody regeneration method described above may 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 its fluorescent spectrum in the visible light region.
  • HPTS 8-hydroxypyrene-1,3,6-trisulfonic acid
  • a thin metal film is formed on the surface of the vibrating body, a molecular film of molecular layer order is formed on this thin metal film by sputtering or the like, and the molecular film is modified with an antibody, thereby fixing the antibody to the vibrating body.
  • the thin metal film can be an adhesive layer and a platinum (Pt) film formed on the adhesive layer. Examples of the adhesive layer include Ti and Cr.
  • the molecular film of molecular layer order can be a self-assembled monolayer (hereinafter sometimes referred to as a "SAM film").
  • a titanium (Ti) and platinum (Pt) film is formed to a thickness of 500 ⁇ on one side of the vibrating body (quartz plate) by sputtering.
  • a SAM film is formed and the SAM film is modified with anti-BNP antibody.
  • the amine coupling method can be used to bond the SAM film and the anti-BNP antibody. In this manner, the anti-BNP antibody is immobilized on the surface of the vibrating body 11 as shown in FIG. 8(a).
  • a pretreatment is carried out to convert trisodium 8-hydroxypyrene-1,3,6-trisulfonate into 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride by substituting the functional group.
  • trisodium 8-hydroxypyrene-1,3,6-trisulfonate (20 g, 0.038 mol) was dissolved in NaOH (2.4 g, 0.06 mol) and water (30 mL), and the solution was cooled to about 0°C.
  • Acetic anhydride (5 g, 4.8 mL, 0.48 mol) is then added dropwise to the solution and the reaction mixture is stirred for 2 hours.
  • 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 heated.
  • the heated mixture is refluxed for 8 hours to distill off the mixture of toluene and excess oxalyl chloride (30 mL).
  • the sodium chloride precipitate is then filtered off and the filtrate is freed of solvent under reduced pressure.
  • the solid residue is then dried in vacuum for 24 hours to give 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride (4 g) (yield 81.5%).
  • the vibrating body 11 on which the anti-BNP antibody is immobilized is incubated for 24 hours in a mixture of 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride and pyridine obtained in the above pretreatment, and then exposed to a saturated sodium bicarbonate solution of the phenol functional group.
  • the anti-BNP antibody and trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate can be immobilized on the surface of the vibrating body 11.
  • the method for measuring the concentration of a marker complex in a test sample in which a marker and an anti-marker antibody are bound is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead method, and a method using a resonance type mass sensor that detects the resonance frequency associated with the mass change when a cardiac disease marker adheres to an oscillator vibrating at a constant frequency.
  • the method using a resonance type mass sensor is preferred because it does not require replenishment of reagents and dissociation solution, the device can be made smaller, the anti-marker antibody can be regenerated simply by irradiating the photoacid with light, the anti-marker antibody can be used repeatedly, and time-series monitoring can be realized.
  • a method using a resonant mass sensor for example, when an oscillator having an anti-marker antibody that specifically binds to a marker and a photoacid immobilized thereon 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.
  • the marker dissociation step is a step in which the photoacid is irradiated with light to dissociate the marker from the marker complex.
  • the anti-marker antibody can be regenerated by irradiating the photoacid with light to dissociate the marker from the marker complex formed by the marker and the anti-marker antibody binding thereto.
  • the light is preferably a laser light.
  • 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.
  • the other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, a cleaning step, a control step, etc. can be mentioned.
  • FIG. 9 are schematic diagrams showing the principle of the diagnostic method.
  • photoacid 3 is immobilized near anti-marker antibody 14 that specifically binds to the marker to be measured on the vibrating body 11, and a test sample 2 is provided.
  • marker 15 in the test sample reacts with anti-marker antibody 14 to form a marker complex 16.
  • marker 15 in the test sample is captured by anti-marker antibody 14, and the mass of vibrating body 11 increases.
  • the resonant frequency of vibrating body 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 vibrating body 11 can be measured using a laser displacement meter.
  • the pH of the test sample 2 is changed by irradiating the photoacid 3 with light of a specific wavelength, and the marker 15 is dissociated from the marker complex 16.
  • the anti-marker antibody 14 can be regenerated as shown in Fig. 9(a) and can be used repeatedly.
  • the diagnostic method of the present invention includes a method for quantifying markers contained in a test sample, and a method for evaluating markers contained in a test sample.
  • 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.
  • the surface is provided with an anti-marker antibody and photoacid that specifically binds to the marker, and a piezoelectric crystal, etc., can be used.
  • a piezoelectric crystal is a material that exhibits an electrical response when strained, and conversely, has the property of generating strain when a voltage is applied, and examples of such a material 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 vibrating body surface is not particularly limited and can be appropriately selected depending on the purpose, and can be carried out in the same manner as the above-mentioned diagnostic method.
  • the vibrating body is placed on a vibrator, and it is preferable that the vibrating body and the vibrator are not mechanically coupled, and that the vibrating body is not mechanically coupled to any member. This is preferable because it does not inhibit the resonance phenomenon due to power supply or fixation, it eliminates design constraints on the shape and dimensions of the vibrating body, and it allows mass changes to be measured with high sensitivity.
  • the detector is a means for detecting the resonant frequency of the vibrating body, and may be an optical detector consisting of a light-emitting element and a light-receiving element.
  • the optical detector may measure any one of the frequency, displacement, velocity, and acceleration of the vibrating body.
  • the detection unit may be a laser displacement meter, etc.
  • the laser displacement meter can measure the change in the resonance frequency of the vibrating body in a non-contact manner.
  • the light irradiating unit is a means for irradiating the photoacid with light, and is preferably a laser light irradiating unit.
  • the laser light irradiating unit has, for example, a laser light source, a diffusion lens, and a power source.
  • Examples of the other means include an input means, a recording means, a display means, a communication means, and a maintenance means.
  • FIG. 10 is a schematic diagram showing an example of a diagnostic device.
  • the diagnostic device 10 in FIG. 10 has a vibrator 11 having an anti-marker antibody 14 that specifically binds to the marker and photoacid 3 on its surface, a detection unit 12 that detects the resonance frequency of the vibrator 11, a light irradiation unit 13 that irradiates the photoacid 3 with light, and a power source 17 that drives the vibrator 11.
  • the vibrating body 11 there are no particular limitations on the vibrating body 11, and it can be used alone, but it is preferable to place the vibrating body 11 on a vibrator.
  • the vibrating body 11 and the vibrator are not mechanically connected, and the vibrating body 11 is not mechanically connected to any member; in other words, the vibrating body 11 has a structure that is not fixed anywhere, which is preferable in that there is no inhibition of the resonance phenomenon due to power supply or fixing, there are no design constraints on the shape and dimensions of the vibrating body 11, and mass changes can be measured with high sensitivity.
  • the vibrating body 11 is machined 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 any frequency, the vibrating body 11 can vibrate at its own resonant frequency.
  • a vibrator such as a piezoelectric element
  • the vibrating body 11 resonates at its own natural resonant frequency. In this way, there are no restrictions on the material or shape of the vibrating body, and design freedom can be dramatically improved.
  • the vibration mode of the vibrating body 11 is set so as not to be a vibration mode such as a uniaxial vibration that moves in a certain direction like a standing wave type ultrasonic motor.
  • 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 top of the vibrator.
  • the resonant mass sensor having the vibrating body and the vibrator reference can be made to the description in Japanese Patent No. 6,086,347, for example.
  • the vibrating body 11 on which the anti-marker antibody 14 and photoacid 3 that specifically bind to the marker are immobilized is provided to a test sample
  • the marker 15 in the test sample is captured by the anti-marker antibody 14, and the mass of the vibrating body 11 increases.
  • the resonant frequency of the vibrating body 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 vibrating body 11 is detected by the detecting unit 12.
  • the detecting unit 12 uses a laser displacement meter as a means for measuring the resonant frequency of the vibrating body 11.
  • Fig. 12 (a) is a schematic diagram showing an example of a bio-attachable cardiac disease diagnostic device that can measure cardiac disease markers quickly, on the spot, and repeatedly.
  • This bio-attachable cardiac disease diagnostic device 22 can be attached to the patient's wrist to repeatedly measure the BNP concentration at intervals. By transmitting the BNP concentration measured by the bio-attachable cardiac disease diagnostic device 22 to the smartphone 21 at any time, the BNP concentration can be displayed in chronological order as shown in Fig. 12 (b), making it possible to accurately and quickly diagnose cardiac disease.
  • the diagnostic method and diagnostic device of the present invention dissociate the marker from the marker complex by placing a photoacid near an anti-marker antibody that specifically binds to the marker and irradiating the photoacid with light. This makes it possible to reuse the anti-marker antibody with only physical stimulation, and since the anti-marker antibody can be used repeatedly, it is possible to realize chronological monitoring of the marker.
  • Example 1 Dissociation of a cardiac marker (BNP) from a cardiac marker complex by irradiation with photoacid (HPTS) was experimentally demonstrated, and the effect of changes in HPTS concentration on the degree of dissociation of BNP was evaluated.
  • BNP cardiac marker
  • HPTS photoacid
  • 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.) as a substrate (1) by a method using a self-assembled monolayer, and 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 incubated for 1 hour to form a cardiac disease marker complex (16).
  • BNP Nemopia (registered trademark) BNP control for BNP, manufactured by Sekisui Medical Co., Ltd.
  • 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)).
  • HPTS (30080, manufactured by Cayman Chemical Company) was dissolved in distilled water to prepare 1.0 ⁇ 10 ⁇ 3 mol/L to 7.0 ⁇ 10 ⁇ 3 mol/L aqueous HPTS solutions (see Table 1). Next, after washing the microplate, an aqueous HPTS solution prepared to the above-mentioned predetermined concentration was added, and the pH of the aqueous HPTS solution was reduced by irradiating the plate with laser light for 30 minutes, as shown in FIG. 13(d), whereby BNP (15) and the enzyme-labeled detection antibody (7) were dissociated from the anti-BNP antibody (14).
  • FIG. 14 shows a method of irradiating a microplate with laser light.
  • 34 is a variable output power source.
  • a TMB solution (8a) (E102, manufactured by Bethyl Laboratories, Inc.) that reacts with the enzyme labeled to the detection antibody (7) to produce color was added to each well after irradiation with the laser light, and the wells were incubated for 1 hour.
  • the absorbance of the colored TMB solution (8b) was measured using a plate reader (Wallac 1420, manufactured by PerkinElmer Inc.) to measure the percentage of BNP dissociation (measurement wavelength: 355 nm).
  • the results are shown in Table 2 and FIG. 15. Note that the higher the absorbance of the TMB solution, the greater the amount of BNP bound, and the lower the absorbance, the more BNP dissociated and the less BNP bound.
  • Example 15 in Example 1, in which the HPTS aqueous solution was added and then irradiated with laser light, the absorbance decreased with an increase in the concentration of the HPTS aqueous solution. This indicates that when the HPTS aqueous solution was irradiated with laser light, hydrogen ions (H + ) were released, lowering the pH of the HPTS aqueous solution, and as a result, dissociation of BNP occurred. In contrast, in Comparative Example 1, in which laser light was not irradiated after addition of the HPTS aqueous solution, no change in absorbance was observed with increasing concentration of the HPTS aqueous solution, indicating that the HPTS aqueous solution alone does not affect the antigen-antibody reaction.
  • Example 2 In order to verify the method for diagnosing heart disease, a sensor for analyzing BNP was constructed and the effect of changes in BNP concentration on the luminescence intensity was evaluated.
  • a stainless steel mesh (SUS304, wire diameter 160 ⁇ m, mesh coarseness 30 mesh, E9107, manufactured by Ky ⁇ ho Metal Works, Ltd.) was cut into a circle with a diameter of 6 mm, and a gold thin film with a thickness of 100 nm was formed using a sputtering device (MSP-20UM, manufactured by Vacuum Device Co., Ltd.) to obtain an Au mesh substrate (see FIG. 16A).
  • the light emitted by the light reaches not only the front side of the substrate but also the HPTS immobilized on the back side, improving the sensor performance.
  • an optical signal detection unit consisting of a light emitting element and a light receiving element can be installed on the back side.
  • the method for forming the Au thin film is not limited to vapor deposition, and plating or other methods may be used.
  • the mesh was immersed in a 10 ⁇ mol/L solution of BNP capture antibody (4BNP2cc-50E1cc, HyTest, Ltd.) dissolved in PBS at pH 7.8 for 30 minutes to immobilize the antibody on the mesh surface.
  • BNP capture antibody 4BNP2cc-50E1cc, HyTest, Ltd.
  • PBS pH 7.8 for 30 minutes
  • ethanolamine 012-12455, Fujifilm Wako Pure Chemical Industries, Ltd.
  • a blocking agent skim milk solution UKB80, KAC Co., Ltd.
  • a luminescent substrate (Chemiluminescent AP Microwell, manufactured by SurModics) was added, and 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.

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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

診断方法、診断装置、及び抗体再生方法Diagnostic method, diagnostic device, and antibody regeneration method
　本発明は、診断方法、診断装置、及び抗体再生方法に関する。 The present invention relates to a diagnostic method, a diagnostic device, and an antibody regeneration method.
　抗原抗体反応は、抗体さえ入手できればほとんどのタンパク質等の抗原に適用可能な優れた分子認識技術であり、臨床検査における主流な分析方法となっている。しかし、従来技術では、抗体を再生して繰り返し使用するためには、解離液により抗原抗体複合物から抗原を解離する必要があり（例えば、特許文献１参照）、試薬の補充が発生し、装置が大型化するため生体に装着して時系列的なモニタリングを実現するデバイス化が困難であるという課題がある。 Antigen-antibody reactions are an excellent molecular recognition technology that can be applied to most antigens, such as proteins, as long as the antibodies are available, and are the mainstream analytical method in clinical testing. However, with conventional technology, in order to regenerate the antibodies for repeated use, it is necessary to dissociate the antigen from the antigen-antibody complex using a dissociation liquid (see, for example, Patent Document 1), which necessitates the need to replenish 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 chronological monitoring.
　光酸を用いた抗体再生に関連する先行技術としては、例えば、非特許文献１には、光酸をセンサに用いてＤＮＡのハイブリダイゼーションの再生に応用することが報告されている。しかし、非特許文献１に記載されているのは、ＤＮＡのハイブリダイゼーション／デハイブリダイゼーションプロセスの局所制御であり、光酸を抗体再生に用いることについて開示していない。 As an example of prior art related to antibody regeneration using photoacid, Non-Patent Document 1 reports the use of photoacid as a sensor for the regeneration of DNA hybridization. However, Non-Patent Document 1 describes only the local control of the DNA hybridization/dehybridization process, and does not disclose the use of photoacid for antibody regeneration.
　また、非特許文献２には、光酸をタンパク質の濃度センサに応用したことが報告されている。しかし、非特許文献２は、光酸を標的タンパク質の選択的分離（収集）に利用するものであり、光酸を抗体再生に用いた濃度センサを意図したものではない。 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.
特開平３－２１４０５１号公報Japanese Patent Application Laid-Open No. 3-214051
　本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、マーカーを認識する検出部の抗マーカー抗体を再生でき、その結果としてマーカーを繰り返して測定することができ、マーカーの時系列的なモニタリングを実現できる診断方法、診断装置、及び簡易に抗体を再生して繰り返し使用することができる抗体再生方法を提供することを目的とする。 The present invention aims to solve the above-mentioned problems in the past and to achieve the following objectives. That is, the present invention aims to provide a diagnostic method and diagnostic device that can regenerate the anti-marker antibody in the detection unit that recognizes the marker, thereby enabling repeated measurement of the marker and realizing time-series monitoring of the marker, as well as an antibody regeneration method that can easily regenerate antibodies for repeated use.
　前記課題を解決するための手段としては、以下の通りである。即ち、
　＜１＞
　基材上に、マーカーと特異的に結合する抗マーカー抗体及び光酸を配し、検査試料を供することにより前記検査試料中のマーカーと前記抗マーカー抗体とが結合したマーカー複合物の濃度を測定するマーカー複合物濃度測定工程と、
　前記光酸に光を照射して前記マーカー複合物から前記マーカーを解離させるマーカー解離工程と、
を含むことを特徴とする診断方法である。
　＜２＞
　間隔をあけて前記マーカー複合物の濃度を繰り返し測定する、前記＜１＞に記載の診断方法である。
　＜３＞
　前記基材が、振動体である、前記＜１＞又は＜２＞に記載の診断方法である。
　＜４＞
　前記光が、レーザー光である、前記＜１＞から＜３＞のいずれかに記載の診断方法である。
　＜５＞
　前記マーカーが、心疾患マーカーであり、
　前記抗マーカー抗体が、抗心疾患マーカー抗体であり、
　前記心疾患マーカーが、脳性ナトリウム利尿ペプチド（ＢＮＰ）及び脳性ナトリウム利尿ペプチド前駆体Ｎ端フラグメント（ＮＴ－ｐｒｏＢＮＰ）の少なくともいずれかである、前記＜１＞から前記＜４＞のいずれかに記載の診断方法である。
　＜６＞
　前記光酸が、８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）である、前記＜１＞から＜５＞のいずれかに記載の診断方法である。
　＜７＞
　マーカーと特異的に結合する抗マーカー抗体及び光酸を表面に配した振動体と、
　前記振動体の共振周波数を検出する検出部と、
　前記光酸に光を照射する光照射部と、
を有することを特徴とする診断装置である。
　＜８＞
　前記振動体が振動子上に載置され、前記振動体と前記振動子が機械的に結合されておらず、かつ前記振動体がいずれの部材にも機械的に結合されていない、前記＜７＞に記載の診断装置である。
　＜９＞
　基材上に、測定対象である抗原と特異的に結合する抗体及び光酸を配し、検査試料を供することにより前記抗原と前記抗体とが結合した抗原抗体複合物を形成する抗原抗体複合物形成工程と、
　前記光酸に光を照射して前記抗原抗体複合物から前記抗原を解離させる抗原解離工程と、
を含むことを特徴とする抗体再生方法である。 The means for solving the above problems are as follows.
<1>
a marker complex concentration measuring step of disposing 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 formed by binding a marker to the anti-marker antibody;
a marker dissociation step of dissociating the marker from the marker complex by irradiating the photoacid with light;
The diagnostic method is characterized by comprising:
＜2＞
This is a diagnostic method described in <1> above, in which the concentration of the marker complex is repeatedly measured at intervals.
＜3＞
The diagnostic method according to <1> or <2>, wherein the substrate is a vibrating body.
＜4＞
The diagnostic method according to any one of <1> to <3>, wherein the light is laser light.
＜5＞
the marker is a cardiac disease marker,
the anti-marker antibody is an anti-cardiac disease marker antibody,
The method for diagnosis according to any one of <1> to <4>, wherein the cardiac disease marker is at least one of brain natriuretic peptide (BNP) and N-terminal fragment of a precursor of brain natriuretic peptide (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 vibrator having an anti-marker antibody and a photoacid on its surface that specifically bind to the marker;
A detection unit that detects a resonance frequency of the vibrator;
A light irradiation unit that irradiates the photoacid with light;
The diagnostic device is characterized by having:
＜8＞
The diagnostic device described in <7> above, wherein the vibrating body is placed on a transducer, the vibrating body and the transducer are not mechanically coupled, and the vibrating body is not mechanically coupled to any member.
＜9＞
an antigen-antibody complex formation step of forming an antigen-antibody complex by providing an antibody that specifically binds to an antigen to be measured and a photoacid on a substrate and then providing a test sample to form an antigen-antibody complex in which the antigen and the antibody are bound to each other;
an antigen dissociation step of irradiating the photoacid with light to dissociate the antigen from the antigen-antibody complex;
The present invention relates to a method for regenerating an antibody, the method comprising the steps of:
　本発明によると、従来における前記諸問題を解決し、前記目的を達成することができ、マーカーを認識する検出部の抗マーカー抗体を再生でき、その結果としてマーカーを繰り返して測定することができ、マーカーの時系列的なモニタリングを実現できる診断方法、診断装置、及び簡易に抗体を再生して繰り返し使用することができる抗体再生方法を提供することができる。 The present invention can solve the above-mentioned problems in the prior art and achieve the above-mentioned objective, and can provide a diagnostic method and diagnostic device that can regenerate the anti-marker antibody in the detection section that recognizes the marker, thereby enabling repeated measurement of the marker and realizing time-series monitoring of the marker, as well as an antibody regeneration method that can easily regenerate antibodies for repeated use.
図１の（ａ）～（ｃ）は、抗体再生方法の原理を示す模式図である。1(a) to (c) are schematic diagrams illustrating the principle of the antibody regeneration method. 図２は、光酸としての８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）を光励起した際の化学反応式を示す図である。FIG. 2 is a diagram showing a chemical reaction formula when 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) as a photoacid is photoexcited. 図３は、ＨＰＴＳの蛍光スペクトル図である。FIG. 3 is a fluorescence spectrum of HPTS. 図４は、ＨＰＴＳ溶液に波長４０５ｎｍのレーザー光を照射した際のｐＨの経時変化を示す図である。FIG. 4 is a graph showing the change in pH over time when a HPTS solution is irradiated with a laser beam having a wavelength of 405 nm. 図５は、ＨＰＴＳ溶液に波長４０５ｎｍのレーザー光を照射した際のレーザー照射強度とｐＨ変化率との関係を示す図である。FIG. 5 is a graph showing the relationship between the laser irradiation intensity and the rate of pH change when a HPTS solution is irradiated with a laser beam having a wavelength of 405 nm. 図６は、ＨＰＴＳ溶液のレーザー光照射によるｐＨ制御能の再現性を示す図である。FIG. 6 is a graph showing the reproducibility of the pH control ability of the HPTS solution by irradiating it with laser light. 図７は、ＨＰＴＳの前処理の一例を示す図である。FIG. 7 is a diagram showing an example of pre-processing of HPTS. 図８は、抗ＢＮＰ抗体及びＨＰＴＳの振動体表面への固相化方法の一例を示す模式図である。FIG. 8 is a schematic diagram showing an example of a method for immobilizing anti-BNP antibody and HPTS on the vibrator surface. 図９の（ａ）～（ｄ）は、診断方法の原理を示す模式図である。9(a) to (d) are schematic diagrams showing the principle of the diagnostic method. 図１０は、診断装置の一例を示す概略図である。FIG. 10 is a schematic diagram showing an example of a diagnostic device. 図１１は、心疾患マーカーであるＢＮＰを繰り返し時系列的に測定しないと、ＢＮＰの高値及び低値が見過ごされてしまい、正常と判断され心疾患の診断を誤ってしまうリスクがあることを示すグラフである。FIG. 11 is a graph showing that unless BNP, a cardiac disease marker, is repeatedly measured over time, high and low BNP values may be overlooked, leading to a risk of being judged as normal and an erroneous diagnosis of cardiac disease. 図１２の（ａ）及び（ｂ）は、生体装着型の心疾患診断装置を腕に装着し、心疾患マーカーを時系列的に計測し、表示した状態を示す概略図である。12A and 12B are schematic diagrams showing a state in which a body-worn cardiac diagnosis device is worn on the arm and cardiac disease markers are measured and displayed in chronological order. 図１３の（ａ）～（ｆ）は、実施例におけるＢＮＰの測定方法を示す模式図である。FIG. 13(a) to (f) are schematic diagrams showing the method for measuring BNP in the examples. 図１４は、マイクロプレートへのレーザー光の照射方法を示す概略図である。FIG. 14 is a schematic diagram showing a method of irradiating a microplate with laser light. 図１５は、実施例１及び比較例１におけるＨＰＴＳ濃度とＴＭＢ溶液の吸光度との関係を示すグラフである。FIG. 15 is a graph showing the relationship between the HPTS concentration and the absorbance of the TMB solution in Example 1 and Comparative Example 1. 図１６Ａは、実施例２におけるＡｕメッシュ基材を示す画像である。FIG. 16A is an image showing the Au mesh substrate in Example 2. 図１６Ｂは、実施例２におけるＡｕメッシュ基材への抗ＢＮＰ抗体固相化と、抗原抗体に基づくＢＮＰ検出反応の模式図である。FIG. 16B is a schematic diagram of immobilization of anti-BNP antibodies on an Au mesh substrate and a BNP detection reaction based on an antigen and antibody in Example 2. 図１７は、実施例２におけるＢＮＰ濃度と発光強度との関係を示すグラフである。FIG. 17 is a graph showing the relationship between BNP concentration and luminescence intensity in Example 2.
（抗体再生方法）
　本発明の抗体再生方法は、抗原抗体複合物形成工程と、抗原解離工程とを含み、更に必要に応じてその他の工程を含む。 (Method of regenerating antibodies)
The antibody regeneration method of the present invention includes an antigen-antibody complex formation step and an antigen dissociation step, and may further include other steps as necessary.
　本発明においては、測定対象である抗原と特異的に結合する抗体の近傍に光酸を配し、前記光酸に光を照射することによって、抗原抗体複合物から抗原を解離させることができ、物理刺激だけで抗体の再利用が可能となり、抗体を繰り返し使用することができる。この抗体再生方法の原理を利用することによって、後述するように、マーカーを繰り返して測定することができ、マーカーの時系列的なモニタリングを実現できる。 In the present invention, a photoacid is placed near an antibody that specifically binds to the antigen to be measured, and the photoacid is irradiated with light, thereby dissociating the antigen from the antigen-antibody complex, making it possible to reuse the antibody with just a physical stimulus, 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 to realize chronological monitoring of the marker.
＜抗原抗体複合物形成工程＞
　抗原抗体複合物形成工程は、基材上に、測定対象である抗原と特異的に結合する抗体及び光酸を配し、検査試料を供することにより前記抗原と前記抗体とが結合した抗原抗体複合物を形成する工程である。 <Antigen-antibody complex formation step>
The antigen-antibody complex formation process is a process 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 antibody are bound to each other.
－基材－
　基材としては、形状、大きさ、構造、材質などについて特に制限はなく、目的に応じて適宜選択することができる。
　前記基材の形状としては、例えば、梁状、棒状、板状、膜状、シート状、マイクロプレート状などが挙げられる。
　前記基材の大きさとしては、特に制限はなく、用途等に応じて適宜選択することができる。
　前記基材の構造としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、単層構造であっても複数層構造であっても構わない。
　前記基材の材質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、樹脂、セラミックス、結晶、金属などが挙げられる。 -Base material-
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 a beam, a rod, a plate, a film, a sheet, and a microplate.
The size of the substrate is not particularly limited and can be appropriately selected depending on the application.
The structure of the substrate is not particularly limited and may be appropriately selected depending on the purpose. For example, the substrate may have 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. Examples of the material include resins, ceramics, crystals, and metals.
　前記樹脂としては、例えば、ポリスチレン樹脂、アクリル樹脂、ポリカーボネート樹脂、ポリフッ化ビニリデン（ＰＶＤＦ）、ポリ乳酸、ポリエチレンテレフタレート樹脂、ポリ塩化ビニル樹脂、ポリスチレン樹脂などが挙げられる。
　前記セラミックスとしては、例えば、シリコン、チタン酸バリウム、チタン酸鉛、チタン酸ジルコン酸鉛（ＰＺＴ）、ニオブ酸カリウム、ニオブ酸リチウム、タングステン酸ナトリウム、タンタル酸リチウムなどが挙げられる。
　前記結晶としては、例えば、水晶などが挙げられる。
　前記金属としては、例えば、鉄、ケイ素鋼、フェライト、コバルト、ニッケル、アルミニウム、アルニコなどが挙げられる。 Examples of the resin 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.
The crystals include, for example, quartz crystal.
Examples of the metal include iron, silicon steel, ferrite, cobalt, nickel, aluminum, and alnico.
－抗原－
　抗原としては、検査試料中に含まれ、抗原抗体反応によって測定対象となる成分を意味し、例えば、アミノ酸、ペプチド、タンパク質、核酸、脂質、糖質、電解質、ウイルス、細菌、花粉、又はその他の低分子代謝産物などが挙げられる。 -antigen-
An antigen refers to a component contained in a test sample that is to be measured through an antigen-antibody reaction, and examples of such components include amino acids, peptides, proteins, nucleic acids, lipids, carbohydrates, electrolytes, viruses, bacteria, pollen, or other low molecular weight metabolic products.
－抗体－
　抗体としては、測定対象である抗原と特異的に結合する、免疫グロブリン分子自体の他、パパインやペプシン等の蛋白質分解酵素、或いは化学的分解により生じるＦａｂ、Ｆ（ａｂ’）２フラグメント等の分解産物も包含される。また、前記抗体は、ポリクローナル抗体及びモノクローナル抗体のどちらを用いても構わない。前記抗体の取得方法については、特に制限はなく、通常使用される方法を用いることができる。 -antibody-
The antibody includes not only the immunoglobulin molecule itself that specifically binds to the antigen to be measured, but also decomposition products such as Fab and F(ab') 2 fragments generated by proteolytic enzymes such as papain and pepsin, or by chemical decomposition. The antibody may be either a polyclonal antibody or a monoclonal antibody. There is no particular limitation on the method for obtaining the antibody, and any commonly used method may be used.
－光酸－
　光酸としては、光の照射によって酸を発生する化合物であれば特に制限はなく、目的に応じて適宜選択することができる。前記光酸としては、酸強度（ｐＫａ）値が５～１０の範囲であるものが好ましい。一重項電子状態では、より強い酸になる。最も一般的な光酸はオキソ塩であるが、酸強度が光励起によって増強できるのは、その一部である。
　前記光酸としては、例えば、８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）、フェノール、７－シアノ－２－ナフトール、トリプトファンなどが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。これらの中でも、酸強度が大きい点及び可視光領域の蛍光スペクトルを有する点から、８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）が好ましい。 - Photoacid -
The photoacid is not particularly limited as long as it is a compound that generates an acid by irradiation with light, and can be appropriately selected according to 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 photoacid is an oxo salt, but the acid strength of only some of them can be increased by photoexcitation.
Examples of the photoacid include 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), phenol, 7-cyano-2-naphthol, tryptophan, etc. These may be used alone or in combination of two or more. Among these, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is preferred because it has a high acid strength and a fluorescent spectrum in the visible light region.
　前記基材上に光酸及び抗体を配する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、抗体を基材上に自己組織化単分子膜を用いる方法により固相化し、固相化された抗体と抗体の隙間を埋めるブロッキング処理の代わりに光酸を固相化する方法などが挙げられる。なお、前記基材上に光酸及び抗体を配する方法の詳細については後述する。 The method for disposing the photoacid and antibody on the substrate is not particularly limited and can be appropriately selected depending on the purpose. For example, 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 gap between the immobilized antibody and the antibody. The method for disposing the photoacid and antibody on the substrate will be described in detail later.
－検査試料－
　検査試料としては、特に制限はなく、目的に応じて適宜選択することができるが、光酸から放出される水素イオンによってｐＨを下げる点から液体の検査試料が好ましい。
　前記液体の検査試料としては、例えば、全血（凝固前）、血漿、血清、羊水、上下気道の粘膜上皮からの分泌物、唾液、滲出液、組織液、分泌液、母乳、汗、鼻汁、漿液、漏出液、嚢胞液、尿、脳脊髄液、涙、胃液、胆汁、膵液、粘液などが挙げられる。
　前記検査試料が液体である場合には、前処理を行わないで直接そのまま分析することができる。これにより、簡便かつ迅速に分析でき、検査試料の分解や酸化などの変性を避けることができるので、検査試料に含まれる成分を正確かつ精度良く分析することができる。
　なお、液体の検査試料は、必要に応じて、希釈、除蛋白、脂質溶出、濃縮等の前処理を行ってもよい。 - Test sample -
The test sample is not particularly limited and can be appropriately selected depending on the purpose, but a liquid test sample is preferred because the pH is lowered by hydrogen ions released from the photoacid.
Examples of the 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, exudates, tissue fluids, secretions, breast milk, sweat, nasal mucus, serous fluid, transudate, cyst fluid, urine, cerebrospinal fluid, tears, gastric juice, bile, pancreatic juice, mucus, etc.
When the test sample is liquid, it can be directly analyzed without pretreatment, which allows for simple and rapid analysis and prevents the test sample from being decomposed or oxidized, thereby enabling accurate and precise analysis of the components contained in the test sample.
Liquid test samples may be subjected to pretreatment such as dilution, deproteinization, lipid elution, and concentration, as necessary.
＜抗原解離工程＞
　抗原解離工程は、光酸に光を照射して抗原抗体複合物から抗原を解離させる工程である。
　本発明の抗体再生方法では、検査試料に含まれる抗原抗体複合物の濃度の測定が終了した後、光酸に光を照射して抗原抗体複合物から抗原を解離させることにより、抗体を再生することができる。
　前記検査試料に含まれる抗原の濃度の測定方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　Ｉｍｍｕｎｏ　Ｓｏｒｂｅｎｔ　Ａｓｓａｙ）法、イムノクロマト法、表面プラズモン共鳴法、磁気ビーズ法、一定周波数で振動している振動体に抗原が付着した際の質量変化に伴う共振周波数を検出する共振型質量センサを用いる方法などが挙げられる。これらの中でも、試薬及び解離液の補充が不要であり、デバイスの小型化が図れる点から共振型質量センサが好ましい。 <Antigen dissociation step>
The antigen dissociation step is a step in which the 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, and examples thereof include ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead method, and a method using a resonance type mass sensor that detects the resonance frequency associated with the mass change when an antigen adheres to a vibrating body vibrating at a constant frequency. Among these, the resonance type mass sensor is preferable because it does not require refilling of reagents and dissociation liquid and can reduce the size of the device.
　前記光酸に照射する光はレーザー光であることが好ましい。
　前記レーザー光の波長としては、特に制限はなく、光酸の種類に応じて適宜選択することができるが、４００ｎｍ以上１,０００ｎｍ以下が好ましく、４００ｎｍ以上８００ｎｍ以下がより好ましい。
　前記レーザー光の出力としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍＷ以上が好ましい。
　光酸にレーザー光を照射することにより、光酸が励起状態となり水素イオンが放出され、検査試料のｐＨが下がることによって抗原抗体複合物から抗原を解離させ、抗体を再生することができる。 The light irradiated to the photoacid is preferably laser light.
The wavelength of the laser light is not particularly limited and can be appropriately selected depending on the type of photoacid, but is preferably from 400 nm to 1,000 nm, and more preferably from 400 nm to 800 nm.
The output of the laser light is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1 mW or more.
By irradiating the photoacid with laser light, the photoacid becomes excited and releases hydrogen ions, lowering the pH of the test sample, dissociating the antigen from the antigen-antibody complex and regenerating the antibody.
＜その他の工程＞
　その他の工程としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、洗浄工程、制御工程などが挙げられる。 <Other processes>
The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, a cleaning step, a control step, etc. can be mentioned.
　ここで、図１の（ａ）～（ｃ）は、抗体再生方法の原理を示す模式図である。図１の（ａ）に示すように、基材１上に固相化した測定対象である抗原と特異的に結合する抗体４の近傍に光酸３を固相化し、検査試料２を供する。すると、図１の（ｂ）に示すように、検査試料中の抗原５が抗体４と抗原抗体反応し、抗原抗体複合物６が形成される。次に、図１の（ｃ）に示すように、光酸３に対して特定波長の光を数秒照射することにより、光酸から水素イオンが放出され検査試料２のｐＨが変化し、極めて短い時間で抗原抗体複合物６から抗原５が解離する。その後、検査試料２を廃液すると、抗体４を再生することができ、抗体を繰り返し使用できる。 Here, (a) to (c) of FIG. 1 are schematic diagrams showing the principle of the antibody regeneration method. As shown in (a) of FIG. 1, 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 provided. Then, as shown in (b) of FIG. 1, antigen 5 in the test sample reacts with antibody 4 to form antigen-antibody complex 6. Next, as shown in (c) of FIG. 1, photoacid 3 is irradiated with light of a specific wavelength for several seconds, causing hydrogen ions to be released from photoacid, changing the pH of test sample 2 and causing antigen 5 to dissociate from antigen-antibody complex 6 in an extremely short time. After that, when test sample 2 is discharged, antibody 4 can be regenerated and the antibody can be used repeatedly.
　図２は、光酸としての８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）を光励起した際の反応式を示す。基底状態のＨＰＴＳに波長４０５ｎｍのレーザー光を照射するとＨＰＴＳが励起状態となり、水素イオンを放出し、波長５１２ｎｍの蛍光を発する。ＨＰＴＳから効率的に水素イオンを放出させるのに有効な励起光波長を検証するために、ＨＰＴＳの蛍光分光特性を計測した（ＦＰ８６００、日本分光株式会社製）。結果を図３に示す。図３に示すＨＰＴＳの蛍光スペクトルより、励起光波長４００ｎｍ～４３０ｎｍで蛍光ピークが観察され、ＨＰＴＳの最適波長は４０５ｎｍであることがわかった。 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 with a wavelength of 405 nm, HPTS becomes excited, releases hydrogen ions, and emits fluorescence with a wavelength of 512 nm. In order to verify the effective excitation light wavelength 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, and it was found that the optimal wavelength for HPTS is 405 nm.
　次に、ＨＰＴＳ溶液のレーザー光照射によるｐＨ制御能を検証するために、ＨＰＴＳ溶液のｐＨの経時的な変化を測定した。まず、ＨＰＴＳ（３００８０、Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ　Ｃｏｍｐａｎｙ製）を蒸留水に溶解し、１．０×１０－３ｍｏｌ／ＬのＨＰＴＳ水溶液を調製した。
　次に、ｐＨメーター（ＰＡＬ－ｐＨ、株式会社アタゴ製）をプレートシェイカーの上に固定し、ｐＨメーターの電極部に上記調製したＨＰＴＳ水溶液１ｍＬを滴下した。
　次に、レーザー照射装置（Ｄ４０５Ｃ－３００－１１－１Ｃ－１１、京セラＳＯＣ株式会社製）と拡散レンズ（ＡＬ１２１０Ｍ－Ａ、Ｔｈｏｒｌａｂｓ　Ｉｎｃ．製）を設置し、滴下したＨＰＴＳ水溶液に波長４０５ｎｍのレーザー光を照射した。このとき、ＨＰＴＳ水溶液全体にレーザー光を照射するために、拡散レンズとｐＨメーター間の距離を１００ｍｍとした。プレートシェイカーを３００ｒｐｍに設定し、攪拌しながらｐＨの測定を行った。その結果を図４及び図５に示す。
　図４に示すＨＰＴＳ水溶液のｐＨの経時変化の結果から、ＨＰＴＳ水溶液に波長４０５ｎｍのレーザー光を連続照射することにより、ＨＰＴＳ溶液のｐＨを６から３近傍まで低下させることを確認できた。また、図５の結果から、ＨＰＴＳ水溶液のｐＨの変化率は、レーザー光の照射強度に比例して増加し、照射強度が５０ｍＷ以上のレーザー光を照射することによって、抗原抗体複合物から抗原を解離できるｐＨ変化が得られることがわかった。 Next, in order to verify the pH controllability 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 to prepare a 1.0×10 −3 mol/L HPTS aqueous solution.
Next, a pH meter (PAL-pH, manufactured by Atago Co., Ltd.) was fixed onto a plate shaker, and 1 mL of the above-prepared HPTS aqueous solution was dropped 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 dropped HPTS aqueous solution was irradiated with a laser beam having a wavelength of 405 nm. At this time, in order to irradiate the entire HPTS aqueous solution with the laser beam, the distance between the diffusion lens and the pH meter was set to 100 mm. The plate shaker was set to 300 rpm, and the pH was measured while stirring. The results are shown in Figures 4 and 5.
From the results of the time-dependent change in pH of the HPTS aqueous solution 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 reduced the pH of the HPTS solution from 6 to approximately 3. Moreover, 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 capable of dissociating the antigen from the antigen-antibody complex.
　続いて、ＨＰＴＳ溶液のレーザー光照射によるｐＨ制御能の再現性を検証した。１ｍｍｏｌ／ＬのＨＰＴＳ（３００８０、Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ　Ｃｏｍｐａｎｙ製）水溶液１ｍＬを、ｐＨメーター（ＰＡＬ－ｐＨ、株式会社アタゴ製）の検出部に加えた。検出部のＨＰＴＳ溶液に対して、４０５ｎｍレーザー（Ｄ４０５Ｃ－３００－１１、京セラＳＯＣ株式会社製）の照射（出力５０ｍＷ）及び停止を繰り返してｐＨ変化を測定した。その結果を図６に示す。図６から、レーザー光照射によって、ＨＰＴＳからＨ＋の解離（脱プロトン化）が生じてｐＨが低下し、レーザー光照射を止めると速やかに元の分子構造に戻ることでｐＨが上昇することが確認された。即ち、光励起のＯＮ／ＯＦＦに伴うＨ＋の放出／吸収が起こっていることが示された。 Next, the reproducibility of the pH control ability of the HPTS solution by laser light irradiation was verified. 1 mL of 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, to measure pH changes. The results are shown in FIG. 6. From FIG. 6, it was confirmed that the dissociation (deprotonation) of H + from HPTS occurs due to laser light irradiation, causing the pH to decrease, and that when the laser light irradiation is stopped, the pH increases due to the rapid return to the original molecular structure. That is, it was shown that the release/absorption of H + occurs with the ON/OFF of photoexcitation.
（診断方法）
　本発明の診断方法は、マーカー複合物濃度測定工程と、マーカー解離工程とを含み、更に必要に応じてその他の工程を含む。 (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.
＜マーカー複合物濃度測定工程＞
　マーカー複合物濃度測定工程は、基材上に、マーカーと特異的に結合する抗マーカー抗体及び光酸を配し、検査試料を供することにより前記検査試料中のマーカーと前記抗マーカー抗体とが結合したマーカー複合物の濃度を測定する工程である。 <Marker Compound Concentration Measurement Step>
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 in the test sample is bound to the anti-marker antibody.
　前記マーカーとしては、特に制限はなく目的に応じて適宜選択することができ、例えば、心疾患マーカー、サイトカイン類（ＩＬ－１β、ＩＬ－６、ＩＬ－４、ＩＮＦγ、ＴＮＦ等）、タンパク質類（ゾヌリン（Ｚｏｎｕｌｉｎ）、ＬＢＰ（リポ多糖結合タンパク質）等）などが挙げられる。これらの中でも、心疾患マーカーであることが好ましい。 The marker is not particularly limited and can be appropriately selected depending on the purpose. Examples include heart disease markers, cytokines (IL-1β, IL-6, IL-4, INFγ, TNF, etc.), proteins (zonulin, LBP (lipopolysaccharide binding protein), etc.). Among these, heart disease markers are preferable.
－心疾患マーカー－
　心疾患マーカーとしては、例えば、脳性ナトリウム利尿ペプチド（ＢＮＰ）、脳性ナトリウム利尿ペプチド前駆体Ｎ端フラグメント（ＮＴ－ｐｒｏＢＮＰ）、トロポニンＴ（ＴｎＴ）、ミオグロビン、ＣＫ－ＭＢ等の生化学的心筋マーカー、心臓型脂肪酸結合蛋白などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。
　これらの中でも、ＢＮＰとＮＴ－ｐｒｏＢＮＰは心不全のマーカー、トロポニンは心筋梗塞のマーカーとして認知されており、初期診断で検査する項目であり、心疾患特異性が高く、最も重要な心疾患の診断情報となりうるものである。 - Heart disease markers -
Examples of cardiac disease markers include biochemical cardiac markers such as brain natriuretic peptide (BNP), N-terminal fragment of brain natriuretic peptide precursor (NT-proBNP), troponin T (TnT), myoglobin, and CK-MB, heart-type fatty acid binding protein, etc. These may be used alone or in combination of two or more.
Among these, BNP and NT-proBNP are recognized as markers for heart failure, and troponin is recognized as a marker for myocardial infarction. They are items tested for early diagnosis, have high specificity for heart disease, and can be the most important diagnostic information for heart disease.
　前記ＢＮＰと前記ＮＴ－ｐｒｏＢＮＰの生成は、同じＢＮＰ遺伝子に由来する。ＢＮＰ遺伝子からは転写・翻訳後、ＢＮＰ前駆体（ｐｒｏＢＮＰ［１－１０８］）が生成される。その後、生理的に非活性のＮＴ－ｐｒｏＢＮＰ（ｐｒｏＢＮＰのＮ端から７６個のアミノ酸［１－７６］）と生理活性を有する成熟型ＢＮＰ（残りの３２個のアミノ酸［７７－１０８］）に切断される。即ち、ＢＮＰとＮＴ－ｐｒｏＢＮＰは、心筋から等モルで分泌される。
　前記ＢＮＰと前記ＮＴ－ｐｒｏＢＮＰは、主として心室における壁応力（進展ストレス）に応じて遺伝子発現が亢進し、速やかに生成・分泌される。したがって、壁応力が増大する心不全では、その重症度に応じて前記ＢＮＰと前記ＮＴ－ｐｒｏＢＮＰの血中濃度が増加する。 The BNP and NT-proBNP are produced from the same BNP gene. After transcription and translation, the BNP precursor (proBNP [1-108]) is produced from the BNP gene. It 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 cardiac muscle.
The gene expression of the BNP and NT-proBNP is enhanced mainly in response to wall stress (progressive stress) in the ventricle, and the BNP and NT-proBNP are rapidly produced and secreted. Therefore, in heart failure in which wall stress increases, the blood concentrations of the BNP and NT-proBNP increase depending on the severity of the condition.
　前記抗マーカー抗体としては、特に制限はなく目的に応じて適宜選択することができ、例えば、抗心疾患マーカー抗体、サイトカイン（ＩＬ－１β、ＩＬ－６、ＩＬ－４、ＩＮＦγ、ＴＮＦ等）を抗原とする抗体、タンパク質（ゾヌリン（Ｚｏｎｕｌｉｎ）、ＬＢＰ（リポ多糖結合タンパク質）等）を抗原とする抗体などが挙げられる。これらの中でも、抗心疾患マーカー抗体であることが好ましい。 The anti-marker antibody is not particularly limited and can be appropriately selected depending on the purpose. Examples include anti-heart 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-heart disease marker antibodies are preferable.
－抗心疾患マーカー抗体－
　抗心疾患マーカー抗体としては、心疾患マーカーと特異的に結合する、免疫グロブリン分子自体の他、パパインやペプシン等の蛋白質分解酵素、或いは化学的分解により生じるＦａｂ、Ｆ（ａｂ’）２フラグメント等の分解産物も包含される。また、前記抗心疾患マーカー抗体は、ポリクローナル抗体及びモノクローナル抗体のどちらを用いても構わない。前記抗心疾患マーカー抗体の取得方法も通常使用される方法を用いることができる。
　例えば、心疾患マーカーがＢＮＰである場合には抗ＢＮＰモノクローナル抗体が用いられる。また、心疾患マーカーがＮＴ－ｐｒｏＢＮＰである場合には抗ＮＴ－ｐｒｏＢＮＰモノクローナル抗体が用いられる。 - Anti-cardiac disease marker antibodies -
The anti-cardiac disease marker antibody includes not only the immunoglobulin molecule itself that specifically binds to the cardiac disease marker, but also decomposition products such as Fab and F(ab') 2 fragments generated by proteolytic enzymes such as papain and pepsin, or by chemical decomposition. The anti-cardiac disease marker antibody may be either a polyclonal antibody or a monoclonal antibody. The anti-cardiac disease marker antibody may be obtained by a commonly used method.
For example, when the cardiac disease marker is BNP, an anti-BNP monoclonal antibody is used, and when the cardiac disease marker is NT-proBNP, an anti-NT-proBNP monoclonal antibody is used.
－基材－
　基材としては、特に制限はなく、目的に応じて適宜選択することができ、上記抗体再生方法と同様な基材を用いることができる。これらの中でも、基材として振動体を用いることが、振動体の共振周波数を測定することによってマーカーの濃度を測定できる点から好ましい。 -Base material-
The substrate is not particularly limited and can be appropriately selected depending on the purpose, and the same substrate as that used in the antibody regeneration method can be used. Among these, it is preferable to use a vibrator as the substrate, because the concentration of the marker can be measured by measuring the resonance frequency of the vibrator.
－光酸－
　光酸としては、特に制限はなく、目的に応じて適宜選択することができ、上記抗体再生方法と同様な光酸を用いることができる。これらの中でも、酸強度が大きい点及び可視光領域の蛍光スペクトルを有する点から、光酸として８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）を用いることが好ましい。 - Photoacid -
The photoacid is not particularly limited and may be appropriately selected depending on the purpose, and the same photoacids as those used in the antibody regeneration method described above may 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 its fluorescent spectrum in the visible light region.
　ここで、基材としての振動体表面に、抗心疾患マーカー抗体としての抗ＢＮＰ抗体及び光酸としてのＨＰＴＳを固相化する方法について説明する。
　振動体の表面に金属薄膜を形成し、この金属薄膜上に分子層オーダーの分子膜をスパッタ法等により形成し、この分子膜に抗体を修飾することにより振動体に抗体を固定することができる。前記金属薄膜としては、密着層と密着層上に形成される白金（Ｐｔ）膜を用いることができる。前記密着層としては、例えば、Ｔｉ、Ｃｒなどが挙げられる。前記分子層オーダーの分子膜としては、自己組織化単分子膜（以下、「ＳＡＭ膜」と称することがある）を用いることができる。
　具体的には、振動体（水晶板）の一面に、チタン（Ｔｉ）と白金（Ｐｔ）を、スパッタ法により５００Åに成膜する。白金（Ｐｔ）膜表面に抗ＢＮＰ抗体を固相化するには、ＳＡＭ膜を形成し、ＳＡＭ膜に抗ＢＮＰ抗体を修飾する。ＳＡＭ膜と抗ＢＮＰ抗体の結合には、アミンカップリング法を用いることができる。
　以上により、図８の（ａ）に示すように振動体１１の表面に、抗ＢＮＰ抗体を固相化する。 Here, a method for immobilizing anti-BNP antibody as an anti-cardiac disease marker antibody and HPTS as a photoacid on the surface of a vibrator as a substrate will be described.
A thin metal film is formed on the surface of the vibrating body, a molecular film of molecular layer order is formed on this thin metal film by sputtering or the like, and the molecular film is modified with an antibody, thereby fixing the antibody to the vibrating body. The thin metal film can be an adhesive layer and a platinum (Pt) film formed on the adhesive layer. Examples of the adhesive layer include Ti and Cr. The molecular film of molecular layer order 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 side of the vibrating body (quartz plate) by sputtering. To immobilize anti-BNP antibody on the platinum (Pt) film surface, a SAM film is formed and the SAM film is modified with anti-BNP antibody. The amine coupling method can be used to bond the SAM film and the anti-BNP antibody.
In this manner, the anti-BNP antibody is immobilized on the surface of the vibrating body 11 as shown in FIG. 8(a).
　次に、図７に示すように、８－ヒドロキシピレン－１，３，６－トリスルホン酸三ナトリウムの４つの基（３つのＳＯ３Ｎａと１つのＯＨ）のうち、ＯＨをアセチル基で保護しておき、３つのＳＯ３Ｎａの１つをＣｌに置換し、図８の（ｃ）に示すように、振動体１１表面のＮＨ２ －末端に８－アセトキシ－ピレン－１，３，６－トリスルホン酸三ナトリウムを固相化する。 Next, as shown in FIG. 7, of the four groups (three SO 3 Na and one OH) of trisodium 8-hydroxypyrene-1,3,6-trisulfonate, the OH is protected with an acetyl group, one of the three SO 3 Na is replaced with Cl, and trisodium 8-acetoxypyrene-1,3,6-trisulfonate is solid-phased at the NH 2 -terminus on the surface of the vibrating body 11 as shown in FIG. 8(c).
－前処理（官能基の置換）－
　図７に示す反応式により、８－ヒドロキシピレン－１，３，６－トリスルホン酸三ナトリウムの官能基を置換して８－アセトキシ－ピレン－１，３，６－トリスルホニルクロリドとする前処理をする。
　まず、８－ヒドロキシピレン－１，３，６－トリスルホン酸三ナトリウム（２０ｇ、０．０３８ｍｏｌ）をＮａＯＨ（２．４ｇ、０．０６ｍｏｌ）の水（３０ｍＬ）に溶解し、約０℃に冷却する。
　次に、無水酢酸（５ｇ、４．８ｍＬ、０．４８ｍｏｌ）を溶液に滴下し、反応混合物を２時間撹拌する。
　次に、エタノール（２０ｍＬ）を添加して沈殿を完了させ、沈殿物を濾過によって回収し、エタノール（３×１０ｍＬ）で洗浄し、減圧下で２４時間乾燥させて、黄色の固体である８－アセトキシ－ピレン－１，３，６－トリスルホン酸三ナトリウム（１７ｇ、収率８０％）を得る。
　次に、８－アセトキシ－ピレン－１，３，６－トリスルホン酸三ナトリウム（５ｇ、０．００８８ｍｏｌ）とトルエン（１５０ｍＬ）の混合物を、自動水分離器（ディーンスタークトラップ）と冷却器を備えた０．２５リットルの丸底フラスコに入れる。
　次に、混合物を２時間加熱還流して、反応混合物を乾燥させる。
　次に、乾燥した反応混合物を６０℃に冷却し、塩化オキサリル（６ｍＬ）及びＮ，Ｎ－ジメチルホルムアミド（ＤＭＦ）（２滴）を添加する。混合物を加熱する。加熱した混合物を８時間加熱還流し、トルエンと過剰の塩化オキサリル（３０ｍＬ）の混合物を蒸留する。
　次に、塩化ナトリウムの沈殿物を濾取し、濾液から溶媒を減圧下で除去する。
　次に、固体残留物を真空中で２４時間乾燥させて、８－アセトキシ－ピレン－１，３，６－トリスルホニルクロリド（４ｇ）を得る（収率８１．５％）。 - Pretreatment (substitution of functional groups) -
According to the reaction formula shown in FIG. 7, a pretreatment is carried out to convert trisodium 8-hydroxypyrene-1,3,6-trisulfonate into 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride by substituting the functional group.
First, trisodium 8-hydroxypyrene-1,3,6-trisulfonate (20 g, 0.038 mol) was dissolved in NaOH (2.4 g, 0.06 mol) and water (30 mL), and the solution was cooled to about 0°C.
Acetic anhydride (5 g, 4.8 mL, 0.48 mol) is then added dropwise to the solution and the reaction mixture is stirred for 2 hours.
Ethanol (20 mL) is then added to complete the precipitation, and the precipitate is collected by filtration, washed with ethanol (3×10 mL), and dried under reduced pressure for 24 hours to give 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 heated. The heated mixture is refluxed for 8 hours to distill off the mixture of toluene and excess oxalyl chloride (30 mL).
The sodium chloride precipitate is then filtered off and the filtrate is freed of solvent under reduced pressure.
The solid residue is then dried in vacuum for 24 hours to give 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride (4 g) (yield 81.5%).
－振動体表面への固相化－
　図８の（ａ）に示すように抗ＢＮＰ抗体を固相化した振動体１１を、上記前処理で得た８－アセトキシ－ピレン－１，３，６－トリスルホニルクロリドとピリジンの混合液中で２４時間インキュベーションした後、フェノール官能基の飽和炭酸水素ナトリウム溶液へ曝露する。
　以上により、図８の（ｃ）に示すように、振動体１１の表面に抗ＢＮＰ抗体及び８－アセトキシ－ピレン－１，３，６－トリスルホン酸三ナトリウムを固相化することができる。 - Solidification onto the vibrating body surface -
As shown in FIG. 8(a), the vibrating body 11 on which the anti-BNP antibody is immobilized is incubated for 24 hours in a mixture of 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride and pyridine obtained in the above pretreatment, and then exposed to a saturated sodium bicarbonate solution of the phenol functional group.
As a result of the above, as shown in FIG. 8(c), the anti-BNP antibody and trisodium 8-acetoxy-pyrene-1,3,6-trisulfonate can be immobilized on the surface of the vibrating body 11.
－マーカー複合物の濃度測定－
　検査試料中のマーカーと抗マーカー抗体とが結合したマーカー複合物の濃度を測定方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　Ｉｍｍｕｎｏ　Ｓｏｒｂｅｎｔ　Ａｓｓａｙ）法、イムノクロマト法、表面プラズモン共鳴法、磁気ビーズ法、一定周波数で振動している振動体に心疾患マーカーが付着した際の質量変化に伴う共振周波数を検出する共振型質量センサを用いる方法などが挙げられる。これらの中でも、試薬及び解離液の補充が不要であり、デバイスの小型化が図れ、光酸に光を照射するだけで抗マーカー抗体の再生が可能となり、抗マーカー抗体を繰り返し使用でき、時系列的なモニタリングを実現できる点から、共振型質量センサを用いる方法が好ましい。
　共振型質量センサを用いる方法としては、例えば、マーカーと特異的に結合する抗マーカー抗体及び光酸が固相化された振動体が検査試料に供されると、検査試料中のマーカーが抗マーカー抗体に捕捉され、振動体の質量が増加し、振動体の共振周波数が低下する。その結果、共振周波数の変化割合から検査試料中のマーカーの濃度を測定することができる。 - Measurement of marker compound concentration -
The method for measuring the concentration of a marker complex in a test sample in which a marker and an anti-marker antibody are bound is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, surface plasmon resonance, magnetic bead method, and a method using a resonance type mass sensor that detects the resonance frequency associated with the mass change when a cardiac disease marker adheres to an oscillator vibrating at a constant frequency. Among these, the method using a resonance type mass sensor is preferred because it does not require replenishment of reagents and dissociation solution, the device can be made smaller, the anti-marker antibody can be regenerated simply by irradiating the photoacid with light, the anti-marker antibody can be used repeatedly, and time-series monitoring can be realized.
In a method using a resonant mass sensor, for example, when an oscillator having an anti-marker antibody that specifically binds to a marker and a photoacid immobilized thereon 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.
＜マーカー解離工程＞
　マーカー解離工程は、光酸に光を照射してマーカー複合物からマーカーを解離させる工程である。
　本発明の診断方法では、検査試料に含まれるマーカーの濃度の測定が終了した後、光酸に光を照射してマーカーと前記抗マーカー抗体とが結合したマーカー複合物からマーカーを解離させることにより、抗マーカー抗体を再生することができる。
　前記光は、レーザー光であることが好ましい。レーザー光を光酸に照射することにより、光酸から水素イオンが放出され、液体の検査試料のｐＨが低下することによってマーカー複合物からマーカーを解離させ、抗マーカー抗体を再生することができる。 <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 anti-marker antibody can be regenerated by irradiating the photoacid with light to dissociate the marker from the marker complex formed by the marker and the anti-marker antibody binding thereto.
The light is preferably a laser light. By irradiating the photoacid with the laser light, hydrogen ions are released from the photoacid, and the pH of the liquid test sample is lowered, whereby the marker is dissociated from the marker complex and the anti-marker antibody is regenerated.
　本発明においては、抗マーカー抗体を再生し、間隔をあけてマーカー複合物の濃度を繰り返し測定することにより、マーカーの時系列的なモニタリングを実現することができる。 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.
＜その他の工程＞
　その他の工程としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、洗浄工程、制御工程などが挙げられる。 <Other processes>
The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, a cleaning step, a control step, etc. can be mentioned.
　ここで、図９の（ａ）～（ｄ）は、診断方法の原理を示す模式図である。図９の（ａ）に示すように、振動体１１上に測定対象であるマーカーと特異的に結合する抗マーカー抗体１４の近傍に光酸３を固相化し、検査試料２を供する。すると、図９の（ｂ）に示すように、検査試料中のマーカー１５が抗マーカー抗体１４と抗原抗体反応し、マーカー複合物１６が形成される。次に、図９の（ｃ）に示すように、検査試料中のマーカー１５が抗マーカー抗体１４によって捕捉され、振動体１１の質量が増加する。その結果、振動体１１の共振周波数が低下し、共振周波数の変化割合から検査試料中のマーカーの濃度を測定することができる。振動体１１の共振周波数の変化は、レーザー変位計を用いて測定することができる。
　次に、図９の（ｄ）に示すように、光酸３に対して特定波長の光を照射することにより検査試料２のｐＨが変化し、マーカー複合物１６からマーカー１５が解離する。その後、検査試料２を廃液すると、図９の（ａ）に示すように抗マーカー抗体１４を再生することができ、繰り返し使用できる。 Here, (a) to (d) of FIG. 9 are schematic diagrams showing the principle of the diagnostic method. As shown in (a) of FIG. 9, photoacid 3 is immobilized near anti-marker antibody 14 that specifically binds to the marker to be measured on the vibrating body 11, and a test sample 2 is provided. Then, as shown in (b) of FIG. 9, marker 15 in the test sample reacts with anti-marker antibody 14 to form a marker complex 16. Next, as shown in (c) of FIG. 9, marker 15 in the test sample is captured by anti-marker antibody 14, and the mass of vibrating body 11 increases. As a result, the resonant frequency of vibrating body 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 vibrating body 11 can be measured using a laser displacement meter.
Next, as shown in Fig. 9(d), the pH of the test sample 2 is changed by irradiating the photoacid 3 with light of a specific wavelength, and the marker 15 is dissociated from the marker complex 16. When the test sample 2 is then discharged, the anti-marker antibody 14 can be regenerated as shown in Fig. 9(a) and can be used repeatedly.
　本発明の診断方法は、上述した通り、検査試料に含まれるマーカーの定量方法、及び検査試料に含まれるマーカーの評価方法を含む。 As described above, the diagnostic method of the present invention includes a method for quantifying markers contained in a test sample, and a method for evaluating markers contained in a test sample.
（診断装置）
　本発明の診断装置は、マーカーと特異的に結合する抗マーカー抗体及び光酸を表面に配した振動体と、前記振動体の共振周波数を検出する検出部と、前記光酸に光を照射する光照射部とを有し、更に必要に応じてその他の手段を有する。 (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.
＜振動体＞
　マーカーと特異的に結合する抗マーカー抗体及び光酸を表面に配しており、圧電結晶などを用いることができる。圧電結晶は、歪みを与えると電気的な応答を示し、逆に、電圧を加えると歪みが生じる性質を持つ材料であり、例えば、水晶、チタン酸バリウム、チタン酸鉛、チタン酸ジルコン酸鉛（ＰＺＴ）、ニオブ酸カリウム、ニオブ酸リチウム、タングステン酸ナトリウム、タンタル酸リチウムなどが挙げられる。
　振動体表面に、抗マーカー抗体及び光酸を配する方法としては、特に制限はなく、目的に応じて適宜選択することができ、上記診断方法と同様の方法により行うことができる。 <Vibration body>
The surface is provided with an anti-marker antibody and photoacid that specifically binds to the marker, and a piezoelectric crystal, etc., can be used. A piezoelectric crystal is a material that exhibits an electrical response when strained, and conversely, has the property of generating strain when a voltage is applied, and examples of such a material 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 vibrating body surface is not particularly limited and can be appropriately selected depending on the purpose, and can be carried out in the same manner as the above-mentioned diagnostic method.
　前記振動体は振動子上に載置され、前記振動体と前記振動子が機械的に結合されておらず、かつ前記振動体がいずれの部材にも機械的に結合されていないことが好ましい。これにより、給電や固定による共振現象の阻害がなく、振動体の形状寸法の設計の制約を無くすことができ、質量変化を高感度で測定できる点から好ましい。 The vibrating body is placed on a vibrator, and it is preferable that the vibrating body and the vibrator are not mechanically coupled, and that the vibrating body is not mechanically coupled to any member. This is preferable because it does not inhibit the resonance phenomenon due to power supply or fixation, it eliminates design constraints on the shape and dimensions of the vibrating body, and it allows mass changes to be measured with high sensitivity.
＜検出部＞
　検出部は、振動体の共振周波数を検出する手段であり、発光素子と受光素子からなる光学的な検出手段を使用することができる。前記光学的な検出手段は、振動体の周波数、変位、速度、及び加速度のいずれかを測定してもよい。
　前記検出部としては、レーザー変位計などを用いることができる。前記レーザー変位計によれば、振動体の共振周波数の変化を非接触で計測することができる。 <Detection unit>
The detector is a means for detecting the resonant frequency of the vibrating body, and may be an optical detector consisting of a light-emitting element and a light-receiving element. The optical detector may measure any one of the frequency, displacement, velocity, and acceleration of the vibrating body.
The detection unit may be a laser displacement meter, etc. The laser displacement meter can measure the change in the resonance frequency of the vibrating body in a non-contact manner.
＜光照射部＞
　光照射部は、光酸に光を照射する手段であり、レーザー光照射部であることが好ましい。レーザー光照射部は、例えば、レーザー光源と、拡散レンズと、電源とを有する。 <Light irradiation unit>
The light irradiating unit is a means for irradiating the photoacid with light, and is preferably a laser light irradiating unit. The laser light irradiating unit has, for example, a laser light source, a diffusion lens, and a power source.
＜その他の手段＞
　その他の手段としては、例えば、入力手段、記録手段、表示手段、通信手段、メンテナンス手段などが挙げられる。 <Other measures>
Examples of the other means include an input means, a recording means, a display means, a communication means, and a maintenance means.
　ここで、図１０は、診断装置の一例を示す概略図である。この図１０の診断装置１０は、マーカーと特異的に結合する抗マーカー抗体１４及び光酸３を表面に配した振動体１１と、振動体１１の共振周波数を検出する検出部１２と、光酸３に光を照射する光照射部１３と、振動体１１を駆動する電源１７とを有する。 Here, FIG. 10 is a schematic diagram showing an example of a diagnostic device. The diagnostic device 10 in FIG. 10 has a vibrator 11 having an anti-marker antibody 14 that specifically binds to the marker and photoacid 3 on its surface, a detection unit 12 that detects the resonance frequency of the vibrator 11, a light irradiation unit 13 that irradiates the photoacid 3 with light, and a power source 17 that drives the vibrator 11.
　振動体１１としては、特に制限はなく、振動体単独で用いることができるが、振動体１１を振動子上に載置して用いることが好ましい。この場合、振動体１１と振動子は機械的に結合されておらず、かつ振動体１１はいずれの部材にも機械的に結合されていない、つまり、振動体１１はどこにも固定化されていない構造を有していることが、給電や固定による共振現象の阻害がなく、振動体１１の形状寸法の設計の制約を無くすことができ、質量変化を高感度で測定できる点から好ましい。 There are no particular limitations on the vibrating body 11, and it can be used alone, but it is preferable to place the vibrating body 11 on a vibrator. In this case, the vibrating body 11 and the vibrator are not mechanically connected, and the vibrating body 11 is not mechanically connected to any member; in other words, the vibrating body 11 has a structure that is not fixed anywhere, which is preferable in that there is no inhibition of the resonance phenomenon due to power supply or fixing, there are no design constraints on the shape and dimensions of the vibrating body 11, and mass changes can be measured with high sensitivity.
　振動体１１は、共振現象に最適な形状寸法に加工されている。振動体１１は、圧電素子などの振動子の上に非拘束状態で載置されており、圧電素子が任意の周波数で振動すると、振動体１１が、振動体１１自身の共振周波数で振動できる構造になっている。圧電素子に梁状の振動体の固有周波数付近の交流電圧を印加すると、振動体は自らの固有の共振周波数で共振する。このようにすれば、振動体は材質や形状寸法に何ら制約を受けることが無くなるので、設計の自由度は飛躍的に改善できる。 The vibrating body 11 is machined 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 any 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 of the vibrating body, and design freedom can be dramatically improved.
　振動体１１の振動モードは、定在波型超音波モータのように、ある一定の方向へ移動するような１軸振動等の振動モードとならないように設定される。つまり、振動体１１の振動モードは、ある一定の方向へ移動するような振動モードではなく、かつ、その振幅もナノメーター（ｎｍ）オーダーと小さいので、振動体１１が振動子の上から脱離することはない。
　なお、前記振動体及び前記振動子を有する共振型質量センサの詳細については、例えば、特許第６０８６３４７号公報の記載を参照することができる。 The vibration mode of the vibrating body 11 is set so as not to be a vibration mode such as a uniaxial vibration that moves in a certain direction like a standing wave type 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 top of the vibrator.
For details of the resonant mass sensor having the vibrating body and the vibrator, reference can be made to the description in Japanese Patent No. 6,086,347, for example.
　マーカーと特異的に結合する抗マーカー抗体１４及び光酸３が、固相化された振動体１１が検査試料に供されると、検査試料中のマーカー１５が抗マーカー抗体１４に捕捉され、振動体１１の質量が増加する。その結果、振動体１１の共振周波数が低下し、共振周波数の変化割合から検査試料中のマーカー１５の濃度を測定することができる。振動体１１の共振周波数の変化は、検出部１２により検知される。検出部１２は、振動体１１の共振周波数の計測手段としてレーザー変位計を用いている。
　次に、検査試料に含まれるマーカー１５の濃度の測定が終了すると、光酸３に光照射部１３からレーザー光を照射する。すると、光酸から放出される水素イオンによって検査試料のｐＨが低下し、マーカー複合物１６からマーカー１５が解離し、抗マーカー抗体１４を再生することができる。 When the vibrating body 11 on which the anti-marker antibody 14 and photoacid 3 that specifically bind to the marker are immobilized is provided to a test sample, the marker 15 in the test sample is captured by the anti-marker antibody 14, and the mass of the vibrating body 11 increases. As a result, the resonant frequency of the vibrating body 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 vibrating body 11 is detected by the detecting unit 12. The detecting unit 12 uses a laser displacement meter as a means for measuring the resonant frequency of the vibrating body 11.
Next, when the measurement of the concentration of marker 15 contained in the test sample is completed, laser light is irradiated onto photoacid 3 from light irradiator 13. Then, hydrogen ions released from photoacid lower the pH of the test sample, causing marker 15 to dissociate from marker complex 16, and allowing anti-marker antibody 14 to be regenerated.
　市販のポータブル式・卓上式等のスタンドアロン型心疾患診断装置は、小規模な医療機関での初期診断には非常に有用であるが、即時性のある情報取得（ＰＯＣＴ）では専門医師が常駐している施設で行われているのが一般的であり、ポータブル式・卓上式の心疾患診断装置の普及は限定的である。
　また、心疾患マーカーとしてのＢＮＰ濃度の測定は、図１１に示すように、生体装着型の心疾患診断装置を用い、間隔をあけて繰り返し時系列的に測定しないと、ＢＮＰの高値及び低値が見過ごされてしまい、正常と判断され、心疾患の診断を誤ってしまうリスクがある。
　図１２の（ａ）は、心疾患マーカーを迅速に、その場で、繰り返し計測できる生体装着型の心疾患診断装置の一例を示す概略図である。この生体装着型の心疾患診断装置２２は、患者の手首に装着することにより、ＢＮＰ濃度を、間隔をあけて繰り返し測定することができる。そして、生体装着型の心疾患診断装置２２で測定したＢＮＰ濃度をスマートフォン２１に随時送信することにより、図１２の（ｂ）に示すように、ＢＮＰ濃度を時系列的に表示することができるので、心疾患の診断を正確かつ迅速に行うことが可能となる。 Commercially available portable, desktop and other standalone cardiac disease diagnosis devices are very useful for initial diagnosis in small medical institutions. However, immediate information acquisition (POCT) is generally performed at facilities where specialist doctors are on-site, and the spread of portable and desktop cardiac disease diagnosis devices is limited.
Furthermore, unless the BNP concentration as a cardiac disease marker is measured repeatedly over time at intervals using a cardiac disease diagnosis device that is attached to the body as shown in Figure 11, high and low BNP values may be overlooked and determined to be normal, resulting in a risk of an erroneous diagnosis of cardiac disease.
Fig. 12 (a) is a schematic diagram showing an example of a bio-attachable cardiac disease diagnostic device that can measure cardiac disease markers quickly, on the spot, and repeatedly. This bio-attachable cardiac disease diagnostic device 22 can be attached to the patient's wrist to repeatedly measure the BNP concentration at intervals. By transmitting the BNP concentration measured by the bio-attachable cardiac disease diagnostic device 22 to the smartphone 21 at any time, the BNP concentration can be displayed in chronological order as shown in Fig. 12 (b), making it possible to accurately and quickly diagnose cardiac disease.
　本発明の診断方法及び診断装置によると、マーカーと特異的に結合する抗マーカー抗体の近傍に光酸を配し、前記光酸に光を照射することによって、マーカー複合物からマーカーを解離させることができ、物理刺激だけで抗マーカー抗体の再利用が可能となり、抗マーカー抗体を繰り返し使用することができるので、マーカーの時系列的なモニタリングを実現することができる。 The diagnostic method and diagnostic device of the present invention dissociate the marker from the marker complex by placing a photoacid near an anti-marker antibody that specifically binds to the marker and irradiating the photoacid with light. This makes it possible to reuse the anti-marker antibody with only physical stimulation, and since the anti-marker antibody can be used repeatedly, it is possible to realize chronological monitoring of the marker.
　以下、本発明の実施例を説明するが、本発明は、これらの実施例に何ら限定されるものではない。 The following describes examples of the present invention, but the present invention is not limited to these examples.
（実施例１）
　光酸（ＨＰＴＳ）への光照射による心疾患マーカー複合物からの心疾患マーカー（ＢＮＰ）の解離を実験により実証すると共に、ＨＰＴＳ濃度の変化によるＢＮＰの解離度の影響を評価した。 Example 1
Dissociation of a cardiac marker (BNP) from a cardiac marker complex by irradiation with photoacid (HPTS) was experimentally demonstrated, and the effect of changes in HPTS concentration on the degree of dissociation of BNP was evaluated.
　まず、図１３の（ａ）に示すように、抗ＢＮＰ抗体（１４）（４ＢＮＰ２ｃｃ－５０Ｅ１ｃｃ、ＨｙＴｅｓｔ　Ｌｔｄ．製）を基材（１）としてのマイクロプレート（ＳｐｅｃｔｒａＰｌａｔｅ－３８４　ＨＢ、ＰｅｒｋｉｎＥｌｍｅｒ　Ｉｎｃ．製）のウェルに自己組織化単分子膜を用いる方法により固相化後、表１に示す条件に基づき、心疾患マーカーとしてのＢＮＰ（１５）（ナノピア（登録商標）ＢＮＰ用ＢＮＰコントロール、積水メディカル株式会社製）を添加し、１時間インキュベートし、心疾患マーカー複合物（１６）を形成させた。 First, as shown in FIG. 13(a), 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.) as a substrate (1) by a method using a self-assembled monolayer, and 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 incubated for 1 hour to form a cardiac disease marker complex (16).
　次に、マイクロプレートを洗浄した後、図１３の（ｂ）に示すように、酵素標識された検出抗体（７）（４ＢＮＰ２ｃｃ－２４Ｃ５ｃｃ、ＨｙＴｅｓｔ　Ｌｔｄ．製）を添加し、１時間インキュベートし、抗ＢＮＰ抗体（１４）、ＢＮＰ（１５）、及び酵素標識された検出抗体（７）の順でマイクロプレートのウェルに結合させた（図１３の（ｃ）参照）。 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)).
　ＨＰＴＳ（３００８０、Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ　Ｃｏｍｐａｎｙ製）を蒸留水に溶解し、１．０×１０－３ｍｏｌ／Ｌ～７．０×１０－３ｍｏｌ／ＬのＨＰＴＳ水溶液を調製した（表１参照）。
　次に、マイクロプレートを洗浄した後、図１３の（ｄ）に示すように、上記所定の濃度に調製したＨＰＴＳ水溶液を添加し、３０分間レーザー光照射することによりＨＰＴＳ水溶液のｐＨが低下し、抗ＢＮＰ抗体（１４）からＢＮＰ（１５）及び酵素標識された検出抗体（７）が解離した。
　図１４は、マイクロプレートへのレーザー光の照射方法を示した。レーザー照射装置（３３）（Ｄ４０５Ｃ－３００－１１－１Ｃ－１１、京セラＳＯＣ株式会社製）により波長４０５ｎｍのレーザー光をｆ＝１０拡散レンズ（３２）（ＡＬ１２１０Ｍ－Ａ、Ｔｈｏｒｌａｂｓ　Ｉｎｃ．製）に通し、マイクロプレート（３１）の底面に拡散したレーザー光を照射した。ｆ＝１０拡散レンズ（３２）とマイクロプレート（３１）の距離を変更することにより、レーザー光が照射されるマイクロプレート（３１）のウェルの数を調節することができる。図１４中３４は出力可変電源である。本実施例では、１０ウェルへのレーザー光照射を行うために、ｆ＝１０拡散レンズ３２とマイクロプレート３１の距離を２００ｍｍとした（照射直径４０ｍｍ）。 HPTS (30080, manufactured by Cayman Chemical Company) was dissolved in distilled water to prepare 1.0×10 −3 mol/L to 7.0×10 −3 mol/L aqueous HPTS solutions (see Table 1).
Next, after washing the microplate, an aqueous HPTS solution prepared to the above-mentioned predetermined concentration was added, and the pH of the aqueous HPTS solution was reduced by irradiating the plate with laser light for 30 minutes, as shown in FIG. 13(d), whereby BNP (15) and the enzyme-labeled detection antibody (7) were dissociated from the anti-BNP antibody (14).
FIG. 14 shows a method of irradiating a microplate with laser light. A laser irradiation device (33) (D405C-300-11-1C-11, manufactured by Kyocera SOC Corporation) irradiated a 405 nm wavelength laser light through an f=10 diffusion lens (32) (AL1210M-A, manufactured by Thorlabs Inc.) and irradiated the diffused laser light onto the bottom surface of the microplate (31). By changing the distance between the f=10 diffusion lens (32) and the microplate (31), the number of wells of the microplate (31) irradiated with the laser light can be adjusted. In FIG. 14, 34 is a variable output power source. In this embodiment, in order to irradiate 10 wells with laser light, the distance between the f=10 diffusion lens 32 and the microplate 31 was set to 200 mm (irradiation diameter 40 mm).
　次に、図１３の（ｅ）に示すように、レーザー光を照射後の各ウェルに検出抗体（７）に標識した酵素に反応し発色するＴＭＢ溶液（８ａ）（Ｅ１０２、Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｉｎｃ．製）を添加し、１時間インキュベートした。 Next, as shown in FIG. 13(e), a TMB solution (8a) (E102, manufactured by Bethyl Laboratories, Inc.) that reacts with the enzyme labeled to the detection antibody (7) to produce color was added to each well after irradiation with the laser light, and the wells were incubated for 1 hour.
　次に、図１３の（ｆ）に示すように、発色したＴＭＢ溶液（８ｂ）の吸光度をプレートリーダー（Ｗａｌｌａｃ　１４２０、ＰｅｒｋｉｎＥｌｍｅｒ　Ｉｎｃ．製）で計測し、ＢＮＰ解離の割合を測定した（計測波長３５５ｎｍ）。結果を表２及び図１５に示した。なお、ＴＭＢ溶液の吸光度が高いほどＢＮＰの結合量が多く、吸光度が低いほどＢＮＰが解離しＢＮＰの結合量が少ないことを示す。 Next, as shown in FIG. 13(f), the absorbance of the colored TMB solution (8b) was measured using a plate reader (Wallac 1420, manufactured by PerkinElmer Inc.) to measure the percentage of BNP dissociation (measurement wavelength: 355 nm). The results are shown in Table 2 and FIG. 15. Note that the higher the absorbance of the TMB solution, the greater the amount of BNP bound, and the lower the absorbance, the more BNP dissociated and the less BNP bound.
（比較例１）
　実施例１において、ＨＰＴＳ水溶液を添加した後、レーザー光を照射しなかった以外は、実施例１と同様にして、ＢＮＰ解離の割合を測定した。結果を表２及び図１５に示した。 (Comparative Example 1)
The rate of BNP dissociation was measured in the same manner as in Example 1, except that laser light was not irradiated after the addition of the HPTS aqueous solution in Example 1. The results are shown in Table 2 and FIG.
　表２及び図１５の結果から、ＨＰＴＳ水溶液を添加した後、レーザー光を照射した実施例１は、ＨＰＴＳ水溶液の濃度上昇に伴い、吸光度が低下している。これは、ＨＰＴＳ水溶液にレーザー光を照射した際に水素イオン（Ｈ＋）が放出されＨＰＴＳ水溶液のｐＨが低下し、その結果、ＢＮＰの解離が起こっていることを示している。
　これに対して、ＨＰＴＳ水溶液を添加した後、レーザー光を照射しなかった比較例１は、ＨＰＴＳ水溶液の濃度上昇に伴う吸光度の変化は観察されなかった。これは、ＨＰＴＳ水溶液のみでは抗原抗体反応に影響を与えないことを示している。 15, in Example 1, in which the HPTS aqueous solution was added and then irradiated with laser light, the absorbance decreased with an increase in the concentration of the HPTS aqueous solution. This indicates that when the HPTS aqueous solution was irradiated with laser light, hydrogen ions (H + ) were released, lowering the pH of the HPTS aqueous solution, and as a result, dissociation of BNP occurred.
In contrast, in Comparative Example 1, in which laser light was not irradiated after addition of the HPTS aqueous solution, no change in absorbance was observed with increasing concentration of the HPTS aqueous solution, indicating that the HPTS aqueous solution alone does not affect the antigen-antibody reaction.
（実施例２）
　心疾患の診断方法を実証するために、ＢＮＰ分析用センサを構成し、ＢＮＰ濃度変化による発光強度の影響を評価した。
　（１）Ａｕメッシュ基材の作製
　ステンレスメッシュ（ＳＵＳ３０４、線径１６０μｍ、メッシュ粗さ３０メッシュ、Ｅ９１０７、株式会社久宝金属製作所製）を直径６ｍｍの円状に切り取り、スパッタリング装置（ＭＳＰ－２０ＵＭ、株式会社真空デバイス製）を用いて膜厚１００ｎｍの金薄膜を形成して、Ａｕメッシュ基材を得た（図１６Ａ参照）。
　このようなメッシュ形状を有する基材を用いると、基材表側だけでなく裏側に固相化されたＨＰＴＳにも光照射による光が到達するため、センサ性能を向上させることができる。また、発光素子と受光素子とからなる光学的な信号検出部を裏側に設置することもできる。なお、Ａｕ薄膜の形成方法は、蒸着に限定されるものではなく、メッキ等の方法を用いてもよい。 Example 2
In order to verify the method for diagnosing heart disease, a sensor for analyzing BNP was constructed and the effect of changes in BNP concentration on the 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 circle with a diameter of 6 mm, and a gold thin film with a thickness of 100 nm was formed using a sputtering device (MSP-20UM, manufactured by Vacuum Device Co., Ltd.) to obtain an Au mesh substrate (see FIG. 16A).
When a substrate having such a mesh shape is used, the light emitted by the light reaches not only the front side of the substrate but also the HPTS immobilized on the back side, improving the sensor performance. Also, an optical signal detection unit consisting of a light emitting element and a light receiving element can be installed on the back side. The method for forming the Au thin film is not limited to vapor deposition, and plating or other methods may be used.
　（２）ＢＮＰ抗体の固相化
　作製したＡｕメッシュ基材をピラニア溶液でタンパク洗浄後、エタノールで溶解した１０ｍｍｏｌ／Ｌの６－Ａｍｉｎｏ－１－ｈｅｘａｎｅｔｈｉｏｌ（Ａ４２５、株式会社同仁化学研究所製）に２５℃で１６時間浸漬し、自己組織化単分子（ＳＡＭ）膜を形成した。その後、ｐＨ７．８の緩衝液（Ｐｈｏｓｐｈａｔｅ　ｂｕｆｆｅｒｅｄ　ｓａｌｉｎｅ、ＰＢＳ）で溶解した１００ｍｍｏｌ／Ｌのｂｉｓ（ｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｙｌ）ｓｕｂｅｒａｔｅ（ＢＳ３、品番Ｂ５７４、株式会社同仁化学研究所製）溶液に３０分間浸漬し、ＳＡＭ膜を活性化した。さらに、ｐＨ７．８のＰＢＳで溶解した１０μｍｏｌ／ＬのＢＮＰキャプチャー抗体（４ＢＮＰ２ｃｃ－５０Ｅ１ｃｃ，ＨｙＴｅｓｔ，Ｌｔｄ．）溶液に３０分間浸漬し、抗体をメッシュ表面に固相化した。ブロッキング処理として、ｐＨ７．８のＰＢＳで溶解したエタノールアミン（０１２－１２４５５、富士フイルム和光純薬株式会社製）溶液に６０分間浸漬し、その後ブロッキング剤であるスキムミルク溶液（ＵＫＢ８０、株式会社ケー・エー・シー製）に１２０分間浸漬した。 (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-hexanethiol (A425, Dojindo Laboratories, Inc.) dissolved in ethanol at 25° C. for 16 hours to form a self-assembled monolayer (SAM) film. The substrate was then immersed for 30 minutes in a 100 mmol/L bis(sulfosuccinimidyl) suberate (BS3, product number B574, Dojindo Laboratories, Inc.) solution dissolved in a pH 7.8 buffer solution (phosphate buffered saline, PBS) to activate the SAM film. Further, the mesh was immersed in a 10 μmol/L solution of BNP capture antibody (4BNP2cc-50E1cc, HyTest, Ltd.) dissolved in PBS at pH 7.8 for 30 minutes to immobilize the antibody on the mesh surface. As a blocking treatment, the mesh was immersed in a solution of ethanolamine (012-12455, Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in PBS at pH 7.8 for 60 minutes, and then in a blocking agent skim milk solution (UKB80, KAC Co., Ltd.) for 120 minutes.
　（３）ＢＮＰ濃度測定の工程
　０ｐｇ／ｍＬ、１ｐｇ／ｍＬ、及び１００ｐｇ／ｍＬのＢＮＰ標準液（ナノピア（登録商標）ＢＮＰ用ＢＮＰコントロール、積水メディカル株式会社製）に抗体を固相化したメッシュを３０分間浸漬し、洗浄液で洗浄後、ＡＬＰで標識したＢＮＰ検出抗体（４ＢＮＰ２ｃｃ－２４Ｃ５ｃｃ、ＨｙＴｅｓｔ，Ｌｔｄ．）に３０分間浸漬した（図１６Ｂ参照）。メッシュを洗浄後、ホルダーに移し、発光基質（Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　ＡＰ　Ｍｉｃｒｏｗｅｌｌ、ＳｕｒＭｏｄｉｃｓ社製）を５０μＬ加え、光電子増倍管とカウンティングユニットからなる検出器で９００秒間、発光強度を測定した。結果を図１７に示した。 (3) BNP concentration measurement process The mesh with the antibody immobilized 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 washing solution, and then immersed in ALP-labeled BNP detection antibody (4BNP2cc-24C5cc, HyTest, Ltd.) for 30 minutes (see FIG. 16B). After washing the mesh, it was transferred to a holder, 50 μL of a luminescent substrate (Chemiluminescent AP Microwell, manufactured by SurModics) was added, and 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.
　図１７の結果から、このようなセンサ構成・工程とすれば，ＢＭＰ濃度に依存して検出信号が増大し、ＢＮＰセンサとして機能することが示された。 The results in Figure 17 show that with this sensor configuration and process, the detection signal increases depending on the BMP concentration, and the sensor functions as a BNP sensor.
　本国際出願は２０２２年１１月１１日に出願した日本国特許出願２０２２－１８０７２７号に基づく優先権を主張するものであり、日本国特許出願２０２２－１８０７２７号の全内容を本国際出願に援用する。 This international application claims priority to Japanese Patent Application No. 2022-180727, filed on November 11, 2022, and the entire contents of Japanese Patent Application No. 2022-180727 are incorporated by reference into this international application.
　　　１　　　基材
　　　２　　　検査試料
　　　３　　　光酸
　　　４　　　抗体
　　　５　　　抗原
　　　６　　　抗原抗体複合物
　　　７　　　酵素標識された検出抗体
　　８ａ　　　ＴＭＢ
　　８ｂ　　　発色したＴＭＢ
　　１０　　　心疾患診断装置
　　１１　　　振動体
　　１２　　　検出部
　　１３　　　光照射部
　　１４　　　抗マーカー抗体
　　１５　　　マーカー
　　１６　　　マーカー複合物
　　１７　　　電源
　　２０　　　生体装着型の心疾患診断装置
　　２１　　　スマートフォン
　　３１　　　マイクロプレート
　　３２　　　拡散レンズ
　　３３　　　レーザー照射装置
　　３４　　　出力可変電源

  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
REFERENCE SIGNS LIST 10 Cardiac disease diagnostic device 11 Vibrator 12 Detection unit 13 Light irradiation unit 14 Anti-marker antibody 15 Marker 16 Marker compound 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 (9)

  1. 　基材上に、マーカーと特異的に結合する抗マーカー抗体及び光酸を配し、検査試料を供することにより前記検査試料中のマーカーと前記抗マーカー抗体とが結合したマーカー複合物の濃度を測定するマーカー複合物濃度測定工程と、
    　前記光酸に光を照射して前記マーカー複合物から前記マーカーを解離させるマーカー解離工程と、
    を含むことを特徴とする診断方法。     a marker complex concentration measuring step of disposing 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 formed by binding a marker to the anti-marker antibody;
    a marker dissociation step of dissociating the marker from the marker complex by irradiating the photoacid with light;
    A diagnostic method comprising the steps of:
  2. 　間隔をあけて前記マーカー複合物の濃度を繰り返し測定する、請求項１に記載の診断方法。 The diagnostic method of claim 1, wherein the concentration of the marker complex is repeatedly measured at intervals.
  3. 　前記基材が、振動体である、請求項１又は２に記載の診断方法。 The diagnostic method according to claim 1 or 2, wherein the substrate is a vibrating body.
  4. 　前記光が、レーザー光である、請求項１から３のいずれかに記載の診断方法。 The diagnostic method according to any one of claims 1 to 3, wherein the light is laser light.
  5. 　前記マーカーが、心疾患マーカーであり、
    　前記抗マーカー抗体が、抗心疾患マーカー抗体であり、
    　前記心疾患マーカーが、脳性ナトリウム利尿ペプチド（ＢＮＰ）及び脳性ナトリウム利尿ペプチド前駆体Ｎ端フラグメント（ＮＴ－ｐｒｏＢＮＰ）の少なくともいずれかである、請求項１から４のいずれかに記載の診断方法。     the marker is a cardiac disease marker,
    the anti-marker antibody is an anti-cardiac disease marker antibody,
    5. The method according to claim 1, wherein 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. 　前記光酸が、８－ヒドロキシピレン－１，３，６－トリスルホン酸（ＨＰＴＳ）である、請求項１から５のいずれかに記載の診断方法。 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. 　マーカーと特異的に結合する抗マーカー抗体及び光酸を表面に配した振動体と、
    　前記振動体の共振周波数を検出する検出部と、
    　前記光酸に光を照射する光照射部と、
    を有することを特徴とする診断装置。     A vibrator having an anti-marker antibody and a photoacid on its surface that specifically bind to the marker;
    A detection unit that detects a resonance frequency of the vibrator;
    A light irradiation unit that irradiates the photoacid with light;
    A diagnostic device comprising:
  8. 　前記振動体が振動子上に載置され、前記振動体と前記振動子が機械的に結合されておらず、かつ前記振動体がいずれの部材にも機械的に結合されていない、請求項７に記載の診断装置。 The diagnostic device according to claim 7, wherein the vibrating body is placed on a transducer, the vibrating body and the transducer are not mechanically coupled, and the vibrating body is not mechanically coupled to any member.
  9. 　基材上に、測定対象である抗原と特異的に結合する抗体及び光酸を配し、検査試料を供することにより前記抗原と前記抗体とが結合した抗原抗体複合物を形成する抗原抗体複合物形成工程と、
    　前記光酸に光を照射して前記抗原抗体複合物から前記抗原を解離させる抗原解離工程と、
    を含むことを特徴とする抗体再生方法。

          an antigen-antibody complex formation step of forming an antigen-antibody complex by providing an antibody that specifically binds to an antigen to be measured and a photoacid on a substrate and then providing a test sample to form an antigen-antibody complex in which the antigen and the antibody are bound to each other;
    an antigen dissociation step of irradiating the photoacid with light to dissociate the antigen from the antigen-antibody complex;
    A method for regenerating an antibody, comprising:
PCT/JP2023/040540 2022-11-11 2023-11-10 Diagnosis method, diagnosis device, and antibody regeneration method WO2024101447A1 (en)

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