WO2014080519A1 - Clinical test using nanocarbon - Google Patents
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- WO2014080519A1 WO2014080519A1 PCT/JP2012/080479 JP2012080479W WO2014080519A1 WO 2014080519 A1 WO2014080519 A1 WO 2014080519A1 JP 2012080479 W JP2012080479 W JP 2012080479W WO 2014080519 A1 WO2014080519 A1 WO 2014080519A1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/158—Carbon nanotubes
- C01B32/168—After-treatment
- C01B32/174—Derivatisation; Solubilisation; Dispersion in solvents
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/586—Liposomes, microcapsules or cells
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
- G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
Definitions
- the present invention relates to a clinical examination technique using a labeled probe that absorbs or emits light in the near infrared region.
- cell components such as blood cells (red blood cells, white blood cells, platelets, mesothelial cells, dropped cells) are removed by centrifugation or filter filtration.
- blood cells red blood cells, white blood cells, platelets, mesothelial cells, dropped cells
- the light of the probe molecules used for detecting the disease biomarker is disturbed and absorbed, and the disease detection sensitivity is lowered.
- cells such as blood cell components, and proteins contained in body cavity fluid and bile emit nonspecifically, which causes noise when detecting probe luminescence. Interference absorption and self-emission noise of probe luminescence due to such components hinders high-sensitivity detection of various disease biomarkers for the purpose of ultra-early diagnosis, and are very serious obstacles to their practical application.
- the present invention can detect only a detection target component with high sensitivity even if biological substances such as blood cells and protein components other than the detection target remain in a clinical test using biological samples such as blood, body cavity fluid, and bile as a sample.
- An object is to provide a possible detection method.
- the present inventors have found that nanocarbons such as single-walled carbon nanotubes (SWCNT) absorb near-infrared light, emit near-infrared light by photoexcitation, or have a peak of Raman scattered light in the near-infrared region. Focusing on the fact that the detection probe is labeled with nanocarbon, the near-infrared light emitted by the detected object is used for detection.
- the present invention has been completed by finding that even if biological substances such as blood cell components other than the detection target remain, only the detection target component can be detected with high sensitivity.
- near-infrared light has high biological permeability
- technological development has been attempted with the goal of increasing the sensitivity of in-vivo diagnostics such as in vivo imaging and axillary lymph node examination.
- in-vivo diagnostics such as in vivo imaging and axillary lymph node examination.
- a probe that efficiently emits light in the near-infrared wavelength region is required.
- SWCNT emits light at 700-2300 nm by photoexcitation at 450-1350 nm (FIG. 2 shows an emission map of a part of the wavelength range).
- This wavelength range includes a wavelength range (wavelength 600-1300 nm) that is difficult to be absorbed by a biological substance or has little self-emission.
- SWCNTs have an optical durability that is more than 10 times that of near-infrared dye molecules and near-infrared quantum dots, and can be chemically modified. In other words, SWCNT has an unparalleled superiority as a near-infrared light emitting probe, and is a powerful new material that enables the realization of next-generation clinical tests using near-infrared light.
- SWCNT luminescence was discovered in 2002, and in vivo SWCNT imaging has been attempted (Non-patent Document 3), but clinical studies that have been the most promising application have not been studied at all. .
- an apparatus that detects weak near-infrared light is also required.
- an InGaAs photodiode array connected to a silicon charge integrating amplifier is usually used.
- the photodiode has a large dark current and is allowed by a silicon IC. With capacitance, sufficient integration time cannot be secured.
- the circuit needs to be integrated into the IC, and sufficient measures have not been taken.
- the present inventors By connecting a lock-in amplifier array using silicon ICs to each element of the photodiode array, the present inventors made femtowatt level faint light detection possible even in a room temperature array (the above-mentioned WO2010 / 041756A1). .
- nanocarbon such as SWCNT that emits light with near infrared light is used as a labeling substrate to detect a target object.
- a blood biochemical specimen test using a fluorescent label in the near-infrared wavelength band is made possible.
- HbA1c quantification and blood glucose level measurement can be performed in parallel with the light absorption / transmission characteristics of glycated hemoglobin and glucose in red blood cells, preventing adult diseases. Contributes greatly to screening.
- ⁇ 1> A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method, which uses the unique optical properties of nanocarbon.
- ⁇ 2> The antigen-antibody reaction or saccharide according to ⁇ 1>, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by light excitation at a wavelength of 450-1350 nm are used as an absorption or emission label.
- SWCNT single-walled carbon nanotubes
- nanographene that emit light at a wavelength of 700-2300 nm by light excitation at a wavelength of 450-1350 nm are used as an absorption or emission label.
- ⁇ 3> Absorption or luminescence label containing nanocarbon.
- ⁇ 4> The light-absorbing or light-emitting label according to ⁇ 3>, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 700-2300 nm by photoexcitation at a wavelength of 450-1350 nm.
- a nanocarbon is bound to an antibody that recognizes a specific antigen or a lectin that recognizes a specific sugar chain, and an antigen-antibody reaction with the specific antigen or a reaction between the specific sugar chain and the lectin is nano
- the light-absorbing or light-emitting label according to ⁇ 3> or ⁇ 4> which is used for detection by measurement of carbon fluorescence, Raman light, and / or light absorption.
- Streptavidin or a related molecule of streptavidin is bonded to nanocarbon, and a biotinylated antibody bound to a specific antigen or a reaction product of biotinylated lectin bound to a specific sugar chain and streptavidin is converted to nanocarbon.
- the light-absorbing or light-emitting label according to ⁇ 3> or ⁇ 4> which is used for detection by measurement of fluorescence, Raman light, and / or light absorption.
- ⁇ 7> A binding reaction between an antibody bound to a specific antigen and an immunoglobulin binding protein formed by binding nanocarbon to an immunoglobulin binding protein by measuring fluorescence, Raman light, and / or light absorption of the nanocarbon.
- the light-absorbing or light-emitting label according to ⁇ 3> or ⁇ 4> which is used for detection.
- ⁇ 8> The light-absorbing or light-emitting label according to ⁇ 7>, wherein the immunoglobulin binding protein is protein G or protein A.
- An antigen-enriched sample is prepared by immobilizing the antigen to the carrier using antibodies against various antigens bound to the carrier, and the antigen on the carrier is labeled with a nanocarbon-labeled antibody for the sample. Or by labeling the sugar chain of the antigen on the carrier with the nanocarbon-labeled lectin, and then measuring the near-infrared light absorption, near-infrared light emission, or near-infrared Raman scattered light of the nanocarbon. Alternatively, a method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to ⁇ 1> or ⁇ 2>, wherein a sugar chain of an antigen is detected.
- ⁇ 11> The method for measuring an antigen-antibody reaction or the reaction between sugar chain lectins or the clinical test method according to ⁇ 10>, wherein the carrier is magnetic beads, Sepharose or Sephadex.
- ⁇ 12> Labeling of the antigen with a nanocarbon-labeled antibody recognizes the antigen and binds the antibody according to ⁇ 5> labeled with nanocarbon, or recognizes the antigen and biotin
- the labeled antibody is bound and labeled with the nanocarbon label according to ⁇ 6>, or the antibody that recognizes the antigen is bound and labeled with the nanocarbon label according to ⁇ 7> or ⁇ 8>
- the lectin according to ⁇ 5> wherein the labeling of the sugar chain of the antigen with the nanocarbon-labeled lectin recognizes the sugar chain of the antigen to the sugar chain of the antigen and is labeled with the nanocarbon.
- a disease marker antigen or sugar chain is labeled with a nanocarbon-labeled antibody or nanocarbon-labeled lectin, collected by electrophoresis, and then absorbed by near-infrared light, near-infrared light emission, or near-red
- ⁇ 14> A method for measuring an antigen-antibody reaction or reaction between sugar chain lectins using absorption or luminescence using the nanocarbon according to ⁇ 1>, ⁇ 2> or ⁇ 9> to ⁇ 13> in a single specimen, or It is characterized by detecting a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands by combining a clinical test method or a measurement method or clinical test method using other light absorption or luminescence.
- ⁇ 15> The immunoassay method or clinical test method according to ⁇ 14>, wherein the light absorption or emission wavelength band covers the ultraviolet, visible, near infrared, and / or infrared region.
- Characterizing a plurality of antigens and / or sugar chains using a ratio of light absorption or emission intensity from a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands in a single specimen ⁇ 14> or ⁇ 15> The method for measuring the antigen-antibody reaction or the reaction between sugar chain lectins or a clinical test method.
- ⁇ 17> In the measurement of the ultraviolet, visible, near-infrared, or infrared emission region of ⁇ 15> or ⁇ 16>, single or plural excitation lights are modulated, and absorption or emission is detected in synchronization with the modulation. Anti-antibody reaction or reaction between sugar chain lectins using a lock-in-amplifier photodetection method, or clinical laboratory equipment.
- a biological substance such as a blood cell component other than the detection target is present in a clinical test using a biological substance such as blood as a sample.
- a biological substance such as blood as a sample.
- only the detection target component can be detected with high sensitivity.
- the clinical test equipment can be greatly expanded from the conventional wavelength range of 400-500 nm to the near infrared range of 400-2300 nm, and the obtained information is further increased.
- the use of near-infrared light that is highly permeable to biological substances enables examination of biological samples derived from living organisms without separating them into serum or plasma. The information that was lost can be obtained.
- the biomarker-derived signal light absorption and non-specific noise due to the mixed blood cells and platelet components can be suppressed, making it possible to detect trace biomarkers, and extremely early such as cancer and Alzheimer's disease. It can greatly contribute to the development of medicine and medical care by enabling discovery and early treatment.
- the clinical testing system using near-infrared emission of nanocarbon of the present invention will bring near infrared light to clinical testing for the first time in the world, and will bring revolutionary progress to the diagnostics and diagnostic equipment industries.
- FIG. 14 is a comparison diagram of a whole blood fluorescence analysis spectrum by the apparatus of FIG.
- FIG. 13 is a schematic diagram of an automatic clinical laboratory test apparatus according to the present invention.
- Examples of the nanocarbon used in the present invention include single-walled carbon nanotubes (SWCNT), multi-walled carbon nanotubes, carbon nanohorns, nanographene, and other nanocarbons such as carbon nanorods, carbon nanocones, carbon nanocups, and fullerenes. It is done. As described above, SWCNT absorbs light having a wavelength of 700-1350 nm in the near infrared region, emits fluorescence at a wavelength of 700-2300 nm in the near infrared region, and, as shown in Example 1 described later, It has a Raman scattering peak near 1585 cm -1 in the region.
- SWCNT single-walled carbon nanotubes
- multi-walled carbon nanotubes carbon nanohorns
- nanographene and other nanocarbons such as carbon nanorods, carbon nanocones, carbon nanocups, and fullerenes. It is done.
- SWCNT absorbs light having a wavelength of 700-1350 nm in the near infrared
- multi-walled carbon nanotubes and carbonnanhorn absorb light in the near-infrared region and have a Raman scattering peak at 1580-1600 cm ⁇ 1 in the near-infrared region.
- Nanographene absorbs light in the near infrared region, has a Raman scattering peak at 1800-1900 cm ⁇ 1 in the near infrared region, and emits fluorescence in the near infrared region from visible light.
- the other nanocarbons described above also have the same optical characteristics as multi-walled carbon nanotubes. As described above, nanocarbon has optical characteristics such as light absorption, emission in the near-infrared region, or peak of Raman scattering light. In the present invention, these unique optical properties of nanocarbon are included.
- the detection target is detected by labeling a probe such as an antibody that specifically binds to the detection target with nanocarbon, binding the labeled probe to the detection target, and detecting light emission of the label.
- the labeling of the probe with nanocarbon is performed by, for example, coating nanocarbon adsorbed or chemically bonded using the later-described DSPE-PEG-NHS or the like with a PEG moiety, and directly or indirectly covering the probe such as an antibody. It can be performed by bonding.
- various antigens present in blood and body cavity fluids that are targeted for detection by the method of the present invention are associated with cancer. Examples include antigens and disease biomarkers.
- SWCNT-DSPE-PEG-IgG SWCNT-labeled antibody
- IgG antibody was labeled with SWCNT.
- SWCNT is dispersed with DSPE-PEG-NHS
- SWCNT coated with DSPE-PEG-NHS SWCNT-DSPE-PEG-NHS
- IgG is mixed with SWCNT-DSPE-PEG-NHS NHS.
- the supernatant contains SWCNTs that are individually separated and surface coated with DSPE-PEG-NHS.
- n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.
- Centrifugation supernatant containing individually separated SWCNTs is filtered using a centrifugal filtration device (Nanosep300K, manufactured by Pall) for 15 minutes at 12000 rpm, and the same centrifugation conditions twice using 100 ⁇ L phosphate buffer. Washed under. In this way, SWCNT-DSPE-PEG-NHS was obtained.
- SWCNT-DSPE-PEG-IgG structure SWCNT-DSPE-PEG-IgG Raman spectrum (FIG. 5), characteristic peaks of SWCNT, i.e., G-band of 1585 cm -1, D band of 1335cm -1 And 270 cm -1 breathing mode (RBM) appeared.
- the lower energy side of the G band corresponds to the Fano line that appears when SWCNT has a small diameter ( ⁇ 1 nm) and is metallic.
- the diameter of CNT estimated from the peak position of RBM was about 0.9 nm.
- the weak D band indicates that the amount of amorphous carbon impurities is very small, or the number of structural defects in the CNT is very small.
- SWCNT-DSPE-PEG-IgG SWCNT-labeled antibody
- IgG antibodies SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) IgG antibodies are known to specifically bind to protein G. Therefore, in order to confirm that SWCNT-DSPE-PEG-IgG retains the activity as an IgG antibody, SWCNT-DSPE-PEG-IgG was mixed with protein G magnetic beads by the following procedure. It was confirmed by spectroscopic measurement that SWCNT-DSPE-PEG-IgG was deposited on protein G beads.
- Example 3 Confirmation that coexistence of hemoglobin does not affect the antibody activity of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) To confirm that hemoglobin does not interfere with protein G of SWCNT-DSPE-PEG-IgG
- hemoglobin was added to the SWCNT-DSPE-PEG-IgG solution, and an immunoprecipitation experiment was performed. 2.5 ⁇ L of 5 wt% hemoglobin solution (trade name JCCRM622-1) was mixed with 42.5 ⁇ L of SWCNT-DSPE-PEG-IgG solution.
- Example 4 Confirmation that coexistence of hemoglobin does not affect the fluorescence spectrum measurement of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) Even if hemoglobin coexists with SWCNT-DSPE-PEG-IgG / protein G beads, In order to confirm that the measurement of the fluorescence spectrum is not hindered, the supernatant obtained at the end of the process of Example 2 (1) was collected, and then 20 ⁇ L of 5 wt% hemoglobin solution (trade name JCCRM622-1) was added. The optical spectrum was measured.
- the SWCNT fluorescence spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads Should not interfere with the measurement. This is because the fluorescence peak shown in the SWCNT spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads measured in the presence of hemoglobin (Fig. 10) is not present in the presence of hemoglobin. This was confirmed by the coincidence of the characteristics with those observed with IgG / protein G beads (FIG. 8).
- FIG. 5 Confirmation that SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) bound to protein G beads can be recovered from protein G beads
- SWCNT-DSPE-PEG-IgG SWCNT-labeled antibody bound to protein G beads
- one of the methods for detecting antigens in samples using labeled antibodies And a method of detecting the label by capturing the captured antigen with an antibody immobilized on protein G beads, labeling the captured antigen with a labeled antibody, and the like.
- the antigen is usually quantified by eluting the antigen-antibody complex produced on the protein G beads from the protein G beads and analyzing the binding amount of the label in a solution system.
- SWCNT-labeled antibodies can be used in such detection methods, specifically, SWCNT-DSPE-PEG-IgG bound to protein G beads can be easily eluted from protein G beads.
- SWCNT-DSPE-PEG-IgG bound to protein G beads is pulled away from protein G beads, and SWCNT-DSPE-PEG-IgG is put into solution. After dispersion, the fluorescence spectrum was measured. Separation of SWCNT-DSPE-PEG-IgG from protein G beads was performed by adding 10 ⁇ L of SDS aqueous solution (5 wt%) and heating to boiling (10 minutes).
- the fluorescence spectrum of the obtained SWCNT-DSPE-PEG-IgG eluate is shown in FIG. From the peak intensity of the fluorescence spectrum shown in FIG. 11, the recovery rate of SWCNT-DSPE-PEG-IgG is estimated to be about 90%, which means that SWCNT labeling is suitable for antigen quantification by immunoprecipitation as described above. Was confirmed.
- the SWCNT-labeled antibody retains the activity as an antibody and retains the fluorescence emission property in the near infrared region, and is interfered by blood components such as hemoglobin. It is shown that the antibody can bind to the target component to which the antibody specifically binds, and that the fluorescence can be detected. Further, the antigen can be quantified by immunoprecipitation using a carrier such as protein G beads. It was shown to be suitable for.
- Example 6 Detection of antigen-antibody reaction by near-infrared emission using SWCNT 1
- antigen-antibody reaction is performed using ovalbumin (OVA) as a model antigen, and the antigen-antibody reaction is detected by near-infrared emission.
- OVA ovalbumin
- a monoclonal antibody against a model antigen is prepared and immobilized on an ELISA plate. After reacting the sample solution containing the model antigen on a solid-phased plate at room temperature for 2 hours, discard the supernatant and wash well with phosphate buffer or Tris buffer.
- a polyclonal antibody probe against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours.
- the plate is washed thoroughly with phosphate buffer or Tris buffer to remove unreacted SWCNT-labeled antibody, and detection of SWCNT-derived near-infrared fluorescence on the plate detects antigen-antibody reaction. .
- 2) In addition to a system using an antibody immobilized on an ELISA plate, it is also possible to bind an antibody to a protein G bead and use it in batch processing. When protein G beads are used, the antibody can be bound to the beads through binding between protein G bound to the beads and the Fc part of the antibody.
- the antibody-immobilized beads are reacted in a sample solution containing a model antigen at room temperature for 2 hours, and then the supernatant is discarded and washed thoroughly with phosphate buffer or Tris buffer.
- a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours.
- SWCNT-labeled antibody against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours.
- the preparation of the SWCNT-labeled antibody used in these antigen-antibody reactions can also be performed by the following procedure.
- (1) Binding of DSPE-PEG with antibody i) Dissolve 125 ⁇ g of antibody in 6 ⁇ L of 50 mM phosphate buffer (pH 7.2), add 5 ⁇ g of DSPE-PEG of the following [Chemical Formula 2] and stir.
- n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.
- WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) catalyst is added and stirred with a stirrer at 4 ° C. for 10 hours. Filter through a filter, and collect the reactant remaining on the filter by dissolving it in a buffer. Or (Ii) 125 ⁇ g of antibody is dissolved in 2.5 ⁇ L of NaHCO 3 buffer.
- PEG-DSPE-NHS of [Chemical Formula 1] below is dissolved in 5 ⁇ g of pure water and added to the antibody solution.
- n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.
- SDBS sodium dodecylbenzenesulfonate
- the detailed procedure of the antigen-antibody reaction is as follows. Reaction system measurement on ELISA plate Using 50 ⁇ L of anti-OVA antibody solution adjusted to 20 ⁇ g / mL with PBS or 100 mM bicarbonate / carbonate coating buffer, coat the plate for 2 hours at room temperature. After thoroughly washing with PBS, 200 ⁇ L of 5% BSA solution dissolved in PBS is put into a well and blocked for 30 minutes at room temperature. After thoroughly washing with PBS, add the sample, react at room temperature for 2 hours, and then wash thoroughly with PBS. Next, the SWCNT-antibody probe diluted to 20 ⁇ g / mL with PBS is reacted at room temperature for 2 hours, and then washed thoroughly with PBS.
- detection of luminescence from the labeled probe is performed as follows.
- magnetic beads-antigen-SWCNT-DSPE-PEG-antibody with a detection wavelength of 660 nm, light emission at a wavelength of about 1200 nm can be observed from SWCNT.
- Its near-infrared emission spectrum is measured by a spectroscopic system equipped with an InGaAs detector.
- FIG. 12 shows a high-sensitivity infrared light detection array module in which a lock-in amplifier and an AD converter are connected to each cell of an InGaAs near-infrared phototransistor array having an internal amplification function. Magnetic beads-antigen-SWCNT-DSPE-PEG- The example used for the detection of an antibody is shown. In this embodiment, since the influence of dark current can be suppressed, faint watt level faint light can be detected without using a cryogen.
- Example 7 Preparation of a device for detection of modified isomers by three wavelengths
- the degree of posttranslational modification of proteins present in serum is measured to detect and evaluate the presence and progression of disease, and treatment policy Get important information to determine.
- Examples include 1) quantitative detection of glycated proteins represented by glycated hemoglobin, and 2) modification with characteristic sugar chain structures that appear in relation to diseases such as fetal cancer antigen (AFP) and MUC1. Quantitative detection of the glycoprotein produced can be mentioned.
- Glycated protein is a substance resulting from the non-enzymatic glycation reaction of glucose, a) intracellular proteins such as hemoglobin A and lens crystallin, b) plasma proteins such as albumin, apoprotein, transferrin, haptoglobin, c) Examples thereof include enzymes such as cathepsins and pancreatic riboases, and d) glycated products of membrane proteins such as vascular endothelium and erythrocyte membrane proteins. Detection of glycated hemoglobin and glycated albumin is effective for detection evaluation and diagnosis of impaired glucose tolerance and diabetes, as well as cirrhosis of the liver.
- AFP-L3 test using Lens culimaris agglutinin-A (LCA) to detect fucosylation of AFP is a highly accurate hepatocyte
- LCA Lens culimaris agglutinin-A
- WFA Wisteria floribunda agglutinin
- D) Aspergillus Oryzae I-fucose specific lectin (AOL) and Ricinus Communis Agglutinin (RCA-I) in ⁇ -1 acid glycoprotein Fibrosis occurring in the liver can be evaluated from the binding rate, and e) detection of sialylated sugar chain MUC1 in blood is an index for detecting interstitial pneumonia.
- AOL Aspergillus Oryzae I-fucose specific lectin
- RCA-I Ricinus Communis Agglutinin
- glycated albumin According to the protocol of Dynabeads Protein G, 200 ⁇ L of anti-human albumin antibody adjusted to 5 ⁇ g / mL with PBS is reacted with 1.5 mg of Dynabeads Protein G for 10 minutes to prepare antibody-bound Dynabeads. Next, after reacting with 500 ⁇ L of a blood sample solution containing glycated albumin at room temperature for 2 hours, the antigen is recovered with Dynabeads to which the antibody is bound. After washing well, react with 100 ⁇ g of SWCNT-antibody human albumin antibody at room temperature for 2 hours, and then wash well with PBS.
- SWCNT-antibody bound to Dynabeads Quantitatively measure the near-infrared emission of SWCNT derived from human albumin antibody to quantify the amount of albumin in serum.
- the ratio of glycated albumin to albumin is calculated using light absorption characteristic of albumin and glycated albumin captured at 1000 nm to 1400 nm.
- ⁇ -1 acid glycoprotein antibody to which ⁇ -1 By collecting acid glycoproteins and reacting them in advance with SWCNT-added streptavidin with different excitation wavelengths and biotinylated AOL, biotinylated MAL-II, and biotinylated RCA-I at the same time, Three modified isomers can be quantitatively evaluated.
- SWCNT separated into three types having different emission wavelengths and using three types of SWCNT-antibodies obtained by adding specific antibodies to the three types of antigens to be detected, It is possible to determine the abundance in the specimen for various types of proteins.
- Anti-human ⁇ -1 acid glycoprotein antibody, anti-M2BP antibody, and anti-MUC1 antibody-bound Dynabeads collect the antigens against them, and the detection antibodies for detecting the recovered antigens are labeled with SWCNTs with different excitation wavelengths. By using it, the three antigen amounts can be quantitatively evaluated at the same time.
- FIG. 13 is a schematic cross-sectional view of a whole blood spectroscopic fluorescence apparatus equipped with a small near infrared spectrometer.
- the whole blood sample to which the SWCNT infrared fluorescent label is added is intermittently irradiated with near infrared light by a light emitting diode, the SWCNT emits fluorescence in synchronization with the light emitting diode light in the infrared wavelength band.
- the fluorescent light is condensed while removing the wavelength component of the illumination light with a low-pass filter and spectrally splitting into the near-infrared light detection array with the concave grating.
- three systems of spectroscopic measurement systems having different spectral wavelength bands are arranged, and fluorescence and absorption in three wavelength bands are measured simultaneously in the same sample.
- FIG. 14 compares the spectrum measured by the apparatus shown in FIG. 13 with the spectrum in the conventional method.
- the emission wavelength of the fluorescent label is at most 800 nm
- the self-emission spectrum of hemoglobin will cover the fluorescence due to the label as background light, so first of all, using a large pretreatment device such as a centrifuge Need to remove red blood cells.
- the fluorescence detection device can detect fluorescence at a wavelength longer than the emission band of hemoglobin with high sensitivity, so that the sample can be introduced into the analysis device as whole blood, which is complicated. There is a feature that pre-treatment is unnecessary, that there is no influence of hemolysis or the like at the time of centrifugation or filtering, and as a result, sensitivity is improved by several tens of times.
- FIG. 15 shows an example in which three sets of light emitting diodes, filters, and a single light receiving element are arranged instead of a small spectroscope. Since this device is simple and inexpensive, it can be used for clinical examinations in clinics and private hospitals.
- FIG. 16 shows a schematic diagram of an apparatus for automatically processing the sample processing such as sample dispensing, antibody addition, and magnetic bead washing described in the present invention, and detecting weak fluorescence at near infrared wavelengths. Background light caused by hemoglobin can be removed by a relatively simple process.
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Abstract
Provided is a detection method whereby, in a clinical test using as a sample a material of a biological origin such as blood, an object component to be detected alone can be detected at a high sensitivity even though a biological material such as a blood cell component other than the object component remains. Nanocarbon such as a single-walled carbon nanotube (SWCNT) absorbs near infrared rays and emits the near infrared rays upon light excitation, or has a Raman scattering light peak in the near infrared region. Owing to these optical properties in the near infrared region of nanocarbon, an object component to be detected alone can be detected at a high sensitivity even though a biological material such as a blood cell component other than the object component remains, by, for example, labeling a detection probe with nanocarbon and detecting the object with the use of near infrared rays emitted by the object.
Description
本発明は、近赤外領域で吸光または発光する標識プローブを用いる臨床検査技術に関する。
The present invention relates to a clinical examination technique using a labeled probe that absorbs or emits light in the near infrared region.
昨今の技術開発を背景として、各種疾患バイオマーカー探索が進められており、「がん」等の疾患の早期発見に役立つ可能性が高い候補分子が相次いで報告されている。産業技術総合研究所の糖鎖医工学研究センターでも、本発明者らの一人である池原らが糖鎖分析技術のアドバンテージを最大限に生かして、疾患の発生と進展に伴って出現する未完成品糖タンパク質(図1)(＝糖鎖修飾異性体)を疾患バイオマーカーとする研究を進めて来た。未完成品の糖タンパク質などの疾病マーカーを検出することで、がんや生活習慣病を早期発見できる。
最近、池原らは、肝炎に伴って生じる線維化を測定できる未完成糖鎖バイオマーカーを発見し、侵襲性の高い生検に代わって血液検査での検出評価を可能とした。さらに、胃がんの腹腔内再発や、胆管がん、卵巣がんのように発見が極めて難しい腫瘍の存在や進展についても、評価できる未完成糖鎖バイオマーカーの発見に成功している。今後、同様のアプローチで開発を進めると、動脈硬化やアルツハイマー病の検査診断にも展開可能であると予想される。しかし、血液・体腔液・胆汁などに存在する疾患マーカー分子を検出することで、これらの疾病の超早期診断を達成するためには、現在の臨床検査で一般に利用される検出技術では、十分な感度を得ることは難しい。 Searches for biomarkers of various diseases are being promoted against the background of recent technological development, and candidate molecules that are likely to be useful for early detection of diseases such as “cancer” have been reported one after another. Ikehara et al., One of the inventors of the present invention, at the Glycotechnology Research Center of the National Institute of Advanced Industrial Science and Technology makes the most of the advantages of glycan analysis technology, and it appears that it appears as the disease develops and progresses We have been conducting research using the glycoprotein (Fig. 1) (= glycosylated isomer) as a disease biomarker. By detecting disease markers such as unfinished glycoproteins, cancer and lifestyle-related diseases can be detected early.
Recently, Ikehara et al. Discovered an incomplete glycan biomarker that can measure fibrosis caused by hepatitis and enabled detection evaluation in blood tests instead of highly invasive biopsy. Furthermore, we have succeeded in discovering unfinished glycan biomarkers that can be evaluated for the recurrence of gastric cancer in the abdominal cavity and the presence and progression of extremely difficult tumors such as bile duct cancer and ovarian cancer. In the future, if development is carried out using the same approach, it is expected to be applicable to laboratory diagnosis of arteriosclerosis and Alzheimer's disease. However, detection techniques commonly used in current clinical tests are not sufficient to achieve ultra-early diagnosis of these diseases by detecting disease marker molecules present in blood, body cavity fluid, bile, etc. It is difficult to obtain sensitivity.
最近、池原らは、肝炎に伴って生じる線維化を測定できる未完成糖鎖バイオマーカーを発見し、侵襲性の高い生検に代わって血液検査での検出評価を可能とした。さらに、胃がんの腹腔内再発や、胆管がん、卵巣がんのように発見が極めて難しい腫瘍の存在や進展についても、評価できる未完成糖鎖バイオマーカーの発見に成功している。今後、同様のアプローチで開発を進めると、動脈硬化やアルツハイマー病の検査診断にも展開可能であると予想される。しかし、血液・体腔液・胆汁などに存在する疾患マーカー分子を検出することで、これらの疾病の超早期診断を達成するためには、現在の臨床検査で一般に利用される検出技術では、十分な感度を得ることは難しい。 Searches for biomarkers of various diseases are being promoted against the background of recent technological development, and candidate molecules that are likely to be useful for early detection of diseases such as “cancer” have been reported one after another. Ikehara et al., One of the inventors of the present invention, at the Glycotechnology Research Center of the National Institute of Advanced Industrial Science and Technology makes the most of the advantages of glycan analysis technology, and it appears that it appears as the disease develops and progresses We have been conducting research using the glycoprotein (Fig. 1) (= glycosylated isomer) as a disease biomarker. By detecting disease markers such as unfinished glycoproteins, cancer and lifestyle-related diseases can be detected early.
Recently, Ikehara et al. Discovered an incomplete glycan biomarker that can measure fibrosis caused by hepatitis and enabled detection evaluation in blood tests instead of highly invasive biopsy. Furthermore, we have succeeded in discovering unfinished glycan biomarkers that can be evaluated for the recurrence of gastric cancer in the abdominal cavity and the presence and progression of extremely difficult tumors such as bile duct cancer and ovarian cancer. In the future, if development is carried out using the same approach, it is expected to be applicable to laboratory diagnosis of arteriosclerosis and Alzheimer's disease. However, detection techniques commonly used in current clinical tests are not sufficient to achieve ultra-early diagnosis of these diseases by detecting disease marker molecules present in blood, body cavity fluid, bile, etc. It is difficult to obtain sensitivity.
一般に血液・体腔液・胆汁等の生体由来物をサンプルとする臨床検査では、血球等の細胞成分(赤血球や白血球、血小板、中皮、脱落細胞)を遠心分離やフィルター濾過により除去する。しかし、いずれの操作でも除ききれないので、これら生体物質が少なからず混入する事となり、疾患バイオマーカー検出のために使うプローブ分子の光を妨害吸収してしまい疾病検出感度を下げてしまう。同時に、血球成分等の細胞や、体腔液や胆汁に含まれるタンパクが非特異的に発光し、プローブ発光検出の際にノイズの原因ともなる。こうした成分によるプローブ発光の妨害吸収や自家発光ノイズは、超早期診断を目的としたさまざまな疾患バイオマーカーの高感度検出を邪魔し、その実用化において非常に大きな障害となっている。
Generally, in a clinical test using biological substances such as blood, body cavity fluid, and bile as samples, cell components such as blood cells (red blood cells, white blood cells, platelets, mesothelial cells, dropped cells) are removed by centrifugation or filter filtration. However, since it cannot be removed by any operation, not a few of these biological substances are mixed, and the light of the probe molecules used for detecting the disease biomarker is disturbed and absorbed, and the disease detection sensitivity is lowered. At the same time, cells such as blood cell components, and proteins contained in body cavity fluid and bile emit nonspecifically, which causes noise when detecting probe luminescence. Interference absorption and self-emission noise of probe luminescence due to such components hinders high-sensitivity detection of various disease biomarkers for the purpose of ultra-early diagnosis, and are very serious obstacles to their practical application.
本発明は、血液・体腔液・胆汁などの生体由来物をサンプルとする臨床検査において、検出対象以外の血球やタンパク成分などの生体物質が残存しても、検出対象成分のみを高感度で検出可能な、検出方法を提供することを課題とする。
The present invention can detect only a detection target component with high sensitivity even if biological substances such as blood cells and protein components other than the detection target remain in a clinical test using biological samples such as blood, body cavity fluid, and bile as a sample. An object is to provide a possible detection method.
本発明者らは、単層カーボンナノチューブ(SWCNT)などのナノカーボンが近赤外光を吸収し、光励起により近赤外光を発光し、あるいは、近赤外領域にラマン散乱光のピークを有することに着目し、検出プローブをナノカーボンにより標識することにより、検出された対象が発する近赤外光を検出に利用するなど、ナノカーボンの有する近赤外領域の光学的特性を利用することで、検出対象以外の血球成分などの生体物質が残存しても、検出対象成分のみを高感度で検出することが可能であることを見出し、本発明を完成した。
The present inventors have found that nanocarbons such as single-walled carbon nanotubes (SWCNT) absorb near-infrared light, emit near-infrared light by photoexcitation, or have a peak of Raman scattered light in the near-infrared region. Focusing on the fact that the detection probe is labeled with nanocarbon, the near-infrared light emitted by the detected object is used for detection. The present invention has been completed by finding that even if biological substances such as blood cell components other than the detection target remain, only the detection target component can be detected with high sensitivity.
近赤外光は生体透過性が高いため、in vivoイメージングや腋窩リンパ節検査などの体内診断の高感度化をゴールに、技術開発が試みられている。血液や体腔液を対象として検索する臨床検査においても、近赤外光が利用可能となれば、高感度化と多種類の疾病マーカーの同時検出が可能となるが、実現していない。実現のためには、近赤外波長領域で効率良く発光するプローブが必要である。
SWCNTは、450－1350nmの光励起により700－2300nmで発光する(図２にその一部の波長範囲の発光マップを示す)。この波長範囲は、生体物質によって吸収されにくいあるいは自家発光の少ない波長範囲(波長600-1300nm)をふくむ
。SWCNTは、近赤外色素分子や近赤外量子ドットに比べて、10倍以上の光学的耐久性があり、化学修飾も可能である。つまり、SWCNTは近赤外発光プローブとしての優位性は比類なく、近赤外光を利用した次世代臨床検査の実現を可能にする強力な新規材料である。SWCNTの発光は2002年に発見され、これまで、生体内のSWCNTイメージングなども試みられた(非特許文献３)ものの、そのもっとも有力な応用である臨床検査への検討はこれまで全くなされていない。 Since near-infrared light has high biological permeability, technological development has been attempted with the goal of increasing the sensitivity of in-vivo diagnostics such as in vivo imaging and axillary lymph node examination. Even in clinical examinations that search for blood and body cavity fluids, if near-infrared light can be used, high sensitivity and simultaneous detection of many types of disease markers are possible, but this has not been realized. For realization, a probe that efficiently emits light in the near-infrared wavelength region is required.
SWCNT emits light at 700-2300 nm by photoexcitation at 450-1350 nm (FIG. 2 shows an emission map of a part of the wavelength range). This wavelength range includes a wavelength range (wavelength 600-1300 nm) that is difficult to be absorbed by a biological substance or has little self-emission. SWCNTs have an optical durability that is more than 10 times that of near-infrared dye molecules and near-infrared quantum dots, and can be chemically modified. In other words, SWCNT has an unparalleled superiority as a near-infrared light emitting probe, and is a powerful new material that enables the realization of next-generation clinical tests using near-infrared light. SWCNT luminescence was discovered in 2002, and in vivo SWCNT imaging has been attempted (Non-patent Document 3), but clinical studies that have been the most promising application have not been studied at all. .
SWCNTは、450－1350nmの光励起により700－2300nmで発光する(図２にその一部の波長範囲の発光マップを示す)。この波長範囲は、生体物質によって吸収されにくいあるいは自家発光の少ない波長範囲(波長600-1300nm)をふくむ
。SWCNTは、近赤外色素分子や近赤外量子ドットに比べて、10倍以上の光学的耐久性があり、化学修飾も可能である。つまり、SWCNTは近赤外発光プローブとしての優位性は比類なく、近赤外光を利用した次世代臨床検査の実現を可能にする強力な新規材料である。SWCNTの発光は2002年に発見され、これまで、生体内のSWCNTイメージングなども試みられた(非特許文献３)ものの、そのもっとも有力な応用である臨床検査への検討はこれまで全くなされていない。 Since near-infrared light has high biological permeability, technological development has been attempted with the goal of increasing the sensitivity of in-vivo diagnostics such as in vivo imaging and axillary lymph node examination. Even in clinical examinations that search for blood and body cavity fluids, if near-infrared light can be used, high sensitivity and simultaneous detection of many types of disease markers are possible, but this has not been realized. For realization, a probe that efficiently emits light in the near-infrared wavelength region is required.
SWCNT emits light at 700-2300 nm by photoexcitation at 450-1350 nm (FIG. 2 shows an emission map of a part of the wavelength range). This wavelength range includes a wavelength range (wavelength 600-1300 nm) that is difficult to be absorbed by a biological substance or has little self-emission. SWCNTs have an optical durability that is more than 10 times that of near-infrared dye molecules and near-infrared quantum dots, and can be chemically modified. In other words, SWCNT has an unparalleled superiority as a near-infrared light emitting probe, and is a powerful new material that enables the realization of next-generation clinical tests using near-infrared light. SWCNT luminescence was discovered in 2002, and in vivo SWCNT imaging has been attempted (Non-patent Document 3), but clinical studies that have been the most promising application have not been studied at all. .
近赤外光の発光により、微量の検出対象を生体に由来する夾雑物が存在するサンプル中で検出するためには、また、微弱な近赤外光を検出する装置が必要とされる。
波長1000～2000nmの近赤外光の検出には、通常シリコン電荷積分アンプに接続されたInGaAsフォトダイオードアレイを使用するが、室温付近においては、フォトダイオードの暗電流が大きくシリコンICで許容されるキャパシタンスでは、十分な積分時間が確保できない。この点は、フォトダイオードを液体窒素冷却することで解決可能であるが、価格や操作性の問題を鑑みると、臨床検査室での実用化は難しい。これに対して本発明者らの一人である小倉は、先に、極微弱光の計測が室温付近で可能となるよう、表面電流ブロック層を設けたヘテロバイポーラフォトトランジスタを開発した(WO2010/041756A1)。
この素子の特色として、内部増幅作用が大きいので(電流増幅率β～数千)、外部電気回路によるノイズの影響を受けにくいこと、ベース領域に光励起キャリアの蓄積効果を持つため、電荷積分機能を内蔵していること、可視から赤外まで広い波長範囲に感度を有することなどが挙げられる。
更に、蛍光や透過スペクトラム計測の場合、光源を変調し、検出系を同期検波する(ロックイン 検波)ことにより暗電流成分を除去することが可能であるが、アレイ検出器に対しては、電子回路をIC化する必要があり、十分な対策が取られていない。
本発明者らは、フォトダイオードアレイのそれぞれの素子に、シリコンICによるロックインアンプアレイを接続することにより、室温アレイにおいてもフェムトワットレベルの微弱光検出を可能にした(上述のWO2010/041756A1)。 In order to detect a very small amount of detection target in a sample in which impurities derived from a living body exist by emitting near-infrared light, an apparatus that detects weak near-infrared light is also required.
For detection of near-infrared light with a wavelength of 1000 to 2000 nm, an InGaAs photodiode array connected to a silicon charge integrating amplifier is usually used. However, near room temperature, the photodiode has a large dark current and is allowed by a silicon IC. With capacitance, sufficient integration time cannot be secured. Although this point can be solved by cooling the photodiode with liquid nitrogen, it is difficult to put it into practical use in a clinical laboratory in view of problems of price and operability. On the other hand, Ogura, one of the inventors of the present invention, previously developed a heterobipolar phototransistor provided with a surface current blocking layer so that measurement of extremely weak light is possible near room temperature (WO2010 / 041756A1). ).
As a feature of this element, since the internal amplification function is large (current amplification factor β to several thousand), it is difficult to be affected by noise from the external electric circuit, and it has the effect of accumulating photoexcited carriers in the base region. It is built-in, and has sensitivity in a wide wavelength range from visible to infrared.
Furthermore, in the case of fluorescence or transmission spectrum measurement, the dark current component can be removed by modulating the light source and synchronously detecting the detection system (lock-in detection). The circuit needs to be integrated into the IC, and sufficient measures have not been taken.
By connecting a lock-in amplifier array using silicon ICs to each element of the photodiode array, the present inventors made femtowatt level faint light detection possible even in a room temperature array (the above-mentioned WO2010 / 041756A1). .
波長1000～2000nmの近赤外光の検出には、通常シリコン電荷積分アンプに接続されたInGaAsフォトダイオードアレイを使用するが、室温付近においては、フォトダイオードの暗電流が大きくシリコンICで許容されるキャパシタンスでは、十分な積分時間が確保できない。この点は、フォトダイオードを液体窒素冷却することで解決可能であるが、価格や操作性の問題を鑑みると、臨床検査室での実用化は難しい。これに対して本発明者らの一人である小倉は、先に、極微弱光の計測が室温付近で可能となるよう、表面電流ブロック層を設けたヘテロバイポーラフォトトランジスタを開発した(WO2010/041756A1)。
この素子の特色として、内部増幅作用が大きいので(電流増幅率β～数千)、外部電気回路によるノイズの影響を受けにくいこと、ベース領域に光励起キャリアの蓄積効果を持つため、電荷積分機能を内蔵していること、可視から赤外まで広い波長範囲に感度を有することなどが挙げられる。
更に、蛍光や透過スペクトラム計測の場合、光源を変調し、検出系を同期検波する(ロックイン 検波)ことにより暗電流成分を除去することが可能であるが、アレイ検出器に対しては、電子回路をIC化する必要があり、十分な対策が取られていない。
本発明者らは、フォトダイオードアレイのそれぞれの素子に、シリコンICによるロックインアンプアレイを接続することにより、室温アレイにおいてもフェムトワットレベルの微弱光検出を可能にした(上述のWO2010/041756A1)。 In order to detect a very small amount of detection target in a sample in which impurities derived from a living body exist by emitting near-infrared light, an apparatus that detects weak near-infrared light is also required.
For detection of near-infrared light with a wavelength of 1000 to 2000 nm, an InGaAs photodiode array connected to a silicon charge integrating amplifier is usually used. However, near room temperature, the photodiode has a large dark current and is allowed by a silicon IC. With capacitance, sufficient integration time cannot be secured. Although this point can be solved by cooling the photodiode with liquid nitrogen, it is difficult to put it into practical use in a clinical laboratory in view of problems of price and operability. On the other hand, Ogura, one of the inventors of the present invention, previously developed a heterobipolar phototransistor provided with a surface current blocking layer so that measurement of extremely weak light is possible near room temperature (WO2010 / 041756A1). ).
As a feature of this element, since the internal amplification function is large (current amplification factor β to several thousand), it is difficult to be affected by noise from the external electric circuit, and it has the effect of accumulating photoexcited carriers in the base region. It is built-in, and has sensitivity in a wide wavelength range from visible to infrared.
Furthermore, in the case of fluorescence or transmission spectrum measurement, the dark current component can be removed by modulating the light source and synchronously detecting the detection system (lock-in detection). The circuit needs to be integrated into the IC, and sufficient measures have not been taken.
By connecting a lock-in amplifier array using silicon ICs to each element of the photodiode array, the present inventors made femtowatt level faint light detection possible even in a room temperature array (the above-mentioned WO2010 / 041756A1). .
すなわち、本発明は、近赤外光を高感度に検出できるシステム機器の基盤技術の存在のもと、近赤外光で発光するSWCNTなどのナノカーボンを標識基材として、目的物の検出に必要な抗体などのプローブを修飾することにより、従来の可視波長帯域に加えて、近赤外波長帯域における蛍光標識を用いた血液生化学の検体検査を可能とするものである。更に、検出波長帯域を２μmまで拡張することにより、赤血球中の糖化ヘモグロビンやグルコースの光吸収・透過特性による、HbA1c定量や血糖値計測も並行して行うことが可能となり、成人病の予防的な検診に大きく貢献する。
That is, in the present invention, in the presence of the fundamental technology of system equipment that can detect near infrared light with high sensitivity, nanocarbon such as SWCNT that emits light with near infrared light is used as a labeling substrate to detect a target object. By modifying necessary probes such as antibodies, in addition to the conventional visible wavelength band, a blood biochemical specimen test using a fluorescent label in the near-infrared wavelength band is made possible. Furthermore, by extending the detection wavelength band to 2 μm, HbA1c quantification and blood glucose level measurement can be performed in parallel with the light absorption / transmission characteristics of glycated hemoglobin and glucose in red blood cells, preventing adult diseases. Contributes greatly to screening.
本出願は、具体的には、以下の発明を提供する。
〈１〉ナノカーボンの特異な光学的性質を利用することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈２〉波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンを吸光または発光標識として用いることを特徴とする、〈１〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈３〉ナノカーボンを含有する吸光または発光標識。
〈４〉ナノカーボンが、波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンであることを特徴とする、〈３〉に記載の吸光または発光標識。
〈５〉特定の抗原を認識する抗体または特定の糖鎖を認識するレクチンにナノカーボンを結合させて成り、当該特定の抗原との抗原抗体反応または当該特定の糖鎖とレクチンとの反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈６〉ストレプトアビジンまたはストレプトアビジンの関連分子にナノカーボンを結合させて成り、特定の抗原に結合したビオチン化抗体または特定の糖鎖と結合したビオチン化レクチンとストレプトアビジンの反応物を、ナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈７〉イムノグロブリン結合タンパクにナノカーボンを結合させて成り、特定の抗原に結合した抗体と当該イムノグロブリン結合タンパクの結合反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈８〉イムノグロブリン結合タンパクがプロテインＧまたはプロテインＡであることを特徴とする、〈７〉に記載の吸光または発光標識。
〈９〉特定の抗原または特定の糖鎖が、正常もしくは病的な状態になった際、血液や体腔液中に存在する各種の抗原または糖鎖であることを特徴とする、〈５〉～〈８〉のいずれかに記載の吸光または発光標識。
〈１０〉担体に結合させた各種抗原に対する抗体をもちいて抗原を担体に固定化することにより、抗原がエンリッチ化したサンプルを作成し、当該サンプルについて、担体上の抗原をナノカーボン標識抗体により標識した後、または、担体上の抗原の有する糖鎖をナノカーボン標識レクチンにより標識した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより抗原または抗原の有する糖鎖を検出することを特徴とする、〈１〉または〈２〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１１〉担体が磁気ビーズ、セファロースまたはセファデックスであることを特徴とする、〈１０〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１２〉抗原のナノカーボン標識抗体による標識が、当該抗原に、当該抗原を認識し、ナノカーボンで標識された〈５〉に記載の抗体を結合させるか、あるいは、当該抗原を認識し、ビオチン化された抗体を結合させ、〈６〉に記載のナノカーボン標識で標識するか、もしくは、当該抗原を認識する抗体を結合させ、〈７〉または〈８〉に記載のナノカーボン標識で標識することにより行われ、抗原の有する糖鎖のナノカーボン標識レクチンによる標識が、当該抗原の有する糖鎖に、当該抗原の有する糖鎖を認識し、ナノカーボンで標識された〈５〉に記載のレクチンを結合させるか、あるいは、当該抗原を認識し、ビオチン化されたレクチンを結合させ、〈６〉に記載のナノカーボン標識で標識することにより行われることを特徴とする、〈１０〉または〈１１〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１３〉疾病マーカーである抗原または糖鎖をナノカーボン標識抗体またはナノカーボン標識レクチンにより標識し、電気泳動により捕集した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより当該抗原または糖鎖を検出することを特徴とする、〈１〉または〈２〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１４〉単一検体において、〈１〉、〈２〉または〈９〉～〈１３〉に記載のナノカーボンを用いる吸光または発光を利用した抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法、あるいはこれと他の吸光または発光を利用した測定方法あるいは臨床検査方法とを組み合わせて、異なった吸光または発光波長帯にて複数の抗原および／または糖鎖を検出することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１５〉吸光または発光波長帯が、紫外、可視、近赤外および／または赤外領域に及ぶ、〈１４〉に記載の免疫測定方法あるいは臨床検査方法。
〈１６〉単一検体において、異なった吸光または発光波長帯における複数の抗原および／または糖鎖からの吸光または発光強度比を用いて、複数の抗原および／または糖鎖の定量を行うことを特徴とする、〈１４〉または〈１５〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１７〉〈１５〉または〈１６〉の紫外、可視、近赤外、赤外発光領域の計測において、単一あるいは複数の励起光を変調して、それに同期して吸光または発光を検出する、ロックインアンプ光検出法を用いた抗原抗体反応または糖鎖レクチン間の反応の測定あるいは臨床検査装置。 Specifically, the present application provides the following inventions.
<1> A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method, which uses the unique optical properties of nanocarbon.
<2> The antigen-antibody reaction or saccharide according to <1>, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by light excitation at a wavelength of 450-1350 nm are used as an absorption or emission label. A method for measuring the reaction between chain lectins or a clinical test method.
<3> Absorption or luminescence label containing nanocarbon.
<4> The light-absorbing or light-emitting label according to <3>, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 700-2300 nm by photoexcitation at a wavelength of 450-1350 nm.
<5> A nanocarbon is bound to an antibody that recognizes a specific antigen or a lectin that recognizes a specific sugar chain, and an antigen-antibody reaction with the specific antigen or a reaction between the specific sugar chain and the lectin is nano The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection by measurement of carbon fluorescence, Raman light, and / or light absorption.
<6> Streptavidin or a related molecule of streptavidin is bonded to nanocarbon, and a biotinylated antibody bound to a specific antigen or a reaction product of biotinylated lectin bound to a specific sugar chain and streptavidin is converted to nanocarbon. The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection by measurement of fluorescence, Raman light, and / or light absorption.
<7> A binding reaction between an antibody bound to a specific antigen and an immunoglobulin binding protein formed by binding nanocarbon to an immunoglobulin binding protein by measuring fluorescence, Raman light, and / or light absorption of the nanocarbon. The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection.
<8> The light-absorbing or light-emitting label according to <7>, wherein the immunoglobulin binding protein is protein G or protein A.
<9> Various antigens or sugar chains that are present in blood or body cavity fluid when a specific antigen or a specific sugar chain becomes normal or pathological, <5> to <8> The light-absorbing or light-emitting label according to any one of the above.
<10> An antigen-enriched sample is prepared by immobilizing the antigen to the carrier using antibodies against various antigens bound to the carrier, and the antigen on the carrier is labeled with a nanocarbon-labeled antibody for the sample. Or by labeling the sugar chain of the antigen on the carrier with the nanocarbon-labeled lectin, and then measuring the near-infrared light absorption, near-infrared light emission, or near-infrared Raman scattered light of the nanocarbon. Alternatively, a method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to <1> or <2>, wherein a sugar chain of an antigen is detected.
<11> The method for measuring an antigen-antibody reaction or the reaction between sugar chain lectins or the clinical test method according to <10>, wherein the carrier is magnetic beads, Sepharose or Sephadex.
<12> Labeling of the antigen with a nanocarbon-labeled antibody recognizes the antigen and binds the antibody according to <5> labeled with nanocarbon, or recognizes the antigen and biotin The labeled antibody is bound and labeled with the nanocarbon label according to <6>, or the antibody that recognizes the antigen is bound and labeled with the nanocarbon label according to <7> or <8> The lectin according to <5>, wherein the labeling of the sugar chain of the antigen with the nanocarbon-labeled lectin recognizes the sugar chain of the antigen to the sugar chain of the antigen and is labeled with the nanocarbon. Or by recognizing the antigen, binding a biotinylated lectin, and labeling with the nanocarbon label according to <6>, 10> or <11> Measurement method or clinical test method of the reaction between the antigen-antibody reaction or a sugar chain lectin described.
<13> A disease marker antigen or sugar chain is labeled with a nanocarbon-labeled antibody or nanocarbon-labeled lectin, collected by electrophoresis, and then absorbed by near-infrared light, near-infrared light emission, or near-red The method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to <1> or <2>, wherein the antigen or sugar chain is detected by measuring external Raman scattered light .
<14> A method for measuring an antigen-antibody reaction or reaction between sugar chain lectins using absorption or luminescence using the nanocarbon according to <1>, <2> or <9> to <13> in a single specimen, or It is characterized by detecting a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands by combining a clinical test method or a measurement method or clinical test method using other light absorption or luminescence. A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method.
<15> The immunoassay method or clinical test method according to <14>, wherein the light absorption or emission wavelength band covers the ultraviolet, visible, near infrared, and / or infrared region.
<16> Characterizing a plurality of antigens and / or sugar chains using a ratio of light absorption or emission intensity from a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands in a single specimen <14> or <15> The method for measuring the antigen-antibody reaction or the reaction between sugar chain lectins or a clinical test method.
<17> In the measurement of the ultraviolet, visible, near-infrared, or infrared emission region of <15> or <16>, single or plural excitation lights are modulated, and absorption or emission is detected in synchronization with the modulation. Anti-antibody reaction or reaction between sugar chain lectins using a lock-in-amplifier photodetection method, or clinical laboratory equipment.
〈１〉ナノカーボンの特異な光学的性質を利用することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈２〉波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンを吸光または発光標識として用いることを特徴とする、〈１〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈３〉ナノカーボンを含有する吸光または発光標識。
〈４〉ナノカーボンが、波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンであることを特徴とする、〈３〉に記載の吸光または発光標識。
〈５〉特定の抗原を認識する抗体または特定の糖鎖を認識するレクチンにナノカーボンを結合させて成り、当該特定の抗原との抗原抗体反応または当該特定の糖鎖とレクチンとの反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈６〉ストレプトアビジンまたはストレプトアビジンの関連分子にナノカーボンを結合させて成り、特定の抗原に結合したビオチン化抗体または特定の糖鎖と結合したビオチン化レクチンとストレプトアビジンの反応物を、ナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈７〉イムノグロブリン結合タンパクにナノカーボンを結合させて成り、特定の抗原に結合した抗体と当該イムノグロブリン結合タンパクの結合反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、〈３〉または〈４〉に記載の吸光または発光標識。
〈８〉イムノグロブリン結合タンパクがプロテインＧまたはプロテインＡであることを特徴とする、〈７〉に記載の吸光または発光標識。
〈９〉特定の抗原または特定の糖鎖が、正常もしくは病的な状態になった際、血液や体腔液中に存在する各種の抗原または糖鎖であることを特徴とする、〈５〉～〈８〉のいずれかに記載の吸光または発光標識。
〈１０〉担体に結合させた各種抗原に対する抗体をもちいて抗原を担体に固定化することにより、抗原がエンリッチ化したサンプルを作成し、当該サンプルについて、担体上の抗原をナノカーボン標識抗体により標識した後、または、担体上の抗原の有する糖鎖をナノカーボン標識レクチンにより標識した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより抗原または抗原の有する糖鎖を検出することを特徴とする、〈１〉または〈２〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１１〉担体が磁気ビーズ、セファロースまたはセファデックスであることを特徴とする、〈１０〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１２〉抗原のナノカーボン標識抗体による標識が、当該抗原に、当該抗原を認識し、ナノカーボンで標識された〈５〉に記載の抗体を結合させるか、あるいは、当該抗原を認識し、ビオチン化された抗体を結合させ、〈６〉に記載のナノカーボン標識で標識するか、もしくは、当該抗原を認識する抗体を結合させ、〈７〉または〈８〉に記載のナノカーボン標識で標識することにより行われ、抗原の有する糖鎖のナノカーボン標識レクチンによる標識が、当該抗原の有する糖鎖に、当該抗原の有する糖鎖を認識し、ナノカーボンで標識された〈５〉に記載のレクチンを結合させるか、あるいは、当該抗原を認識し、ビオチン化されたレクチンを結合させ、〈６〉に記載のナノカーボン標識で標識することにより行われることを特徴とする、〈１０〉または〈１１〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１３〉疾病マーカーである抗原または糖鎖をナノカーボン標識抗体またはナノカーボン標識レクチンにより標識し、電気泳動により捕集した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより当該抗原または糖鎖を検出することを特徴とする、〈１〉または〈２〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１４〉単一検体において、〈１〉、〈２〉または〈９〉～〈１３〉に記載のナノカーボンを用いる吸光または発光を利用した抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法、あるいはこれと他の吸光または発光を利用した測定方法あるいは臨床検査方法とを組み合わせて、異なった吸光または発光波長帯にて複数の抗原および／または糖鎖を検出することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１５〉吸光または発光波長帯が、紫外、可視、近赤外および／または赤外領域に及ぶ、〈１４〉に記載の免疫測定方法あるいは臨床検査方法。
〈１６〉単一検体において、異なった吸光または発光波長帯における複数の抗原および／または糖鎖からの吸光または発光強度比を用いて、複数の抗原および／または糖鎖の定量を行うことを特徴とする、〈１４〉または〈１５〉に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。
〈１７〉〈１５〉または〈１６〉の紫外、可視、近赤外、赤外発光領域の計測において、単一あるいは複数の励起光を変調して、それに同期して吸光または発光を検出する、ロックインアンプ光検出法を用いた抗原抗体反応または糖鎖レクチン間の反応の測定あるいは臨床検査装置。 Specifically, the present application provides the following inventions.
<1> A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method, which uses the unique optical properties of nanocarbon.
<2> The antigen-antibody reaction or saccharide according to <1>, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by light excitation at a wavelength of 450-1350 nm are used as an absorption or emission label. A method for measuring the reaction between chain lectins or a clinical test method.
<3> Absorption or luminescence label containing nanocarbon.
<4> The light-absorbing or light-emitting label according to <3>, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 700-2300 nm by photoexcitation at a wavelength of 450-1350 nm.
<5> A nanocarbon is bound to an antibody that recognizes a specific antigen or a lectin that recognizes a specific sugar chain, and an antigen-antibody reaction with the specific antigen or a reaction between the specific sugar chain and the lectin is nano The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection by measurement of carbon fluorescence, Raman light, and / or light absorption.
<6> Streptavidin or a related molecule of streptavidin is bonded to nanocarbon, and a biotinylated antibody bound to a specific antigen or a reaction product of biotinylated lectin bound to a specific sugar chain and streptavidin is converted to nanocarbon. The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection by measurement of fluorescence, Raman light, and / or light absorption.
<7> A binding reaction between an antibody bound to a specific antigen and an immunoglobulin binding protein formed by binding nanocarbon to an immunoglobulin binding protein by measuring fluorescence, Raman light, and / or light absorption of the nanocarbon. The light-absorbing or light-emitting label according to <3> or <4>, which is used for detection.
<8> The light-absorbing or light-emitting label according to <7>, wherein the immunoglobulin binding protein is protein G or protein A.
<9> Various antigens or sugar chains that are present in blood or body cavity fluid when a specific antigen or a specific sugar chain becomes normal or pathological, <5> to <8> The light-absorbing or light-emitting label according to any one of the above.
<10> An antigen-enriched sample is prepared by immobilizing the antigen to the carrier using antibodies against various antigens bound to the carrier, and the antigen on the carrier is labeled with a nanocarbon-labeled antibody for the sample. Or by labeling the sugar chain of the antigen on the carrier with the nanocarbon-labeled lectin, and then measuring the near-infrared light absorption, near-infrared light emission, or near-infrared Raman scattered light of the nanocarbon. Alternatively, a method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to <1> or <2>, wherein a sugar chain of an antigen is detected.
<11> The method for measuring an antigen-antibody reaction or the reaction between sugar chain lectins or the clinical test method according to <10>, wherein the carrier is magnetic beads, Sepharose or Sephadex.
<12> Labeling of the antigen with a nanocarbon-labeled antibody recognizes the antigen and binds the antibody according to <5> labeled with nanocarbon, or recognizes the antigen and biotin The labeled antibody is bound and labeled with the nanocarbon label according to <6>, or the antibody that recognizes the antigen is bound and labeled with the nanocarbon label according to <7> or <8> The lectin according to <5>, wherein the labeling of the sugar chain of the antigen with the nanocarbon-labeled lectin recognizes the sugar chain of the antigen to the sugar chain of the antigen and is labeled with the nanocarbon. Or by recognizing the antigen, binding a biotinylated lectin, and labeling with the nanocarbon label according to <6>, 10> or <11> Measurement method or clinical test method of the reaction between the antigen-antibody reaction or a sugar chain lectin described.
<13> A disease marker antigen or sugar chain is labeled with a nanocarbon-labeled antibody or nanocarbon-labeled lectin, collected by electrophoresis, and then absorbed by near-infrared light, near-infrared light emission, or near-red The method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to <1> or <2>, wherein the antigen or sugar chain is detected by measuring external Raman scattered light .
<14> A method for measuring an antigen-antibody reaction or reaction between sugar chain lectins using absorption or luminescence using the nanocarbon according to <1>, <2> or <9> to <13> in a single specimen, or It is characterized by detecting a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands by combining a clinical test method or a measurement method or clinical test method using other light absorption or luminescence. A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method.
<15> The immunoassay method or clinical test method according to <14>, wherein the light absorption or emission wavelength band covers the ultraviolet, visible, near infrared, and / or infrared region.
<16> Characterizing a plurality of antigens and / or sugar chains using a ratio of light absorption or emission intensity from a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands in a single specimen <14> or <15> The method for measuring the antigen-antibody reaction or the reaction between sugar chain lectins or a clinical test method.
<17> In the measurement of the ultraviolet, visible, near-infrared, or infrared emission region of <15> or <16>, single or plural excitation lights are modulated, and absorption or emission is detected in synchronization with the modulation. Anti-antibody reaction or reaction between sugar chain lectins using a lock-in-amplifier photodetection method, or clinical laboratory equipment.
本発明により、近赤外光を発光するナノカーボンで検出対象を標識することによって、血液などの生体由来物をサンプルとする臨床検査において、検出対象以外の血球成分などの生体物質が存在しても、検出対象成分のみを高感度で検出することができる。
フローサイトメーターや共焦点レーザー顕微鏡では、利用できる波長域の拡大によって、得られる情報量が格段に増えてきた。本発明により、臨床検査機器も、今までの利用波長域400-500nmから、400-2300nmと近赤外領域にまで大幅に拡張することができ、得られる情報はさらに増加する。たとえば、異なる励起発光プローブを組み合わせる事で、１回のアッセイで同時に、数種類のバイオマーカーを検出評価できるようになる。本発明により、生体物質に対して透過性の高い近赤外光を使用することにより、生体由来の臨床サンプルを血清や血漿へと分離せずに検査ができるので、血球・血小板成分の分離操作によって失われていた情報を取得できるようになる。しかも、血清検査の際でも、混入した血球・血小板成分によるバイオマーカー由来のシグナル光吸収と非特異的ノイズが抑えられるので、微量バイオマーカーの検出が可能となり、がんやアルツハイマー病等の超早期発見・早期治療を可能とするなど、医学・医療の発展に大きく貢献できる。
本発明のナノカーボンの近赤外発光を利用した臨床検査システムにより、世界ではじめて近赤外光を臨床検査へ持ち込むこととなり、診断薬・診断機器産業界に革命的な進歩をもたらす。 According to the present invention, by labeling a detection target with nanocarbon that emits near-infrared light, a biological substance such as a blood cell component other than the detection target is present in a clinical test using a biological substance such as blood as a sample. In addition, only the detection target component can be detected with high sensitivity.
With flow cytometers and confocal laser microscopes, the amount of information that can be obtained has increased dramatically due to the expansion of the usable wavelength range. According to the present invention, the clinical test equipment can be greatly expanded from the conventional wavelength range of 400-500 nm to the near infrared range of 400-2300 nm, and the obtained information is further increased. For example, by combining different excitation luminescence probes, several kinds of biomarkers can be detected and evaluated simultaneously in one assay. According to the present invention, the use of near-infrared light that is highly permeable to biological substances enables examination of biological samples derived from living organisms without separating them into serum or plasma. The information that was lost can be obtained. Moreover, even during serum testing, the biomarker-derived signal light absorption and non-specific noise due to the mixed blood cells and platelet components can be suppressed, making it possible to detect trace biomarkers, and extremely early such as cancer and Alzheimer's disease. It can greatly contribute to the development of medicine and medical care by enabling discovery and early treatment.
The clinical testing system using near-infrared emission of nanocarbon of the present invention will bring near infrared light to clinical testing for the first time in the world, and will bring revolutionary progress to the diagnostics and diagnostic equipment industries.
フローサイトメーターや共焦点レーザー顕微鏡では、利用できる波長域の拡大によって、得られる情報量が格段に増えてきた。本発明により、臨床検査機器も、今までの利用波長域400-500nmから、400-2300nmと近赤外領域にまで大幅に拡張することができ、得られる情報はさらに増加する。たとえば、異なる励起発光プローブを組み合わせる事で、１回のアッセイで同時に、数種類のバイオマーカーを検出評価できるようになる。本発明により、生体物質に対して透過性の高い近赤外光を使用することにより、生体由来の臨床サンプルを血清や血漿へと分離せずに検査ができるので、血球・血小板成分の分離操作によって失われていた情報を取得できるようになる。しかも、血清検査の際でも、混入した血球・血小板成分によるバイオマーカー由来のシグナル光吸収と非特異的ノイズが抑えられるので、微量バイオマーカーの検出が可能となり、がんやアルツハイマー病等の超早期発見・早期治療を可能とするなど、医学・医療の発展に大きく貢献できる。
本発明のナノカーボンの近赤外発光を利用した臨床検査システムにより、世界ではじめて近赤外光を臨床検査へ持ち込むこととなり、診断薬・診断機器産業界に革命的な進歩をもたらす。 According to the present invention, by labeling a detection target with nanocarbon that emits near-infrared light, a biological substance such as a blood cell component other than the detection target is present in a clinical test using a biological substance such as blood as a sample. In addition, only the detection target component can be detected with high sensitivity.
With flow cytometers and confocal laser microscopes, the amount of information that can be obtained has increased dramatically due to the expansion of the usable wavelength range. According to the present invention, the clinical test equipment can be greatly expanded from the conventional wavelength range of 400-500 nm to the near infrared range of 400-2300 nm, and the obtained information is further increased. For example, by combining different excitation luminescence probes, several kinds of biomarkers can be detected and evaluated simultaneously in one assay. According to the present invention, the use of near-infrared light that is highly permeable to biological substances enables examination of biological samples derived from living organisms without separating them into serum or plasma. The information that was lost can be obtained. Moreover, even during serum testing, the biomarker-derived signal light absorption and non-specific noise due to the mixed blood cells and platelet components can be suppressed, making it possible to detect trace biomarkers, and extremely early such as cancer and Alzheimer's disease. It can greatly contribute to the development of medicine and medical care by enabling discovery and early treatment.
The clinical testing system using near-infrared emission of nanocarbon of the present invention will bring near infrared light to clinical testing for the first time in the world, and will bring revolutionary progress to the diagnostics and diagnostic equipment industries.
本発明に用いられるナノカーボンとしては、単層カーボンナノチューブ(SWCNT)、多層カーボンナノチューブ、カーボンナノホーン、ナノグラフェン、及び、カーボンナノロッド、カーボンナノコーン、カーボンナノカップ、フラーレンなどの、その他のナノカーボンが挙げられる。
SWCNTは、上述のとおり、近赤外領域の波長700-1350nmの光を吸収し、近赤外領域の波長700-2300nmにおいて蛍光を発し、また、後述の実施例１において示すとおり、近赤外領域の1585cm-1付近にラマン散乱ピークを有する。多層カーボンナノチューブおよびカーボンナンホーンは、同様に、近赤外領域の光を吸収し、また、近赤外領域の1580-1600cm-1にラマン散乱ピークを有する。ナノグラフェンは、近赤外領域の光を吸収し、近赤外領域の1800-1900cm-1にラマン散乱ピークを有し、また、可視光から近赤外領域において蛍光を発する。また、上述のその他のナノカーボンも、多層カーボンナノチューブ等と同様の光特性を有する。
このようにナノカーボンは、近赤外領域において光吸収し、発光し、あるいはラマン散乱光のピークを有するという光特性を有しており、本発明においては、ナノカーボンの有するこれらの特異な光学的性質を利用することにより、血液成分などが共存していても、ナノカーボンにより標識された検出対象のみを高感度で検出することができる。
具体的には、検出対象に対し特異的に結合する抗体などのプローブをナノカーボンにより標識し、当該標識プローブを検出対象に結合させて、標識の発光等を検出することにより、検出対象を検出する。
ナノカーボンによるプローブの標識は、例えば、後述のDSPE-PEG-NHSなどを用いて吸着あるいは化学結合させたナノカーボンをPEG部分により被覆し、これを抗体などのプローブに直接、あるいは、間接的に結合させること、などにより行うことができる。
また、本発明の方法の検出対象とされる、正常もしくは病的な状態になった際、血液や体腔液中に存在する各種の抗原としては、例えば、タンパクや糖鎖等の、がん関連抗原、疾患バイオマーカーが挙げられる。 Examples of the nanocarbon used in the present invention include single-walled carbon nanotubes (SWCNT), multi-walled carbon nanotubes, carbon nanohorns, nanographene, and other nanocarbons such as carbon nanorods, carbon nanocones, carbon nanocups, and fullerenes. It is done.
As described above, SWCNT absorbs light having a wavelength of 700-1350 nm in the near infrared region, emits fluorescence at a wavelength of 700-2300 nm in the near infrared region, and, as shown in Example 1 described later, It has a Raman scattering peak near 1585 cm -1 in the region. Similarly, multi-walled carbon nanotubes and carbonnanhorn absorb light in the near-infrared region and have a Raman scattering peak at 1580-1600 cm −1 in the near-infrared region. Nanographene absorbs light in the near infrared region, has a Raman scattering peak at 1800-1900 cm −1 in the near infrared region, and emits fluorescence in the near infrared region from visible light. The other nanocarbons described above also have the same optical characteristics as multi-walled carbon nanotubes.
As described above, nanocarbon has optical characteristics such as light absorption, emission in the near-infrared region, or peak of Raman scattering light. In the present invention, these unique optical properties of nanocarbon are included. By utilizing the intrinsic properties, only the detection target labeled with nanocarbon can be detected with high sensitivity even if blood components and the like coexist.
Specifically, the detection target is detected by labeling a probe such as an antibody that specifically binds to the detection target with nanocarbon, binding the labeled probe to the detection target, and detecting light emission of the label. To do.
The labeling of the probe with nanocarbon is performed by, for example, coating nanocarbon adsorbed or chemically bonded using the later-described DSPE-PEG-NHS or the like with a PEG moiety, and directly or indirectly covering the probe such as an antibody. It can be performed by bonding.
In addition, various antigens present in blood and body cavity fluids that are targeted for detection by the method of the present invention, such as proteins and sugar chains, are associated with cancer. Examples include antigens and disease biomarkers.
SWCNTは、上述のとおり、近赤外領域の波長700-1350nmの光を吸収し、近赤外領域の波長700-2300nmにおいて蛍光を発し、また、後述の実施例１において示すとおり、近赤外領域の1585cm-1付近にラマン散乱ピークを有する。多層カーボンナノチューブおよびカーボンナンホーンは、同様に、近赤外領域の光を吸収し、また、近赤外領域の1580-1600cm-1にラマン散乱ピークを有する。ナノグラフェンは、近赤外領域の光を吸収し、近赤外領域の1800-1900cm-1にラマン散乱ピークを有し、また、可視光から近赤外領域において蛍光を発する。また、上述のその他のナノカーボンも、多層カーボンナノチューブ等と同様の光特性を有する。
このようにナノカーボンは、近赤外領域において光吸収し、発光し、あるいはラマン散乱光のピークを有するという光特性を有しており、本発明においては、ナノカーボンの有するこれらの特異な光学的性質を利用することにより、血液成分などが共存していても、ナノカーボンにより標識された検出対象のみを高感度で検出することができる。
具体的には、検出対象に対し特異的に結合する抗体などのプローブをナノカーボンにより標識し、当該標識プローブを検出対象に結合させて、標識の発光等を検出することにより、検出対象を検出する。
ナノカーボンによるプローブの標識は、例えば、後述のDSPE-PEG-NHSなどを用いて吸着あるいは化学結合させたナノカーボンをPEG部分により被覆し、これを抗体などのプローブに直接、あるいは、間接的に結合させること、などにより行うことができる。
また、本発明の方法の検出対象とされる、正常もしくは病的な状態になった際、血液や体腔液中に存在する各種の抗原としては、例えば、タンパクや糖鎖等の、がん関連抗原、疾患バイオマーカーが挙げられる。 Examples of the nanocarbon used in the present invention include single-walled carbon nanotubes (SWCNT), multi-walled carbon nanotubes, carbon nanohorns, nanographene, and other nanocarbons such as carbon nanorods, carbon nanocones, carbon nanocups, and fullerenes. It is done.
As described above, SWCNT absorbs light having a wavelength of 700-1350 nm in the near infrared region, emits fluorescence at a wavelength of 700-2300 nm in the near infrared region, and, as shown in Example 1 described later, It has a Raman scattering peak near 1585 cm -1 in the region. Similarly, multi-walled carbon nanotubes and carbonnanhorn absorb light in the near-infrared region and have a Raman scattering peak at 1580-1600 cm −1 in the near-infrared region. Nanographene absorbs light in the near infrared region, has a Raman scattering peak at 1800-1900 cm −1 in the near infrared region, and emits fluorescence in the near infrared region from visible light. The other nanocarbons described above also have the same optical characteristics as multi-walled carbon nanotubes.
As described above, nanocarbon has optical characteristics such as light absorption, emission in the near-infrared region, or peak of Raman scattering light. In the present invention, these unique optical properties of nanocarbon are included. By utilizing the intrinsic properties, only the detection target labeled with nanocarbon can be detected with high sensitivity even if blood components and the like coexist.
Specifically, the detection target is detected by labeling a probe such as an antibody that specifically binds to the detection target with nanocarbon, binding the labeled probe to the detection target, and detecting light emission of the label. To do.
The labeling of the probe with nanocarbon is performed by, for example, coating nanocarbon adsorbed or chemically bonded using the later-described DSPE-PEG-NHS or the like with a PEG moiety, and directly or indirectly covering the probe such as an antibody. It can be performed by bonding.
In addition, various antigens present in blood and body cavity fluids that are targeted for detection by the method of the present invention, such as proteins and sugar chains, are associated with cancer. Examples include antigens and disease biomarkers.
以下に示す実施例において、反応生成物等の構造、物性の確認は、ラマンおよび蛍光スペクトル法により行った。共鳴ラマンスペクトルは、光学顕微鏡(対物レンズ ×50)を備えた堀場JY社製T64000システムを用いて室温で測定した。励起源としてAr+レーザー(λ＝514.5nm)を用いた。Micro-HR分光器(堀場JY社製)と光学顕微鏡(オリンパス社製、商品名BX51、使用した対物レンズ：×50)を組み合わせた蛍光測定システムを用いて、近赤外領域における蛍光スペクトルを測定した。蛍光検出のために、液体窒素により冷却されたInGaAs検知器(堀場JY社製)を用い、励起光は660nmの半導体レーザー(Coherent社製)を使用した。
In the following examples, the structure and physical properties of reaction products and the like were confirmed by Raman and fluorescence spectroscopy. The resonance Raman spectrum was measured at room temperature using a T64000 system manufactured by Horiba JY Co., Ltd. equipped with an optical microscope (objective lens × 50). An Ar + laser (λ = 514.5 nm) was used as an excitation source. Fluorescence spectrum in the near infrared region is measured using a fluorescence measurement system that combines a Micro-HR spectrometer (manufactured by Horiba JY) and an optical microscope (manufactured by Olympus, product name BX51, objective lens used: x50). did. For fluorescence detection, an InGaAs detector (Horiba JY) cooled by liquid nitrogen was used, and a 660 nm semiconductor laser (Coherent) was used as the excitation light.
実施例１．SWCNT標識抗体(SWCNT-DSPE-PEG-IgG)の調製
SWCNTが免疫分析のための近赤外蛍光標識として有用であることを示すために、IgG抗体をSWCNTで標識した。以下の手順で、SWCNTをDSPE-PEG-NHSとともに分散させ、DSPE-PEG-NHSで被覆されたSWCNT(SWCNT-DSPE-PEG-NHS)を調製し、IgGをSWCNT-DSPE-PEG-NHSのNHS基に結合させて、SWCNT標識抗体、SWCNT-DSPE-PEG-IgGを得た。
（１）SWCNT-DSPE-PEG-IgGの調製
（ｉ）10ｍLの50ｍMリン酸緩衝液(pH8.0)中に、1mgのSWCNT(商品名CoMoCAT CG，Sigma Aldrich社製)と10mgの下記［化１］の３-(Ｎ－サクシンイミジロキシグルタリル)アミノプロピル，ポリエチレングリコール－カルバミルジステアロイルホスファチジル－エタノールアミン(商品名DSPE-PEG-NHS、Sunbright社製)を加え、チップ型超音波混合器を用いて、10分間、超音波混合し、室温において145000gで2時間遠心分離した。上清には、個々に分離され、DSPE-PEG-NHSで表面コーティングされたSWCNTが含まれている。
(式中、ｎはPEG鎖の分子量が2000になる-O-CH2CH2－単位の繰り返し数を表す。)
個々に分離したSWCNTを含む遠心分離上清を、遠心濾過装置(Nanosep300K、Pall社製)を用いて、12000rpmで15分間ろ過し、100μLのリン酸緩衝液を用いて２回、同じ遠心分離条件下で洗浄した。このようにして、SWCNT-DSPE-PEG-NHSを得た。
（ii）SWCNT-DSPE-PEG-NHSを、Nanosepを用いて、100μLのリン酸緩衝液中でウサギIgG(Sigma,15006)と混合し、反応させるために、室温で1時間放置し、次いで、リン酸緩衝液を除去した。Nanosep中の反応物を100μLのリン酸緩衝液を用いて３回洗浄し、12000rpmで15分間、遠心分離した。最後に、反応物SWCNT-DSPE-PEG-IgGを100μLのリン酸緩衝液中に分散させ、ガラス瓶に入れ保管した。以上の手順を図４に図示する。 Example 1. Preparation of SWCNT -labeled antibody (SWCNT-DSPE-PEG-IgG) In order to show that SWCNT is useful as a near-infrared fluorescent label for immunoassay, IgG antibody was labeled with SWCNT. In the following procedure, SWCNT is dispersed with DSPE-PEG-NHS, SWCNT coated with DSPE-PEG-NHS (SWCNT-DSPE-PEG-NHS) is prepared, and IgG is mixed with SWCNT-DSPE-PEG-NHS NHS. By coupling to a group, a SWCNT-labeled antibody, SWCNT-DSPE-PEG-IgG, was obtained.
(1) Preparation of SWCNT-DSPE-PEG-IgG (i) In 10 mL of 50 mM phosphate buffer (pH 8.0), 1 mg of SWCNT (trade name CoMoCAT CG, manufactured by Sigma Aldrich) and 10 mg 1] 3- (N-succinimidyloxyglutaryl) aminopropyl, polyethylene glycol-carbamyl distearoyl phosphatidyl-ethanolamine (trade name DSPE-PEG-NHS, manufactured by Sunbright) and a chip-type ultrasonic mixer Was sonicated for 10 minutes and centrifuged at 145000 g for 2 hours at room temperature. The supernatant contains SWCNTs that are individually separated and surface coated with DSPE-PEG-NHS.
(In the formula, n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.)
Centrifugation supernatant containing individually separated SWCNTs is filtered using a centrifugal filtration device (Nanosep300K, manufactured by Pall) for 15 minutes at 12000 rpm, and the same centrifugation conditions twice using 100 μL phosphate buffer. Washed under. In this way, SWCNT-DSPE-PEG-NHS was obtained.
(Ii) SWCNT-DSPE-PEG-NHS is mixed with rabbit IgG (Sigma, 15006) in 100 μL phosphate buffer using Nanosep and allowed to react for 1 hour at room temperature, then The phosphate buffer was removed. The reaction in Nanosep was washed 3 times with 100 μL phosphate buffer and centrifuged at 12000 rpm for 15 minutes. Finally, the reaction product SWCNT-DSPE-PEG-IgG was dispersed in 100 μL of phosphate buffer and stored in a glass bottle. The above procedure is illustrated in FIG.
SWCNTが免疫分析のための近赤外蛍光標識として有用であることを示すために、IgG抗体をSWCNTで標識した。以下の手順で、SWCNTをDSPE-PEG-NHSとともに分散させ、DSPE-PEG-NHSで被覆されたSWCNT(SWCNT-DSPE-PEG-NHS)を調製し、IgGをSWCNT-DSPE-PEG-NHSのNHS基に結合させて、SWCNT標識抗体、SWCNT-DSPE-PEG-IgGを得た。
（１）SWCNT-DSPE-PEG-IgGの調製
（ｉ）10ｍLの50ｍMリン酸緩衝液(pH8.0)中に、1mgのSWCNT(商品名CoMoCAT CG，Sigma Aldrich社製)と10mgの下記［化１］の３-(Ｎ－サクシンイミジロキシグルタリル)アミノプロピル，ポリエチレングリコール－カルバミルジステアロイルホスファチジル－エタノールアミン(商品名DSPE-PEG-NHS、Sunbright社製)を加え、チップ型超音波混合器を用いて、10分間、超音波混合し、室温において145000gで2時間遠心分離した。上清には、個々に分離され、DSPE-PEG-NHSで表面コーティングされたSWCNTが含まれている。
個々に分離したSWCNTを含む遠心分離上清を、遠心濾過装置(Nanosep300K、Pall社製)を用いて、12000rpmで15分間ろ過し、100μLのリン酸緩衝液を用いて２回、同じ遠心分離条件下で洗浄した。このようにして、SWCNT-DSPE-PEG-NHSを得た。
（ii）SWCNT-DSPE-PEG-NHSを、Nanosepを用いて、100μLのリン酸緩衝液中でウサギIgG(Sigma,15006)と混合し、反応させるために、室温で1時間放置し、次いで、リン酸緩衝液を除去した。Nanosep中の反応物を100μLのリン酸緩衝液を用いて３回洗浄し、12000rpmで15分間、遠心分離した。最後に、反応物SWCNT-DSPE-PEG-IgGを100μLのリン酸緩衝液中に分散させ、ガラス瓶に入れ保管した。以上の手順を図４に図示する。 Example 1. Preparation of SWCNT -labeled antibody (SWCNT-DSPE-PEG-IgG) In order to show that SWCNT is useful as a near-infrared fluorescent label for immunoassay, IgG antibody was labeled with SWCNT. In the following procedure, SWCNT is dispersed with DSPE-PEG-NHS, SWCNT coated with DSPE-PEG-NHS (SWCNT-DSPE-PEG-NHS) is prepared, and IgG is mixed with SWCNT-DSPE-PEG-NHS NHS. By coupling to a group, a SWCNT-labeled antibody, SWCNT-DSPE-PEG-IgG, was obtained.
(1) Preparation of SWCNT-DSPE-PEG-IgG (i) In 10 mL of 50 mM phosphate buffer (pH 8.0), 1 mg of SWCNT (trade name CoMoCAT CG, manufactured by Sigma Aldrich) and 10 mg 1] 3- (N-succinimidyloxyglutaryl) aminopropyl, polyethylene glycol-carbamyl distearoyl phosphatidyl-ethanolamine (trade name DSPE-PEG-NHS, manufactured by Sunbright) and a chip-type ultrasonic mixer Was sonicated for 10 minutes and centrifuged at 145000 g for 2 hours at room temperature. The supernatant contains SWCNTs that are individually separated and surface coated with DSPE-PEG-NHS.
Centrifugation supernatant containing individually separated SWCNTs is filtered using a centrifugal filtration device (Nanosep300K, manufactured by Pall) for 15 minutes at 12000 rpm, and the same centrifugation conditions twice using 100 μL phosphate buffer. Washed under. In this way, SWCNT-DSPE-PEG-NHS was obtained.
(Ii) SWCNT-DSPE-PEG-NHS is mixed with rabbit IgG (Sigma, 15006) in 100 μL phosphate buffer using Nanosep and allowed to react for 1 hour at room temperature, then The phosphate buffer was removed. The reaction in Nanosep was washed 3 times with 100 μL phosphate buffer and centrifuged at 12000 rpm for 15 minutes. Finally, the reaction product SWCNT-DSPE-PEG-IgG was dispersed in 100 μL of phosphate buffer and stored in a glass bottle. The above procedure is illustrated in FIG.
（２）SWCNT-DSPE-PEG-IgGの構造
SWCNT-DSPE-PEG-IgGのラマンスペクトル(図５)には、SWCNTの特徴的ピーク、すなわち、1585cm-１のＧバンド、1335cm-１のＤバンドおよび270cm-１の呼吸モード(RBM)が現れた。Ｇバンドの低エネルギー側の裾野は、SWCNTの直径が小さく(<1nm)、金属的であるときに現れるファノ・ラインに相当する。RBMのピーク位置から推測したCNTの直径は、約0.9nmであった。弱いＤバンドは、アモルファス・カーボン不純物の量が大変少ないか、あるいは、CNT中の構造欠陥の数が極めて少ないことを示している。
SWCNT-DSPE-PEG-IgGの蛍光スペクトル(図６)には、それぞれ、(７，５)および(７，６)SWCNTに対応する1047nmおよび1142nmの２つの突出したピークが観測された。
これらの観察結果は、上述の反応操作による調製物SWCNT-DSPE-PEG-IgGに、実際にSWCNTが結合しており、当該調製物が近赤外領域の蛍光を発することができることを示している。 (2) The SWCNT-DSPE-PEG-IgG structure SWCNT-DSPE-PEG-IgG Raman spectrum (FIG. 5), characteristic peaks of SWCNT, i.e., G-band of 1585 cm -1, D band of 1335cm -1 And 270 cm -1 breathing mode (RBM) appeared. The lower energy side of the G band corresponds to the Fano line that appears when SWCNT has a small diameter (<1 nm) and is metallic. The diameter of CNT estimated from the peak position of RBM was about 0.9 nm. The weak D band indicates that the amount of amorphous carbon impurities is very small, or the number of structural defects in the CNT is very small.
In the fluorescence spectrum of SWCNT-DSPE-PEG-IgG (FIG. 6), two protruding peaks of 1047 nm and 1142 nm corresponding to (7,5) and (7,6) SWCNT were observed, respectively.
These observation results indicate that SWCNT is actually bound to the preparation SWCNT-DSPE-PEG-IgG prepared by the above reaction procedure, and that the preparation can emit near-infrared fluorescence. .
SWCNT-DSPE-PEG-IgGのラマンスペクトル(図５)には、SWCNTの特徴的ピーク、すなわち、1585cm-１のＧバンド、1335cm-１のＤバンドおよび270cm-１の呼吸モード(RBM)が現れた。Ｇバンドの低エネルギー側の裾野は、SWCNTの直径が小さく(<1nm)、金属的であるときに現れるファノ・ラインに相当する。RBMのピーク位置から推測したCNTの直径は、約0.9nmであった。弱いＤバンドは、アモルファス・カーボン不純物の量が大変少ないか、あるいは、CNT中の構造欠陥の数が極めて少ないことを示している。
SWCNT-DSPE-PEG-IgGの蛍光スペクトル(図６)には、それぞれ、(７，５)および(７，６)SWCNTに対応する1047nmおよび1142nmの２つの突出したピークが観測された。
これらの観察結果は、上述の反応操作による調製物SWCNT-DSPE-PEG-IgGに、実際にSWCNTが結合しており、当該調製物が近赤外領域の蛍光を発することができることを示している。 (2) The SWCNT-DSPE-PEG-IgG structure SWCNT-DSPE-PEG-IgG Raman spectrum (FIG. 5), characteristic peaks of SWCNT, i.e., G-band of 1585 cm -1, D band of 1335cm -1 And 270 cm -1 breathing mode (RBM) appeared. The lower energy side of the G band corresponds to the Fano line that appears when SWCNT has a small diameter (<1 nm) and is metallic. The diameter of CNT estimated from the peak position of RBM was about 0.9 nm. The weak D band indicates that the amount of amorphous carbon impurities is very small, or the number of structural defects in the CNT is very small.
In the fluorescence spectrum of SWCNT-DSPE-PEG-IgG (FIG. 6), two protruding peaks of 1047 nm and 1142 nm corresponding to (7,5) and (7,6) SWCNT were observed, respectively.
These observation results indicate that SWCNT is actually bound to the preparation SWCNT-DSPE-PEG-IgG prepared by the above reaction procedure, and that the preparation can emit near-infrared fluorescence. .
実施例２．SWCNT標識抗体(SWCNT-DSPE-PEG-IgG)の抗体活性の確認
IgG抗体はプロテインＧに特異的に結合することが知られている。そこで、SWCNT-DSPE-PEG-IgGがIgG抗体としての活性を保持していることを確認するために、以下の手順で、SWCNT-DSPE-PEG-IgGをプロテインＧ磁性ビーズと混合し、これにより、SWCNT-DSPE-PEG-IgGがプロテインＧビーズ上に沈着することを分光測定により確認した。
（１）SWCNT-DSPE-PEG-IgGとプロテインＧとの結合の形成
20μLのプロテインＧ磁性ビーズ溶液(Ademtech社製)を100μLのリン酸緩衝液(pH8)中、0.5％のTween20を用いて前処理し、上清を除いた。50μLのリン酸緩衝液中に分散したSWCNT-DSPE-PEG-IgGを、プロテインＧビーズと混合し、室温で30分放置した後、上清を除去し、スペクトル測定のために、0.5％Tween20を100μLの50ｍＭリン酸緩衝液(pH8.0)に溶解した溶液でビーズを３回洗浄した。
（２）SWCNT-DSPE-PEG-IgGの抗体活性の確認
SWCNT-DSPE-PEG-IgGで処理した後のビーズのラマンスペクトルは1585cm-1のＧバンド、ファノ・ラインおよび260cm-1の呼吸モードを示した(図７)。これらのスペクトルの特徴は、SWCNT-DSPE-PEG-IgGの特徴(図５および図６)と同様であった。一方、プロテインＧビーズ単独では、いかなるＧおよびＤバンドも示さなかった。
プロテインＧ上に沈着したSWCNT-DSPE-PEG-IgGの蛍光を測定した。図８に示す蛍光スペクトルは1060nmおよび1160nm付近の２つの主要なピークを有していた。
これらの観察結果は、SWCNT-DSPE-PEG-IgGがIgG抗体としての活性を保持しており、これによりプロテインＧに付着したこと、そして、このようにして生成したSWCNT-DSPE-PEG-IgGとプロテインＧビーズの複合体が近赤外領域の蛍光を発することができることを示すものである。 Example 2 Confirmation of antibody activity of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) IgG antibodies are known to specifically bind to protein G. Therefore, in order to confirm that SWCNT-DSPE-PEG-IgG retains the activity as an IgG antibody, SWCNT-DSPE-PEG-IgG was mixed with protein G magnetic beads by the following procedure. It was confirmed by spectroscopic measurement that SWCNT-DSPE-PEG-IgG was deposited on protein G beads.
(1) Formation of a bond between SWCNT-DSPE-PEG-IgG and protein G A 20 μL protein G magnetic bead solution (manufactured by Ademtech) was previously used in 100 μL phosphate buffer (pH 8) with 0.5% Tween20. Processed and removed supernatant. SWCNT-DSPE-PEG-IgG dispersed in 50 μL of phosphate buffer is mixed with protein G beads and allowed to stand at room temperature for 30 minutes. The supernatant is removed, and 0.5% Tween20 is added for spectral measurement. The beads were washed three times with a solution dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0).
(2) G-band of the confirmation of SWCNT-DSPE-PEG-IgG antibody activity Raman spectrum of the beads after treatment with SWCNT-DSPE-PEG-IgG is 1585 cm -1, the breathing mode of the Fano line and 260 cm -1 Shown (FIG. 7). The characteristics of these spectra were the same as the characteristics of SWCNT-DSPE-PEG-IgG (FIGS. 5 and 6). On the other hand, protein G beads alone did not show any G and D bands.
The fluorescence of SWCNT-DSPE-PEG-IgG deposited on protein G was measured. The fluorescence spectrum shown in FIG. 8 had two main peaks around 1060 nm and 1160 nm.
These observation results show that SWCNT-DSPE-PEG-IgG retains the activity as an IgG antibody, and thereby adheres to protein G, and the SWCNT-DSPE-PEG-IgG thus produced This shows that the complex of protein G beads can emit fluorescence in the near infrared region.
IgG抗体はプロテインＧに特異的に結合することが知られている。そこで、SWCNT-DSPE-PEG-IgGがIgG抗体としての活性を保持していることを確認するために、以下の手順で、SWCNT-DSPE-PEG-IgGをプロテインＧ磁性ビーズと混合し、これにより、SWCNT-DSPE-PEG-IgGがプロテインＧビーズ上に沈着することを分光測定により確認した。
（１）SWCNT-DSPE-PEG-IgGとプロテインＧとの結合の形成
20μLのプロテインＧ磁性ビーズ溶液(Ademtech社製)を100μLのリン酸緩衝液(pH8)中、0.5％のTween20を用いて前処理し、上清を除いた。50μLのリン酸緩衝液中に分散したSWCNT-DSPE-PEG-IgGを、プロテインＧビーズと混合し、室温で30分放置した後、上清を除去し、スペクトル測定のために、0.5％Tween20を100μLの50ｍＭリン酸緩衝液(pH8.0)に溶解した溶液でビーズを３回洗浄した。
（２）SWCNT-DSPE-PEG-IgGの抗体活性の確認
SWCNT-DSPE-PEG-IgGで処理した後のビーズのラマンスペクトルは1585cm-1のＧバンド、ファノ・ラインおよび260cm-1の呼吸モードを示した(図７)。これらのスペクトルの特徴は、SWCNT-DSPE-PEG-IgGの特徴(図５および図６)と同様であった。一方、プロテインＧビーズ単独では、いかなるＧおよびＤバンドも示さなかった。
プロテインＧ上に沈着したSWCNT-DSPE-PEG-IgGの蛍光を測定した。図８に示す蛍光スペクトルは1060nmおよび1160nm付近の２つの主要なピークを有していた。
これらの観察結果は、SWCNT-DSPE-PEG-IgGがIgG抗体としての活性を保持しており、これによりプロテインＧに付着したこと、そして、このようにして生成したSWCNT-DSPE-PEG-IgGとプロテインＧビーズの複合体が近赤外領域の蛍光を発することができることを示すものである。 Example 2 Confirmation of antibody activity of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) IgG antibodies are known to specifically bind to protein G. Therefore, in order to confirm that SWCNT-DSPE-PEG-IgG retains the activity as an IgG antibody, SWCNT-DSPE-PEG-IgG was mixed with protein G magnetic beads by the following procedure. It was confirmed by spectroscopic measurement that SWCNT-DSPE-PEG-IgG was deposited on protein G beads.
(1) Formation of a bond between SWCNT-DSPE-PEG-IgG and protein G A 20 μL protein G magnetic bead solution (manufactured by Ademtech) was previously used in 100 μL phosphate buffer (pH 8) with 0.5% Tween20. Processed and removed supernatant. SWCNT-DSPE-PEG-IgG dispersed in 50 μL of phosphate buffer is mixed with protein G beads and allowed to stand at room temperature for 30 minutes. The supernatant is removed, and 0.5% Tween20 is added for spectral measurement. The beads were washed three times with a solution dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0).
(2) G-band of the confirmation of SWCNT-DSPE-PEG-IgG antibody activity Raman spectrum of the beads after treatment with SWCNT-DSPE-PEG-IgG is 1585 cm -1, the breathing mode of the Fano line and 260 cm -1 Shown (FIG. 7). The characteristics of these spectra were the same as the characteristics of SWCNT-DSPE-PEG-IgG (FIGS. 5 and 6). On the other hand, protein G beads alone did not show any G and D bands.
The fluorescence of SWCNT-DSPE-PEG-IgG deposited on protein G was measured. The fluorescence spectrum shown in FIG. 8 had two main peaks around 1060 nm and 1160 nm.
These observation results show that SWCNT-DSPE-PEG-IgG retains the activity as an IgG antibody, and thereby adheres to protein G, and the SWCNT-DSPE-PEG-IgG thus produced This shows that the complex of protein G beads can emit fluorescence in the near infrared region.
実施例３．ヘモグロビンの共存がSWCNT標識抗体(SWCNT-DSPE-PEG-IgG)の抗体活性に影響を与えないことの確認
ヘモグロビンがSWCNT-DSPE-PEG-IgGのプロテインＧとの結合を妨げないこと確認するために、以下の手順で、プロテインＧビーズと混合する前に、ヘモグロビンをSWCNT-DSPE-PEG-IgG溶液に添加して、免疫沈降実験を行った。
5重量％ヘモグロビン溶液(商品名JCCRM622-1)2.5μLをSWCNT-DSPE-PEG-IgG溶液42.5μLに混合した。この溶液へプロテインＧビーズ溶液20μLを加え、混合し、この混合物からビーズを取り出し、洗浄した後、ビーズをリン酸緩衝溶液20μLに分散させ、蛍光スペクトルを測定した。スペクトルは、SWCNTの特徴的なピークを示した(図９)。
このことは、ヘモグロビンの共存がSWCNT-DSPE-PEG-IgGとプロテインＧとの結合を妨害しないことを示している。 Example 3 Confirmation that coexistence of hemoglobin does not affect the antibody activity of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) To confirm that hemoglobin does not interfere with protein G of SWCNT-DSPE-PEG-IgG In the following procedure, before mixing with protein G beads, hemoglobin was added to the SWCNT-DSPE-PEG-IgG solution, and an immunoprecipitation experiment was performed.
2.5 μL of 5 wt% hemoglobin solution (trade name JCCRM622-1) was mixed with 42.5 μL of SWCNT-DSPE-PEG-IgG solution. To this solution, 20 μL of protein G bead solution was added and mixed, and the beads were taken out from this mixture and washed. Then, the beads were dispersed in 20 μL of a phosphate buffer solution, and the fluorescence spectrum was measured. The spectrum showed a characteristic peak of SWCNT (FIG. 9).
This indicates that coexistence of hemoglobin does not interfere with the binding of SWCNT-DSPE-PEG-IgG and protein G.
ヘモグロビンがSWCNT-DSPE-PEG-IgGのプロテインＧとの結合を妨げないこと確認するために、以下の手順で、プロテインＧビーズと混合する前に、ヘモグロビンをSWCNT-DSPE-PEG-IgG溶液に添加して、免疫沈降実験を行った。
5重量％ヘモグロビン溶液(商品名JCCRM622-1)2.5μLをSWCNT-DSPE-PEG-IgG溶液42.5μLに混合した。この溶液へプロテインＧビーズ溶液20μLを加え、混合し、この混合物からビーズを取り出し、洗浄した後、ビーズをリン酸緩衝溶液20μLに分散させ、蛍光スペクトルを測定した。スペクトルは、SWCNTの特徴的なピークを示した(図９)。
このことは、ヘモグロビンの共存がSWCNT-DSPE-PEG-IgGとプロテインＧとの結合を妨害しないことを示している。 Example 3 Confirmation that coexistence of hemoglobin does not affect the antibody activity of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) To confirm that hemoglobin does not interfere with protein G of SWCNT-DSPE-PEG-IgG In the following procedure, before mixing with protein G beads, hemoglobin was added to the SWCNT-DSPE-PEG-IgG solution, and an immunoprecipitation experiment was performed.
2.5 μL of 5 wt% hemoglobin solution (trade name JCCRM622-1) was mixed with 42.5 μL of SWCNT-DSPE-PEG-IgG solution. To this solution, 20 μL of protein G bead solution was added and mixed, and the beads were taken out from this mixture and washed. Then, the beads were dispersed in 20 μL of a phosphate buffer solution, and the fluorescence spectrum was measured. The spectrum showed a characteristic peak of SWCNT (FIG. 9).
This indicates that coexistence of hemoglobin does not interfere with the binding of SWCNT-DSPE-PEG-IgG and protein G.
実施例４．ヘモグロビンの共存がSWCNT標識抗体(SWCNT-DSPE-PEG-IgG)の蛍光スペクトル測定に影響を与えないことの確認
ヘモグロビンがSWCNT-DSPE-PEG-IgG/プロテインＧビーズと共存していても、CNTの蛍光スペクトル測定を妨げないことを確認するため、実施例２の(１)のプロセスの最後に得られた上清を分取後、5重量％ヘモグロビン溶液(商品名JCCRM622-1)を20μL加え、光学的スペクトルを測定した。
ヘモグロビンは、近赤外領域において吸光も発光もしないので、たとえSWCNT-DSPE-PEG-IgG/プロテインＧビーズと共存していても、SWCNT-DSPE-PEG-IgG/プロテインＧビーズにおけるSWCNTの蛍光スペクトル測定を妨げないはずである。このことは、実際にヘモグロビンの共存下で測定したSWCNT-DSPE-PEG-IgG/プロテインＧビーズのSWCNTのスペクトル(図１０)に示す蛍光ピークが、ヘモグロビンが共存しないときにSWCNT-DSPE-PEG-IgG/プロテインＧビーズで観察されたもの(図８)と、特徴が一致することにより、確認された。 Example 4 Confirmation that coexistence of hemoglobin does not affect the fluorescence spectrum measurement of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) Even if hemoglobin coexists with SWCNT-DSPE-PEG-IgG / protein G beads, In order to confirm that the measurement of the fluorescence spectrum is not hindered, the supernatant obtained at the end of the process of Example 2 (1) was collected, and then 20 μL of 5 wt% hemoglobin solution (trade name JCCRM622-1) was added. The optical spectrum was measured.
Since hemoglobin does not absorb or emit light in the near-infrared region, even if it coexists with SWCNT-DSPE-PEG-IgG / Protein G beads, the SWCNT fluorescence spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads Should not interfere with the measurement. This is because the fluorescence peak shown in the SWCNT spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads measured in the presence of hemoglobin (Fig. 10) is not present in the presence of hemoglobin. This was confirmed by the coincidence of the characteristics with those observed with IgG / protein G beads (FIG. 8).
ヘモグロビンがSWCNT-DSPE-PEG-IgG/プロテインＧビーズと共存していても、CNTの蛍光スペクトル測定を妨げないことを確認するため、実施例２の(１)のプロセスの最後に得られた上清を分取後、5重量％ヘモグロビン溶液(商品名JCCRM622-1)を20μL加え、光学的スペクトルを測定した。
ヘモグロビンは、近赤外領域において吸光も発光もしないので、たとえSWCNT-DSPE-PEG-IgG/プロテインＧビーズと共存していても、SWCNT-DSPE-PEG-IgG/プロテインＧビーズにおけるSWCNTの蛍光スペクトル測定を妨げないはずである。このことは、実際にヘモグロビンの共存下で測定したSWCNT-DSPE-PEG-IgG/プロテインＧビーズのSWCNTのスペクトル(図１０)に示す蛍光ピークが、ヘモグロビンが共存しないときにSWCNT-DSPE-PEG-IgG/プロテインＧビーズで観察されたもの(図８)と、特徴が一致することにより、確認された。 Example 4 Confirmation that coexistence of hemoglobin does not affect the fluorescence spectrum measurement of SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) Even if hemoglobin coexists with SWCNT-DSPE-PEG-IgG / protein G beads, In order to confirm that the measurement of the fluorescence spectrum is not hindered, the supernatant obtained at the end of the process of Example 2 (1) was collected, and then 20 μL of 5 wt% hemoglobin solution (trade name JCCRM622-1) was added. The optical spectrum was measured.
Since hemoglobin does not absorb or emit light in the near-infrared region, even if it coexists with SWCNT-DSPE-PEG-IgG / Protein G beads, the SWCNT fluorescence spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads Should not interfere with the measurement. This is because the fluorescence peak shown in the SWCNT spectrum of SWCNT-DSPE-PEG-IgG / Protein G beads measured in the presence of hemoglobin (Fig. 10) is not present in the presence of hemoglobin. This was confirmed by the coincidence of the characteristics with those observed with IgG / protein G beads (FIG. 8).
実施例５．プロテインＧビーズに結合したSWCNT標識抗体(SWCNT-DSPE-PEG-IgG)がプロテインＧビーズから回収できることの確認
一般に標識抗体を用いてサンプル中の抗原を検出する方法の態様の一つとして、サンプル中の抗原をプロテインＧビーズに固定した抗体により捕捉し、当該捕捉された抗原を標識抗体を用いて標識し、当該標識を検出する方法を挙げることができる。そして、このような方法では、抗原の定量は、通常、プロテインＧビーズ上に生成した抗原抗体複合体をプロテインＧビーズから溶出させ、溶液系で標識の結合量を分析することで行われる。
そこで、SWCNT標識抗体がこのような検出方法に用い得るかどうか、具体的には、プロテインＧビーズに結合したSWCNT-DSPE-PEG-IgGがプロテインＧビーズから容易に溶出でき、また、溶出したSWCNT-DSPE-PEG-IgGについて蛍光スペクトル測定が行えるかどうかを確認するため、プロテインＧビーズに結合したSWCNT-DSPE-PEG-IgGをプロテインＧビーズから引き離し、SWCNT-DSPE-PEG-IgGを溶液中に分散させ、その蛍光スペクトルを測定した。
SWCNT-DSPE-PEG-IgGのプロテインＧビーズからの引き離しは、SDS水溶液(5重量％)を10μL添加し、加熱沸騰(10分間)することにより行った。得られたSWCNT-DSPE-PEG-IgG溶出液の蛍光スペクトルを図１１に示す。図１１の蛍光スペクトルのピーク強度から、SWCNT-DSPE-PEG-IgGの回収率は90％程度と見積もられ、これによりSWCNT標識が上述のような免疫沈降法による抗原の定量に適していることが確認された。 Example 5 FIG. Confirmation that SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) bound to protein G beads can be recovered from protein G beads In general, one of the methods for detecting antigens in samples using labeled antibodies And a method of detecting the label by capturing the captured antigen with an antibody immobilized on protein G beads, labeling the captured antigen with a labeled antibody, and the like. In such a method, the antigen is usually quantified by eluting the antigen-antibody complex produced on the protein G beads from the protein G beads and analyzing the binding amount of the label in a solution system.
Therefore, whether SWCNT-labeled antibodies can be used in such detection methods, specifically, SWCNT-DSPE-PEG-IgG bound to protein G beads can be easily eluted from protein G beads. In order to confirm whether fluorescence spectrum measurement can be performed for -DSPE-PEG-IgG, SWCNT-DSPE-PEG-IgG bound to protein G beads is pulled away from protein G beads, and SWCNT-DSPE-PEG-IgG is put into solution. After dispersion, the fluorescence spectrum was measured.
Separation of SWCNT-DSPE-PEG-IgG from protein G beads was performed by adding 10 μL of SDS aqueous solution (5 wt%) and heating to boiling (10 minutes). The fluorescence spectrum of the obtained SWCNT-DSPE-PEG-IgG eluate is shown in FIG. From the peak intensity of the fluorescence spectrum shown in FIG. 11, the recovery rate of SWCNT-DSPE-PEG-IgG is estimated to be about 90%, which means that SWCNT labeling is suitable for antigen quantification by immunoprecipitation as described above. Was confirmed.
一般に標識抗体を用いてサンプル中の抗原を検出する方法の態様の一つとして、サンプル中の抗原をプロテインＧビーズに固定した抗体により捕捉し、当該捕捉された抗原を標識抗体を用いて標識し、当該標識を検出する方法を挙げることができる。そして、このような方法では、抗原の定量は、通常、プロテインＧビーズ上に生成した抗原抗体複合体をプロテインＧビーズから溶出させ、溶液系で標識の結合量を分析することで行われる。
そこで、SWCNT標識抗体がこのような検出方法に用い得るかどうか、具体的には、プロテインＧビーズに結合したSWCNT-DSPE-PEG-IgGがプロテインＧビーズから容易に溶出でき、また、溶出したSWCNT-DSPE-PEG-IgGについて蛍光スペクトル測定が行えるかどうかを確認するため、プロテインＧビーズに結合したSWCNT-DSPE-PEG-IgGをプロテインＧビーズから引き離し、SWCNT-DSPE-PEG-IgGを溶液中に分散させ、その蛍光スペクトルを測定した。
SWCNT-DSPE-PEG-IgGのプロテインＧビーズからの引き離しは、SDS水溶液(5重量％)を10μL添加し、加熱沸騰(10分間)することにより行った。得られたSWCNT-DSPE-PEG-IgG溶出液の蛍光スペクトルを図１１に示す。図１１の蛍光スペクトルのピーク強度から、SWCNT-DSPE-PEG-IgGの回収率は90％程度と見積もられ、これによりSWCNT標識が上述のような免疫沈降法による抗原の定量に適していることが確認された。 Example 5 FIG. Confirmation that SWCNT-labeled antibody (SWCNT-DSPE-PEG-IgG) bound to protein G beads can be recovered from protein G beads In general, one of the methods for detecting antigens in samples using labeled antibodies And a method of detecting the label by capturing the captured antigen with an antibody immobilized on protein G beads, labeling the captured antigen with a labeled antibody, and the like. In such a method, the antigen is usually quantified by eluting the antigen-antibody complex produced on the protein G beads from the protein G beads and analyzing the binding amount of the label in a solution system.
Therefore, whether SWCNT-labeled antibodies can be used in such detection methods, specifically, SWCNT-DSPE-PEG-IgG bound to protein G beads can be easily eluted from protein G beads. In order to confirm whether fluorescence spectrum measurement can be performed for -DSPE-PEG-IgG, SWCNT-DSPE-PEG-IgG bound to protein G beads is pulled away from protein G beads, and SWCNT-DSPE-PEG-IgG is put into solution. After dispersion, the fluorescence spectrum was measured.
Separation of SWCNT-DSPE-PEG-IgG from protein G beads was performed by adding 10 μL of SDS aqueous solution (5 wt%) and heating to boiling (10 minutes). The fluorescence spectrum of the obtained SWCNT-DSPE-PEG-IgG eluate is shown in FIG. From the peak intensity of the fluorescence spectrum shown in FIG. 11, the recovery rate of SWCNT-DSPE-PEG-IgG is estimated to be about 90%, which means that SWCNT labeling is suitable for antigen quantification by immunoprecipitation as described above. Was confirmed.
以上、実施例１～５により、SWCNTで標識された抗体が、抗体としての活性を保持し、かつ、近赤外領域の蛍光発光特性を保持しており、ヘモグロビンなどの血液成分により妨害されることなく、抗体が特異的に結合する対象成分と結合することができ、また、その蛍光を検出することができることが示され、さらに、プロテインＧビーズなどの担体を用いる免疫沈降法による抗原の定量に適していることが示された。
As described above, according to Examples 1 to 5, the SWCNT-labeled antibody retains the activity as an antibody and retains the fluorescence emission property in the near infrared region, and is interfered by blood components such as hemoglobin. It is shown that the antibody can bind to the target component to which the antibody specifically binds, and that the fluorescence can be detected. Further, the antigen can be quantified by immunoprecipitation using a carrier such as protein G beads. It was shown to be suitable for.
実施例６．SWCNTを用いた近赤外発光による抗原抗体反応の検出
１）本実施例では、卵白アルブミン(OVA)をモデル抗原として抗原抗体反応を行い、抗原抗体反応を近赤外発光で検出する。
まず、モデル抗原に対するモノクローナル抗体を準備し、ELISAプレートに固相化する。モデル抗原を含むサンプル溶液を固相化したプレート上で、室温において2時間反応させた後に上澄を捨て、リン酸バッファーやトリスバッファーで良く洗う。次いで、SWCNTで標識したモデル抗原に対するポリクローナル抗体プローブ(SWCNT-標識抗体)をリン酸バッファーやトリスバッファー中で、室温で2時間反応させる。プレートをリン酸バッファーやトリスバッファーで良く洗うことで、未反応のSWCNT-標識抗体を、洗浄除去し、プレート上のSWCNT由来の近赤外蛍光を検出することで、抗原抗体反応の検出を行なう。
２）ELISAプレートに固相化した抗体を用いる系のほかに、プロテインＧビーズに抗体を結合させてバッチ処理で使用することも可能である。プロテインＧビーズを用いた場合、ビーズに結合しているプロテインＧと抗体のFc部との結合を介して、抗体はビーズに結合できる。抗体を固相化したビーズを、モデル抗原を含むサンプル溶液中で、室温において2時間反応させた後に上澄を捨て、リン酸バッファーやトリスバッファーで良く洗う。次いで、SWCNTで標識したモデル抗原に対するポリクローナル抗体プローブ(SWCNT-標識抗体)をリン酸バッファーやトリスバッファー中で、室温で2時間反応させる。ビーズをよく洗うことで、未反応のSWCNT-標識抗体を除去し、ビーズ上のSWCNT由来の近赤外蛍光を検出することで、抗原抗体反応の検出を行なう。 Example 6 Detection of antigen-antibody reaction by near-infrared emission using SWCNT 1) In this example, antigen-antibody reaction is performed using ovalbumin (OVA) as a model antigen, and the antigen-antibody reaction is detected by near-infrared emission.
First, a monoclonal antibody against a model antigen is prepared and immobilized on an ELISA plate. After reacting the sample solution containing the model antigen on a solid-phased plate at room temperature for 2 hours, discard the supernatant and wash well with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. The plate is washed thoroughly with phosphate buffer or Tris buffer to remove unreacted SWCNT-labeled antibody, and detection of SWCNT-derived near-infrared fluorescence on the plate detects antigen-antibody reaction. .
2) In addition to a system using an antibody immobilized on an ELISA plate, it is also possible to bind an antibody to a protein G bead and use it in batch processing. When protein G beads are used, the antibody can be bound to the beads through binding between protein G bound to the beads and the Fc part of the antibody. The antibody-immobilized beads are reacted in a sample solution containing a model antigen at room temperature for 2 hours, and then the supernatant is discarded and washed thoroughly with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. By washing the beads thoroughly, unreacted SWCNT-labeled antibodies are removed, and by detecting near-infrared fluorescence derived from SWCNTs on the beads, antigen-antibody reaction is detected.
１）本実施例では、卵白アルブミン(OVA)をモデル抗原として抗原抗体反応を行い、抗原抗体反応を近赤外発光で検出する。
まず、モデル抗原に対するモノクローナル抗体を準備し、ELISAプレートに固相化する。モデル抗原を含むサンプル溶液を固相化したプレート上で、室温において2時間反応させた後に上澄を捨て、リン酸バッファーやトリスバッファーで良く洗う。次いで、SWCNTで標識したモデル抗原に対するポリクローナル抗体プローブ(SWCNT-標識抗体)をリン酸バッファーやトリスバッファー中で、室温で2時間反応させる。プレートをリン酸バッファーやトリスバッファーで良く洗うことで、未反応のSWCNT-標識抗体を、洗浄除去し、プレート上のSWCNT由来の近赤外蛍光を検出することで、抗原抗体反応の検出を行なう。
２）ELISAプレートに固相化した抗体を用いる系のほかに、プロテインＧビーズに抗体を結合させてバッチ処理で使用することも可能である。プロテインＧビーズを用いた場合、ビーズに結合しているプロテインＧと抗体のFc部との結合を介して、抗体はビーズに結合できる。抗体を固相化したビーズを、モデル抗原を含むサンプル溶液中で、室温において2時間反応させた後に上澄を捨て、リン酸バッファーやトリスバッファーで良く洗う。次いで、SWCNTで標識したモデル抗原に対するポリクローナル抗体プローブ(SWCNT-標識抗体)をリン酸バッファーやトリスバッファー中で、室温で2時間反応させる。ビーズをよく洗うことで、未反応のSWCNT-標識抗体を除去し、ビーズ上のSWCNT由来の近赤外蛍光を検出することで、抗原抗体反応の検出を行なう。 Example 6 Detection of antigen-antibody reaction by near-infrared emission using SWCNT 1) In this example, antigen-antibody reaction is performed using ovalbumin (OVA) as a model antigen, and the antigen-antibody reaction is detected by near-infrared emission.
First, a monoclonal antibody against a model antigen is prepared and immobilized on an ELISA plate. After reacting the sample solution containing the model antigen on a solid-phased plate at room temperature for 2 hours, discard the supernatant and wash well with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. The plate is washed thoroughly with phosphate buffer or Tris buffer to remove unreacted SWCNT-labeled antibody, and detection of SWCNT-derived near-infrared fluorescence on the plate detects antigen-antibody reaction. .
2) In addition to a system using an antibody immobilized on an ELISA plate, it is also possible to bind an antibody to a protein G bead and use it in batch processing. When protein G beads are used, the antibody can be bound to the beads through binding between protein G bound to the beads and the Fc part of the antibody. The antibody-immobilized beads are reacted in a sample solution containing a model antigen at room temperature for 2 hours, and then the supernatant is discarded and washed thoroughly with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT is reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. By washing the beads thoroughly, unreacted SWCNT-labeled antibodies are removed, and by detecting near-infrared fluorescence derived from SWCNTs on the beads, antigen-antibody reaction is detected.
これらの抗原抗体反応において使用するSWCNT-標識抗体の作製は、実施例１に記載した方法に加え、以下の手順でも行うことができる。
（１）DSPE-PEGと抗体との結合
（ｉ）抗体125μgを50mMリン酸バッファー(pH7.2)6μLに溶かし、下記［化２］のDSPE-PEG 5μgを加え攪拌する。
(式中、ｎはPEG鎖の分子量が2000になる-O-CH2CH2－単位の繰り返し数を表す。)
次に、WSC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)触媒を8μg加え、4℃で10時間、スターラーで攪拌する。フィルターでろ過し、フィルター上に残った反応物をバッファーに溶かして回収する。
あるいは、
（ii）抗体125μgをNaHCO3緩衝液2.5μLに溶かす。純水5μgに下記［化１］のPEG-DSPE-NHSを溶かし、抗体溶液に添加する。ボルテックスを用いて4℃、6時間で攪拌し、反応させる。反応溶液をゲル濾過にて精製し、回収する。
(式中、ｎはPEG鎖の分子量が2000になる-O-CH2CH2－単位の繰り返し数を表す。)
（２）SWCNTの処理
まず、約1mgのカーボンナノチューブ(SWCNT)を約20mLの1重量％ドデシルベンゼンスルホン酸ナトリウム(SDBS)水溶液中に浸し、超音波破砕機によって分散させる。得られた分散液を171000gで2.5時間超遠心処理し、半透明な上澄み液(約10mL)を得る。
（３）SWCNT-標識抗体プローブの作製
このSWCNT分散液の上澄み液1mLあたり、抗体を結合させた1mgのDSPE-PEGを加え、3分間バス型超音波洗浄機にかけ、抗体結合DSPE-PEGを完全に溶解させる。その後、分子量3500以下の分子を通過させる透析膜に入れ、5日間透析作業をおこなう。この透析作業によってSDBSを抗体結合DSPE-PEGに置き換える。 In addition to the method described in Example 1, the preparation of the SWCNT-labeled antibody used in these antigen-antibody reactions can also be performed by the following procedure.
(1) Binding of DSPE-PEG with antibody (i) Dissolve 125 μg of antibody in 6 μL of 50 mM phosphate buffer (pH 7.2), add 5 μg of DSPE-PEG of the following [Chemical Formula 2] and stir.
(In the formula, n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.)
Next, 8 μg of WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) catalyst is added and stirred with a stirrer at 4 ° C. for 10 hours. Filter through a filter, and collect the reactant remaining on the filter by dissolving it in a buffer.
Or
(Ii) 125 μg of antibody is dissolved in 2.5 μL of NaHCO 3 buffer. PEG-DSPE-NHS of [Chemical Formula 1] below is dissolved in 5 μg of pure water and added to the antibody solution. Stir using a vortex at 4 ° C for 6 hours to react. The reaction solution is purified by gel filtration and collected.
(In the formula, n represents the number of repeating -O-CH2CH2- units in which the molecular weight of the PEG chain is 2000.)
(2) Treatment of SWCNT First, about 1 mg of carbon nanotubes (SWCNT) is immersed in about 20 mL of a 1 wt% sodium dodecylbenzenesulfonate (SDBS) aqueous solution and dispersed by an ultrasonic crusher. The obtained dispersion is ultracentrifuged at 171000 g for 2.5 hours to obtain a translucent supernatant (about 10 mL).
(3) Preparation of SWCNT- labeled antibody probe For 1 mL of the supernatant of this SWCNT dispersion, add 1 mg of DSPE-PEG to which antibody is bound, and apply it to a bath-type ultrasonic cleaner for 3 minutes to complete the antibody-bound DSPE-PEG. Dissolve in. After that, put it in a dialysis membrane that allows molecules with a molecular weight of 3500 or less to pass through and perform dialysis for 5 days. This dialysis operation replaces SDBS with antibody-bound DSPE-PEG.
（１）DSPE-PEGと抗体との結合
（ｉ）抗体125μgを50mMリン酸バッファー(pH7.2)6μLに溶かし、下記［化２］のDSPE-PEG 5μgを加え攪拌する。
次に、WSC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)触媒を8μg加え、4℃で10時間、スターラーで攪拌する。フィルターでろ過し、フィルター上に残った反応物をバッファーに溶かして回収する。
あるいは、
（ii）抗体125μgをNaHCO3緩衝液2.5μLに溶かす。純水5μgに下記［化１］のPEG-DSPE-NHSを溶かし、抗体溶液に添加する。ボルテックスを用いて4℃、6時間で攪拌し、反応させる。反応溶液をゲル濾過にて精製し、回収する。
（２）SWCNTの処理
まず、約1mgのカーボンナノチューブ(SWCNT)を約20mLの1重量％ドデシルベンゼンスルホン酸ナトリウム(SDBS)水溶液中に浸し、超音波破砕機によって分散させる。得られた分散液を171000gで2.5時間超遠心処理し、半透明な上澄み液(約10mL)を得る。
（３）SWCNT-標識抗体プローブの作製
このSWCNT分散液の上澄み液1mLあたり、抗体を結合させた1mgのDSPE-PEGを加え、3分間バス型超音波洗浄機にかけ、抗体結合DSPE-PEGを完全に溶解させる。その後、分子量3500以下の分子を通過させる透析膜に入れ、5日間透析作業をおこなう。この透析作業によってSDBSを抗体結合DSPE-PEGに置き換える。 In addition to the method described in Example 1, the preparation of the SWCNT-labeled antibody used in these antigen-antibody reactions can also be performed by the following procedure.
(1) Binding of DSPE-PEG with antibody (i) Dissolve 125 μg of antibody in 6 μL of 50 mM phosphate buffer (pH 7.2), add 5 μg of DSPE-PEG of the following [Chemical Formula 2] and stir.
Next, 8 μg of WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) catalyst is added and stirred with a stirrer at 4 ° C. for 10 hours. Filter through a filter, and collect the reactant remaining on the filter by dissolving it in a buffer.
Or
(Ii) 125 μg of antibody is dissolved in 2.5 μL of NaHCO 3 buffer. PEG-DSPE-NHS of [Chemical Formula 1] below is dissolved in 5 μg of pure water and added to the antibody solution. Stir using a vortex at 4 ° C for 6 hours to react. The reaction solution is purified by gel filtration and collected.
(2) Treatment of SWCNT First, about 1 mg of carbon nanotubes (SWCNT) is immersed in about 20 mL of a 1 wt% sodium dodecylbenzenesulfonate (SDBS) aqueous solution and dispersed by an ultrasonic crusher. The obtained dispersion is ultracentrifuged at 171000 g for 2.5 hours to obtain a translucent supernatant (about 10 mL).
(3) Preparation of SWCNT- labeled antibody probe For 1 mL of the supernatant of this SWCNT dispersion, add 1 mg of DSPE-PEG to which antibody is bound, and apply it to a bath-type ultrasonic cleaner for 3 minutes to complete the antibody-bound DSPE-PEG. Dissolve in. After that, put it in a dialysis membrane that allows molecules with a molecular weight of 3500 or less to pass through and perform dialysis for 5 days. This dialysis operation replaces SDBS with antibody-bound DSPE-PEG.
また、上記抗原抗体反応の詳細な手順は、以下のとおりである。
ELISAプレートでの反応系計測
PBSもしくは100mM Bicarbonate/carbonate coating bufferで20μg/mLに調整した抗OVA抗体溶液を50μL使用して、プレートを2時間室温でコートする。PBSでよく洗浄したのち、PBSに溶解した5%BSA溶液200μLをウエルに入れて、室温で30分間、ブロッキングする。PBSでよく洗浄した後、サンプルを加えて室温で2時間反応させたのち、PBSでよく洗浄する。次にPBS で20μg/mLに希釈したSWCNT-抗体プローブを室温で2時間反応させたのち、PBSでよく洗浄する。
プレートを乾かした後、発光を検出する。
磁気ビーズを用いたバッチ処理での反応系計測
Dynabeads ProteinG のプロトコールに従って、PBSで5μg/mLに調整した抗OVA抗体200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調整する。次に、モデル抗原を含むサンプル溶液500μLと室温で2時間反応させて、抗体の結合したDynabeadsで抗原を回収する。
つぎに、PBSでよく洗浄したのち、次にPBSで20μg/mLに希釈したSWCNT-抗体プローブ100μLと、室温で2時間反応させたのち、PBSでよく洗浄する。
Bufferを除いたのち、SWCNTの発光を検出する。 The detailed procedure of the antigen-antibody reaction is as follows.
Reaction system measurement on ELISA plate Using 50 μL of anti-OVA antibody solution adjusted to 20 μg / mL with PBS or 100 mM bicarbonate / carbonate coating buffer, coat the plate for 2 hours at room temperature. After thoroughly washing with PBS, 200 μL of 5% BSA solution dissolved in PBS is put into a well and blocked for 30 minutes at room temperature. After thoroughly washing with PBS, add the sample, react at room temperature for 2 hours, and then wash thoroughly with PBS. Next, the SWCNT-antibody probe diluted to 20 μg / mL with PBS is reacted at room temperature for 2 hours, and then washed thoroughly with PBS.
After the plate is dried, luminescence is detected.
Reaction system measurement in batch processing using magnetic beads According to the protocol of Dynabeads Protein G, 200 μL of anti-OVA antibody adjusted to 5 μg / mL with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes. adjust. Next, 500 μL of the sample antigen-containing sample solution is reacted at room temperature for 2 hours, and the antigen is recovered with Dynabeads to which the antibody is bound.
Next, after thoroughly washing with PBS, the mixture is reacted with 100 μL of SWCNT-antibody probe diluted to 20 μg / mL with PBS for 2 hours at room temperature, and then thoroughly washed with PBS.
After removing Buffer, SWCNT luminescence is detected.
ELISAプレートでの反応系計測
PBSもしくは100mM Bicarbonate/carbonate coating bufferで20μg/mLに調整した抗OVA抗体溶液を50μL使用して、プレートを2時間室温でコートする。PBSでよく洗浄したのち、PBSに溶解した5%BSA溶液200μLをウエルに入れて、室温で30分間、ブロッキングする。PBSでよく洗浄した後、サンプルを加えて室温で2時間反応させたのち、PBSでよく洗浄する。次にPBS で20μg/mLに希釈したSWCNT-抗体プローブを室温で2時間反応させたのち、PBSでよく洗浄する。
プレートを乾かした後、発光を検出する。
磁気ビーズを用いたバッチ処理での反応系計測
Dynabeads ProteinG のプロトコールに従って、PBSで5μg/mLに調整した抗OVA抗体200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調整する。次に、モデル抗原を含むサンプル溶液500μLと室温で2時間反応させて、抗体の結合したDynabeadsで抗原を回収する。
つぎに、PBSでよく洗浄したのち、次にPBSで20μg/mLに希釈したSWCNT-抗体プローブ100μLと、室温で2時間反応させたのち、PBSでよく洗浄する。
Bufferを除いたのち、SWCNTの発光を検出する。 The detailed procedure of the antigen-antibody reaction is as follows.
Reaction system measurement on ELISA plate Using 50 μL of anti-OVA antibody solution adjusted to 20 μg / mL with PBS or 100 mM bicarbonate / carbonate coating buffer, coat the plate for 2 hours at room temperature. After thoroughly washing with PBS, 200 μL of 5% BSA solution dissolved in PBS is put into a well and blocked for 30 minutes at room temperature. After thoroughly washing with PBS, add the sample, react at room temperature for 2 hours, and then wash thoroughly with PBS. Next, the SWCNT-antibody probe diluted to 20 μg / mL with PBS is reacted at room temperature for 2 hours, and then washed thoroughly with PBS.
After the plate is dried, luminescence is detected.
Reaction system measurement in batch processing using magnetic beads According to the protocol of Dynabeads Protein G, 200 μL of anti-OVA antibody adjusted to 5 μg / mL with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes. adjust. Next, 500 μL of the sample antigen-containing sample solution is reacted at room temperature for 2 hours, and the antigen is recovered with Dynabeads to which the antibody is bound.
Next, after thoroughly washing with PBS, the mixture is reacted with 100 μL of SWCNT-antibody probe diluted to 20 μg / mL with PBS for 2 hours at room temperature, and then thoroughly washed with PBS.
After removing Buffer, SWCNT luminescence is detected.
また、標識プローブからの発光の検出は、以下のように行う。
磁気ビーズ-抗原-SWCNT-DSPE-PEG-抗体の検出
波長660nmの光照射により、SWCNTから波長1200nm前後の発光が観測できる。その近赤外発光スペクトルをInGaAs検出器を搭載した分光システムによって測定する。
更に、蛍光測定の場合、励起光を変調することが可能であることから、変調周期に対応して同期検波を行うロックインアンプ検出法を用いて微弱光の検出レベルを、暗電流相当レベル(数pW)からショットノイズ限界(～数十fW)まで向上することが可能である。図１２に、内部増幅作用を有するInGaAs近赤外フォトトランジスタアレイの各セルにロックインアンプおよびAD変換器を接続した高感度赤外光検出アレイモジュールを磁気ビーズ-抗原-SWCNT-DSPE-PEG-抗体の検出に用いた例を示す。この実施例の場合、暗電流の影響を抑制できるため、寒剤を使用せずにフェムトワットレベルの微弱光が検出可能である。 In addition, detection of luminescence from the labeled probe is performed as follows.
By irradiating magnetic beads-antigen-SWCNT-DSPE-PEG-antibody with a detection wavelength of 660 nm, light emission at a wavelength of about 1200 nm can be observed from SWCNT. Its near-infrared emission spectrum is measured by a spectroscopic system equipped with an InGaAs detector.
Furthermore, in the case of fluorescence measurement, since excitation light can be modulated, the detection level of faint light using a lock-in amplifier detection method that performs synchronous detection corresponding to the modulation period is set to a dark current equivalent level ( It is possible to improve from a few pW) to a shot noise limit (up to several tens of fW). Figure 12 shows a high-sensitivity infrared light detection array module in which a lock-in amplifier and an AD converter are connected to each cell of an InGaAs near-infrared phototransistor array having an internal amplification function. Magnetic beads-antigen-SWCNT-DSPE-PEG- The example used for the detection of an antibody is shown. In this embodiment, since the influence of dark current can be suppressed, faint watt level faint light can be detected without using a cryogen.
磁気ビーズ-抗原-SWCNT-DSPE-PEG-抗体の検出
波長660nmの光照射により、SWCNTから波長1200nm前後の発光が観測できる。その近赤外発光スペクトルをInGaAs検出器を搭載した分光システムによって測定する。
更に、蛍光測定の場合、励起光を変調することが可能であることから、変調周期に対応して同期検波を行うロックインアンプ検出法を用いて微弱光の検出レベルを、暗電流相当レベル(数pW)からショットノイズ限界(～数十fW)まで向上することが可能である。図１２に、内部増幅作用を有するInGaAs近赤外フォトトランジスタアレイの各セルにロックインアンプおよびAD変換器を接続した高感度赤外光検出アレイモジュールを磁気ビーズ-抗原-SWCNT-DSPE-PEG-抗体の検出に用いた例を示す。この実施例の場合、暗電流の影響を抑制できるため、寒剤を使用せずにフェムトワットレベルの微弱光が検出可能である。 In addition, detection of luminescence from the labeled probe is performed as follows.
By irradiating magnetic beads-antigen-SWCNT-DSPE-PEG-antibody with a detection wavelength of 660 nm, light emission at a wavelength of about 1200 nm can be observed from SWCNT. Its near-infrared emission spectrum is measured by a spectroscopic system equipped with an InGaAs detector.
Furthermore, in the case of fluorescence measurement, since excitation light can be modulated, the detection level of faint light using a lock-in amplifier detection method that performs synchronous detection corresponding to the modulation period is set to a dark current equivalent level ( It is possible to improve from a few pW) to a shot noise limit (up to several tens of fW). Figure 12 shows a high-sensitivity infrared light detection array module in which a lock-in amplifier and an AD converter are connected to each cell of an InGaAs near-infrared phototransistor array having an internal amplification function. Magnetic beads-antigen-SWCNT-DSPE-PEG- The example used for the detection of an antibody is shown. In this embodiment, since the influence of dark current can be suppressed, faint watt level faint light can be detected without using a cryogen.
実施例７．3波長による修飾異性体検出のための装置の作製
臨床検査に於いて、血清中に存在するタンパクの翻訳後修飾の程度を測定する事により、疾病の存在や進展を検出評価して、治療方針を決定するための重要な情報を得ている。その例として、１）糖化ヘモグロビンに代表される糖化タンパクの定量的検出、および、２）胎児性がん抗原(AFP)やMUC1などの疾患に関連して出現する特徴的な糖鎖構造で修飾された糖タンパクの定量的検出を挙げる事ができる。
１）糖化タンパクは、グルコースの非酵素的な糖化反応の結果生じる物質で、ａ)ヘモグロビンAや水晶体クリスタリン等の細胞内タンパク、ｂ)アルブミン、アポタンパク、トランスフェリン、ハプトグロビンなどの血漿タンパク、ｃ)カテプシンや膵リボアーゼなどの酵素類、ｄ)血管内皮や赤血球膜タンパク質などの膜タンパクの糖化物が、その例として知られている。糖化ヘモグロビンや糖化アルブミンの検出は、耐糖能異常や糖尿病の他、肝硬変などの検出評価および診断に有効である。
２）疾患に関連した糖鎖で修飾されたタンパクとして例えば、ａ)AFPのフコシル化を検出するためLens culimaris agglutinin-A(LCA)を活用して行なわれるAFP-L3検査は精度の高い肝細胞がんの検査であるし、ｂ)Wisteria floribunda agglutinin (WFA)が反応するMUC1の検出は、胆道系に由来するがんのスクリーニングに有効であるし、ｃ)WFAの反応するM2BPの検出は、ウイルス性肝炎で進行する線維化の進行を評価するのに有効な検査であるし、ｄ)α-1 acid glycoproteinにおけるAspergillus Oryzae I-fucose specific lectin(AOL)とRicinus Communis Agglutinin (RCA-I)の結合率から、肝臓で生じる線維化を評価する事が可能であるし、ｅ)血液中のシアル化糖鎖MUC1の検出は、間質性肺炎を検出する指標となっている。
以上いずれのバイオマーカー検査も、特定のタンパクに生じる翻訳後の修飾をそれぞれ、定量的に検出測定した後、疾患に特徴的な翻訳後修飾が生じている割合を算出することで、疾患の進行度を評価しているものである。２ないし３項目の測定結果をもとに計算される理由は、一つの反応系で同時に、複数の修飾異性体タンパクを測定できないことが理由である。このような状況を鑑み、以下、１）の実施例では、糖化血清アルブミンの直接検出を、２）では糖タンパクにおける複数種の糖鎖修飾について、同時検出を行なう。 Example 7 Preparation of a device for detection of modified isomers by three wavelengths In clinical examination, the degree of posttranslational modification of proteins present in serum is measured to detect and evaluate the presence and progression of disease, and treatment policy Get important information to determine. Examples include 1) quantitative detection of glycated proteins represented by glycated hemoglobin, and 2) modification with characteristic sugar chain structures that appear in relation to diseases such as fetal cancer antigen (AFP) and MUC1. Quantitative detection of the glycoprotein produced can be mentioned.
1) Glycated protein is a substance resulting from the non-enzymatic glycation reaction of glucose, a) intracellular proteins such as hemoglobin A and lens crystallin, b) plasma proteins such as albumin, apoprotein, transferrin, haptoglobin, c) Examples thereof include enzymes such as cathepsins and pancreatic riboases, and d) glycated products of membrane proteins such as vascular endothelium and erythrocyte membrane proteins. Detection of glycated hemoglobin and glycated albumin is effective for detection evaluation and diagnosis of impaired glucose tolerance and diabetes, as well as cirrhosis of the liver.
2) As a protein modified with a sugar chain related to a disease, for example, a) AFP-L3 test using Lens culimaris agglutinin-A (LCA) to detect fucosylation of AFP is a highly accurate hepatocyte B) Detection of MUC1 to which Wisteria floribunda agglutinin (WFA) reacts is effective for screening for cancer derived from the biliary system, and c) Detection of M2BP to which WFA reacts. It is an effective test to evaluate the progression of fibrosis that progresses due to viral hepatitis. D) Aspergillus Oryzae I-fucose specific lectin (AOL) and Ricinus Communis Agglutinin (RCA-I) in α-1 acid glycoprotein Fibrosis occurring in the liver can be evaluated from the binding rate, and e) detection of sialylated sugar chain MUC1 in blood is an index for detecting interstitial pneumonia.
In any of the biomarker tests described above, each post-translational modification that occurs in a specific protein is quantitatively detected and measured, and then the ratio of the post-translational modification that is characteristic of the disease is calculated. The degree is evaluated. The reason for calculation based on the measurement results of 2 to 3 items is that a plurality of modified isomer proteins cannot be measured simultaneously in one reaction system. In view of such circumstances, in the following 1), glycated serum albumin is directly detected, and in 2), multiple types of sugar chain modifications in glycoprotein are simultaneously detected.
臨床検査に於いて、血清中に存在するタンパクの翻訳後修飾の程度を測定する事により、疾病の存在や進展を検出評価して、治療方針を決定するための重要な情報を得ている。その例として、１）糖化ヘモグロビンに代表される糖化タンパクの定量的検出、および、２）胎児性がん抗原(AFP)やMUC1などの疾患に関連して出現する特徴的な糖鎖構造で修飾された糖タンパクの定量的検出を挙げる事ができる。
１）糖化タンパクは、グルコースの非酵素的な糖化反応の結果生じる物質で、ａ)ヘモグロビンAや水晶体クリスタリン等の細胞内タンパク、ｂ)アルブミン、アポタンパク、トランスフェリン、ハプトグロビンなどの血漿タンパク、ｃ)カテプシンや膵リボアーゼなどの酵素類、ｄ)血管内皮や赤血球膜タンパク質などの膜タンパクの糖化物が、その例として知られている。糖化ヘモグロビンや糖化アルブミンの検出は、耐糖能異常や糖尿病の他、肝硬変などの検出評価および診断に有効である。
２）疾患に関連した糖鎖で修飾されたタンパクとして例えば、ａ)AFPのフコシル化を検出するためLens culimaris agglutinin-A(LCA)を活用して行なわれるAFP-L3検査は精度の高い肝細胞がんの検査であるし、ｂ)Wisteria floribunda agglutinin (WFA)が反応するMUC1の検出は、胆道系に由来するがんのスクリーニングに有効であるし、ｃ)WFAの反応するM2BPの検出は、ウイルス性肝炎で進行する線維化の進行を評価するのに有効な検査であるし、ｄ)α-1 acid glycoproteinにおけるAspergillus Oryzae I-fucose specific lectin(AOL)とRicinus Communis Agglutinin (RCA-I)の結合率から、肝臓で生じる線維化を評価する事が可能であるし、ｅ)血液中のシアル化糖鎖MUC1の検出は、間質性肺炎を検出する指標となっている。
以上いずれのバイオマーカー検査も、特定のタンパクに生じる翻訳後の修飾をそれぞれ、定量的に検出測定した後、疾患に特徴的な翻訳後修飾が生じている割合を算出することで、疾患の進行度を評価しているものである。２ないし３項目の測定結果をもとに計算される理由は、一つの反応系で同時に、複数の修飾異性体タンパクを測定できないことが理由である。このような状況を鑑み、以下、１）の実施例では、糖化血清アルブミンの直接検出を、２）では糖タンパクにおける複数種の糖鎖修飾について、同時検出を行なう。 Example 7 Preparation of a device for detection of modified isomers by three wavelengths In clinical examination, the degree of posttranslational modification of proteins present in serum is measured to detect and evaluate the presence and progression of disease, and treatment policy Get important information to determine. Examples include 1) quantitative detection of glycated proteins represented by glycated hemoglobin, and 2) modification with characteristic sugar chain structures that appear in relation to diseases such as fetal cancer antigen (AFP) and MUC1. Quantitative detection of the glycoprotein produced can be mentioned.
1) Glycated protein is a substance resulting from the non-enzymatic glycation reaction of glucose, a) intracellular proteins such as hemoglobin A and lens crystallin, b) plasma proteins such as albumin, apoprotein, transferrin, haptoglobin, c) Examples thereof include enzymes such as cathepsins and pancreatic riboases, and d) glycated products of membrane proteins such as vascular endothelium and erythrocyte membrane proteins. Detection of glycated hemoglobin and glycated albumin is effective for detection evaluation and diagnosis of impaired glucose tolerance and diabetes, as well as cirrhosis of the liver.
2) As a protein modified with a sugar chain related to a disease, for example, a) AFP-L3 test using Lens culimaris agglutinin-A (LCA) to detect fucosylation of AFP is a highly accurate hepatocyte B) Detection of MUC1 to which Wisteria floribunda agglutinin (WFA) reacts is effective for screening for cancer derived from the biliary system, and c) Detection of M2BP to which WFA reacts. It is an effective test to evaluate the progression of fibrosis that progresses due to viral hepatitis. D) Aspergillus Oryzae I-fucose specific lectin (AOL) and Ricinus Communis Agglutinin (RCA-I) in α-1 acid glycoprotein Fibrosis occurring in the liver can be evaluated from the binding rate, and e) detection of sialylated sugar chain MUC1 in blood is an index for detecting interstitial pneumonia.
In any of the biomarker tests described above, each post-translational modification that occurs in a specific protein is quantitatively detected and measured, and then the ratio of the post-translational modification that is characteristic of the disease is calculated. The degree is evaluated. The reason for calculation based on the measurement results of 2 to 3 items is that a plurality of modified isomer proteins cannot be measured simultaneously in one reaction system. In view of such circumstances, in the following 1), glycated serum albumin is directly detected, and in 2), multiple types of sugar chain modifications in glycoprotein are simultaneously detected.
１）糖化アルブミンの検出：
Dynabeads ProteinGのプロトコールに従って、PBSで5μg/mLに調整した抗ヒトアルブミン抗体200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調整する。
次に、糖化アルブミンを含む血液サンプル溶液500μLと室温で2時間反応させた後、抗体の結合したDynabeadsで抗原を回収する。よく洗浄した後、SWCNT-抗体ヒトアルブミン抗体100μgと、室温で2時間反応させたのち、PBSでよく洗浄する。Dynabeadsに結合しているSWCNT-抗体ヒトアルブミン抗体に由来するSWCNTの近赤外発光を定量的に測定して、血清中のアルブミン量を定量する。また、アルブミンに対する糖化アルブミンの比率は、1000nm-1400nmで捉えられるアルブミンと糖化アルブミンに特徴的な光吸収を利用して算出する。 1) Detection of glycated albumin:
According to the protocol of Dynabeads Protein G, 200 μL of anti-human albumin antibody adjusted to 5 μg / mL with PBS is reacted with 1.5 mg of Dynabeads Protein G for 10 minutes to prepare antibody-bound Dynabeads.
Next, after reacting with 500 μL of a blood sample solution containing glycated albumin at room temperature for 2 hours, the antigen is recovered with Dynabeads to which the antibody is bound. After washing well, react with 100 μg of SWCNT-antibody human albumin antibody at room temperature for 2 hours, and then wash well with PBS. SWCNT-antibody bound to Dynabeads Quantitatively measure the near-infrared emission of SWCNT derived from human albumin antibody to quantify the amount of albumin in serum. The ratio of glycated albumin to albumin is calculated using light absorption characteristic of albumin and glycated albumin captured at 1000 nm to 1400 nm.
Dynabeads ProteinGのプロトコールに従って、PBSで5μg/mLに調整した抗ヒトアルブミン抗体200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調整する。
次に、糖化アルブミンを含む血液サンプル溶液500μLと室温で2時間反応させた後、抗体の結合したDynabeadsで抗原を回収する。よく洗浄した後、SWCNT-抗体ヒトアルブミン抗体100μgと、室温で2時間反応させたのち、PBSでよく洗浄する。Dynabeadsに結合しているSWCNT-抗体ヒトアルブミン抗体に由来するSWCNTの近赤外発光を定量的に測定して、血清中のアルブミン量を定量する。また、アルブミンに対する糖化アルブミンの比率は、1000nm-1400nmで捉えられるアルブミンと糖化アルブミンに特徴的な光吸収を利用して算出する。 1) Detection of glycated albumin:
According to the protocol of Dynabeads Protein G, 200 μL of anti-human albumin antibody adjusted to 5 μg / mL with PBS is reacted with 1.5 mg of Dynabeads Protein G for 10 minutes to prepare antibody-bound Dynabeads.
Next, after reacting with 500 μL of a blood sample solution containing glycated albumin at room temperature for 2 hours, the antigen is recovered with Dynabeads to which the antibody is bound. After washing well, react with 100 μg of SWCNT-antibody human albumin antibody at room temperature for 2 hours, and then wash well with PBS. SWCNT-antibody bound to Dynabeads Quantitatively measure the near-infrared emission of SWCNT derived from human albumin antibody to quantify the amount of albumin in serum. The ratio of glycated albumin to albumin is calculated using light absorption characteristic of albumin and glycated albumin captured at 1000 nm to 1400 nm.
２）疾患に関連した糖鎖で修飾されたタンパクの検出：
Dynabeads ProteinGのプロトコールに従って、PBSで5μg/mLに調整した抗ヒトα-1 acid glycoprotein抗体(または、抗M2BP抗体、抗MUC1抗体)200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調製する。次に、血液サンプル溶液500μLと室温で2時間反応させた後、抗体の結合したDynabeadsで抗原を回収する。回収したDynabeadsを良く洗浄した後、Biotin化したWFAまたは、MAL-II、AOL、RCA-Iを室温で20分間反応させる。PBSで良く洗浄した後、SWCNT-付加ストレプトアビジンを反応させて、上記レクチンの結合状態を発光により測定する。
このような方法では、サンプルに特段の前処理をする必要がないので、１つの生体サンプルについて、複数のレクチンの反応性を検出することが可能である。
例えば、α-1 acid glycoproteinにみられる異なる３種類の糖鎖修飾異性体を同時に検出するためにはまず、抗ヒトα-1 acid glycoprotein抗体を結合させたDynabeadsで血液等の検体からα-1 acid glycoproteinを回収し、あらかじめそれぞれに反応させておいた励起波長の異なるSWCNT-付加ストレプトアビジンとビオチン化AOL，ビオチン化 MAL-II、ビオチン化RCA-Iを後に、同時に反応させることで、一度に３つの修飾異性体を定量評価することができる。
また、SWCNTを発光波長の異なる３種類に分離して用い、各々に検出すべき３種類の抗原に特異的な抗体を付加することにより得られた３種類のSWCNT－抗体を用いることで、３種類のタンパクに付いて検体中の存在量を決定することができる。抗ヒトα-1 acid glycoprotein抗体、抗M2BP抗体、抗MUC1抗体を結合させたDynabeadsでこれらに対する抗原を回収し、回収された抗原を検出するための検出抗体をそれぞれ、励起波長の異なるSWCNTによりラベルしておいて使用することで、同時に、３つの抗原量を定量評価することができる。 2) Detection of proteins modified with sugar chains related to diseases:
According to the Dynabeads Protein G protocol, 200 μL of anti-human α-1 acid glycoprotein antibody (or anti-M2BP antibody or anti-MUC1 antibody) adjusted to 5 μg / mL with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes. Prepare bound Dynabeads. Next, after reacting with 500 μL of the blood sample solution at room temperature for 2 hours, the antigen is recovered with Dynabeads to which the antibody is bound. The collected Dynabeads are thoroughly washed, and then biotinylated WFA or MAL-II, AOL, RCA-I is reacted at room temperature for 20 minutes. After thoroughly washing with PBS, SWCNT-added streptavidin is reacted, and the binding state of the lectin is measured by luminescence.
In such a method, since it is not necessary to perform a special pretreatment on the sample, it is possible to detect the reactivity of a plurality of lectins in one biological sample.
For example, in order to simultaneously detect three different types of sugar chain-modified isomers found in α-1 acid glycoprotein, first of all, α-1 acid glycoprotein antibody to which α-1 By collecting acid glycoproteins and reacting them in advance with SWCNT-added streptavidin with different excitation wavelengths and biotinylated AOL, biotinylated MAL-II, and biotinylated RCA-I at the same time, Three modified isomers can be quantitatively evaluated.
In addition, by using SWCNT separated into three types having different emission wavelengths, and using three types of SWCNT-antibodies obtained by adding specific antibodies to the three types of antigens to be detected, It is possible to determine the abundance in the specimen for various types of proteins. Anti-human α-1 acid glycoprotein antibody, anti-M2BP antibody, and anti-MUC1 antibody-bound Dynabeads collect the antigens against them, and the detection antibodies for detecting the recovered antigens are labeled with SWCNTs with different excitation wavelengths. By using it, the three antigen amounts can be quantitatively evaluated at the same time.
Dynabeads ProteinGのプロトコールに従って、PBSで5μg/mLに調整した抗ヒトα-1 acid glycoprotein抗体(または、抗M2BP抗体、抗MUC1抗体)200μLを、1.5mgのDynabeads ProteinGと10分間反応させて、抗体の結合したDynabeadsを調製する。次に、血液サンプル溶液500μLと室温で2時間反応させた後、抗体の結合したDynabeadsで抗原を回収する。回収したDynabeadsを良く洗浄した後、Biotin化したWFAまたは、MAL-II、AOL、RCA-Iを室温で20分間反応させる。PBSで良く洗浄した後、SWCNT-付加ストレプトアビジンを反応させて、上記レクチンの結合状態を発光により測定する。
このような方法では、サンプルに特段の前処理をする必要がないので、１つの生体サンプルについて、複数のレクチンの反応性を検出することが可能である。
例えば、α-1 acid glycoproteinにみられる異なる３種類の糖鎖修飾異性体を同時に検出するためにはまず、抗ヒトα-1 acid glycoprotein抗体を結合させたDynabeadsで血液等の検体からα-1 acid glycoproteinを回収し、あらかじめそれぞれに反応させておいた励起波長の異なるSWCNT-付加ストレプトアビジンとビオチン化AOL，ビオチン化 MAL-II、ビオチン化RCA-Iを後に、同時に反応させることで、一度に３つの修飾異性体を定量評価することができる。
また、SWCNTを発光波長の異なる３種類に分離して用い、各々に検出すべき３種類の抗原に特異的な抗体を付加することにより得られた３種類のSWCNT－抗体を用いることで、３種類のタンパクに付いて検体中の存在量を決定することができる。抗ヒトα-1 acid glycoprotein抗体、抗M2BP抗体、抗MUC1抗体を結合させたDynabeadsでこれらに対する抗原を回収し、回収された抗原を検出するための検出抗体をそれぞれ、励起波長の異なるSWCNTによりラベルしておいて使用することで、同時に、３つの抗原量を定量評価することができる。 2) Detection of proteins modified with sugar chains related to diseases:
According to the Dynabeads Protein G protocol, 200 μL of anti-human α-1 acid glycoprotein antibody (or anti-M2BP antibody or anti-MUC1 antibody) adjusted to 5 μg / mL with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes. Prepare bound Dynabeads. Next, after reacting with 500 μL of the blood sample solution at room temperature for 2 hours, the antigen is recovered with Dynabeads to which the antibody is bound. The collected Dynabeads are thoroughly washed, and then biotinylated WFA or MAL-II, AOL, RCA-I is reacted at room temperature for 20 minutes. After thoroughly washing with PBS, SWCNT-added streptavidin is reacted, and the binding state of the lectin is measured by luminescence.
In such a method, since it is not necessary to perform a special pretreatment on the sample, it is possible to detect the reactivity of a plurality of lectins in one biological sample.
For example, in order to simultaneously detect three different types of sugar chain-modified isomers found in α-1 acid glycoprotein, first of all, α-1 acid glycoprotein antibody to which α-1 By collecting acid glycoproteins and reacting them in advance with SWCNT-added streptavidin with different excitation wavelengths and biotinylated AOL, biotinylated MAL-II, and biotinylated RCA-I at the same time, Three modified isomers can be quantitatively evaluated.
In addition, by using SWCNT separated into three types having different emission wavelengths, and using three types of SWCNT-antibodies obtained by adding specific antibodies to the three types of antigens to be detected, It is possible to determine the abundance in the specimen for various types of proteins. Anti-human α-1 acid glycoprotein antibody, anti-M2BP antibody, and anti-MUC1 antibody-bound Dynabeads collect the antigens against them, and the detection antibodies for detecting the recovered antigens are labeled with SWCNTs with different excitation wavelengths. By using it, the three antigen amounts can be quantitatively evaluated at the same time.
図１３は、小型近赤外分光器を装着した全血分光蛍光装置の断面模式図である。SWCNT赤外蛍光標識を付加された全血サンプルに対して発光ダイオードにより間欠的に近赤外光を照射すると、SWCNTは、赤外波長帯域において、発光ダイオード光に同期して蛍光を発する。この蛍光を、ロウパスフィルターにより照明光波長成分を除去し、凹面グレーティングにより、近赤外光検出アレイに分光しつつ集光する。同図では、それぞれ分光波長帯域の異なる３系統の分光計測システムを配置し、同一サンプルにおいて、同時に３つの波長帯における蛍光および吸収を測定する。
FIG. 13 is a schematic cross-sectional view of a whole blood spectroscopic fluorescence apparatus equipped with a small near infrared spectrometer. When the whole blood sample to which the SWCNT infrared fluorescent label is added is intermittently irradiated with near infrared light by a light emitting diode, the SWCNT emits fluorescence in synchronization with the light emitting diode light in the infrared wavelength band. The fluorescent light is condensed while removing the wavelength component of the illumination light with a low-pass filter and spectrally splitting into the near-infrared light detection array with the concave grating. In the figure, three systems of spectroscopic measurement systems having different spectral wavelength bands are arranged, and fluorescence and absorption in three wavelength bands are measured simultaneously in the same sample.
現在のバイオマーカーの検出は、シグナル検出に利用する光波長の制限があるため、臨床検査では通常、ｉ）採血した全血から血球成分(赤血球や白血球、血小板)を除いた血漿、または、ii）血液を凝固させて血球成分と線維素を除いた血清をサンプルとして測定する。フィルター濾過により除去する方法も取り入れられているが、いずれの操作でもこれらの成分が除ききれない他、操作で生じる赤血球溶血によるヘモグロビンの混入がおこる。これらの生体由来物質混入は、疾患バイオマーカー検出評価で利用する発光(蛍光)を妨害吸収してしまい疾病検出感度を下げてしまう。同時に、血球成分が非特異的に発光し、プローブ発光検出の際にノイズの原因ともなる。こうした血球成分によるプローブ発光の妨害吸収や自家発光ノイズは、早期診断を目的とした疾患バイオマーカーの高感度検出を邪魔し、その実用化において非常に大きな障害となっている。
図１４は、図１３で示した装置により測定されたスペクトラムを、従来方法におけるスペクトラムと比較したものである。従来技術では、蛍光標識の発光波長は、高々800nmであるため、そのままではヘモグロビンの自家発光スペクトラムがバックグラウンド光として標識による蛍光を覆ってしまうので、まず、遠心分離器など大型の前処理装置により、赤血球を取り除く必要がある。また、蛍光標識の発光波長域が狭いので、通常１回の検出には１波長のみを使用するため、複数の抗原を検出するためには、それと同数の検体を用意する必要がある。
一方、本発明による蛍光検出装置では、ヘモグロビンによる発光帯よりも長波長における蛍光を高感度にて検出することができるため、サンプルは全血のままで解析装置に導入することができ、煩雑な前処理が不要であること、これにより遠心分離やフィルタリングの際の溶血などの影響が無いこと、その結果感度が数十倍向上すること、などの特徴がある。
また、肝機能、癌マーカーに加え、波長2μmまでの光の吸収を計測することにより、生活習慣病の監視に不可欠な血糖値の計測が安価に可能である。
さらにまた、可視、赤外の複数の発光帯で分光計測を行うことが可能であり、数種類の抗原をまとめて１つの反応ポットで検出を行うことができる。その結果、検体必要量が少ない、ランニングコストが安いなどの特徴がある。
図１５は、小型分光器の代わりに、発光ダイオードとフィルターおよび単体の受光素子を３組配置した例を示す。この装置は、構造が簡単で安価であるため、診療所や個人病院における臨床検査に使用できる。
図１６は、本発明で説明した検体分注、抗体添加、磁気ビーズ洗浄などの検体の処理を自動的に処理し、近赤外波長にて微弱蛍光を検出する装置の模式図を示す。比較的簡単な処理により、ヘモグロビンによる背景光を除去できる。 Since detection of current biomarkers is limited by the light wavelength used for signal detection, in clinical examinations, i) plasma obtained by removing blood cell components (red blood cells, white blood cells, and platelets) from collected blood, or ii ) Serum obtained by coagulating blood to remove blood cell components and fibrin is measured as a sample. Although the method of removing by filter filtration is also taken in, these components cannot be removed by any operation, and hemoglobin is mixed by erythrocyte hemolysis caused by the operation. The contamination of these bio-derived substances obstructs and absorbs the luminescence (fluorescence) used in the disease biomarker detection evaluation and lowers the disease detection sensitivity. At the same time, blood cell components emit light non-specifically, which causes noise when detecting probe luminescence. Such interference and absorption of probe luminescence by autologous blood cell components and self-luminous noise interfere with high-sensitivity detection of disease biomarkers for the purpose of early diagnosis, and are very serious obstacles to their practical application.
FIG. 14 compares the spectrum measured by the apparatus shown in FIG. 13 with the spectrum in the conventional method. In the prior art, since the emission wavelength of the fluorescent label is at most 800 nm, the self-emission spectrum of hemoglobin will cover the fluorescence due to the label as background light, so first of all, using a large pretreatment device such as a centrifuge Need to remove red blood cells. In addition, since the emission wavelength range of the fluorescent label is narrow, normally only one wavelength is used for one detection, and therefore it is necessary to prepare the same number of specimens in order to detect a plurality of antigens.
On the other hand, the fluorescence detection device according to the present invention can detect fluorescence at a wavelength longer than the emission band of hemoglobin with high sensitivity, so that the sample can be introduced into the analysis device as whole blood, which is complicated. There is a feature that pre-treatment is unnecessary, that there is no influence of hemolysis or the like at the time of centrifugation or filtering, and as a result, sensitivity is improved by several tens of times.
In addition to liver function and cancer markers, by measuring light absorption up to a wavelength of 2 μm, it is possible to inexpensively measure blood glucose levels that are essential for monitoring lifestyle-related diseases.
Furthermore, spectroscopic measurement can be performed in a plurality of visible and infrared emission bands, and several types of antigens can be collected and detected in one reaction pot. As a result, there are features such as a small amount of specimen required and a low running cost.
FIG. 15 shows an example in which three sets of light emitting diodes, filters, and a single light receiving element are arranged instead of a small spectroscope. Since this device is simple and inexpensive, it can be used for clinical examinations in clinics and private hospitals.
FIG. 16 shows a schematic diagram of an apparatus for automatically processing the sample processing such as sample dispensing, antibody addition, and magnetic bead washing described in the present invention, and detecting weak fluorescence at near infrared wavelengths. Background light caused by hemoglobin can be removed by a relatively simple process.
図１４は、図１３で示した装置により測定されたスペクトラムを、従来方法におけるスペクトラムと比較したものである。従来技術では、蛍光標識の発光波長は、高々800nmであるため、そのままではヘモグロビンの自家発光スペクトラムがバックグラウンド光として標識による蛍光を覆ってしまうので、まず、遠心分離器など大型の前処理装置により、赤血球を取り除く必要がある。また、蛍光標識の発光波長域が狭いので、通常１回の検出には１波長のみを使用するため、複数の抗原を検出するためには、それと同数の検体を用意する必要がある。
一方、本発明による蛍光検出装置では、ヘモグロビンによる発光帯よりも長波長における蛍光を高感度にて検出することができるため、サンプルは全血のままで解析装置に導入することができ、煩雑な前処理が不要であること、これにより遠心分離やフィルタリングの際の溶血などの影響が無いこと、その結果感度が数十倍向上すること、などの特徴がある。
また、肝機能、癌マーカーに加え、波長2μmまでの光の吸収を計測することにより、生活習慣病の監視に不可欠な血糖値の計測が安価に可能である。
さらにまた、可視、赤外の複数の発光帯で分光計測を行うことが可能であり、数種類の抗原をまとめて１つの反応ポットで検出を行うことができる。その結果、検体必要量が少ない、ランニングコストが安いなどの特徴がある。
図１５は、小型分光器の代わりに、発光ダイオードとフィルターおよび単体の受光素子を３組配置した例を示す。この装置は、構造が簡単で安価であるため、診療所や個人病院における臨床検査に使用できる。
図１６は、本発明で説明した検体分注、抗体添加、磁気ビーズ洗浄などの検体の処理を自動的に処理し、近赤外波長にて微弱蛍光を検出する装置の模式図を示す。比較的簡単な処理により、ヘモグロビンによる背景光を除去できる。 Since detection of current biomarkers is limited by the light wavelength used for signal detection, in clinical examinations, i) plasma obtained by removing blood cell components (red blood cells, white blood cells, and platelets) from collected blood, or ii ) Serum obtained by coagulating blood to remove blood cell components and fibrin is measured as a sample. Although the method of removing by filter filtration is also taken in, these components cannot be removed by any operation, and hemoglobin is mixed by erythrocyte hemolysis caused by the operation. The contamination of these bio-derived substances obstructs and absorbs the luminescence (fluorescence) used in the disease biomarker detection evaluation and lowers the disease detection sensitivity. At the same time, blood cell components emit light non-specifically, which causes noise when detecting probe luminescence. Such interference and absorption of probe luminescence by autologous blood cell components and self-luminous noise interfere with high-sensitivity detection of disease biomarkers for the purpose of early diagnosis, and are very serious obstacles to their practical application.
FIG. 14 compares the spectrum measured by the apparatus shown in FIG. 13 with the spectrum in the conventional method. In the prior art, since the emission wavelength of the fluorescent label is at most 800 nm, the self-emission spectrum of hemoglobin will cover the fluorescence due to the label as background light, so first of all, using a large pretreatment device such as a centrifuge Need to remove red blood cells. In addition, since the emission wavelength range of the fluorescent label is narrow, normally only one wavelength is used for one detection, and therefore it is necessary to prepare the same number of specimens in order to detect a plurality of antigens.
On the other hand, the fluorescence detection device according to the present invention can detect fluorescence at a wavelength longer than the emission band of hemoglobin with high sensitivity, so that the sample can be introduced into the analysis device as whole blood, which is complicated. There is a feature that pre-treatment is unnecessary, that there is no influence of hemolysis or the like at the time of centrifugation or filtering, and as a result, sensitivity is improved by several tens of times.
In addition to liver function and cancer markers, by measuring light absorption up to a wavelength of 2 μm, it is possible to inexpensively measure blood glucose levels that are essential for monitoring lifestyle-related diseases.
Furthermore, spectroscopic measurement can be performed in a plurality of visible and infrared emission bands, and several types of antigens can be collected and detected in one reaction pot. As a result, there are features such as a small amount of specimen required and a low running cost.
FIG. 15 shows an example in which three sets of light emitting diodes, filters, and a single light receiving element are arranged instead of a small spectroscope. Since this device is simple and inexpensive, it can be used for clinical examinations in clinics and private hospitals.
FIG. 16 shows a schematic diagram of an apparatus for automatically processing the sample processing such as sample dispensing, antibody addition, and magnetic bead washing described in the present invention, and detecting weak fluorescence at near infrared wavelengths. Background light caused by hemoglobin can be removed by a relatively simple process.
Claims (17)
- ナノカーボンの特異な光学的性質を利用することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical examination method, which uses the unique optical properties of nanocarbon.
- 波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンを吸光または発光標識として用いることを特徴とする、請求項１に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 2. The antigen-antibody reaction or between sugar chain lectins according to claim 1, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by photoexcitation at a wavelength of 450-1350 nm are used as an absorption or emission label. Method of measuring the response of or the method of clinical examination.
- ナノカーボンを含有する吸光または発光標識。 An absorption or emission label containing nanocarbon.
- ナノカーボンが、波長450-1350nmの光励起により波長700-2300nmで発光する単層カーボンナノチューブ(SWCNT)あるいはナノグラフェンであることを特徴とする、請求項３に記載の吸光または発光標識。 The light-absorbing or light-emitting label according to claim 3, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 700-2300 nm by photoexcitation at a wavelength of 450-1350 nm.
- 特定の抗原を認識する抗体または特定の糖鎖を認識するレクチンにナノカーボンを結合させて成り、当該特定の抗原との抗原抗体反応または当該特定の糖鎖とレクチンとの反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、請求項３または４に記載の吸光または発光標識。 Nanocarbon is bound to an antibody that recognizes a specific antigen or a lectin that recognizes a specific sugar chain, and the antigen-antibody reaction with the specific antigen or the reaction between the specific sugar chain and the lectin is the fluorescence of the nanocarbon. 5. The light-absorbing or light-emitting label according to claim 3 or 4, which is used for detection by measurement of Raman light and / or light absorption.
- ストレプトアビジンまたはストレプトアビジンの関連分子にナノカーボンを結合させて成り、特定の抗原に結合したビオチン化抗体または特定の糖鎖と結合したビオチン化レクチンとストレプトアビジンの反応物を、ナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、請求項３または４に記載の吸光または発光標識。 Streptavidin or related molecules of streptavidin are combined with nanocarbon, biotinylated antibody bound to a specific antigen or biotinylated lectin bound to a specific sugar chain and the reaction product of streptavidin, the fluorescence of nanocarbon, The light-absorbing or light-emitting label according to claim 3 or 4, which is used for detection by measurement of Raman light and / or light absorption.
- イムノグロブリン結合タンパクにナノカーボンを結合させて成り、特定の抗原に結合した抗体と当該イムノグロブリン結合タンパクの結合反応をナノカーボンの蛍光、ラマン光、および／または、光吸収の計測により検出するために用いることを特徴とする、請求項３または４に記載の吸光または発光標識。 To detect the binding reaction between an antibody bound to a specific antigen and the immunoglobulin binding protein by measuring the fluorescence, Raman light, and / or light absorption of the nanobinding carbon. The light-absorbing or light-emitting label according to claim 3 or 4, wherein the light-absorbing or light-emitting label is used.
- イムノグロブリン結合タンパクがプロテインＧまたはプロテインＡであることを特徴とする、請求項７に記載の吸光または発光標識。 The light-absorbing or light-emitting label according to claim 7, wherein the immunoglobulin-binding protein is protein G or protein A.
- 特定の抗原または特定の糖鎖が、正常もしくは病的な状態になった際、血液や体腔液中に存在する各種の抗原または糖鎖であることを特徴とする、請求項５～８のいずれか１項に記載の吸光または発光標識。 9. The antigen according to claim 5, wherein the specific antigen or the specific sugar chain is various antigens or sugar chains present in blood or body cavity fluid when normal or pathological condition is reached. The light-absorbing or luminescent label according to claim 1.
- 担体に結合させた各種抗原に対する抗体をもちいて抗原を担体に固定化することにより、抗原がエンリッチ化したサンプルを作成し、当該サンプルについて、担体上の抗原をナノカーボン標識抗体により標識した後、または、担体上の抗原の有する糖鎖をナノカーボン標識レクチンにより標識した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより抗原または抗原の有する糖鎖を検出することを特徴とする、請求項１または２に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 By immobilizing the antigen to the carrier using antibodies against various antigens bound to the carrier, an antigen-enriched sample is created, and for the sample, the antigen on the carrier is labeled with a nanocarbon-labeled antibody, Alternatively, after the sugar chain of the antigen on the carrier is labeled with nanocarbon-labeled lectin, the near-infrared light absorption, near-infrared light emission, or near-infrared Raman scattered light of the nanocarbon is measured to measure the antigen or antigen. 3. A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to claim 1, wherein the sugar chain is detected.
- 担体が磁気ビーズ、セファロースまたはセファデックスであることを特徴とする、請求項１０に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 The method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to claim 10, wherein the carrier is magnetic beads, Sepharose or Sephadex.
- 抗原のナノカーボン標識抗体による標識が、当該抗原に、当該抗原を認識し、ナノカーボンで標識された請求項５に記載の抗体を結合させるか、あるいは、当該抗原を認識し、ビオチン化された抗体を結合させ、請求項６に記載のナノカーボン標識で標識するか、もしくは、当該抗原を認識する抗体を結合させ、請求項７または８に記載のナノカーボン標識で標識することにより行われ、抗原の有する糖鎖のナノカーボン標識レクチンによる標識が、当該抗原の有する糖鎖に、当該抗原の有する糖鎖を認識し、ナノカーボンで標識された請求項５に記載のレクチンを結合させるか、あるいは、当該抗原を認識し、ビオチン化されたレクチンを結合させ、請求項６に記載のナノカーボン標識で標識することにより行われることを特徴とする、請求項１０または１１に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 6. Labeling of the antigen with a nanocarbon-labeled antibody recognizes the antigen to the antigen and binds the antibody according to claim 5 labeled with nanocarbon, or recognizes the antigen and is biotinylated It is performed by binding an antibody and labeling with the nanocarbon label according to claim 6, or by binding an antibody that recognizes the antigen and labeling with the nanocarbon label according to claim 7 or 8, Labeling the sugar chain of the antigen with the nanocarbon-labeled lectin recognizes the sugar chain of the antigen to the sugar chain of the antigen and binds the lectin according to claim 5 labeled with nanocarbon, Alternatively, it is carried out by recognizing the antigen, binding a biotinylated lectin, and labeling with the nanocarbon label according to claim 6, Measurement method or clinical examination methods of the reaction between the antigen-antibody reaction or a sugar chain-lectin according to Motomeko 10 or 11.
- 疾病マーカーである抗原または糖鎖をナノカーボン標識抗体またはナノカーボン標識レクチンにより標識し、電気泳動により捕集した後に、ナノカーボンの近赤外光吸収、近赤外発光、もしくは近赤外ラマン散乱光を計測することにより当該抗原または糖鎖を検出することを特徴とする、請求項１または２に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 After labeling a disease marker antigen or sugar chain with a nanocarbon-labeled antibody or nanocarbon-labeled lectin and collecting it by electrophoresis, the near-infrared light absorption, near-infrared light emission, or near-infrared Raman scattering of the nanocarbon 3. The method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical examination method according to claim 1, wherein the antigen or sugar chain is detected by measuring light.
- 単一検体において、請求項１、２または請求項９～１３に記載のナノカーボンを用いる吸光または発光を利用した抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法、あるいはこれと他の吸光または発光を利用した測定方法あるいは臨床検査方法とを組み合わせて、異なった吸光または発光波長帯にて複数の抗原および／または糖鎖を検出することを特徴とする、抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 In a single sample, a method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins using a nanocarbon according to claim 1, 2, or 9 to 13, or a reaction between sugar chain lectins, a clinical test method, and An antigen-antibody reaction or saccharide characterized by detecting a plurality of antigens and / or sugar chains in different light absorption or emission wavelength bands in combination with another measurement method using light absorption or luminescence or a clinical examination method A method for measuring a reaction between chain lectins or a clinical test method.
- 吸光または発光波長帯が、紫外、可視、近赤外および／または赤外領域に及ぶ、請求項１４に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 The method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins or a clinical test method according to claim 14, wherein the light absorption or emission wavelength band covers the ultraviolet, visible, near infrared and / or infrared region.
- 単一検体において、異なった吸光または発光波長帯における複数の抗原および／または糖鎖からの吸光または発光強度比を用いて、複数の抗原および／または糖鎖の定量を行うことを特徴とする、請求項１４または１５に記載の抗原抗体反応または糖鎖レクチン間の反応の測定方法あるいは臨床検査方法。 Quantifying a plurality of antigens and / or sugar chains using a ratio of absorbance or emission intensity from a plurality of antigens and / or sugar chains in different absorbance or emission wavelength bands in a single specimen, A method for measuring an antigen-antibody reaction according to claim 14 or a reaction between sugar chain lectins or a method for clinical examination.
- 請求項１５または１６の紫外、可視、近赤外、赤外発光領域の計測において、単一あるいは複数の励起光を変調して、それに同期して吸光または発光を検出する、ロックインアンプ光検出法を用いた抗原抗体反応または糖鎖レクチン間の反応の測定あるいは臨床検査装置。 17. Lock-in-amplifier light detection, wherein in the measurement of the ultraviolet, visible, near-infrared, or infrared light emitting region according to claim 15 or 16, single or plural excitation light is modulated and light absorption or light emission is detected in synchronization therewith. A method for measuring an antigen-antibody reaction or a reaction between sugar chain lectins using a method, or a clinical test apparatus.
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