Patent Application
20200264170
This application claims priority from prior Japanese Patent Application No. 2019-028495, filed on Feb. 20, 2019, entitled “METHOD FOR ACQUIRING INFORMATION ON ANALYTE, AND METHOD FOR CAPTURING ANALYTE”, the entire contents of which are incorporated herein by reference.
The present invention relates to: a method for acquiring information on a polypeptide that is an analyte.
In the pathological diagnosis and the decision of the strategy for a treatment, it is useful to acquire information on the quantity or structure of an analyte in a clinical specimen collected from a subject. For example, in Alzheimer's disease, the quantity and structure of an analyte, e.g., amyloid β (AP), vary with the evolution of the disease. Therefore, the clinical condition can be understood accurately by acquiring information on the quantity and structure of the analyte. Examples of a disease associated with the denaturation of a protein include, in addition to Alzheimer's disease, Huntington's disease, Parkinson's disease, prion disease and amyotrophic lateral sclerosis (ALS).
As one example of a method for acquiring information on the quantity or structure of an analyte in a clinical specimen, it is disclosed that AP can be detected by immobilizing AP contained in a cerebrospinal fluid (CSF) onto a cover glass, then immunostaining the AP by indirect immunofluorescence, and then imaging the AP with a super-resolving microscope in Zhang W. I. et. al., Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics, J Alzheimers Dis, 2015, vol. 46, p. 1007-1020. In an acquired image, AP is detected as a spot having a size and strength corresponding to the degree of aggregation.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
In the method disclosed in Zhang W. I. et. al., Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics, J Alzheimers Dis, 2015, vol. 46, p. 1007-1020, a CSF is dropped onto a cover glass, and therefore a contaminant in the CSF, as well as Aβ, is also immobilized on the cover glass. Therefore, a primary antibody and a fluorescently labeled secondary antibody which are used in the immunostaining is sometimes bonded to the contaminant immobilized on the cover glass in a non-specific manner. Or the fluorescently labeled secondary antibody is sometimes bonded to the cover glass in a non-specific manner. When the contaminant and the cover glass, as well as Aβ, are also labeled with a labeling antibody, information on the quantity or structure of Aβ that is an analyte cannot be acquired with high accuracy.
In a clinical specimen, e.g., a CSF, collected from a subject, an analyte is not necessarily to occur in an enough quantity for detection. Therefore, for the measurement of an analyte in a clinical specimen, it is demanded to improve detection sensitivity. Actually, the present inventors have found that, even in a measurement system which can detect a synthetic polypeptide that is an analyte satisfactorily, when an analyte contained in a clinical specimen is measured, there is still a room for improvement with respect to detection sensitivity.
The present inventors have found that a polypeptide, which is an analyte, in a clinical specimen can be detected with higher sensitivity by using a capture substance capable of binding to a C-terminal region of the polypeptide in an immune complex transfer (ICT) method. This finding leads to the accomplishment of the present invention.
The present invention provides a method for acquiring information on an analyte, comprising the steps of: bringing a polypeptide that is the analyte into contact with a capture substance that binds to the polypeptide to form a complex comprising the polypeptide and the capture substance; bonding the capture substance in the complex to a first solid phase to immobilize the complex onto the first solid phase; collecting the first solid phase on which the complex immobilized; releasing the complex from the collected first solid phase, and then bonding the capture substance in the released complex to a second solid phase to immobilize the complex onto the second solid phase; and acquiring information on the polypeptide from the complex immobilized on the second solid phase, wherein the capture substance binds to a C-terminal region of the polypeptide.
The present invention also provides a method for acquiring information on an analyte, comprising the steps of: bringing a polypeptide that is the analyte into contact with a first capture substance that binds to the polypeptide and a second capture substance that binds to the polypeptide to form a complex comprising the polypeptide, the first capture substance and the second capture substance; bonding the first capture substance in the complex to a first solid phase to immobilize the complex onto the first solid phase; collecting the first solid phase on which the complex immobilized; releasing the complex from the collected first solid phase, and then bonding the second capture substance in the released complex to a second solid phase to immobilize the complex onto the second solid phase; and acquiring information on the polypeptide from the complex immobilized on the second solid phase, wherein the first capture substance and/or the second capture substance binds to a C-terminal region of the polypeptide.
The present invention also provides a method for acquiring information on an analyte, comprising the step of acquiring information of a polypeptide that is the analyte from a complex comprising the polypeptide and a capture substance that binds to the polypeptide, wherein the complex is produced by: contacting the polypeptide with the capture substance to form a complex comprising the polypeptide and the capture substance; bonding the capture substance in the complex to a first solid phase to immobilize the complex onto the first solid phase; collecting the first solid phase comprising the complex immobilized on the first solid phase; and releasing the complex from the collected first solid phase, and then bonding the capture substance in the released complex to a second solid phase, wherein the capture substance binds to a C-terminal region of the polypeptide.
Referring to
In the method according to the present embodiment, the analyte is a polypeptide. The term “polypeptide” as used herein also includes a protein within the scope thereof. The polypeptide may be an artificially synthesized polypeptide or an organism-derived polypeptide that is contained in a biological sample or the like. A preferred example of the analyte is an organism-derived polypeptide. Examples of the biological sample include a clinical specimen collected from a living organism and cultured cells. Examples of the clinical specimen include: a body fluid such as a cerebrospinal fluid, blood (whole blood, plasma, serum), a tissue fluid and urine; and a tissue specimen such as a brain tissue.
Because the formation of the complex is generally carried out in a solution, it is preferred that the sample containing the polypeptide has a liquid form. The liquid sample is not limited to a solution, and may be a liquid suspension or a sol. In the case where a solid sample such as a tissue specimen is used, it is preferred to carry out a treatment for making the sample into a liquid form prior to the method according to the present embodiment. The type of the treatment can be selected appropriately from known methods depending on the type of the sample to be used. For example, in the case where the sample is a solid tissue, the solid tissue is homogenized in a solution containing a surfactant, then solid components including debris are separated from the resultant solution by centrifugation to obtain a supernatant containing a polypeptide.
The type of the polypeptide is not particularly limited, and the type of the polypeptide may be selected arbitrarily from polypeptides that cause diseases and disorders. Examples of the polypeptide of this type include Aβ, tau protein, huntingtin, prion and α-synuclein. Among these polypeptides, Aβ is particularly preferred. Aβ is a polypeptide normally composed of 39 to 43 amino acid residues. The term “amyloid β” or “Aβ” as used herein includes an Aβ polypeptide having any length, unless otherwise stated.
In the present embodiment, the polypeptide may have a form of a multimer. A multimer is also called as a “polymer”, and a multimer is formed by the physical or chemical polymerization or aggregation of a plurality of monomeric polypeptides. The multimer is only needed to contain a plurality of monomeric polypeptides, and the multimer may also contain another molecule. In the multimer, it is not needed for the monomeric polypeptides to be bonded strongly to each other via a covalent bond. An aggregate that is a cluster of a plurality of monomeric polypeptides bonded to each other through a more moderate bond is also included within the scope of the multimer. For example, Aβ has a property of being aggregated to form an insoluble amyloid fibril. Examples of the multimer of the polypeptide include an Aβ oligomer formed by the polymerization of Aβ monomers and a tau oligomer formed by the polymerization of tau proteins.
The term “capture substance” as used herein refers to a substance which can bind to a polypeptide that is the analyte specifically and can be immobilized on a solid phase to capture the polypeptide onto the solid phase. Examples of the capture substance include an antibody and an aptamer. Hereinafter, an antibody that can be used as the capture substance is also referred to as a “capture antibody”. The term “antibody” as used herein includes, within the scope thereof, an antigen-binding antibody fragment such as Fab, F(ab′)2 and Fab′ and derivatives thereof. The capture antibody may be any one of a monoclonal antibody and polyclonal antibody, and the capture antibody is preferably a monoclonal antibody.
The term “solid phase” as used herein refers to an insoluble support for immobilizing the capture substance thereon. The wording “a capture substance is immobilized onto a solid phase” means that the capture substance and the solid phase are bonded to each other directly or indirectly so that the capture substance can be captured on the solid phase. When the capture substance that is bonded specifically to the polypeptide is immobilized onto the solid phase, the polypeptide is captured on the solid phase through the capture substance.
The method according to the present embodiment relies on the ICT method. As mentioned below, in the method, a complex of the capture substance and the polypeptide is transferred from a first solid phase to a second solid phase. According to this transfer of the complex, the influence by contaminants or the like is reduced, and therefore the polypeptide can be measured with higher sensitivity. A single type of the capture substance may be used, or two types of the capture substances may be used. In an embodiment in which a single type of the capture substance is used, the capture substance is one capable of binding to both of the first solid phase and the second solid phase. In an embodiment in which two types of the capture substances are used, the capture substances are a combination of a capture substance capable of binding to the first solid phase (also referred to as a “first capture substance”, hereinafter) and a capture substance capable of binding to the second solid phase (also referred to as a “second capture substance”, hereinafter. Alternatively, it is possible to use a combination of the first capture substance or the second capture substance and a capture substance capable of binding to both of the first solid phase and the second solid phase.
In the present embodiment, a capture substance capable of binding to at least one of the first solid phase and the second solid phase can bind to a C-terminal region of the polypeptide. Preferably, the capture substance capable of binding to the first solid phase can bind to a C-terminal region of the polypeptide. The C-terminal region of a polypeptide does not have a critical boundary, and may be, for example, a region lying between an amino acid residue located at a position in the vicinity of the center and an amino acid residue located at the C-terminal in the amino acid sequence for the monomeric polypeptide. When the full length of the monomeric polypeptide is 2n residues or 2n+1 residue (wherein n represents an integer of 1 or more), the amino acid residue located in the vicinity of the center may be an amino acid residue at position-(n+1). For example, when the polypeptide is composed of 50 amino acid residue, the C-terminal region may be a region lying between position-26 and an amino acid residue located at position-50. Alternatively, when a specific C-terminal region of the polypeptide is defined by the structure, physical property, function or the like thereof in the art, it is possible to follow the definition. For example, in Aβ1-40 and Aβ1-42, it is known that a region lying between an amino acid residue located the N-terminal and an amino acid residue located at position-28 is hydrophilic and a region lying between an amino acid residue located at position-29 and an amino acid residue located at the C-terminal is hydrophobic. When the polypeptide is Aβ1-40 or Aβ1-42, it is possible to define a region lying between an amino acid residue located at position-29 and an amino acid residue located at the C-terminal as the C-terminal region.
In the present embodiment, the capture substance capable of binding to the C-terminal region of the polypeptide may recognize an epitope present in the C-terminal region of the polypeptide or the three-dimensional structure of the C-terminal region of the polypeptide to bind specifically to the polypeptide. Hereinafter, the epitope or the three-dimensional structure recognized by the capture substance is also referred to as a “recognition site of the capture substance”. For example, in the case where the polypeptide is Aβ (particularly Aβ1-42), an antibody capable of recognizing an epitope present in a region lying between an amino acid residue located at position-22, preferably position-29, more preferably position-33, in Aβ and an amino acid residue located at the C-terminal of Aβ can be used as the capture substance capable of binding to the C-terminal region of the polypeptide. An anti-Aβ monoclonal antibody capable of binding to the C-terminal region of Aβ is generally available. For example, antibodies against clones H31L21 (epitope: 36 to 42), G2-11 (epitope: 33 to 42), 16C11 (epitope: 33 to 42), 21F12 (epitope: 34 to 42) and the like are commercially available. Among these, H31L21 is particularly preferred.
In the present embodiment, it is preferred that a substance for detection having a labeling substance and capable of binding to the polypeptide is further brought into contact with the polypeptide to form a complex containing the polypeptide, the capture substance and the substance for detection. The term “substance for detection” as used herein refers to a substance which can bond specifically to the polypeptide that is the analyte to provide a signal that can be detected through the labeling substance. It is preferred for the substance for detection not to be immobilized onto a solid phase. Examples of the substance for detection include an antibody and an aptamer. The antibody that can be used as the substance for detection is also referred to as an “detection antibody” hereinafter. The detection antibody may be any one of a monoclonal antibody and a polyclonal antibody, and the detection antibody is preferably a monoclonal antibody.
In the present embodiment, the substance for detection may be bonded to a C-terminal region of the polypeptide or a N-terminal region of the polypeptide. Preferably, the substance for detection has a labeling substance and can bind to a N-terminal region of the polypeptide. The N-terminal region of a polypeptide does not have a critical boundary, and may be, for example, a region lying between an amino acid residue located at the N-terminal in the amino acid sequence for the monomeric polypeptide and an amino acid residue located at a position in the vicinity of the center in the amino acid sequence. For example, when the polypeptide is composed of 50 amino acid residue, the N-terminal region may be a region lying between position-1 and an amino acid residue located at position-25. Alternatively, when a specific N-terminal region of the polypeptide is defined by the structure, physical property, function or the like thereof in the art, it is possible to follow the definition.
In the present embodiment, the substance for detection can be produced by labeling a substance capable of recognizing an epitope present in the polypeptide (preferably a N-terminal region thereof) or the three-dimensional structure of the polypeptide (preferably a N-terminal region thereof) to bind specifically to the polypeptide with a known labeling substance that has been used conventionally in immunological techniques. Hereinafter, the epitope or the three-dimensional structure recognized by the substance for detection is also referred to as a “recognition site of the substance for detection”. For example, in the case where the polypeptide is Aβ (particularly Aβ1-42), an antibody capable of recognizing an epitope present in a region lying between an amino acid residue located at the N-terminal and an amino acid residue located at position-28, preferably position-21, more preferably position-16, in Aβ can be used as the substance for detection. An anti-Aβ monoclonal antibody capable of binding to the N-terminal region of Aβ is generally available. For example, antibodies against clones 82E1 (epitope: 1 to 16), 6E10 (epitope: 3 to 8), WO-2 (epitope: 4 to 10) and 2H4 (epitope: 1 to 8) are commercially available. Among these, 82E1 is particularly preferred.
The method for labeling the antibody or the aptamer with a labeling substance is known in the art, and the method can be selected appropriately depending on the type of the labeling substance to be used. For example, the antibody or the aptamer can be bonded or linked to the labeling substance using a proper cross-linking agent, a commercially available labeling kit or the like.
The labeling substance is not particularly limited, as long as the labeling substance can generate a detectable signal directly or indirectly. Examples of the labeling substance include an enzyme, a fluorescent substance and a radioactive isotope. Examples of the enzyme include alkaline phosphatase, β-galactosidase, peroxidase, glucose oxidase, tyrosinase, acid phosphatase and luciferase. Examples of the fluorescent substance include: a fluorescent dye such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered tradename); and a fluorescent protein such as green fluorescent protein (GFP). Examples of the radioactive isotope include 125I, 14C and 32P.
The timing of adding the substance for detection can be determined depending on, for example, whether or not a recognition site of the substance for detection and a recognition site of the capture substance are overlapped with each other. The wording “recognition sites are overlapped with each other” as used herein refers to the matter that recognition sites of substances capable of bonding specifically to the polypeptide are the identical to each other or partially coincide with each other. In the case where the recognition site of the substance for detection and the recognition site of the capture substance are not overlapped with each other, i.e., in the case where both of the substance for detection and the capture substance can bind to the monomeric polypeptide, the substance for detection may be added in the complex formation step, or the substance for detection may be added during the below-mentioned step of immobilizing onto the first (or second) solid phase or the below-mentioned step of collecting the first solid phase. In the case where the recognition site of the substance for detection and the recognition site of the capture substance are overlapped with each other, i.e., in the case where only one of the substance for detection and the capture substance can bind to the monomeric polypeptide, it is preferred that the substance for detection is added in the complex formation step. In this case, finally information on a polypeptide that is a multimer higher than a dimer can be acquired. In a preferred embodiment, the substance for detection is added in the complex formation step.
The complex formation step can be carried out by, for example, mixing a sample containing the polypeptide with a solution containing the capture substance. As the result of the mixing, the polypeptide is contacted with and bonded to the capture substance. In this manner, a complex containing the “(polypeptide)-(capture substance)” can be formed. In the case where the substance for detection is added in the complex formation step, a sample containing the polypeptide, a solution containing the capture substance and a solution containing the substance for detection are mixed together. As the result of the mixing, the substance for detection, the polypeptide and the capture substance are contacted with one another and are bonded together. In this manner, a sandwich complex containing the “(substance for detection)-(polypeptide)-(capture substance)” is formed. The order of the mixing of the polypeptide, the capture substance and the substance for detection is not particularly limited, and it is preferred that these components are mixed substantially simultaneously. The reaction temperature and the reaction time to be employed in the complex formation step are not particularly limited. In general, a reaction solution is allowed to leave or stirred gently at a temperature of 20 to 45° C. for 15 minutes to 1 hour.
In an embodiment in which two types of capture substances are used, a recognition site of a first capture substance and a recognition site of a second capture substance may be or may not be overlapped with each other. In the case where the recognition site of the first capture substance and the recognition site of the second capture substance are overlapped with each other, only one of the first capture substance and the second capture substance can bind to the monomeric polypeptide. In this case, finally information on a polypeptide that is a multimer higher than a dimer can be acquired.
(Step of Immobilizing onto First Solid Phase)
Referring to
The first solid phase can be selected from known solid phases that have been used conventionally in immunological procedures. Examples of the form of the first solid phase include a particle, a thin film, a membrane, a plate, a micro tube and a test tube. The plate may be a plate with a plural well or may be a planar plate without a well. The material to be used for the solid phase can be selected from an organic polymeric compound, an inorganic compound, a biological polymer and the like. Examples of the organic polymeric compound include a latex, a rubber, polyethylene, polypropylene, polystyrene, a styrene-butadiene copolymer, poly(vinyl chloride), poly(vinyl acetate), polyacrylamide, polymethacrylate, a styrene-methacrylate copolymer, poly(glycidyl methacrylate), an acrolein-(ethylene glycol dimethacrylate) copolymer, a poly(vinylidene difluoride) (PVDF) and a silicone. Examples of the inorganic compound include a magnetic material (e.g., iron oxide, chromium oxide, cobalt, nickel, ferrite, magnetite), a glass, silica and alumina. Examples of the biological polymer include insoluble agarose, insoluble dextran, gelatin and cellulose. It is possible to use a combination of two or more of these materials. In the present embodiment, the first solid phase is preferably an insoluble particle such as a magnetic particle and a latex particle, particularly preferably a magnetic particle.
The bonding between the capture substance and the first solid phase in the complex is only needed to be a dissociable bonding. In a preferred embodiment, the capture substance and the first solid phase in the complex are bonded to each other indirectly through another substance. As the substance, a combination of two substances that can be bonded specifically to each other and are dissociable is preferred. Hereinafter, the two substances are called as a “bonding substance” and a “bonding partner”, respectively. The combination of the bonding substance and the bonding partner is known in the art, and examples of the combination include a combination of an antigen (excluding the analyte) and an antibody thereof, a combination of a ligand and a receptor thereof, a combination of an oligonucleotide and a strand complementary thereto, a combination of a biotin-type compound (including biotin and a biotin analogue such as desthiobiotin) and an avidin-type compound (including avidin and an avidin analogue such as streptavidin), a combination of nickel and a histidine tag, and a combination of glutathione and glutathione-S-transferase. Preferred examples of the combination of an antigen and an antibody thereof include a combination of a hapten and an anti-hapten antibody, and a combination of biotin (or desthiobiotin) and an anti-biotin antibody (or an anti-desthiobiotin antibody). A particularly preferred example of the combination of a hapten and an anti-hapten antibody is a combination of 2,4-dinitrophenyl (DNP) group and an anti-DNP antibody.
In an embodiment in which a single type of capture substance is used, it is preferred that the capture substance has both of the first bonding substance and the second bonding substance and can bind to a C-terminal region of the polypeptide. In the embodiment in which two types of capture substances are used, it is preferred that the capture substance includes a first capture substance having the first bonding substance and a second capture substance having the second bonding substance and at least one of the first capture substance and the second capture substance can bind to a C-terminal region of the polypeptide. In both of the embodiments, it is preferred that the first solid phase has a first bonding partner capable of bonding specifically to a first bonding substance and the below-mentioned second solid phase has a second bonding partner capable of bonding specifically to a second bonding substance.
In the present embodiment, the capture substance and the first solid phase in the complex bind to each other through a specific bonding between the first bonding substance and the first bonding partner. As a result, the complex is immobilized onto the first solid phase. In a preferred embodiment, the first bonding substance is a DNP group and the first bonding partner is an anti-DNP antibody. The method for bonding the substance to the capture substance and solid phase is known in the art. For example, in the case where biotin or DNP is bonded to an antibody, a method is known in which a cross-linking agent capable of reacting with an amino group or a thiol group in the antibody (e.g., maleimide, N-hydroxysuccinimide) is used. Alternatively, a commercially available labeling kit may be used. As the method for bonding the bonding substance or the bonding partner to a solid phase, a physical adsorption method, a covalent bonding method, an ionic bonding method and the like are known.
The immobilization step can be carried out by bringing the complex into contact with the first solid phase. For example, in the case where the first solid phase has a particulate form, a liquid containing the complex is mixed with the first solid phase to cause the contact between the complex and the first solid phase. In the case where the substance for detection is added in the immobilization step, the complex, the substance for detection and the first solid phase are brought into contact with one another. The reaction temperature and the reaction time are not particularly limited. In general, the reaction solution is allowed to leave or stirred gently at a temperature of 20 to 45° C. for 15 minutes to 3 hours.
Referring to
The method for collecting the first solid phase having the complex immobilized thereon is known in the art, and the method can be selected appropriately depending on the type of the first solid phase used. For example, in the case where a magnetic particle is used, the magnetic particle can be collected by magnetic separation. More specifically, a magnet is brought close to the wall surface of a container containing a magnetic particle each the complex immobilized thereto to immobilize the magnetic particle onto the wall surface of the container, and then a liquid is removed by sucking to collect the magnetic particle having the complex immobilized thereon. In the case where an insoluble particle such as a latex particle is used, the particle is precipitated by centrifugation and then a liquid is removed by sucking to collect the insoluble particle having the complex immobilized thereon.
In the present embodiment, the step of washing the collected first solid phase can be further included. The washing of the first solid phase can be carried out by, for example, adding a wash solution to the collected first solid phase and then removing the wash solution from the first solid phase. As the wash solution, a buffer solution that cannot impair the complex immobilized on the first solid phase is preferred. As the wash solution of this type, a buffer solution containing a surfactant is particularly preferred, and examples of the buffer solution include TBST (a Tris-buffered saline containing 0.05% of Tween20) and PBST (a phosphate-buffered saline containing 0.05% of Tween20). A commercially available wash solution, such as HISCL wash solution (Sysmex Corporation), can also be used. By the washing, a component that is adsorbed non-specifically onto the first solid phase or the complex can be removed.
(Step of Immobilizing onto Second Solid Phase)
Referring to
The method for releasing the complex on the solid phase is known in the art. For example, a method can be mentioned, in which a substance capable of dissociating the bonding between the capture substance in the complex and the first solid phase (also referred to as a “releasing agent”, hereinafter). The releasing agent is known in the art, and the releasing agent can be selected appropriately depending on the type of the bonding between the capture substance and the first solid phase. For example, in the case where the capture substance in the complex and the first solid phase are bonded to each other through physical adsorption, the complex can be released from the first solid phase by using a solution containing a surfactant as the releasing agent. In the case where the capture substance in the complex and the first solid phase are bonded to each other through ionic bonding, the complex can be released from the first solid phase by using a solution containing an ion.
In the case where the capture substance and the first solid phase are bonded indirectly through specific bonding between the first bonding substance and the first bonding partner, the complex can be released by using a releasing agent capable of dissociating the bonding between the first bonding substance and the first bonding partner. The releasing agent is also known in the art, and the releasing agent can be selected appropriately depending on the combination of the bonding substance and the bonding partner. For example, in the case of the bonding between a hapten and an anti-hapten antibody, the hapten or a derivative thereof can be used as the releasing agent. For example, in the case of the bonding between a DNP group and an anti-DNP antibody, a dinitrophenyl amino acid can be used as the releasing agent. In the case of the bonding between biotin (or desthiobiotin) and avidin (or streptavidin), biotin can be used as the releasing agent.
In the case where the complex is released using the releasing agent, the treatment temperature and the treatment time can be preset appropriately depending on the type of the releasing agent to be used. In general, the reaction solution may be allowed to leave or stirred gently at a temperature of 20 to 45° C. for 3 to 15 minutes after the addition of the releasing agent.
After the addition of the releasing agent, it is preferred that the complex released from the first solid phase and the first solid phase are separated from each other, and a liquid containing the released complex is collected. For example, in the case where a particle is used as the first solid phase, the complex is released from the first solid phase, and then the first solid phase is collected on the wall surface or the bottom of a container by a magnetic force or centrifugation in the same manner as mentioned with respect to the collection step. Subsequently, a liquid containing the complex is collected.
The immobilization of the complex onto the second solid phase can be carried out by, for example, bringing the complex and the second solid phase into contact with each other. For example, in the case where the second solid phase has a particulate form, a liquid containing the complex is mixed with the second solid phase to cause the complex and the second solid phase to contact with each other. In the case where the second solid phase has a thin-plate-like form, a liquid containing the complex is dropped onto the second solid phase to cause the complex and the second solid phase to contact with each other. The reaction temperature and the reaction time are not particularly limited. In general, the reaction solution is allowed to leave or stirred gently at a temperature of 20 to 45° C. for 15 minutes to 3 hours.
As mentioned above, when the complex released from the first solid phase is brought into contact with the second solid phase that is different from the second solid phase, the capture substance in the complex is bonded to the second solid phase to cause the complex to be transferred onto the second solid. The term “second phase that is different from the first solid phase” as used herein refers to a novel solid phase which is different from the first solid phase that is present from the point of time at which the complex is added in the step of immobilizing onto the first solid phase. Namely, in the step of immobilizing the complex onto the second solid phase, it is not intended to bond the complex released from the first solid phase to the first solid phase again. In a preferred embodiment, a liquid containing the complex released from the first solid phase is collected, and the collected liquid is brought into contact with a newly provided second solid phase.
The material and the form of the second solid phase are the same as those materials and forms mentioned above with respect to the first solid phase. The material for the second solid phase may be the same as or different from the material for the first solid phase. Examples of the form of the second solid phase include a particle, a thin film, a membrane, a plate, a micro tube and a test tube. The plate may be a plate with a plural well or may be a planar plate without a well. The material and the form of the solid phase may be selected appropriately depending on the type of the measurement means to be employed. For example, in the case where the polypeptide is observed with a microscope, a solid phase made from a material through which light can pass and a thin-plate-like form (e.g., a glass slide) is preferred.
The mode of the binding between the capture substance in the complex and the second solid phase is not particularly limited. For example, the capture substance and the second solid phase may be bonded to each other directly through physical adsorption, ionic bonding or the like. Alternatively, the capture substance and the second solid phase may be bonded to each other indirectly through another substance. As the substance, a combination of the bonding substance and the bonding partner can be mentioned. The combination of the second bonding substance and the second bonding partner is preferably different from the combination of the first bonding substance and the first bonding partner. In the case where a single type of capture substance is used, a capture substance having a biotin-type compound and a DNP group, a first solid phase having an anti-DNP antibody immobilized on the surface thereof and a second solid surface having an avidin-type compound immobilized on the surface thereof can be used, for example. In the case where two types of capture substances are used, a first capture substance having a DNP group, a second capture substance having a biotin-type compound, a first solid phase having an anti-DNP antibody immobilized on the surface thereof and a second solid phase having an avidin-type compound immobilized on the surface thereof can be used, for example.
In a preferred embodiment, the step of washing the first solid phase having the complex immobilized thereon is further included. The washing of the first solid phase can be carried out in the same manner as mentioned with respect to the washing of the second solid phase.
Hereinbelow, the process from the step of forming the complex to the step of immobilizing the complex onto the second solid phase will be described with reference to drawings. These drawings only illustrate one example of the present embodiment, and is not intended to limit the present disclosure. As shown in
Referring to
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In
In the complex formation step shown in
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In
In
In the present embodiment, each step or all steps in a sequence of steps from the step of forming the complex to the step of releasing and collecting the complex from the first solid phase may be carried out by a manual procedure or using a device. Alternatively, each step or all steps in a sequence of steps from the formation of the complex to the immobilization of the complex onto the second solid phase may be carried out by a manual procedure or using a device. Examples of the device include an automatic sample treatment device, an automatic immunoassay device and a device for manufacturing a glass slide for microscopic observation use.
Referring to
The information on the quantity of the polypeptide may be quantitative information or quantitative information. An example of the quantitative information is the presence or absence of the polypeptide. Examples of the quantitative information include the concentration of the polypeptide, the content (weight) of the polypeptide, and a measurement value thereof. The quantitative information also includes semi-quantitative information which indicates the quantity of the polypeptide in a graded manner, such as “a small quantity”, “a medium quantity” and “a large quantity”.
Examples of the information on the structure of the polypeptide include information on the size of the polypeptide, information on the morphology of the polypeptide, and information on the aggregation degree of the polypeptide. In a preferred embodiment, information on the structure of the polypeptide is acquired by imaging the polypeptide on the second solid phase with a microscope to obtain an image of the polypeptide. The type of the microscope is not particularly limited, as long as an image of the peptide can be obtained. Examples of the microscope include a fluorescence microscope, a super-resolving microscope, a Raman microscope, a probe microscope and an electron microscope.
In the present embodiment, information on the polypeptide is acquired by measuring a signal coming from the labeling substance in the complex immobilized on the second solid phase. In the case where information on the quantity of the polypeptide is to be acquired, it is preferred to measure the intensity of the signal or the like in terms of a numerical value. For example, information on the concentration or content of a polypeptide can be acquired by assigning an obtained measurement value to a calibration curve produced from measurement values for a polypeptide having a known concentration. In the case where information on the structure of the polypeptide is to be acquired, it is preferred to obtain an image associated with the signal.
The method for measuring a signal coming from a labeling substance is known in the art. In the present embodiment, a proper method can be selected depending on the signal coming from the labeling substance. For example, in the case where the labeling substance is an enzyme, it is possible that the enzyme in the complex is reacted with a substrate for the enzyme and then a signal, e.g., light, color, generated from a reaction produce produced as the result of the enzymatic reaction can be measured using a known device. Examples of the device include a spectrophotometer and a luminometer.
The substrate for an enzyme can be selected appropriately from known substrates depending on the type of the enzyme to be used. For example, in the case where alkaline phosphatase is used as the enzyme, examples of the substrate for the enzyme include: a chemiluminescent substrate such as CDP-Star (registered tradename) (disodium 4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate) and CSPD (registered tradename) (disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate); a luminescent substrate such as p-nitrophenyl phosphate, a 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazolium chloride (NBT) and iodonitrotetrazolium (INT); a fluorescent substrate such as 4-methylumbelliferylphosphate (4MUP); and a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate and p-nitrophenylphosphate. In the case where β-galactosidase is used as the enzyme, the substrate for the enzyme is, for example, 4-methylumbelliferyl-β-D-galactopyranoside.
In the case where the labeling substance is a fluorescent substance, it is possible that the complex is irradiated with excitation light, and then fluorescence generated from the fluorescent substance in the complex is measured using a known device such as a fluorescence microplate reader. In the case where the labeling substance is a radioactive isotope, radioactive ray generated from a radioactive isotope in the complex can be measured using a known device such as a scintillation counter.
Hereinbelow, the case where information on the structure of the polypeptide is acquired using a super-resolving fluorescence microscope that is one type of super-resolving microscope will be described with reference to drawings. A super-resolving microscope is a microscope having resolution performance beyond the limit of diffraction of light.
In
Referring to
In step S102, during the irradiation of the fluorescent dye 51 with the excitation light, generated fluorescent light is imaged to acquire an image of the fluorescent dye 51. In step S102, the imaging is repeated during the irradiation of the fluorescent dye 51 with the excitation light. In this manner, 3000 images, for example, can be acquired. Since the distribution of the fluorescent dye 51 in the light-emitting state is changed with elapse of time, the distributions of fluorescent light on the acquired images are different among the acquired images.
In step S103, a predetermined period of time is elapsed, and it is determined as to whether or not the acquisition of a desired image is completed. Until the predetermined period of time is elapsed, the imaging is repeated in step S102. When the predetermined period of time is elapsed and the acquisition of the desired image is completed, the procured proceeds to step S104. In this manner, the image is acquired, it becomes possible to acquire information on the structure of the polypeptide 11 in the subsequent step.
Alternatively, an image may also be acquired by employing a step based on a technique such as STORM (Stochastic optical reconstruction microscopy), PALM (Photoactivated localization microscopy), STED (Stimulated emission depletion) or SIM (Structured illumination microscopy) in place of step S101 to S103. In the case where the image is acquired by a step based on STORM, the fluorescent dye 51 is so configured that the state of the fluorescent dye 51 can be switched between an active state where fluorescent light is generated and an inactive state where fluorescent light is not generated. By switching the state of the fluorescent dye 51 The distribution of the fluorescent light between the active state and the inactive state with two types of light, a plurality of images having different fluorescent light distributions from each other can be acquired.
Subsequently, in step S104, a super-resolved image is produced. As shown in
Accordingly, in the case where 3000 fluorescent images are obtained in step S102, the super-resolved image of the fluorescent images is produced by extracting bright points from the 3000 fluorescent images and superposing the bright point regions of the extracted bright points on each other.
Now taking a look back over
In step S105, the information on the structure of the polypeptide 11 is acquired by the following procedure. As shown in
The method for classifying bright points into a single group is not limited to this method, and may be another clustering technique. For example, a region having a pixel value equal to or larger than a specified threshold value on a fluorescent image obtained by adding all of fluorescent images together may be deemed as a single cluster of aggregates. Alternatively, a region having a pixel value equal to or larger than a specified threshold value on a fluorescent image obtained imaging fluorescent light excited from all molecules of the fluorescent dye 51 immediately after the start of step S101 in the information acquisition step may be deemed as a single cluster of aggregates.
Subsequently, the following types of information are acquired on every aggregates of the polypeptide 11 on the basis of a super-resolved image. Namely, with respect to the size of the polypeptide 11, length in the longer length direction, a length in the shorter length direction, a periphery, an area and the like can be acquired. With respect to the morphology of the polypeptide 11, an aspect ratio, circularity, the number of branches, a branching angle and the like can be acquired. The aspect ratio can be obtained by, for example, dividing a length in the longer length direction by a length in the shorter length direction. With respect to the structure of the polypeptide 11, information on which a cluster of aggregates of the polypeptide 11 is among a primary structure, a secondary structure, a third-order structure and a four-order structure, can be obtained. With respect to the aggregation degree of the polypeptide 11, the number of monomers forming a cluster of aggregates can be obtained. The number of the monomers can be obtained by comparing between the standard size of the monomer and the size of the cluster of the aggregates.
In step S105, the information on the structure of the polypeptide 11 is acquired on the basis of the super-resolved image. However, the method for acquiring the information is not limited to this procedure, and the information may also be acquired on the basis of a fluorescent image obtained by imaging fluorescent light generated from the fluorescent dye 51. For example, the information on the structure of the polypeptide 11 may be obtained on the basis of a fluorescent image obtained by imaging fluorescent light generated from all molecules of the fluorescent dye 51 immediately after the start of step S101. In this case, however, it is impossible to analyze at resolution performance beyond the diffraction limit of light. Therefore, it is preferred to acquire the information on the structure of the polypeptide 11 on the basis of the super-resolved image, as mentioned above.
Now taking a look back over
Referring to
In the present embodiment, the information acquisition step may be performed automatically using a detection device 100 shown in
The information acquisition unit 101 is provided with a light source unit 110, a shutter 121, a quarter-wave retarder 122, a beam expander 123, a condenser lens 124, a dichroic mirror 125, an objective lens 126, a condenser lens 127, a stage 130, an imaging unit 140, a shutter driving mechanism 151, and a stage driving mechanism 152. On the stage 130, a plate 80 having a polypeptide 11 immobilized thereon is mounted.
The light source unit 110 is provided with a light source 111 and a mirror 112. The light source 111 can emit excitation light. As the light source 111, a laser beam source can be used preferably, and a mercury lamp, a xenon lamp, a LED or the like may also be used. The excitation light emitted from the light source 111 can change the state of fluorescent dye 51 bonded to the polypeptide 11 into a light emitting state or a quenched state, and can excite the fluorescent dye 51 in a light emitting state to generate fluorescent light. The mirror 112 can reflect the excitation light coming from the light source 111 to guide the excitation light to the shutter 121.
In the case where the fluorescent dye 51 is so configured that the state of the fluorescent dye 51 can be switched between an active state where fluorescent light is generate and an inactive state where fluorescent light is not generated, the light source unit 110 is so configured as to have two light sources, a mirror and a dichroic minor. In this case, one of the light sources can emit light capable of shifting the state of the fluorescent dye 51 into an active state, and the other can emit light capable of shifting the state of the fluorescent dye 51 into an inactive state. Optical axes of two types of light respectively emitted from the two light sources are made coincides with each other by means of the mirror and the dichroic mirror.
The shutter 121 can be driven by the shutter driving mechanism 151, and can be switched between a state where the excitation light emitted from the light source unit 110 can pass through the shutter 121 and a state where the excitation light emitted from the light source unit 110 can be blocked. In this manner, the time of irradiation of the analyte 11 with the excitation light can be adjusted. The shutter driving mechanism 151 is composed of, for example, a motor, a spring or the like. The quarter-wave retarder 122 can convert the excitation light having linear polarization which is emitted from the light source unit 110 to circularly polarized light. The fluorescent dye 51 can respond to excitation light having a specific polarization direction. Therefore, when the excitation light emitted from the light source unit 110 is converted to circularly polarized light, it becomes easier for the polarization direction of the excitation light to coincide with the polarization direction to which the fluorescent dye 51 responds. As a result, fluorescent light from the fluorescent dye 51 can be excited with high efficiency. The beam expander 123 can expand the irradiation region of the excitation light on the plate 80. The condenser lens 124 can condense the excitation light in such a manner that parallel light can be emitted from the objective lens 126 toward the plate 80.
The dichroic mirror 125 can reflect the excitation light emitted from the light source unit 110 and can permit the passage of the excitation light generated from the fluorescent dye 51 therethrough. The objective lens 126 can guide the excitation light reflected on the dichroic mirror 125 to the plate 80. The stage 130 can be driven the stage driving mechanism 152 to move the stage 130 in the planar direction. The stage driving mechanism is composed of, for example, a motor, a shaft, a nut or the like. The fluorescent light generated from the fluorescent dye 51 on the plate 80 passes through the objective lens 126 and penetrates through the dichroic mirror 125. The condenser lens 127 can condense fluorescent light that penetrates through the dichroic mirror 125 and guide the fluorescent light to a light-receiving surface 141 of the imaging unit 140. The imaging unit 140 can take an image of fluorescent light emitted to the light-receiving surface 141 to produce a fluorescent image. The imaging unit 140 is composed of, for example, a CCD or the like.
The information processing unit 102 is provided with a processing unit 161, a storage unit 162, a display unit 163, an input unit 164 and an interface 165.
The processing unit 161 is, for example, a CPU. The storage unit 162 is a ROM, a RAM, a hard disk or the like. The processing unit 161 can control each part of the information processing unit 102, the light source 111 in the light source unit 120, the imaging unit 140, the shutter driving mechanism 151 and the stage driving mechanism 152 through the interface 165 in accordance with a program stored in the storage unit 162.
The processing unit 161 can perform the information acquisition step shown in
The display unit 163 is used for displaying processing results and the like produced by the processing unit 161, and the display unit 163 is a liquid crystal display, a plasma display, a cathode ray tube (CRT) display or the like. The display unit 163 displays a screen 90 shown in
In the information acquisition step shown in
With respect to the information on the structure of the polypeptide, the size, morphology and aggregation degree of the polypeptide can be obtained when the polypeptide 11 is measured with a super-resolving fluorescence microscope, a Raman microscope, a probe microscope or an electron microscope. Particularly the structure of the polypeptide, among the information on the structure of the polypeptide, can be obtained by measuring the polypeptide 11 with a super-resolving fluorescence microscope, a Raman microscope or a probe microscope.
In the case where the polypeptide 11 is measured with a Raman microscope, a Raman spectrum reflecting molecules or atoms constituting the polypeptide 11 or an image reflecting the shape of the polypeptide 11 is acquired. Therefore, when a Raman microscope is used, the chemical bond can also be acquired in addition to the size, morphology, structure and aggregation degree of the polypeptide as the information on the structure of the polypeptide. More specifically, as the chemical bond of the polypeptide 11, the type, number, concentration, content ratio and the like of a molecule or atoms constituting the polypeptide 11 can be acquired. In this case, the chemical bond in the polypeptide 11 is also displayed, in addition to the size, morphology, structure or aggregation degree of the polypeptide 11, in a region 93 in the screen 90 shown in
The present disclosure includes, within the scope thereof, a method for capturing an analyte. In the method, a polypeptide that is an analyte in a sample can be captured by carrying out the process from step S1 to step S4 shown in
The present disclosure includes, within the scope thereof, a method for acquiring information on an analyte, including the step of acquiring information on a polypeptide that is an analyte from a complex containing the polypeptide and a capture substance capable of binding to the polypeptide. Details about the information acquisition step are similar to those mentioned with respect to the method for acquiring information on an analyte according to the present embodiment. In the method, a complex to be used for the measurement for information acquisition purpose can be obtained by the process from step S1 to step S4 shown in
Hereinafter, the present disclosure will be described in more detail with reference to examples. However, the present disclosure is not limited to these examples.
It was examined as to whether or not Aβ contained in a clinical specimen collected from a subject was able to be measured with high sensitivity by the method according to the present embodiment which utilized an immune complex transfer method (ICT) and a fluorescence microscope.
As a sample containing Aβ, a cerebrospinal fluid (CSF) collected from an Alzheimer's disease patient who agreed to the donation of a specimen was used. In Example 1, a pool CFS prepared by mixing CSFs collected from a plurality of patients (also referred to as a “clinical specimen 1”, hereinafter) was used.
As a first solid phase, a magnetic particle having an anti-DNP antibody immobilized on the surface thereof (also simply referred to as a “magnetic particle”, hereinafter) was produced in the following manner. An anti-DNP antibody (Sysmex Corporation) was immobilized onto a magnetic particle (product name: MAG2201, manufactured by JSR Corporation) having a particle diameter of 2.2 μm by a conventional technique. In this manner, a suspension of the magnetic particles each having the anti-DNP antibody immobilized on the surface thereof (particle concentration: 2.5%) was produced.
As a second solid phase, a glass plate having streptavidin immobilized on the surface thereof (also simply referred to as a “plate”, hereinafter) was produced in the following manner. Though-holes each having a diameter of 6 mm were formed in a silicon rubber sheet (SR-50, manufactured by Tigers Polymer Corporation), and then the silicon rubber sheet was attached to a MAS coat glass (Matsunami Glass Ind., Ltd.). Thirty μg/mL-biotin-binding bovine serum albumin (BSA) (0.5 μL) was dropped onto a glass placed inside of the silicon rubber sheet, and then the glass was allowed to leave at room temperature for 1 hour. The glass was washed by pipetting with HISCL wash solution (Sysmex Corporation) (40 μL). The washing was carried out twice in total. The glass was further washed by pipetting using PBS (40 μL). The washing was carried out twice in total. A 1% BSA/PBS solution (40 μL) was dropped onto the glass, and the glass was allowed to leave at 4° C. overnight. The glass was washed by pipetting with HISCL wash solution (Sysmex Corporation) (40 μL). The washing was carried out twice in total. The glass was further washed by pipetting using PBS (40 μL). The washing with PBS was carried out twice in total. A (10 μg/mL streptavidin)/(1% BSA/PBS) solution (40 μL) was dropped onto the glass, and the glass was stirred at room temperature for 1 hour. The glass was washed by pipetting with HISCL wash solution (Sysmex Corporation) (40 μL). The washing was carried out twice in total. The glass was further washed by pipetting using PBS (40 μL). The washing with PBS was carried out twice in total.
In Example 1, as a capture substance capable of binding to an Aβ peptide, a combination of a rabbit anti-amyloid β42 monoclonal antibody (Life Technologies Corporation, clone name: H31L21) (also referred to as “H31L21 antibody”, hereinafter) and a murine anti-human amyloid β monoclonal antibody (IBL Co., Ltd., clone name: 82E1) (also referred to as “82E1 antibody”, hereinafter) was used. The H31L21 antibody is an antibody capable of binding to a C-terminal region of Aβ peptide and can recognize a region lying between position-36 and position-42 in Aβ. The 82E1 antibody is an antibody capable of binding to a N-terminal region in Aβ peptide, and can recognize a region lying between position-1 to position-16 in Aβ. In Example 1, DNP was used as a first bonding substance and biotin was used as a second bonding substance. More specifically, each of the antibodies was labeled with DNP or biotin, thereby producing a first capture substance and a second capture substance.
Using N-succinimide S-acetylthioacetate (SATA) (Thermo Fischer Scientific), a thio group was introduced into each of the H31L21 antibody and the 82E1 antibody. Using N-(6-maleimidocaproyloxy)succinimide (EMCS), N-(2,4-dinitrophenyl)-L-lysine (DNP-Lys) (Tokyo Chemical Industry Co., Ltd.) was maleimdated. Each of the antibodies each having a thiol group introduced therein was mixed with and reacted with the maleimdated DNP-Lys. In this manner, as first capture substances for immobilizing Aβ onto the first solid phase (the above-mentioned magnetic particle), a DNP-labeled H31L21 antibody and a DNP-labeled 82E1 antibody were produced.
Using SATA (Thermo Fischer Scientific), a thiol group was introduced into each of the H31L21 antibody and the 82E1 antibody. Each of the antibodies each having a thiol group introduced therein was mixed with and reacted with Biotin-PEAC5-maleimide (6-[N′-[2-(N-maleimide)ethyl]-N-piperazinylamide]hexyl D-biotinamide hydrochloric acid salt). In this manner, as a second capture substance for immobilizing Aβ onto the second solid phase (the above-mentioned plate), a biotin-labeled H31L21 antibody and a biotin-labeled 82E1 antibody were produced.
A 82E1 antibody was labeled with a silylrhodamine-type fluorescent dye to produce a fluorescently labeled antibody for Aβ detection. The fluorescent dye was synthesized in accordance with the description of Grimm J. B. et. al., “A general method to improve fluorophores for live-cell and single-molecule microscopy”, Nature Methods, vol. 12, (2015) p. 244-250.
As a releasing agent for dissociating the bonding between DNP in the first capture substance and the anti-DNP antibody in the first solid phase (magnetic particle), a 2.5-mM DNP-Lys solution was used. The DNP-Lys solution was prepared by diluting N-(2,4-dinitrophenyl)-L-lysine (Tokyo Chemical Industry Co., Ltd.) with a buffer (0.1 Tris-HCl (pH 7.5), 2% sodium caseinate, 0.1% NaN3 and DMSO) so that the concentration became 2.5 mM.
Any one of the two types of capture substances and any one of the two types of the substances for detection were mixed together in each of the combinations A to D shown in Table 1 in a HISCL R3 diluent (Sysmex Corporation) to produce an antibody solution. The antibody solutions were prepared in such a manner that the content of each of the antibodies in each of the antibody solutions became 300 fmol/assay. In the table, “(N)” represents the matter that the 82E1 antibody was an antibody capable of binding to a N-terminal region of Aβ, “(C)” represents the matter that the H31L21 antibody was an antibody capable of binding to a C-terminal region of Aβ. In Table 1, combination A of the antibodies corresponds to
Each of the antibody solutions, the first solid phase (magnetic particle) suspension, the HISCL wash solution and the releasing agent (DNP-Lys solution) were placed on Magtration System 6GC (Precision System Science Co., Ltd.) that was a full-automatic sample treatment device. The sample was treated by the device, and a complex containing Aβ and the antibodies was collected. Specific treatment was as follows. The sample (80 μL) was mixed with the antibody solution (80 μL), and the resultant mixed solution was incubated at 37° C. for 30 minutes. The magnetic particle suspension (20 μL) was further mixed with the mixed solution, and the resultant solution was incubated at 37° C. for 15 minutes. The magnetic particles were magnetically collected to remove a supernatant, and the HISCL wash solution (600 μL) was added thereto to wash the magnetic particles. The washing was carried out twice in total. The washing was carried out twice in total. Subsequently, the HISCL wash solution (150 μL) was further added to the washed magnetic particles to wash the magnetic particles. The washing was carried out once in total. After the washing, the DNP-Lys solution (30 μL) was added to the magnetic particles, and then the resultant solution was stirred at 37° C. for 10 minutes. The magnetic particles were magnetically collected, and a supernatant was collected.
The collected supernatant was dropped onto the plate, and was then stirred at room temperature for 3 hours. The plate was washed by pipetting using a HISCL wash solution (Sysmex Corporation) (40 μL). The washing was carried out twice in total. The glass was further washed by pipetting using PBS (40 μL). The washing with PBS was carried out twice in total. A fluorescent image of Aβ on the plate was taken with an inverted microscope equipped with a laser. Fluorescent images in 64 viewing fields were superposed on each other with Max intensity to obtain results.
The obtained fluorescent images are shown in
As shown in Table 2, it was suggested that Aβ in a clinical specimen was able to be detected with high sensitivity when at least one of the first capture substance and the second capture substance was an antibody capable of binding to a C-terminal region of Aβ and the substance for detection was an antibody capable of binding to a N-terminal region of Aβ. Particularly when each of the first capture substance and the second capture substance was an antibody capable of binding to a C-terminal region of Aβ and the substance for detection was an antibody capable of binding to a N-terminal region of Aβ, the sensitivity of the detection of Aβ in a clinical specimen was significantly improved.
In Example 1, two type of capture substances were used in the ICT. In Example 2, it was examined as to whether or not Aβ contained in a clinical specimen was measured with high sensitivity by the method of the present embodiment utilizing an ICT using a single type of capture substance antibody and a fluorescence microscope.
As a sample containing Aβ, a cerebrospinal fluid (CSF) collected from an Alzheimer's disease patient who agreed to the donation of a specimen was used.
In the measurement using two types of capture substances, as in the case of Example 1, a DNP-labeled H31L21 antibody was used as a first capture substance and a biotin-labeled H31L21 antibody was used as a second capture substance. In the measurement using a single type of capture substance, a H31L21 antibody labeled with both of DNP and biotin was used as the capture substance. A H31L21 antibody labeled with both of DNP and biotin was produced as mentioned below with reference to the description of WO 2017/138497 A1. A first solid phase, a second solid phase, a substance for detection (a fluorescently labeled 82E1 antibody) and a releasing agent were the same as those used in Example 1.
A H31L21 antibody was digested with pepsin in the conventional manner to obtain a F(ab′)2 fragment. The F(ab′)2 fragment thus obtained was reduced to produce Fab′. BSA conjugated with DNP and biotin (product name: DNP-BSA-Biotin, LGC Biosearch Technologies) was reacted with a poly(ethylene glycol) (PEG) cross-linker (succinimidyl-[(N-maleimidepropionamide)-octa(ethylene glycol)] ester (product name: SM(PEG)8, Thermo Fisher Scientific)) to bond a linker having a maleimide group to DNP-BSA-Biotin. The DNP-BSA-Biotin having the linker bonded thereto was mixed with the Fab′ to cause the reaction of a thiol group in the Fab′ with a maleimide group in the DNP-BSA-Biotin, thereby producing a DNP/biotin-labeled Fab′. In Example 2, a DNP/biotin-labeled Fab′ derived from a H31L21 antibody was used as a DNP/biotin-labeled capture substance.
Each of combinations of the capture substance and the substance for detection were mixed in a HISCL R3 diluent (Sysmex Corporation) in each of combinations of A and E shown in Table 3, thereby producing an antibody solution. The content of each of the antibodies in the antibody solution was adjusted to 300 fmol/assay. In Table 3, the antibody combination A corresponds to
The measurement of each of the samples was carried out in the same manner as in Example 1, except that the above-mentioned antibody solutions were used. The measurement (n=2) was carried out twice, and the measurement procedures were carried out independently. Typical examples of the acquired fluorescent images are shown in
As shown in Table 4, it was demonstrated that, in the case where an antibody capable of binding to a C-terminal region of Aβ was used as a capture substance, Aβ in a clinical specimen was detected with high sensitivity regardless of the number of the capture substance, i.e., the use of a single type of capture substance or the use of two types of capture substances. As shown in each of panels in
It was examined as to whether or not Aβ in a clinical specimen was able to be measured by the method according to the present embodiment utilizing chemiluminescent immunoassay (CELIA).
As a sample containing Aβ, a CSF collected from an Alzheimer's disease patient who agreed to the donation of a specimen (also referred to as a “clinical specimen 2”, hereinafter) was used. As a positive control, a solution containing a reparation produced by chemically crosslinking aggregates of Beta Amyloid (1-42) (Anaspec) (wherein the preparation was referred to as “synthetic Aβ”, hereinafter) at a concentration of 10 pg/mL was used. The chemical crosslinking was carried out in accordance with the description of Farid Rahimi et. al., “Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides”, J Vis Exp, vol. 23, (2009), 1071. As a negative control, a CO solution (Sysmex Corporation) was used.
As a second solid phase, HISCL R2 reagent (Sysmex Corporation) which was a suspension containing magnetic particles each having streptavidin immobilized on the surface thereof was used. As a substance for detection, a 82E1 antibody labeled with alkaline phosphatase (ALP) was used. The ALP-labeled 82E1 antibody was produced as mentioned below in accordance with the description of WO 2017/138497 A1. A first solid phase, a first capture substance (a DNP-labeled H31L21 antibody), a second capture substance (a biotin-labeled H31L21 antibody and a biotin-labeled 82E1 antibody) and a releasing agent were the same as those used in Example 1. In the measurement of chemiluminescence, HISCL R4 reagent (Sysmex Corporation) which includes a measurement buffer and HISCL R5 reagent (Sysmex Corporation) which include CDP-Star (registered tradename) that was a chemiluminescent substrate for alkaline phosphatase were used.
An 82E1 antibody was digested with pepsin by the conventional technique to obtain a F(ab′)2 fragment. The F(ab′)2 fragment thus obtained was reduced to produce Fab′. ALP was reacted with EMCS to maleimdate the ALP. The Fab′ was mixed with the maleimdated ALP to cause the reaction of a thiol group in the Fab′ with a maleimide group in the ALP, thereby producing an ALP-labeled Fab′. In Example 3, an ALP-labeled Fab′ derived from a 82E1 antibody was used as the ALP-labeled detection antibody.
Each of combinations of the capture substance and the substance for detection were mixed in a HISCL R3 diluent (Sysmex Corporation) in each of combinations of A and B shown in Table 5, thereby producing an antibody solution. The content of each of the antibodies in the antibody solution was adjusted to 300 fmol/assay. In Table 5, the antibody combination A corresponds to
The measurement was carried out using a full-automatic immunoassay device HISCL-800 (Sysmex Corporation) in the following manner. A sample (70 μL) was mixed with an antibody solution (80 μL), and the resultant solution was incubated at 37° C. for 27 minutes. The solution was further mixed with a suspension of a first solid phase (20 μL), and the resultant solution was incubated at 37° C. for 11 minutes. The magnetic particles were magnetically collected, and then a supernatant was removed. A HISCL wash solution (300 μL) was added to the resultant product to wash the magnetic particles. The washing was carried out three times in total. After the washing, a DNP-Lys solution (110 μL) was added to the magnetic particles, and the resultant solution was incubated at 42° C. for 5 minutes. The magnetic particles were magnetically collected, and a supernatant (80 μL) was collected. The collected supernatant was mixed with a second solid phase (HISCL R2 reagent) (30 μL), and the resultant solution was incubated at 37° C. for 5 minutes. The magnetic particles in the mixed solution were magnetically collected, and a supernatant was removed. A HISCL wash solution (300 μL) was added to the resultant product to wash the magnetic particle. The washing was carried out four times in total. The magnetic particles were magnetically collected, and a supernatant was removed. The magnetic particles were mixed with HISCL R4 reagent (30 μL) and HISCL R5 reagent (60 μL), then the resultant solution was incubated at 42° C. for 5 minutes, and then the luminous intensity of the solution was measured.
All of the measurement values of the chemiluminescence intensities exceeded the detection limit. An SN ratio (S/N) was calculated from each of the measurement values in accordance with the below-mentioned formula. The results are shown in
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-028495 | Feb 2019 | JP | national |
