Method and kit for measuring n-terminal fragment of human brain natriuretic peptide precursor

Abstract

To provide a method of measuring an N-terminal fragment of a human brain natriuretic peptide precursor, which reduces the effect of a duration from collection of a sample till detection on separation property of a complex, and to provide a measurement kit for implementing the above method.SOLUTION: A method of measuring a human brain natriuretic peptide precursor N-terminal fragment in a sample is provided, the method comprising separating a complex containing the human brain natriuretic peptide precursor N-terminal fragment and a substance binding to the human brain natriuretic peptide precursor N-terminal fragment in the presence of a polyanionic polymer, and measuring the complex, where the concentration of the polyanionic polymer in a separation medium is 5.1% (w / v) or greater.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for measuring the N-terminal fragment of human brain natriuretic peptide precursor and a kit for measuring the N-terminal fragment of human brain natriuretic peptide precursor. [Background technology]

[0002] Mobility shift assays are useful methods for detecting and quantifying associations between biomolecules. For example, the presence of bound molecules can be confirmed by a change in the retention time of the molecules in an electrophoretic or chromatographic assay. Binding can be specific, such as antibody-antigen interactions, or nonspecific, such as ionic attraction between a positively charged molecule and a negatively charged polymer.

[0003] Meanwhile, microfluidics technology, which utilizes microscale channel devices, is well known for the analysis of DNA, RNA, proteins, and metabolites. Microfluidics technology has the advantages of reduced reagent volume, high resolution, shorter operation time, and easy solution handling.

[0004] Patent Document 1 describes a method for detecting or identifying a target analyte in a sample, including the steps of: (i) contacting a sample containing the analyte with a conjugate of an affinity molecule having affinity for the analyte and a charged carrier molecule in the presence of a polyanionic polymer to form a complex of the analyte and the conjugate; (ii) concentrating the complex in the presence of the polyanionic polymer by using a concentration channel of a microfluidic device including a concentration channel; (iii) separating the complex from the unbound conjugate in the presence of the polyanionic polymer by using a separation channel of a microfluidic device including a separation channel; and (iv) detecting the complex to identify the presence of the analyte or to measure the amount of the analyte in the sample. Patent Document 1 states that the concentration of the charged polymer in the separation channel is typically about 0.01 to 5% (w / v). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4862093 Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors' investigations have revealed that when measuring N-terminal fragment of human brain natriuretic peptide precursor (hereinafter also referred to as NT-proBNP) in stored blood samples, a single peak was detected, but when NT-proBNP was measured in fresh blood samples, a bimodal peak was detected. When the peak becomes bimodal, a lower value (deviation) of the measured value occurs, increasing the possibility of a false negative, and therefore improvement is desired. Patent Document 1 does not describe measuring NT-proBNP, nor does it describe any problems specific to NT-proBNP when measuring fresh blood.

[0007] An object of the present invention is to provide a method for measuring the N-terminal fragment of human brain natriuretic peptide precursor, which reduces the effect of the time from sample collection to detection on the separation characteristics of the complex. Another object of the present invention is to provide a measurement kit for carrying out the above measurement method. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above problems, the present inventors have found that when measuring the N-terminal fragment of human brain natriuretic peptide precursor in a sample, the detection peak in fresh blood measurement can be reduced to a single peak by adding an anionic polymer to the separation medium at a concentration of 5.1% (w / v) or more. The present invention was completed based on this finding.

[0009] According to the present invention, the following inventions are provided. <1> isolating a complex comprising the N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor in the presence of a polyanionic polymer; and Measuring the complex A method for measuring an N-terminal fragment of a human brain natriuretic peptide precursor in a sample, comprising: <2> The sample is taken within 24 hours. <1> The method described below. <3> The sample is a blood sample. <1> or <2> The method described below. <4> The polyanionic polymer is one or more selected from the group consisting of heparin and its salts, dextran sulfate and its salts, and chondroitin sulfate and its salts. <1> from <3> 1. The method according to claim 1 , <5> The heparin salt is one or more selected from the group consisting of lithium heparin, heparin sulfate, sodium heparin, and potassium heparin. <4> The method described below. <6> A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; a second binding substance that binds to the N-terminal fragment of the human brain natriuretic peptide precursor, the second binding substance being labeled with a detectable marker; It is a complex of <1> from <5> 1. The method according to claim 1 , <7> A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; a binding substance that binds to a complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a detectable marker; It is a complex of <1> from <5> 1. The method according to claim 1 , <8> A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a detectable marker; a binding substance that binds to the complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a charged carrier molecule; It is a complex of <1> from <5> 1. The method according to claim 1 , <9> (1) a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, and (2) A separation medium containing 5.1% (w / v) or more of a polyanionic polymer; A kit for measuring the N-terminal fragment of a human brain natriuretic peptide precursor, comprising: <10> A substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; and a second binding substance that binds to the N-terminal fragment of the human brain natriuretic peptide precursor, the second binding substance being labeled with a detectable marker; It is a combination of <9> The kit according to claim 1. <11> the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a charged carrier molecule; The present invention further includes a binding substance that binds to a complex of an N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a detectable marker. <9> The kit according to claim 1. <12> the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a detectable marker; The present invention further includes a binding substance that binds to the complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a charged carrier molecule. <9> The kit according to claim 1. [Effects of the Invention]

[0010] According to the present invention, it is possible to reduce the effect of the time from sample collection to detection on the separation characteristics of the complex. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a schematic diagram of the chip. Waste is used as a waste reservoir when R2, R3, R4, C1, and the immune reaction solution are introduced into the analytical flow channel. FD contains a fluorescent substance and flows through a focusing flow channel arranged along the side of the analytical flow channel in the detection section (photometric section), allowing the photometric section of the device to recognize the position of the analytical flow channel. [Figure 2] FIG. 2 is a schematic diagram of the immune response. [Figure 3] FIG. 3 is a schematic diagram of the immune response. [Figure 4] FIG. 4 is a schematic diagram of the immune response. [Figure 5]FIG. 5 shows a mobility shift chart of NT-proBNP in fresh blood when 3% (w / v) lithium heparin was added to the separation gel. [Figure 6] FIG. 6 shows a mobility shift chart of NT-proBNP in fresh blood when 4.5% (w / v) lithium heparin was added to the separation gel. [Figure 7] FIG. 7 shows a mobility shift chart of NT-proBNP in fresh blood when 5% (w / v) lithium heparin was added to the separation gel. [Figure 8] FIG. 8 shows a mobility shift chart of NT-proBNP in fresh blood when 5.2% (w / v) lithium heparin was added to the separation gel. [Figure 9] FIG. 9 shows a mobility shift chart of NT-proBNP in fresh blood when 5.5% (w / v) lithium heparin was added to the separation gel. [Figure 10] FIG. 10 shows a mobility shift chart of NT-proBNP in stored blood when 4% (w / v) lithium heparin was added to the separation gel. [Figure 11] FIG. 11 shows a mobility shift chart of NT-proBNP in stored blood when 5.5% (w / v) lithium heparin was added to the separation gel. [Figure 12] FIG. 12 shows a mobility shift chart of NT-proBNP in fresh blood when 4% (w / v) dextran sulfate sodium was added to the separation gel. [Figure 13] FIG. 13 shows a mobility shift chart of NT-proBNP in fresh blood when 5.5% (w / v) dextran sulfate sodium was added to the separation gel. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0013] The present invention relates to a method for measuring the N-terminal fragment of human brain natriuretic peptide precursor in a sample, which comprises separating a complex containing the N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor in the presence of a polyanionic polymer, and measuring the complex, wherein the polyanionic polymer is present in the separation medium at a concentration of 5.1% or more (w / v). In the present invention, measurement may be either qualitative or quantitative.

[0014] <Mobility shift assay> The measurement method of the present invention can be applied to, for example, a mobility shift assay. A mobility shift assay is performed for the purpose of separating and analyzing an analyte and a substance having affinity for the analyte (e.g., an affinity molecule, which may be labeled; hereinafter, also referred to as an affinity substance). These are brought into contact with each other to form a complex between the analyte and the affinity substance. After that, a microfluidic device is used to separate the complex from the affinity substance not involved in the complex (e.g., a free or unbound affinity molecule, which may be labeled) based on the difference in migration speed between the two, and the separated complex is analyzed. That is, in a mobility shift assay, for example, a microfluidic device can be used to detect the difference in migration speed between an affinity molecule and an affinity molecule / analyte complex, particularly between a charged carrier molecule / affinity molecule conjugate bound to the analyte and a charged carrier molecule / affinity molecule conjugate not bound to the analyte. As used herein, the term "affinity substance" refers to an affinity molecule or a labeled affinity molecule. As used herein, the term "conjugate" refers to a conjugate of a charged carrier molecule and an affinity molecule, or a conjugate of a detection marker and an affinity molecule.

[0015] The mobility shift assay can be exemplified by the following method. A method comprising the steps of contacting a sample containing an analyte with an affinity molecule labeled with a detection marker such as a fluorescent marker, and then with an affinity molecule labeled with a charged carrier molecule (i.e., a conjugate of an affinity molecule and a charged carrier molecule) to form a complex between the analyte, the affinity molecule labeled with a detection marker such as a fluorescent marker, and the affinity molecule labeled with the charged carrier molecule, separating the complex from free affinity molecules not involved in the formation of the complex in a separation channel of a microfluidic device, measuring the amount of the separated complex or detecting the presence of the separated complex, and determining the amount of the analyte in the sample based on the measured amount or identifying the presence of the analyte in the sample based on the detected presence.

[0016] The measurement method of the present invention can be carried out, for example, as follows. Specifically, a sample containing the analyte, the N-terminal fragment of human brain natriuretic peptide precursor, is contacted with at least one affinity molecule to form a complex between the analyte and the affinity molecule. The resulting complex is separated from unbound affinity molecules in the presence of a polyanionic polymer using a separation channel of a microfluidic device containing at least one separation channel having at least one microscale dimension of about 0.1 to 500 μm. The presence of the analyte can then be identified or the amount of the analyte can be measured by measuring the complex.

[0017] <Polyanionic polymer> In the measurement method of the present invention, the polyanionic polymer is present in the separation medium at a concentration of 5.1% (w / v) or more.

[0018] When measuring NT-proBNP in stored blood samples, a single peak was detected, but when measuring NT-proBNP in fresh blood samples, a bimodal peak was detected. When the peak becomes bimodal, the measured value becomes lower (deviation), so this needs to be improved.

[0019] In the measurement method of the present invention, the peak position (electrophoretic mobility) varies depending on the molecular weight and charge. When the molecular weight of NT-proBNP antigen in fresh blood samples and stored blood samples was measured by Western blotting, bands showing the same molecular weight were detected. Furthermore, when measuring fresh blood samples and stored blood samples that had been refrigerated for one day or more, the peak shape changed over time from a bimodal to a single peak. These results suggest that NT-proBNP antigens in fresh blood samples may contain antigens with different charge characteristics. In addition, when detecting NT-proBNP in stored blood samples, only the peak size changed due to serum dilution, which is unlikely to be due to interference with the assay by serum components.

[0020] Heparin and its salts are polyanionic polymers with a strong negative charge, and it is thought that by prescribing them at high concentrations, they affect the charge of the NT-proBNP antigen in fresh blood samples, thereby reducing the effect of the time from sample collection to detection on electrophoretic mobility. Other polyanionic polymers such as dextran sulfate have the same effect, and polyanionic polymers with larger molecular weights, i.e., stronger negative charges, are more effective. Therefore, it is thought that the polyanionic polymers suppress the differences in the characteristics of the NT-proBNP antigen that can be separated in the measurement method of the present invention, resulting in a single peak.

[0021] Examples of polyanionic polymers include heparin, heparin salts (e.g., lithium heparin, heparin sulfate, sodium heparin, potassium heparin, etc.), chondroitin sulfate, chondroitin sulfate salts (e.g., sodium chondroitin sulfate, potassium chondroitin sulfate), dextran sulfate, dextran sulfate salts (e.g., sodium dextran sulfate, potassium dextran sulfate), polysaccharides such as polytungstic acid, phosphotungstic acid, hyaluronic acid, dermatan sulfate, and polyanetholesulfonic acid; polynucleotides such as DNA (e.g., plasmid DNA, calf thymus DNA, salmon sperm DNA, DNA bound to cellulose, synthetic DNA, etc.) and RNA; polypeptides such as polyamino acids (e.g., polyaspartic acid, polyglutamic acid, etc.) and synthetic polypeptides; and synthetic polymer compounds such as poly(deoxyinosinic-deoxycytidylic) acid (poly-dIdC), polyvinyl sulfate, and polyacrylate.

[0022] The polyanionic polymer is preferably one or more selected from the group consisting of heparin and its salts, dextran sulfate and its salts, and chondroitin sulfate and its salts, more preferably heparin and its salts, dextran sulfate and its salts, even more preferably heparin and its salts, and particularly preferably heparin. The heparin salt is preferably one or more selected from the group consisting of lithium heparin, heparin sulfate, sodium heparin, and potassium heparin. The chondroitin sulfate is preferably one or more selected from the group consisting of sodium chondroitin sulfate and potassium chondroitin sulfate. The dextran sulfate is preferably one or more selected from the group consisting of sodium dextran sulfate and potassium dextran sulfate.

[0023] In the present invention, the above polyanionic polymers can be used alone or in combination.

[0024] To separate an analyte / affinity substance complex from a free affinity substance (e.g., a free affinity molecule or a labeled affinity molecule) that is not involved in the formation of the complex in the presence of a polyanionic polymer, the separation can be performed in the presence of the polyanionic polymer. For example, the polyanionic polymer is preferably present in the separation channel of a microfluidic device containing at least one separation channel. Specifically, the polyanionic polymer is preferably added to the separation medium filled in the separation channel. The presence of the polyanionic polymer in the separation medium can reduce carryover of interfering sample components during sample analysis. Alternatively, the polyanionic polymer may be present in a solution containing the analyte and the analyte / affinity substance complex (e.g., water, a buffer used in hybridization assays, immunoassays, etc., such as Tris-buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, SSC buffer, TBE buffer, or TAE buffer), and then the resulting solution containing the polyanionic polymer, analyte, and analyte / affinity substance complex is added to the separation channel. Furthermore, the polyanionic polymer may be present in a solution used to add a solution containing the analyte and the analyte / affinity substance complex to a microfluidic device, and such solutions include, for example, eluents and running buffers used in separation (e.g., water, buffers used in hybridization assays, immunoassays, etc., such as tris-buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, SSC buffer, TBE buffer, TAE buffer, etc.).

[0025] In the above-mentioned method, the polyanionic polymer can be present in a solution containing an analyte and an analyte / affinity substance complex by the following methods: (i) adding a polyanionic polymer to a sample containing an analyte or a solution containing the sample, and contacting the resulting solution containing the analyte and the polyanionic polymer with an affinity substance; (ii) adding a polyanionic polymer to a solution containing an affinity substance, and contacting the resulting solution containing the affinity substance and the polyanionic polymer with a sample containing an analyte or a solution containing the sample; (iii) adding a sample containing an analyte or a solution containing the sample and the affinity substance to a solution containing the polyanionic polymer; or (iv) contacting a sample containing an analyte or a solution containing the sample with an affinity substance, and mixing the resulting solution containing the analyte and the analyte / affinity substance complex with a solution containing the polyanionic polymer. In the above-mentioned method, the polyanionic polymer can be added as a solution or a dry powder.

[0026] The polyanionic polymer is present in the separation medium at a concentration of 5.1% (w / v) or more, typically at a concentration of 5.1% (w / v) to 20% (w / v), preferably at a concentration of 5.2% (w / v) to 20% (w / v), more preferably at a concentration of 5.3% (w / v) to 20% (w / v), even more preferably at a concentration of 5.4% (w / v) to 10% (w / v), and particularly preferably at a concentration of 5.5% (w / v) to 10% (w / v). When the polyanionic polymer is heparin or a salt thereof, the heparin or a salt thereof is present in the separation medium at a concentration of preferably 5.2% (w / v) to 20% (w / v), more preferably 5.3% (w / v) to 10% (w / v), even more preferably 5.4% (w / v) to 10% (w / v), and particularly preferably 5.5% (w / v) to 10% (w / v). When the polyanionic polymer is dextran sulfate or a salt thereof, the dextran sulfate or a salt thereof is present in the separation medium at a concentration of preferably 5.3% (w / v) to 20% (w / v), more preferably 5.4% (w / v) to 10% (w / v), and even more preferably 5.5% (w / v) to 10% (w / v).

[0027] <Analyte> The analyte in the present invention is N-terminal fragment of human brain natriuretic peptide precursor (NT-proBNP: N-Terminal pro Brain Natriuretic Peptide). NT-proBNP is an N-terminal peptide fragment (fragment) generated when proBNP, the precursor of BNP, is cleaved by a proteolytic enzyme, and is a type of hormone mainly secreted by the heart. As cardiac function declines and the burden on the heart increases (depending on the severity), more NT-proBNP is secreted into the blood together with BNP. BNP has strong natriuretic, smooth muscle relaxing, and vasodilatory effects, but is degraded in the blood within a short time and is unstable. On the other hand, inactive NT-proBNP is relatively stable in the blood and, like BNP, is used as a marker reflecting cardiac function, mainly for the treatment of heart failure.

[0028] <Sample> The sample can be any material potentially containing the analyte. Examples of samples include blood samples (e.g., serum, plasma, whole blood, etc.), tissue extracts, cell extracts, nuclear extracts, culture media, microbial culture extracts, members of molecular libraries, clinical samples, saliva specimens, stool specimens, cerebrospinal fluid, urine samples, urethral-genital swabs, throat swabs, environmental samples, and / or the like. Of the above, blood samples are preferred. If the analyte is not free in solution, it can be released into solution by trituration, lysis, extraction, filtration, centrifugation, and other suitable techniques known in the art. Preferably, the sample is within 24 hours of collection.

[0029] <Affinity molecules> The affinity molecule of the present invention is a molecule that binds to the N-terminal fragment of human brain natriuretic peptide precursor, and can be selected from the group consisting of, for example, an antibody, an antibody Fab, F(ab')2, or Fab' fragment, an antibody variable region, and an aptamer.

[0030] In the present invention, the above affinity molecules may be used alone or in appropriate combination. When two or more affinity molecules are used, each affinity molecule binds to the analyte at a position on the analyte that is different from all other affinity molecules.

[0031] In the measurement method of the present invention, the concentration of the affinity molecule can be varied depending on the detection limit of the analyte. It is generally desirable to maintain the affinity molecule at a concentration higher than the concentration at which the affinity molecule can completely bind to the analyte at a concentration corresponding to the specified detection limit in the reaction mixture. The concentration in the reaction mixture is preferably maintained at least twice the detection limit, more preferably at least five times. When two or more affinity molecules are used, the concentration of each affinity molecule is selected from the above concentration range.

[0032] In the present specification, the affinity molecule may be labeled. For example, the affinity molecule may be labeled with a detectable marker. When the affinity molecule is labeled with a detection marker, examples of the detection marker include those conventionally used in the field of the present invention, such as enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), hybridization assay, etc. Examples of such substances include enzymes such as alkaline phosphatase (ALP), β-galactosidase (β-Gal), peroxidase (POD), microperoxidase, glucose oxidase (GOD), glucose-6-phosphate dehydrogenase (G6PDH), malate dehydrogenase, and luciferase; dyes such as Coomassie Brilliant Blue R250 and methyl orange; 99m Tc, 131 I,125 I, 14 C. 3 H, 32 P, 35 radioactive tracers such as S; fluorescein, rhodamine, dansyl, fluorescamine, coumarin, naphthylamine, or derivatives thereof, cyanine-type fluorescent dyes or oxazine-type fluorescent dyes [e.g., Cy series dyes (Cy3, Cy5, and Cy5.5, etc.: Amersham Biosciences Corp.), Alexa Fluor series dyes (Alexa Fluor 647, 488, 594, etc.: Molecular Probes, Inc.), DY series dyes (DY-630, 633, 635, 640, 650, 655, 656, 780, 550, etc.: MoBiTec GmbH, Goettingen Germany), EVOblue® 30 (MoBiTec GmbH, Goettingen Germany), rare earth fluorescent dyes [e.g., combinations of rare earth metals such as samarium (Sm), europium (Eu), terbium (Tb), or dysprosium (Dy) with chelating compounds such as 4,4'-bis(1”,1”,1”,2”,2”,3”,3”-heptafluoro-4”,6”-hexadion-6”-yl)chlorosulfo-o-terphenyl (BHHCT), 4,7-bis(chlorosulfonyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), and β-naphthyltrifluoroacetic acid (β-NTA)]; nucleic acid-binding fluorescent dyes; fluorescent proteins; luciferin, isoluminol, luminol, bis(2,4,6- trifluoro-phenyl) oxalate and other luminescent dyes, luminescent proteins or particles; ultraviolet absorbing substances such as phenol, naphthol, anthracene, or derivatives thereof; substances having properties as spin labeling agents [for example, compounds having an oxyl group such as 4-amino-2,2,6,6-tetramethyl-piperidine-1-oxyl, 3-amino-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl, 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy], phosphorescent dyes, chemiluminescent compounds, redox mediators, electrogenic compounds, gold colloid particles, silver particles, and the like.

[0033] Nucleic acid-binding fluorescent dyes emit strong fluorescence upon binding to nucleic acid chains. Examples of such nucleic acid-binding fluorescent dyes include so-called intercalator dyes that are incorporated between the bases of nucleic acid chains [for example, acridine dyes such as acridine orange, ethidium bromide, ethidium homodimer 1 (EthD-1), ethidium homodimer 2 (EthD-2), ethidium bromide monoazide (EMA), dihydroethidium, and other ethidium compounds, iodide compounds such as propidium iodide and hexidium iodide, 7-amino-actinomycin D (7-AAD), POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, etc. (all from Molecular cyanine dimer dyes such as PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3, LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, etc. (all trade names of Molecular Probes Inc., Eugene, OR); cyanine monomer dyes such as SYBR Gold, SYBR Green I and SYBR Green II, SYTOX Green, SYTOX Blue, SYTOX Orange, etc. (all trade names of Molecular Probes); dyes that bind to the minor groove of the DNA double helix [e.g., 4',6-diamino-2-phenylindole (DAPI, trade name of Molecular Probes), pentahydrate (bis-benzimide) (Hoechst 33258, trade name of Molecular Probes), trihydrochloride (Hoechst 33342, trade name of Molecular Probes), and bisbenzimide dyes (Hoechst 34580: trade name of Molecular Probes), etc.); and those that specifically bind to the adenine-thymine sequence (AT) [for example, acridine dyes such as 9-amino-6-chloro-2-methoxyacridine (ACMA) and bis-(6-chloro-2-methoxy-9-acridinyl)spermine (acridine homodimer); for example, hydroxystilbamidine, etc.].

[0034] Labeling of affinity molecules with detection markers can be carried out by any of the conventional methods commonly used in the art, such as known labeling methods commonly used in EIA, RIA, FIA, hybridization assays, etc., or conventional methods utilizing the reaction of avidin (or streptavidin) with biotin.

[0035] <Contacting the sample with the affinity molecule, which may be labeled> To contact an analyte-containing sample with an affinity molecule, which may be labeled, a contacting step can be performed to form a complex between the analyte and the affinity molecule. The method for this is not particularly limited. For example, the analyte-containing sample and the affinity molecule can be dissolved, dispersed, or suspended in water or a buffer such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, SSC buffer, TBE buffer, or TAE buffer to obtain liquid materials, and then these liquid materials can be mixed and contacted with each other. Alternatively, the sample and the affinity molecule can be dissolved, dispersed, or suspended simultaneously. If the analyte-containing sample is liquid, the affinity molecule can be mixed directly with the sample. If the analyte-containing sample is liquid as described above, it does not need to be dissolved, dispersed, or suspended in, for example, water or a buffer. In the above method, the buffer concentration can be selected from the range commonly used in the field of the present invention.

[0036] The pH and temperature for forming a complex between the analyte and the affinity molecule can be selected according to conventional methods commonly used in the field of the present invention (e.g., known EIA, RIA, FIA, or hybridization assays) as long as they do not interfere with the formation of the complex. The contact can be carried out at a pH of typically about 2 to 10, preferably 5 to 9, and at a temperature of typically 0 to 90°C, preferably 5 to 40°C. The reaction time required for complex formation can range from a few seconds to a few hours.

[0037] <Affinity substances> The affinity substance of the present invention is a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor (e.g., an optionally labeled molecule that binds to the N-terminal fragment of human brain natriuretic peptide precursor). For example, it can be selected from the group consisting of an antibody, an antibody Fab, F(ab')2, or Fab' fragment, an antibody variable region, and an aptamer, all of which may be labeled.

[0038] <Form of a complex containing the N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor> In a first aspect of the present invention, A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; The complex is a second binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a detectable marker.

[0039] In a second aspect of the present invention, A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; a binding substance that binds to a complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a detectable marker; It is a complex of

[0040] In a third aspect of the present invention, A complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, Human brain natriuretic peptide precursor N-terminal fragment, a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a detectable marker; a binding substance that binds to the complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a charged carrier molecule; It is a complex of

[0041] <Charged carrier molecules> To improve or increase the separation efficiency between the analyte / affinity molecule complex and the free affinity molecule and to analyze the analyte with sufficient accuracy, an affinity molecule bound to a charged carrier molecule (e.g., a conjugate of an affinity molecule and a charged carrier molecule) can be used. A sample containing the analyte can be contacted with the affinity molecule / charged carrier molecule conjugate to form a complex between the analyte and the conjugate, and the resulting complex can be separated from the unbound conjugate in the presence of a polyanionic polymer using a separation channel of a microfluidic device comprising at least one separation channel. The presence of the analyte can then be identified or the amount of the analyte in the sample can be measured by detecting the complex.

[0042] When a conjugate of an affinity molecule (e.g., an antibody) and a charged carrier molecule is used in an assay format, the charged carrier molecule can carry the affinity molecule and the bound analyte while providing high resolution and a detectable signal in an assay that separates and analyzes molecules according to their size. The charged carrier molecule of the conjugate, for example, provides high resolution and sensitivity, while the affinity molecule, for example, provides specificity for the mobility shift assay of the present invention. The charged carrier molecule has a high charge-to-mass ratio and minimal variation in molecular structure, allowing for high resolution in the separation medium. The use of charged carrier molecules offers many advantages in mobility shift assays.

[0043] Charged carrier molecules include molecules that bind to analytes via affinity molecules to form complexes of the analyte, affinity molecules, and charged carrier molecules, thereby causing a change in the separation (e.g., migration) properties of the analytes.

[0044] Charged carrier molecules can have a net positive or negative charge, with carrier molecules having a net negative charge being preferred.

[0045] The charged carrier molecule may be selected from inorganic metal oxides such as silica and alumina; metals such as gold, titanium, iron, and nickel; inorganic metal oxides having functional groups introduced therein by a silane coupling method or the like; organisms such as various microorganisms and eukaryotic cells; polysaccharides such as agarose, cellulose, and insoluble dextran; synthetic polymer compounds such as polystyrene latex, styrene-butadiene copolymer, styrene-methacrylate copolymer, acrolein-ethylene glycol dimethacrylate copolymer, styrene-styrene sulfonate latex, polyacrylamide, polyglycidyl methacrylate, polyacrolein-coated particles, crosslinked polyacrylonitrile, acrylic acid or acrylic acid ester copolymer, acrylonitrile-butadiene, vinyl chloride-acrylic acid ester, and polyvinyl acetate-acrylate; and biomolecules such as red blood cells, sugars, nucleic acid chains (e.g., DNA, RNA), polypeptides or derivatives thereof (e.g., sulfonated polypeptides), proteins, and lipids. As a charged carrier molecule having a negative net charge, a nucleic acid chain (e.g., DNA, RNA) or a sulfonated polypeptide is preferred, with DNA or RNA being more preferred. In the present invention, an anionic molecule comprising a nucleic acid chain (e.g., DNA, RNA) or a sulfonated polypeptide is most preferred. DNA is particularly preferred because the molecule is stable and there is a wealth of experience in the field in terms of synthesis and binding chemistry.

[0046] Nucleic acid chains are composed of nucleotide residues containing a purine or pyrimidine base, a pentose sugar moiety, and phosphate as a basic unit. Each nucleotide is linked to the 3' and 5' carbons of the sugar moiety via phosphate to form a polynucleotide chain (e.g., RNA, where the sugar moiety is ribose, and / or DNA, where the sugar moiety is deoxyribose). Nucleic acid chains may be single-stranded, double-stranded, or multi-stranded. The nucleic acid chains used in the present invention can be produced by conventional methods, such as chemical synthesis, extraction and purification of cells derived from microorganisms, insects, animals, plants, etc., incubating the cells with an appropriate vector gene (e.g., plasmid, phage, cosmid), and extracting and purifying the propagated vector, or by gene amplification techniques such as PCR (e.g., Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press). The resulting nucleic acid chains are chemically degraded or disrupted with nucleases such as restriction enzymes, and then optionally purified to form nucleic acid chains of the desired length. In the present invention, the above-mentioned charged carrier molecules may be used alone or in appropriate combination.

[0047] For example, any type of modified nucleotide known to enhance the stability of nucleotides against various nuclease activities can be used to create the charged carrier molecule. For example, phosphorothioate analogs of nucleotides, nucleotides containing methylene groups instead of oxygen in the ribose ring, or nucleotides in which the 2'-sugar deoxy substituent has been replaced with 2'-fluoro, 2'-O-methyl, 2-O-alkoxyl, and 2'-O-allyl modifications can be used. Such modifications are described, for example, in Susan M. Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443.

[0048] The size of the charged carrier molecule can be, for example, typically about 0.6 kDa to 70,000 kDa, preferably about 3 kDa to 7,000 kDa, and more preferably about 6 kDa to about 400 kDa. The size of the carrier molecule can be optimized to provide useful sensitivity and resolution, depending on, for example, the type of separation medium, the separation cutoff of the separation medium, the size of the analyte, the size of the affinity molecule, etc. In particular, when a nucleic acid chain is used as the charged carrier molecule, the length of the nucleic acid chain can be typically about 1 bp to 100,000 bp, preferably 5 bp to 10,000 bp, more preferably 10 bp to 1,000 bp, and most preferably 10 bp to 500 bp, as long as the object of the present invention can be achieved. The nucleic acid chain used in the present invention can be appropriately modified with an appropriate group within the scope of achieving the object of the present invention.

[0049] The binding of a charged carrier molecule to an affinity molecule can be carried out in the same manner as the labeling of an analyte or affinity molecule with the above-mentioned detection marker. For example, the binding of a charged carrier molecule to an affinity molecule can be carried out directly or via a linker [e.g., 4-(p-maleimidophenyl)butyric acid sulfosuccinimidyl (sulfo-SMPB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimidyl (sulfo-SMCC), N-(ε-maleimido-caproyloxy)succinimide (EMCS), N-hydroxysuccinimide ester (NHS), etc.] using the respective functional groups of the affinity molecule and the charged carrier molecule. Binding can be carried out by a known labeling method used in known EIA, RIA, FIA or hybridization assays [e.g., Medical Chemistry Experimental Lectures, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody Methods, by Kawao Akira, 1st Edition, Soft Sciences, 1983; Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc., EP1088592A2, EP1061370A2, etc.] or a conventional method commonly used in the field, such as a conventional method utilizing the reaction of avidin (or streptavidin) with biotin.

[0050] After the reactive functional group is introduced into the charged carrier molecule in advance, the affinity molecule can be bound to the charged carrier molecule containing the reactive functional group by the above-mentioned binding method. In particular, when a nucleic acid chain is used as the charged carrier molecule, the reactive functional group can be introduced into the nucleic acid chain by a method known per se. For example, a method of introducing a reactive functional group into the 5' triphosphate group located at the end of a nucleic acid by forming a phosphoramidite bond using a compound having a reactive functional group (e.g., a compound having an amino group such as N-trifluoroacetylaminoalkylamine, a compound having a thiol group such as cystamine, a compound having biotin such as N-biotinylaminoalkylamine, a compound having a maleimide group such as maleimidealkylamine, etc.) in the presence of a condensing agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydrochloride (WSC), etc.) [Nucleic Acid Res. (1988) 16, 3671, Chu, BC et al.]; a method in which a reactive functional group is introduced into the 3' hydroxyl group located at the end of a nucleic acid by forming an ester bond using a compound having a reactive functional group (e.g., a compound having an amino group such as N-trifluoroacetylaminoalkylcarboxylic acid, a compound having biotin such as N-biotinylaminoalkyl-carboxylic acid, or a compound having a maleimide group such as maleimidealkylcarboxylic acid) in the presence of a condensing agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydrochloride (WSC), etc.), or by directly reacting the activated ester [Nucleic Acid Res. (1986) 14, 6115, Jabloski et al.]; a method in which an amino-reactive linker is introduced into the end (sticky end, sticky end) of a fragment cleaved with a restriction enzyme, where an amino-containing base (adenine, cytosine) protrudes as a single strand (e.g., a protruding end) [Chemistry of Proteins and Crosslinking, Shan S.Wong, (1991) CRC Press]; a method in which a nucleotide monomer having a reactive functional group is incorporated into a fragment cleaved with a restriction enzyme that forms a single-stranded overhanging end using a blunting enzyme (T4 DNA polymerase, DNA blunting enzyme, etc.) (Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc.); a method in which a reactive functional group is introduced into the 5' end of an oligonucleotide having a sequence complementary to the single-stranded portion of a fragment cleaved with a restriction enzyme that forms a single-stranded overhanging end using hybridization, and then hybridized to the single-stranded overhanging end of the fragment cleaved with the restriction enzyme (Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc.); a method in which a PCR primer with a reactive functional group introduced into its 5' end is used in PCR to obtain a nucleic acid chain with a reactive functional group introduced into its 5' end as a PCR product (Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc.). Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, etc.). In this way, a reactive functional group can be introduced into the end of a nucleic acid. When a single-stranded nucleic acid is used, a nucleic acid chain into which a reactive functional group has been introduced can also be prepared by hybridizing an oligonucleotide having a sequence complementary to the 5'-end of the nucleic acid chain and a reactive functional group introduced into the 5'-end to the single-stranded nucleic acid (Molecular Cloning, A Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T.(Maniatis, Cold Spring Harbor Laboratory Press, etc.) Examples of such reactive functional groups include hydroxyl groups, halogenated alkyl groups, isothiocyanate groups, avidin groups, biotin groups, carboxyl groups, ketone groups, maleimide groups, active ester groups, sulfonyl halide groups, carboxylic acid halide groups, amino groups, sulfonic acid groups, pyridyldithio groups, and aldehyde groups.

[0051] If the number of nucleic acid strands bound to affinity molecules is non-uniform, the number of nucleic acid strands present in the formed complex will also be non-uniform, resulting in non-specific separation of the complex. Therefore, it is preferable to make the number of nucleic acid strands bound to affinity molecules uniform. For the same reason, it is appropriate to make the number of affinity molecules bound to one nucleic acid strand one molecule.

[0052] In the above-mentioned binding method, when the nucleic acid chain has functional groups at both ends to which affinity molecules can be bound, the nucleic acid chain can be enzymatically or chemically degraded in advance to introduce a reactive functional group at one end, and then bound to the affinity molecule. Alternatively, the nucleic acid chain can be bound to the affinity molecule to generate an intermediate with affinity molecules bound to both ends, and the nucleic acid chain bound to the intermediate can be enzymatically or chemically degraded to obtain a product with an affinity molecule bound to one end of the nucleic acid.

[0053] Affinity molecules can be linked to charged carrier molecules to form conjugates using linking chemistry. Linking chemistry can utilize reactions with amino groups, thiols, carboxyl groups, imidazole groups, succinimide groups, etc. For example, DNA can be crosslinked to affinity molecules by mixing a DNA carrier containing nucleotides modified to have amine groups with affinity molecules and NHS linkers at both ends in solution. Other techniques for binding, associating, or interacting affinity molecules with carrier molecules are disclosed in detail in WO02 / 082083, "Electrophoresis Methods."

[0054] The conjugate of an affinity molecule and a charged carrier molecule is preferably a conjugate of at least one affinity molecule selected from the group consisting of an antibody, Fab, F(ab')2 or Fab' fragment, and an antibody variable region, with at least one charged carrier molecule selected from the group consisting of a nucleic acid chain (e.g., DNA, RNA) and a sulfonated polypeptide. Most preferred is a conjugate of at least one affinity molecule selected from an antibody, Fab or Fab' fragment, with a nucleic acid chain, particularly DNA, as the charged carrier molecule.

[0055] In the present invention, the step of contacting a sample containing an analyte with a conjugate of a charged carrier molecule and an affinity molecule can be carried out in the same manner as in the contact of a sample containing an analyte with an affinity molecule as described above. The reaction conditions (e.g., pH, temperature, reaction time, etc.) are the same as those for contacting a sample with an affinity molecule.

[0056] <Concentration> In the present invention, a microfluidic device may be used to concentrate an analyte in a sample for the purpose of applying a high concentration of the analyte to a mobility shift assay. Various concentration methods, such as online sample concentration techniques, can be used with the microfluidic device to concentrate the analyte in the sample. For online sample concentration or sample stacking, electrophoretic concentration methods (e.g., FASS, FASI, ITP, IF, etc.) that utilize differences in electrophoretic mobility of sample components in a capillary can be used.

[0057] For example, the following concentration method can be used. (i) FASS (Field Amplification Sample Stacking), which utilizes the difference in electrical conductivity between the concentrated and separated domains; (ii) FASI (Field Amplification Sample Injection), which inserts a tiny water plug between the enrichment and separation domains in FASS; (iii) ITP (Isotachophoresis), which utilizes the difference in ion mobility in a domain sandwiched between a leading solution and a trailing solution; and (iv) IF (Isoelectric Focusing) utilizes the difference in isoelectric points between substances.

[0058] In particular, it is preferable to use ITP or FASS based on the following principle. ITP is a method based on the principle of concentrating an analyte by placing it between two ions: a leading ion with a faster electrophoretic mobility than the analyte and a trailing ion with a slower electrophoretic mobility than the analyte. FASS is a method based on the principle that the conductivity of the separation domain is made higher than that of the concentration domain, and when a substance in the concentration domain reaches the boundary between the separation domain and the concentration domain, the electrophoretic mobility of the analyte decreases, and the substance is then concentrated.

[0059] In the present invention, all buffers, fillers, various reagents (e.g., treatment solutions), etc. commonly used in the above-mentioned concentration methods can be used. The concentrations of these materials can be selected according to known concentration methods, as the case may be. Concentration conditions (e.g., pH, temperature, applied voltage, time, etc.) can be appropriately selected according to known methods.

[0060] <Separation operation> The resulting complex between the analyte and the affinity substance is separated from the free affinity substance that is not involved in the formation of the complex. A separation method capable of separating the complex and the free affinity substance based on the difference in migration speed can be applied. For example, the B / F separation method used in this field can be used. Specific examples include electrical separation methods that utilize electricity, such as electrophoresis (e.g., isoelectric focusing, SDS-polyacrylamide electrophoresis, agarose gel electrophoresis, acrylamide electrophoresis) and dielectrophoresis. In particular, electrophoresis such as isoelectric focusing, SDS-polyacrylamide electrophoresis, agarose gel electrophoresis, and acrylamide electrophoresis, or electrical separation methods such as dielectrophoresis, are preferred. More specifically, capillary electrophoresis or dielectrophoresis is preferred because it can be performed with high separation efficiency under efficient cooling conditions and high voltage.

[0061] Particularly when separations are performed using microfluidic devices and systems, the analyte is often present in the sample in very low concentrations and in very small amounts. The amount of analyte is often reduced to at, near, or below the detection threshold of the microfluidic analytical system. Therefore, it may be preferable to use one or more online sample concentration or sample stacking operations (as described above and below) in the microfluidic device to increase the sensitivity of analyte detection. One particularly useful example of an online sample concentration technique that can be used in practicing the methods of the present invention is isotachophoresis (ITP), as described in Everaerts, F.M., Geurts, M., Mikkers, F.E.P., Verheggen, TPEM J. Chromatagr. 1976, 119, 129-155; Mikkers, F.E.P., Everaerts, F.M., Peek, J.A.F.J. Chromatogr. 1979, 168, 293-315; and Mikkers, F.E.P., Everaerts, F.M., Peek, J.A.F.J. Chromatogr. 1979, 168, 317-332, the entire disclosures of which are incorporated herein by reference. In ITP, the sample is inserted between leading and terminal electrolytes, typically of sufficiently high and sufficiently low electrophoretic mobility, respectively. However, other combinations of leading and terminal electrolytes can be placed before or after the sample plug. See, for example, Hirokawa, T., Okamoto, H., Ikuta, N., and Gas, B., "Optimization of Operational Modes for Transient Isotachophoresis Preconcentration-CZE," Analytical Sciences 2001, Vol. 17, Supplement 1185. According to well-known moving boundary principles, a steady-state configuration is eventually reached, with all sample zones migrating at the same rate. The sample concentration in each migrating zone self-adjusts relative to the concentration of the leading electrolyte.A variety of other sample concentration techniques for use in capillary electrophoresis other than ITP can be used to practice the methods of the present invention, including field-amplified sample stacking (FASS) and solid phase extraction (SPE). For example, FASS in a microfluidic chip using simultaneous multiport pressure and electrokinetic fluid control is described in U.S. patent application Ser. No. 10 / 206,386, entitled "Microfluidic Methods, Devices and Systems for In Situ Material Concentration," the entire disclosure of which is incorporated herein by reference. Additionally, various other recently developed sample concentration techniques can be used to practice the methods of the present invention, such as altering the pH of the leading / terminal electrolyte to form a sample stacking region (see, e.g., Weiss, DJ, Saunders, K., Lunte, CE Electrophoresis 2001, 22, 59-65; Britz-McKibbin, P., Bebault, GM, Chen, DDY Anal Chem. 2000, 72, 1729-1735, the entire disclosures of which are incorporated herein by reference) and / or equilibrating the electrophoretic velocity of the analyte against the bulk flow of the solution in the presence of a temperature gradient (see, e.g., Ross, D., Locascio, LE Anal Chem. 2002, 71, 5137-5145, the entire disclosures of which are incorporated herein by reference). The present invention can utilize various reagents, such as buffers, packing materials, and process solutions, all of which are conventionally used in such separation methods. The concentrations of these materials can be selected, if necessary, according to known separation methods. Separation conditions (for example, pH, temperature, applied voltage, time, etc.) can be appropriately selected according to known methods.

[0062] <Microfluidic Devices> In the present invention, separation of analyte-affinity substance complexes from free affinity substances not involved in complex formation can be carried out using a microfluidic system, typically including a microfluidic device and a detector based on the above-described separation method. The method of the present invention is suitable for application in a microfluidic device. A sample can be introduced into a microfluidic device for rapid and accurate mobility shift assays using minimal volumes of reagents and sample. The sample and affinity substance mixture can be introduced into a low-salt buffer, while the buffer in the separation medium has a high salt concentration to provide a "stacking" effect that accumulates assay mixture components at the front of the sample bolus, resulting in higher sensitivity and better resolution. For example, samples can be screened in a high-throughput screening format by aspirating (shipping) samples from a sample library chip or multiwell plate (e.g., standard 96-, 384-, or other larger multiwell plate) into a microfluidic device (e.g., chip) for rapid screening, data acquisition, and data analysis. The microfluidic device can have, for example, one or more separation channels that contain a separation medium and flow into a detection channel region or separate detection channel where the effluent is monitored by a detector. The microfluidic device can include, for example, a detector for detecting separated components in a sample. Such detectors can include, for example, a gel scanner, a fluorescence detector, or a fluorescence polarization detector.

[0063] A microfluidic device typically has a body structure containing and / or housing at least one fluidic component (e.g., a channel, chamber, well, etc.) having at least one cross-sectional dimension of about 0.1 to about 500 μm. These channels and / or chambers often have at least one cross-sectional dimension of about 0.1 μm to 200 μm, sometimes about 0.1 μm to 100 μm, and often about 0.1 μm to 20 μm. Such cross-sectional dimensions include, for example, width, depth, height, diameter, etc. Structures having these dimensions are typically referred to as "microscale." A microfluidic device typically contains at least one, and preferably two or more, channels and / or chambers disposed within a single body structure. Such channels / chambers may be individually isolated or may be fluidly connected. Such fluid connections can be provided by channels, channel intersections, valves, etc. Channel intersections can exist in a number of formats, including cross intersections, "T" intersections, or a number of other structures that place two channels in fluid communication.

[0064] The body structure of a microfluidic device typically consists of a collection of two or more separate components that, when properly combined or assembled, form a microfluidic device containing channels and / or chambers. Microfluidic devices are typically fabricated as a collection of substrate layers. In particular, such preferred devices comprise an upper portion, a lower portion, and an inner portion, with the inner portion substantially defining the channels and chambers of the device. A variety of substrate materials can be utilized for the lower portion. Because the devices are typically microfabricated, substrate materials are selected based on their compatibility with known microfabrication techniques (e.g., photolithography, wet chemical etching, laser ablation, air ablation techniques, injection molding, embossing, and other techniques). Substrate materials are typically also selected based on their compatibility with all conditions to which the microfluidic device may be exposed (e.g., pH, temperature, salt concentration, and electric field limits). Accordingly, in certain preferred aspects, substrate materials typically include materials typically associated with the semiconductor industry, where such microfabrication techniques are routinely utilized (e.g., glass, quartz, silica substrates such as silicon or polysilicon, and other substrate materials such as gallium arsenide). In the case of semiconductor materials, it is often desirable to provide an insulating coating or layer (eg, silicon oxide) over the substrate material, particularly in applications where an electric field is applied to the device or its contents.

[0065] In additional preferred aspects, the substrate material comprises a polymeric material, such as plastics like polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON®), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, polystyrene, polymethylenepentene, polypropylene, polyethylene, polyvinylidene fluoride, ABS (acrylonitrile-butadiene-styrene copolymer), and the like. Such polymeric substrates are readily fabricated using available microfabrication techniques, as described above, or from microfabricated masters using well-known molding techniques, such as injection molding, embossing, or stamping. Such polymeric substrate materials are preferred because they are easy to manufacture, low-cost, easy to dispose of, and generally resistant to even the most extreme reaction conditions. Furthermore, these polymeric materials can include engineered surfaces (e.g., derivatized or coated surfaces) to enhance their utility in microfluidic systems, e.g., to improve fluid flow, as described, for example, in U.S. Pat. No. 5,885,470, the disclosure of which is incorporated herein by reference in its entirety.

[0066] The microfluidic system preferably includes a detector that monitors elution from the separation channel, allowing detection of elution of affinity material / analyte complexes before free affinity material reaches the detector. Microfluidic devices such as the Agilent DNA 500 LabChip® can rapidly analyze multiple samples with high sensitivity and resolution.

[0067] The detector can be configured to detect free affinity material and / or affinity material / analyte complexes as they elute from the separation medium in the separation channel. The affinity material can be detected without modification, or a detection marker can be attached to the affinity material to increase detection sensitivity. The affinity material can include a predefined charged carrier molecule, such as a polymer, that can be detected by its characteristic absorbance properties. For example, DNA as a charged carrier molecule has a strong absorbance at about 260 nm that can be detected by a spectrophotometer as it elute from the separation medium.

[0068] The detector can be positioned, for example, at the outlet end of the separation channel to monitor the elution of a detectable peak. Optionally, the detector can scan the separation medium, such as a polyacrylamide gel, to detect the relative positions of the separated complex and the free affinity substance. The detector can be of any type appropriate for the detectable marker, including an absorbance detector, a fluorescence detector, a fluorescence polarization detector, a spectrophotometer, a phosphorimager, a voltmeter, a scintillation counter, a refractometer, and / or the like. Such detectors can generate digital or analog output signals that can be analyzed to identify and / or quantify the analyte.

[0069] Analysis of the detector output over time can be used, for example, to determine the presence of an analyte and / or to quantify the amount of analyte present in a sample. Peak parameters such as retention time, migration rate, peak height, peak position, and peak ratio can be analyzed to identify the presence of an analyte and / or to quantify the amount of analyte in a sample. Standard analytical techniques, such as the use of reference samples, standards, and regression analysis, can be utilized to analyze the analytical results.

[0070] Various fluid delivery methods can be used in microfluidic devices. For example, fluid flow can be induced through the channels of a device by applying a pressure differential across the desired channel. This can be accomplished by applying positive pressure to one end of the channel or negative pressure to the other. In complex channel networks, the flow rate can be controlled throughout each interconnected channel, for example, by inserting valves or the like within the device structure to stop and start flow in a given channel. Alternatively, a single pressure differential, e.g., applying a vacuum to a single channel port, can also be adjusted to adjust the channel resistance to control the rate, timing, and / or volume of material movement through each channel.

[0071] Alternatively, controlled electrokinetic transport systems may be used. This type of electrokinetic transport is described in detail in U.S. Pat. No. 5,858,195 (Ramsey), the entire disclosure of which is incorporated herein by reference for all purposes. Such electrokinetic material transport and direction systems include those that rely on the electrophoretic mobility of charged species within an electric field applied to the structure. These systems are specifically referred to as electrophoretic material transport systems. Other electrokinetic material direction and transport systems rely on electroosmotic flow of fluids and materials inside channel or chamber structures induced by applying an electric field to these structures. In summary, when a fluid is placed into an etched glass channel or glass microcapillary with charged functional groups (e.g., hydroxyl groups) on its surface, these groups can be ionized. In the case of hydroxyl functional groups, for example, this ionization occurs at neutral pH, resulting in the release of protons from the surface into the fluid interior, leading to a concentration of protons near the fluid / surface interface or the formation of a positively charged sheath around the bulk fluid within the channel. When a voltage gradient is applied along the length of the channel, the proton sheath moves in the direction of the voltage drop, i.e., toward the negative electrode.

[0072] "Controlled electrokinetic material transport and direction" refers to an electrokinetic system, as described above, that utilizes active control of voltages applied to multiple, i.e., three or more, electrodes. In other words, such a controlled electrokinetic system simultaneously controls the voltage gradients applied to at least two intersecting channels. In particular, preferred microfluidic devices and systems described herein include a body structure containing at least two intersecting channels or fluid conduits (e.g., interconnected closed chambers), the channels having at least three non-intersecting ends. An intersection of two channels refers to a point at which two or more channels are in fluid communication with one another, including a "T" intersection, a cross intersection, a multi-channel "wheel" intersection, or any other channel arrangement in which two or more channels are in such fluid communication. A non-intersecting end of a channel is a terminal point at which a channel does not intersect with another channel, such as a "T" intersection. In a preferred aspect, the device includes at least three intersecting channels with at least four non-intersecting ends. In a basic cross-channel configuration, where one horizontal channel crisscrosses one vertical channel, controlled electrokinetic material transport functions to control the flow of material through the intersection by providing constraining flow from the other channel at the intersection. For example, assume it is desired to transport a first material through the horizontal channel across its intersection with the vertical channel, e.g., from left to right. Simple electrokinetic material flow of this material through the intersection can be achieved by applying a voltage gradient along the length of the horizontal channel, i.e., applying a first voltage to the left end of the channel and a second, lower voltage to the right end of the channel, or by allowing the right end to float (no voltage applied). However, due to the inherent diffusive nature of the material being transported in the medium used and convective effects at the intersection, a substantial amount of diffusion occurs at the intersection as a result of this type of material flow through the intersection.

[0073] In controlled electrokinetic material transport, material transported through an intersection is constrained by a low-level flow from a side channel (e.g., the upper or lower channel). This can be achieved, for example, by applying a small voltage gradient along the path of the material flow from the top or bottom end of a vertical channel toward the right end. This results in the material flow being "pinched" at the intersection, preventing the material from diffusing into the vertical channel. The pinched volume of material at the intersection can then be injected into the vertical channel by applying a voltage gradient along the length of the vertical channel, i.e., from the top end to the bottom end. To prevent material from flowing out of the horizontal channel during this injection, the low-level flow is forced back into the side channel, "pulling" the material back from the intersection.

[0074] In addition to the pinch injection scheme, controlled electrokinetic material transport can be easily utilized to create a virtual valve without mechanical or moving parts. Specifically, with regard to the cross-junction mentioned above, the flow of material from one channel segment to another (e.g., from the left arm to the right arm of a horizontal channel) can be efficiently controlled, stopped, and restarted by controlled flow from the vertical channel (e.g., from the lower arm to the upper arm of a vertical channel). Specifically, in the "off" mode, by applying a voltage gradient between the left and upper ends, material is transported from the left arm through the intersection to the upper arm. By applying a similar voltage gradient along this path (from the lower end to the upper end), a constraining flow is introduced from the lower arm to the upper arm. Next, by reversing the applied voltage gradient from left to top to left to right, a metered amount of material is dispensed from the left arm to the right arm of the horizontal channel. The amount of material dispensed depends on the amount of time and the applied voltage gradient. Although described for illustrative purposes with respect to a four-way cross-junction, these controlled electrokinetic material transport systems can be readily applied to more complex interconnected channel networks (e.g., arrays of interconnected parallel channels).

[0075] When the migration of affinity materials through a separation medium is induced by a voltage potential, as in electrophoresis, a large loading volume can be added to a low-salt buffer to improve sensitivity while maintaining sufficient resolution. When a sample contains only low concentrations of analytes or when a highly diluted mixture is used, it is desirable to load a large sample volume onto the separation medium to achieve good sensitivity in mobility shift analysis. However, a large sample volume can enter the separation medium as a broad bolus that elutes as a broad, poorly resolved peak. This problem can be reduced by loading the sample into a low-salt buffer and performing electrophoresis in a running buffer with a high salt concentration. Because low-salt samples are relatively deficient in charge carriers relative to the electrophoretic current, charged sample components migrate rapidly and accumulate at the front of the sample bolus. When they reach the high-salt running buffer, which is rich in charge carriers, they accumulate as a sharp band at the front of the sample bolus. Thus, a large volume of diluted sample can be loaded onto the electrophoretic separation medium, allowing for the detection of stronger peaks without a substantial loss of resolution.

[0076] The detector of the system can include any device appropriate for the signal of interest. If the affinity material has a useful light absorption spectrum, the detector can be a spectrophotometer. If the affinity material has an attached detectable marker, the detector can be of a type appropriate for the marker. For example, a fluorometer can be used for fluorescent markers, a scintillation counter can be used for radioactive markers, or a photodiode tube can be used for chemiluminescent markers. If the separation is performed by polyacrylamide gel electrophoresis (PAGE), the detector can include, for example, a scanner that detects the extent of gel migration of free affinity material and / or affinity material / analyte complex bands. If the separation is performed by chromatography or capillary electrophoresis, the detector can be, for example, a suitable detector that focuses the effluent stream from the separation medium to detect free affinity material and / or affinity material / analyte complex as it elutes over time. Such detectors can generate analog or digital output signals that can be analyzed by the logic circuitry of the present invention.

[0077] The logic circuitry of the device can receive, for example, a quantitative signal from a detector, representing, for example, the amount of affinity material detected in the gel position or chromatography effluent over time. The logic circuitry can be as simple as a chart recorder that plots signal amplitude on moving chart paper, or it can be a sophisticated digital computer / software system. Commonly available software, such as the Agilent Technologies 2100 Bioanalyzer-Biosizing [DNA 7500], can perform, for example, peak identification, peak height, peak area integration, background subtraction, and regression analysis to identify and quantify analytes.

[0078] <Separation medium> In the present invention, it is preferable to use a separation medium such as a polymer having a molecular sieve effect in the separation channel of a microfluidic device, and to perform separation using the separation medium. The separation medium (e.g., packing material) filled in the separation channel is not particularly limited.

[0079] Specifically, separation of the free affinity material and the analyte / affinity material complex is preferably performed by capillary gel electrophoresis, for example, in a separation medium disposed in a separation channel of a microfluidic device, where the separation medium is, for example, a restrictive matrix of linear or cross-linked polymers that can block the flow of large molecules while allowing the free flow of small molecules.

[0080] Examples of such separation media include polyethers such as polyethylene oxide (PEO), polyethylene glycol (PEG), and polypropylene oxide; polyalkyleneimines such as polyethyleneimine; polyacrylic acid-type polymers such as polyacrylic acid, polyacrylic acid esters, and polymethyl acrylate; polyamide-type polymers such as polyacrylamide, polymethacrylamide, and poly-n,n-dimethylacrylamide (pDMA); polymethacrylic acid-type polymers such as polymethacrylic acid, polymethacrylic acid esters, and polymethyl methacrylate; polyvinyl-type polymers such as polyvinyl acetate, polyvinylpyrrolidone (PVP), and polyvinyloxazolidone; water-soluble hydroxyl polymers such as pullulan, elsinan, xanthan, dextran, guar gum, and agarose gel; water-soluble celluloses such as methylcellulose, hydroxyethyl cellulose (HEC), and hydroxypropyl cellulose; water-soluble copolymers such as copolymers of sucrose and epichlorohydrin [e.g., Ficoll (trade name, Pharmacia)]; and derivatives thereof, as well as copolymers containing multiple types of monomer units that constitute these polymers. Separation media can be used alone or in combination. Polyacrylamide gel, polyethylene glycol (PEG), polyethylene oxide (PEO), sucrose-epichlorohydrin copolymer (Ficoll), polyvinylpyrrolidone (PVP), hydroxyethyl cellulose (HEC), poly-N,N-dimethylacrylamide (pDMA), and agarose gel are particularly preferred. Poly-N,N-dimethylacrylamide (pDMA) is the most preferred.

[0081] Such media can be packed into separation channels of microfluidic devices to perform, for example, rapid, high-throughput separations. In the present invention, separations can also be performed using only water or buffers without using the above-mentioned separation media.

[0082] The molecular weight of the separation medium is typically about 500 to 6,000 kDa, preferably 1 to 1,000 kDa, and more preferably 100 to 1,000 kDa. The concentration of the separation medium used as described above is selected from the range commonly used in the field of the present invention, i.e., typically about 0.01 to 40% (w / v), preferably 0.01 to 20% (w / v), and more preferably 0.1 to 10% (w / v). Typically, the separation medium is packed into the separation channel of a microfluidic device together with a buffer.

[0083] The type of buffer that can be used in the method of the present invention is not particularly limited. Examples of buffers that can be used include many of those used in fields such as hybridization assays and immunoassays, such as tris-buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, SSC buffer, TBE buffer, and TAE buffer. These buffers can typically be used at a concentration of about 0.1 mM to 10 M, preferably 1 mM to 5 M, and more preferably 5 mM to 1 M. The pH of the buffer can be set to any range that does not adversely affect substance separation, typically about 2 to 13, preferably 4 to 11, and more preferably 5 to 9. These parameters can be optimized, if desired, to perform electric field-amplified stacking. When the above-mentioned separation medium is added to the buffer, the viscosity of the buffer typically becomes about 2 to 1,000 centipoise, preferably 5 to 200 centipoise, and more preferably 10 to 100 centipoise.

[0084] <Measurement> The analyte / affinity substance complex, or the free affinity substance not involved in the formation of the complex separated by the above-mentioned separation method, can be measured, for example, by a detection marker bound to the affinity molecule. In this way, the analyte in the sample can be measured. That is, according to the above-mentioned separation method, the analyte / affinity substance complex is separated from the free affinity molecule not involved in the formation of the complex, or the analyte / affinity substance complex is separated from the free affinity substance not involved in the formation of the complex. The resulting complex can be measured, for example, by a detection marker. Therefore, the amount of the analyte in the sample can be measured or the presence of the analyte in the sample can be identified with high sensitivity in a short time.

[0085] The presence of an analyte in a sample can be identified, for example, by detecting a mobility shift of the analyte and an affinity molecule, which may be labeled. A negative control sample containing no precipitate can be analyzed in the assay, and the elution time or separation medium migration rate of such labeled molecules and / or their complexes can be measured. A positive control sample containing a detectable amount of a reference analyte can be tested in the assay, and the elution time or separation medium migration rate of the labeled molecules and / or their complexes can be measured. When an unknown sample is tested in the same assay, the presence of the analyte can be identified by detecting a peak with the same retention time or migration rate as the labeled molecule and / or its complex peak. To ensure that the identified peak does not represent background noise in the assay, standard method validation techniques can be used to determine a peak height or peak area threshold that provides statistical confidence that a real signal above background is being detected.

[0086] Internal markers can be added to each sample to provide a reference frame for peak identification or to adjust elution times to account for intra-assay variability, allowing for rigorous comparison between assay runs. For example, detectable high and low molecular weight markers can be added to samples to separate peaks within a reference frame. If elution times vary between analyses, peaks can also be identified by their relative positions among internal markers, as known to those skilled in the art.

[0087] The amount of analyte present in a sample can be determined by comparing the peak height or peak area of ​​the identified analyte / affinity material complex to a standard curve. A standard curve can be, for example, a calculation formula that displays the peak height or area values ​​of one or more standard samples with known amounts of analyte. Peak height or area values ​​from unknown samples can be input into the formula to determine the amount of analyte in the sample. Peak height or area values ​​can be adjusted by subtracting a negative control background to increase the accuracy of the measurement.

[0088] The analyte may be quantified by correlating the peak height or peak area ratio of the free conjugate and complex peaks to a formula or chart of values. The formula or chart of concentration versus analyte concentration can be calculated or empirically derived for the assay, as known in the art. The addition of an internal marker can improve the quantitative data by comparing the results obtained from each run of the assay with a known amount of analyte.

[0089] <Kit> The reagents and other materials used to carry out the assay method of the present invention can be prepared as a kit to enable the assay method of the present invention to be carried out. Specifically, the present invention provides a kit for measuring the N-terminal fragment of human brain natriuretic peptide precursor, which comprises (1) a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, and (2) a separation medium containing 5.1% (w / v) or more of a polyanionic polymer.

[0090] Specific examples and preferred embodiments of substances that bind to the N-terminal fragment of human brain natriuretic peptide precursor are as described herein. Specific examples and preferred embodiments of the polyanionic polymer are as described herein.

[0091] In a first embodiment, the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a combination of a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a charged carrier molecule, and a second binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a detectable marker.

[0092] In a second embodiment, the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a charged carrier molecule, and the kit further includes a binding substance that binds to a complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance and is labeled with a detectable marker.

[0093] In a third embodiment, the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor and is labeled with a detectable marker, and the kit further includes a binding substance that binds to a complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance and is labeled with a charged carrier molecule.

[0094] The above kits can be used in combination with a microfluidic device, which may be sold as part of the kit.

[0095] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the examples, mM means mmol / L, nM means nmol / L, and % means mass % unless otherwise specified. [Example]

[0096] Example 1: Evaluation of the sample effect of lithium heparin on NT-proBNP measurement in fresh blood NT-proBNP-containing serum samples (fresh blood samples) refrigerated within 24 hours of collection were measured using the Mutas Wako i50 (Fujifilm Wako Pure Chemical Industries, Ltd.), and the effect of lithium heparin on peak shape was evaluated. (1) Reagents used in the measurement The following reagents were prepared and used for the measurements.

[0097] Running buffer 1 (R1) 77 mM Tris (Fujifilm Wako Pure Chemical Industries, Ltd.) / 55 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.) / 9 mM MES (Dojindo Laboratories, Ltd.) / 0.01% BSA (Nihon Millipore) / 3.4% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 7.8)

[0098] Running buffer 2 (R2): Separating gel 0.7% PEG20000 (Fujifilm Wako Pure Chemical Industries, Ltd.) / 42 mM Tris (Fujifilm Wako Pure Chemical Industries, Ltd.) / 105 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.) / 0.01% BSA (Nihon Millipore) / 1% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 8.75)

[0099] Running buffer 3 (R3) 0.8% PEG20000 (Fujifilm Wako Pure Chemical Industries, Ltd.) / 77 mM Tris (Fujifilm Wako Pure Chemical Industries, Ltd.) / 138 mM HEPES (Dojindo Laboratories, Inc.) / 0.01% BSA (Nihon Millipore) / 3.4% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 7.5)

[0100] Running buffer 4 (R4) 77 mM Tris (Fujifilm Wako Pure Chemical Industries, Ltd.) / 0.01% BSA (Nihon Millipore) / 3% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 7.5)

[0101] Preparation of DNA-labeled anti-NT-proBNP antibody (DNA-Fab'(NT-proBNP)) Pepsin (Roche) was added to anti-human NT-proBNP mouse monoclonal antibody (Anti NT-proBNP monoclonal antibody, Medix Biochemica) to a final concentration of 0.002%, and the mixture was incubated at 37°C for 30 minutes to obtain anti-NT-proBNP antibody (Fab)2 fragment. Next, 2-aminoethanethiol hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 50 mM, and the mixture was reduced by incubation in a refrigerator for 30 minutes to obtain anti-NT-proBNP antibody Fab' fragment.

[0102] A 250 bp DNA fragment (Applide DNA Sciences) with an NH2 group introduced at the 5' end was purified (purified terminally aminated DNA), and the NH2 group previously introduced into this DNA fragment was mixed with an N-(6-maleimidocaproyloxy)succinimide (EMCS) linker (a linker with a maleimide group, Dojindo Chemical Industries) to react with each other. After that, gel filtration was performed to remove unreacted linkers, and a 250 bp DNA fragment with a linker attached was obtained. The resulting linker-bound 250 bp DNA fragment was reacted with a previously prepared anti-NT-proBNP antibody Fab' fragment, and the resulting reaction product was purified using a DEAE column to prepare an anti-NT-proBNP antibody Fab' fragment bound to the 250 bp DNA fragment (hereinafter abbreviated as "DNA-Fab'(NT-proBNP)").

[0103] Labeled antibody solution 1 (C1) 150 nM DNA-Fab' (NT-proBNP) (Fujifilm Healthcare Manufacturing Co., Ltd.) / 0.8% PEG20000 (Fujifilm Wako Pure Chemical Industries, Ltd.) / 77 mM Bis-Tris (Dojindo Laboratories, Ltd.) / 51 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.) / 0.5% EDTA·2Na (Dojindo Laboratories, Ltd.) / 0.01% BSA (Nihon Millipore) / 3% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 6.0)

[0104] Preparation of fluorescently labeled anti-NT-proBNP antibody (Hilyte-Fab'(NT-proBNP)) Mice were immunized with a synthetic peptide of NT-proBNP sequence 15-25 as an immunogen to obtain an anti-human NT-proBNP monoclonal antibody. This antibody recognizes an epitope of NT-proBNP that is different from that of the anti-NT-proBNP monoclonal antibody (Medix Biochemica). We confirmed that this antibody can sandwich the full-length NT-proBNP antigen in combination with the anti-NT-proBNP monoclonal antibody (Medix Biochemica).

[0105] Pepsin (Roche) was added to this antibody to a final concentration of 0.002%, and the mixture was incubated at 37°C for 30 minutes to obtain anti-NT-proBNP antibody (Fab)2 fragment. Next, 2-aminoethanethiol hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 50 mM, and the mixture was incubated in a refrigerator for 30 minutes for reduction to obtain anti-NT-proBNP antibody Fab' fragment. The fluorescent substance HiLyte647 (manufactured by AnaSpec) to which an NH2 group had been introduced was purified (purified terminally aminated HiLyte647). Next, the NH2 group previously introduced into this fluorescent substance HiLyte647 was reacted with an N-(6-maleimidocaproyloxy)succinimide (EMCS) linker (a linker having a maleimide group, manufactured by Dojindo Chemical Industries) by mixing, and then the product was purified using a DEAE column to remove unreacted linkers, thereby obtaining the fluorescent substance HiLyte647 to which the linker had been attached. The resulting linker-bound fluorescent substance HiLyte647 was reacted with a previously prepared anti-NT-proBNP antibody Fab' fragment, and the resulting reaction product was purified using a DEAE column to prepare a HiLyte647-labeled anti-NT-proBNP antibody Fab' fragment (hereinafter abbreviated as "Hilyte-Fab'(NT-proBNP)").

[0106] Labeled antibody solution 2 (C2) 2200 nM Hilyte-Fab' (NT-proBNP) (Fujifilm Healthcare Manufacturing Co., Ltd.) / 51 mM Bis-Tris (Dojindo Laboratories Co., Ltd.) / 152 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.) / 7 mM sodium benzoate (Fujifilm Wako Pure Chemical Industries, Ltd.) / 0.05% BSA (Nihon Millipore Corporation) (pH: 5.55)

[0107] Fluorescent liquid (FD) 50 mM Bis-Tris (Dojindo Laboratories) / 30 nM Hilyte solution (Fujifilm Healthcare Manufacturing Co., Ltd.) / 20% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 6.0)

[0108] (2) Measurement of NT-proBNP This assay was performed using the reagents prepared in (1) on a fully automated fluorescent immunoassay system, the Mutas Wako i50 (Fujifilm Wako Pure Chemical Industries), based on the LBA (Liquid-phase Binding Assay) method, which separates and measures the immune complexes formed after an antigen-antibody reaction in the liquid phase without requiring a solid phase in the reaction system. The samples used were NT-proBNP-containing serum samples (hereinafter sometimes abbreviated as fresh blood samples) that had been refrigerated within 24 hours of collection.

[0109] First, 8 μL of a fresh blood sample was contacted with 4 μL of labeled antibody solution 2 (C2), and 28 μL of electrophoresis buffer 1 (R1) to form NT-proBNP-fluorescent-Fab' (NT-proBNP), yielding an immunoreaction mixture. The resulting immunoreaction mixture was dispensed into designated wells on the Mu-TAS Wako i50 chip. Next, 20.6 μL of electrophoresis buffer 2 (R2), 11 μL of electrophoresis buffer 3 (R3), 5.4 μL of electrophoresis buffer 4 (R4), 3 μL of labeled antibody solution 1 (C1), and 7 μL of fluorescent solution (FD) were dispensed into designated wells on the chip (Figure 1). After dispensing the reagents, they were introduced into the analytical flow path of the chip by pressure application (Figure 2).

[0110] After introduction, when voltage is applied, DNA-Fab' (NT-proBNP) migrates toward the anode, concentrating according to the principle of isotachophoresis. The concentrated DNA-Fab' (NT-proBNP) then migrates to the sample zone, where a complex of DNA-Fab' (NT-proBNP)-NT-proBNP-fluorescent-Fab' (NT-proBNP) is formed (Figure 3). As electrophoresis continues, the DNA-Fab' (NT-proBNP)-NT-proBNP-fluorescent-Fab' (NT-proBNP) complex and DNA-Fab' (NT-proBNP) migrate to the zone electrophoresis zone due to the anionic charge. Unreacted fluorescent-Fab' (NT-proBNP) is uncharged and does not migrate to the zone electrophoresis zone. Therefore, in this measurement system, B / F separation (bound / unbound separation) occurs in the isotachophoresis zone. Furthermore, since unreacted DNA-Fab'(NT-proBNP) does not emit fluorescence, B / F separation is not necessary (Figure 4). Next, the DNA-Fab'(NT-proBNP)-NT-proBNP-fluorescence-Fab'(NT-proBNP) complex migrates through the zone electrophoresis zone, and the peak area of ​​fluorescence intensity is measured in the photometric section.

[0111] The peak area of ​​the fluorescence intensity derived from the DNA-Fab'(NT-proBNP)-NT-proBNP-fluorescence-Fab'(NT-proBNP) complex measured as described above was compared with the peak area of ​​the fluorescence intensity obtained by measuring a standard solution with a known concentration of NT-proBNP, to determine the NT-proBNP concentration in the fresh blood sample. Figures 5 to 9 show mobility shift charts of NT-proBNP in fresh blood when 3% (w / v), 4.5% (w / v), 5% (w / v), 5.2% (w / v), or 5.5% (w / v) lithium heparin was added to the separation gel.

[0112] Example 2: Evaluation of the effect of lithium heparin on NT-proBNP measurement in stored blood The NT-proBNP concentration was determined by the same method as in Example 1, except that instead of the "NT-proBNP-containing serum sample (fresh blood sample) refrigerated within 24 hours after collection" in Example 1, a "sample (preserved blood) that had been refrigerated for one day or more" was used, and the heparin concentration in the separation gel was 4% (w / v) or 5.5% (w / v). 10 and 11 show mobility shift charts of NT-proBNP in stored blood when 4% (w / v) or 5.5% (w / v) lithium heparin was added to the separation gel, respectively.

[0113] The results of Example 2 show that in the measurement of stored blood, the NT-proBNP peak was a single peak in either the presence of 4% (w / v) or 5.5% (w / v) lithium heparin in the separation gel. On the other hand, the results of Example 2 showed that in the measurement of fresh blood samples, a bimodal peak was observed when 3% (w / v) or 4.5% (w / v) lithium heparin was present in the separation gel. On the other hand, a single peak was observed when 5.5% (w / v) lithium heparin was present in the separation gel. This peak shape is similar to the peak shape observed in the measurement of stored blood in Example 2. In other words, it was found that the influence of the time from sample collection to measurement can be suppressed by performing measurement in the presence of 5.1% (w / v) or more lithium heparin.

[0114] Example 3: Evaluation of the effect of dextran sulfate sodium on NT-proBNP measurement NT-proBNP in fresh blood samples was measured in the same manner as in Example 1, except that 4% (w / v) or 5.5% (w / v) dextran sulfate sodium was used instead of lithium heparin in the separation gel in Example 1. Specifically, the following separation gel was used:

[0115] Running buffer 2 (R2): Separating gel 0.7% PEG20000 (Fujifilm Wako Pure Chemical Industries, Ltd.) / 42 mM Tris (Fujifilm Wako Pure Chemical Industries, Ltd.) / 105 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.) / 0.01% BSA (Nihon Millipore) / 1% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) (pH: 8.75)

[0116] When measuring NT-proBNP-containing serum samples (fresh blood samples) that have been refrigerated within 24 hours of collection, adding 5.5% or more polyanions to the separation gel can suppress changes in antigen characteristics.The mobility shift chart for NT-proBNP when 4% dextran sodium sulfate is added to the separation gel and when 5.5% dextran sodium sulfate is added to the separation medium shows that dextran sodium sulfate binds nonspecifically to sample components, thereby suppressing separation due to differences in antigen characteristics.

[0117] Figures 12 and 13 show mobility shift charts of NT-proBNP in fresh blood when 4% (w / v) dextran sulfate sodium was added to the separation gel and when 5.5% (w / v) dextran sulfate sodium was added, respectively.

[0118] The results of Example 3 show that the influence of the time from sample collection to measurement can also be suppressed by using dextran sulfate sodium as a polyanionic polymer.

Claims

1. isolating a complex comprising the N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor in the presence of a polyanionic polymer; and measuring the complex A method for measuring an N-terminal fragment of a human brain natriuretic peptide precursor in a sample, comprising:

2. 10. The method of claim 1, wherein the sample is taken within 24 hours.

3. The method of claim 1 , wherein the sample is a blood sample.

4. 2. The method of claim 1, wherein the polyanionic polymer is one or more selected from the group consisting of heparin and its salts, dextran sulfate and its salts, and chondroitin sulfate and its salts.

5. 5. The method of claim 4, wherein the heparin salt is one or more selected from the group consisting of lithium heparin, heparin sulfate, sodium heparin, and potassium heparin.

6. a complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, a human brain natriuretic peptide precursor N-terminal fragment; a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; A second binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the second binding substance being labeled with a detectable marker. The method of claim 1, wherein the compound is a complex with

7. a complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, a human brain natriuretic peptide precursor N-terminal fragment; a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; A binding substance that binds to a complex of an N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a detectable marker. The method of claim 1, wherein the compound is a complex with

8. a complex comprising an N-terminal fragment of human brain natriuretic peptide precursor and a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, a human brain natriuretic peptide precursor N-terminal fragment; a first binding substance that binds to an N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a detectable marker; a binding substance that binds to a complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a charged carrier molecule; The method of claim 1, wherein the compound is a complex with

9. (1) a substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, and (2) A separation medium containing 5.1% (w / v) or more of a polyanionic polymer; A kit for measuring an N-terminal fragment of a human brain natriuretic peptide precursor, comprising:

10. A substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; and A second binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the second binding substance being labeled with a detectable marker. The kit according to claim 9, which is a combination of

11. the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a charged carrier molecule; The present invention further includes a binding substance that binds to a complex of an N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a detectable marker. The kit of claim 9.

12. the substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor is a first binding substance that binds to the N-terminal fragment of human brain natriuretic peptide precursor, the first binding substance being labeled with a detectable marker; The present invention further includes a binding substance that binds to the complex of the N-terminal fragment of human brain natriuretic peptide precursor and the first binding substance, the binding substance being labeled with a charged carrier molecule. The kit of claim 9.

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