WO2017094825A1 - Éprouvette immunochromatographique - Google Patents

Éprouvette immunochromatographique Download PDF

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Publication number
WO2017094825A1
WO2017094825A1 PCT/JP2016/085690 JP2016085690W WO2017094825A1 WO 2017094825 A1 WO2017094825 A1 WO 2017094825A1 JP 2016085690 W JP2016085690 W JP 2016085690W WO 2017094825 A1 WO2017094825 A1 WO 2017094825A1
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Prior art keywords
immunochromatographic test
test piece
control line
antibody
detection reagent
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PCT/JP2016/085690
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English (en)
Japanese (ja)
Inventor
裕 川南
裕二 辻
圭三 米田
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東洋紡株式会社
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Priority to JP2017554170A priority Critical patent/JP6834979B2/ja
Publication of WO2017094825A1 publication Critical patent/WO2017094825A1/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

Definitions

  • the present invention relates to a test piece and a detection method used for detection of a detection target substance contained in a biological sample by an immunochromatography method, a kit for detection, and a method for producing the kit.
  • a biological sample is developed from one end of a porous support on which an antibody that specifically binds to the detection target substance is immobilized, moving through the porous support by capillary action, and is labeled in the process.
  • This is an immunoassay method for determining the presence or absence of a substance to be detected in a biological sample by accumulating with the passage of time by binding the substance to be detected and an antibody by an immune reaction and locally developing color.
  • Immunochromatography method includes simple operation and visual determination.
  • In vitro diagnostic agents such as pregnancy test diagnostic agents and influenza diagnostic agents that make use of these advantages are widespread worldwide, and are attracting attention as POCT (PointPOf Care Testing) (for example, Patent Document 1).
  • POCT PointPOf Care Testing
  • POCT refers to a clinical test performed by a medical worker alongside the subject. Unlike clinical tests performed in central laboratories of large-scale hospitals, POCT has a growing need in the medical field because test results can be obtained instantaneously on the spot.
  • Sandwich method is a typical quantification method.
  • two types of antibodies having different epitopes in the detection target substance are used.
  • One antibody is used as a detection reagent sensitized with detection particles such as colloidal gold, colored latex, and fluorescent particles.
  • the other antibody is used for measurement of the test line as a capture antibody fixed to one end of the porous support.
  • an antibody that specifically binds to the detection antibody is immobilized on one end opposite to the porous support and used for measurement of the control line.
  • the detection target substance contained in the biological sample develops from one end of the porous support, moves while forming an immune complex with the detection antibody, and is captured and colored by contacting the capture antibody on the test line.
  • the free detection reagent that has not formed an immune complex with the detection target substance passes through the test line, and is captured by the control line antibody and develops color.
  • the concentration of the detection target substance can be quantified by using these color development intensities by using a device such as a chromatographic reader.
  • a common detection reagent is used to detect the test line and the control line, so there is a problem that the color intensity of the control line changes due to the concentration of the detection target substance. It was.
  • a high-concentration detection target substance is present in a biological sample, a large amount of detection particles form an immune complex with the detection target substance and are captured by the upstream test line, and the color intensity of the test line increases.
  • the amount of free detection reagent is small, there is a problem that the color intensity of the control line is low.
  • control line may be used to measure the amount of detection reagent flowing into the porous support. Therefore, when a certain amount of detection reagent develops on the porous support. It is desired that the color intensity is always constant. As described above, if the color intensity of the control line changes due to the concentration of the detection target substance, the measurement accuracy may be greatly affected.
  • Patent Document 1 describes a method in which the position of a control line arranged downstream from a test line is determined first, and then a test line is detected based on the position.
  • this method takes a long time to measure, and in addition, if the test particles and the control line have the same detection particles, the color intensity of the downstream control line changes due to the color intensity of the upstream test line. As a result, measurement accuracy may be affected.
  • An object of the present invention is to provide an immunochromatographic analysis method that is faster and more accurate than before, an immunochromatographic test piece and kit using the analysis method, and a method for producing the kit.
  • the present inventor uses a control line detection reagent that is not affected by the concentration of a detection target substance in a biological sample as a detection reagent for detecting a control line. It was found that the color development intensity of the control line can be stabilized. Further, the present inventors have found that the measurement time can be shortened by arranging the control line upstream of the test line and detecting the color intensity of the control line first as the configuration of the immunochromatographic test piece. Based on this knowledge, the inventor further made an invention related to an immunochromatographic test strip containing a control line detection reagent, a method for producing the test strip, and a method for quantifying a detection target substance in a biological sample using the immunochromatographic test strip. completed.
  • An immunochromatographic test piece for quantifying a detection target substance in a biological sample a) a conjugation pad impregnated with a detection reagent that specifically binds to the detection target substance and a control line detection reagent; and b) A porous membrane pad on which an antibody for specifically capturing the control line detection reagent is immobilized on the upstream side and an antibody for capturing the detection target substance is immobilized on the downstream side.
  • An immunochromatographic test piece comprising: (2) The immunochromatographic test strip according to (1), wherein the control line detection reagent contains detection particles bound with a biotin-labeled protein.
  • the immunochromatographic test piece according to (2) wherein the protein is a protein derived from a microorganism or an animal.
  • the immunochromatographic test piece according to (3) wherein the protein derived from the microorganism is Blocking Peptide Fragment.
  • the immunochromatographic test piece according to (3) wherein the animal-derived protein is bovine serum albumin or casein.
  • An immunochromatographic analysis kit comprising the immunochromatographic test strip according to any one of (1) to (6), a sample diluent, and an analyzer. (9) The method for producing an immunochromatographic test piece according to any one of (1) to (6).
  • the color intensity of the control line can be stabilized without depending on the concentration of the detection target substance in the biological sample, an immunochromatographic test strip with high measurement accuracy is provided quickly. be able to.
  • An embodiment of the immunochromatographic test piece of the present invention is an immunochromatographic test strip including a detection reagent that specifically binds to a detection target substance and a control line detection reagent.
  • control line detection reagent is a detection particle to which a biotin-labeled protein is bound.
  • a biotin labeling method is the N-hydroxysuccinimide method.
  • the detection particles are not particularly limited, and examples thereof include gold colloids, latex particles, and fluorescent particles. Examples of the method for binding the biotin-labeled protein and the detection particles include a physical adsorption by a hydrophobic bond or a binding method via a covalent bond.
  • the protein used for the biotin-labeled protein is not particularly limited, but is preferably a protein derived from a microorganism or a protein derived from an animal.
  • a protein derived from microorganisms Blocking Peptide Fragment is preferable, and as a protein derived from animals, bovine serum albumin or casein is preferable. If these proteins are commercially available, they may be used, or may be produced by separately known methods.
  • the molecular size is not particularly limited, the average molecular weight is preferably 100 kDa or less. Generally, as the molecular size of the protein is smaller, the amount of protein binding to one detection particle increases, so the performance such as sensitivity becomes higher.
  • the type of the detection particles is not particularly limited, but color developing particles and fluorescent particles can be used.
  • the color developing particles include metal particles and latex particles.
  • the metal particles include gold colloid, silver colloid, and platinum colloid.
  • the particle size of the metal particles is preferably 1 nm to 100 nm. More preferably, it is 20 to 80 nm, and further preferably 40 to 60 nm.
  • latex particles include those made of materials such as polystyrene, polymethyl methacrylate, and acrylic acid polymer.
  • the particle size of the latex particles is preferably 25 nm to 500 nm. More preferably, it is 50 to 250 nm, and still more preferably 80 to 200 nm.
  • a fluorescent particle What consists of materials, such as a polystyrene, polymethyl methacrylate, polyvinyl toluene, a silica, can be illustrated. Although it does not restrict
  • the biotin labeling method of the biotin-labeled protein is not particularly limited, and an N-hydroxysuccinimide method can be exemplified.
  • an N-hydroxysuccinimide method can be exemplified.
  • the carboxyl group of biotin is condensed in the presence of an N-hydroxyamine compound in the presence of a dehydrating condensing agent, selectively activated, and labeled via the amino group and amide bond of the protein. Can do.
  • N-hydroxyamine compounds examples include N-hydroxysuccinimide, N-hydroxynorbornene-2,3-dicarboxylic acid imide, 2-hydroxyimino-2-cyanoacetic acid ethyl ester, and 2-hydroxyimino.
  • -2-Cyanoacetamide N-hydroxypiperidine, N-hydroxyphthalimide, N-hydroxyimidazole, N-hydroxymaleimide and the like. Two or more of these compounds may be used.
  • N-hydroxysuccinimide hereinafter sometimes referred to as NHS
  • NHS is more preferable because it is relatively inexpensive, easily available, and has a track record in the field of peptide synthesis.
  • Examples of the dehydrating condensing agent used in the condensation reaction include 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride, 1-cyclohexyl- (2-morpholin-4-ethyl) -carbodiimide, meso p-toluenesulfonate, and the like. Can be mentioned. Among them, 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (hereinafter sometimes referred to as EDC / HCl) is more preferable because it has a track record as a general-purpose water-soluble condensing agent in the field of peptide synthesis. It is.
  • the amount of biotin introduced into the biotin-labeled protein can be controlled by adjusting the molar ratio of protein to biotin. In order to improve performance such as sensitivity, it is desirable that the amount of biotin introduced is high. More specifically, the number of moles of biotin relative to the number of moles of protein is preferably 1.0 mole times or more, more preferably 5.0 mole times or more, and 10.0 mole times or more. More preferably. Since the molar ratio is an example, it may be appropriately increased or decreased according to the type of protein and the actual sensitivity.
  • the method for binding the biotin-labeled protein and the detection particles is not particularly limited, and examples include a physical adsorption by a hydrophobic bond or a binding method via a covalent bond.
  • the treatment is preferably performed at a pH near the isoelectric point of the biotin-labeled protein.
  • covalent bonding the bonding method varies depending on the functional group on the surface of the detection particle. For example, when the functional group present on the surface of the detection particle is an amino group, bonding is performed using the N-hydroxysuccinimide method described above. can do.
  • the reaction temperature is not particularly limited, but is preferably 10 ° C or higher, more preferably 20 ° C or higher. Moreover, 50 degrees C or less is preferable and 40 degrees C or less is more preferable.
  • the reaction time varies depending on the reaction temperature, but is preferably 1 hour or longer, more preferably 2 hours or longer. Moreover, Preferably it is 24 hours or less, More preferably, it is 12 hours or less.
  • the unreacted N-hydroxyamine compound and the dehydrating condensing agent contained in the reaction solution can be easily separated from the aqueous solvent by filtration or centrifugation.
  • the detection reagent that specifically binds to the detection target substance is an antibody that can bind to the detection target substance in a biological sample (hereinafter, sometimes referred to as a detection antibody) bound to detection particles.
  • a detection antibody varies depending on the type of the substance to be detected, but if it is commercially available, it may be used, or may be produced by a known method.
  • the molecular size is not particularly limited.
  • an antibody for capturing a detection target substance may be a commercially available one, or may be produced by a known method. May be.
  • a recognition site of the capture antibody for the detection target substance an antibody that recognizes a site different from the detection antibody is preferably used.
  • the molecular size is not particularly limited.
  • Examples of capture antibodies for measuring human chorionic gonadotropin include, for example, Anti hCG — 1646157 (V-24), Human (Rabbit) (manufactured by Santa Cruz Biotechnology, Inc.), Anti hCG / Chorionic Gonadotropin (Study) Biosciences, Inc.), Anti CGB / hCG ⁇ , Human (Mouse) (LifeSpan Biosciences, Inc.), Anti-Corionic Gonadotropin, Human (Rabbit), HC Laboratories, Inc. (EY Laboratories, Inc.) Mouse) (manufactured by Boster Immunoleader), Anti CG ⁇ ( ⁇ HCG), Human (Rabbit) (manufactured by R & D Systems Inc.), and the like.
  • an antibody that captures the control line detection reagent is preferably an antibody that captures biotin (hereinafter sometimes referred to as an anti-biotin antibody).
  • an anti-biotin antibody As the anti-biotin antibody, if it is commercially available, it may be used, or may be produced by a known method. The molecular size is not particularly limited.
  • Anti-biotin antibodies include, for example, Anti-Biotin antibody (Genetex, Inc.), Anti-Biotin, Goat-Poly (Betyl Laboratories, Inc.), and IgG Fractional Monoclonal Mouse In-Chemical Anti-Biot ) And the like.
  • porous membrane pad In the present invention, by fixing the anti-biotin antibody on the upstream side (control line) of the porous membrane pad and the capture antibody on the downstream side (test line), a porous membrane pad usable for immunochromatography or the like is obtained. Can be produced. These antibodies can be easily immobilized on a porous membrane pad generally used in immunochromatography and the like.
  • the material of the porous membrane pad is not particularly limited, and examples thereof include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, and nylon. Membranes, fabrics, fibrous or non-woven matrices made of these materials are suitable. A cellulose derivative membrane is preferably used, and a nitrocellulose membrane is more preferably used.
  • 1 is a porous membrane pad
  • 2 is a sample pad
  • 3 is a conjugation pad
  • 4 is an absorption pad
  • 5 is an anti-biotin antibody immobilization site (control line)
  • 6 is an immobilization of a capture antibody.
  • part (test line) and 7 show an adhesive sheet.
  • the porous membrane pad is made of an elongated test piece-like nitrocellulose membrane having a width of 4 mm and a length of 60 mm.
  • the conjugation pad of the immunochromatographic test strip is impregnated with a detection reagent that specifically binds to the detection target substance and a control line detection reagent.
  • a control line 5 in which an anti-biotin antibody is immobilized is formed at a position of about 10 mm from the end of the nitrocellulose membrane on the chromatographic development start side.
  • a test line 6 in which a capture antibody is immobilized is formed at a position of about 15 mm.
  • the porous membrane pad 1 is attached to the middle position of the pressure-sensitive adhesive sheet 7, and a part of the conjugation pad 3 overlaps the upstream side of the porous membrane pad 1.
  • the sample pad 2 is connected to the upstream side of the conjugation pad 3 so as to partially overlap
  • the absorbent pad 4 is connected to the downstream side of the porous membrane pad 1 so as to partially overlap. Can be produced.
  • the sample pad 2 is not particularly limited as long as it is a material that can rapidly absorb and spread a biological sample, and examples thereof include cellulose filter paper, nonwoven fabric, and porous materials such as polyethylene and polypropylene. . Among these, filter paper is preferable.
  • the conjugation pad 3 is not particularly limited as long as it is made of a material that can hold the detection reagent in a dry state and can quickly release the detection reagent along with the development of the biological sample.
  • a material that can hold the detection reagent in a dry state and can quickly release the detection reagent along with the development of the biological sample examples thereof include glass fiber, cellulose filter paper, and polyester non-woven fabric. Of these, glass fiber is preferred.
  • the absorbent pad 4 is not particularly limited as long as it is made of a material that can rapidly absorb and hold a biological sample, and examples thereof include cellulose filter paper, nonwoven fabric, and porous materials such as polyethylene and polypropylene. . Among these, cellulose filter paper is preferable.
  • the immunochromatographic test piece has an appropriate opening portion for injecting a biological sample and an opening portion for quantifying the amount of detected particles at the positions of the sample pad 2, the control line 5 and the test line 6, respectively. It may be housed in a plastic housing case.
  • the method for producing the immunochromatographic test piece of the present invention is not particularly limited.
  • a conjugation pad can be prepared by uniformly applying a certain amount of a detection reagent to a band-shaped glass fiber and then drying it at a suitable temperature in a thermostat for a certain period of time.
  • a certain amount of the capture antibody and the anti-biotin antibody may be applied in a line on the porous membrane pad.
  • coating on a porous membrane pad is not specifically limited, A commercially available immunochromatography dispenser can be used.
  • the coating amount of the capture antibody and the anti-biotin antibody is preferably 10 ⁇ l or less per 10 cm line length, more preferably 8 ⁇ l or less, and even more preferably 6 ⁇ l or less. Moreover, it is preferably 4 ⁇ l or more.
  • the concentration of the capture antibody and the anti-biotin antibody is preferably 0.1 to 2.0 mg / ml, more preferably 0.2 to 1.8 mg / ml, and still more preferably 0.3 to 1.6 mg / ml.
  • the capture antibody and anti-biotin antibody coated on the porous membrane pad can be immobilized very easily simply by drying.
  • the drying temperature is not particularly limited, but is preferably 20 ° C. to 80 ° C., more preferably 30 to 70 ° C., and further preferably 40 to 60 ° C.
  • the drying time varies depending on the drying temperature, but is 5 to 120 minutes, preferably 10 to 60 minutes.
  • a method for performing measurement using the immunochromatographic test piece as described above is not particularly limited.
  • the following method is exemplified.
  • a biological sample is mixed with a suitable developing solvent (specimen diluent) as necessary to obtain a developable mixed solution, and then the mixed solution is injected (dropped) onto the sample pad 2.
  • the mixed solution passes through the sample pad 2 and develops on the conjugation pad 3.
  • the mixed solution dissolves the detection reagent impregnated in the conjugation pad 3 and the control line detection reagent, and the detection target substance and the detection reagent in the biological sample form an immune complex and spread on the porous membrane pad. .
  • the control line detection reagent is captured and collected by the anti-biotin antibody, and the control line 5 develops color. Further, the liquid mixture passes through the control line 5 and reaches the test line 6. Here, the immune complex in the mixed solution is captured and collected by the capture antibody, and the test line 6 develops color. The remaining liquid mixture is finally absorbed by the absorbent pad.
  • the color intensity of the control line is not particularly limited, but may be any color intensity that can be detected visually or with an immunochromatographic reader. It is preferably 10 mAbs to 500 mAbs, more preferably 50 mAbs to 450 mAbs, and still more preferably 100 mAbs to 400 mAbs.
  • the immunochromatographic analysis kit of the present invention includes, in addition to an immunochromatographic test piece, a specimen diluent and an analyzer for the purpose of pretreatment and dilution of a biological sample.
  • the immunochromatographic test piece may be housed in a plastic housing case having a specimen dropping part for dropping a biological sample and an observation window for measuring the color intensity of the detection line.
  • Example 1 Preparation of hCG detection reagent
  • WRGH1 colloidal gold solution
  • 10 mM Tris-HCl pH 9.2 50 ⁇ l of 10 mM Tris-HCl pH 9.2 was added and vortexed.
  • a commercially available anti-hCG monoclonal antibody product name: Anti CGB / hCG ⁇ , Human (Mouse), product number: LS-C196902-100, manufactured by LifeSpan Biosciences, Inc.
  • test line having a line width of about 1 mm at a position of about 15 mm from one end (upstream side) of the nitrocellulose membrane
  • 0.5 mg / ml of an anti-hCG monoclonal antibody product name: Anti-Chorionic Gondotropin, Human (Rabbit), Product number: AT-2601-2, manufactured by EY Laboratories, Inc.
  • 50 mM KH 2 PO 4 , pH 7.0 + 5 wt% sucrose 50 mM KH 2 PO 4 , pH 7.0 + 5 wt% sucrose
  • the test piece for hCG measurement was prepared with a slide configuration generally used in an immunochromatograph. Specifically, a sample pad (trade name: Surewick (Merck Millipore Corporation)), a conjugation pad (trade name: Surewick (Merck Millipore Corporation)), a nitrocellulose membrane (trade name: Hi-Flow) Plus type HF120 (manufactured by Merck Millipore) and absorption pad (trade name: Surewick (manufactured by Merck Millipore)) were connected in series. Next, the sample was cut into a test piece having a width of about 4 mm and a length of about 60 mm using a cutter.
  • the prepared hCG measurement specimen was placed on a horizontal base. Next, the prepared 100 IU / L hCG sample was taken with a 40 ⁇ l pipette and dropped onto the sample pad. The color intensity was measured using an immunochromatography reader (C10060-10 (manufactured by Hamamatsu Photonics)) every 2 minutes after the sample was dropped. The results are shown in FIG. The color intensity of the control line reached 310 mAbs 12 minutes after the start of measurement, and was 308 mAbs after 14 minutes. Therefore, it was judged that the control line had stabilized 14 minutes after the start of measurement. Next, the color intensity of the test line after 14 minutes was 303 mAbs. The time required for measuring the control line and the test line was about 14 minutes.
  • control line is arranged upstream of the test line by using an immunochromatographic test piece in which the detection reagent that specifically binds to the detection target substance and the control line detection reagent are impregnated in the conjugation pad. Even so, it was possible to perform quantitative analysis quickly, easily, and with high measurement accuracy.

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Abstract

Le problème décrit par la présente invention est de fournir une éprouvette immunochromatographique contenant un agent de détection de ligne témoin sur lequel la concentration d'une substance à détecter n'influe pas. La solution selon l'invention porte sur une éprouvette immunochromatographique permettant de doser une substance à détecter dans un échantillon biologique, l'éprouvette immunochromatographique étant caractérisée en ce qu'un réactif de détection de ligne témoin et un réactif de détection de liaison spécifique à la substance à détecter sont imprégnés conjointement dans un tampon de conjugaison, des anticorps de capture spécifique du réactif de détection de ligne témoin sont immobilisés sur un côté amont d'un tampon à membrane poreuse de l'éprouvette immunochromatographique, et des anticorps de capture de la substance à détecter sont immobilisés sur le côté aval de ce dernier.
PCT/JP2016/085690 2015-12-02 2016-12-01 Éprouvette immunochromatographique WO2017094825A1 (fr)

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WO2019138898A1 (fr) * 2018-01-09 2019-07-18 東洋紡株式会社 Éprouvette d'immunochromatographie, kit de mesure et procédé de mesure
WO2019138899A1 (fr) * 2018-01-11 2019-07-18 東洋紡株式会社 Liquide de dilution d'échantillon de mesure, trousse, et procédé de mesure
WO2020166699A1 (fr) * 2019-02-15 2020-08-20 東洋紡株式会社 Pièce de test immunochromatographique et procédé d'immunochromatographie l'utilisant

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WO2015080286A1 (fr) * 2013-11-29 2015-06-04 積水メディカル株式会社 Procédé de détection assistée par immunochromatographie

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WO2019138898A1 (fr) * 2018-01-09 2019-07-18 東洋紡株式会社 Éprouvette d'immunochromatographie, kit de mesure et procédé de mesure
JPWO2019138898A1 (ja) * 2018-01-09 2021-01-07 東洋紡株式会社 イムノクロマト試験片および測定キットおよび測定方法
JP7352831B2 (ja) 2018-01-09 2023-09-29 東洋紡株式会社 イムノクロマト試験片および測定キットおよび測定方法
WO2019138899A1 (fr) * 2018-01-11 2019-07-18 東洋紡株式会社 Liquide de dilution d'échantillon de mesure, trousse, et procédé de mesure
US11543419B2 (en) 2018-01-11 2023-01-03 Toyobo Co., Ltd. Measurement sample dilution liquid, kit, and measurement method
WO2020166699A1 (fr) * 2019-02-15 2020-08-20 東洋紡株式会社 Pièce de test immunochromatographique et procédé d'immunochromatographie l'utilisant

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