WO2018185942A1 - タンパク質修飾蛍光体集積粒子の精製物を製造する方法、蛍光染色液の製造方法、タンパク質修飾蛍光体集積粒子の精製物、蛍光染色液およびタンパク質修飾蛍光体集積粒子精製用フィルター - Google Patents
タンパク質修飾蛍光体集積粒子の精製物を製造する方法、蛍光染色液の製造方法、タンパク質修飾蛍光体集積粒子の精製物、蛍光染色液およびタンパク質修飾蛍光体集積粒子精製用フィルター Download PDFInfo
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- 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
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- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- 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
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- G01N2001/302—Stain compositions
Definitions
- the present invention relates to a method for producing a purified product of protein-modified phosphor integrated particles, a method for producing a fluorescent staining solution, a purified product of protein-modified phosphor integrated particles, a fluorescent staining solution, and a filter for purifying protein-modified phosphor integrated particles.
- a specimen slide is prepared using tissues and cells collected from a patient, and the morphology of cells or tissues is observed based on a stained image obtained by staining with a predetermined method, and a specific living body is observed.
- evaluating the expression state of a substance it is a method for determining various events such as whether or not the patient suffers from a specific disease or whether a specific therapeutic drug is successful.
- HER2 gene (HER2 / neu, c-erbB-2) which is a kind of oncogene and / or a membrane protein produced from the HER2 gene
- HER2 / neu, c-erbB-2 a HER2 gene which is a kind of oncogene and / or a membrane protein produced from the HER2 gene
- the HER2 protein is thought to perform signal transduction by forming a homodimer or a heterodimer bound to activated EGFR (HER1), HER3 or HER4, and as a receptor for cancer cell growth factor. Presumed to be functional (to date no endogenous ligand binding to HER2 is known).
- Examples of HER2 testing methods for tumor tissues include immunohistochemistry (IHC) method for staining HER2 protein and fluorescent in situ hybridization (FISH) method for staining HER2 gene.
- IHC immunohistochemistry
- FISH fluorescent in situ hybridization
- ASCO testing procedures and criteria
- the ASCO / CAP HER2 testing guidelines published in 2007 and revised in 2013 published by the American Society of Clinical Oncology and the American Pathology Society are widely used in various countries.
- a HER2 examination guide (HER2 examination guide fourth edition, HER2 examination pathology committee, April 2014) compliant with the revised ASCO / CAP HER2 examination guideline is used.
- an enzyme-labeled antibody is brought into contact with a specimen, and a target protein and antibody are bound directly or indirectly using an antigen-antibody reaction, and then a substrate corresponding to the enzyme is added.
- a method of developing a color by reacting is generally used. For example, a DAB staining method using peroxidase as an enzyme and diaminobenzidine as a substrate is widely adopted.
- the staining method based on the reaction between the enzyme and the substrate greatly depends on conditions such as temperature and time, so that the amount of protein that is the actual target can be accurately determined from the coloring concentration. There was a problem that it was difficult to estimate.
- nano-sized particles in which a plurality of fluorescent materials such as organic fluorescent dyes and semiconductor nanoparticles (quantum dots) are integrated that is, phosphor integrated particles (PID), “fluorescence”
- PID phosphor integrated particles
- fluorescence A method using “substance-integrated nanoparticles” or the like is proposed and is being put to practical use.
- phosphor-aggregated particles die resin particles in which the particle surface is modified with streptavidin are prepared, and an antigen (HER2 protein) on a tissue section is prepared.
- a primary antibody anti-HER2 rabbit monoclonal antibody
- biotin anti-rabbit IgG antibody
- streptavidin-modified phosphor-aggregated particles are collected.
- a fluorescence labeling method avidin-biotin combined secondary antibody method
- production of streptavidin-modified phosphor-aggregated particles is carried out by the following method.
- Patent Document 2 International Publication No. 2016/129444
- an appropriate number of disulfide bonds (—S—S—) are reduced to form a thiol group (—SH) by treatment with a specific reducing agent.
- a specific reducing agent for example, sodium thiol group
- a binding group capable of reacting with the thiol group for example, maleimide group
- an appropriate number of antibodies per unit area of the particle surface can be bound.
- Antibody-bound phosphor-integrated nanoparticles have been disclosed.
- Such antibody-bound phosphor-aggregated nanoparticles can suppress aggregation and precipitation during storage of the particles by inhibiting the binding of a plurality of phosphor-aggregated nanoparticles to one antibody. it can.
- the antibody may be a primary antibody used in a direct method in the IHC method, a secondary antibody used in an indirect method, or the like.
- Patent Document 2 an embodiment in which HER2 immunostaining is performed using antibody-modified fluorescent dye-encapsulated silica nanoparticles prepared by the following method is disclosed.
- Fluorescent dye-encapsulated silica nanoparticles are formed using 3-aminopropyltrimethoxysilane and 5-carboxytetramethylrhodamine (registered trademark “TAMRA”) as raw materials, and succinimidyl as a linker on the silica nanoparticles -[(N-maleimidopropionamide) -dodecaethylene glycol] ester (product name “SM (PEG) 12 ”) is reacted to introduce maleimide groups on the particle surface.
- TAMRA 3-aminopropyltrimethoxysilane and 5-carboxytetramethylrhodamine
- a primary antibody (anti-HER2 rabbit monoclonal antibody) or secondary antibody (anti-rabbit IgG antibody) is treated with 2-mercaptoethanol or the like as a reducing agent to reduce a disulfide bond to generate a thiol group. .
- Patent Document 3 (International Publication No. 2015/163209) contains a buffer solution, a protein, a surfactant, and the like that are used to preserve antibody-assembled phosphor-aggregated particles until they are used for tissue staining.
- the storage solution is described, and by improving the dispersion stability during storage of the antibody-binding phosphor-aggregated nanoparticles during storage, sedimentation and / or aggregation is suppressed, and coarse particles of the particles are removed in tissue staining. There exists an effect that it can control.
- Such phosphor-aggregated particles whose surface is modified with a biological substance-binding protein such as an antibody or streptavidin are generally produced after production. There is a problem that sedimentation or aggregation is likely to occur during storage until use. When a specimen such as a tissue section is immunostained using a fluorescent staining solution prepared from a solution containing such a sediment or aggregate, it is thought that aggregated protein-modified phosphor-aggregated particles are the cause in the acquired fluorescence image. In some cases, a large lump of bright spots was generated, which hindered the correct measurement of the number of bright spots.
- An object of one embodiment of the present invention is to provide a means for improving dispersion stability during storage of protein-modified phosphor-aggregated particles.
- a solution containing protein-modified phosphor-aggregated particles and a foreign substance derived from the production process is used for a substance having reversible binding ability to the biological substance-binding protein (this specification) Purification of protein-modified phosphor-aggregated particles, comprising a purification step of separating the protein-modified phosphor-aggregated particles from the contaminants by contacting a filter carrying the protein).
- the present invention relates to a method for manufacturing a product.
- the biological substance-binding protein is preferably an antibody, and the protein-binding substance is preferably protein A, protein G, or protein L.
- the protein-modified phosphor-aggregated particle includes a phosphor-aggregated particle modified with a first reactive substance, and a biological substance-binding protein modified with a second reactive substance. Fluorescent premix particles that are linked by the interaction of the second reactive substance.
- the first reactive substance is preferably streptavidin, and the second reactive substance is preferably biotin.
- the protein-modified phosphor-aggregated particles are preferably particles having 1000 to 5000 biological substance-binding proteins per phosphor-aggregated particle.
- the average particle diameter of the phosphor-aggregated particles is preferably 30 to 300 nm.
- One embodiment of the present invention relates to a method for producing a fluorescent staining solution containing a purified product of protein-modified phosphor-aggregated particles obtained by the production method.
- One embodiment of the present invention relates to a purified product of protein-modified phosphor-aggregated particles obtained by the production method.
- another embodiment of the present invention relates to a fluorescent staining solution containing the purified product.
- one embodiment of the present invention is a protein-modified phosphor-aggregated particle purification comprising a filter having pores of a size that allows protein-modified phosphor-aggregated particles to pass through, and a protein-binding substance supported on the filter.
- a filter having pores of a size that allows protein-modified phosphor-aggregated particles to pass through, and a protein-binding substance supported on the filter.
- a purified product of protein-modified phosphor-aggregated particles that can suppress aggregation and sedimentation when storing protein-modified phosphor-aggregated particles and has high dispersion stability can be obtained.
- a fluorescent staining solution can be quickly prepared, and a coarse lump of bright spots based on particle agglomerates that hinders the quantification of biological substances. It is possible to obtain a fluorescent image in which the suppression is suppressed.
- FIG. 1 is an example of an image obtained by photographing a dispersion (non-purified product) of anti-HER2 antibody-directed TAMRA-integrated silica particles in ⁇ Evaluation of Dispersion Stability> in an example with a fluorescence microscope. A bright spot (aggregate) with extremely high brightness is observed in the circle.
- FIG. 2 is an image obtained by photographing a staining slide prepared using a fluorescent staining solution prepared from the dispersion obtained in Comparative Example 2 in ⁇ Evaluation of staining performance> of the example with a fluorescence microscope. Cluster-like bright spots (aggregates) are observed in the circle.
- a method for producing a purified product of protein-modified phosphor-aggregated particles includes a solution containing protein-modified phosphor-aggregated particles and contaminants derived from the production process.
- the present inventor as one of the factors that reduce the dispersion stability during storage of protein-modified phosphor-aggregated particles, is that various contaminants derived from the production process are mixed, and biological material binding According to the purified product obtained here, contaminants can be removed at a very high level by bringing the solution of protein-modified phosphor-aggregated particles into contact with a filter carrying a substance having a reversible binding ability to a protein. It has been found that a decrease in dispersion stability during storage can be suppressed.
- a biological substance-binding protein is formed on the surface of the phosphor-aggregated particles.
- a step of repeating centrifugation and dispersion in a pH buffer solution is performed a plurality of times.
- impurities in the solution containing the particles are sufficiently removed. It was thought to be removed.
- contaminants that could not be removed by such a method contribute to aggregation and sedimentation that occur during storage of the particles, and removal of such contaminants generates during storage of the particles.
- the present inventor has found that aggregation and sedimentation can be suppressed. It was also surprising to those skilled in the art that this can suppress aggregation and sedimentation that occur during storage of particles.
- Contaminants that could not be sufficiently removed in the conventional washing process for example, particle raw materials that were not consumed in the particle formation reaction due to secondary interactions with impurities contained in the raw materials and reagents, cannot be excluded by washing
- Surfactants proteins that have been denatured and become non-reactive and have not been bound to particles. These impurities are presumed to have an adverse effect on the dispersion stability of the protein-modified phosphor-aggregated particles, and further on the staining property of the fluorescent (immune) staining.
- the production method according to one embodiment of the present invention is a novel embodiment, specifically, an embodiment disclosed in Patent Document 1 (second antibody combined with avidin-biotin). Dispersion of particles when storing a solution containing protein-modified phosphor-aggregated particles obtained by pre-reacting (premixing) streptavidin-modified phosphor-aggregated particles with a biotin-labeled secondary antibody. It was also found to be extremely effective for improving the performance.
- the protein-binding substance accumulated in the filter is captured by the filter by binding the protein-binding substance supported on the filter and the biological substance-binding protein constituting the protein-modified phosphor accumulation particle. Contaminants that do not react with the protein-binding substance flow out without being captured by the filter, so that both can be separated.
- a desorption step of separating the protein-modified phosphor-aggregated particles captured by the filter and the filter is further included.
- This desorption step can be performed, for example, by flowing an eluent through a filter.
- the eluent is not particularly limited, but is preferably a liquid that can sufficiently desorb the protein-modified phosphor-aggregated particles from the filter. It is preferable that the liquid does not lose its function.
- An appropriate liquid can be used depending on the constituent components of the protein-modified phosphor-aggregated particles, the type of protein-binding substance contained in the filter, and the like. For example, glycine-HCl or the like is preferably used.
- the protein-binding substance carried on the filter is not only protein-modified phosphor-aggregated particles, but also captures the protein when the unreacted biological substance-binding protein is contained in the solution in contact with the filter.
- the protein-modified phosphor-aggregated particles and the unreacted biological substance-binding protein can be appropriately separated by the molecular sieving function or the like of the filter.
- protein-modified phosphor-aggregated particles are usually larger in size than biological substance-binding proteins, and are particles in which a plurality of biological substance-binding proteins are linked per particle. Compared to biological substance-binding protein, it tends to be captured more strongly by the filter and is difficult to pass through the filter. Therefore, when the eluent is flowed, the unreacted biological substance-binding protein has a fast outflow time from the filter, whereas the protein-modified phosphor-aggregated particles have a long outflow time from the filter. For this reason, a “purified product” having a low content of unreacted biological material-binding protein can be obtained by fractionating a fraction containing a large amount of protein-modified phosphor-aggregated particles (late elution phase).
- the purified product of the protein-modified phosphor-aggregated particles according to an embodiment of the present invention is obtained by a method including the purification step.
- the remaining amount of contaminants in the purified product is extremely small, and the amount of contaminants present before carrying out the production method is also very small, both of which are quantified with sufficient accuracy by known means.
- it in order to solve the above-mentioned problem, it must be said that it is impossible or impractical to specify the amount of impurities in the purified product. . Therefore, it is reasonable to specify that the purified product is obtained by the production method.
- the protein-modified phosphor-aggregated particle is a phosphor-aggregated particle whose surface is modified with a biological material binding protein.
- the modification method of the phosphor-aggregated particles with the biological substance binding protein may be appropriately selected according to the fluorescent labeling method of the biological substance targeted for fluorescent staining.
- the protein-modified phosphor-aggregated particles the phosphor-aggregated particles modified with the first reactive substance and the biological substance-binding protein modified with the second reactive substance are the first reactive substance and the second reactive substance.
- Fluorescent premix particles which are particles connected by the interaction of reactive substances, are preferred.
- the protein-modified phosphor-aggregated particles are preferably particles in which the number of biological substance-binding proteins linked per phosphor-aggregated particle is preferably 1000 or more, more preferably 1000 to 50000.
- particles having such a number of biological substance-binding proteins can be obtained.
- the number of biological substance-binding proteins linked per phosphor-aggregated particle can be calculated by, for example, the method described in International Publication No. 2015/15977. In the case of the fluorescent premix particle, these can be determined by the following methods (i) to (iv).
- the biological material-binding protein is a protein having a binding ability to a biological material, and is used for directly or indirectly binding phosphor-aggregated particles to a target biological material that is an object of fluorescent staining.
- a biological substance-binding protein may be appropriately selected depending on the fluorescent labeling method of the target biological substance and the modification of the phosphor-aggregated particles, but is preferably an antibody.
- an antibody that specifically binds to the target biological material is bound to the biological material. It can be a protein.
- the immunostaining method when the phosphor-aggregated particles are indirectly bound to the target biological substance, (i) an antibody that specifically binds to the primary antibody (secondary antibody), and (ii) a secondary antibody that is specific. That specifically binds to a higher-order antibody that is higher than the antibody (tertiary antibody) that binds specifically, or (iii) a reactive substance (second reactive substance) that modifies the primary antibody or the secondary antibody
- the reactive substance (first reactive substance) can be a biological substance-binding protein.
- the surface of the phosphor-aggregated particles is modified with a biological substance-binding protein means that not only the surface of the phosphor-aggregated particles is directly bound to the biological substance-binding protein, but also any binding. In addition, a case where a biological substance-binding protein is bound is also included.
- a biological material binding protein is bound most distally from the surface of the phosphor-aggregated particle.
- an antibody is preferably used as the biological substance-binding protein.
- the antibody is not particularly limited as long as it is an antibody that can bind directly or indirectly to the target biological substance in the immunostaining method.
- the antibody may be any of the primary antibody, the secondary antibody, and the tertiary antibody higher than the tertiary antibody.
- a primary antibody is an antibody that recognizes and binds a unique epitope to an antigen.
- a primary antibody is a mixture of two or more monoclonal antibodies, which are preferably monoclonal antibodies over polyclonal antibodies. When used, combinations of monoclonal antibodies that specifically bind for different epitopes from antibody to antibody are preferred.
- the immunized animal producing the primary antibody is not particularly limited and can be selected from general animals such as mouse, rat, guinea pig, rabbit, goat, sheep and the like.
- the secondary antibody or higher antibody is an epitope unique to the primary antibody or lower antibody, preferably a region not involved in the binding of the antibody to a target biological substance (antigen) or the like (Fc etc.) It recognizes and binds to an epitope present in
- the secondary antibody or the like is preferably a monoclonal antibody from the viewpoint of the stability of quantification of the target biological substance, but a polyclonal antibody may be used from an economical viewpoint.
- An immunized animal that produces a secondary antibody or the like is an animal species that produces a primary antibody or the like or an animal species that forms a region such as Fc from among the animal species or the like exemplified as an immunized animal that produces a primary antibody.
- an appropriate animal may be selected. For example, when a natural mouse antibody (IgG produced by a mouse) is used as a primary antibody, an antibody that specifically binds to mouse IgG produced by an immunized animal other than a mouse (such as a rabbit) as a secondary antibody It is appropriate to use
- the antibody may be of any isotype, but is usually IgG or IgM, with IgG being particularly preferred.
- the antibody may be a natural antibody such as full-length IgG as long as it has the ability to specifically recognize and bind to the target biological substance or lower-order antibody, or Fab, Fab ′, F (ab ') 2 , non-natural antibodies such as antibody fragments such as Fv and scFv, or artificial antibodies that are multifunctional (multivalent or multispecific) using these antibody fragments Good.
- it may be a natural antibody derived from a specific immunized animal (for example, a mouse antibody produced by a mouse), a chimeric antibody or a humanized antibody prepared by an artificial means using a vector or the like.
- it may be a fully human antibody.
- the binding affinity (affinity) between the antibody and the antibody differs depending on the antibody site, animal species, subclass, etc., and it is necessary to use an appropriate antibody. There is.
- protein A and protein G mainly recognize and bind to the Fc region, it is preferable to use a full-length antibody or antibody fragment having the Fc region (because protein G binds weakly to the Fab region, In some cases, antibody fragments containing the Fab region may be used).
- protein L recognizes and binds to a light chain ( ⁇ light chain)
- antibody fragments such as scFv and Fab that do not have an Fc region can also be used.
- Protein A for example, binds strongly to human IgG1, IgG2, and IgG4, but hardly binds to human IgG3 and binds strongly to mouse IgG2a, IgG2b, and IgG3, but binding to mouse IgG1 is Since it is weak in a buffer under biological conditions (Tris-HCl or sodium phosphate buffer), it is preferable to use a subclass antibody having strong binding property depending on the animal species.
- the first reactive substance and the second reactive substance are a combination of substances that specifically bind to each other by interaction, both of which are a biological substance-binding protein and a target biological substance to which it binds (target protein, or A substance that does not cross-react with specific binding to a secondary antibody or a higher-order antibody higher than a tertiary antibody).
- the first reactive substance and the second reactive substance are not limited, but for example, when the target biological substance (antigen, etc.) and the labeling substance (phosphor, etc.) are indirectly bound in a known staining method. You can choose from those used in
- an avidin such as avidin, streptavidin or neutravidin, preferably streptavidin is selected as the first reactive substance
- Biotin can be selected as the reactive substance.
- the target biological substance can be detected with higher sensitivity.
- a hapten which is not immunogenic but exhibits antigenicity and has a relatively low molecular weight capable of reacting with an antibody
- an anti-hapten antibody for example, dicoxygenin and Anti-dicoxygenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antibodies
- dicoxygenin and Anti-dicoxygenin antibodies for example, dicoxygenin and Anti-dicoxygenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antibodies
- FITC fluorescein isothiocyanate
- the phosphor-aggregated particles are not particularly limited, but phosphors (for example, fluorescent dyes) are formed inside or on the surface of the base particles made of organic or inorganic substances. Nano-sized particles having a structure in which a plurality of particles are fixed and accumulated (diameter of 1 ⁇ m or less) are preferable, and particles that can emit fluorescence with sufficient luminance are preferable.
- Such phosphor-aggregated particles have a higher fluorescence intensity (brightness) when labeling the target biological substance compared to the case where the phosphor is used alone (one molecule without being accumulated). It is preferably used in fluorescent staining because it is highly discriminable from noise such as autofluorescence and other dyes, and is less susceptible to deterioration by irradiation with excitation light (higher light resistance) than a phosphor alone.
- the phosphor accumulated on the phosphor-aggregated particles is not particularly limited, and various known organic fluorescent dye molecules and semiconductor nanoparticles (sometimes referred to as quantum dots) can be used, for example.
- organic fluorescent dye integrated particles phosphor integrated particles when organic fluorescent dyes are used as phosphors
- semiconductor nanoparticles are used as phosphors
- inorganic phosphor integrated particles phosphor integrated particles when semiconductor nanoparticles are used as phosphors
- a phosphor that emits fluorescence having a desired wavelength (color) may be selected according to a desired use.
- Organic fluorescent dye integrated particles are preferably nano-sized fluorescent particles in which a plurality of organic fluorescent dyes are integrated inside or on the surface of a substance serving as a base of the particles.
- organic fluorescent dye examples include a fluorescein dye, a rhodamine dye, an Alexa Fluor (registered trademark, manufactured by Invitrogen) dye, a BODIPY (registered trademark, manufactured by Invitrogen) dye, and a cascade (registered trademark, Invitrogen).
- Low molecular organic compounds such as dyes, coumarin dyes, NBD (registered trademark) dyes, pyrene dyes, cyanine dyes, perylene dyes, oxazine dyes, and pyromethene dyes ).
- rhodamine dyes such as sulforhodamine 101 and its hydrochloride, such as TexasRed (registered trademark), perylene dyes such as perylene diimide, and pyromethene dyes such as pyromethene 556 are preferable because of their relatively high light resistance.
- Examples of the matrix constituting the organic fluorescent dye integrated particles include substances that can integrate organic fluorescent dyes with physical or chemical bonding force, such as resin and silica.
- resin and silica substances that can integrate organic fluorescent dyes with physical or chemical bonding force, such as resin and silica.
- Hydrophobic compounds such as polystyrene, polymelamine, and silica, particularly melamine resins and styrene resins, are preferred because they facilitate the production of fluorescent dye-aggregated particles and provide particles with high emission intensity.
- organic fluorescent dye integrated particles produced using a fluorescent dye such as perylene diimide, sulforhodamine 101 or its hydrochloride (Texas Red), pyromethene as a phosphor, and a resin such as melamine resin or styrene resin as a matrix. From the viewpoint of excellent labeling performance and the like, the phosphor-aggregated particles are preferable.
- the inorganic phosphor aggregated particles are preferably nano-sized phosphor particles in which a plurality of semiconductor nanoparticles are accumulated inside or on the surface of a substance serving as a base of the particles.
- the semiconductor nanoparticles are not particularly limited, and examples thereof include II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements, and particle dots such as CdSe exemplified in International Publication No. 2012/130347. It is done.
- quantum dots having semiconductor nanoparticles as a core and a shell around the core.
- the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS.
- semiconductor nanoparticles having a shell include CdSe / ZnS exemplified in International Publication No. 2012/130347.
- the semiconductor nanoparticles may be surface-treated with an organic polymer or the like as necessary.
- an organic polymer or the like for example, CdSe / ZnS (manufactured by Invitrogen) whose particle surface is modified with a carboxy group, CdSe / ZnS (manufactured by Invitrogen) whose particle surface is modified with an amino group, and the like can be used.
- Examples of the matrix constituting the inorganic phosphor aggregated particles include substances that can integrate semiconductor nanoparticles with physical or chemical bonding force, such as resin and silica.
- Examples of the resin include thermosetting resins such as melamine resin, urea resin, benzoguanamine resin, phenol resin, xylene resin; styrene resin, (meth) acrylic resin, polyacrylonitrile, AS resin (acrylonitrile-styrene copolymer). And various types of homopolymers and copolymers prepared using one or more monomers such as ASA resin (acrylonitrile-styrene-methyl acrylate copolymer).
- organic fluorescent dye-aggregated particles and the inorganic phosphor aggregates are publicly known, and details such as the phosphor used for the production thereof and the matrix and the production method, and specific examples of the embodiments are described in, for example, International Publication No. 2013/035703, Reference may be made to International Publication No. 2013/147081, International Publication No. 2014/136776, and the like.
- the average particle diameter of the phosphor-aggregated particles is preferably 30 to 300 nm, more preferably 40 to 160 nm. In general, the smaller the particle size, the larger the specific surface area and the higher the binding force with the specimen. However, when the average particle size is less than 30 nm, the bright spot that should be observed in the fluorescence observation due to the phosphor integrated particles May not be observed at all or may be difficult to observe. Conversely, when the average particle diameter of the phosphor-aggregated particles exceeds 300 nm, it is difficult to accurately count the bright spots without separating the bright spots, such as too many bright spots observed in fluorescence observation. There is a case.
- the coefficient of variation indicating the variation in the particle diameter of the phosphor integrated particles is not particularly limited, but is preferably about 20% or less.
- the particle diameter of the phosphor integrated particles is a diameter obtained by taking an image using a scanning electron microscope (SEM), measuring the cross-sectional area of the resin particles for fluorescent labeling, and setting the measured value as the area of a corresponding circle. It can be measured as (area circle equivalent diameter). After measuring the particle diameter of each of the phosphor-aggregated particles contained in a sufficient number (for example, 1000) of the population, the average particle diameter is calculated as the arithmetic average thereof, and the coefficient of variation is expressed by the formula: 100 ⁇ grains Calculated by standard deviation of diameter / average particle diameter.
- the average particle diameter of the phosphor-aggregated particles can be adjusted within a desired range by adjusting the conditions during the production.
- an emulsion polymerization method specifically, (co) polymerizing monomers for synthesizing a resin (thermoplastic resin or thermosetting resin) as a base material of the particles.
- a resin thermoplastic resin or thermosetting resin
- the phosphor is added and the phosphor is incorporated into or on the surface of the (co) polymer.
- micelles having an aqueous phase on the outside and an oil phase on the inside are formed by the surfactant, and the monomer constituting the resin is included in the oil phase inside the micelle.
- a polymerization reaction is performed inside.
- the average particle diameter is 30 to 30%. 300 nm particles can be produced.
- the average particle size of the fluorescent dye-aggregated particles can also be changed by changing the ratio of the resin raw material and the phosphor used in the production of the fluorescent dye-aggregated particles to the entire reaction system. The diameter can be adjusted.
- the average particle size of the inorganic phosphor aggregated particles is, for example, after producing the inorganic phosphor aggregated particles, classified by a size selective precipitation method, and collecting a fraction of the inorganic phosphor aggregated particles having a predetermined particle size.
- it can be within a predetermined range.
- an adsorbate having a lipophilic group is adsorbed on the surface of the inorganic phosphor aggregated particle in advance, and then the inorganic phosphor aggregated particle is dispersed in the lipophilic solvent, and amphiphilic addition is added to the solvent.
- the agent is added little by little to cause precipitation.
- the dispersibility of the inorganic phosphor aggregated particles strongly depends on the interaction between the adsorbing group on the particle surface and the solvent, so that by gradually adding the additive, aggregated precipitates are formed in order from the large-sized inorganic phosphor aggregated particles.
- the precipitate is recovered by centrifugation and redispersed in a solvent, whereby inorganic phosphor-aggregated particles having a narrow particle size distribution can be obtained.
- examples of the adsorbate having a lipophilic group include compounds having an alkyl group such as heptane, octane, and dodecane, and compounds having 8 to 12 carbon atoms are preferable.
- examples of the lipophilic solvent include pyridine and hexane
- examples of the amphiphilic additive include chloroform and methanol.
- lipophilic groups that can be adsorbed on the surface of inorganic phosphors such as quantum dots include phosphino groups such as trioctylphosphine (TOP), phosphine oxide groups such as trioctylphosphine oxide (TOPOT), phosphate groups, amino groups, etc. Is mentioned.
- phosphino groups such as trioctylphosphine (TOP)
- phosphine oxide groups such as trioctylphosphine oxide (TOPOT)
- phosphate groups amino groups, etc. Is mentioned.
- phosphor-aggregated particles in which a primary antibody and a secondary antibody are linked as biological substance-binding proteins, respectively are used as the third and fourth embodiments of the immunostaining method.
- phosphor-aggregated particles in which a first reactive substance (for example, streptavidin, anti-hapten antibody) is linked as a biological substance-binding protein are used as protein-modified phosphor-aggregated particles.
- protein-modified phosphor-aggregated particles whose biological substance-binding protein is an antibody
- the phosphor-aggregated particles and the antibody In this reaction, an excess amount of antibody is usually added, so in addition to the protein-modified phosphor-aggregated particles generated in the solution after the reaction, unreacted antibodies (not bound to the particles) May be included.
- These protein-modified phosphor-aggregated particles can be produced according to a known method.
- the reactive site of the synthesized fluorescent particle and the biological substance binding protein may be directly linked by covalent bonding or the like. Specifically, by using a linker having reactive functional groups at both ends, each reactive functional group of the linker reacts with a reactive site possessed by the phosphor-aggregated particles and a reactive site possessed by the biological substance binding protein. Then, the phosphor-aggregated particles and the biological material-binding protein may be linked via a linker by forming a covalent bond.
- Examples of reactive sites possessed by biological substance-binding proteins include amino groups, carboxy groups, and thiol groups that are possessed by general proteins.
- the reactive site may be a site originally possessed by the biological substance-binding protein, or is introduced into the biological substance-binding protein by using a treatment agent (compound) other than the linker (eg, silane coupling). It may be a site introduced to the particle surface by reacting with a compound such as an agent.
- a treatment agent compound
- silane coupling e.g, silane coupling
- a disulfide bond (-S--) originally possessed by a biological substance-binding protein can be obtained by introducing a thiol group into an amino group that has been present or by using a reducing agent such as dithiothreitol (DTT).
- DTT dithiothreitol
- a method of cleaving S-) to generate a thiol group can be mentioned.
- the reactive functional group that the linker used for binding the phosphor-aggregated particles and the biological substance-binding protein should have a thiol group.
- those capable of reacting with, for example, maleimide groups are preferred.
- the linker only needs to have a reactive functional group at one or both ends capable of reacting with the reactive site of such a biological substance-binding protein, and the reactive functional groups at both ends may be the same or different. May be.
- the linker may be a compound having a chain structure such as a polyoxyalkylene moiety in the molecule, typically a PEG (polyethylene glycol) chain.
- the function of the linker may be performed by the silane coupling agent itself as described below.
- silanol groups on the surface of the silica particles may be used as reactive sites of the phosphor accumulation particles (silica particles), or a silane coupling agent is used. Then, reactive sites other than silanol groups introduced on the surface of the silica particles may be used.
- the silane coupling agent used here is a compound having a hydrolyzable group such as an alkoxy group, an acetoxy group or a halogen atom at one end and a functional group such as an amino group, a mercapto group or an epoxy group at the other end.
- a hydrolyzable group such as an alkoxy group, an acetoxy group or a halogen atom at one end
- a functional group such as an amino group, a mercapto group or an epoxy group at the other end.
- a functional group such as the amino group described above can be introduced.
- the introduced functional group such as an amino group may be used for reacting itself with a reactive site of the biological substance-binding protein, and if necessary, a reactive functional group at one end of the linker.
- the reactive functional group at the other end of the linker may be used to react with the reactive site of the biological material binding protein.
- a mercapto group or amino group introduced by a silane coupling agent is used as a reactive site of the phosphor-aggregated particles
- a maleimide group or an N-hydroxysuccinimide (NHS) group is formed at one end as a reactive functional group for the reactive site. It is preferable to use a linker having
- the reactive sites of the phosphor-aggregated particles are synthesized by the raw materials (monomers, etc.) used to synthesize the resin. It may be a functional group remaining afterwards, or may have a predetermined structure formed by a resin synthesis reaction, or by using a treatment agent (compound) other than the linker described above, the phosphor
- aggregation particles originally have may be sufficient.
- a silane coupling agent having a hydrolyzable group for example, trialkoxy group
- a silane coupling agent having a hydrolyzable group for example, trialkoxy group
- introducing amino groups as reactive functional groups corresponding to the reactive sites of biological substance-binding proteins on the surface of melamine resin particles by reacting with or by reacting linkers with amino groups at both ends can do.
- one amino group of the linker reacts with the reactive site of the melamine resin, for example, melamine and formaldehyde used in the synthesis of the melamine resin in advance. It is a reactive functional group for the methylol group (—CH 2 OH) possessed by methylol melamine that is produced when it is made to react, and the etherified product (—CH 2 OR) produced by further reaction with alcohol.
- phosphor-aggregated particles whose base material is a styrene resin
- a monomer having a functional group such as an amino group or an epoxy group in the side chain that can be copolymerized with styrene is used when the styrene resin is synthesized.
- styrene resin particles having a functional group such as an amino group or an epoxy group on the surface as a reactive site for reacting with the reactive functional group at one end of the linker can be obtained.
- linker and silane coupling agent for the reaction as described above those having various reactive functional groups at both ends are commercially available and can be easily obtained, and desired reactive functional groups are provided at both ends. Those possessed can also be synthesized according to a known method.
- the linker can be reacted with the biological substance-binding protein and / or phosphor-aggregated particles according to a known protocol. Reaction conditions between the linker and the biological material-binding protein and / or phosphor-aggregated particles, for example, the number (density) of reactive sites of the biological material-binding protein and / or phosphor-aggregated particles, the linker used in the reaction and the living body Fluorescence by biological substance-binding protein depends on the ratio of the number of molecules to the substance-binding protein and / or phosphor-aggregated particles (concentration and volume of each solution), type and amount of reaction reagent, reaction temperature, reaction time, etc. Since the modification state of the body-aggregated particles can vary, these may be adjusted appropriately.
- the reaction between the linker and the biological substance-binding protein and the reaction between the linker and the phosphor-aggregated particles may be performed sequentially (or simultaneously if possible).
- the phosphor-aggregated particles and the linker are first bound, and then the linker whose one end is bound to the phosphor-aggregated particles and the biological material binding protein may be bound.
- fluorescent premix particles in which a primary antibody or a secondary antibody is linked as a biological material-binding protein are used as protein-modified phosphor integrated particles.
- Such fluorescent premix particles are obtained by combining phosphor-aggregated particles modified with a first reactive substance and antibodies modified with a second reactive substance with each other between the first reactive substance and the second reactive substance. It can produce by connecting by an effect
- the fluorescent premix particles can be produced by the following first to third steps.
- the first step is a step for preparing phosphor aggregated particles modified with a first reactive substance. This step can be the same embodiment as the step for producing the phosphor-aggregated particles modified with the biological substance-binding protein described in ⁇ Method for producing protein-modified phosphor-aggregated particles >>.
- a purification treatment may be performed by contacting a filter carrying a protein binding substance in order to immediately remove unreacted substances and impurities.
- Second step Step of preparing antibody modified with second reactive substance
- the second step is a step for preparing an antibody modified with the second reactive substance.
- each reactive functional group of the linker is reacted with a reactive site of the antibody and a reactive site of the second reactive substance.
- the linker By forming a covalent bond, the antibody and the second reactive substance are linked via a linker.
- this step is a step for preparing an antibody modified with biotin used in an immunostaining method (ABC method) using a conventional avidin-biotin complex. It can be set as the same embodiment.
- the reaction mode between the linker and the second reactive substance (preferably biotin) and the reaction mode between the linker and the antibody are the reactions between the linker and the protein, both of the above-described linker and biological substance are used. It may be the same as the reaction pattern with the binding protein, and may be modified as necessary.
- a linker having biotin bonded to one end in advance and a reactive functional group (for example, a maleimide group to a thiol group) for the reactive site of the antibody at the other end includes so-called biotin labeling, including necessary reaction reagents. It is commercially available as a product such as a kit and is commonly used. In the case of using such a kit, the second step only needs to carry out a reaction for binding a biotinylated linker to the antibody.
- the reaction between the linker and the second reactive substance (not required when using a product such as the kit described above) and the reaction between the linker and the antibody are sequentially performed (or simultaneously if possible). Just do it.
- a purification treatment may be performed by contacting a filter carrying a protein binding substance in order to immediately remove unreacted substances and impurities.
- Third step a reaction step of the first reactive substance-modified phosphor-aggregated particle and the second reactive substance-modified antibody
- the third step is a first reactive substance-modified phosphor-aggregated particle prepared in the first step.
- the second reactive substance-modified antibody prepared in the second step to finally produce fluorescent premix particles.
- the first reactive substance-modified phosphor-aggregated particles and the second reactive substance-modified antibody are mixed in an appropriate solvent (for example, in a buffer solution such as PBS) for a predetermined time. It can be performed by reacting.
- an appropriate solvent for example, in a buffer solution such as PBS
- a solution containing protein-modified phosphor-aggregated particles and contaminants derived from the production process typically binds the surface of the phosphor-aggregated particles to the biological material to produce protein-modified phosphor-aggregated particles. It is a solution (reaction solution) after the reaction for modifying with a sex protein is performed.
- reaction solution a solution after the reaction for modifying with a sex protein is performed.
- the protein-modified phosphor-aggregated particles are produced through a long process such as the synthesis of the phosphor-aggregated particles and the surface modification thereof. Contaminants such as impurities contained in the raw materials of the reaction and the raw materials and reagents are included.
- a filter for purifying protein-modified phosphor-aggregated particles includes a filter having pores of a size through which protein-modified phosphor-aggregated particles can pass, and a protein-binding substance carried on the filter. Prepare.
- the filter is preferably used in the production method.
- the filter before supporting the protein binding substance is not particularly limited, and filters of various materials such as a porous polymer made of a crosslinked copolymer can be used.
- filters of various materials such as a porous polymer made of a crosslinked copolymer can be used.
- a porous polymer filter comprising a cross-linked copolymer of dextran or a derivative thereof (such as allyl dextran) and acrylamide or a derivative thereof (N, N-methylenebisacrylamide or the like) is preferable.
- filters examples include (eg, trade name “Sephacryl® S-1000® SF”, manufactured by GE Healthcare Japan, Inc.) and the like.
- the size of the pores of the filter is not particularly limited, but is preferably a size through which protein-modified phosphor particles composed of phosphor-aggregated particles having an average particle diameter of 30 to 300 nm can pass.
- a filter widely used for antibody purification for example, protein A-supported resin “TOYOPEARL AF-rProtein A HC-650F” has an average pore diameter of about 5 nm and is not a filter through which protein-modified phosphor aggregated particles can pass. .
- the method of loading the protein binding substance on the filter before loading the protein binding substance is not particularly limited, but in general, the cross-linked copolymer of the filter reacts with the functional group of each protein binding substance. And may be covalently bonded.
- a carboxymethyl group can be introduced by a treatment of reacting bromoacetic acid with a hydroxyl group of the copolymer.
- protein A, protein G or protein L is selected as the protein binding substance
- the amino group of these proteins can be used.
- WSC water-soluble carbodiimide
- the protein-binding substance-carrying filter can be used, for example, for purification of protein-modified phosphor-aggregated particles by packing in a column similar to that for gel filtration that has been used in conventional purification processes.
- the protein-binding substance has binding ability to a biological substance-binding protein (a biological substance-binding protein linked to the fluorescent substance-integrating particle) included in the protein-modified fluorescent substance-integrating particle, While it can be captured, the protein-modified phosphor-aggregated particles (and biological substance-binding protein) can be desorbed by weakening the binding (that is, having reversible binding performance).
- a biological substance-binding protein a biological substance-binding protein linked to the fluorescent substance-integrating particle
- an appropriate substance may be selected according to the biological substance-binding protein.
- protein A when purifying protein-modified phosphor-aggregated particles having an antibody as a biological material-binding protein, protein A, protein G or protein L, which is known to have strong reversible binding performance to the antibody, is used. It is preferably used as a protein binding substance.
- protein A When protein A, protein G, or protein L is used as a protein binding substance, the affinity (affinity) for the antibody differs depending on the site of the antibody, animal species, subclass, etc., and is thus linked to the protein-modified phosphor-integrated particle. It is necessary to select an appropriate one corresponding to the antibody used.
- protein A binds strongly to, for example, human IgG1, IgG2, and IgG4, but hardly binds to human IgG3, and binds strongly to mouse IgG2a, IgG2b, and IgG3, but binding to mouse IgG1 is close to biological conditions. It has the property of binding only weakly in a buffer (Tris-HCl or sodium phosphate buffer).
- protein A and protein G mainly recognize and bind to the Fc region, so that a protein for purifying protein-modified phosphor-aggregated particles to which a full-length antibody or antibody fragment having the Fc region is linked is used.
- Preferred as a binding substance Protein G binds weakly to the Fab region, and may be used for purification of protein-modified phosphor-aggregated particles to which antibody fragments containing the Fab region are linked.
- protein L recognizes and binds to the light chain ( ⁇ light chain), it is used to purify protein-modified phosphor-aggregated particles that do not have an Fc region and to which antibody fragments such as scFv and Fab are linked.
- Preferred as a protein binding substance Preferred as a protein binding substance.
- the fluorescent staining liquid includes a purified product of the protein-modified phosphor-aggregated particles, and is usually a dispersion of the purified product.
- the fluorescent staining solution may be the liquid itself when the purified product of the protein-modified phosphor-aggregated particles is a liquid, and the purified product of the protein-modified phosphor-aggregated particles is dispersed in an appropriate dispersion medium. It may be a liquid.
- the dispersion medium include PBS (phosphate buffered saline) containing 1% BSA.
- the fluorescent staining liquid when used in an embodiment in which two or more kinds of target biological substances are targeted for fluorescent labeling, it may contain two or more kinds of protein-modified phosphor integrated particles corresponding to each target biological substance. Good.
- the two or more kinds of protein-modified phosphor-aggregated particles have mutually different fluorescence wavelength peaks so as not to adversely affect the distinguishability of the fluorescence (bright spot) of the protein-modified phosphor-aggregated particles that label each target biological substance. It is preferable that they are sufficiently separated, for example, 100 nm or more is preferable.
- such a fluorescent staining liquid using a plurality of target biological substances as targets may be a one-pack type in which two or more kinds of protein-modified phosphor integrated particles are contained in the same pack (dispersion).
- a multi-pack type in which each protein-modified phosphor-aggregated particle is contained in a separate pack may be used.
- the fluorescent staining liquid is a pack of other reagents (for example, a staining liquid for cell morphology observation) in addition to the one-pack or multi-pack pack of protein-modified phosphor-aggregated particles. May be included.
- the target biological substance which is the target of the fluorescent staining, is preferably at least one biological substance contained in the specimen, particularly preferably a protein. Further, mainly in pathological diagnosis, protein quantification or detection may be performed. Most preferably, it is a protein (antigen) that is the subject of immunostaining. Typically, for example, proteins (so-called biomarkers) related to cancer pathological diagnosis are preferable.
- PD-L1 Programmed cell death1 ligand 1
- CTLA4 cytotoxic T lymphocyte antigen-4
- CD8 CD30, CD48, CD59
- EGFR HER1 (Epidermal Growth Factor Receptor: epithelium) Growth factor receptor
- HER2 Human Epidermal Growth Factor Receptor
- HER3, HER4, VEGFR Vasular Endothelial Growth Factor Receptor
- IGFR Insulin-like Growth Factor Receptor
- HGFR hepatocyte growth factor receptor
- growth factor receptor receptor
- important inhibitory immune checkpoint molecules on the T cell surface Receptors of the immune system such as PD-1 (Programmed cell death 1), which is the PD-L1 receptor A protein
- the fluorescent staining solution can be used for various fluorescent staining methods.
- a target protein contained in the specimen is fluorescently labeled by immunostaining, Used to make stained slides.
- the fluorescence staining method includes specimen pretreatment steps (deparaffinization treatment, antigen activation treatment, cell fixation treatment, washing treatment, etc.), staining steps (immunostaining treatment, morphological observation staining treatment, phosphor-aggregated particles). Step treatment, washing treatment, blocking treatment, etc.), sample post-treatment steps (encapsulation treatment, penetration treatment, dehydration treatment, etc.), etc.
- the analysis method using the completed stained slide can be performed by an observation / photographing process, an image processing / analysis process using the photographed image, and the like.
- Such fluorescent staining methods and analysis methods include, for example, International Publication No. 2013/035688, Japanese Patent Application Laid-Open No. 2015-117980, International Publication No. 2014/136985, International Publication No. 2016/129444, International Publication No. It is possible to implement appropriately by referring to patent documents such as 163209 and based on general or known technical matters.
- the fluorescent staining method performed using the fluorescent staining solution is not particularly limited as long as the target biological substance can be fluorescently labeled to achieve a predetermined purpose, but representative examples include the following first to sixth embodiments.
- a fluorescent labeled primary antibody in which phosphor-aggregated particles and a primary antibody are linked is prepared, and the target biological substance (target protein) is directly fluorescent with the fluorescent labeled primary antibody.
- the primary antibody corresponds to a biological material-binding protein
- the fluorescently labeled primary antibody corresponds to a protein-modified phosphor-aggregated particle.
- a primary antibody and a fluorescently labeled secondary antibody in which phosphor-aggregated particles and a secondary antibody are linked are prepared, and the primary antibody reacts with a target biological substance (target protein). Then, the target antibody is indirectly fluorescently labeled and stained by reacting the primary antibody with a fluorescently labeled secondary antibody (secondary antibody method).
- the secondary antibody corresponds to the biological material-binding protein
- the fluorescently labeled secondary antibody corresponds to the protein-modified phosphor-aggregated particle.
- the first reactive substance-modified primary antibody in which the primary antibody and the second reactive substance (for example, biotin and hapten) are linked, and the phosphor-aggregated particles and the first reactivity are used.
- a first reactive substance-modified phosphor-aggregated particle linked with a substance for example, avidin, anti-hapten antibody
- a target biological substance target protein
- the target protein is indirectly fluorescently labeled and stained by reacting the first reactive substance-modified phosphor-aggregated particles by utilizing the specific binding of the first reactive substance to the second reactive substance.
- the first reactive substance corresponds to the biological substance-binding protein
- the first reactive substance-modified phosphor integrated particle corresponds to the protein-modified phosphor integrated particle.
- a primary antibody, a secondary antibody-modified secondary antibody in which a secondary antibody and a second reactive substance are linked, and phosphor-aggregated particles and the first reactive substance are combined.
- First reactive substance-modified phosphor-integrated particles linked to each other, after reacting the target biological substance (target protein) with the primary antibody and then reacting with the second reactive substance-modified secondary antibody A method of indirectly fluorescently labeling and staining a target protein by reacting the first reactive substance-modified phosphor-aggregated particles using specific binding of the first reactive substance to the second reactive substance (Avidin-biotin combined secondary antibody method and hapten-anti-hapten antibody combined secondary antibody method).
- the first reactive substance corresponds to the biological substance-binding protein
- the first reactive substance-modified phosphor integrated particle corresponds to the protein-modified phosphor integrated particle.
- the fifth embodiment of the fluorescent staining method is a modification of the third embodiment described above, and is a method using “fluorescent premix particles” having a primary antibody (primary antibody type fluorescent premix particle method).
- the second reactive substance-modified primary antibody in which the primary antibody and the second reactive substance are linked and the first reactive substance-modified phosphor in which the phosphor-aggregated particles and the first reactive substance are linked. Particles are prepared, these are reacted in advance, and phosphor-aggregated particles to which primary antibodies are linked are prepared (premixed) through the reaction between the first reactive substance and the second reactive substance.
- the target biological material is then fluorescently labeled and stained by reacting fluorescent premix particles, which are phosphor-aggregated particles with primary antibodies linked by premix.
- fluorescent premix particles which are phosphor-aggregated particles with primary antibodies linked by premix.
- the primary antibody corresponds to the biological material-binding protein
- the fluorescent premix particle corresponds to the protein-modified phosphor-aggregated particle.
- the sixth embodiment of the fluorescent staining method is a modification of the above-described fourth embodiment, and is a method using a “fluorescent premix particle” having a secondary antibody (secondary antibody type fluorescent premix particle method).
- a second reactive substance-modified secondary antibody in which a secondary antibody and a second reactive substance are linked, and a first reactive substance-modified phosphor in which phosphor-aggregated particles and a first reactive substance are linked Particles are prepared, these are reacted in advance, and phosphor-aggregated particles to which secondary antibodies are linked are prepared (premixed) through the reaction between the first reactive substance and the second reactive substance.
- the primary antibody is reacted with fluorescent premix particles, which are phosphor-aggregated particles in which the secondary antibody is linked by premix. Fluorescently labeled and stained.
- the secondary antibody corresponds to the biological material-binding protein
- the fluorescent premix particle corresponds to the protein-modified fluorescent substance integrated particle.
- each of two or more kinds of target biological substances contained in the specimen may be modified so as to be fluorescently labeled with different types of protein-modified phosphor integrated particles.
- the target biological substances may be fluorescently labeled one by one, or all target biological substances may be fluorescently labeled simultaneously.
- TAMRA-Accumulated Silica Particles According to the following steps (1-1a) to (1-1d), TAMRA (registered trademark) (5-carboxytetramethylrhodamine), which is an organic fluorescent dye, is used as a phosphor. Integrated (encapsulated) TAMRA integrated silica particles were produced.
- Step (1-1c) The mixture prepared in Step (1-1a) was added to the mixture prepared in Step (1-1b) while stirring at room temperature. Stirring was performed at room temperature for 12 hours from the start of addition.
- the average particle diameter of the TAMRA-integrated silica particles was 100 nm, and the variation coefficient of the average particle diameter was 15%. there were.
- PBS phosphate buffered saline
- EDTA ethylenediaminetetraacetic acid
- SM (PEG) 12 manufactured by Thermo Scientific, succinimidyl-[(N-maleimidopropionamid)-
- the amount (mole number) of mercapto groups was measured. Further, 1 ⁇ L of the same amount of antibody was separately collected, and the mass of the collected anti-HER2 antibody was quantified by performing the BCA method. From this mass and the molecular weight of the anti-HER2 antibody, the number of moles of the fractionated antibody was calculated. Furthermore, when the number of mercapto groups per antibody was calculated by the formula “number of moles of mercapto group / number of moles of antibody”, it was 1.5.
- the anti-HER2 antibody-directed TAMRA-integrated silica particles had an average of 3000 antibodies on the surface of one particle and a particle surface of 1 mm. When converted to 2 units, it is estimated that 9.555 ⁇ 10 16 antibodies are bound.
- Example 1 Production of purified product of anti-HER2 antibody-directed TAMRA-aggregated silica particles using a protein A-binding resin column After the same production steps 1-1 to 1-4 as in Comparative Example 1, the following production step 1 was performed. Using the protein A-binding resin column produced in -5, purification treatment by the following production step 1-6 was performed.
- Thermo Fisher Scientific, “21184”) was reacted, and the amino group of protein A was bound (immobilized) to the active esterified carboxyl group by dehydration reaction. .
- the protein A-binding resin thus obtained was packed in a column (1 mL syringe) to produce a protein A-binding resin column.
- Example 1 ′ Production of purified product of anti-HER2 antibody-directed TAMRA-aggregated silica particles using protein G-binding resin column
- Example 1 instead of protein A-binding resin column, produced by the following production steps 1-7
- a purified product of anti-HER2 antibody-directed TAMRA-integrated silica particles was produced in the same manner as in Example 1 except that the protein G-binding resin column was used.
- 0.6 mL of the obtained organoalkoxysilane compound was mixed with 48 mL of 99% ethanol, 0.6 mL of tetraethoxysilane (TEOS), 2 mL of ultrapure water, and 2.0 mL of 28% by mass of ammonia water at 5 ° C. for 3 hours. .
- TEOS tetraethoxysilane
- the mixed solution prepared in the above step was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. To this precipitate, ethanol was added to disperse the precipitate, and rinsing was performed again by centrifugation. Further, the same rinsing was repeated twice to obtain Texas Red integrated silica particles (excitation wavelength: 590 nm, emission wavelength: 620 nm). When 1000 particles of the obtained particles were observed with an SEM, the particle diameter was measured, and the average particle diameter was calculated.
- the obtained reaction solution was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. PBS containing 2 mM EDTA was added thereto, the precipitate was dispersed, and centrifuged again under the same conditions. By performing washing by the same procedure three times, maleimide group-introduced Texas Red integrated silica particles were obtained.
- streptavidin manufactured by Wako Pure Chemical Industries, Ltd.
- 1 mg / mL was added to 210 ⁇ L of borate buffer, and then 70 ⁇ L of 2-iminothiolane hydrochloride (Sigma Aldrich) adjusted to 64 mg / mL. And reacted at room temperature for 1 hour.
- a mercapto group was introduced with respect to the amino group of streptavidin (—NH—C ( ⁇ NH 2+ Cl ⁇ ) —CH 2 —CH 2 —CH 2 —SH).
- the obtained solution was desalted by passing through a gel filtration column (Zaba Spin Desaling Columns: manufactured by Funakoshi Co., Ltd.) to obtain a mercapto group-introduced streptavidin capable of binding to maleimide group-introduced Texas red integrated silica particles.
- a gel filtration column Zaba Spin Desaling Columns: manufactured by Funakoshi Co., Ltd.
- the total amount of the obtained mercapto group-introduced streptavidin (0.04 mg) and maleimide group-introduced Texas red integrated silica particles so that the concentration of the silica particles is 0.67 nM 740 ⁇ L of the liquid mixed with was prepared and reacted at room temperature for 1 hour. Thereafter, 10 mM mercaptoethanol was added to stop the reaction.
- reaction solution 1 a purified streptavidin-modified Texas Red-integrated silica particle dispersion having a concentration of the particles adjusted to 0.02 nM was prepared.
- the obtained streptavidin-modified Texas Red integrated silica particles were found to have a density of 0.0311 particles / nm 2 on the surface of the Texas Red integrated silica particles. Are estimated to be connected.
- linker reagent “maleimide-PEG 2 -biotin” (manufactured by Thermo Scientific, product number 21901) was adjusted to 0.4 mM using DMSO. 8.5 ⁇ L of the obtained linker reagent solution was added to the primary antibody solution, mixed, and reacted at 37 ° C. for 30 minutes to bind biotin to the anti-HER2 antibody via the PEG chain. The obtained reaction solution was passed through a desalting column to purify the biotin-modified anti-HER2 antibody.
- biotin-modified primary antibody biotin-modified anti-HER2 antibody
- the absorbance at a wavelength of 300 nm is measured using a spectrophotometer (Hitachi, “F-7000”), whereby the protein in the solution (biotin The concentration of the modified primary antibody) was calculated.
- a biotin-modified primary antibody solution (reaction solution 2) was obtained by adjusting the concentration of the biotin-modified primary antibody to 6 ⁇ g / mL using a 50 mM Tris solution.
- Example 2 Production of purified product of anti-HER2 antibody-linked Texas red integrated silica particles using protein A-binding resin column After the production steps 2-1 to 2-4, the same as the production step 1-5 described above Using the protein A-binding resin column produced as described above, purification treatment by the following production step 2-5 was performed.
- Example 2 ′ Production of purified product of anti-HER2 antibody-linked Texas Red-aggregated silica particles using protein G-binding resin column
- Example 2 instead of the protein A-binding resin column, the production steps 1-7 and A purified product of anti-HER2 antibody-linked Texas Red integrated silica particles was produced in the same manner as in Example 2 except that a protein G-binding resin column produced in the same manner was used.
- the protein-modified phosphor-aggregated particles can be recovered with a sufficiently high recovery rate when using either the protein A-binding resin column or the protein G-binding resin column.
- Each of the fluorescent staining solutions immediately after preparation (before storage) or after storage for 1 month or 3 months was subjected to ultrasonic dispersion treatment, and 40 ⁇ L was weighed and placed on an APS-coated glass slide. Then, it was left to stand at room temperature for about 2 hours to remove moisture and dry.
- the objective lens was set to 40 times, and the dried fluorescent fluorescent solution was irradiated with excitation light.
- the image was taken with an exposure time of 500 ms, focusing on the stage height at which bright spots with uniform brightness were seen.
- the number of bright spots with extremely high brightness was visually counted for each slide image.
- the results are shown in Table 4.
- the fluorescent staining solution using the dispersion obtained in Comparative Examples 1 and 2 that has not been subjected to purification using a protein A-supported column has many aggregates remaining after ultrasonic dispersion, whereas the purification treatment It can be seen that the fluorescent staining solution using the dispersion liquid obtained in Examples 1 and 2 in which almost no agglomerates exist even after storage.
- the sample pretreatment step (deparaffinization treatment, activation) was performed using the obtained fluorescent staining solution in the procedure as shown in (1), (2) and (3) below.
- Treatment staining step (immuno-staining treatment), and specimen post-treatment step (washing treatment and encapsulation treatment), thereby producing a staining slide based on the immunostaining method. Thereafter, using the prepared staining slide, observation and imaging were performed in the procedure as shown in (4) below.
- Specimen pretreatment step (1-1) Deparaffinization Tissue array slide (CB-A712) (HER2) manufactured by Cosmo Bio, whose FISH score was calculated in advance using Pathvision HER-2 DNA probe kit (Abbott) Positive staining control specimens) were used.
- the tissue array slide was deparaffinized by the following procedures (i) to (iii).
- Observation / Imaging Fluorescence is emitted by irradiating the stained slide after the encapsulation process with predetermined excitation light (excitation light having a wavelength corresponding to the excitation wavelength of TAMRA or Texas Red used as a fluorescent dye).
- excitation light having a wavelength corresponding to the excitation wavelength of TAMRA or Texas Red used as a fluorescent dye.
- the stained slide in this state was observed and imaged using a fluorescence microscope (Olympus, “BX-53”) and a digital camera for microscope (Olympus, “DP73”).
- the excitation light was passed through an optical filter, and was set to 545 to 565 nm for TAMRA and 575 to 600 nm for Texas Red.
- the fluorescence to be observed was also set to 570 to 590 nm for TAMRA and 612 to 692 nm for Texas Red by passing through an optical filter.
- the conditions of the excitation wavelength at the time of microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W / cm 2 in excitation at 550 nm for TAMRA and 580 nm for Texas Red.
- the exposure time at the time of image acquisition was set to 200 milliseconds so that the luminance of the image was not saturated.
- the bright spot was measured by an ImageJ FindMaxims method based on an image taken at 400 times.
- Table 5 shows the presence / absence of cluster-like bright spots (aggregates) and the average luminance per pixel in the photographed image of the prepared stained slide.
- FIG. 2 shows a fluorescent staining image obtained by photographing a slide stained with a fluorescent staining solution prepared from the dispersion obtained in Comparative Example 2.
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Abstract
Description
本発明の一実施形態に係るタンパク質修飾蛍光体集積粒子の精製物を製造する方法は、タンパク質修飾蛍光体集積粒子と、その作製工程に由来する夾雑物とを含有する溶液を、タンパク質結合性物質を担持したフィルターと接触させることにより、前記タンパク質修飾蛍光体集積粒子を前記夾雑物から分離する、精製工程を含む。
本発明の一実施形態に係るタンパク質修飾蛍光体集積粒子の精製物は、前記精製工程を含む方法により得られる。
前記タンパク質修飾蛍光体集積粒子は、表面が生体物質結合性タンパク質で修飾された蛍光体集積粒子である。蛍光体集積粒子の生体物質結合性タンパク質による修飾方法は、蛍光染色の目的とする生体物質の蛍光標識方法に応じて適宜選択すればよい。前記タンパク質修飾蛍光体集積粒子としては、第1反応性物質で修飾された蛍光体集積粒子と、第2反応性物質で修飾された生体物質結合性タンパク質とが、第1反応性物質と第2反応性物質の相互作用により連結している粒子である、蛍光プレミックス粒子が好ましい。
(ii)その第1反応性物質1つに対して、最大いくつの第2反応性物質(で修飾された生体物質結合性タンパク質)が結合するか、係数を決定する(例えば、第1反応性物質がアビジン等である場合は4であり、第1反応性物質が抗ハプテン抗体である場合は1である)
(iii)前記(i)の1粒子あたりの数に、前記(ii)の係数を乗じた値を、蛍光体集積粒子1粒子あたりに連結し得る生体物質結合性タンパク質の数の最大値とみなす
(iv)反応のために添加した第2反応性物質で修飾された生体物質結合性タンパク質の量が、(iii)の最大値よりも多いときは、前記最大値を、蛍光体集積粒子1粒子あたりの生体物質結合性タンパク質の数とみなし、(iii)の最大値よりも少ないときは、第2反応性物質で修飾された生体物質結合性タンパク質の添加総数を蛍光体集積粒子の数で除した値を、蛍光体集積粒子1粒子あたりの生体物質結合性タンパク質の数とみなす。
生体物質結合性タンパク質は、生体物質への結合性能を有するタンパク質であって、蛍光染色の対象である目的生体物質に、蛍光体集積粒子を直接的または間接的に結合させるために用いられる。このような生体物質結合性タンパク質は、目的生体物質の蛍光標識方法に応じて、また、蛍光体集積粒子の修飾の実施形態に応じて適宜選択すればよいが、抗体であることが好ましい。
前記生体物質結合性タンパク質としては、抗体(免疫グロブリン)を用いることが好ましい。該抗体は、免疫染色法において目的生体物質と直接的にまたは間接的に結合できる抗体であれば特に限定されない。該抗体としては、前記1次抗体、2次抗体および3次抗体以上の高次抗体のいずれであってもよい。
第1反応性物質および第2反応性物質は、相互作用により互いに特異的に結合する物質同士の組み合わせであって、いずれも生体物質結合性タンパク質とそれが結合する目的生体物質(目的タンパク質、または2次抗体もしくは3次抗体以上の高次抗体)との特異的な結合と交差して反応することのない物質から選択される。
前記蛍光体集積粒子(生体物質結合性タンパク質で修飾される前の粒子)としては、特に制限されないが、有機物または無機物でできた母体となる粒子の内部または表面に、蛍光体(例えば蛍光色素)を複数個固定して集積した構造を有するナノサイズの(直径が1μm以下の)粒子であることが好ましく、一粒子で十分な輝度の蛍光を発することができる粒子であることが好ましい。
有機蛍光色素集積粒子は、粒子の母体となる物質の内部または表面に有機蛍光色素を複数個集積した、ナノサイズの蛍光粒子であることが好ましい。
無機蛍光体集積粒子は、粒子の母体となる物質の内部または表面に半導体ナノ粒子を複数個集積した、ナノサイズの蛍光粒子であることが好ましい。
蛍光体集積粒子の平均粒子径は、好ましくは30~300nm、より好ましくは40~160nmである。一般的に、粒子径が小さくなるほど比表面積が大きくなり、検体との結合力が高まるが、平均粒子径が30nmを下回ると、蛍光体集積粒子に起因して蛍光観察で観察されるべき輝点が全く観察されないか、または観察されにくい場合がある。逆に、蛍光体集積粒子の平均粒子径が300nmを上回ると、蛍光観察において観察される輝点が多くなりすぎる等、輝点同士が分離されずに正確に輝点をカウントすることが困難となる場合がある。
後述する免疫染色法の第1および第2実施形態では、それぞれ生体物質結合性タンパク質として1次抗体および2次抗体が連結された蛍光体集積粒子を、免疫染色法の第3および第4実施形態では、ともに生体物質結合性タンパク質として第1反応性物質(例えばストレプトアビジン、抗ハプテン抗体)が連結された蛍光体集積粒子を、タンパク質修飾蛍光体集積粒子として用いる。
後述する、免疫染色法の第5および第6実施形態では、それぞれ生体物質結合性タンパク質として1次抗体または2次抗体が連結された蛍光プレミックス粒子を、タンパク質修飾蛍光体集積粒子として用いる。そのような蛍光プレミックス粒子は、第1反応性物質で修飾された蛍光体集積粒子と、第2反応性物質で修飾された抗体とを、第1反応性物質と第2反応性物質の相互作用により連結させることにより作製することができる。典型的には、蛍光プレミックス粒子は、下記第1~第3工程により作製することができる。
前記第1工程は、第1反応性物質で修飾された蛍光体集積粒子を調製するための工程である。この工程は、≪タンパク質修飾蛍光体集積粒子の作製方法≫において説明した、生体物質結合性タンパク質で修飾された蛍光体集積粒子を作製するための工程と同様の実施形態とすることができる。
前記第2工程は、第2反応性物質で修飾された抗体を調製するための工程である。典型的には、両末端に反応性官能基を有するリンカーを用いて、リンカーのそれぞれの反応性官能基を、抗体が有する反応性部位および第2反応性物質が有する反応性部位と反応させて共有結合を形成することにより、抗体と第2反応性物質とをリンカーを介して連結する。例えば、第2反応性物質としてビオチンを用いる場合、この工程は、従来のアビジン-ビオチン複合体を利用した免疫染色法(ABC法)において用いられる、ビオチンで修飾された抗体を調製するための工程と同様の実施形態とすることができる。
前記第3工程は、第1工程において調製された第1反応性物質修飾蛍光体集積粒子と、第2工程において調製された第2反応性物質修飾抗体とを反応させて、最終的に蛍光プレミックス粒子を作製するための工程である。
タンパク質修飾蛍光体集積粒子と、その作製工程に由来する夾雑物とを含有する溶液は、典型的には、タンパク質修飾蛍光体集積粒子を作製するために、蛍光体集積粒子の表面を生体物質結合性タンパク質で修飾する反応が行われた後の溶液(反応後溶液)である。タンパク質修飾蛍光体集積粒子は前述のように、蛍光体集積粒子の合成やその表面修飾など、長いプロセスを経て作製され、反応後溶液には、反応により生成したタンパク質修飾蛍光体集積粒子とともに、未反応の原料、原料や試薬に含まれる不純物などの夾雑物が含まれる。
本発明の一実施形態に係るタンパク質修飾蛍光体集積粒子精製用フィルターは、タンパク質修飾蛍光体集積粒子が通過可能なサイズの細孔を有するフィルターと、該フィルターに担持されたタンパク質結合性物質とを備える。
該フィルターは、前記製造方法に好適に用いられる。
前記タンパク質結合性物質は、タンパク質修飾蛍光体集積粒子が有する生体物質結合性タンパク質(蛍光体集積粒子に連結されている生体物質結合性タンパク質)に対する結合性能を有してタンパク質修飾蛍光体集積粒子を捕捉することができる一方、その結合を弱めることで、タンパク質修飾蛍光体集積粒子(および生体物質結合性タンパク質)を脱離させることができる(つまり可逆的な結合性能を有する)。このようなタンパク質結合性物質は、生体物質結合性タンパク質に応じて、適切な物質を選択すればよい。
本発明の一実施形態に係る蛍光染色液は、前記タンパク質修飾蛍光体集積粒子の精製物を含み、通常、該精製物の分散液である。
前記蛍光染色の対象である、目的生体物質は、検体に含まれる少なくとも1種の生体物質であることが好ましく、タンパク質であることが特に好ましく、さらに主に病理診断において、タンパク質の定量ないし検出のために行われる免疫染色の対象であるタンパク質(抗原)であることが最も好ましい。典型的には、例えば、がんの病理診断に関係するタンパク質(いわゆるバイオマーカー)が好ましい。具体的には、PD-L1(Programmed cell death1 ligand 1)、CTLA4(細胞傷害性Tリンパ球抗原-4)、CD8、CD30、CD48、CD59、あるいは、EGFR(HER1)(Epidermal Growth Factor Receptor:上皮増殖因子受容体)、HER2(Human Epidermal Growth Factor Receptor:ヒト上皮増殖因子受容体)、HER3、HER4、VEGFR(Vasular Endothelial Growth Factor Receptor:血管内皮細胞増殖因子受容体)、IGFR(Insulin-like Growth Factor Receptor:インスリン様増殖因子受容体)、HGFR(Hepatocyte Growth Factor Receptor:肝細胞増殖因子受容体)といった増殖因子の受容体(レセプター)や、T細胞表面上にある重要な抑制性の免疫チェックポイント分子であって前記PD-L1の受容体であるPD-1(Programmed cell death 1)などの免疫系の受容体であるタンパク質が例示できる。
前記蛍光染色液は、様々な蛍光染色法に利用することができるが、典型的には、検体である組織切片から作製した組織スライドにおいて、検体に含まれる目的タンパク質を免疫染色により蛍光標識した、染色スライドを作製するために利用される。
下記製造工程1-1~1-4により、抗HER2抗体直結TAMRA集積シリカ粒子を製造した。
下記工程(1-1a)~(1-1d)により、蛍光体として有機蛍光色素であるTAMRA(登録商標)(5-カルボキシテトラメチルローダミン)を集積化(内包)したTAMRA集積シリカ粒子を作製した。
下記工程(1-2a)~(1-2g)により、製造工程1-1で得られたTAMRA集積シリカ粒子に対してマレイミド基を導入した。
下記工程(1-3a)~(1-3c)により、抗HER2抗体にメルカプト基(-SH)を生成させたメルカプト基導入抗HER2抗体を調製し、該抗体中のメルカプト基の量を定量した。
下記工程(1-4a)~(1-4c)により、マレイミド基導入TAMRA集積シリカ粒子とメルカプト基導入抗HER2抗体とを結合させることで、抗HER2抗体直結TAMRA集積シリカ粒子を製造した。
比較例1と同様製造工程1-1~1-4を行った後、下記製造工程1-5により作製したプロテインA結合樹脂カラムを用いて、下記製造工程1-6による精製処理を行った。
ゲル濾過担体「Sephacryl S-1000 SF」(GEヘルスケア・ジャパン株式会社製、アリルデキストランとN,N-メチレンビスアクリルアミドが共有架橋結合した樹脂マトリックス)のデキストラン水酸基とブロモ酢酸とを16時間反応させることにより、担体表面をカルボキシメチル化した(Monchaux, E., and Vermette, P. (2008). Cell adhesion resistance mechanisms using arrays of dextran-derivative layers. J Biomed Mater Res A 85, 1052-1063参照)。なお、「Sephacryl S-1000 SF」は、粒径230nmの物体(リポソーム)が侵入し、通過できる程度の空隙を有する多孔質体と考えられる(http://lifesciencedb.jp/dbsearch/Literature/get_pne_cgpdf.php?year=1990&number=3511&file=2Qgovzb50x7RW4IcCUhKPw==参照)。
製造工程1-5により作製されたプロテインA結合樹脂カラムに、5mLの緩衝液A(0.1Mグリシン+1.2M酒石酸ナトリウム、pH9.0)を3回流すことによって平衡化した。製造工程1-4により得られた抗HER2抗体直結TAMRA集積シリカ粒子の分散液1.5mLを、1.5mLの緩衝液Aで希釈して、平衡化したプロテインA結合樹脂カラムに添加し、抗HER2抗体直結TAMRA集積シリカ粒子における抗HER2抗体(IgG)をプロテインA結合樹脂カラムにおけるプロテインAに結合させた。10mLの緩衝液Aでカラムを洗浄した後、溶離液として3mLの0.1Mグリシン-HCl(pH2.8)を流し、抗HER2抗体直結TAMRA集積シリカ粒子を脱着させ、精製物を回収した。
実施例1において、プロテインA結合樹脂カラムの代わりに、下記製造工程1-7により作製したプロテインG結合樹脂カラムを用いた以外は、実施例1と同様にして、抗HER2抗体直結TAMRA集積シリカ粒子の精製物を製造した。
プロテインAの代わりにプロテインG(サーモフィッシャーサイエンティフィック社製、「21193」)を用いたこと以外は製造工程1-5と同様にして、プロテインG結合樹脂カラムを作製した。
下記製造工程2-1~2-4により、蛍光プレミックス粒子を製造した。
蛍光体として赤色の有機蛍光色素である「テキサスレッド-X」(Sulforhodamine 101-X、シグマアルドリッチ社製)3.4mgと、3-アミノプロピルトリメトキシシラン(信越シリコーン社製、KBM903)3μLとを、N,N-ジメチルホルムアミド(DMF)中で混合し、オルガノアルコキシシラン化合物を得た。
製造工程2-1で得られたテキサスレッド集積シリカ粒子を、2mMのEDTA(エチレンジアミン四酢酸)を含有するPBSを用いて3nMの濃度に調整し、得られた液体に、リンカーとしてSM(PEG)12を最終濃度が10mMとなるように添加、混合し、5℃で1時間反応させた。
目的タンパク質HER2に対する1次抗体、すなわち抗HER2抗体として、ウサギモノクローナル抗体であるAnti-Erb 2 antibody[EP1045Y](abcam社製)を用いた。この抗HER2抗体を50mMのTris溶液に溶解して1次抗体溶液を調製した。
製造工程2-2で得られた0.02nMのストレプトアビジン修飾テキサスレッド集積シリカ粒子分散液(反応液1)25μLと、製造工程2-3で得られた濃度6μg/mLのビオチン修飾1次抗体溶液(反応液2)25μLとを混合し、室温で1時間反応させることにより、抗HER2抗体連結テキサスレッド集積シリカ粒子(蛍光プレミックス粒子)の分散液を作製した。
製造工程2-1~2-4を行った後、前記製造工程1-5と同様にして作製したプロテインA結合樹脂カラムを用いて、下記製造工程2-5による精製処理を行った。
プロテインA結合樹脂カラムに、5mLの緩衝液A(0.1Mグリシン+1.2M酒石酸ナトリウム、pH9.0)を3回流すことによって平衡化した。製造工程2-4で得られた抗HER2抗体連結テキサスレッド集積シリカ粒子の分散液1.5mLを、1.5mLの緩衝液Aで希釈して、平衡化したプロテインA結合樹脂カラムに添加し、抗HER2抗体連結テキサスレッド集積シリカ粒子における抗HER2抗体(IgG)をプロテインA結合樹脂カラムにおけるプロテインAに結合させた。10mLの緩衝液Aでカラムを洗浄した後、溶離液として3mLの0.1Mグリシン-HCl(pH2.8)を流し、抗HER2抗体連結テキサスレッド集積シリカ粒子を脱着させ、製造工程1-6と同様に、溶出後期の画分を取得することによって、抗HER2抗体連結テキサスレッド集積シリカ粒子の精製物を回収した。
実施例2において、プロテインA結合樹脂カラムの代わりに、前記製造工程1-7と同様にして作製したプロテインG結合樹脂カラムを用いた以外は、実施例2と同様にして、抗HER2抗体連結テキサスレッド集積シリカ粒子の精製物を製造した。
実施例1および1’ならびに実施例2および2’の精製処理によるタンパク質修飾蛍光体集積粒子の回収率を測定した。回収率は、カラムに添加する前のタンパク質修飾蛍光体集積粒子分散液に含まれるタンパク質修飾蛍光体集積粒子の量と、得られた精製物に含まれるタンパク質修飾蛍光体集積粒子の量とから算出した。結果を表3に示す。回収率は、精製前後における蛍光染色液の蛍光強度の維持率と相関する。
比較例1および2、ならびに、実施例1および2で得られた分散液を、タンパク質修飾蛍光体集積粒子の濃度が0.002nMとなるよう、1%BSA含有PBSで希釈することで蛍光染色液を調製し、遮光下、冷蔵保存した。
前記分散安定性の評価と同様にして、得られた蛍光染色液を用い、下記(1)、(2)および(3)に示すような手順で、標本前処理工程(脱パラフィン処理、賦活化処理)、染色工程(免疫染色処理)および標本後処理工程(洗浄処理および封入処理)を行うことにより、免疫染色法に基づく染色スライドを作製した。その後、作製された染色スライドを用いて、下記(4)に示すような手順で観察および撮像を行った。
(1-1)脱パラフィン処理
予めパスビジョンHER-2 DNAプローブキット(アボット)を用いてFISHスコアを算出したコスモバイオ社製の組織アレイスライド(CB-A712)(HER2陽性染色対照標本)を用いた。該組織アレイスライドに対し、以下の(i)~(iii)の手順で脱パラフィン処理を行った。
脱パラフィン処理した組織アレイスライドを、以下の(i)~(iv)の手順で賦活化処理した。
賦活化処理(1-2)を行った組織アレイスライドに、前記調製した蛍光染色液を滴下し、4℃で一晩反応させた。
免疫染色処理を行ったスライドに、常温でエンテランニュー(メルク社製)を滴下した後、カバーガラスを被せ、常温で10分間風乾することで、封入処理を行った。その後、シグナルの計測まで、封入処理を終えた染色スライドを遮光して保存した。
封入処理を終えた染色スライドに対して所定の励起光(蛍光色素として用いたTAMRAまたはテキサスレッドの励起波長に対応した波長を有する励起光)を照射して、蛍光を発光させた。その状態の染色スライドを、蛍光顕微鏡(オリンパス社製、「BX-53」)、顕微鏡用デジタルカメラ(オリンパス社製、「DP73」)を用いて観察および撮像した。前記励起光は、光学フィルターに通すことで、TAMRAに対しては545~565nm、テキサスレッドに対しては575~600nmに設定した。また、観察する蛍光についても、光学フィルターを通すことで、TAMRAに対しては570~590nm、テキサスレッドに対しては612~692nmに設定した。
Claims (13)
- タンパク質修飾蛍光体集積粒子と、その作製工程に由来する夾雑物とを含有する溶液を、タンパク質結合性物質を担持したフィルターに接触させることにより、前記タンパク質修飾蛍光体集積粒子を前記夾雑物から分離する、精製工程を含み、
前記タンパク質修飾蛍光体集積粒子が、表面が生体物質結合性タンパク質で修飾された蛍光体集積粒子であり、
前記タンパク質結合性物質が、前記生体物質結合性タンパク質への可逆的な結合性能を有する物質である、
タンパク質修飾蛍光体集積粒子の精製物を製造する方法。 - 前記生体物質結合性タンパク質が抗体であり、前記タンパク質結合性物質がプロテインA、プロテインGまたはプロテインLである、請求項1に記載の製造方法。
- 前記タンパク質修飾蛍光体集積粒子が蛍光プレミックス粒子であり、
前記蛍光プレミックス粒子が、第1反応性物質で修飾された蛍光体集積粒子と、第2反応性物質で修飾された生体物質結合性タンパク質とが、第1反応性物質と第2反応性物質の相互作用により連結している粒子である、
請求項1または2に記載の製造方法。 - 前記第1反応性物質がストレプトアビジンであり、前記第2反応性物質がビオチンである、請求項3に記載の製造方法。
- 前記タンパク質修飾蛍光体集積粒子が、蛍光体集積粒子1粒子当たり、生体物質結合性タンパク質を1000~5000個有する粒子である、請求項1~4のいずれか一項に記載の製造方法。
- 前記蛍光体集積粒子の平均粒子径が30~300nmである、請求項1~5のいずれか一項に記載の製造方法。
- 請求項1~6のいずれか一項に記載の製造方法で得られた、タンパク質修飾蛍光体集積粒子の精製物を含有する蛍光染色液の製造方法。
- 請求項1~6のいずれか一項に記載の製造方法により得られたタンパク質修飾蛍光体集積粒子の精製物。
- 請求項8に記載のタンパク質修飾蛍光体集積粒子の精製物を含有する、蛍光染色液。
- タンパク質修飾蛍光体集積粒子が通過可能なサイズの細孔を有するフィルターと、該フィルターに担持されたタンパク質結合性物質とを備え、
前記タンパク質修飾蛍光体集積粒子が、表面が生体物質結合性タンパク質で修飾された蛍光体集積粒子であり、
前記タンパク質結合性物質が、前記生体物質結合性タンパク質への可逆的な結合性能を有する物質である、
タンパク質修飾蛍光体集積粒子精製用フィルター。 - 前記フィルターが、デキストランまたはその誘導体と、アクリルアミドまたはその誘導体との架橋共重合体である、請求項10に記載のフィルター。
- 前記タンパク質結合性物質がプロテインA、プロテインGまたはプロテインLである、請求項10または11に記載のフィルター。
- 前記蛍光体集積粒子の平均粒子径が30~300nmである、請求項10~12のいずれか一項に記載のフィルター。
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CN111289336B (zh) * | 2019-12-13 | 2023-06-13 | 山东源科生物科技股份有限公司 | 一种真菌荧光染色液及其制备方法 |
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EP3608665A4 (en) | 2020-04-22 |
JP6897761B2 (ja) | 2021-07-07 |
US20200003764A1 (en) | 2020-01-02 |
JPWO2018185942A1 (ja) | 2020-02-13 |
EP3608665A1 (en) | 2020-02-12 |
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