WO2019182130A1 - 標識化抗体分散液、spfs用キット - Google Patents
標識化抗体分散液、spfs用キット Download PDFInfo
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- WO2019182130A1 WO2019182130A1 PCT/JP2019/012158 JP2019012158W WO2019182130A1 WO 2019182130 A1 WO2019182130 A1 WO 2019182130A1 JP 2019012158 W JP2019012158 W JP 2019012158W WO 2019182130 A1 WO2019182130 A1 WO 2019182130A1
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- antibody
- labeled antibody
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
Definitions
- the present invention relates to a labeled antibody dispersion and a SPFS kit.
- biomarkers eg, antigens such as specific proteins contained in blood, urine, and other biological samples (specimens) of humans and animals are detected to detect diseases early. There is a wide range of research to quantify.
- biomarker contained in a specimen is extremely small, and accuracy is required for its detection in a clinical field. Therefore, immunoassay methods for accurate detection and quantification of biomarkers have been studied.
- the immunoassay method includes a sandwich assay using an antibody for capturing an antigen (biomarker) and an antibody in which a labeling substance and an antibody are combined to detect the antigen (also referred to as a labeled antibody in the present invention),
- a radioassay using a label with a radioactive substance has been widely practiced.
- a sandwich assay is excellent in detecting a very small amount of antigen, and thus is a useful method for detecting and quantifying a biomarker.
- surface plasmon excitation enhanced fluorescence spectroscopy which is a method that can detect analytes with high accuracy by applying surface plasmon resonance phenomenon using binding of ligand and analyte.
- SPFS Surface Plasmon-field enhanced Fluorescence Spectroscopy
- SPFS is a method for quantifying an analyte, which is a substance that binds to a ligand, using a sensor chip on which the ligand is immobilized.
- surface plasmon light (coherent wave) is generated on the surface of the metal thin film under the condition that excitation light such as laser light emitted from the light source is attenuated and totally reflected (ATR) on the surface of the metal thin film on the sensor chip.
- excitation light such as laser light emitted from the light source is attenuated and totally reflected (ATR) on the surface of the metal thin film on the sensor chip.
- an analyte for example, an antigen
- an antibody labeled with a fluorescent substance that specifically binds to the analyte a labeled anti-antibody, wherein the labeling substance is a fluorescent substance; hereinafter also referred to as a fluorescently labeled antibody
- the fluorescently labeled fluorescent substance bound to the analyte trapped on the metal thin film on the sensor chip by the ligand is efficiently excited by the enhanced localized field light, so the fluorescent signal derived from this fluorescent substance is detected. By doing so, it is possible to detect an extremely small amount and an extremely low concentration of the analyte.
- Patent Document 1 research for preventing protein aggregation in protein-containing preparations such as antibodies using polyoxyethylene (POE) sorbitan and polyethylene glycol (PEG) has been made (for example, Patent Document 1). What is being conducted is a study on a normal antibody that is not labeled, and its aggregation suppressing effect is not sufficient. In addition, no studies on labeled antibodies have been made in this document.
- POE polyoxyethylene
- PEG polyethylene glycol
- the present inventors have intensively studied a labeled antibody in which a labeling substance is bound to a thiol group generated by cleaving a disulfide bond contained in an antibody molecule.
- a labeled antibody tend to agglomerate and precipitate in a dispersion because the three-dimensional structure becomes unstable due to cleavage of disulfide bonds, which is important for maintaining the three-dimensional structure of the antibody. It has been found that the storage stability is lower than that of the dispersed dispersion.
- the present invention has an object to provide a labeled antibody dispersion in which a labeled antibody is suitably dispersed, and a SPFS kit including the dispersion.
- the present invention provides labeled antibody dispersions and SPFS kits shown, for example, in [1] to [8] below.
- a labeled antibody dispersion liquid comprising a labeled antibody obtained by cleaving a part of disulfide bonds of an antibody molecule and binding a labeling substance to a thiol group generated by the cleavage, and a nonionic surfactant.
- the nonionic surfactant is a polyoxyethylene-based surfactant.
- the labeled antibody dispersion liquid according to one.
- the labeling substance is a fluorescent dye, a fluorescent nanoparticle, an aggregation-induced luminescent molecule, an enzyme / coenzyme, a chemiluminescent substance, or a radioactive substance.
- Labeled antibody dispersion Any one of [1] to [6], wherein the antibody molecule is an anti-troponin I (cTnI) antibody, an anti-troponin T (cTnT) antibody, an anti-BNP antibody, or an anti-D-dimer antibody
- the labeled antibody dispersion according to the description.
- a SPFS kit comprising the labeled antibody dispersion according to any one of [1] to [7] and a sensor chip dedicated to surface plasmon excitation enhanced fluorescence spectroscopy (SPFS).
- SPFS surface plasmon excitation enhanced fluorescence spectroscopy
- a labeled antibody dispersion and a SPFS kit with high storage stability can be provided.
- FIG. 1 is a graph showing the results of comparing the initial performance due to the difference in the surfactant in the labeled antibody dispersion.
- the vertical axis represents the S / N ratio, and the horizontal axis represents the type of surfactant.
- FIG. 2 is a graph showing the results of comparison of storage stability depending on the surfactant in the labeled antibody dispersion.
- the vertical axis shows the blank increase rate (%) after storage at 30 ° C. for 5 days, and the horizontal axis shows the type of surfactant.
- FIG. 3 is a graph showing the variation rate (%) of the blank value when the labeled antibody dispersions in Example 3-1 and Comparative Example 3-1 were stored at 4 ° C. for 0 to 29 days.
- FIG. 4 is a graph showing the fluctuation rate (%) of the signal value when the labeled antibody dispersions in Example 3-2 and Comparative Example 3-2 were stored at 4 ° C. for 0 to 29 days.
- the vertical axis shows the rate of change (%) of the signal value, and the horizontal axis shows the storage days.
- FIG. 5 is a graph showing the variation rate (%) of the blank value when the labeled antibody dispersions in Example 3-3 and Comparative Example 3-3 are stored at 30 ° C. for 0 to 29 days.
- the vertical axis shows the fluctuation rate (%) of the blank value, and the horizontal axis shows the storage days.
- Example 6 is a graph showing the fluctuation rate (%) of the signal value when the labeled antibody dispersions in Example 3-4 and Comparative Example 3-4 are stored at 30 ° C. for 0 to 29 days.
- the vertical axis shows the rate of change (%) of the signal value, and the horizontal axis shows the storage days.
- the labeled antibody dispersion of the present invention is a label obtained by cleaving a part of disulfide bonds (—S—S—) of an antibody molecule and binding a labeling substance to a thiol group (—SH HS—) generated by this cleavage.
- the labeled antibody dispersion of the present invention can be used for immunoassay such as sandwich assay.
- a sandwich assay is a method in which a substance that specifically binds to a detection target substance is immobilized in advance in a measurement region such as a well plate or a sensor chip, captures the detection target substance, and then is specific to the detection target substance.
- This is a method of detecting using a labeling substance in which a labeling substance is bound to a substance that binds automatically.
- a sandwich immunoassay is performed using a protein (antigen) as a detection target substance, an antibody against the detection target substance as a substance that specifically binds to the detection target substance, and a labeled antibody as a labeling substance. It is done.
- an anti-cardiac troponin I antibody (anti-cTnI antibody) can be used as the antibody to be immobilized.
- an anti-cardiac troponin I antibody (anti-cTnI antibody)
- an anti-cardiac troponin I antibody (anti-cTnI antibody)
- the labeled antibody a labeled antibody obtained by binding a labeling substance to an anti-cTnI antibody can be used.
- the anti-cTnI antibody used as a supplementary substance and the anti-cTnI antibody used as a labeled antibody are preferably antibodies that recognize different epitopes on cTnI.
- the labeled antibody is not necessarily a primary antibody, and may be an n-order antibody such as a secondary antibody or a tertiary antibody (n is an integer of 2 or more, preferably an integer of 2 to 4).
- n is an integer of 2 or more, preferably an integer of 2 to 4.
- the labeled antibody is preferably an anti-IgG antibody that specifically recognizes the anti-cTnI antibody.
- a fluorescence measurement method such as SPFS method, which is often performed when detecting a very small amount of a detection target substance (antigen)
- SPFS method the detection target substance captured by the supplementary substance is fluorescently labeled on the surface of the measurement region of the sensor chip, and the antigen is measured by detecting the fluorescent signal.
- a fluorescent labeled antibody in which a fluorescent substance and an antibody are combined may be used.
- a dispersion of fluorescently labeled antibodies is referred to as a fluorescently labeled antibody dispersion.
- the present inventors cleaved a disulfide bond in a part of an antibody molecule, and a labeled antibody in which a labeling substance is bound to a thiol group generated by this cleavage is dispersed in a nonionic surfactant to label it. It has been found that the labeling substance is not dropped from the labeled antibody, the labeled antibody is fragmented, and aggregation, precipitation, oxidation, denaturation, etc. are less likely to occur, and as a result, an increase in the blank value in the measurement by the SPFS method is suppressed. It was.
- the fluctuation rate of the signal value in the measurement by the SPFS method before and after storage may be within ⁇ 20%. preferable.
- the signal value is a value of the amount of fluorescence obtained by measuring a specimen containing a detection target substance (antigen) when performing a fluorescence measurement method such as SPFS method.
- the blank value is a value of the fluorescence amount obtained by measuring a specimen that does not contain the detection target substance (antigen).
- the medium of the labeled antibody dispersion of the present invention is preferably a buffer solution from the viewpoint of pH stabilization.
- a buffer solution acetate buffer solution, phosphate buffer solution, Tris buffer solution, HEPES buffer solution, citrate buffer solution, citrate phosphate buffer solution, borate buffer solution and the like can be mentioned.
- Buffers include phosphate buffered saline, Tris buffered solution, and HEPES buffered solution. Phosphate buffered saline (PBS), Tris buffered saline (TBS), and HEPES buffered saline that are almost isotonic with body fluids. Is preferred.
- the labeled antibody dispersion of the present invention may contain a metal salt.
- a metal salt for example, sodium chloride and potassium chloride are preferable because they are components contained in blood and the like that can be measured.
- the labeled antibody dispersion of the present invention preferably contains a labeled antibody at 0.5 to 10 ⁇ g / mL, more preferably 1.0 to 5.0 ⁇ g / mL.
- the labeled antibody dispersion of the present invention contains a labeled antibody in which a labeling substance and an antibody are bound.
- the labeled antibody includes a labeled antibody obtained by cleaving a disulfide bond in a part of an antibody molecule and binding a labeling substance to a thiol group generated by the cleavage.
- the labeled antibody used in the present invention is obtained by binding a labeling substance to a thiol group generated by cleaving a part of disulfide bonds important for maintaining the three-dimensional structure of the antibody among the disulfide bonds of the antibody.
- a labeled antibody as described above is unstable in three-dimensional structure, and thus causes aggregation or precipitation when it is fragmented or the light chain and the labeling substance are dropped.
- it is usually preferable that a thiol group produced by cleaving a disulfide bond constituting the hinge part of the antibody is bound to a labeling substance.
- a thiol group having a disulfide bond other than the hinge portion cleaved and a labeling substance may be bonded.
- the labeling substance when the labeling substance is a substance that emits fluorescence, such as a fluorescent dye described later, the labeling substance may be referred to as a fluorescent labeling substance.
- the labeling substance used in the present invention has a functional group capable of binding to a thiol group derived from a disulfide bond of an antibody molecule as described above, or has a functional group appropriately introduced by a known method May be used in accordance with the purpose of detection.
- fluorescent dyes, fluorescent nanoparticles, aggregation-induced luminescent molecules, enzymes / coenzymes, chemiluminescent substances, or radioactive substances can be used.
- the labeling substance used in the present invention is preferably a fluorescent dye or a fluorescent nanoparticle from the viewpoint of reducing the number of reaction steps. It is preferable that the fluorescent dye and the fluorescent nanoparticle include a substance that emits fluorescence when excited using irradiation of predetermined excitation light or a field effect.
- the fluorescence has a broad meaning and includes phosphorescence having a relatively long emission lifetime in which the emission lasts and fluorescence in a narrow sense having a relatively short emission lifetime.
- the type of the fluorescent dye is not particularly limited.
- fluorescent dyes Alexa Fluor (registered trademark) dye series (Invitrogen Corporation), fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan, Inc.) )), Hexachlorofluorescein family fluorescent dyes (Applied Biosystems Japan), coumarin family fluorescent dyes (Invitrogen), rhodamine family fluorescent dyes (GE Healthcare Biosciences), Cyanine family fluorescent dye, indocarbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanum Family of fluorescent dyes, BODIPY (registered trademark) family of fluorescent dyes (Invitrogen Corp.), naphthalenesulfonic acid family of fluorescent dyes, pyrene family of fluorescent dyes, triphenylmethane family of fluorescent dyes, etc.
- An organic fluorescent dye is mentioned.
- the fluorescent dye is not limited to the organic fluorescent dye.
- a rare earth complex phosphor such as Eu or Tb can also be used.
- Rare earth complexes generally have a large wavelength difference between the excitation wavelength (about 310 to 340 nm) and the emission wavelength (about 615 nm for the Eu complex and about 545 nm for the Tb complex), and the fluorescence lifetime is usually several hundred microseconds or more. There is a feature that it is long.
- An example of a commercially available rare earth complex phosphor is ATBTA-Eu 3+ .
- semiconductor nanoparticles containing a II-VI group compound, a III-V group compound, or a group IV element as a component or core-shell type semiconductor nanoparticles having a semiconductor nanoparticle as a core and a shell provided around the semiconductor nanoparticle Can also be used.
- the fluorescent nanoparticles can be used without particular limitation as long as they are nano-sized (a diameter of 1 ⁇ m or less) particulate phosphor and can emit fluorescence with sufficient luminance with one particle.
- it has a structure in which particles made of an organic substance or an inorganic substance are used as a base, and a plurality of phosphors (organic fluorescent dyes or semiconductor nanoparticles) are encapsulated therein and / or adsorbed on the surface thereof. , Nano-sized particles.
- fluorescent nanoparticles include organic fluorescent dye integrated nanoparticles and inorganic phosphor (semiconductor) integrated nanoparticles.
- the aggregation-inducing luminescent molecule may be used without particular limitation as long as it is a fluorescent substance that has the property of agglomerating to form aggregates to increase the quantum yield and thereby emit strong fluorescence or increase fluorescence intensity. be able to.
- the aggregation-induced luminescent molecules include maleimide-based aggregation-induced luminescent molecules, aminobenzopyranoxanthene (ABPX) -based aggregation-induced luminescent molecules, benzofuro-oxazolo-carbazole-based aggregation-induced luminescent molecules, and carborane-based aggregation-induced luminescent molecules.
- rhodamine-based aggregation-induced luminescent molecules tetraphenylethylene-based aggregation-induced luminescent molecules, silole-based aggregation-induced luminescent molecules, aromatic ring-containing metal complex compounds, BODIPY-based boron imine complex aggregation-induced luminescent molecules, other Examples include hetero compounds.
- Alexa Fluor 647 Invitrogen Corp.
- fluorescent dyes, fluorescent nanoparticles, aggregated organic light emitting molecules, etc. having a maximum fluorescence wavelength in the outer region Invitrogen Corp.
- the antibody used in the present invention may be any antibody or antibody fragment that can specifically recognize an antigen contained in a specimen and bind to the antigen, and is appropriately selected according to the use. Examples thereof include natural polyclonal antibodies or monoclonal antibodies, recombinant antibodies obtained by gene recombination, and fragments thereof.
- troponin I cTnI
- troponin T cTnT
- CK-MB myoglobin
- H-FABP H-FABP
- BNP BNP
- NT-proBNP D-dimer
- the labeled antibody used in the present invention is a labeled antibody in which a labeling substance and an antibody are bound.
- a part of the disulfide bond (—S—S—) of the antibody molecule is cleaved by reduction treatment, which will be described later, with respect to any of the above antibodies, resulting from the cleaved disulfide bond.
- a labeled antibody can be prepared by binding a labeling substance to two thiol groups (-SH HS-).
- Examples of reducing agents that can be used to cleave the disulfide bond of an antibody include 2-mercaptoethanol, 3-mercapto-1,2-propanediol, glutathione ( ⁇ -L-glutamyl-L-cysteinylglycine) , Tris (2-carboxyethyl) phosphine hydrochloride and cysteine, 2-mercaptoethylamine and the like.
- the binding between the reduced antibody and the labeling substance can be performed by mixing in a buffer solution.
- a buffer that can be used as a medium of the above-mentioned labeled antibody dispersion can be used.
- the labeled antibody used in the present invention can be prepared in the same manner as a complex (conjugate) of a fluorescent substance and an antibody used in general immunoassay methods.
- a commercially available kit for example, using Alexa Fluor protein labeling kit, Invitrogen
- the functional group introduced into the fluorescent substance and the functional group possessed by the anti-troponin antibody are reacted in the presence of a predetermined reagent.
- a labeled antibody of fluorescent substance-antitroponin antibody can be prepared.
- the labeled antibody dispersion of the present invention is a dispersion containing a nonionic surfactant in which the labeled antibody is dispersed.
- the labeled antibody dispersion of the present invention preferably contains 0.001 to 1% by mass of nonionic surfactant, more preferably 0.05 to 0.5% by mass, and more preferably 0.1 to 0. It is particularly preferable to contain 3% by mass.
- the nonionic surfactant is preferably a polyoxyethylene-based surfactant, more preferably polyoxyethylene sorbitan fatty acid esters or polyoxyethylene octylphenyl ethers.
- polyoxysorbitan fatty acid ester examples include Tween (registered trademark) 20, Tween (registered trademark) 40, Tween (registered trademark) 60, Tween (registered trademark) 65, Tween (registered trademark) 80, and Tween (registered trademark) 85.
- Tween (registered trademark) 20 is preferred because of its high hydrophilicity.
- polyoxyethylene octylphenyl ether examples include Triton (registered trademark) X-100, Triton (registered trademark) X-114, Triton (registered trademark) X-405, etc.
- Triton® X-100 is preferred because it is commonly used in assays.
- the present inventors collect nonionic surfactant around the labeled antibody, so that the labeled antibody is hardly oxidized or denatured. Therefore, it was speculated that the storage stability would be improved.
- a nonionic surfactant is contained at a concentration within the above range, denaturation and fragmentation of the labeled antibody can be suppressed, aggregation and precipitation can be more effectively prevented, and storage can be performed. It has been found that the stability of time is further increased.
- the labeled antibody dispersion of the present invention can be used when immunoassay such as sandwich immunoassay is performed on a sample that may contain a detection target substance (antigen).
- the specimen may be a specimen that actually contains a detection target substance, or a specimen that does not actually contain a detection target substance.
- the specimen is typically collected from a human, but a model animal of a human disease, for example, a mammal other than a human such as a mouse, rat, guinea pig, rabbit, goat, cat, dog, pig, monkey. There may be.
- specimens examples include biological substances such as blood, urine, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (for example, biopsy specimens).
- biological substances such as blood, urine, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (for example, biopsy specimens).
- blood and urine are preferable as samples used in the present invention because they are highly likely to contain glycoproteins that can be used as diagnostic markers.
- Liquid samples such as blood, serum, plasma, urine, spinal fluid, and saliva may be used as samples as they are, or those appropriately diluted with an appropriate sample dilution solution may be used as samples.
- a suspension homogenized with an appropriate buffer solution of about 2 to 10 times the volume of the specimen, or a supernatant thereof, is used directly or further. It can be used as a sample after being diluted with a diluting solution.
- blood is used as a specimen.
- the blood may be whole blood, or serum or plasma prepared from whole blood by a known method.
- whole blood it is preferable to use whole blood as a sample for the purpose of rapid measurement, and for the purpose of accurate quantification, blood cell components are removed from whole blood by centrifugation, etc.
- plasma it is preferable to prepare plasma and use it as a specimen.
- an anticoagulant is usually added to whole blood at the time of blood collection.
- the whole blood is diluted to an appropriate concentration or a necessary reagent. Etc. are preferably added. Therefore, such an anticoagulant and other reagents may be added to the specimen used in the present invention as necessary.
- antigen a protein that is a detection target substance contained in a specimen.
- troponin I cTnI
- troponin T cTnT
- CK-MB myoglobin
- H-FABP H-FABP
- BNP NT-proBNP
- D-dimer D-dimer
- a specimen that can contain them can be used, and a commercially available standard antigen can also be used as a control for the purpose of more accurately measuring the amount of antigen in the specimen.
- SPFS Surface plasmon excitation enhanced fluorescence spectroscopy
- an analyte (antigen) is brought into contact with a metal thin film on a sensor chip on which a ligand (for example, an antibody) that specifically binds to the analyte is immobilized, thereby causing the sensor chip to capture the analyte.
- a ligand for example, an antibody
- an antibody labeled with a fluorescent substance that specifically binds to the analyte (a labeled antibody, the labeled substance being a fluorescent substance; hereinafter also referred to as a fluorescent labeled antibody) is brought into contact.
- a biomarker such as myocardial infarction
- analyte a detection target substance
- analyte an antibody
- an antibody for example, anti-cTnI antibody
- SPFS fluorescent-labeled anti-cTnI antibody dispersed in the labeled antibody dispersion of the present invention as the fluorescent-labeled antibody.
- the SPFS kit of the present invention comprises a labeled antibody dispersion and a sensor chip dedicated to SPFS.
- the SPFS kit of the present invention can be used for immunoassay such as the above-mentioned sandwich immunoassay.
- a sensor chip dedicated for surface plasmon excitation enhanced fluorescence spectroscopy is set in a measuring apparatus by the SPFS method, and the labeled antibody dispersion is used.
- the target substance to be detected (antigen) can be detected.
- cTnI when used as a detection target substance (antigen), a surface in which a hydrophilic polymer layer is formed with carboxymethyldextran (CMD) in a measurement region and an anti-cTnI IgG monoclonal antibody is immobilized thereon.
- CMD carboxymethyldextran
- a sensor chip dedicated to plasmon excitation enhanced fluorescence spectroscopy (SPFS) is set in an SPFS measuring apparatus, and cTnI can be detected using a fluorescence labeled antibody dispersion.
- the sensor chip dedicated to SPFS used in the present invention is preferably a sensor chip in which an antibody is immobilized on the surface of a metal thin film on a transparent support.
- the immobilized antibody is immobilized on a solid phase.
- an antibody also called an antibody.
- the method for immobilizing the antibody include a method in which a hydrophilic polymer layer is formed on the surface of the metal thin film, and the antibody prepared at an appropriate concentration is allowed to react with the hydrophilic polymer layer.
- the antibody immobilized on the sensor chip is appropriately selected for the antigen that is the detection target substance.
- the support on which the gold thin film was formed was immersed in 10 mL of an ethanol solution of 10-amino-1-decanethiol prepared to 1 mM for 24 hours to form a measurement region on the surface of the gold thin film. Thereafter, the support was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun.
- MES buffered saline MES
- CMD carboxymethyldextran
- NHS N-hydroxysuccinimide
- WSC water-soluble carbodiimide
- the support on which the measurement region was formed was immersed for 1 hour, and CMD was solid-phased in the measurement region to form a hydrophilic polymer layer.
- the succinic acid ester was hydrolyzed by immersing in 1M NaOH aqueous solution for 30 minutes.
- the average film thickness of the CMD layer was 70 nm, and the density was 5.0 ng / mm 2 .
- the support was immersed in MES containing NHS 50 mM and WSC 100 mM for 1 hour, and then immersed in an anti-cTnI IgG1 monoclonal antibody (560; 2.5 ⁇ g / mL, Hytest) solution for 30 minutes. By soaking, the antibody was immobilized on the measurement region on the support.
- the measurement region on which the antibody is immobilized is also referred to as a measurement region.
- nonspecific adsorption prevention treatment to the measurement region was performed by circulating the solution for 30 minutes in PBS containing 1% by mass of bovine serum albumin (BSA) and 1M aminoethanol.
- BSA bovine serum albumin
- Example 1 and Comparative Example 1 Preparation of labeled antibody dispersion ⁇ A fluorescently labeled antibody was prepared by the following method.
- an anti-cTnI IgG monoclonal antibody (19C7; manufactured by Hytest) was dispersed in phosphate buffered saline (PBS) to prepare 1 mg / mL to obtain an antibody dispersion.
- PBS phosphate buffered saline
- CF registered trademark
- 660R manufactured by Biotium
- TCEP Tris (2-carboxyethyl) phosphine
- the fluorescence-labeled anti-cTnI antibody dispersion After measuring the absorbance of the above-mentioned fluorescence-labeled anti-cTnI antibody dispersion and quantifying its concentration, it was diluted with a PBS solution containing surfactants of different types and concentrations, and the concentration of the fluorescence-labeled anti-cTnI antibody was 5 ⁇ g. / ML to prepare.
- the fluorescence-labeled anti-cTnI antibody dispersion is referred to as a fluorescence-labeled antibody dispersion.
- the types and concentrations of the surfactants in the fluorescently labeled antibody dispersion are as follows.
- Example 1-1 and 1-2 the nonionic surfactant Tween 20 (registered trademark) (manufactured by Nacalai Tesque Co., Ltd.) was 0.15% by mass, Triton X-100 (registered trademark) (Fuji Film). A fluorescently labeled dispersion containing 0.15% by mass of Wako Pure Chemical Industries, Ltd. was used.
- Table 1 shows the types and concentrations of the surfactants used in each example and comparative example.
- a PBS solution containing 11 ng / L of a commercially available cTnI control reagent (Biorad) was sent to the measurement region of the SPFS sensor chip. Subsequently, after removing the cTnI solution, Tris-buffered saline (TBS) containing 0.05% by mass of Tween 20 was fed and circulated for 10 minutes for washing. Thereafter, the PBS solution containing 5 ⁇ g / mL of the labeled antibody dispersion prepared above was fed and removed from the measurement region, and then Tris-buffered saline (TBS) containing 0.05% by mass of Tween 20 was fed. And washed.
- TBS Tris-buffered saline
- Table 2 shows the classification of each surfactant in the labeled antibody dispersion used in each Example and Comparative Example, the initial performance value (S / N ratio), and the case where each dispersion was stored at 30 ° C. for 5 days. The result about preservability is shown.
- FIG. 1 is a graph showing the results of comparing the initial performance due to the difference in the surfactant in the labeled antibody dispersion.
- the vertical axis represents the S / N ratio, and the horizontal axis represents the type of surfactant.
- FIG. 2 is a graph showing the results of comparison of storage stability depending on the surfactant in the labeled antibody dispersion.
- the vertical axis shows the blank increase rate after storage at 30 ° C. for 5 days, and the horizontal axis shows the type of surfactant.
- Example 2 and Comparative Example 2 ⁇ Preparation of labeled antibody dispersion ⁇ A labeled antibody dispersion was prepared in the same manner as in Experimental Example 1, except that Tween 20 and Triton X-100, which are nonionic surfactants, were used in the amounts shown in Table 3, respectively.
- Tween 20 and Triton X-100 which are nonionic surfactants
- Table 3 shows the results of the type and concentration of the nonionic surfactant, the initial performance value (S / N ratio), and the storage stability of each dispersion when stored at 30 ° C. for 5 days.
- Example 3 and Comparative Example 3 ⁇ Preparation of labeled antibody dispersion ⁇
- a fluorescently labeled antibody dispersion was prepared in the same manner as in Experimental Example 1, except that 20 mol of the fluorescent dye dispersion was mixed with 1 mol of the reduced antibody dispersion.
- 0.15% Tween20-PBS was used to adjust the concentration of the fluorescently labeled antibody dispersion, and PBS was used in the comparative example.
- the labeling rate was 5.1.
- Table 4 shows the measurement results of the Tween 20 concentration and the blank value (0 ng / L) and signal value (9.5 ng / L) for each storage period when stored at 4 ° C. or 30 ° C. for 0 to 29 days.
- the S / N ratio was calculated in the same manner as in Experimental Example 1 using the blank value and the signal value.
- Table 5 shows the concentration of Tween 20 and the S / N ratio at each storage day when stored at 4 ° C. or 30 ° C. for 0 to 29 days.
- Table 6 shows the concentration of Tween 20 and the fluctuation rate (%) of the blank value and the signal value in each storage day when stored at 4 ° C. or 30 ° C. for 0 to 29 days.
- FIGS. 3 to 6 show graphs of the blank value and the fluctuation rate (%) of the signal value, based on the storage period of 0 days, as described in Table 6 above.
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Abstract
Description
[1]抗体分子の一部のジスルフィド結合を切断し、この切断により生じたチオール基に標識物質を結合させた標識化抗体と、非イオン性界面活性剤とを含む、標識化抗体分散液。[2]前記非イオン性界面活性剤が、ポリオキシエチレン系の界面活性剤である、[1]に記載の標識化抗体分散液。
[3]前記非イオン系界面活性剤が、ポリオキシエチレンソルビタン脂肪酸エステルまたはポリオキシエチレンオクチルフェニルエーテルである、[1]または[2]に記載の標識化抗体分散液。
[4]試料に含まれる抗原を、表面プラズモン励起増強蛍光分光法(SPFS)で検出するための、[1]~[3]のいずれか1つに記載の標識化抗体分散液。
[5]標識化抗体分散液100質量%中に、前記ポリオキシエチレンソルビタン脂肪酸エステル類またはポリオキシエチレンオクチルフェニルエーテル類を0.001~1質量%含む、[1]~[4]のいずれか1つに記載の標識化抗体分散液。
[6]前記標識物質が蛍光色素、蛍光ナノ粒子、凝集誘起発光性分子、酵素・補酵素、化学発光物質、または放射性物質である、[1]~[5]のいずれか1つに記載の標識化抗体分散液。
[7]前記抗体分子が抗トロポニンI(cTnI)抗体、抗トロポニンT(cTnT)抗体、抗BNP抗体、または、抗D-dimer抗体である、[1]~[6]のいずれか1つに記載の標識化抗体分散液。
[8][1]~[7]のいずれか1つに記載の標識化抗体分散液と、表面プラズモン励起増強蛍光分光法(SPFS)専用のセンサーチップとからなる、SPFS用キット。
≪標識化抗体分散液≫
本発明の標識化抗体分散液は、抗体分子の一部のジスルフィド結合(-S-S-)を切断し、この切断により生じたチオール基(-SH HS-)に標識物質を結合させた標識化抗体と、非イオン性界面活性剤とを含む、標識化抗体分散液である。
本発明の標識化抗体分散液は、標識物質と抗体が結合した標識化抗体を含む。前記標識化抗体は、抗体分子の一部のジスルフィド結合を切断し、この切断により生じたチオール基に標識物質を結合させた標識化抗体を含む。
(標識物質)
本発明で用いられる標識物質は、上述するような抗体分子のジスルフィド結合に由来するチオール基に結合可能な官能基を有しているもの、または適宜公知の方法で官能基が導入されているものを、検出の目的にあわせて用いればよく、例えば、蛍光色素、蛍光ナノ粒子、凝集誘起発光性分子、酵素・補酵素、化学発光物質、もしくは放射性物質を用いることができる。
(抗体)
本発明で用いられる抗体は、検体中に含有される抗原を特異的に認識して抗原に結合し得る抗体または抗体断片であればよく、用途に応じて適宜選択される。例えば、天然のポリクローナル抗体またはモノクローナル抗体、遺伝子組換えにより得られるリコンビナント抗体、およびそれらの断片などが挙げられる。
(標識化抗体の作成方法)
本発明で用いられる標識化抗体は、標識物質と抗体とが結合している標識化抗体である。
本発明の標識化抗体分散液は、前記標識化抗体を分散させた非イオン性界面活性剤を含む分散液である。
本発明の標識化抗体分散液は、検出対象物質(抗原)を含む可能性のある検体についてサンドイッチイムノアッセイ等の免疫測定を行う際に用いることができる。
本明細書においては、検体に含まれる検出対象物質であるタンパク質のことを抗原と記す。
SPFSは、リガンドを固定したセンサーチップを用いて、リガンドと結合する物質であるアナライトを定量する方法である。
本発明のSPFS用キットは、標識化抗体分散液とSPFS専用のセンサーチップとからなる。本発明のSPFS用キットは、前述のサンドイッチイムノアッセイ等の免疫測定を行う際に用いることができる。
本発明で用いる、SPFS専用のセンサーチップは、透明支持体上の金属薄膜表面に抗体が固相化されたセンサーチップであることが好ましく、この場合、固相化された抗体を、固相化抗体とも称する。抗体を固相化する方法としては、金属薄膜表面に親水性高分子層を形成させ、その箇所に適当な濃度に調製した抗体を反応させる方法などが挙げられる。センサーチップ上に固相化する抗体は、検出対象物質である抗原に対して適宜選択する。
≪SPFS用センサーチップの調製≫
屈折率〔nd〕1.72、厚さ1mmのガラス製の透明支持体(株式会社オハラ製:S-LAL 10)をプラズマ洗浄し、該支持体の片面にクロム薄膜をスパッタリング法により形成した後、その表面にさらにスパッタリング法により金属部材である金薄膜を形成した。クロム薄膜の厚さは1~3nm、金薄膜の厚さは42~47nmであった。
分子量50万のカルボキシメチルデキストラン(CMD)を1mg/mL、N-ヒドロキシコハク酸イミド(NHS) 0.5mM、水溶性カルボジイミド(WSC) 1mMを含むpH7.4のMES緩衝生理食塩水(MES)(イオン強度:10mM)に、前記測定領域を形成した支持体を1時間浸漬し、測定領域にCMDを固相化して親水性高分子層を形成した。その後1MのNaOH水溶液に30分間浸漬することでコハク酸エステルを加水分解させた。CMD層の平均膜厚は70nmであり、密度は5.0ng/mm2であった。
実施例1および比較例1
≪標識化抗体分散液の調製≫
以下の手法で蛍光標識化抗体を作成した。
SPFS用センサーチップの測定領域に、市販のcTnIコントロール試薬(Biorad社製)を11ng/L含むPBS溶液を送液した。続いて、当該cTnI溶液を除去した後、Tween20を0.05質量%含むトリス緩衝生理食塩水(TBS)を送液し、10分間循環させて洗浄した。その後、上記で調製した標識化抗体分散液5μg/mLを含むPBS溶液を送液し、測定領域から除去した後、Tween20を0.05質量%含むトリス緩衝生理食塩水(TBS)を送液して洗浄した。
≪S/N比の算出≫
各標識化抗体分散液の初期性能を示す指標として、上述のように得られたシグナル値(S)およびブランク値(N)から下記式(I)を用いてS/N比を算出した。
≪ブランク上昇率(%)の算出≫
各標識化抗体分散液を30℃で5日間保存した場合の保存性の検討として、下記式(II)を用いて、ブランク上昇率(%)を算出した。
表2に、それぞれの実施例および比較例で用いた標識化抗体分散液における各界面活性剤の分類、初期性能値(S/N比)および各分散液の30℃で5日間保存した場合の保存性についての結果を示す。
〔実験例2〕
実施例2および比較例2
≪標識化抗体分散液の調製≫
非イオン性界面活性剤である、Tween20とTritonX-100を、それぞれ表3に記載の量用いた以外は、実験例1と同様の手法にて標識化抗体分散液を調製した。
≪測定の実施・S/N比およびブランク上昇率(%)の算出≫
実験例1と同様に測定を行い、得られたシグナル値およびブランク値からS/N比およびブランク上昇率(%)を算出した。
実施例3および比較例3
≪標識化抗体分散液の調製≫
還元処理された抗体分散液1モルに対して、前記蛍光色素分散液を20モル等量混合した以外は、実験例1と同様に蛍光標識化抗体分散液を調製した。なお、実施例においては蛍光標識化抗体分散液の濃度の調整において0.15%のTween20-PBSを用い、比較例においてはPBSを用いた。標識率は5.1であった。
測定領域に、市販のcTnIコントロール試薬(Biorad社製)を9.5ng/L含むPBS溶液を送液したこと以外は、実験例1と同様の操作を行った。
標識化抗体分散液の保存日数が0日のブランク値を基準として、4℃または30℃で、0~29日間保存した場合の各保存日数におけるブランク値(%)を、下記式(III)を用いて、算出した。
シグナル値の変動率(%)は、上記式(III)のブランク値をシグナル値に変えて、同様に算出した。
Claims (8)
- 抗体分子の一部のジスルフィド結合を切断し、この切断により生じたチオール基に標識物質を結合させた標識化抗体と、非イオン性界面活性剤とを含む、標識化抗体分散液。
- 前記非イオン性界面活性剤が、ポリオキシエチレン系の界面活性剤である、請求項1に記載の標識化抗体分散液。
- 前記非イオン系界面活性剤が、ポリオキシエチレンソルビタン脂肪酸エステル類またはポリオキシエチレンオクチルフェニルエーテル類である、請求項1または2に記載の標識化抗体分散液。
- 試料に含まれる抗原を、表面プラズモン励起増強蛍光分光法(SPFS)で検出するための、請求項1~3のいずれか一項に記載の標識化抗体分散液。
- 標識化抗体分散液100質量%中に、前記ポリオキシエチレンソルビタン脂肪酸エステル類またはポリオキシエチレンオクチルフェニルエーテル類を0.001~1質量%含む、請求項1~4のいずれか一項に記載の標識化抗体分散液。
- 前記標識物質が蛍光色素、蛍光ナノ粒子、凝集誘起発光性分子、酵素・補酵素、化学発光物質、または放射性物質である、請求項1~5のいずれか一項に記載の標識化抗体分散液。
- 前記抗体分子が抗トロポニンI(cTnI)抗体、抗トロポニンT(cTnT)抗体、抗BNP抗体、または、抗D-dimer抗体である、請求項1~6のいずれか一項に記載の標識化抗体分散液。
- 請求項1~7のいずれか1項に記載の標識化抗体分散液と、表面プラズモン励起増強蛍光分光法(SPFS)専用のセンサーチップとからなる、SPFS用キット。
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US16/981,202 US20200408688A1 (en) | 2018-03-23 | 2019-03-22 | Labeled antibody dispersion liquid and kit for spfs |
JP2020507943A JP7171702B2 (ja) | 2018-03-23 | 2019-03-22 | 標識化抗体分散液、spfs用キット |
EP19770386.1A EP3745129A4 (en) | 2018-03-23 | 2019-03-22 | MARKED ANTIBODY DISPERSION LIQUID AND KIT FOR SPFS |
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