WO2015194350A1 - 標識化レクチンを用いるサンドイッチ型アッセイおよびそのためのキット - Google Patents
標識化レクチンを用いるサンドイッチ型アッセイおよびそのためのキット Download PDFInfo
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- WO2015194350A1 WO2015194350A1 PCT/JP2015/065731 JP2015065731W WO2015194350A1 WO 2015194350 A1 WO2015194350 A1 WO 2015194350A1 JP 2015065731 W JP2015065731 W JP 2015065731W WO 2015194350 A1 WO2015194350 A1 WO 2015194350A1
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- lectin
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- sugar chain
- detection target
- glycoprotein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
- G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4728—Details alpha-Glycoproteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
Definitions
- the present invention relates to a sandwich type assay using a labeled lectin and a kit for the same. More specifically, the present invention provides a sandwich-type assay that includes a treatment that suppresses the binding of labeled lectin to contaminants that are non-specifically adsorbed to a measurement region, and a sandwich-type assay that includes such a treatment. It relates to a suitable kit.
- Post-translational modifications such as glycosylation play an extremely important role in order for proteins, which are the main players responsible for vital functions of living organisms, to exhibit their functions in an orderly manner in the living body.
- post-translational modifications the following has been revealed in recent years.
- Most proteins in the body are modified by sugar chains, and the sugar chain added to the protein is protein stability, binding to hormones, binding to toxins, virus infection, mycoplasma infection, bacterial infection It plays an important role in various life phenomena such as protozoan parasitism, fertilization, developmental differentiation, cancer cell metastasis, and apoptosis.
- Even proteins with the same amino acid sequence and the same name have a variety of modified sugar chains, and the structure of the sugar chain varies depending on the state of the cell that produces the protein, and the role in vivo is different. .
- Patent Document 1 describes the following for a prostate-specific antigen (hereinafter referred to as PSA) indicating that it is suffering from a prostate disease. That is, in a blood sample derived from a patient with prostate cancer, a prostate-specific antigen having a specific sugar residue, N-acetyl-D-galactosamine ⁇ 1-4N acetylglucosamine (hereinafter referred to as LacdiNAc) residue in the sugar chain.
- PSA prostate-specific antigen
- LacdiNAc N-acetyl-D-galactosamine ⁇ 1-4N acetylglucosamine
- LacdiNAc-PSA LacdiNAc-PSA
- PSA sugar chains are changed due to the onset of prostate cancer, and PSA having the specific sugar residue increases, and the concentration of PSA having the specific sugar residue is high in the blood sample of prostate cancer patients. It is observed.
- the change of the sugar chain does not occur even when prostatic hypertrophy develops, no change is observed in the concentration of PSA having the specific sugar residue in the prostatic hypertrophy patient.
- Patent Document 1 it is possible to discriminate between a patient with prostate cancer and a patient with benign prostatic hypertrophy by measuring the concentration of PSA having the specific sugar residue in the blood sample.
- a method for distinguishing prostate cancer by sugar chain fraction measurement is disclosed.
- Patent Document 2 and Non-Patent Document 1 a method for differentiating liver cancer by measuring ⁇ -fetoprotein (AFP) sugar chain fractions, and carcinoembryonic antigen as disclosed in Non-Patent Documents 2 and 3 (CEA) A method for identifying adenocarcinoma by sugar chain fraction measurement is known.
- AFP ⁇ -fetoprotein
- CEA carcinoembryonic antigen
- lectin having the ability to specifically recognize and bind to the sugar residue is widely used. ing. This is because an antibody having a sugar chain as an epitope, particularly an antibody having a specific sugar residue as an epitope, is very difficult to produce and is difficult to obtain. Lectins are inexpensive and can be obtained in large quantities, have excellent stability, and can be stored for a long time.
- nodafuji lectin (Wisteria floribunda Agglutinin: hereinafter referred to as WFA) is known to have N-acetylgalactosamine as the main linking sugar residue.
- WFA Wisteria floribunda Agglutinin: hereinafter referred to as WFA
- WFA having such characteristics is bound to a carrier, packed in a column, fractionated PSA having a LacdiNAc residue in the side chain of an asparagine-linked sugar chain, and then subjected to ELISA or the like.
- a method for quantifying minutes is described.
- Patent Document 3 uses a solid-phased anti-PSA antibody and fluorescently labeled WFA to form a sandwich-type complex with PSA having a LacdiNAc residue in the side chain of a sugar chain, and SPFS (A method for quantifying PSA having this specific sugar residue by Surface Plasmon-field enhanced Fluorescence Spectroscopy (surface plasmon resonance excitation enhanced fluorescence spectroscopy) is described.
- nodafuji lectin uses N-acetylgalactosamine residues as the main binding site, but also binds slightly to galactose residues. Therefore, when a sugar chain containing a galactose residue is present in the reaction system, nodafuji lectin binds to that sugar chain, so that only the sugar chain containing an N-acetylgalactosamine residue should be specifically bound. I can't.
- a sandwich-type assay that can easily and quantitatively analyze a protein (glycoprotein) having a specific sugar chain as a detection target substance has been performed.
- An antibody that specifically binds to the protein portion of a glycoprotein is immobilized on a substrate and used as a solid-phase antibody, and a lectin whose main binding target is a sugar residue contained in the sugar chain of the glycoprotein is used as a labeling agent. Ligated and used as a labeled lectin.
- this technique is effective when the glycoprotein that is the detection target substance is purified to some extent, but it is a glycoprotein other than the detection target substance, such as blood and urine that are used as specimens in normal disease diagnosis.
- the labeled lectin binds not only to the sugar chain of the detection target substance but also to the sugar chain of the contaminant, resulting in increased background (noise) and sensitivity.
- the performance deteriorates remarkably in terms of quantitativeness. Therefore, it is extremely difficult to accurately perform quantitative analysis by sandwich type assay using lectin and antibody in general diagnosis using blood or the like as a specimen. It is only used for diagnosis when a limited type of glycoprotein having a significantly high concentration (about several ⁇ g / mL) is targeted for detection.
- blocking agents such as bovine serum albumin (BSA) and casein are used to suppress the adsorption of serum contaminants to the surface of the support on which the antibody is immobilized.
- BSA bovine serum albumin
- casein casein
- a method to be used Patent Document 4
- glycosaminoglycan glycosaminoglycan, heparin, etc.
- Patent Documents 5 and 6 A method of adding an absorbent such as a polymer or a sugar chain complex (Patent Documents 5 and 6), a specific salt strength, a surfactant, etc. to efficiently remove impurities adsorbed on the support
- a method using a cleaning solution having the composition (Patent Document 4) and the like has been studied.
- the present applicant first uses a labeling lectin that binds to a plurality of types of sugar chains including a detection target sugar chain and a non-detection target sugar chain, and a detection target substance having the detection target sugar chain in a sample.
- (Analyte) detection method wherein a labeling treatment in which the labeling lectin is brought into contact with the detection target substance before the detection step of detecting the detection target substance bound to the labeling lectin, and at least 1
- a method for detecting a detection target substance comprising: performing mask processing for bringing a sugar chain recognition molecule for a mask that binds to the non-detection target sugar chain of a species into contact with a contaminant having the non-detection target sugar chain.
- the masking glycan recognition molecule masks the non-detection target sugar chain of the contaminants to which the labeling lectin is secondarily bound, whereby the labeling lectin becomes the detection target sugar chain of the detection target substance. It becomes possible to dominate the binding, and the background noise can be reduced and the detection sensitivity and quantitativeness of the detection target substance can be improved.
- the mask sugar chain recognition molecule a lectin of a type different from the labeling lectin that mainly recognizes and binds to the non-detection target sugar chain rather than the detection target sugar chain can be used. *
- the detection method of the detection target substance described in Patent Document 7 is applicable when a contaminant has a sugar chain that is recognized by the same lectin but includes a sugar residue different from the detection target substance. . Further, when such impurities are present in a large amount in the specimen, in the invention described in Patent Document 7, a sugar chain recognition molecule for mask (a lectin different from the labeling lectin) is added at a high concentration. This necessitates an increase in reagent cost, or a reduction in mask efficiency.
- Patent Document 7 does not disclose that the measurement sensitivity and quantitativeness of the substance to be detected can be actually improved by using it.
- Patent Document 8 A non-specific signal suppression method that performs at least one of pretreatment for adding metal ions and pretreatment for heating a specimen has also been proposed.
- Patent Document 8 By these pretreatments, nonspecific adsorption to the sensor unit can be suppressed by changing (denaturing) the structure of the contaminants.
- Patent Document 8 also effectively suppresses the influence when a sample contains a large amount of contaminants (glycoprotein, etc.) having a sugar chain containing the same sugar residue as the detection target substance. There is no disclosure of possible means.
- the present invention is a sandwich-type assay for quantifying a glycoprotein as a detection target substance in a specimen using a labeled lectin, and introduces a simple process to thereby influence influences from contaminants, that is, detection. It is an object of the present invention to provide a kit in which noise with respect to a quantitative value of a target substance is suppressed, and a kit suitable for performing such a sandwich type assay.
- the present inventors are one of the typical diagnostic methods applying a sandwich type assay using a labeled lectin, which is a WFA-binding PSA (ie, a sugar containing an N-acetylgalactosamine residue) contained in a serum sample.
- a labeled lectin which is a WFA-binding PSA (ie, a sugar containing an N-acetylgalactosamine residue) contained in a serum sample.
- a WFA-binding PSA ie, a sugar containing an N-acetylgalactosamine residue
- glycoproteins such as IgM contained in serum to the measurement region are a factor that raises the value in healthy subjects. Since these glycoproteins have a sugar chain containing N-acetylgalactosamine residues as well as specific PSA, it can be seen that they are WFA-binding, and the content of each glycoprotein varies from individual to individual, but from specific PSA It was also revealed that it was contained in serum at a high and constant concentration.
- the inventors have maintained the binding of the labeled lectin to the specific PSA (substance to be detected) and adsorbed nonspecifically to the measurement region, such as a glycoprotein (contaminant) such as IgM having the same sugar chain as the specific PSA. If the binding of WFA (labeled lectin) to the product) can be suppressed, a large signal (background) that is noisy with respect to the signal for quantifying the specific PSA is suppressed, and the problem as described above I thought that could be solved.
- WFA labeled lectin
- the present inventors as a treatment for suppressing the binding of WFA to the same sugar chain as the specific PSA possessed by contaminants such as IgM, are substances that exhibit the action of cleaving disulfide bonds of glycoproteins, and representative examples include
- a reducing agent such as sodium sulfite
- the background noise
- Such a process is not limited to a measurement system in which specific PSA, IgM, and the like and WFA are combined, and both the detection target substance and the contaminant share the sugar chain that is the main recognition target of the lectin. It has been found that the present invention can be applied to other measurement systems, and that the same effects can be achieved, and the present invention has been completed.
- the present invention is a sandwich type assay for quantifying a glycoprotein as a detection target substance using a labeled lectin and a capture substance immobilized on the measurement area, wherein the measurement area
- a sandwich type assay comprising a treatment that suppresses the binding of labeled lectin to the same sugar chain as the detection target substance possessed by the non-specifically adsorbed contaminant.
- the present invention provides, as a detection target substance, a labeled lectin suitable for use in carrying out the sandwich-type assay of the present invention and a capture substance immobilized on a measurement region.
- a kit for carrying out a sandwich-type assay for quantifying the glycoprotein of the present invention, comprising a substance exhibiting an action of cleaving a disulfide bond for treating the glycoprotein as a contaminant is provided.
- the present invention first, a relatively large amount of contaminants having the same sugar chain as the detection target substance is contained in the sample, and the binding of the labeled lectin to the contaminants adsorbed nonspecifically to the measurement region is suppressed.
- a highly sensitive sandwich type assay such as SPFS.
- the binding of the detection lectin to the contaminant is suppressed by releasing the domain containing the same sugar chain as the detection target substance from other domains of the contaminant by, for example, cleaving the disulfide bond using a reducing agent. It became clear that we could do it.
- the labeled lectin Even if other domains of contaminants remain in the measurement region due to non-specific adsorption, the labeled lectin will no longer be bound there, so the background (noise) derived from the contaminants will be increased. (The labeled lectin may be bound to the sugar chain of the released domain, but they can be removed relatively easily by washing or the like).
- the sandwich-type assay of the present invention that exhibits such effects, even when a specimen containing a relatively large amount of glycoprotein as a contaminant such as blood (serum) is used, tumor markers and the like can be used. It becomes possible to accurately quantify the detection target substance. In particular, since an abnormally high value of a measurement value obtained when measuring based on SPFS can be significantly suppressed, a highly reliable diagnosis with excellent specificity and sensitivity can be performed.
- FIG. 1 is a diagram schematically showing the state of a measurement region in one embodiment of a measurement process based on SPFS.
- FIG. 2 is a schematic view schematically showing the overall configuration of a measuring apparatus for SPFS suitable for use in the present invention.
- FIG. 3 is a partially enlarged view around the measurement region of the SPFS measuring apparatus shown in FIG.
- FIG. 4 is a graph showing background values (B), signal values (S), and S / B ratios measured in Examples and Comparative Examples.
- FIG. 5 shows measured values of SPFS signals (left) for specific PSA contained in prostate cancer specimens, prostate hypertrophy specimens and healthy subject specimens (left), and prostate hypertrophy specimens and healthy subjects, which were performed in Reference Example 1. It is a graph showing the result of the measured value (right) of the ELISA which made all PSA a detection object about what has a high SPFS signal measured value among human specimens.
- FIG. 4 is a graph showing background values (B), signal values (S), and S / B ratios measured in Examples and Comparative Examples.
- FIG. 5 shows measured values of SPFS signals (left) for specific PSA contained in prostate cancer specimens, prostate hypertrophy specimens and healthy subject specimens (left), and prostate hypertrophy specimens and healthy subjects, which were performed in Reference Example 1. It is a graph showing the result of the measured value (right) of the ELISA which made all PSA
- FIG. 6 is a photograph showing the results of the treatment of a healthy subject sample performed in Reference Example 1 using a WFA column and the electrophoresis analysis of the bound fraction.
- FIG. 7 shows preparations relating to inter- ⁇ trypsin inhibitor (I ⁇ I), ⁇ 2-macroglobulin ( ⁇ 2M), haptoglobin (HP), immunoglobulin M (IgM) and immunoglobulin G (IgG), which were performed in Reference Example 1. It is a graph showing the measured value of SPFS signal of the sialidase processed material of a reagent.
- FIG. 1 inter- ⁇ trypsin inhibitor
- ⁇ 2M ⁇ 2-macroglobulin
- HP haptoglobin
- IgM immunoglobulin M
- IgG immunoglobulin G
- FIG. 8 is a graph showing the measured values of the SPFS signal for the normal fraction, the pass-through fraction, and the bound fraction when the healthy subject sample was treated with the IgM purification column, which was performed in Reference Example 1.
- FIG. 9 is a diagram showing the structure of immunoglobulin M (IgM), one of the glycoproteins that can be a contaminant in the present invention. Immunoglobulin G (IgG) is the same as the IgG-like molecule that is a degradation product of IgM shown on the right side.
- FIG. 10 is a diagram showing the structure of an inter- ⁇ trypsin inhibitor (I ⁇ I), which is one of the glycoproteins that can be contaminated in the present invention (cited from J. Biol. Chem.
- FIG. 11 is a diagram showing the structure of ⁇ 2-macroglobulin ( ⁇ 2M), which is one of the glycoproteins that can be contaminated in the present invention (cited from protein nucleic acid enzyme Vol.7 No.9).
- FIG. 12 is a diagram showing the structure of haptoglobin (HP), which is one of the glycoproteins that can be a contaminant in the present invention.
- the sandwich-type assay of the present invention is a sandwich-type assay for quantifying glycoprotein as a detection target substance using a labeled lectin and a capture substance immobilized on a measurement region, and is non-specific to the measurement region.
- the “measurement region”, which will be described in detail later, is a region (space) in which the intensity of fluorescence emitted therefrom is measured, and is a solid-phased capture substance, a support carrying it, Furthermore, the area
- the treatment for suppressing the binding of the labeled lectin to the same sugar chain as the detection target substance that the non-specifically adsorbed contaminant has in the measurement region is not particularly limited, and the type of the contaminant (glycoprotein or Or glycolipids), and can be modified according to the detection target substance, capture substance, labeled lectin, measurement system, and the like.
- the present invention will be described mainly based on an embodiment in which the contaminant is a “glycoprotein” having the same sugar chain as the detection target substance.
- the contaminant is the same sugar chain as the detection target substance.
- the contaminant is the same sugar chain as the detection target substance.
- the contaminant nonspecifically adsorbed on the measurement region is a glycoprotein having the same sugar chain as the detection target substance.
- the treatment for suppressing the binding of the labeled lectin to the sugar chain is a glycoprotein in which a substance exhibiting an action of cleaving a disulfide bond (referred to herein as an “SS bond cleaving agent”) is a contaminant. It is preferable that the treatment includes releasing the domain containing the same sugar chain as that of the detection target substance by contacting with the.
- the substance exhibiting the action of cleaving the disulfide bond of impurities is not particularly limited.
- a reducing agent that is, two sulfhydryls of —SH HS— having a cross-linking structure of —SS—
- a compound exhibiting an action of reducing to a group (thiol group) is preferred.
- the reducing agent is not particularly limited, and can be selected from various reducing agents used for cleaving disulfide bonds of proteins.
- Examples of such a reducing agent include lower oxygen salts such as sulfurous acid, hyposulfite, phosphorous acid, and hypophosphorous acid, alkali hydroxides, and thiol salts that serve as nucleophiles.
- sodium metabisulfite, thioglycerol, sodium toluenethiosulfinate, 2-mercaptoethanol, dithiothreitol (DTT), 2-mercaptoethylamine hydrochloride, 2-aminoethylisothiouronium bromide odor Hydrohydride, tris (2-carboxyethyl) phosphine and the like can be used, and among these, sodium metabisulfite is particularly preferable.
- disulfide reductase (disulfide reductase) can be used in addition to the above compounds.
- a detection target substance is a glycoprotein having a sugar chain containing a specific sugar residue that is recognized and bound by an appropriate lectin and having a site (particularly a protein portion) that is recognized and bound by an appropriate antibody.
- a typical example of a glycoprotein as a detection target substance is a marker molecule contained in a specimen and used for pathological diagnosis.
- cancer antigens / tumor markers such as PSA, AFP, and CEA are suitable.
- glycoproteins such as signaling substances and hormones can be used as detection target substances.
- a PSA having a sugar chain containing an N-acetyl-D-galactosamine ⁇ 1-4N acetylglucosamine (abbreviated herein as “LacdiNAc”) residue (herein (Abbreviated as “LacdiNAc-PSA”)).
- the substance to be detected contains the same sugar chain as the contaminant and the binding site of the capture substance in the same domain, that is, the domain containing the same sugar chain as the contaminant and the domain containing the binding site of the capture substance are SS bond cleavage It is preferably a glycoprotein that is not separated by the agent.
- the substance to be detected is such, even if the SS bond cleaving agent for the contaminant comes into contact with the substance to be detected and a part of the domain of the substance to be detected dissociates, the same sugar as the contaminant Since both the capture substance and the labeled lectin bind to the domain having the binding site with the chain and the capture substance, and a sandwich-type complex can be formed in the measurement region, the treatment targeting the contaminants can be performed. It is possible to avoid affecting the quantification of the detection target substance.
- the impurities to be targeted for suppressing the influence on the quantification of the detection target substance are substances having the same sugar chain as the detection target substance.
- biologically relevant substances such as small molecules having glycoproteins, glycolipids, and other sugar chains other than the detection target substance that are present in the specimen together with the detection target substance can be such impurities.
- glycoproteins such as immunoglobulin M (IgM), inter- ⁇ trypsin inhibitor (I ⁇ I), ⁇ 2-macroglobulin ( ⁇ 2M), haptoglobin (HP), immunoglobulin G (IgG), Main contaminants to be controlled.
- IgM immunoglobulin M
- I ⁇ I inter- ⁇ trypsin inhibitor
- ⁇ 2M ⁇ 2-macroglobulin
- HP haptoglobin
- IgG immunoglobulin G
- Each of the above five types of glycoproteins has a plurality of types of sugar chains having different sugar residues, but at least N-acetyl-D-galactosamine ⁇ 1-4N acetylglucosamine residues. It has a sugar chain that contains it.
- the composition ratios of the above five types of glycoproteins in serum and the composition ratios of sugar chains (sugar residues) contained in each glycoprotein are determined depending on the specimen, that is, whether or not it is affected by a specific disease. It was different due to individual differences, and it was the cause of noise (background) that was difficult to suppress uniformly in the past.
- IgM is a molecule usually present in blood at a concentration of 0.3 to 2.5 mg / mL, and has 50 or more N-linked sugar chains in the molecule.
- IgM is a multimer (pentamer or hexamer) having a structure in which five or six IgG-like molecules are connected by a disulfide bond.
- I ⁇ I is a molecule usually present in blood at a concentration of about 0.5 mg / mL, and has four N-linked sugar chains and four O-linked sugar chains in the molecule. I ⁇ I has three domains of H1 heavy chain, H2 heavy chain and bikunin linked by one chondroitin sulfate chain, and has four disulfide bonds in the molecule.
- ⁇ 2M is a macromolecule associated with four subunits having a molecular weight of 160,800, usually present in serum at a concentration of 1 to 2.5 mg / mL, and eight N-linked sugars in each subunit. Has a chain. Each subunit of ⁇ 2M is connected by a disulfide bond, and a large number of disulfide bonds exist in the subunit.
- HP is a molecule usually present in the blood at a concentration of 0.1 to 1.7 mg / mL, and two ⁇ chains and two ⁇ chains each having four N-linked sugar chains are linked by a disulfide bond. It has a structured. There are two types of ⁇ chains, ⁇ 1 and ⁇ 2 and one type of ⁇ chain, and there are three genotypes, 1-1 type, 2-1 type and 2-2 type, depending on the combination.
- IgG is a molecule usually present in blood at a high concentration of 8.7 to 18.1 mg / mL, and has two or more N-linked sugar chains in the molecule. IgG has three disulfide bonds in the molecule, and exists in a state in which two heavy chains (heavy chain) and two light chains (light chain) are bound.
- glycoproteins contain SS bonds and can be fragmented or deformed by cleaving them, so that treatment with a disulfide bond cleaving agent prevents nonspecific adsorption. It becomes an effective means for suppressing.
- an SS bond cleaving agent is allowed to act on IgM
- the disulfide bond connecting IgG-like molecules is cleaved and broken down into individual IgG-like molecules.
- the strength of the action of the SS bond cleaving agent it is cleaved to the three disulfide bonds present in the IgG-like molecule, and finally the IgG-like molecule is divided into two light chains and two heavy chains. Is broken down.
- Other glycoproteins can be similarly degraded to subunits or domains as described for each.
- the degradation or deformation of the contaminants due to the cleavage of the SS bond causes the non-specific adsorption force of the contaminants to be lost in the measurement region and, at the same time, the partial decomposition products of the contaminants are non-specific in the measurement region. Even when adsorbed on the glycan, noise can be prevented from being generated if the site modified with a sugar chain containing a specific sugar residue that can react with the labeled lectin is dissociated from the partial degradation product.
- the site modified with a sugar chain containing a specific sugar residue that is recognized and bound by the lectin (part or all) is located in a region separated from the antigen recognition site with at least one disulfide bond sandwiched between them. It is considered to exist.
- the other substances exemplified above are also effective when an SS bond cleaving agent is allowed to act, so that the lectin recognizes and binds to the non-specifically adsorbed site in the measurement region. It is considered that the site (part or all) modified with a sugar chain containing a specific sugar residue exists at a distance between at least one disulfide bond.
- IgM In the reference examples described below, attention is focused on IgM, but the effects of the present invention are not construed as being limited to cases where IgM is a contaminant. It has been shown that contaminants other than IgM such as I ⁇ I, ⁇ 2M, HP, and IgG cause noise to the SPFS signal to some extent. It is also effective to suppress noise. In addition, as described above, the constituent ratio of contaminants or the constituent ratio of sugar chains of a certain contaminant varies depending on the specimen. For specimens different from those used in Reference Examples described later, I ⁇ I, ⁇ 2M, HP There is a possibility that impurities other than IgM, such as IgG, may be a major cause of noise.
- the present invention has a relatively large amount in IgM, I ⁇ I, ⁇ 2M, HP, IgG, and other specimens such as serum that have the same sugar chain as the substance to be detected and to which the same labeled lectin may bind. It should be understood that the effects can be exerted on all the impurities contained in.
- a specimen that may contain a detection target substance and impurities is used.
- the specimen that may contain the detection target substance and contaminants may be a specimen that actually contains them, or a specimen that does not actually contain them. Specifically, for example, it may be a specimen derived from a patient with a specific disease that is likely to contain a detection target substance (and impurities), or may contain a detection target substance (and impurities). It may be a specimen derived from a low healthy person.
- the specimen is typically collected from a human, but a human disease model animal such as a mouse, rat, guinea pig, rabbit, goat, cat, dog, pig, monkey or other non-human mammal. There may be.
- specimens examples include biological samples and biological samples such as blood, urine, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (for example, biopsy specimens).
- biological samples and biological samples such as blood, urine, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (for example, biopsy specimens).
- blood and urine are specimens that may contain glycoproteins that can be used as diagnostic markers and that may contain relatively large amounts of glycoproteins as contaminants. It is preferable as a specimen to be used.
- Liquid samples such as blood, serum, plasma, urine, spinal fluid and saliva can be diluted with an appropriate buffer and used.
- a solid sample such as a cell, tissue, or organ is homogenized with an appropriate buffer that is about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant is diluted as it is or further diluted. Can be used.
- blood is used as a specimen.
- the blood may be whole blood, or serum or plasma prepared from whole blood.
- whole blood may be used as a sample for the purpose of rapid measurement, and for the purpose of accurate quantification, blood cell components are removed from whole blood by centrifugation, etc.
- plasma may be prepared and used as a specimen.
- an anticoagulant is usually added to whole blood at the time of blood collection, and whole blood, serum, and plasma are diluted with a diluent (buffer solution, etc.) to adjust to an appropriate concentration when used for measurement of SPFS, etc.
- necessary reagents and the like are added.
- whole blood, serum, and plasma as specimens to which such an anticoagulant, diluent, other reagents, etc. may be added as necessary are referred to as “blood specimens”.
- a lectin that recognizes and binds to a specific sugar residue contained in the sugar chain of the detection target substance is labeled and used for a sandwich type assay.
- the lectin includes a substance called agglutinin, but in this specification, the term lectin is used.
- the lectin used in the present invention is suitable for a specific sugar residue contained in the sugar chain of the detection target substance as a main recognition and binding target. That is, since lectins generally have different binding strengths for a plurality of types of sugar residues, it is appropriate to use a lectin that exhibits the strongest binding strength for the sugar residue of the detection target substance. .
- lectins derived from various animals, plants, fungi, bacteria or viruses, which can be obtained by separating, extracting and purifying from these materials, and are also commercially available as commercial products. Yes. Specific examples of known lectins are as follows: ACA (sennin clectin), BPL (purple lectin lectin), ConA (spotted bean lectin), DBA (horsegram lectin), and DSA (Hopaceae morning glory).
- WFA having a LacdiNAc residue as a main binding target is used to produce a labeled lectin.
- the LacdiNAc residue is possessed by the specific PSA (LacdiNAc-PSA) described above and also has IgM, which is a contaminant that greatly affects the quantification of the residue, so that WFA is compatible with both LacdiNAc-PSA and IgM. Has strong reactivity.
- SBA seybean lectin
- VVL rowd pea lectin
- TJA-II kirasouri lectin
- a labeled lectin can be prepared by binding a desired label to the lectin using a known general technique, and in that case, various commercially available kits (labeling kits) should be used. You can also.
- fluorescent dyes are generally used in SPFS, and enzymes are generally used in ELISA.
- fluorescent substances other than fluorescent dyes fluorescent proteins, fluorescent fine particles, etc.
- chemiluminescent substances radioactive substances It is possible to use a known label such as a substance.
- fluorescent dyes examples include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), hexachlorofluorescein family Fluorescent dyes (Applied Biosystems Japan), Coumarin family fluorescent dyes (Invitrogen Corporation), Rhodamine family fluorescent dyes (GE Healthcare Biosciences), Cyanine family fluorescent dyes, India Carbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide family fluorescent dye, BODIPY® family Fluorescent dyes (Invitrogen Corporation), naphthalenesulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, Alexa Fluor (registered trademark) dye series (Invitrogen Corporation), etc.
- Organic fluorescent dyes, and rare earth complex fluorescent dyes such as Eu and Tb (for example,
- HRP horseradish-derived peroxidase
- ALP alkaline phosphatase
- a substance that specifically binds to a glycoprotein as a detection target substance is used by immobilizing it in the measurement region.
- the detection target substance is captured in the measurement region, and a complex (sandwich complex) of the capture substance-detection target substance-labeled lectin can be formed.
- the capture substance is not particularly limited as long as it is an appropriate substance corresponding to the detection target substance.
- an antibody, an aptamer, a synthetic peptide, or the like can be used.
- a glycoprotein as a detection target substance particularly an antibody that specifically recognizes and binds to the protein part and does not interfere with sugar chain recognition by a lectin is used as a capture substance.
- the antibody against the detection target substance may be any antibody that can be prepared by a general technique when the detection target substance is used as an antigen, and is not particularly limited. However, from a polyclonal antibody due to measurement stability (reproducibility). Monoclonal antibodies are also preferred. In addition, an antibody having an epitope on a protein portion rather than a sugar chain of a glycoprotein is preferable so as not to prevent the labeled lectin from binding to a specific sugar residue in the sugar chain.
- a cancer antigen / tumor marker is a detection target substance
- monoclonal antibodies against various cancer antigens / tumor markers have been developed and are commercially available.
- PSA as a monoclonal antibody against PSA known as a marker for prostate cancer (anti-PSA monoclonal antibody), PS2, PS3, PS4, PS5, PS6, PS15, 2H9, 3B4, 5A6, 5G6, 8G4, 9A8, 9G2, PS1, 8A6, 2H9, 1H12, no. 79 and the like.
- antibody is used in a broad sense to include not only complete antibodies but also antibody fragments and derivatives.
- antibody fragments such as Fab, Fab ′ 2 , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv) or the like It is possible to carry out using derivatives.
- a “support” supporting them may be used as necessary.
- the support may be selected according to the measurement system, and is not particularly limited.
- insoluble polysaccharides such as agarose, cellulose, dextran, silicon resin, polystyrene resin, polyacrylamide resin, nylon resin
- synthetic resin such as polycarbonate resin and an insoluble support such as glass.
- the support can have a desired shape corresponding to the measurement system, such as a bead (mainly spherical) or a plate (mainly flat).
- a bead mainly spherical
- a plate mainly flat
- the beads a column filled with magnetic beads, resin beads, or the like can be used.
- a multiwell plate 96-well multiwell plate or the like
- a biosensor chip or the like can be used.
- a known immobilization mode such as chemical bonding or physical adsorption can be used.
- a functional group amino group, carboxyl group, etc.
- the reactivity of the antibody with a functional group is required.
- a modifying group having a silane coupling agent, a linker, a support or the like By introducing a modifying group having a silane coupling agent, a linker, a support or the like, and reacting the modifying group with a functional group of an antibody in the presence of a predetermined reaction reagent as necessary, The antibody can be immobilized to the support by covalent bonding.
- a layer made of a polysaccharide such as a hydrophilic polymer such as carboxymethyldextran (CMD) is first formed on the surface of the metal thin film as a support, and a reactive group possessed by the hydrophilic polymer such as carboxymethyldextran is formed.
- CMD carboxymethyldextran
- a sandwich type assay In a sandwich type assay, a measurement region is provided on the surface of a measurement member, and a capture substance-detection target substance-labeled lectin complex (sandwich complex) is formed in the measurement region. The intensity of the signal emitted from the labeled lectin is measured, and the content (concentration) of the detection target substance in the sample is quantified based on the measured value.
- Such sandwich-type assays generally include a “pre-measurement step” in which various processes are performed to form a sandwich-type complex before measuring the signal, and a signal is generated after the formation of the sandwich-type complex. It is roughly divided into “measurement process” for performing various processes for measurement.
- the pre-measurement step includes a process of causing a substance exhibiting an action of cleaving a disulfide bond (SS bond cleaving agent) to act on the contaminant. Is preferred.
- the pre-measurement process generally includes the following three processes (A) to (C): (A) a process of bringing a labeled lectin into contact with a detection target substance (labeling process); (B) a treatment for bringing an SS bond cleaving agent into contact with a contaminant (cleavage treatment); and (C) The process (capture process) which makes a detection target substance (and impurities) contact a capture substance.
- the labeling process (A), the cleavage process (B), and the capture process (C) can be performed in various orders depending on the embodiment of the pre-measurement process. In any of the embodiments, even when a non-specifically adsorbed contaminant is present in the measurement region at the stage where the complex of the capture substance-detectable substance-labeled lectin is formed, it is detected from the contaminant. A domain having the same sugar chain as the target substance is excised.
- a specific example of the order of the labeling process, the cleavage process and the capturing process is as follows. The symbol “+” indicates that the processing on the left side and the right side is performed simultaneously, and the symbol “ ⁇ ” indicates that the processing on the right side is performed after the processing on the left side is performed. For example, “(labeling process + cleavage process) ⁇ capture process” represents that the capture process is performed after the labeling process and the cleavage process are performed simultaneously.
- the SS bond cleaving agent is brought into contact with the contaminant before contacting the contaminant (glycoprotein) with the capture substance.
- a SS bond cleaving agent in the form of a solution
- Add an SS bond cleaving agent
- an SS bond cleaving agent and a labeled lectin may be simultaneously added to a specimen and the specimen may be brought into contact with a capture substance.
- a labeled lectin is added to the specimen, and then an SS bond cleaving agent is further added.
- the SS bond cleaving agent is added to the specimen or the like.
- a labeled lectin is further added, and in any case, the specimen or the like may be brought into contact with the capture substance thereafter.
- the SS bond cleaving agent is brought into contact with the contaminant.
- impurities in a state in which the domain having a predetermined sugar chain is not cleaved may be nonspecifically adsorbed to the measurement region, and by cleaving the nonspecifically adsorbed impurities.
- the domain having a predetermined sugar chain is dissociated from the support.
- the order of (5) After adding a labeled lectin to a specimen or the like, the specimen or the like is brought into contact with a capture substance, and then a solution of the SS bond cleaving agent is further brought into contact with the capture substance.
- a sample or the like is brought into contact with a capture substance, and then a solution in which both the SS bond cleaving agent and the labeled antibody are dissolved is brought into contact therewith.
- the order of (7) the specimen or the like is brought into contact with the capture substance, then the labeled antibody solution is brought into contact therewith, and then the SS bond cleaving agent solution is further brought into contact therewith.
- the order of (8) is carried out, the specimen or the like is brought into contact with the capture substance, then the SS bond cleaving agent solution is brought into contact therewith, and then the labeled antibody solution is further brought into contact therewith.
- the SS bond cleavage agent acts on the immobilized antibody on the support.
- the ability to capture the detection target substance may be affected. Therefore, in the present invention, at least in the case of group (I) in which the cleavage treatment is performed before the capture treatment, that is, in the order of (1) to (4), before the contaminant (glycoprotein) is brought into contact with the capture substance.
- an SS bond cleaving agent such as a reducing agent
- the SS bond cleaving agent for example, when a reducing agent is added to a sample or the like in the order (1) to (4), it is generally added to a sample such as serum at a final concentration of 1 mM to 1M. It may be added and allowed to react for 1 to 60 minutes. If the added amount of the SS bond cleaving agent is large, the cleavage of the SS bond of the foreign substance proceeds rapidly, and it tends to be a fragment having a small molecular weight.
- the measurement step is a step of measuring the intensity of the signal emitted from the labeled lectin, and its embodiment is based on a measurement system using a sandwich type assay.
- a lectin linked to a fluorescent dye is generally used as a labeled lectin. Therefore, in the measurement process, excitation light corresponding to the fluorescent dye is irradiated and the intensity of the generated fluorescence is determined. Measure with a detector.
- a lectin linked to an enzyme is used as a labeled lectin. Therefore, in the measurement process, a substrate corresponding to the enzyme is added, and the intensity of the generated fluorescence is measured by a detector. To.
- the sandwich-type assay of the present invention is preferably carried out according to an SPFS (Surface Plasmon-field enhanced fluorescence spectroscopy) measurement system.
- SPFS Surface Plasmon-field enhanced fluorescence spectroscopy
- SPFS can measure the intensity of the fluorescence emitted from the labeled lectin with high sensitivity, but the measurement value of the fluorescence intensity (signal: S) is bound to the detection target substance accordingly.
- S the measurement value of the fluorescence intensity
- B background
- the cleavage process performed in the pre-measurement process as described above significantly reduces such noise, thereby improving the S / B ratio that is an index of the quantitative value of the detection target substance and more accurately detecting the detection target substance. It becomes possible to quantify.
- the evanescent wave transmitted through the metal thin film is several tens of times due to resonance with the surface plasmon.
- This is a method for efficiently exciting a fluorescent substance labeled with a substance to be measured trapped in the vicinity of a metal thin film by utilizing the fact that it is enhanced several hundred times.
- the intensity of the fluorescence emitted from the fluorescent substance can be measured, and the detection target substance can be quantified by the measured value. If necessary, it can be converted into the concentration of the detection target substance in the sample by comparing with the measured value of the fluorescence intensity when measured using a standard sample with a known concentration. Since such SPFS is extremely sensitive compared to a general fluorescent labeling method or the like, even when the concentration of the detection target substance in the sample is extremely low, the concentration can be measured.
- a metal thin film 14 is formed on a dielectric member 12, and a support 54 made of a hydrophilic polymer such as carboxymethyl dextran carrying a trapping substance 50 is provided as a reaction layer.
- the measurement area 38 is constructed.
- FIG. 1A shows a state where the cleavage treatment is not performed (conventional embodiment).
- the detection target substance 60 binds to the capture substance 50 carried on the support 54, and the labeled lectin 52 having the fluorescent substance 52a binds to the sugar chain 62a of the detection target substance 60 to form a complex.
- a contaminant 62 having a sugar chain 52a containing the same sugar residue as the sugar chain 62a of the detection target substance 60 is adsorbed non-specifically on the support 54 and the capture substance 50, and the sugar chain 62a.
- the labeled lectin 52 binds. Therefore, the fluorescence emitted from the reaction region 38 includes fluorescence (background) derived from nonspecifically adsorbed impurities as well as fluorescence (signal) indicating the presence of the detection target substance 60.
- FIG. 1B shows a state when the cleavage process is performed (embodiment of the present invention).
- the detection target substance 60 binds to the capture substance 50 carried on the support 54, and the labeled lectin 52 having the fluorescent substance 52a binds to the sugar chain 62a of the detection target substance 60 to form a complex.
- the contaminant 62 is fragmented for each domain 62b by the cleavage treatment, and nonspecific adsorption to the support 54 and the capture substance 50 by the domain 62b having the sugar chain 62a is suppressed.
- the fluorescence emitted from the reaction region 38 is mainly the fluorescence (signal) indicating the presence of the detection target substance 60, and the fluorescence (background) derived from the nonspecifically adsorbed contaminants can be almost eliminated. it can.
- Embodiments of a basic configuration for implementing SPFS that is, a measurement member (sensor chip and the like), a measurement device and system, a measurement procedure, and the like are known (for example, Japanese Patent Application Laid-Open No. 2013-253866: Patent Document 7). , International Publication WO2014 / 088702: see Patent Document 8), and these can be applied to appropriately implement the present invention.
- a measurement member sensor chip and the like
- Patent Document 7 Japanese Patent Application Laid-Open No. 2013-253866
- Patent Document 8 International Publication WO2014 / 088702
- the measurement member for SPFS is a member generally called a sensor chip 16, and a substrate-like member 16 a on which a measurement region which is a place for forming a sandwich type immune complex and performing fluorescence measurement by SPFS is formed. And various solutions (specimens or other samples containing the detection target substance, labeled lectin solution, other necessary reagents, etc.) used for the formation of sandwich-type complexes, etc. are introduced and held on the measurement area.
- a configuration is possible in which the flow path member 16b for constructing the flow path 36 is laminated.
- the sensor chip 16 is attachable to and detachable from a measurement member loading unit 18 included in the SPFS measuring apparatus 10 and is loaded and used at the start of measurement.
- the substrate-like member 16a in the embodiment shown in FIG. 2A includes a prism-shaped dielectric member 12 having a substantially trapezoidal vertical cross-section for introducing excitation light into the back surface of the metal thin film 14, and a horizontal direction of the dielectric member 12.
- the metal thin film 14 for generating surface plasmon resonance formed on the upper surface 12a and the solid phase formed on the upper surface 14a of the metal thin film 14 are solid-phased to capture the detection target substance on the surface of the metal thin film 14.
- a reaction layer containing a trapping substance may be provided.
- the substrate-like member 16a in the embodiment shown in FIG. 2B is different from the embodiment shown in FIG. 2A in that the prism-shaped dielectric member 12 and the transparent flat substrate 13 made of the same material as the dielectric member 12 are separated. Have taken.
- the metal thin film 14 is formed not on the dielectric member 12 but on the surface of the transparent flat substrate 13, and a reaction layer and a spacer layer provided as necessary are also formed on the metal thin film 14.
- the dielectric member 12 is fixedly installed on the measurement apparatus 10 (measurement member loading section 18), and constitutes a part of the measurement apparatus 10.
- the substrate-like member 16a including the transparent flat substrate 13 and the like is combined with the flow path member 16b to constitute the sensor chip 16, and is then used by being mounted on the measuring device 10 (measurement member loading unit 18).
- the other surface (back surface) of the transparent flat substrate 13 is stacked so as to be in close contact with the upper surface 12a of the dielectric member 12, and measurement can be performed as in the embodiment shown in FIG. 2A.
- the part where the reaction layer on the metal thin film is formed corresponds to the measurement region 38.
- a reaction layer may be formed over the entire bottom surface of the flow path 36 (or well) to form the measurement region 38, or the reaction layer may be formed on only a part of the bottom surface by a desired patterning if necessary. It is good.
- the area of the measurement region 38 can be adjusted in consideration of the irradiation area of the incident light 22 (excitation light) generally irradiated as laser light. For example, if the spot diameter of the incident light 22 is about 1 mm ⁇ , the measurement region 38 is usually designed to have an area of at least several mm square.
- the channel member 16b for forming the channel is, for example, A film-like “flow cell” with holes that form the wall of the road and a “top plate” with a liquid feed inlet and a liquid feed outlet at positions corresponding to the holes in the flow cell can be constructed.
- the flow path 36 can be formed by closely contacting and fixing the substrate-like member 16a.
- the surface of the sensor chip at a position corresponding to the hole of the flow cell forms the bottom surface of the flow path 36, and a measurement region 38 is formed there.
- a groove having a depth corresponding to the height of the flow path is carved, and a member provided with through holes corresponding to the liquid feeding inlet and the liquid feeding outlet at both ends is used as the flow path member 16b. It is also possible to construct the flow path 36 by stacking them so that they are on the inside and fixing them closely.
- various liquids can be fed into the flow channel from the liquid feed inlet and discharged from the liquid feed outlet using a liquid feeding means including a pump and a tube.
- a liquid feeding means including a pump and a tube.
- reciprocating and circulating liquid feeding can also be performed.
- Conditions such as the feeding speed and feeding (circulation) time include the amount of sample, the concentration of the detection target substance in the sample, the size of the flow channel or well, the mode of the reaction layer (capture substance density, etc.), and the pump performance It is possible to make appropriate adjustments while considering the above.
- the SPFS system can be a “well type” in which various solutions are stored in a space wider than the flow path.
- a “well member” having a through hole for forming a well may be stacked and fixed on the substrate-like member 16a.
- various liquids can be added to the well and removed using, for example, a pipette-shaped member.
- the flow cell can be made of, for example, a sheet-like polydimethylsiloxane (PDMS).
- the top plate is made of a material having transparency so that fluorescence emitted from the measurement region 38 can be measured, and can be made of, for example, plate-shaped polymethyl methacrylate (PMMA).
- PMMA plate-shaped polymethyl methacrylate
- the flow cell and the top plate can be made of plastic having a desired shape by molding or photolithography.
- Means for fixing the flow cell or well member in close contact with the substrate-like member 16a is not particularly limited, and generally pressure may be physically applied from above and below, and if necessary, transparent support is provided. You may use the adhesive agent, matching oil, transparent adhesive sheet, etc. which have the same optical refractive index as a body.
- the SPFS measurement device 10 basically irradiates excitation light (preferably laser light) having an appropriate wavelength according to the measurement member loading unit 18 to which the SPFS measurement member can be attached and detached, and the phosphor to be used.
- Light source 20 light receiving means 26 (receiver and movable means) that receives light reflected by the metal thin film and measures the intensity thereof, a lens for collecting the fluorescence emitted from the phosphor, and the intensity of the fluorescence
- Light detection means 32 detector, etc.
- various filters for transmitting only light having a predetermined wavelength from excitation light and fluorescence, and cutting other light, flow of SPFS measurement member
- a liquid feeding means (not shown) for supplying various solutions to the channel or well is provided.
- a light source 20 is disposed on the side surface 12 b below the dielectric member 12.
- the light source 20 may be any light source that can emit excitation light corresponding to the fluorescent dye of the labeled lectin.
- a laser diode (LD) can be used as the light source 20.
- the light source 20 is provided with incident angle adjusting means (not shown) that can appropriately change the incident angle ⁇ 1 of the incident light 22 with respect to the metal thin film 14.
- Incident light 22 is incident from one side 12 b below the dielectric member 12 and is irradiated toward the metal thin film 14 through the dielectric member 12 at a suitable incident angle that maximizes surface plasmon resonance. Can be done.
- the incident light 22 (laser light) emitted from the light source 20 is P-polarized light that efficiently generates surface plasmons on the metal thin film 14.
- a polarizing filter may be provided.
- light receiving means 26 for receiving the reflected light 24 of the incident light 22 reflected by the metal thin film 14 is provided.
- the reflected light 24 is attenuated more than the incident light 22 depending on the degree, so that the amount of light measured by the light receiving means 26 Can determine whether or not the incident angle ⁇ 1 is appropriate.
- the light receiving means 26 is also provided with a movable means (not shown), and the reflected light 24 is reliably received even when the incident angle ⁇ 1 and the reflection angle are changed in synchronization with the incident angle adjusting means of the light source 20. Can be done.
- the light detection means 32 positions the light detector above the measurement region 38 so that the fluorescence emitted from the measurement region 38 of the sensor chip 16 can be measured.
- the photodetector for example, a photomultiplier tube (PMT) can be used.
- the light detection means 32 is usually provided with a condensing lens and a filter for condensing only fluorescence of a specific wavelength and excluding other fluorescence.
- the light receiving means 26 and the light detection means 32 are respectively connected to the control calculation means 40, and the amount of reflected light 24 received by the light receiving means 26 and the amount of fluorescence 30 received by the light detection means 32 are controlled. It is configured to be transmitted to the calculation means 40 for calculation processing. For example, it is possible to automatically calculate the S / B ratio based on the light amount data of the fluorescence 30 measured in a predetermined process. Moreover, the control calculation means 40 can also have the function to operate the structural member of the SPFS measuring apparatus 10 appropriately according to a predetermined program.
- the unit comprised by the sensor chip 16, the light source 20, and the light detection means 32 may be called the SPFS measurement part 34 for performing the fluorescence measurement based on SPFS.
- a unit constituted by the sensor chip 16, the light source 20 and the light receiving means 26 may be referred to as an SPR measurement unit 28.
- the sandwich-type assay of the present invention can be used for various uses that require quantification of a substance to be detected, and the purpose of the use is not particularly limited.
- the sandwich type assay of the present invention when a quantification is performed by a sandwich type assay using a glycoprotein serving as a marker of various diseases as a detection target substance, the measurement result is used for diagnosis of the disease.
- the sandwich type assay of the present invention performed according to SPFS or the like can significantly reduce noise and obtain a signal or S / B ratio that more accurately represents the amount of the detection target substance. . By using such data, it becomes possible to more accurately diagnose whether or not the subject who provided the specimen is a patient with the disease.
- PSA having a sugar chain containing a LacdiNAc residue is affected by prostate cancer or not (affected by benign prostatic hyperplasia or healthy)
- the measured value becomes abnormally high due to noise derived from contaminants. Therefore, it is possible to reduce the risk of misdiagnosis that the patient is not afflicted with prostate cancer, that is, increase the specificity for prostate cancer.
- the kit of the present invention is a kit capable of performing a sandwich type assay for quantifying a glycoprotein as a detection target substance using a labeled lectin and a capture substance immobilized on a measurement region, It contains at least a substance (SS bond cleaving agent) that acts to cleave disulfide bonds for treating glycoproteins as contaminants.
- SS bond cleaving agent a substance that acts to cleave disulfide bonds for treating glycoproteins as contaminants.
- Such a kit of the present invention is suitable for efficiently performing the sandwich type assay of the present invention including the cleavage treatment as described above.
- the kit of the present invention may contain reagents and instruments used for carrying out the sandwich-type assay of the present invention in addition to the SS bond cleaving agent such as a reducing agent.
- a substrate-like member dielectric member or transparent flat substrate
- a reaction layer containing a solid-phased capture substance (antibody etc.) a solid-phased capture substance (antibody etc.)
- Flow channel member flow cell and top plate
- specimen dilution solution labeled lectin
- washing solution fluorescence measurement solution
- fluorescence measurement solution or these solutions are loaded and used in a sealed SPFS device. It may contain at least one kind, preferably all selected from reagent containers that can be used.
- an unmodified substrate-like member instead of a substrate-like member provided with a reaction layer, etc., an unmodified substrate-like member, a capture substance (antibody, etc.) for producing it, and if necessary, for fixing a capture substance on the surface of a metal thin film Silane coupling agents, SAMs, hydrophilic polymers such as carboxymethyl dextran and reaction reagents, and preparation tools may be included in the kit.
- a labeled lectin a lectin for preparing it, a label such as a fluorescent dye, a reaction reagent and a solvent for linking them, and a preparation tool may be included in the kit.
- a cleaning agent and a solvent for preparing it and a preparation tool may be included in the kit.
- the kit of the present invention may contain instructions for use showing procedures for performing the sandwich type assay of the present invention.
- the kit of the present invention includes nodaphuji lectin (WFA), soybean lectin (SBA), citrus lectin-II (TJA-II), which mainly contains LacdiNAc residues. ) Or a labeled lectin using a crow pea lectin (VVL).
- WFA nodaphuji lectin
- SBA soybean lectin
- TJA-II citrus lectin-II
- VVL crow pea lectin
- the kit of the present invention preferably contains a specimen diluent that can be used by dissolving it together with an SS bond cleaving agent, or a specimen diluent in which an SS bond cleaving agent is dissolved in advance.
- FIGS. 2B and 3B A measurement apparatus for SPFS having the configuration shown in FIGS. 2B and 3B was manufactured (the reference numerals in the following description are the same as those in FIGS. 2B and 3B). Further, a 60-degree prism manufactured by Sigma Kogyo Co., Ltd. is used as the dielectric member 12, and the top surface of the dielectric member 12 is manufactured using the transparent flat substrate 13 obtained in Preparation Example 2 described below.
- the sensor chip 16 provided with the flow path 36, on which the anti-PSA antibody was solid-phased, was fixed in the measurement region 38.
- the light source 20 a laser diode (LD) capable of irradiating light with a wavelength of 635 nm was used.
- LD laser diode
- a light reduction filter (neutral density filter) is provided as an optical filter between the light source 20 and the dielectric member 12 so that the amount of photons can be adjusted.
- An objective lens is provided as a condensing lens on the upper part of the measurement region 38 of the sensor chip 16, and a photomultiplier tube (PMT) is provided as a photodetector, thereby providing the light detection means 32.
- PMT photomultiplier tube
- the substrate provided with the gold thin film thus formed is immersed in an ethanol solution containing 1 mM 10-carboxy-1-decanethiol for 24 hours or more, and a SAM film (Self-Assembled Monolayer: self-assembled (Molecular film) was formed on the surface of the gold thin film.
- SAM film Self-Assembled Monolayer: self-assembled (Molecular film) was formed on the surface of the gold thin film.
- the substrate was removed from this solution, washed with ethanol and isopropanol, and then dried using an air gun.
- a sheet made of polydimethylsiloxane (PDMS) having a groove that becomes a flow path with a height of 0.5 mm and through holes at both ends of the groove is formed so that the surface of the CMD film is inside the flow path.
- PDMS polydimethylsiloxane
- a peristaltic pump is connected to the flow path constructed on the plasmon excitation sensor as described above, ultrapure water is added for 10 minutes, and then phosphate buffered saline (PBS) is supplied for 20 minutes at room temperature (25 ° C.). The surface of the plasmon excitation sensor was equilibrated by circulating the solution at 500 ⁇ L / min.
- PBS phosphate buffered saline
- a PBS solution containing 1 wt% bovine serum albumin (BSA) was circulated and fed at a flow rate of 500 ⁇ L / min for 30 minutes to perform nonspecific adsorption prevention treatment (blocking treatment) in the flow path.
- BSA bovine serum albumin
- PSA free pool serum normal human pool serum, Kojin Bio Inc., confirmed that the PSA concentration is 0.01 ng / mL or less by ELISA
- LNCaP human prostate cancer cell culture
- a serum sample solution having a PSA concentration of 1 pg / mL (0.001 ng / mL) or less and showing a non-specific reaction is selected from PSA free pool sera by ELISA, and a background (B) is obtained. It was used as a sample for
- Example 1 When sodium metabisulfite is used as a reducing agent (measurement of signal value) A PBS (pH 7.4) solution containing 3.5 wt% sodium metabisulfite as a reducing agent was prepared and used as a treatment solution. 100 ⁇ L of this treatment solution was added to 50 ⁇ L of the signal acquisition sample, stirred well in the tube, and allowed to stand at room temperature for 5 minutes.
- a fluorescently labeled lectin (Alexa Fluor 647-labeled WFA) solution having a concentration of 1 ⁇ g / mL obtained in Preparation Example 3 was introduced into the flow path, and circulated for 10 minutes at a flow rate of 200 ⁇ L / min. Reacted. Thereafter, a TBS (TBS-T) solution containing 0.05 wt% of “Tween (registered trademark) 20” was introduced into the flow path, and the flow path and the measurement region were washed for 3 minutes. Then, excitation light was irradiated in a state where the flow path was filled with this TBS solution, the fluorescence emission intensity of Alexa Fluor 647 was measured, and the measured value was used as a signal value.
- TBS TBS
- the S / B ratio which is the ratio of the signal value obtained by the above procedure to the background value obtained by the above procedure, was calculated.
- Example 2 When thioglycerol is used as a reducing agent Signal values and background values were obtained in the same procedure as in Example 1 except that a treatment solution to which thioglycerol was added instead of sodium metabisulfite was used. The S / B ratio was calculated.
- Example 3 When sodium toluenethiosulfinate is used as a reducing agent in the same manner as in Example 1 except that a treatment solution containing sodium toluenethiosulfinate instead of sodium metabisulfite was used, the signal value and The background value was obtained and the S / B ratio was calculated.
- the results of Examples 1 to 3 and the comparative example are shown in Table 1 and FIG.
- the signal value (S) in the table is an actual measurement value, and corresponds to the sum of the signal value derived from the specific PSA and the signal value corresponding to the background (B).
- a large S / B ratio is detected when the specimen is derived from a patient containing a large amount of LacdiNAc-PSA, while a small S / B ratio is detected when the specimen is derived from a healthy subject that hardly contains LacdiNAc-PSA.
- S since S is close to B, the S / B ratio is close to 1), the specificity and sensitivity to patient-derived specimens can be improved, and the possibility of misdiagnosing healthy subjects as patients Can be lowered.
- the above five candidate substances are all general serum proteins, and standard reagents are commercially available.
- the sample reagent was treated with sialidase and then added to a PBS buffer, and the SPFS signal was measured. The results are shown in FIG. As for the healthy subject sample used this time, a remarkable SPFS signal that could be noise was confirmed for IgM. In addition, SPFS signals that could be noise but weaker than IgM were also confirmed for I ⁇ I, ⁇ 2M, HP, and IgG.
- each healthy subject sample was treated with an IgM purification column, before the column, each of the flow-through fraction (the fraction from which IgM was removed) and the bound fraction (the fraction from which IgM was eluted).
- SPFS signal was measured. The results are shown in FIG.
- the measurement signal is significantly reduced in the pass-through fraction, while the measurement signal is high in the bound fraction. Therefore, non-specific adsorption of IgM is a factor for increasing the signal value of the healthy subject described at the beginning, and it is highly possible that the background can be significantly reduced by removing this. It is shown.
- SPFS measuring apparatus Dielectric member 12a Upper surface 12b Side surface 12c Side surface 13 Transparent flat substrate 14 Metal thin film 14a Upper surface 16 Sensor chip 16a Substrate-like member 16b Flow path member 18 Measuring member loading part 20 Light source 22 Incident light 24 Reflected light 26 Light reception Means 28 SPR measurement section 30 Fluorescence 32 Photodetection means 34 SPFS measurement section 36 Flow path 38 Measurement region 40 Quantitative calculation means 50 Capture substance 52 Labeled lectin 52a Fluorescent substance 54 Support body 60 Detection target substance 60a Sugar chain 62 Contaminant 62a Sugar Chain 62b domain (fragmented contaminant)
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Abstract
Description
本発明のサンドイッチ型アッセイは、標識化レクチンおよび測定領域に固相化された捕捉物質を用いて、検出対象物質としての糖タンパク質を定量するためのサンドイッチ型アッセイであって、測定領域に非特異的に吸着した夾雑物が有する、検出対象物質と同じ糖鎖への標識レクチンの結合を抑制する処理を含む。なお、「測定領域」は、詳細は後述するが、そこから発せられる蛍光の強度が測定される領域(空間)であって、固相化された捕捉物質、それを担持している支持体、さらにそれらを載せている部材の表面などを含む領域を指す。
本発明の典型的な実施形態において、測定領域に非特異的に吸着した夾雑物は、検出対象物質と同じ糖鎖を有する糖タンパク質である。この場合、その糖鎖への標識化レクチンの結合を抑制する処理は、ジスルフィド結合を開裂する作用を呈する物質(本明細書において「SS結合開裂剤」と称する。)を夾雑物としての糖タンパク質に接触させることにより、検出対象物質と同じ糖鎖を含むドメインを遊離させる処理であることが好適である。
本発明では、適切なレクチンが認識して結合する特定の糖残基を含む糖鎖を有し、かつ適切な抗体が認識して結合する部位(特にタンパク質部分)を有する糖タンパク質を検出対象物質とする。
本発明において、検出対象物質の定量に対する影響を抑制するための対象とする夾雑物は、検出対象物質と同じ糖鎖を有する物質である。具体的には、検出対象物質とともに検体中に存在する、検出対象物質以外の糖タンパク質、糖脂質、その他の糖鎖を有する生体関連物質(低分子等)がそのような夾雑物となり得る。
本発明では、検出対象物質および夾雑物を含む可能性のある検体を用いる。検出対象物質および夾雑物を含む可能性のある検体は、現実にそれらを含む検体であってもよいし、現実にはそれらを含まない検体であってもよい。具体的には、たとえば、検出対象物質(および夾雑物)を含む可能性の高い特定の疾患の患者に由来する検体であってもよいし、検出対象物質(および夾雑物)を含む可能性の低い健常者に由来する検体であってもよい。また、検体を採取する対象は、典型的にはヒトであるが、ヒトの疾患のモデル動物、たとえばマウス、ラット、モルモット、ウサギ、ヤギ、ネコ、イヌ、ブタ、サルといったヒト以外の哺乳動物であってもよい。
本発明では、検出対象物質が有する糖鎖に含まれる特定の糖残基を認識しそれに結合できるレクチンを標識化して、サンドイッチ型アッセイのために用いる。なお、レクチンには、凝集素〔agglutinin〕と呼ばれている物質も含まれるが、本明細書においてはレクチンという呼び方で統一する。
本発明では、検出対象物質としての糖タンパク質に特異的に結合する物質を、測定領域に固相化して用いる。それにより、測定領域に検出対象物質を捕捉し、捕捉物質-検出対象物質-標識化レクチンの複合体(サンドイッチ型複合体)を形成することができるようになる。
サンドイッチ型アッセイでは、測定部材の表面に測定領域を設け、そこに捕捉物質-検出対象物質-標識化レクチンの複合体(サンドイッチ型複合体)を形成させたときに、測定領域の内部に存在する標識化レクチンから発せられるシグナルの強度を測定し、その測定値によって検体中の検出対象物質の含有量(濃度)を定量する。そのようなサンドイッチ型アッセイは、一般的に、シグナルを測定する前にサンドイッチ型複合体を形成させるための各種の処理を行う「測定前工程」と、サンドイッチ型複合体を形成した後、シグナルを測定するための各種の処理を行う「測定工程」とに大別される。
夾雑物が検出対象物質と同じ糖鎖を有する「糖タンパク質」である場合、測定前工程は、ジスルフィド結合を開裂する作用を呈する物質(SS結合開裂剤)を夾雑物に作用させる処理を含むことが好ましい。この場合、測定前工程は一般的に、下記(A)~(C)の3つの処理を含むことになる:
(A)標識化レクチンを検出対象物質に接触させる処理(標識処理);
(B)SS結合開裂剤を夾雑物に接触させる処理(開裂処理);および、
(C)捕捉物質に検出対象物質(および夾雑物)を接触させる処理(捕捉処理)。
(1)(標識処理+開裂処理)→捕捉処理
(2)標識処理→開裂処理→捕捉処理
(3)開裂処理→標識処理→捕捉処理
(4)開裂処理→捕捉処理→標識処理
(5)標識処理→捕捉処理→開裂処理
(6)捕捉処理→(標識処理+開裂処理)
(7)捕捉処理→標識処理→開裂処理
(8)捕捉処理→開裂処理→標識処理
測定前工程における開裂処理の順序は、(1)~(4)の順序に対応する、捕捉処理を行う前に開裂処理を行うグループ(I)と、(5)~(8)の順序に対応する、捕捉処理を行った後に開裂処理を行うグループ(II)の2つに大きく分けることができる。
測定工程は、標識化レクチンから発せられるシグナルの強度を測定する工程であり、その実施形態はサンドイッチ型アッセイを用いた測定系に準じたものとなる。
本発明のサンドイッチ型アッセイは、SPFS(Surface Plasmon-field enhanced fluorescence spectroscopy:表面プラズモン共鳴励起増強蛍光分光法)測定系に準じて実施されることが好適である。以下に述べるように、SPFSは標識化レクチンから発せられる蛍光の強度を高感度で測定することができるが、その分、蛍光強度の測定値(シグナル:S)には、検出対象物質に結合した標識化レクチンに由来する測定されるべきシグナルだけでなく、夾雑物に結合した標識化レクチンなどに由来する、測定されるべきでないバックグラウンド(B)も含まれやすくなる。前述したような測定前工程で行われる開裂処理は、そのようなノイズを著しく低減することにより、検出対象物質の定量値の指標となるS/B比を向上させ、検出対象物質をより正確に定量することができるようになる。
本発明のサンドイッチ型アッセイは、検出対象物質の定量が必要とされる様々な用途に対して利用することができるものであり、その利用の目的は特に限定されるものではない。
本発明のキットは、標識化レクチンおよび測定領域に固相化された捕捉物質を用いて、検出対象物質としての糖タンパク質を定量するためのサンドイッチ型アッセイを実施することのできるキットであって、少なくとも、夾雑物としての糖タンパク質を処理するためのジスルフィド結合を開裂する作用を呈する物質(SS結合開裂剤)を含む。このような本発明のキットは、前述したような開裂処理を含む本発明のサンドイッチ型アッセイを効率的に実施するために好適なものとなっている。
図2Bおよび図3Bに示す構成を有するSPFS用測定装置を作製した(以下の記載中の符号は図2Bおよび図3Bと同じである)。また、誘電体部材12としてシグマ光機(株)製の60度プリズムを用い、この誘電体部材12の上面に、次に述べる作製例2により得られた、透明平面基板13を用いて作製され、測定領域38に抗PSA抗体が固相化された、流路36を備えたセンサーチップ16を固定した。光源20としては波長635nmの光を照射することができるレーザーダイオード(LD)を用いた。光源20と誘電体部材12との間には、光学フィルタとして減光フィルタ(中性濃度フィルタ)を設けてフォトン量を調整できるようにした。センサーチップ16の測定領域38の上部に、集光レンズとして対物レンズを備え付け、さらに光検出器として光電子増倍管(PMT)を備え付け、光検出手段32とした。
屈折率1.72、厚さ1mmのガラス製の透明平面基板「S-LAL 10」((株)オハラ)をプラズマ洗浄し、この基板の片面にクロム薄膜をスパッタリング法によって形成した。その後、その表面にさらに金薄膜をスパッタリング法によって形成した。クロム薄膜の厚さは1~3nm、金薄膜の厚さは44~52nmであった。
上記のようにしてプラズモン励起センサーの上に構築した流路にペリスタポンプを接続し、超純水を10分間、その後リン酸緩衝生理食塩水(PBS)を20分間、室温(25℃)で、流量500μL/分で循環送液させて、プラズモン励起センサーの表面を平衡化した。
蛍光標識化レクチン(Alexa Fluor 647標識WFA)を、蛍光物質ラベリングキット「Alexa Fluor(商標名)647タンパク質ラベリングキット」(インビトロジェン社製)を利用して作製した。WFA(ノダフジレクチン)「L-1350」(Vector社)100μg相当と、0.1M重炭酸ナトリウムと、Alexa Fluor 647 reactive dyeとを混合し、室温で1時間反応させた後、ゲル濾過クロマトグラフィーおよび限外濾過を行い、標識に利用されなかったAlexa Fluor 647 reactive dyeを取り除いて、蛍光標識化WFAを回収した。得られた蛍光標識化WFA溶液の吸光度を測定して濃度を定量し、PBSで希釈して1μg/mLの濃度となるよう調整した。
陽性試料として、PSAフリープール血清(正常ヒトプール血清、コージンバイオ社、ELISAにてPSA濃度が0.01ng/mL以下であることを確認)に、LNCaP(ヒト前立腺癌細胞培養株)培養上清を、全PSA濃度が2ng/mLとなるように添加した血清サンプルを調製し、シグナル(S)を取得するための試料として用いた。
(シグナル値の測定)
還元剤としてメタ重亜硫酸ナトリウムを3.5wt%含有するPBS(pH7.4)溶液を調製し、処理溶液として用いた。この処理液100μLを、シグナル取得用試料50μLに添加し、チューブ内でよく撹拌して、室温にて5分間放置した。
シグナル取得用試料の代わりにバックグラウンド取得用試料を用いたこと以外は上記「シグナル値の測定」と同様の手順で、Alexa Fluor 647の蛍光の発光強度を測定し、その測定値をバックグラウンド値とした。
上記の手順で取得したバックグラウンド値に対する、上記の手順で取得したシグナル値の比の値であるS/B比を算出した。
メタ重亜硫酸ナトリウムの代わりにチオグリセロールを添加した処理溶液を用いたこと以外は実施例1と同様の手順でシグナル値およびバックグラウンド値を取得し、S/B比を算出した。
メタ重亜硫酸ナトリウムの代わりにトルエンチオスルフィン酸ナトリウムを添加した処理溶液を用いたこと以外は実施例1と同様の手順でシグナル値およびバックグラウンド値を取得し、S/B比を算出した。
メタ重亜硫酸ナトリウムを添加していない処理溶液を用いたこと以外は実施例1と同様の手順でシグナル値およびバックグラウンド値を取得し、S/B比を算出した。
実施例1~3および比較例の結果を表1および図4に示す。表中のシグナル値(S)は実測値であり、特定PSA由来のシグナル値とバックグラウンド(B)に相当するシグナル値の合計に相当する。
前立腺癌患者由来のサンプル35例、前立腺肥大症患者由来のサンプル(前立腺肥大症検体)30例、および健常者由来のサンプル(健常者検体)25例について、まず、還元剤を用いない比較例1と同様にして、SPFSのシグナル値を求めた。結果を図5(左)に示す。次に、そのSPFSのシグナル値が高値であった、前立腺肥大症検体および健常者検体について、ELISA法に準じて全PSA量を定量した。結果を図5(右)に示す。前立腺肥大症検体および健常者検体の結果について、図5(左)の四角枠内のデータを対比すると、健常者検体は前立腺肥大症検体に比べて、全PSA含量が低いにもかかわらず、特定の糖鎖を有するPSA(LacdiNAc-PSA)を測定しようとしたSPFSのシグナルが高値化していることが分かる。
12 誘電体部材
12a 上面
12b 側面
12c 側面
13 透明平面基板
14 金属薄膜
14a 上面
16 センサーチップ
16a 基板状部材
16b 流路部材
18 測定部材装填部
20 光源
22 入射光
24 反射光
26 受光手段
28 SPR測定部
30 蛍光
32 光検出手段
34 SPFS測定部
36 流路
38 測定領域
40 定量演算手段
50 捕捉物質
52 標識化レクチン
52a 蛍光物質
54 支持体
60 検出対象物質
60a 糖鎖
62 夾雑物
62a 糖鎖
62b ドメイン(断片化された夾雑物)
Claims (8)
- 標識化レクチンおよび測定領域に固相化された捕捉物質を用いて、検出対象物質としての糖タンパク質を定量するためのサンドイッチ型アッセイであって、前記測定領域に非特異的に吸着した夾雑物が有する、検出対象物質と同じ糖鎖への標識化レクチンの結合を抑制する処理を含む、サンドイッチ型アッセイ。
- 前記測定領域に非特異的に吸着した夾雑物が、前記検出対象物質と同じ糖鎖を有する糖タンパク質であって、その糖鎖への標識化レクチンの結合を抑制する前記処理が、ジスルフィド結合を開裂する作用を呈する物質を前記夾雑物としての糖タンパク質に接触させることにより、前記検出対象物質と同じ糖鎖を含むドメインを遊離させる処理である、請求項1に記載のサンドイッチ型アッセイ。
- 前記検出対象物質が、前記夾雑物としての糖タンパク質と同じ糖鎖および前記捕捉物質との結合部位を同一のドメイン内に含む糖タンパク質である、請求項2に記載のサンドイッチ型アッセイ。
- 前記検出対象物質および夾雑物が、N-アセチル-D-ガラクトサミンβ1-4Nアセチルグルコサミン残基を含む糖鎖を有する糖タンパク質であって、前記標識化レクチンが、ノダフジレクチン(WFA)、ダイズレクチン(SBA)、キカラスウリレクチン-II(TJA-II)またはカラスノエンドウレクチン(VVL)を標識化したものである、請求項2または3に記載のサンドイッチ型アッセイ。
- 前記夾雑物が、免疫グロブリンM(IgM)、インターαトリプシンインヒビター(IαI)、α2-マクログロブリン(α2M)、ハプトグロビン(HP)および免疫グロブリンG(IgG)からなる群より選ばれる少なくとも一種である、請求項2~4のいずれか一項に記載のサンドイッチ型アッセイ。
- 前記夾雑物としての糖タンパク質を前記捕捉物質に接触させる前に、前記ジスルフィド結合を開裂する作用を呈する物質を前記夾雑物としての糖タンパク質と接触させるようにする、請求項2~5のいずれか一項に記載のサンドイッチ型アッセイ。
- 固相化抗体および標識化レクチンを用いて、検出対象物質としての糖タンパク質を定量するためのサンドイッチ型アッセイを実施するためのキットであって、夾雑物としての糖タンパク質を処理するためのジスルフィド結合を開裂する作用を呈する物質を含むキット。
- さらに、ノダフジレクチン(WFA)、ダイズレクチン(SBA)、キカラスウリレクチン-II(TJA-II)またはカラスノエンドウレクチン(VVL)を用いた標識化レクチンを含む、請求項7に記載のキット。
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WO2019171967A1 (ja) | 2018-03-07 | 2019-09-12 | コニカミノルタ株式会社 | 検体希釈液、試料の製造方法、試料、およびサンドイッチ法 |
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