WO2014077142A1 - Spécimen de test de chromatographie, procédé de test de chromatographie, et membrane - Google Patents

Spécimen de test de chromatographie, procédé de test de chromatographie, et membrane Download PDF

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Publication number
WO2014077142A1
WO2014077142A1 PCT/JP2013/079740 JP2013079740W WO2014077142A1 WO 2014077142 A1 WO2014077142 A1 WO 2014077142A1 JP 2013079740 W JP2013079740 W JP 2013079740W WO 2014077142 A1 WO2014077142 A1 WO 2014077142A1
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Prior art keywords
membrane
marker
chromatographic test
test piece
sample
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PCT/JP2013/079740
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English (en)
Japanese (ja)
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鶴紀 田村
幸司 宮崎
新一 村松
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コニカミノルタ株式会社
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Priority to JP2014546938A priority Critical patent/JPWO2014077142A1/ja
Publication of WO2014077142A1 publication Critical patent/WO2014077142A1/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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N21/47—Scattering, i.e. diffuse reflection
    • 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
    • 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
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/86—Investigating moving sheets
    • 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
    • G01N33/54386—Analytical elements

Definitions

  • the present invention relates to a chromatographic test piece, a chromatographic test method, and a membrane.
  • a chromatographic test piece that is supplied with a sample and developed in a predetermined development direction is used.
  • the chromatographic test piece includes a housing case that opens upward, and a membrane that is accommodated in the housing case and that allows the sample to be transferred and developed in a predetermined development direction.
  • test lines and control lines are provided in a strip shape at intervals as a plurality of detection target areas for detecting the presence or absence or amount of the analyte.
  • the test line is a line on which a capture ligand (antigen-specific antibody) that specifically captures a complex of an analyte (eg, antigen) and a labeled ligand (eg, antibody) in a sample is immobilized.
  • the control line is a line on which a capture ligand (labeled antibody-specific antibody) that specifically captures the labeled ligand is immobilized.
  • test line when performing a chromatographic test, if the position of the test line is not known, there is a problem that it takes time to obtain a test result. Specifically, when the test line is not colored, it takes time to confirm that there is no color in the area other than the test line unless the position of the test line is known. Further, when a test line is colored, if there are a plurality of test lines, it takes time to confirm which test line is colored by data analysis or image analysis.
  • the first technique has been proposed in the technical field of chromatographic tests in which the detection or quantification of an analyte is performed by detecting the fluorescence generated from the detection target region, and the fluorescence is transmitted from the control line regardless of the presence or amount of the analyte. Utilizing this phenomenon, the position of the control line is detected by fluorescence detection, and then the position of the test line is specified using this position as a reference position (see, for example, Patent Document 1).
  • a marker for specifying a position is provided in advance in a housing case for storing a membrane, and the position of the test line is specified using the position of the marker as a reference position.
  • the position of the control line that is, the reference position is not accurately specified, and as a result, the position of the test line is accurately specified. It will disappear.
  • the positional relationship between the marker position (reference position) and the test line changes, and the position of the test line is changed. It will not be accurately identified.
  • the detection method of the marker and the detection method of the color in the detection target region are different, for example, when detecting the color of the detection target region visually while detecting the marker with fluorescence generated from the marker, When detecting the color of the detection target region with the fluorescence generated from the detection target region while visually detecting the marker, the detection of the marker and the detection of the color of the detection target region are performed by separate work / equipment. This must be done, increasing the cost of the test.
  • a marker for specifying the position is provided on the membrane separately from the control line.
  • the marker is simply provided on the membrane, the marker component flows due to the development of the sample.
  • the exact position of the marker cannot be specified, and the position of the test line cannot be specified correctly, or the void in the membrane is blocked with the marker component, and the sample development due to capillary action is hindered.
  • the complexation reaction between the analyte in the sample and the labeled ligand and the complex capture reaction in the test line are hindered, resulting in a decrease in analyte detection accuracy.
  • the present invention has been made in view of the circumstances as described above, and a chromatographic test piece, a chromatogram capable of accurately specifying the position of each detection target region without reducing the detection accuracy of the analyte. It is an object of the present invention to provide a graphic test method and a membrane.
  • a chromatographic test piece reflecting one aspect of the present invention, Used for detecting or quantifying analytes in a sample in a chromatographic test using a fluorescently labeled ligand.
  • the sample is supplied and developed in a predetermined development direction, and a scan is performed to detect the presence or absence of fluorescence.
  • a plurality of detection target areas for detecting the presence or absence or amount of the analyte, arranged in a state of being separated in the development direction A marker that includes a cationic material to produce fluorescence; Is provided.
  • chromatographic test strip of the present invention is used to detect or quantify the analyte in the sample.
  • the membrane reflecting one aspect of the present invention Used for detecting or quantifying analytes in a sample in a chromatographic test using a fluorescently labeled ligand.
  • the sample is supplied and developed in a predetermined development direction, and a scan is performed to detect the presence or absence of fluorescence.
  • a membrane provided in a chromatographic test piece The membrane
  • the sample to be supplied is propagated and developed in the development direction, In the scan area where scanning is performed, A plurality of detection target areas for detecting the presence or absence or amount of the analyte, arranged in a state of being separated in the development direction, A marker that includes a cationic material to produce fluorescence; Is provided.
  • FIG. 1A is a perspective view showing an example of an external configuration of a chromatographic test piece 9 (hereinafter referred to as a test piece 9), and FIG. 1B is a side view.
  • the test piece 9 is used for detecting or quantifying the analyte in the sample in the chromatographic test.
  • the test piece 9 is supplied with the sample and developed, and in the chromatography test apparatus 1 (see FIG. 2) described later. A scan for detecting the presence or absence of fluorescence is performed.
  • the test piece 9 is formed in a rectangular plate shape that is long in the development direction K of the sample. As shown in FIG. 1B, the test piece 9 is in a case (not shown) that opens upward. And a test strip 95.
  • test strip 95 is formed in a rectangular sheet shape that is long in the developing direction K, and a sample pad 90 is provided at one end of the upper surface, and an absorption pad 91 is provided at the other end.
  • the sample pad 90 is a portion where a liquid sample is dropped and supplied. Further, the absorption pad 91 is a part that absorbs the sample dropped on the sample pad 90 and develops the sample in the deployment direction K by capillary action.
  • a conjugate pad 92 is disposed on the downstream side in the development direction K with respect to the sample pad 90.
  • the conjugate pad 92 includes a fluorescently labeled ligand that binds to the analyte contained in the sample. While moving the sample supplied from the sample pad 90 to the downstream side in the development direction K, the analyte in the sample. Is bound to a fluorescently labeled ligand. Details of the sample, the analyte, and the fluorescently labeled ligand will be described later.
  • a membrane 96 extends from the lower surface of the conjugate pad 92 to the lower surface of the absorption pad 91, and the sample supplied from the conjugate pad 92 is transmitted and developed in the development direction K. Yes.
  • the membrane 96 is made of a microporous inactive substance (a substance that does not react with an analyte, various ligands, various phosphors, etc.) that exhibits capillary action and develops the sample as soon as the sample is added.
  • the membrane 96 a fibrous or non-woven fibrous matrix composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose, cellulose derivatives such as cellulose acetate, or the like , Filter paper, glass fiber filter paper, cloth, cotton and the like.
  • a membrane composed of a cellulose derivative or nylon, a filter paper, a glass fiber filter paper, etc. are preferably used, more preferably a nitrocellulose membrane, a mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, a nylon membrane, a filter paper. Is used.
  • the scan area 900 is a rectangular area and is located on the inner side of both ends of the membrane 96 in the development direction K. Further, in FIG. 1 and FIGS. 3 and 4 described later, for the sake of convenience, the detection target region 93 and the marker 94 are shown so as to be visible, but may be provided so as not to be visible.
  • the three detection target regions 93 are formed in a band shape and are spaced apart from each other in the development direction K by a predetermined interval, and are orthogonal to the development direction K, that is, the width direction of the test piece 9 (hereinafter referred to as detection). Extending in the line direction Z).
  • the interval between the detection target areas 93 is constant at about 2 mm, and the length (width) of the detection target areas 93 in the development direction K is about 1 mm to 3 mm and equal to each other. .
  • the two upstream detection target areas 93 are test lines 93a, and the most downstream detection target area 93 is a control line 93b.
  • the test line 93a is an area also called a detection zone.
  • a capture ligand that specifically captures the complex of the analyte and the fluorescently labeled ligand in the sample is immobilized on the test line 93a.
  • one test line 93a captures a complex of an A-type influenza antigen as an analyte and a fluorescently labeled ligand
  • the other test line 93a is an analyte.
  • a complex of a type B influenza antigen as a light and a fluorescently labeled ligand is captured.
  • control line 93b is an area also referred to as a reference zone.
  • a capture ligand that specifically captures the fluorescently labeled ligand is immobilized on the control line 93b.
  • test line 93a and control line 93b are formed on the membrane 96 by the same coating apparatus (for example, an ink jet printer). Further, as the capture ligands in the test line 93a and the control line 93b, conventionally known ligands can be used according to the type of analyte or fluorescent labeling ligand.
  • the marker 94 is a region where fluorescence is generated, and in the present embodiment, the marker 94 is formed in a band shape and extends in a direction orthogonal to the development direction K, that is, the detection line direction Z.
  • the marker 94 is disposed downstream of at least one detection target region 93 in the development direction K among the three detection target regions 93. In the present embodiment, the entire three detection target regions 93 are arranged. It is arrange
  • the distance between the most downstream detection target region 93 and the marker 94 is equal to the interval between the detection target regions 93, and the length (width) of the marker 94 in the development direction K is the length of the detection target region 93. It is equal to (width).
  • the marker 94 described above contains a phosphor, and preferably further contains a carrier carrying the phosphor.
  • these phosphors and carriers those described later as phosphors and carriers (insoluble particles) in the fluorescently labeled ligand can be used.
  • the wavelength region of the fluorescence generated from the marker 94 and the wavelength region of the fluorescence generated from the detection target region 93 overlap at least a part of wavelengths.
  • at least one of the phosphor and the carrier contained in the marker 94 is cationic. Specifically, when the marker 94 does not contain a carrier, the phosphor is cationic, and when the marker 94 contains a carrier, the phosphor is cationic or the carrier is cationic.
  • the phosphor of the marker 94 has an indocyanine skeleton represented by the following chemical structural formula, and emits fluorescence with excitation light in the range of 600 to 800 nm.
  • examples of the cationic carrier include latex particles introduced with a primary amine.
  • examples of such a carrier include “Polybead Amino Microspheres” (particle size 0.20 ⁇ m, manufactured by Polysciences, Inc., where “Polybead” is a registered trademark).
  • cationic phosphors examples include BODIPY dyes, dyes having an indocyanine skeleton, coumarin dyes (such as AMCA), anilinonaphthalene sulfonic acid (ANS) dyes, Prodan dyes, Hoechst dyes, oranges. Yellow, YOYO dye (orange yellow dimer), thiazole orange, TOTO dye (thiazole orange dimer), and the like.
  • phosphors having an indocyanine skeleton include, for example, Alexa Fluor 647 (where "Alexa Fluor” is a registered trademark), Cy5, Cy5.5, “S0830” (manufactured by DKSH Japan Ltd.) It is a cyanine dye.
  • the above markers 94 are formed on the membrane 96 by the same coating apparatus as the test line 93a and the control line 93b.
  • the sample is not particularly limited as long as it can contain proteins, saccharides, nucleic acids, various physiologically active substances and the like as analytes. Examples thereof include biological samples that can contain the target analyte, food extracts, and the like. Biological samples include whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin exudate, tissue, cells, and stool. And the like.
  • the sample may be pretreated with an analyte in order to easily cause a specific binding reaction between the fluorescently labeled ligand or the capture ligand of the test line 93a and the analyte.
  • pretreatment include chemical treatments using various chemicals such as acids, bases, surfactants, and physical treatments using heating, stirring, ultrasonic waves, etc. It may be used.
  • the analyte is a substance that is not normally exposed on the surface, such as an influenza virus NP antigen
  • a nonionic surfactant may be used in consideration of specific binding reaction, for example, binding reactivity between ligand and analyte such as antigen-antibody reaction. .
  • the sample may be appropriately diluted with a solvent (water, physiological saline, buffer, or the like) used in a normal immunological analysis method or a water-miscible organic solvent.
  • a solvent water, physiological saline, buffer, or the like
  • Analytes include tumor markers, signaling substances, hormones and other proteins (including polypeptides, oligopeptides, etc.), nucleic acids (single or double stranded DNA, RNA, polynucleotides, oligonucleotides, PNA ( And other molecules such as lipids, test substances 93a, and other molecules such as lipids, and other molecules such as lipids (including oligosaccharides, polysaccharides, sugar chains, etc.) or sugar chains.
  • CEA carcinoembryonic antigen
  • HER2 protein HER2 protein
  • PSA prostate specific antigen
  • CA19-9 ⁇ -fetoprotein
  • IPA immunity Inhibitory acidic protein
  • CA15-3 CA125
  • estrogen receptor progesterone Scepter
  • IPA immunity Inhibitory acidic protein
  • CA15-3 CA125
  • estrogen receptor progesterone Scepter
  • fecal occult blood troponin I, troponin T, CK-MB, CRP
  • HCG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • syphilis antibody influenza virus, human hemoglobin, chlamydia antigen , Group A ⁇ -streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like.
  • the fluorescently labeled ligand is obtained by labeling a ligand that specifically binds to an analyte with fluorescent particles containing a phosphor.
  • a ligand is a molecule or molecular fragment that recognizes an analyte contained in a sample or is recognized by the analyte and can specifically bind to the analyte.
  • molecules or molecular fragments include nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., or nucleosides, nucleotides and their modified molecules, although they may be single-stranded.
  • protein polypeptide, oligopeptide, etc.
  • amino acid including modified amino acid
  • carbohydrate oligo, etc., polysaccharide, substance containing sugar chain, etc.
  • lipid or these The modified molecule, complex, etc. are mentioned.
  • antibody includes a polyclonal antibody or a monoclonal antibody, an antibody obtained by genetic recombination, and an antibody fragment.
  • the fluorescent particle means a particle in which a phosphor is directly or indirectly fixed to a carrier as an insoluble particle by chemical or physical bonding or adsorption.
  • the fluorescently labeled ligand may be one in which fluorescent particles are directly bonded to the ligand, or one in which the ligand and fluorescent particles are bonded via a linker molecule, The ligand may be fixed to insoluble particles.
  • the insoluble particles (carrier) constituting the fluorescent particles for example, synthetic polymer particles, inorganic compound particles, or polysaccharide particles are used.
  • the synthetic polymer particles are not particularly limited, and examples thereof include latex particles and polylactic acid particles, and latex particles are preferable.
  • the material of the latex particles is not particularly limited.
  • the inorganic compound particles are not particularly limited, and examples thereof include metal particles such as gold, silver and platinum, or metal oxide particles such as metal colloid particles, porous glass particles, silica and alumina.
  • the polysaccharide particles are not particularly limited, and examples thereof include agarose particles, dextran particles, cellulose particles, and chitosan particles.
  • the phosphor contained in the fluorescent particles is not particularly limited.
  • fluorescent particles when excited with light having a wavelength in the range of 200 to 700 nm (ultraviolet to near infrared light) as excitation light, Examples thereof exhibit fluorescence having a wavelength within the range of 400 to 900 nm (visible to near infrared light fluorescence).
  • Specific phosphors include fluorescein dye molecules, rhodamine dye molecules, AlexaFluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes.
  • Examples thereof include a dye molecule, an NBD dye molecule, a pyrene dye molecule, a Texas Red dye molecule, and a cyanine dye molecule. More specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein, 6-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6- Carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor405,
  • phosphors that emit fluorescence with excitation light in the range of 600 to 800 nm have indocyanine skeletons such as Cy3.5, Alixa Fluor 647, Cy5, Cy5.5, AlexaFluor 680, Cy7, AlexaFluor 790.
  • indocyanine skeletons such as Cy3.5, Alixa Fluor 647, Cy5, Cy5.5, AlexaFluor 680, Cy7, AlexaFluor 790.
  • organic dyes such as acriflavine and DDAO
  • cyanine derivatives such as brilliant blue and brilliant green
  • fluorescent proteins such as allophycocyanin.
  • the insoluble particles constituting the fluorescent particles are particles made of a polymer compound such as latex particles
  • the phosphor is exposed to the surface of the particles or as long as it can be detected at the time of fluorescence detection. It may be kneaded so that it exists near the surface of the particle, or may be adsorbed on the surface of the particle.
  • the method of immobilizing the ligand on fluorescent particles or insoluble particles can be roughly classified into a method of physically adsorbing the ligand to the fluorescent particles or insoluble particles and a method of covalent bonding as a chemical bond.
  • the former includes, for example, a method in which the above-mentioned ligand is added to a solution in which silica particles or gold particles are colloidally dispersed as insoluble particles, and then left for a predetermined time for physical adsorption.
  • the method has the advantage of simple operation.
  • the latter includes, for example, a method in which a carboxyl group introduced on the particle surface of fluorescent particles or insoluble particles and an amino group of the above ligand are bonded by an amide bond using a condensing agent, or a fluorescent particle or There is a method of binding an insoluble particle and the ligand, and such a method has an advantage that the ligand can be introduced quantitatively and irreversibly into a fluorescent particle or an insoluble particle.
  • a blocking agent such as bovine serum albumin solution is added to block the particle surface where the antibody is not bound. Is preferred.
  • FIG. 2 is a perspective view showing an example of the external configuration of the chromatography test apparatus 1.
  • the chromatography test apparatus 1 detects or quantifies an analyte on a test piece 9, and includes an insertion slot 10, a switch group 11, a display 12, and a printer unit 13. ing.
  • the insertion port 10 is an opening for inserting the test piece 9 into the chromatography test apparatus 1 or discharging the test piece 9 in the chromatography test apparatus 1.
  • the switch group 11 has a plurality of switches for giving various instructions to the chromatography test apparatus 1.
  • the display 12 displays various types of information such as the analysis result of the test piece 9, and is an LCD (Liquid Crystal Display) in the present embodiment.
  • the printer unit 13 prints out various information such as the analysis result of the test piece 9.
  • FIG. 3 is a schematic diagram showing an example of the internal configuration of the chromatography test apparatus 1.
  • the chromatography test apparatus 1 includes a light source 2, an irradiation optical system 3, a line sensor 4, a detection optical system 5, a scanning device 6, and a control unit 7.
  • the light source 2 emits light and is a laser light source in the present embodiment.
  • the wavelength of the laser light source is preferably in the range of 630 nm to 780 nm, although it depends on the type of fluorescently labeled ligand used.
  • the irradiation optical system 3 is an optical system that guides the light emitted from the light source 2 to the test piece 9 and emits the irradiation light S with respect to the test piece 9.
  • the lenses 30, 32, A band-pass filter 31 and an aperture 33 are included.
  • the irradiation light S refers to a portion of the light irradiated from the light source 2 toward the test piece 9 until it hits the test piece 9 after passing through the irradiation optical system 3.
  • the lens 30 converts the light emitted from the light source 2 into parallel light and makes it incident on the band-pass filter 31.
  • the bandpass filter 31 passes only the frequency components that excite the fluorescently labeled ligand and the marker 94 of the test piece 9 to generate fluorescence among the frequency components of the light emitted from the light source 2.
  • the lens 32 collects the light that has passed through the bandpass filter 31 on the test piece 9.
  • the aperture 33 prevents the unnecessary light from hitting the test piece 9 by blocking the peripheral portion of the convergent light emitted from the lens 32.
  • the line sensor 4 is a light receiving sensor that performs photoelectric conversion by a plurality of pixels arranged in a line, and is arranged extending in a predetermined longitudinal direction Y. As described above, by using the line sensor 4 as the light receiving sensor, it is possible to simplify the control of the signal processing and the configuration of the scanning mechanism as compared with the case where the area sensor or the photodiode is used as the light receiving sensor.
  • a conventionally known sensor such as a line CCD, a line CMOS, or a one-dimensional photodiode can be used.
  • the detection optical system 5 is an optical system that guides the detection target light T generated in the test piece 9 due to the irradiation light S to the line sensor 4.
  • the lenses 50 and 52 and the bandpass filter 51 are used. And have.
  • the detection target light T is a portion of the light generated by the test piece 9 due to the light emitted from the light source 2 until it enters the detection optical system 5 and is excited. It includes fluorescence generated by the fluorescently labeled ligand and the marker 94, reflected light from the test piece 9, and scattered light.
  • the lens 50 is configured to convert the detection target light T generated in the test piece 9 into parallel light and enter the bandpass filter 51.
  • the band-pass filter 51 is configured to pass only the frequency components of the fluorescence generated by the fluorescently labeled ligand and the marker 94 among the frequency components of the detection target light T.
  • the lens 52 condenses the light that has passed through the bandpass filter 51 on the line sensor 4.
  • the scanning device 6 scans at least one optical element in the irradiation optical system 3 or the detection optical system 5 or the test piece 9 in the scanning direction X as a scanning object, so that the irradiation spot of the irradiation light S is irradiated on the test piece 9.
  • a plate-like support base 60 that supports the test piece 9 from below and a motor 61 that reciprocates the support base 60 in the scanning direction X are used.
  • the test piece 9 is scanned in the scanning direction X together with the support base 60 as a scanning object.
  • the motor 61 a conventionally known motor such as a stepping motor, a voice coil motor, or a DC motor can be used.
  • the scanning direction X is parallel to the developing direction K, and is orthogonal to the longitudinal direction Y of the line sensor 4.
  • the control unit 7 executes predetermined processing according to an instruction input from the switch group 11, performs an instruction to each functional unit, data transfer, and the like, and comprehensively controls the chromatography test apparatus 1, Various operations are performed.
  • the control unit 7 detects or quantifies the analyte based on the output signal from the line sensor 4.
  • 3 illustrates a state in which the control unit 7 is connected to the light source 2, the line sensor 4, the motor 61, and the like, but the connection target of the control unit 7 is not limited to these.
  • the test piece 9 is inserted into the insertion port of the chromatography test apparatus 1. 10 is inserted.
  • the time from when the sample is dropped to when the fluorescently labeled ligand is bound to the control line 93b is generally about 10 minutes after the sample is dropped.
  • the control unit 7 performs a scanning process (hereinafter referred to as a preliminary scanning process) for detecting the position and width of the marker 94 on the test piece 9. Specifically, in this preliminary scanning process, first, the control unit 7 drives the motor 61 to move the test piece 9 by one step in the scanning direction X, thereby irradiating the irradiation spot on the test piece 9 with the irradiation light S. Is moved from the downstream end to the upstream end side in the scan area 900.
  • a predetermined amount smaller than the width of the marker 94 in the scanning direction X is set in advance.
  • control unit 7 After the control unit 7 turns on the light source 2 to irradiate the light, it acquires light reception signal data at each pixel from the line sensor 4 and turns off the light source 2. Then, the control unit 7 repeats the above process until the entire area of the marker 94 is detected in the scanning direction X, and then ends the preliminary scanning process.
  • the control unit 7 calculates and specifies the positions of the test line 93a and the control line 93b based on the position of the marker 94 detected by the preliminary scanning process, and also controls the control line. Based on the amount of light at 93b, the power of the light source 2 is changed, the threshold used for detection of the test line 93a is changed, and then scanning processing for detecting the presence or amount of the analyte in the test line 93a, that is, main scanning. Process. As this main scanning process, a conventionally known process can be used.
  • the control unit 7 changes the power of the light source 2 with reference to the light amount at the marker 94 instead of using the light amount at the control line 93b as a reference, and changes the threshold used for detection of the test line 93a. Also good.
  • the reference light amount is not affected by the intensity of the capture reaction of the labeled ligand.
  • the housing case and the membrane 96 are separated from each other. It is possible to prevent the position specifying accuracy of the test line 93a from being affected by the alignment accuracy. Further, since the marker 94 generates fluorescence, the position of the test line 93a is determined by the strength of the capture reaction, unlike the conventional case where the capture region of the fluorescently labeled ligand that flows along with the sample, that is, the position of the control line is the reference position. It is possible to prevent the accuracy from being affected. Accordingly, the position of each detection target region 93 can be accurately specified.
  • the marker 94 since the marker 94 generates fluorescence, the position of the marker 94 can be detected using equipment for detecting fluorescence from the detection target region 93. Therefore, since it is not necessary to provide new equipment for detecting and quantifying the position of the marker 94, it is possible to prevent the test cost from increasing. In addition, since the marker 94 is cationic, it is less likely to be water-soluble in the sample as compared with the case of being anionic.
  • the component of the marker 94 can be prevented from flowing due to the development of the sample, the exact position of the marker 94 cannot be specified, and the position of the test line cannot be specified accurately, or the void in the membrane 96 Is blocked by the component of the marker 94 and the development of the sample due to capillarity is inhibited, the complexation reaction between the analyte in the sample and the fluorescently labeled ligand, and the complex capture reaction in the test line 93a is inhibited. It is possible to prevent the light detection accuracy and quantitative accuracy from being lowered.
  • the marker for detecting the fluorescence from the detection target region 93 is used.
  • the position 94 can be reliably detected. Therefore, it is possible to reliably prevent the cost of the test from being increased by providing new equipment for detecting and quantifying the position of the marker 94.
  • the marker 94 is disposed downstream of the at least one detection target region 93 in the development direction K, the analyte in the sample and the fluorescently labeled ligand that flows along with the sample are located at the position of the marker 94. Even when the dam is blocked, data relating to detection and quantification of the analyte can be obtained in at least one detection target region 93.
  • the marker 94 is disposed on the downstream side in the development direction K from the whole of the plurality of detection target regions 93, the analyte in the sample and the fluorescently labeled ligand that flows along with the sample are the markers 94. Even in the case of being dammed up at the position, data relating to detection and quantification of the analyte can be obtained in all the detection target regions 93.
  • the position specifying accuracy of the test line 93a is affected by the alignment accuracy of each member. Can be prevented.
  • the marker 94 contains a phosphor carrier, it is possible to reliably prevent the components of the marker 94 from flowing due to the development of the sample. Therefore, the exact position of the marker 94 cannot be specified, and the position of the test line 93a cannot be specified accurately, or the complexing reaction between the analyte in the sample and the fluorescently labeled ligand, or the capture reaction of the complex in the test line. It is possible to reliably prevent the detection accuracy and quantitative accuracy of the analyte from being impaired.
  • the phosphor since the phosphor is supported on the carrier, the phosphor is not easily dissolved in water due to the cationic nature, and even if it is difficult to uniformly coat the membrane 96, the phosphor and the carrier
  • the composite can be uniformly dispersed in a solvent and applied uniformly on the membrane 96.
  • the reference light amount is different from the case where the light amount at the control line 93b is used as the reference light amount. Is not affected by the intensity of the capture reaction of the fluorescently labeled ligand, so that the analyte can be detected and quantified accurately.
  • the operation check and calibration of the chromatography test apparatus 1 are performed using this amount of light. Can do.
  • the test piece 9A in the present modification includes a test strip 95A and a transparent cover member 901 disposed on the test strip 95A as shown in FIG. ing.
  • the cover member 901 does not necessarily have to be transparent as long as fluorescence generated in the test strip 95A can be detected from above the cover member 901.
  • the test strip 95A has a membrane 96A.
  • the membrane 96A has the same configuration as the membrane 96 in the above embodiment except that the marker 94 is not provided.
  • the cover member 901 is a member that covers the upper surface of the test strip 95A, and preferably covers the region from the upper surface of the conjugate pad 92 to the most downstream detection target region 93 of the upper surface of the membrane 96A. ing. It is preferable that there is a space between the cover member 901 and the upper surface of the membrane 96A so that a sample containing an analyte can be developed on the membrane 96A, and if necessary, the lower surface of the cover member 901 and / or the membrane 96A. A spacer or the like may be formed on the upper surface. However, the cover member 901 may cover the upper surface of the membrane 96A with an adhesive. For example, in the hydrophilic film, the pressure-sensitive adhesive layer may be formed on the entire surface or the peripheral edge of the surface facing the membrane 96A.
  • a liquid impermeable sheet made of PET or the like may be used, or a hydrophilic film may be used.
  • the hydrophilic film refers to a film in which at least the both surfaces of the film are in contact with the membrane 96A (lower side), and the “hydrophilic” refers to a water contact angle in the air. It means having a surface of 50 ° or less, preferably 40 ° or less, more preferably 30 ° or less.
  • the “water contact angle” is a static contact angle formed between water in the air and the film surface, and more specifically, an angle formed between water and the film surface generated when water is dropped on the film. is there. In other words, the “water contact angle” is an angle formed between the gas-liquid interface of the water droplet and the film surface at the point where the gas-liquid interface of the water droplet contacts the film surface.
  • This water contact angle can be evaluated by measuring the water contact angle in air by various conventionally known methods such as the ⁇ / 2 method. More specifically, the water contact angle is determined by dropping 2 ⁇ l of pure water in an atmosphere of 23 ° C. and 50% RH and using a contact angle meter (Kyowa Interface Chemical Co., Ltd., model: CA-X150). , ⁇ / 2 method.
  • the hydrophilic film is not particularly limited as long as it has a hydrophilic surface as described above.
  • the hydrophilic film may be, for example, a film made of a hydrophilic polymer such as cellophane. Further, it may be a film obtained by adding a modifier to a hydrophobic polymer and obtained from a polymer or a composition imparted with hydrophilicity, or at least a membrane 96A of a film made of a hydrophobic polymer. It may be a film obtained by surface-treating the surface on the side in contact with the surface to impart hydrophilicity.
  • the hydrophobic polymer has a water contact angle of more than 50 ° on the surface, for example, polyester resins such as polyethylene terephthalate and polyethylene naphthalate, polyolefin resins such as polyethylene, polypropylene, polyisobutylene and polybutadiene, polycarbonate
  • polyester resins such as polyethylene terephthalate and polyethylene naphthalate
  • polyolefin resins such as polyethylene, polypropylene, polyisobutylene and polybutadiene
  • polycarbonate examples include polymethyl methacrylate resin, polyvinyl chloride resin, cellulose ester resin, polyimide resin, polyamide resin, polysulfone resin, AS resin, ABS resin, melamine resin, polyvinyl acetate, and polyvinyl fluoride.
  • polyvinyl alcohol polyvinyl pyrrolidone
  • modified polyvinyl alcohol a copolymer with acrylamide, unsaturated carboxylic acid, sulfonic acid monomer, cationic monomer, unsaturated silane monomer, unsaturated ethylene oxide monomer, etc.
  • Modified polyvinylpyrrolidone copolymer with vinyl acetate, unsaturated imidazole, vinyl caprolactone, etc.
  • hydrophilic acrylic polymer polyacrylic acid, polyacrylates, polyacrylamide, polyisopropylacrylamide, polyformamide, polyethylene glycol, Starch, modified starch, gelatin, casein, algin, cellulose compounds (carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose), hydrophilic Hydrophilic polymers such as tan polymers, cellulose compounds, water-soluble nylon, polyvinyl formal, polyvinyl acetal, polyvinyl benz
  • the modifier is a hydrophilic polymer or polysaccharide
  • a crosslinking agent such as a carbodiimide compound, a polyisocyanate compound, an oxazoline compound, or an epoxy compound is added to the hydrophobic polymer together with the modifier to make the polymer hydrophobic.
  • the polymer and the modifier may be cross-linked.
  • inorganic oxide particles may be added to the hydrophobic polymer as the inorganic oxide, or an inorganic oxide in which the inorganic oxide is dispersed in water.
  • a sol may be added.
  • the inorganic oxide as the modifier may have a photocatalytic action such as amorphous titania or hydrous titania.
  • Surface treatment of hydrophobic film includes irradiation with radiation, ultraviolet rays, arc, direct current glow, high frequency, micro-electrochemical treatment such as plasma treatment by corona discharge, etc., coupling with hydrophilic groups such as hydroxyl groups and carboxyl groups Chemical surface treatment with an agent or the like can be mentioned.
  • a coupling agent a silane coupling agent, a titanium coupling agent, etc. are mentioned.
  • the color tone and transparency of the cover member 901 are preferably such that the absorption and scattering of irradiation light and excitation light do not substantially affect the measurement of fluorescence intensity.
  • a hydrophilic transparent film is used as such a cover member 901
  • specific examples thereof include a TAC film having a high light transmittance, and a polyolefin film such as polycarbonate and polypropylene.
  • the sample can be rapidly developed as compared with the case of using the liquid impermeable sheet, and the sample development time is short.
  • the analyte can be detected or quantified with good sensitivity.
  • a marker 94A is provided on the upper surface of the cover member 901 described above.
  • the marker 94 ⁇ / b> A is a region that includes a cationic material and generates fluorescence, like the marker 94 in the above embodiment, and extends in the detection line direction Z. Unlike the marker 94 in the above-described embodiment, the marker 94A does not contact the sample developed on the membrane 96A. It is possible.
  • the detection target region 93 is provided in the membrane 96, and the marker 94 is the cover member 901. Therefore, it is possible to reliably prevent the component of the marker 94 from flowing due to the development of the sample. Accordingly, the exact position of the marker 94 cannot be specified, and the position of the test line 93a cannot be specified correctly, or the void in the membrane 96 is blocked with the components of the marker 94, and the sample development due to capillary action is inhibited. To prevent degradation of analyte detection accuracy and quantification accuracy due to inhibition of the complexation reaction between the analyte in the sample and the fluorescently labeled ligand and the complex capture reaction in the test line 93a. Can do.
  • the detection target areas 93 are described as three, but may be two, four or more. Also, two of the three detection target areas 93 have been described as the test lines 93a and one as the control lines 93b, but all three may be the test lines 93a.
  • test strip 95 has been described as a long rectangular shape, other arbitrary shapes such as an L shape may be used. In this case, the sample is developed along the shape of the test strip 95 from the addition position.
  • the sample is added to one end of the test strip 95, it may be added to the central portion.
  • the sample is developed on the test strip 95 from the addition position toward the end (periphery).
  • the detection target region 93 and the marker 94 have been described as shapes extending in a direction orthogonal to the development direction K, at least one of the extending directions may be the other direction, or a shape that does not extend in one direction, for example, It is good also as a circle.
  • one marker 94 is provided on the test strip 95, a plurality of markers 94 may be provided.
  • the same number of markers 94 as the detection target area 93 may be disposed in the vicinity of the detection target area 93.
  • the marker 94A is described as being provided on the upper surface of the cover member 901. However, as long as the marker 94A is not swept away by the sample developed on the membrane 96A, the marker 94A is disposed on the lower surface of the cover member 901. It may be provided.
  • insoluble carrier dispersion carrying cationic phosphor As a cationic phosphor contained in the marker 94, 2- [5- [1,1-Dimethyl-3- (3-methyl-butyl) -1,3-dihydro-benzo [e] indol-2-ylidene] -penta-1,3-dienyl] -1,1-dimethyl-3- (3-methyl-butyl) -1H-benzo [e] indolium hexafluorophosphate (product number “S0830”, manufactured by DKSH Japan Ltd.) .
  • This phosphor has an indocyanine skeleton.
  • polystyrene latex particles having a particle size of 220 nm (product number “IMMUTEX P2219” (where “IMMUTEX” is a registered trademark), manufactured by JSR Corporation) were used as a water-insoluble carrier carrying a phosphor.
  • polystyrene latex particles “P2219” were suspended in 400 ⁇ L of DMSO (dimethyl sulfoxide) and heated to 120 ° C.
  • DMSO dimethyl sulfoxide
  • 20 ⁇ L of a DMSO solution of a cationic phosphor “S0830” (the phosphor concentration was 100 mg / mL) was added and heated at 120 ° C. for 1 hour.
  • 5 mL of a water / ethanol mixed solvent is added to this suspension to suspend it, and the particles are collected by centrifugation (15000 ⁇ G, 30 min).
  • an aqueous solution of 0.5 wt% Triton-X100 (surfactant) is used.
  • the particles were washed to prepare a dispersion of latex particles carrying a cationic phosphor (however, the concentration of latex particles was 0.1 wt%).
  • test line 93a As the detection target region 93, 11.7 mm from the downstream end in the scan region 900 out of the upper surface of the membrane 96 made of trinitrocellulose (Millipore, white, width 60 mm ⁇ length 350 mm), Separate capture ligands were applied at 14.7 mm and 17.7 mm positions, respectively.
  • the test line 93a formed at the most upstream position is the test line 93a 1
  • the test line 93a formed at the second most upstream position is the test line 93a 2
  • the test is formed at the most downstream position.
  • the capture ligand of the test line 93a 1 is defined as capture ligand (1)
  • the analyte captured by the capture ligand (1) is defined as analyte (1)
  • the capture ligand of the test line 93a 2 is the capture ligand (2)
  • the analyte captured by the capture ligand (2) is the analyte (2)
  • the capture ligand of the test line 93a 3 is the capture ligand (3
  • the analyte captured by the capture ligand (3) is defined as analyte (3).
  • the latex particle dispersion adjusted as described above was applied to a position 20.7 mm from the downstream end in the scan region 900.
  • a positive pressure spray device “BioJet” manufactured by BioDot was used for coating.
  • a test strip (hereinafter referred to as a test strip of the example) 95 was manufactured using this membrane 96.
  • a test strip (hereinafter, referred to as a test strip of the comparative example) 95H in which the marker 94 is not formed was manufactured.
  • the test line 93a is shown so as not to be visible, and in FIG. 5A, the marker 94 is shown so as to be visible.
  • the present invention is suitable for a chromatographic test piece, a chromatographic test method, and a membrane that need to specify the position of the detection target region accurately.

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Abstract

L'invention concerne un spécimen de test de chromatographie (9) qui est mis en œuvre afin de déceler ou de déterminer quantitativement un analyte à l'intérieur d'un échantillon par un test de chromatographie faisant appel à un ligand à marquage fluorescent, et dans lequel un échantillon est fourni et déployé dans une direction de déploiement K prédéfinie, et simultanément une analyse a lieu afin de détecter la présence ou l'absence de fluorescence. Ce spécimen de test de chromatographie (9) est équipé : d'une pluralité de régions objets de détection (93) destinées à détecter la présence/absence ou la quantité de fluorescence, qui est disposée dans un état de séparation dans la direction de déploiement K à l'intérieur d'une région d'analyse (900) dans laquelle l'analyse à lieu ; et d'un marqueur (94) contenant un matériau cationique, et générant une fluorescence.
PCT/JP2013/079740 2012-11-14 2013-11-01 Spécimen de test de chromatographie, procédé de test de chromatographie, et membrane WO2014077142A1 (fr)

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Cited By (5)

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WO2018043687A3 (fr) * 2016-08-31 2018-04-26 積水化学工業株式会社 Particules fluorescentes pour agent de diagnostic et réactif de dosage immunologique l'employant
WO2018168907A1 (fr) * 2017-03-14 2018-09-20 デンカ生研株式会社 Évprouvette immunochromatographique pouvant réguler le développement d'échantillons et servant à extraire et mesurer des antigènes carbohydrates
WO2019058903A1 (fr) * 2017-09-20 2019-03-28 帝人株式会社 Substrat de support de chromatographie, support de chromatographie et bande d'immunochromatographie
JPWO2018179983A1 (ja) * 2017-03-30 2019-04-11 帝人株式会社 イムノクロマトグラフ用血球分離膜及びイムノクロマトグラフ用ストリップ
WO2022210312A1 (fr) * 2021-04-01 2022-10-06 富士フイルム株式会社 Dispositif testeur

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JP2012198083A (ja) * 2011-03-22 2012-10-18 Konica Minolta Advanced Layers Inc クロマトグラフィー分析装置及びクロマトグラフィー分析方法
JP2012215494A (ja) * 2011-04-01 2012-11-08 Konica Minolta Medical & Graphic Inc イムノクロマト測定法ならびにそれに用いられるキットおよびシステム

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JP2001004613A (ja) * 1999-06-18 2001-01-12 Matsushita Electric Ind Co Ltd クロマトグラフィー分析装置
WO2012086376A1 (fr) * 2010-12-20 2012-06-28 コニカミノルタオプト株式会社 Dispositif d'analyse par chromatographie
JP2012198083A (ja) * 2011-03-22 2012-10-18 Konica Minolta Advanced Layers Inc クロマトグラフィー分析装置及びクロマトグラフィー分析方法
JP2012215494A (ja) * 2011-04-01 2012-11-08 Konica Minolta Medical & Graphic Inc イムノクロマト測定法ならびにそれに用いられるキットおよびシステム

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018043687A3 (fr) * 2016-08-31 2018-04-26 積水化学工業株式会社 Particules fluorescentes pour agent de diagnostic et réactif de dosage immunologique l'employant
JPWO2018043687A1 (ja) * 2016-08-31 2019-08-15 積水化学工業株式会社 診断薬用蛍光粒子及びそれを用いた免疫測定試薬
WO2018168907A1 (fr) * 2017-03-14 2018-09-20 デンカ生研株式会社 Évprouvette immunochromatographique pouvant réguler le développement d'échantillons et servant à extraire et mesurer des antigènes carbohydrates
JP2018151331A (ja) * 2017-03-14 2018-09-27 デンカ生研株式会社 検体の展開を制御し得る、糖鎖抗原を抽出し測定するためのイムノクロマト試験片
JPWO2018179983A1 (ja) * 2017-03-30 2019-04-11 帝人株式会社 イムノクロマトグラフ用血球分離膜及びイムノクロマトグラフ用ストリップ
WO2019058903A1 (fr) * 2017-09-20 2019-03-28 帝人株式会社 Substrat de support de chromatographie, support de chromatographie et bande d'immunochromatographie
WO2022210312A1 (fr) * 2021-04-01 2022-10-06 富士フイルム株式会社 Dispositif testeur

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