WO2014097877A1 - Puce de détection et système de mesure d'immunofluorescence par spfs - Google Patents

Puce de détection et système de mesure d'immunofluorescence par spfs Download PDF

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Publication number
WO2014097877A1
WO2014097877A1 PCT/JP2013/082414 JP2013082414W WO2014097877A1 WO 2014097877 A1 WO2014097877 A1 WO 2014097877A1 JP 2013082414 W JP2013082414 W JP 2013082414W WO 2014097877 A1 WO2014097877 A1 WO 2014097877A1
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cardiac troponin
spfs
sensor chip
troponin
antibody
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PCT/JP2013/082414
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English (en)
Japanese (ja)
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真紀子 大谷
高敏 彼谷
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コニカミノルタ株式会社
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Priority to JP2014553064A priority Critical patent/JP6369330B2/ja
Publication of WO2014097877A1 publication Critical patent/WO2014097877A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • the present invention relates to a sensor chip capable of measuring myocardial troponin I and myocardial troponin I autoantibodies with respect to the same specimen using surface plasmon excitation enhanced fluorescence spectroscopy [SPFS; Surface Plasmon-field enhanced Fluorescence Spectroscopy]
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • the present invention relates to an SPFS immunofluorescence measurement system.
  • myocardial troponin I is a biomarker useful in determining whether it is AMI (acute myocardial infarction) or other heart disease (such as angina pectoris) in ACS (acute coronary syndrome). It is also used in places.
  • Troponin I has different amino acid sequences in cardiac muscle and skeletal muscle, and cardiac troponin I (cTnI) has high myocardial specificity. Therefore, cTnI should be specifically measured based on the difference in amino acid sequence. Is useful for diagnosis of myocardial infarction and the like. In recent years, Troponin I has shown many usefulness of high-sensitivity measurement in early diagnosis of myocardial infarction.
  • the cardiac troponin I autoantibody binds to the cardiac troponin I antigen and inhibits the reaction between the cardiac troponin I antibody used for quantification of the antigen and the cardiac troponin I antigen, resulting in false negatives.
  • a myocardial troponin autoantibody assay system is known that provides a cascade of additional diagnostic items such as whether or not a subject subject is a candidate for immunotherapy or the like (see, for example, Patent Document 1). .
  • cardiac troponin I autoantibodies are produced by molecular mimicry of pathogens such as viruses, bacteria or toxins, genetic abnormalities, tissue damage or sudden diseases.
  • Myocardial troponin I autoantibody refers to an antibody that is produced from autoimmune cells and reacts with autologous cardiac troponin I protein.
  • the present invention has been made in view of the above problems, and can detect cardiac troponin I more quickly, accurately and with high sensitivity, and can reduce the amount of sample collected from a subject (patient etc.) (minimally invasive). It is an object of the present invention to provide a sensor chip capable of achieving the above characteristics and a SPFS immunofluorescence measurement system using the sensor chip.
  • a sensor chip reflecting one aspect of the present invention is a sensor chip for SPFS immunofluorescence measurement, SPFS immunofluorescence measurement comprising a first capture means capable of specifically binding to cardiac troponin I contained in a subject-derived specimen, and a second capture means capable of binding to an autoantibody against cardiac troponin I It is a sensor chip for.
  • an SPFS immunofluorescence measurement system reflecting one aspect of the present invention includes the sensor chip, This is an SPFS immunofluorescence measurement system in which the detection of cardiac troponin I and the detection of cardiac troponin I autoantibodies are performed in parallel on the same specimen.
  • a sensor chip capable of detecting myocardial troponin I more quickly and accurately and with high sensitivity and achieving a small amount of sample collected from a subject (patient etc.) (minimally invasive) and An SPFS immunofluorescence measurement system using this can be provided.
  • FIG. 1 is a schematic diagram showing an SPFS immunofluorescence measurement system equipped with a sensor chip according to the present invention.
  • FIG. 2A is a top view of the sensor chip of FIG.
  • FIG. 2B is a top view of another sensor chip according to the present invention.
  • FIG. 3 is a diagram showing a flowchart of reliability evaluation processing by the SPFS immunofluorescence measurement system.
  • FIG. 4 is a diagram showing a state in which immunofluorescence measurement is performed on the specimen with positive cardiac troponin I and positive cardiac troponin I autoantibody using the SPFS immunofluorescence measurement system of FIG.
  • FIG. 5 is a diagram showing a state in which immunofluorescence measurement is performed on a specimen with positive cardiac troponin I and negative cardiac troponin I autoantibody using the SPFS immunofluorescence measurement system of FIG.
  • FIG. 6 is a diagram showing a state in which immunofluorescence measurement is performed on a specimen in which cardiac troponin I is negative and cardiac troponin I autoantibody is positive using the SPFS immunofluorescence measurement system in FIG.
  • FIG. 7 is a diagram showing a state in which immunofluorescence measurement is performed on the specimen with negative cardiac troponin I and negative cardiac troponin I autoantibody using the SPFS immunofluorescence measurement system of FIG.
  • FIG. 6 is a diagram showing a state in which immunofluorescence measurement is performed on a specimen in which cardiac troponin I is negative and cardiac troponin I autoantibody is positive using the SPFS immunofluorescence measurement system in FIG.
  • FIG. 7 is a diagram showing a state in which immunofluorescence measurement is performed on the specimen with negative
  • FIG. 8 is a diagram showing a state in which immunofluorescence measurement is performed on a specimen having positive cardiac troponin I and positive cardiac troponin I autoantibodies using a conventional SPFS immunofluorescence measurement system.
  • FIG. 9 is a diagram showing a state in which immunofluorescence measurement is performed on a specimen with negative cardiac troponin I and positive cardiac troponin I autoantibodies using a conventional SPFS immunofluorescence measurement system.
  • the sensor chip 10 according to the present invention includes a first capture means 1 that can specifically bind to cardiac troponin I contained in a specimen derived from a subject, and a second capture means that can bind to cardiac troponin I autoantibodies. 2 and can be used to detect cardiac troponin I and cardiac troponin I autoantibodies against the same specimen (see FIGS. 1 to 7).
  • a flow path 3 through which the sample solution is circulated may be formed, and the first capture means 1 and the second capture means 2 may be fixed to the flow path 3.
  • the SPFS immunofluorescence measurement system 100 includes the sensor chip 10 and performs the detection of cardiac troponin I and the detection of cardiac troponin I autoantibodies in parallel for the same specimen. (See FIGS. 1-7).
  • the detection of cardiac troponin I autoantibody may be performed using a used specimen after the detection of cardiac troponin I.
  • FIG. 1 shows an example of an SPFS immunofluorescence measurement system according to the present invention.
  • the SPFS immunofluorescence measurement system 100 includes a sensor chip 10 and an SPFS device 10A.
  • the sensor chip 10 is detachably mounted on an SPFS apparatus 10A described later and used for SPFS immunofluorescence measurement.
  • the sensor chip 10 includes a transparent support 5 for passing excitation light during SPFS immunofluorescence measurement, a metal film 4 formed on the transparent support 5, and a metal film 4. Detecting a cardiac troponin I autoantibody, a flow path 3 for circulating a sample solution as a part, a first capturing means 1 provided on the surface of the metal film 4 for detecting cardiac troponin I Second capturing means 2 for the purpose, and optionally further including third capturing means.
  • Reference numeral 6 denotes a pump connection portion for connecting a tip of a liquid feed pump 14 (described later) of the SPFS device 10A when a sample solution or the like is fed to the sensor chip 10.
  • Reference numeral 7 denotes a detachable liquid reservoir for temporarily storing a sample solution and the like after flowing through the sensor chip 10.
  • Reference numeral 15 denotes a flow path forming body such as a flow path top plate or a flow path substrate.
  • the transparent support 5 is used for supporting the structure of the sensor chip 10. As shown in FIG. 1, the transparent support 5 has a flat portion 5a for forming the metal film 4, a prism portion 5b, and the like. The flat surface portion 5a and the prism portion 5b may be separate or integrated.
  • a glass or resin material can be used as a material of the transparent support 5.
  • resin those made of optical resin such as acrylic, polycarbonate (PC), polymethyl methacrylate (PMMA), and cycloolefin polymer (COP) can be used.
  • various inorganic materials such as ceramics, natural polymers, silicon dioxide (SiO 2 ), and titanium dioxide (TiO 2 ) may be used.
  • the refractive index [n d ] of the transparent support 5 is preferably 1.40 to 2.20.
  • the thickness of the flat portion 5a is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.
  • the surface of the transparent support 5 is preferably cleaned with acid and / or plasma before the metal film 4 is formed.
  • As the washing treatment with an acid it is preferable to immerse the transparent support 5 in 0.0001 to 1N hydrochloric acid for 1 to 3 hours.
  • Examples of the plasma cleaning treatment include a method in which the transparent support 5 is immersed in a plasma dry cleaner (“PDC200” manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.
  • the size (length ⁇ width) of the flat portion 5a of the transparent support 5 is not particularly limited as long as it does not adversely affect the SPFS immunofluorescence measurement.
  • the shape of the cross section of the prism portion 5b along the normal direction of the flat surface portion 5a of the transparent support 5 is not limited to the illustrated inverted trapezoidal shape, but may be a triangular shape, a semicircular shape, or an elliptical shape.
  • the prism portion 5b of the transparent support 5 includes an incident surface 5c for allowing excitation light L1 from the light source 19 of the SPFS device to be described later to enter the prism portion 5b, and the metal film 4 on the flat portion 5a of the transparent support 5. It has the output surface 5d which radiate
  • the metal film 4 is a member for amplifying an evanescent wave (enhanced electric field) generated when the excitation light L1 incident on the inside of the prism part 5b under the total reflection condition is totally reflected at the interface between the metal film 4 and the flat part 5a. It is.
  • the metal film 4 formed on the surface of the transparent support 5 is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum, and more preferably made of gold. These metals may be in the form of an alloy thereof. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.
  • Examples of the method for forming the metal film 4 on the transparent support 5 include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since adjustment of the formation conditions of the metal film 4 is easy, it is preferable to form the metal film 4 by sputtering or vapor deposition.
  • the thickness of the metal film 4 is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm.
  • the thickness of the metal film 4 is as follows: gold: 20 to 70 nm, silver: 20 to 70 nm, aluminum: 10 to 50 nm, copper: 20 to 70 nm, platinum: 20 to 70 nm, and alloys thereof: 10 More preferred is ⁇ 70 nm.
  • the thickness of the metal film 4 is within the above range, surface plasmons can be suitably generated.
  • the size (length ⁇ width) of the metal film 4 is not particularly limited as long as it does not adversely affect the SPFS immunofluorescence measurement, similarly to the flat portion 5a.
  • the flow path 3 of the sensor chip 10 is a flow path for circulating a sample solution, such as serum, collected from a subject (human, dog, cat, etc.), pretreated as described below, or a cleaning solution, as necessary. is there.
  • a sample solution such as serum, collected from a subject (human, dog, cat, etc.), pretreated as described below, or a cleaning solution, as necessary. is there.
  • the flow path 3 can be a non-branched flow path 3 in which the first and second capturing means 1 and 2 are provided in series. However, as shown in FIG. It may also be a parallel flow path 3 that is branched at a further downstream position and provided with first and second capturing means 1 and 2 in the branch flow path.
  • the non-branched series flow path 3 of FIG. 2A is preferable in that the amount of sample collected from the subject is smaller (becomes less invasive).
  • the flow path 3 of the sensor chip 10 may be configured such that the upstream side and the downstream side of the flow path 3 can be reversed, such as by forming the sensor chip 10 symmetrically (in FIG. 1). This is because in the case of the serial flow path 3 as shown in FIG. 2A, it is possible to select each time whether priority is given to detection by the first capture means 1 or priority is given to detection by the second capture means 2.
  • the flow direction M (see FIG. 1) of the flow path 3 is fixed in one direction, and the used sample solution discharged from the downstream side is circulated again to the upstream side. It may be a flow path configuration.
  • the used sample solution can be further reacted with the first and second capture means 1 and 2, and the antigen-antibody reaction between the first and second capture means 1 and 2 and the sample.
  • This circulation may be automatically controlled by the control means of the SPFS device 10A described later.
  • the length of the flow path 3 of the sensor chip 10 is preferably as short as possible in order to increase the minimally invasiveness of the subject (patient or the like) to reduce the amount of specimen necessary for the measurement.
  • 2 and the cardiac troponin I and the cardiac troponin I autoantibodies are required to have a length sufficient to secure a binding reaction.
  • the width of the flow path 3 of the sensor chip 10 is such that when the serial flow path 3 is not branched, the spots 1 a and 2 a of the first and second capturing means 1 and 2 and the flow path 3 It is preferable to set so as to eliminate the gap as much as possible.
  • the reason for this is that not only the reaction at each spot is sufficiently performed, but, for example, as shown in FIG. 2A, the amount of myocardial troponin I bound to the first capture means 1 and the spot 1a on the upstream side, 2 because the binding between the capturing means 2 and the cardiac troponin I autoantibody is not adversely affected.
  • the flow path is made of a material that minimizes non-specific binding such as a blocking agent.
  • a material that minimizes non-specific binding such as a blocking agent.
  • the surface treatment for example, plasma treatment (oxygen plasma treatment or the like) or corona is applied to a member (see FIG. 1) such as the metal film 4 or the flow path forming body 15 that forms the flow path 3 together with the metal film 4.
  • a member such as the metal film 4 or the flow path forming body 15 that forms the flow path 3 together with the metal film 4.
  • the discharge treatment or the coating with a hydrophilic polymer, protein, lipid, or the like can be performed, but it is not limited thereto.
  • the blocking agent examples include hydrophilic polymers such as casein, skim milk, albumin (BSA, and the like), polyethylene glycol, and phospholipids, and low molecular compounds such as ethylenediamine and acetonitrile. May be used in combination of two or more. These can be used after diluted with a solvent such as phosphate buffered saline [PBS], HEPES, MEM, RPMI, phosphate buffer or the like.
  • a solvent such as phosphate buffered saline [PBS], HEPES, MEM, RPMI, phosphate buffer or the like.
  • the shape of the flow path 3 of the sensor chip 10 may be a rectangular tube (tube) shape or a round tube (tube) shape, but may be an analyte (myocardial troponin I or myocardial troponin I autoantibody) and primary.
  • the reaction part / measurement part (wide channel part where the spots 1a and 2a are present) that binds the antibody and measures fluorescence is preferably in the form of a rectangular tube from the relationship of allowing light to pass through. It is preferable that the flow path portion used only for liquid feeding has a round cylindrical shape.
  • the material of the flow path forming body 15 (see FIG. 1) constituting the outer wall of the sensor chip 10 is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material; polyolefin such as polyethylene, and the flow path It is preferable to use a polymer such as silicone rubber, Teflon (registered trademark), polyethylene, or polypropylene for the portion facing 3.
  • the portion of the flow path 3 corresponding to the reaction part / measurement part around the spots 1a and 2a In the cross section of the channel 3 (see FIG. 1), it is preferable that the length and the width are about 100 nm to 1 mm, respectively.
  • a flow path height of 0.5 mm is set on the surface side of the sensor chip 10 where the metal film 4 is formed.
  • the sheet made of polydimethylsiloxane [PDMS] is pressure-bonded so as to surround the portion of the sensor chip 10 where the metal thin film 4 is formed, and then the sheet made of polydimethylsiloxane [PDMS] and the sensor chip 10 are bonded to each other with a screw or the like.
  • a method of fixing with a closing tool is preferred (partially not shown).
  • the method of forming the flow path 3 in the sensor chip 10 corresponds to the flow path forming body 15 of the flow path substrate and the flow path top plate in the sensor chip 10.
  • a method of forming a member by integral molding of plastic may be used.
  • the flow path 3 is formed using a flow path forming body 15 such as a flow path top plate or a flow path substrate, but the flow path 3 may be formed without using this.
  • the first capture means 1 is a molecule that specifically binds to cardiac troponin I.
  • the term “specifically binds” refers to molecules that bind at specific sites (for example, generally two polypeptides, one polypeptide and a nucleic acid, as measured by means known in the art). Molecule, or two nucleic acid molecules) is preferentially bound over other molecules.
  • biomolecules that function as the first capturing means 1 include cardiac troponin I antibody and partial fragments of cardiac troponin I antibody.
  • the first capture means 1 is a partial fragment, it is generally an epitope that can bind to cardiac troponin I (cTnI) (cTnI specific and highly conserved N-terminal sequence (aa 13 to 36: PAPAPIRRRSSNYRAYATEPHAK)) or cTnI Having an intermediate part sequence (aa 30-110) having high conservative property of (aa 30-110).
  • the secondary antibody used in combination with the first capture means has an epitope different from that of the first capture means, and specifically recognizes cardiac troponin I captured by the first capture means via this epitope. Antibody.
  • the epitope of the first capture means 1 for detecting cardiac troponin I is: It is necessary to recognize either one of the two epitopes. Further, the epitope of the sandwiched secondary antibody needs to recognize the other epitope.
  • the first capture means 1 is a full-length cardiac troponin I-binding antibody, it may be a wild-type amino acid sequence of the cardiac troponin I-binding antibody or a variant of the amino acid sequence of the corresponding region of the wild-type polypeptide. .
  • the first capturing means 1 may include other amino acid sequences including sequences derived from heterologous proteins in addition to the amino acid sequences described above. Therefore, the first capture means 1 includes a fusion polypeptide in which the amino acid sequence of the biomolecule of the cardiac troponin I antibody is fused to one or more heterologous protein (one or more) amino acid sequences at one or both ends. Is done.
  • additional amino acid sequences to be fused also include a signal sequence that facilitates protein production, and an epitope tag that can be used for immunological detection or affinity purification.
  • the cardiac troponin I antibody can be purchased from each pharmaceutical company (Funakoshi, etc.) (product name “Anti-Troponin-I, Cardiac Human”, etc.).
  • the cardiac troponin I antibody can also be prepared by a known method. For example, by immunizing rodents with myocardial troponin I obtained by gel filtration or purchase based on a standard method (Kohler, Milstein, Nature, 1975, Vol. 256, p495-497), polyclonal Alternatively, cardiac troponin I antibody can be obtained in the form of a monoclonal antibody.
  • the partial fragment of the cardiac troponin I antibody can be obtained by treating the F (ab ′) 2, Fab ′, Fab, Fv antibody fragment by treating the complete antibody with a protease enzyme and optionally reducing it.
  • the cDNA can be isolated from an antibody-producing hybridoma, and the antibody or an antibody fragment thereof or a fusion protein of an antibody fragment and another protein can be produced using an expression vector prepared by genetic modification. it can. In this case, it is preferable to have an epitope tag for affinity purification as described above.
  • an antibody-producing animal is immunized with a partial fragment of cardiac troponin I containing an epitope that is not used for binding between the first capture means 1 and cardiac troponin I. It can be obtained by a method or a method using a hybridoma as described above.
  • the second capture means 2 is a molecule that binds to cardiac troponin I autoantibodies.
  • a cardiac troponin I autoantibody-binding antibody, a partial fragment of the antibody, or the like can be used.
  • the cardiac troponin I autoantibody binding antibody include an anti-human antibody that recognizes and binds to the Fab region and the Fc region, and an antibody obtained by a predetermined method described later.
  • cardiac troponin I autoantibodies increase L-type calcium current, which is closely related to heart rate and cardiac contraction, and is related to the persistence of myocardial injury (Ayumi Kagaku 2008 Vol. 226, No. 1, p. 16-21) Etc.), and is also a causative factor of dilated cardiomyopathy (see Table 2004/091476). Therefore, not only the determination of the reliability of the detection result of cardiac troponin I but also the significance of measurement / detection is high from the above viewpoint.
  • a partial fragment of a cardiac troponin I autoantibody binding antibody has an epitope capable of binding to a cardiac troponin I autoantibody, and is a polyclonal or monoclonal antibody, a humanized antibody, a fully human antibody or a shortened form thereof (for example, F (ab ′ ) 2, Fab ′, Fab, Fv) Any form such as an antibody may be used.
  • the secondary antibody used in combination with the second capture means is an antibody that recognizes and binds to cardiac troponin I bound to the cardiac troponin I autoantibody captured by the second capture means.
  • the troponin I autoantibody has an epitope different from the epitope recognized upon binding to troponin I, and recognizes and binds to cardiac troponin I via this epitope. This is to prevent the secondary antibody from competing with the cardiac troponin I autoantibody bound to the second capture means 2.
  • the second capture means 2 is an anti-human antibody that recognizes and binds to the Fc region of a human antibody
  • “Goat Anti-Human IgG, Fc Fragment Specific” Merck- Millipore
  • Fab region or those that recognize both the Fab region and the Fc region can be purchased in the same manner and used in the present invention.
  • cardiac troponin I autoantibodies in the sample recognize and bind to cardiac troponin I, and the epitope to which the secondary antibody binds (does not compete with cardiac troponin I autoantibodies) is also unknown Therefore, in order to increase the detection sensitivity of cardiac troponin I autoantibodies, it is possible to use a polyclonal antibody capable of comprehensively recognizing each epitope of cardiac troponin I as much as possible, or a combination of such monoclonal antibodies as a secondary antibody. desirable.
  • this secondary antibody As a method for preparing this secondary antibody, known as described above, using a fragment of cardiac troponin I having one or more epitopes of cardiac troponin I so as to cover all the epitopes of cardiac troponin I as much as possible. It can be prepared by inoculating an antibody-producing animal several times by the above method and separating and purifying from the animal. Moreover, the method of preparing using a hybridoma as mentioned above may be used.
  • the above-mentioned fragment of cardiac troponin I having one or more epitopes of cardiac troponin I preferably contains a smaller number of epitopes in order to increase detection sensitivity.
  • the cardiac troponin I or a partial fragment thereof may be fixed to the flow path 3 as the third capturing means.
  • the third capturing means is fixed upstream of the second capturing means. This is because free cardiac troponin I autoantibodies that are not detected even when captured by the second capturing means are removed as much as possible before the second capturing means. Since the third capturing means is cardiac troponin I itself, it is detected by the secondary antibody used in combination with the first and second capturing means, so it is necessary to set the third spot by the spot position setting means. There is.
  • cardiac troponin I for example, as described above, full-length cardiac troponin I can be obtained by protease treatment.
  • cTnI polynucleotide encoding cardiac troponin I
  • a gene fragment from which a part of the sequence has been removed is prepared and used as an expression vector. It can be obtained by incorporating the protein into a host that produces the protein, culturing the host, and purifying the target protein produced from the cultured host.
  • the sample may be pretreated or concentrated by diluting in an appropriate buffer solution as necessary.
  • These pretreatments can use any of a number of standard aqueous buffer solutions, optionally using any of a variety of buffers such as phosphate, Tris, etc. at physiological pH.
  • the sample is not limited to serum, and plasma and whole blood may be used.
  • the method for fixing the first and second capturing means 1 and 2 and the third capturing means to the metal film 4 is not particularly limited, but the following metal binding groups are attached to the first and second capturing means 1 and 2.
  • the third capture means is introduced into a region other than the antigen-binding region, and the first and second capture means or the third capture means are introduced by a conventionally known method (for example, a method described later) through this metal binding group.
  • the metal film 4 can be bonded and fixed.
  • the metal binding group examples include a thiol group (—SH), a tellurium group (—TeH), a selenol group (—SeH), a symmetric or asymmetric diselenide group (—SeSe—), a symmetric or asymmetric disulfide group (—SS—), Thioisocyanide group (—SCN), isonitrile group (—NC), trivalent phosphate group (—PO 4 2 ⁇ ), sulfide group (—SRZ), disulfide group (—SSRZ), selenide group (—SeRZ), diselenide Group (—SeSeRY), xanthate group (—OCSS—), nitro group (—NO 2 ), thiocarbamate group (—SCH), phosphine group (—PR 2 ), thioacid group or dithioacid group (—COSH, — CSSH), carboxyl group (—COOH), silane group (—SH 3 ) and the like.
  • the first and second capturing means 1 and 2 are dissolved on the surface of the metal film 4 on the transparent substrate.
  • the ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories Co., Ltd.) or 10-amino-1-decanethiol was dropped, and the spot was not dried at room temperature for a predetermined time (for example, 24 hours), and after the incubation, each spot is dried with an air gun to complete the fixation.
  • the method of directly fixing the first and second capturing means 1 and 2 and the third capturing means to the metal film 4 has been described.
  • the first and second capturing means 1 and 2 via the self-assembled monolayer (SAM) are described.
  • the second capturing means 1, 2 and the third capturing means are fixed to the metal film 4, and a hydrophilic polymer layer such as carboxymethyl dextran (CMD) is provided on the SAM, and the hydrophilic polymer layer is provided on the hydrophilic polymer layer.
  • CMD carboxymethyl dextran
  • the first and second capturing means 1, 2 and the third capturing means can be fixed, or a conventionally known fixing method can be used.
  • the SPFS device 10A includes a light source 19, a linearly polarizing plate 18, an optical path switching mirror 17, a transparent support 5 as a prism, a neutral density filter 22, a filter replacement means 20, a cut filter 21, and a detection. 23, a surface plasmon resonance [SPR] detector 16, a liquid storage well 13, a liquid feed pump 14, an actuator, a control means, and the like (partly not shown).
  • the liquid storage well 13 may be configured to be detachable from the SPFS device 10A.
  • the light source 19 that is irradiated when measuring the amount of fluorescence is not particularly limited as long as it can cause plasmon excitation in the metal film 4, but the unity of the wavelength distribution and the intensity of light energy are not limited. In this respect, it is preferable to use laser light as a light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism 5 through the optical filter.
  • the surface plasmon is generated on the surface of the metal film 4 under the total reflection attenuation condition [ATR] by the laser light irradiation. Due to the electric field enhancement effect of the surface plasmon, the fluorescent dye 12 (see FIG. 4 and the like) is excited by photons that are increased by several tens to several hundred times the amount of photons irradiated. The amount of increase in photons due to the electric field enhancement effect depends on the refractive index of the prism 5, the metal type of the metal member, and the film thickness thereof, but usually increases about 10 to 20 times when the metal film 4 is a gold film. Amount.
  • the electrons in the molecule are excited by light absorption, move to the first electron excited state in a short time, and return to the ground state from this state (level), the wavelength corresponding to the energy difference. Emits fluorescence.
  • the laser light L1 for example, an LD having a wavelength of 200 to 900 nm, 0.001 to 1,000 mW; a wavelength of 230 to 800 nm (resonance wavelength is determined by the metal species used in the metal film 4), and a semiconductor having a wavelength of 0.01 to 100 mW A laser etc. are mentioned.
  • the linearly polarizing plate 18 converts the laser light, which is excitation light, into P-polarized light that efficiently generates surface plasmons. Thereby, the detection sensitivity at the time of SPFS immunofluorescence measurement also increases.
  • the optical path switching mirror 17 reflects the laser light L1 emitted from the light source 19 to be incident through the incident surface 5c of the prism (transparent support) 5 and receives the reflected light that is reflected and emitted from the emission surface 5d. Based on the information obtained by the surface plasmon resonance [SPR] detector 16, the irradiation angle to the metal film 4 is adjusted.
  • the prism 5 may be integrated with the transparent support 5 of the sensor chip 10 or may be a separate body. In the case of being integrated, it has a flat portion 5a on which the metal film 4 is formed and the other prism portion 5b.
  • the prism 5 is intended to allow the laser light L1 through various filters such as an optical filter, a polarizing filter, and a cut filter used as necessary to be efficiently incident on the metal film 4, and in the case of a separate body.
  • the refractive index of the prism 5 is preferably the same as that of the transparent support 5 of the sensor chip 10. Since various prisms capable of setting the total reflection condition can be selected as appropriate, the angle and shape are not particularly limited, and for example, a 60-degree dispersion prism may be used.
  • the neutral density filter 22 is intended to adjust the amount of light incident on the detection unit 23.
  • the detector 23 having a narrow dynamic range it is preferable to use it for carrying out a highly accurate measurement.
  • the cut filter 21 includes external light (illumination light outside the SPFS device 10A), stray light (scattering component of excitation light at various points), and plasmon scattered light (which originates from excitation light and has a structure or attachment on the surface of the sensor chip 10).
  • the detector 23 is preferably a photomultiplier tube (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) from the viewpoint of ultrahigh sensitivity.
  • the sensitivity is lower than these, but since it can be viewed as an image and noise light can be easily removed, a CCD image sensor capable of multipoint measurement can also be suitably used.
  • the surface plasmon resonance [SPR] detector 16 is an angle variable unit (adjusting the total reflection attenuation [ATR] condition with a servo motor) for adjusting the optimum angle of the photodiode, SPR and SPFS as a light receiving sensor dedicated to SPR.
  • the photodiode and the light source 19 can be synchronized to change the angle of 45 to 85 °.
  • the resolution is preferably 0.01 ° or more.
  • the computer may also be included.
  • the liquid storage well 13 stores a measurement target solution S, a dilution buffer DB, an antibody solution AS, a washing buffer WB, a standard solution (not shown) of myocardial troponin I or myocardial troponin I autoantibody, and the like.
  • Each well has a cartridge type. Further, the solution and buffer in each well are sucked up each time by the automatic pipetting operation of the liquid feeding pump 14.
  • liquid feed pump 14 for example, a micropump suitable for a small amount of liquid feed, a syringe pump that is not applicable to circulating liquid feed but has high feed accuracy and little pulsation, and may be unsuitable for a small amount of liquid feed.
  • a tube pump and the like can be mentioned, but without being limited thereto, various means can be appropriately selected and used depending on the purpose and application.
  • the actuator is provided in each member such as the detector 23 and the optical path switching mirror 17 and drives their operation (not shown).
  • the control unit of the SPFS apparatus 10A includes a display unit, an input unit, a CPU, a memory, and the like (not shown), and has a function as a general PC terminal.
  • the memory stores programs for operating the control means as spot position setting means, reference value setting means, SPFS immunofluorescence measurement means, measurement value comparison means, calibration curve creation means, and the like.
  • the control means performs a spot position setting process, a reference value setting process, an SPFS immunofluorescence measurement process, a reliability evaluation process, and the like based on an input by the user via the input means.
  • This control means may be connected to, for example, a hospital terminal through a line such as the Internet, and may be configured to exchange information with other terminals in a data format such as MML (Medical Markup Language).
  • MML Medical Markup Language
  • the spot position setting means includes a first capture means (myocardial troponin I antibody or the like) 1 and a second capture means (myocardial troponin I autoantibody binding antibody) of the sensor chip 10 set in the SPFS device based on the input of the input means by the user. 2) Optionally, each spot position of the third capturing means is set and recorded in the memory.
  • the spot position setting means for example, in the order of spots in which fluorescence is confirmed from the upstream side of the flow path 3 at the time of SPFS immunofluorescence measurement by the input through the input means by the user, the spot 1a of the first capturing means 1 , (Optionally the spot of the third capturing means), the setting is made to recognize the spot 2a of the second capturing means 2, and the setting information is stored in the memory as spot position information.
  • information indicating that either side of the detection image acquired by the detector 23 is the upstream side of the flow path 3 is recorded in a memory in advance and obtained as information.
  • This spot position information is used for spot identification at the time of measurement as one of the setting conditions regarding the above-mentioned SPFS immunofluorescence measurement.
  • the reference value setting means records a reference value related to the SPFS immunofluorescence measurement in a memory or changes the recorded reference value based on key input information by the user via the input means.
  • This reference value includes a reference value (first reference value) for the measured value of cardiac troponin I and a reference value (second reference value) for the measured value of cardiac troponin I autoantibody.
  • the first reference value is used when a doctor diagnoses a subject (patient or the like).
  • the second reference value is determined by the measured value comparison means of the control unit of the SPFS device 10A by comparing the measured value of the cardiac troponin I with the measured value of the cardiac troponin I autoantibody by SPFS immunofluorescence measurement. It is used for reliability evaluation processing.
  • the first reference value used by the doctor to determine whether cardiac troponin I is positive is 0.04 ng / mL in terms of antigen concentration in the serum and plasma of the patient specimen (in some cases 0.05 ng / mL Other values are used).
  • the cut-off value of the measured value of cardiac troponin I is 0 to the first reference value.
  • the second reference value is because the reliability of measurement of cardiac troponin I is low when immunofluorescence of cardiac troponin I autoantibody exceeding the blank value is detected. Is set to a range that can be taken by the measurement blank signal of the spot 2a of the sensor chip 10 or a value close thereto, for example, slightly higher than the upper limit of the range that the measurement blank can take. In addition, a plurality of other reference values may be provided in a range exceeding the second reference value, and the degree of reliability of the measurement result of cardiac troponin I may be evaluated stepwise.
  • SPFS immunofluorescence measurement means The SPFS immunofluorescence measurement means outputs various control commands to the light source 19 and the like of the SPFS apparatus 10A based on the setting conditions relating to the SPFS immunofluorescence measurement stored in the memory, based on the input by the user via the input means. A series of steps of SPFS immunofluorescence measurement is performed.
  • the measured value comparison means performs the fluorescence measurement value reliability evaluation process described below based on the input by the user via the input means (see FIG. 3).
  • step S1 the presence or absence of a measured value of cardiac troponin I is determined by SPFS immunofluorescence measurement means. Even if the measured value of cardiac troponin I is 0, it exists as data. If the measured value exists and YES, the process proceeds to step S2, and if not, the process moves to step S6 to display an error, and then the reliability evaluation process ends.
  • the “measured value” in the reliability evaluation process is a value obtained by converting the content of the measurement target contained in the sample from each calibration curve from the immunofluorescence measurement signal of the measurement target.
  • step S2 it is determined whether there is a measured value of the cardiac troponin I autoantibody and a second reference value. If both pieces of information exist and YES, the process proceeds to step S3. If neither of them exists, the process moves to step S6 to display an error, and then the reliability evaluation process ends.
  • step S3 the measured value of the cardiac troponin I autoantibody in step S2 is compared with the second reference value. If the measured value of the cardiac troponin I autoantibody is higher than the second reference value and YES, the process moves to step S4. To do. If the measured value of the cardiac troponin I autoantibody is not more than the second reference value and NO, the process moves to step S5.
  • step S4 the display unit displays that the measurement result of myocardial troponin I is low in reliability, and the measurement value information of myocardial troponin I associated with the subject information (patient name, etc.) is trusted.
  • the information indicating that the reliability is low is associated and recorded in the memory, and the reliability evaluation process is terminated.
  • the second reference value used for the comparison when recording in the memory, record the second reference value used for the comparison, the measured value of the cardiac troponin I autoantibody, the information that there is a need for an additional test due to false negative, and the like together. You may make it do. Further, simultaneously with the recording, these data may be transmitted to another terminal such as a hospital (for example, a terminal used by a doctor) to share the data with the other terminal.
  • a hospital for example, a terminal used by a doctor
  • step S5 for example, the display unit displays that the reliability of the measurement result of the cardiac troponin I is high, and the information indicating that the reliability is high with respect to the measurement value of the cardiac troponin I is recorded in the memory, The reliability evaluation process ends.
  • step S4 when recording in the memory, as in step S4, the second reference value used for the comparison, the measured value of the cardiac troponin I autoantibody, and information such as no additional examination are associated together.
  • the above recording may be performed. Further, simultaneously with the recording, these data may be transmitted to another terminal such as a hospital (for example, a terminal used by a doctor) to share the data with the other terminal.
  • a hospital for example, a terminal used by a doctor
  • the calibration curve creation means performs a calibration curve creation process as described below, or reads the existing cardiac muscle troponin I or cardiac troponin I autoantibody calibration curve data and stores it in the memory.
  • cardiac troponin I is obtained from serum derived from a commercially available subject (patient etc.)
  • a calibration curve is prepared in the same manner as in the preparation of the calibration curve for cardiac troponin I in (1) above, for the known concentration of the complex in which the cardiac troponin I autoantibody is bound.
  • the SPFS immunofluorescence measurement includes the following steps (a) to (d), and optionally includes washing steps (1) and (2).
  • the process cleaning step (1) for calculating the amount of the analyte The step of cleaning the inside of the sensor chip obtained through the step (a) using the cleaning liquid (2): the step (b) In the sensor chip obtained through the Washing with washing liquid.
  • Step (a) is a step of bringing the analyte solution into contact with a primary antibody (such as a cardiac troponin I antibody or a cardiac troponin I autoantibody-binding antibody) immobilized on the metal film 4 of the sensor chip.
  • a primary antibody such as a cardiac troponin I antibody or a cardiac troponin I autoantibody-binding antibody
  • the analyte solution is a solution obtained by diluting the analyte with a predetermined buffer, and the solvent used for diluting the analyte (cardiac troponin I or myocardial troponin I autoantibody) is, for example, phosphate buffered saline [ PBS], Tris buffered saline [TBS], HEPES buffered saline [HBS] and the like, but are not particularly limited.
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • HBS HEPES buffered saline
  • the sent analyte solution is circulated back and forth with respect to the flow path 3 as described above, or the flow direction of the flow path 3 is reversed. It is preferable to make it.
  • the temperature and time of the analyte solution at that time vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. for 1 to 60 minutes, preferably 25 ° C. for 5 to 15 minutes. is there.
  • the initial concentration of the analyte (cardiac troponin I and myocardial troponin I autoantibody) that may be contained in the analyte solution is 100 ⁇ g. / ML to 0.001 pg / mL is preferred.
  • the total amount of the analyte solution sent to the flow path 3 is usually 0.001 to 20 mL, preferably 0.1 to 1 mL.
  • the flow rate of the analyte solution fed to the flow path 3 is usually 1 to 5,000 ⁇ L / min, preferably 5,000 to 10000 ⁇ L / min.
  • a cleaning process (1) for cleaning the inside of the sensor chip with the cleaning liquid after the above-described process (a), and a cleaning process (2) for cleaning the inside of the sensor chip with the cleaning liquid after the above-mentioned process (b) are performed. There is.
  • washing solution used in the washing steps (1) and (2) for example, a surfactant such as Tween 20 or Triton X100 is added to the same solvent or buffer used in the reaction of steps (a) and (b). It is preferable to dissolve it so as to contain 0.00001 to 1% by mass, or to contain 10 to 500 mM of a salt such as sodium chloride or potassium chloride.
  • a low pH buffer solution for example, 10 mM Glycine HCl having a pH of 1.5 to 4.0 may be used as the washing solution.
  • the temperature and flow rate of the washing solution in the washing step are preferably the same as the temperature and flow rate at the time of feeding the analyte solution in the step (a).
  • the washing step (washing time with the washing solution) is usually 0.5 to 180 minutes, preferably 2 to 10 minutes.
  • Step (b) In the step (b), after the step (a), preferably after the washing step (1), an analyte (myocardial troponin I or myocardium bound to each primary antibody immobilized on the metal film 4 is further added.
  • Troponin I autoantibodies, etc. is a step of reacting a fluorescent dye-labeled secondary antibody.
  • Fluorescent dye is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or by excitation using electric field effect, and the “fluorescence” includes various kinds of light emission such as phosphorescence. .
  • the usable fluorescent dye is not particularly limited as long as it is not completely quenched due to light absorption by the metal member, and may be any known fluorescent dye.
  • fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.
  • fluorescent dyes examples include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes. (Applied Biosystems Japan Co., Ltd.), Coumarin family fluorescent dye (Invitrogen Co., Ltd.), Rhodamine family fluorescent dye (GE Healthcare ⁇ Bioscience Co., Ltd.), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dyes, oxazine family fluorescent dyes, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelated lanthanide dyes Millie's fluorescent dye, BODIPY® family fluorescent dye (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (Registered trademark) dye series (manufactured by Invitrogen Corp.) and the
  • Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.
  • the fluorescent dye is not limited to the organic fluorescent dye.
  • rare earth complex fluorescent dyes such as Eu and Tb can also be used.
  • rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there.
  • a commercially available rare earth complex fluorescent dye is ATBTA-Eu 3+ .
  • a fluorescent dye having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal contained in the metal film 4 is small.
  • a fluorescent dye having a maximum fluorescence wavelength 600 nm or more in order to minimize the influence of light absorption by the gold member.
  • a fluorescent dye having a maximum fluorescence wavelength in the near-infrared region such as Cy5, Alexa® Fluor (registered trademark) 647.
  • the use of a fluorescent dye having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful.
  • a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more when silver is used as the metal member, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more. These fluorescent dyes may be used alone or in combination of two or more.
  • the secondary antibody may be a monoclonal antibody or a polyclonal antibody.
  • the primary antibody is a monoclonal antibody
  • the secondary antibody is the primary antibody. It is desirable that the antibody is a monoclonal antibody that recognizes an epitope that is not recognized, or a polyclonal antibody.
  • a carboxyl group is added to the fluorescent dye, and the carboxyl group is converted into a water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethyl).
  • WSC water-soluble carbodiimide
  • EDC aminopropyl carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Method of dehydrating and immobilizing Method of immobilizing by reacting a secondary antibody and a fluorescent dye each having an isothiocyanate and an amino group; Reacting and immobilizing a secondary antibody and a fluorescent dye having a sulfonyl halide and an amino group, respectively
  • the concentration of the solution to be sent is preferably 0.001 to 10,000 ⁇ g / mL. 0.1 to 10 ⁇ g / mL is more preferable.
  • the temperature of the solution, the flow rate, and the time of step (b) (liquid feeding time) when sending this solution are the same as those in the above step (a).
  • Step (c) In the step (c), the sensor chip after the step (b) is irradiated with laser light via a prism from the other surface of the support on which the metal member is not formed. In this step, the amount of fluorescence emitted from the fluorescent dye is measured.
  • the actuator adjusts the position / angle of the optical path switching mirror 17 and the position of the detector 23 based on the command of the control means of the SPFS device 10A. L1 is irradiated and the amount of fluorescence emitted from each spot is measured.
  • Step (d) is a step of calculating the amount of analyte contained in the analyte solution from the measurement result obtained in step (c).
  • the amount of the analyte (myocardial troponin I or myocardial troponin I autoantibody) in the sample to be measured is calculated from the measurement signal and the data of the calibration curve based on the calibration curve described above. This is a step of storing these data as measured values in the memory of the control means of the SPFS device 10A.
  • the sensor chip 10 includes first capture means 1 such as a cardiac troponin I antibody that can specifically detect cardiac troponin I in a specimen derived from an animal such as a human or a pet, Since the second capturing means 2 such as an antibody capable of specifically detecting the cardiac troponin I autoantibody is provided, the cardiac troponin I and the cardiac troponin I autoantibody can be detected from the same specimen.
  • first capture means 1 such as a cardiac troponin I antibody that can specifically detect cardiac troponin I in a specimen derived from an animal such as a human or a pet
  • the second capturing means 2 such as an antibody capable of specifically detecting the cardiac troponin I autoantibody
  • the amount of sample collected from the subject can be reduced, and measurement errors due to different specimen lots. Also disappear. For this reason, it is possible to improve the reliability of the measured value of the cardiac troponin I and to improve the reliability of the measured value of the cardiac troponin I. For example, the measurement of the cardiac troponin I can be performed. Even when the value is in the vicinity of the cutoff value, it is possible to contribute to providing data for detecting AMI more quickly and accurately.
  • the flow path 3 is essentially the same.
  • the detection of cardiac troponin I and the cardiac troponin I self Detection can be performed by setting the priority of antibody detection.
  • the first capturing means 1 for detecting myocardial troponin I is installed on the upstream side, which is close to the connection part of the flow path 3 with the syringe pump, and the myocardial troponin I self is located downstream of the flow path 3.
  • the detection accuracy can be improved by installing the second capturing means 2 for detecting the antibody, giving priority to the detection of the cardiac troponin I, and detecting the cardiac troponin I for the sample in a more intact state.
  • the second capturing means 2 for capturing the cardiac troponin I autoantibody is installed upstream of the first capturing means 1.
  • the detection accuracy of the cardiac troponin I autoantibody can be prioritized, and the detection accuracy of the cardiac troponin I autoantibody can be increased for a more intact sample.
  • the flow path area of the serial flow path can be smaller than that of the parallel flow path, and the amount of specimen collected from the subject can be reduced. Etc.) can be improved.
  • the SPFS immunofluorescence measurement system 100 that has the sensor chip 10 and performs the detection of the cardiac troponin I and the detection of the cardiac troponin I autoantibody in parallel on the same specimen will have the same detection.
  • the system can detect myocardial troponin I and myocardial troponin I autoantibodies, and can reduce measurement errors due to different detection devices and detection timings. As a result, the reliability of the measured value of cardiac troponin I can be further improved, which can contribute to the provision of data for AMI detection that is faster, more accurate, and more sensitive.
  • Example 1 When SPFS immunofluorescence measurement and reliability evaluation processing are performed on a serum sample derived from a patient who is positive for cardiac troponin I and positive for cardiac troponin I autoantibody using the SPFS immunofluorescence measurement system 100, first, FIG. As shown in A) and (B), the sample is supplied to the flow path 3 of the sensor chip 10 in the step (a).
  • step (b) the secondary antibody 11 labeled with the fluorescent dye 12 binds to each cardiac troponin I ⁇ 8, and steps (c) and (d) ), Fluorescence of spots 1a and 2a (see FIG. 2A) of both cardiac troponin I 8 and cardiac troponin I autoantibody 9 is observed.
  • the measurement result of the cardiac troponin I of the sample is detected by detecting the cardiac troponin I autoantibody.
  • the low reliability is displayed on the display unit of the SPFS apparatus 10A.
  • Example 2 When SPFS immunofluorescence measurement and reliability evaluation processing are performed on a serum sample derived from a patient who is positive for cardiac troponin I and negative for cardiac troponin I autoantibodies by the SPFS immunofluorescence measurement system 100, first, FIG. As shown in A) and (B), the sample is supplied to the flow path 3 of the sensor chip 10 in the step (a).
  • the myocardial troponin I autoantibody 9 since the myocardial troponin I autoantibody 9 does not exist in the specimen, the myocardial troponin I 8 as an antigen binds only to the cardiac troponin I antibody 1 as shown in FIG. 5 (B). Then, as shown in FIG. 5 (C), in step (b), the secondary antibody 11 labeled with the fluorescent dye 12 is bound to the cardiac troponin I 8 bound to the cardiac troponin I antibody 1. In steps (c) and (d), only the fluorescence of the spot 1a of the cardiac troponin I antibody 1 is observed.
  • the measurement value of the cardiac troponin I autoantibody is lower than the second reference value. Is displayed on the display unit of the SPFS apparatus 10A.
  • Example 3 When the SPFS immunofluorescence measurement system 100 performs SPFS immunofluorescence measurement and reliability evaluation processing on a serum sample derived from a patient who is negative for cardiac troponin I and positive for cardiac troponin I autoantibody, FIG. 6 (A) And as shown to (B), the said test substance is supplied with respect to the flow path 3 of the sensor chip 10 by process (a).
  • the myocardial troponin I 8 serving as an antigen is already bound to the cardiac troponin I autoantibody 9 and is supplied to the flow path 3 in this state.
  • the epitope recognized by the cardiac troponin I antibody 1 is covered with the cardiac troponin I autoantibody 9 and does not bind to the cardiac troponin I antibody 1 as shown in FIG.
  • an epitope recognized by cardiac troponin I antibody 1 that is not covered by cardiac troponin I autoantibody 9 binds to cardiac troponin I antibody 1.
  • the ratio to the whole is low, and illustration in FIG. 6 is omitted.
  • step (b) the secondary antibody 11 labeled with the fluorescent dye 12 is bound to the cardiac troponin I 8 bound to the cardiac troponin I autoantibody 9.
  • steps (c) and (d) only the fluorescence of the spot 2a of the cardiac troponin I autoantibody-bound antibody 2 is observed.
  • the measurement result of the cardiac troponin I of the sample is detected by detecting the cardiac troponin I autoantibody.
  • the low reliability is displayed on the display unit of the SPFS apparatus 10A.
  • Example 4 When the SPFS immunofluorescence measurement system 100 performs SPFS immunofluorescence measurement and reliability evaluation processing on a serum sample derived from a patient having negative cardiac troponin I and negative cardiac troponin I autoantibodies, FIG. And as shown to (B), the said test substance is supplied with respect to the flow path 3 of the sensor chip 10 at a process (a).
  • the measurement value of the cardiac troponin I autoantibody is lower than the second reference value. Is displayed on the display unit of the SPFS apparatus 10A.
  • FIG. 8A shows a sensor chip 50 according to the prior art and an SPFS immunofluorescence measurement system (partially not shown) equipped with the same.
  • the sensor chip 50 has only the cardiac troponin I antibody 1 and does not have the cardiac troponin I autoantibody binding antibody 2 as compared with the sensor chip 10.
  • the SPFS immunofluorescence measurement system according to the prior art does not have a means for performing reliability evaluation processing or the like.
  • FIG. 8 (A) and (B) As shown in FIG. 4, the sample is supplied to the flow path 3 of the sensor chip 50, but a part of the cardiac troponin I 8 combined with the cardiac troponin I autoantibody 9 is not detected, and the cardiac muscle It cannot be determined that the reliability of the troponin I measurement result data is reliable.
  • the cardiac troponin I antibody 8 in which the epitope recognized by the cardiac troponin I antibody 1 is covered with the cardiac troponin I autoantibody 9 does not bind to the cardiac troponin I antibody 1.
  • myocardial troponin I in which the epitope recognized by cardiac troponin I antibody 1 is not obscured by cardiac troponin I autoantibody 9 binds to cardiac troponin I antibody 1.
  • illustration in FIG. 9 is omitted.
  • the first capturing means 1 detects cardiac troponin I, but may also detect other target substances such as cardiac troponin T.
  • SYMBOLS 1 1st acquisition means 1a Spot 2 2nd acquisition means 2a Spot 3 Channel 4 Metal film 5 Transparent support body (prism) 5a Plane portion 5b Prism portion 5c Entrance surface 5d Exit surface 6 Pump connection portion 7 Liquid reservoir 8 Myocardial troponin I 9 Myocardial Troponin I Autoantibody 10 Sensor Chip 10A SPFS Device 11 Secondary Antibody 12 Fluorescent Dye 13 Liquid Storage Well 14 Liquid Pump 15 Channel Forming Body 16 Surface Plasmon Resonance [SPR] Detection Unit 17 Optical Path Switching Mirror 18 Linear Polarizing Plate 19 Light source 20 Filter replacement means 21 Cut filter 22 Neutral filter 23 Detector 50 Sensor chip (prior art) 100 SPFS immunofluorescence measurement system

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Abstract

La présente invention a pour objet de réaliser une puce de détection qui détecte la troponine I cardiaque plus rapidement, plus précisément et avec une haute sensibilité, et au moyen de laquelle la taille de l'échantillon prélevé sur le sujet (patient, etc.) peut être réduite (faible invasivité), et de réaliser un système de mesure d'immunofluorescence par SPFS utilisant ladite puce de détection. La présente puce (10) de détection, destinée à être utilisée lors d'une mesure d'immunofluorescence par SPFS, comporte un premier moyen (1) de capture capable de se fixer spécifiquement à la troponine I cardiaque contenue dans un spécimen provenant du subject, et un deuxième moyen (2) de capture capable de se fixer à un auto-anticorps contre la troponine I cardiaque; le présent système (100) de mesure d'immunofluorescence par SPFS est un système d'immunodiagnostic utilisant ladite puce (10) de détection.
PCT/JP2013/082414 2012-12-19 2013-12-03 Puce de détection et système de mesure d'immunofluorescence par spfs WO2014097877A1 (fr)

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JP2015172512A (ja) * 2014-03-11 2015-10-01 学校法人北里研究所 生体試料中の疾患関連自己抗体の検出方法、キット及び検出装置
WO2016031412A1 (fr) * 2014-08-25 2016-03-03 コニカミノルタ株式会社 Procédé de réaction, procédé de détection et dispositif de détection
JP2018054495A (ja) * 2016-09-29 2018-04-05 コニカミノルタ株式会社 血液検査用センサチップ、血液検査装置及び血液検査方法
JP2018189523A (ja) * 2017-05-08 2018-11-29 国立大学法人電気通信大学 計測用デバイス及び計測センサ
CN111308099A (zh) * 2020-03-17 2020-06-19 北京利德曼生化股份有限公司 一种快速定量检测全血中cTnI的微流控荧光免疫芯片
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