WO2017094825A1 - Immunochromatographic test piece - Google Patents

Immunochromatographic test piece 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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French (fr)
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/en
Publication of WO2017094825A1 publication Critical patent/WO2017094825A1/en

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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.

Abstract

[Problem] To provide an immunochromatographic test piece including a control-line detection agent not affected by the concentration of a substance to be detected. [Solution] An immunochromatographic test piece for assaying a substance to be detected in a biological sample, the immunochromatographic test piece characterized in that a control-line detection reagent and a detection reagent for specifically binding to the substance to be detected are impregnated together in a conjugation pad, antibodies for specifically capturing the control-line detection reagent are immobilized on an upstream side of a porous membrane pad of the immunochromatographic test piece, and antibodies for capturing the substance to be detected are immobilized on the downstream side thereof.

Description

イムノクロマト試験片Immunochromatographic test piece
　本発明は、イムノクロマト法による生体試料中に含まれる検出対象物質の検出に用いる試験片および検出方法、検出のためのキット、当該キットの製造方法に関する。 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.
　イムノクロマト法とは、検出対象物質と特異的に結合する抗体が固定化された多孔質支持体の一端から、生体試料が毛細管現象によって多孔質支持体内を移動しながら展開し、その過程で標識された検出対象物質と抗体が免疫反応によって結合することによって、時間経過に伴って集積し、局所的に発色することで生体試料中の検出対象物質の有無を判定する免疫測定法である。 In immunochromatography, 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.
　イムノクロマト法の利点としては、操作が簡便であることや目視判定が可能であること等が挙げられる。これらの利点を利用した妊娠検査診断薬やインフルエンザ診断薬等の体外診断薬が世界的に普及しており、ＰＯＣＴ（Ｐｏｉｎｔ Ｏｆ Ｃａｒｅ Ｔｅｓｔｉｎｇ）として注目を集めている（例えば、特許文献１）。 Advantages of the immunochromatography method include 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とは、医療従事者が被験者の傍らで行う臨床検査のことをいう。ＰＯＣＴは大規模病院の中央検査室等で行う臨床検査とは異なり、その場で瞬時に検査結果が得られることから医療現場でニーズが高まっている。 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.
　従来のイムノクロマト法は目視判定（定性評価）が一般的であったが、近年、発色強度を測定するクロマトリーダー等の装置を利用することで、生体試料中に含まれる検出対象物質の濃度を定量化する技術が開発されつつある。 In conventional immunochromatography, visual determination (qualitative evaluation) was common, but in recent years, the concentration of the target substance contained in a biological sample has been quantified by using a device such as a chromatographic reader that measures color intensity. Technology is being developed.
　前記定量化の手法として、サンドイッチ法が代表的である。サンドイッチ法では、検出対象物質におけるエピトープの異なる２種類の抗体を利用する。一方の抗体は、金コロイド、着色ラテックス、蛍光粒子等の検出粒子と感作した検出試薬として使用する。他方の抗体は、多孔質支持体の一端に固定した捕捉抗体としてテストラインの測定に使用する。加えて、検出抗体と特異的に結合する抗体を多孔質支持体と反対側の一端に固定化しコントロールラインの測定に使用する。生体試料中に含まれる検出対象物質は、多孔質支持体の一端から展開し、検出抗体と免疫複合体を形成しながら移動し、テストライン上で捕捉抗体と接触して捕捉され発色する。検出対象物質と免疫複合体を形成しなかった遊離の検出試薬は、テストライン上を通過し、コントロールラインの抗体に捕捉され発色する。これらの発色強度をクロマトリーダー等の装置を利用することで検出対象物質の濃度を定量することができる。 サ ン ド イ ッ チ Sandwich method is a typical quantification method. In the sandwich 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. In addition, 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.
　従来のイムノクロマト法では、テストラインとコントロールラインを検出するための検出試薬として共通のものを使用していたので、検出対象物質の濃度に影響を受けてコントロールラインの発色強度が変化する問題があった。生体試料中に高濃度の検出対象物質が存在した場合、多量の検出粒子が検出対象物質と免疫複合体を形成し、上流側のテストラインで捕捉されテストラインの発色強度は高くなる。一方、遊離の検出試薬は少量になるのでコントロールラインの発色強度は低くなるといった問題があった。生体試料中に検出対象物質が低濃度に存在した場合、検出対象物質と免疫複合体を形成した検出粒子が少量になるため、遊離の検出粒子がコントロールラインで多量に捕捉された結果、コントロールラインの発色強度が高くなるといった問題があった。 In the conventional immunochromatography method, 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. When 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. On the other hand, since the amount of free detection reagent is small, there is a problem that the color intensity of the control line is low. When a detection target substance is present in a biological sample at a low concentration, the amount of detection particles that form an immune complex with the detection target substance becomes small, and as a result, a large amount of free detection particles are captured by the control line. There was a problem that the color development intensity of the toner was increased.
　イムノクロマト法を用いた定量化においては、コントロールラインは検出試薬の多孔質支持体への流入量を測定するために使用する場合があるため、一定量の検出試薬が多孔質支持体に展開した場合、発色強度は常に一定であることが望まれる。前記のように検出対象物質の濃度に影響を受けてコントロールラインの発色強度が変化すると測定精度に大きな影響を及ぼすことがある。 In quantification using the immunochromatography method, the 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.
　両ラインの発色強度を迅速に定量する手法としては、ハウジングケースに位置決め用の目印を設けておき、その位置に基づいて両ラインを検出する手法が挙げられる。しかし、この場合、ハウジングケースとイムノクロマト試験片の位置精度が悪いことから、位置決め用の目印と両ラインの位置との間に大きなずれが生じ、正確に検出できないことがあった。他の手法として、特許文献１にはテストラインより下流に配置したコントロールラインの位置を先に決定した後、これに基づいてテストラインを検出する手法が記載されている。しかし、この手法では測定に長時間を要し、加えてテストラインとコントロールラインの検出粒子が共通の場合、下流のコントロールラインの発色強度は上流のテストラインの発色強度の影響を受けて変化することがあるので、測定精度に影響を及ぼすことがあった。 As a method for quickly quantifying the color intensity of both lines, there is a method in which a positioning mark is provided on the housing case and both lines are detected based on the positions. However, in this case, since the positional accuracy of the housing case and the immunochromatographic test piece is poor, there is a case where a large deviation occurs between the positioning mark and the positions of both lines, and the detection cannot be performed accurately. As another method, 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. However, 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.
特開２０１２－１９８０８３号公報JP 2012-198083 A
　本発明の課題は、従来よりも迅速かつ高精度なイムノクロマト分析方法、当該分析方法を用いたイムノクロマト試験片およびキット、キットの製造方法を提供することにある。 SUMMARY OF THE INVENTION 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.
　本発明者は、上記課題を解決するために鋭意研究した結果、コントロールラインを検出するための検出試薬として、生体試料中の検出対象物質の濃度に影響を受けないコントロールライン検出試薬を使用することによりコントロールラインの発色強度を安定化できることを見出した。また、イムノクロマト試験片の構成として、コントロールラインをテストラインよりも上流側に配置し、コントロールラインの発色強度を先に検出することで測定時間を短縮できることを見出した。本発明者はさらに、この知見を基に、コントロールライン検出試薬を含むイムノクロマト試験片、当該試験片の製造方法、当該イムノクロマト試験片を使用した生体試料中の検出対象物質の定量法に係る発明を完成した。 As a result of diligent research to solve the above problems, 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.
　すなわち、代表的な本願発明は以下の通りである。
（１）　生体試料中の検出対象物質を定量するためのイムノクロマト試験片であって、
a)前記検出対象物質と特異的に結合する検出試薬及びコントロールライン検出試薬が含浸されたコンジュゲーションパッド、および
b)上流側にコントロールライン検出試薬を特異的に捕捉するための抗体が固定化されており、かつ下流側に前記検出対象物質を捕捉するための抗体が固定化されている多孔性メンブレンパッド
を有することを特徴とするイムノクロマト試験片。
（２）　前記コントロールライン検出試薬は、ビオチン標識タンパク質が結合した検出粒子を含むことを特徴とする（１）に記載のイムノクロマト試験片。
（３）　前記タンパク質は、微生物由来または動物由来のタンパク質であること特徴とする（２）に記載のイムノクロマト試験片。
（４）　前記微生物由来のタンパク質は、Ｂｌｏｃｋｉｎｇ Ｐｅｐｔｉｄｅ Ｆｒａｇｍｅｎｔであることを特徴とする（３）に記載のイムノクロマト試験片。
（５）　前記動物由来のタンパク質は、ウシ血清アルブミンまたはカゼインであることを特徴とする（３）に記載のイムノクロマト試験片。
（６）　前記コントロールライン検出試薬を特異的に捕捉するための抗体は、抗ビオチン抗体であることを特徴とする（１）～（５）のいずれかに記載のイムノクロマト試験片。
（７）　（１）～（６）のいずれかに記載のイムノクロマト試験片を用いる、生体試料中の検出対象物質を定量する方法。
（８）　（１）～（６）のいずれかに記載のイムノクロマト試験片、検体希釈液および分析装置を含むイムノクロマト分析キット。
（９）　（１）～（６）のいずれかに記載のイムノクロマト試験片の製造方法。 That is, typical inventions of the present application are as follows.
(1) 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.
(3) The immunochromatographic test piece according to (2), wherein the protein is a protein derived from a microorganism or an animal.
(4) The immunochromatographic test piece according to (3), wherein the protein derived from the microorganism is Blocking Peptide Fragment.
(5) The immunochromatographic test piece according to (3), wherein the animal-derived protein is bovine serum albumin or casein.
(6) The immunochromatographic test strip according to any one of (1) to (5), wherein the antibody for specifically capturing the control line detection reagent is an anti-biotin antibody.
(7) A method for quantifying a detection target substance in a biological sample using the immunochromatographic test piece according to any one of (1) to (6).
(8) 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).
　本発明のイムノクロマト試験片によれば、生体試料中の検出対象物質の濃度に依存せず、コントロールラインの発色強度を安定化することができるため、迅速かつ測定精度の高いイムノクロマト試験片を提供することができる。 According to the immunochromatographic test strip of the present invention, since 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.
本発明で使用するイムノクロマト試験片の一例を示す上面図である。It is a top view which shows an example of the immunochromatography test piece used by this invention. 本発明で使用するイムノクロマト試験片の一例を示す側面図である。It is a side view which shows an example of the immunochromatography test piece used by this invention. 本発明における発色強度と測定時間の関係を示すグラフである。It is a graph which shows the relationship between the coloring intensity in this invention, and measurement time. 従来技術における発色強度と測定時間の関係を示すグラフである。It is a graph which shows the relationship between the coloring intensity in a prior art, and measurement time.
　本発明のイムノクロマト試験片の一つの実施形態を説明する。本発明の実施形態としては、検出対象物質と特異的に結合する検出試薬とコントロールライン検出試薬を含むイムノクロマト試験片である。 One embodiment of the immunochromatographic test piece of the present invention will be described. An embodiment 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.
（コントロールライン検出試薬）
　本発明において、コントロールライン検出試薬は、ビオチン標識タンパク質が結合した検出粒子である。ビオチンの標識方法としては、Ｎ－ヒドロキシスクシンイミド法を挙げることができる。検出粒子は、特に制限されないが、金コロイド、ラテックス粒子、蛍光粒子などを例示することができる。ビオチン標識タンパク質と検出粒子の結合方法としては、疎水結合による物理吸着または共有結合を介した結合方法が例示できる。 (Control line detection reagent)
In the present invention, the control line detection reagent is a detection particle to which a biotin-labeled protein is bound. An example of 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.
（タンパク質）
　本発明において、前記ビオチン標識タンパク質に用いるタンパク質は、特に制限されないが、微生物由来のタンパク質や動物由来のタンパク質などが好ましい。微生物由来のタンパク質としては、Ｂｌｏｃｋｉｎｇ Ｐｅｐｔｉｄｅ Ｆｒａｇｍｅｎｔが好ましく、動物由来のタンパク質としては、ウシ血清アルブミンまたはカゼインが好ましい。これらのタンパク質は市販されているものがあればそれを用いても良いし、別途公知の方法で製造しても良い。分子サイズも特に制限されないが、平均分子量で１００ｋＤａ以下が好ましい。一般的に、タンパク質の分子サイズが小さいほど検出粒子１粒子に対するタンパク質の結合量は増加するので、感度などの性能が高くなる。 (protein)
In the present invention, 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. As 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. Although 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.
（検出粒子）
　本発明において、前記検出粒子の種類は、特に制限されないが、発色粒子や蛍光粒子を用いることができる。発色粒子としては、金属粒子、ラテックス粒子などを例示することができる。金属粒子としては、金コロイド、銀コロイド、白金コロイドなどを例示することができる。金属粒子の粒径は、粒径１ｎｍ～１００ｎｍが好ましい。より好ましくは２０～８０ｎｍ、さらに好ましくは４０～６０ｎｍが好ましい。ラテックス粒子としては、ポリスチレン、ポリメタクリル酸メチル、アクリル酸重合体などの材質からなるものを例示することができる。ラテックス粒子の粒径は、粒径２５ｎｍ～５００ｎｍが好ましい。より好ましくは５０～２５０ｎｍ、さらに好ましくは８０～２００ｎｍである。蛍光粒子としては、特に制限されないが、ポリスチレン、ポリメタクリル酸メチル、ポリビニルトルエン、シリカなどの材質からなるものを例示することができる。蛍光色素としては、特に制限されないが、ＦＩＴＣ（フルオレセインイソチオシアネート）、ローダミンＢ誘導体、ヒドロキシクマリンなどを例示することができる。これらの中でも、汎用性が高く、視認性に優れた金コロイドやラテックス粒子を用いることが好ましい。 (Detected particles)
In the present invention, the type of the detection particles is not particularly limited, but color developing particles and fluorescent particles can be used. Examples of the color developing particles include metal particles and latex particles. Examples of 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. Examples of 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. Although it does not restrict | limit especially as 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 | limit especially as a fluorescent dye, FITC (fluorescein isothiocyanate), a rhodamine B derivative, a hydroxycoumarin, etc. can be illustrated. Among these, it is preferable to use gold colloid or latex particles that are highly versatile and excellent in visibility.
（Ｎ－ヒドロキシスクシンイミド法）
　本発明において、前記ビオチン標識タンパク質のビオチンの標識方法は、特に限定されないが、Ｎ－ヒドロキシスクシンイミド法を例示できる。Ｎ－ヒドロキシスクシンイミド法を用いることでビオチンのカルボキシル基をＮ－ヒドロキシアミン系化合物と脱水縮合剤存在下で縮合反応し、選択的に活性化し、タンパク質のアミノ基とアミド結合を介して標識することができる。 (N-hydroxysuccinimide method)
In the present invention, the biotin labeling method of the biotin-labeled protein is not particularly limited, and an N-hydroxysuccinimide method can be exemplified. By using the N-hydroxysuccinimide method, 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－ヒドロキシアミン系化合物）
　前記縮合反応に使用するＮ－ヒドロキシアミン系化合物は、例えば、Ｎ－ヒドロキシスクシンイミド、Ｎ－ヒドロキシノルボルネン－２，３－ジカルボン酸イミド、２－ヒドロキシイミノ－２－シアノ酢酸エチルエステル、２－ヒドロキシイミノ－２－シアノ酢酸アミド、Ｎ－ヒドロキシピペリジン、Ｎ－ヒドロキシフタルイミド、Ｎ－ヒドロキシイミダゾール、Ｎ－ヒドロキシマレイミド等が挙げられる。これら化合物を２種以上用いてもよい。中でも、Ｎ－ヒドロキシスクシンイミド（以下、ＮＨＳと表記する場合がある）が、比較的安価で、入手し易いことやペプチド合成分野等で実績があることから、より好適である。 (N-hydroxyamine compounds)
Examples of the N-hydroxyamine compound used in the condensation reaction 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. Among these, N-hydroxysuccinimide (hereinafter sometimes referred to as NHS) is more preferable because it is relatively inexpensive, easily available, and has a track record in the field of peptide synthesis.
（脱水縮合剤）
　前記縮合反応に使用する脱水縮合剤としては、例えば、１－エチル－３－ジメチルアミノプロピルカルボジイミド塩酸塩、１－シクロヘキシル－（２－モルホニル－４－エチル）－カルボジイミド・メソｐ－トルエンスルホネート等が挙げられる。中でも、１－エチル－３－ジメチルアミノプロピルカルボジイミド塩酸塩（以下、ＥＤＣ・ＨＣｌと表記する場合がある）が、ペプチド合成分野等で汎用的な水溶性縮合剤として実績があることから、より好適である。 (Dehydration condensation agent)
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.
（ビオチンの導入量）
　本発明において、前記ビオチン標識タンパク質におけるビオチンの導入量は、タンパク質とビオチンのモル比を調整することで制御することができる。感度などの性能を高めるためには、ビオチンの導入量は高いものが望ましい。より具体的には、タンパク質のモル数に対してビオチンのモル数が、１．０モル倍以上であることが好ましく、５．０モル倍以上であることがより好ましく、１０．０モル倍以上であることがさらに好ましい。前記モル比は一例であるためタンパク質の種類や現実の感度に応じて適宜増減すれば良い。 (Introduction amount of biotin)
In the present invention, 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.
（ビオチン標識タンパク質と検出粒子の結合）
　本発明において、前記ビオチン標識タンパク質と検出粒子の結合方法は、特に制限されないが、疎水結合による物理吸着あるいは共有結合を介した結合方法が例示できる。疎水結合においては、ビオチン標識タンパク質と検出粒子の表面層で直接的に結合するので、ビオチン標識タンパク質の等電点付近のｐＨで処理することが好ましい。共有結合においては、検出粒子表面の官能基によって結合方法は異なるが、一例を挙げると、検出粒子の表面に存在する官能基がアミノ基の場合は、前述したＮ－ヒドロキシスクシンイミド法を用いて結合することができる。 (Binding of biotin-labeled protein and detection particles)
In the present invention, 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. In the hydrophobic binding, since the biotin-labeled protein is directly bound to the surface layer of the detection particle, the treatment is preferably performed at a pH near the isoelectric point of the biotin-labeled protein. In 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.
　反応温度は、特に限定されないが、好ましくは１０℃以上、より好ましくは２０℃以上である。また、５０℃以下が好ましく、４０℃以下がより好ましい。反応時間は、反応温度によって異なるが、好ましくは１時間以上、より好ましくは２時間以上である。また、好ましくは２４時間以下、より好ましくは１２時間以下である。 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.
　反応液中に含まれる未反応のＮ－ヒドロキシアミン系化合物や脱水縮合剤は、濾過や遠心分離などにより水溶媒から容易に分離することができる。 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.
（検出対象物質と特異的に結合する検出試薬）
　本発明において、前記検出対象物質と特異的に結合する検出試薬は、生体試料中の検出対象物質と結合できる抗体（以下、検出抗体と表記する場合がある）が検出粒子と結合したものである。検出抗体は、検出対象物質の種類によって異なるが、市販されているものがあればそれを用いても良いし、別途公知の方法で製造しても良い。分子サイズも特に制限されない。 (Detection reagent that specifically binds to the target substance)
In the present invention, 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. . The 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.
（検出対象物質を捕捉するための抗体）
　本発明において、検出対象物質を捕捉するための抗体（以下、捕捉抗体と表記する場合がある）は、市販されているものがあればそれを用いても良いし、別途公知の方法で製造しても良い。検出対象物質に対する捕捉抗体の認識部位は、検出抗体とは異なる部位を認識する抗体を用いるのが好ましい。分子サイズも特に制限されない。ヒト絨毛性ゴナドトロピンを測定するための捕捉抗体としては、例えば、Ａｎｔｉ ｈＣＧ_１６４６１５７(Ｖ－２４)，Ｈｕｍａｎ(Ｒａｂｂｉｔ)（Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｉｎｃ．製）、Ａｎｔｉ ｈＣＧ / Ｃｈｏｒｉｏｎｉｃ Ｇｏｎａｄｏｔｒｏｐｉｎ，Ｈｕｍａｎ(Ｍｏｕｓｅ)（ＬｉｆｅＳｐａｎ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ．製）、Ａｎｔｉ ＣＧＢ / ｈＣＧ β ，Ｈｕｍａｎ(Ｍｏｕｓｅ)（ＬｉｆｅＳｐａｎ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ．製）、Ａｎｔｉ Ｃｈｏｒｉｏｎｉｃ Ｇｏｎａｄｏｔｒｏｐｉｎ，Ｈｕｍａｎ(Ｒａｂｂｉｔ)（ＥＹ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ．製）、Ａｎｔｉ β－ＨＣＧ，Ｈｕｍａｎ(Ｍｏｕｓｅ)（Ｂｏｓｔｅｒ Ｉｍｍｕｎｏｌｅａｄｅｒ社製）、Ａｎｔｉ ＣＧ α (α ＨＣＧ)，Ｈｕｍａｎ(Ｒａｂｂｉｔ)（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ Ｉｎｃ．製）等が挙げられる。 (Antibodies for capturing detection target substances)
In the present invention, an antibody for capturing a detection target substance (hereinafter sometimes referred to as a capture antibody) may be a commercially available one, or may be produced by a known method. May be. As the 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 β, Human (Mouse) (LifeSpan Biosciences, Inc.), Anti-Corionic Gonadotropin, Human (Rabbit), HC Laboratories, Inc. (EY Laboratories, Inc.) Mouse) (manufactured by Boster Immunoleader), Anti CG α (α HCG), Human (Rabbit) (manufactured by R & D Systems Inc.), and the like.
（コントロールライン検出試薬を捕捉する抗体）
　本発明において、前記コントロールライン検出試薬を捕捉する抗体は、ビオチンを捕捉する抗体（以下、抗ビオチン抗体と表記する場合がある）を用いるのが好ましい。抗ビオチン抗体としては、市販されているものがあればそれを用いても良いし、別途公知の方法で製造しても良い。分子サイズも特に制限されない。抗ビオチン抗体としては、例えば、Ａｎｔｉ－Ｂｉｏｔｉｎ ａｎｔｉｂｏｄｙ（ＧＥＮＥＴＥＸ，ｉｎｃ．製）、Ａｎｔｉ－Ｂｉｏｔｉｎ， Ｇｏａｔ－Ｐｏｌｙ（Ｂｅｔｈｙｌ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ．製）、ＩｇＧ Ｆｒａｃｔｉｏｎ Ｍｏｎｏｃｌｏｎａｌ Ｍｏｕｓｅ Ａｎｔｉ－Ｂｉｏｔｉｎ（岩井化学薬品株式会社製）等が挙げられる。 (An antibody that captures the control line detection reagent)
In the present invention, 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). 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.
（多孔性メンブレンパッド）
　本発明において、前記抗ビオチン抗体を多孔性メンブレンパッドの上流側（コントロールライン）に、捕捉抗体を下流側（テストライン）に固定化することで、イムノクロマトグラフ等で使用可能な多孔性メンブレンパッドを作製することができる。これらの抗体は、イムノクロマトグラフ等で一般的に使用される多孔性メンブレンパッドに容易に固定化することができる。多孔性メンブレンパッドの材質は、特に限定されないが、セルロース、セルロース誘導体、ニトロセルロース、酢酸セルロース、ポリウレタン、ポリエステル、ポリエチレン、ポリ塩化ビニル、ポリフッ化ビニリデン、ナイロン等が挙げられる。これらの材質で構成された膜、布帛、繊維状又は不織布状マトリックス等が好適である。好ましくはセルロース誘導体の膜が用いられ、より好ましくはニトロセルロース膜が用いられる。 (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.
　続いて、本発明のイムノクロマト試験片の一例を、図面を参照して説明する。図１、２において、１は多孔性メンブレンパッド、２はサンプルパッド、３はコンジュゲーションパッド、４は吸収パッド、５は抗ビオチン抗体の固定化部位（コントロールライン）、６は捕捉抗体の固定化部位（テストライン）、７は粘着シートを示す。 Subsequently, an example of the immunochromatographic test piece of the present invention will be described with reference to the drawings. 1 and 2, 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), and 6 is an immobilization of a capture antibody. A site | part (test line) and 7 show an adhesive sheet.
　図１、２の例では、多孔性メンブレンパッドは、幅４ｍｍ、長さ６０ｍｍの細長い試験片状のニトロセルロース製メンブレンで作製されている。イムノクロマト試験片のコンジュゲーションパッドには、検出対象物質と特異的に結合する検出試薬とコントロールライン検出試薬が含浸されている。ニトロセルロースメンブレンのクロマト展開始点側の末端から約１０ｍｍの位置に抗ビオチン抗体が固定化されたコントロールライン５が形成される。また、同約１５ｍｍの位置に捕捉抗体が固定化されたテストライン６が形成される。 In the example of FIGS. 1 and 2, 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. Further, a test line 6 in which a capture antibody is immobilized is formed at a position of about 15 mm.
　図１、２に示すようなイムノクロマト試験片は、多孔性メンブレンパッド１を粘着シート７の中程の位置に貼着し、多孔性メンブレンパッド１の上流側にコンジュゲーションパッド３の一部が重なるように連接し、さらにコンジュゲーションパッド３の上流側にサンプルパッド２の一部が重なるように連接するとともに、吸収パッド４を多孔性メンブレンパッド１の下流側に一部が重なるように連接することで作製することができる。 In the immunochromatographic test piece as shown in FIGS. 1 and 2, 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. In addition, the sample pad 2 is connected to the upstream side of the conjugation pad 3 so as to partially overlap, and the absorbent pad 4 is connected to the downstream side of the porous membrane pad 1 so as to partially overlap. Can be produced.
　本発明において、サンプルパッド２は、生体試料を速やかに吸収、展開できる材質のものであれば良く、特に制限されないが、セルロース製の濾紙、不織布や、ポリエチレン、ポリプロピレンなどの多孔質材料が挙げられる。この中でも濾紙が好適である。 In the present invention, 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.
　本発明において、コンジュゲーションパッド３は、検出試薬を乾燥状態で保持することができ、生体試料の展開と共に検出試薬を速やかに放出することができる材質のものであれば良く、特に制限されないが、ガラスファイバー、セルロース製の濾紙、ポリエステル製の不織布などが挙げられる。この中でもガラスファイバーが好適である。 In the present invention, 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. Examples thereof include glass fiber, cellulose filter paper, and polyester non-woven fabric. Of these, glass fiber is preferred.
　本発明において、吸収パッド４は、生体試料を速やかに吸収、保持できる材質のものであればよく、特に限定されないが、セルロース製の濾紙、不織布や、ポリエチレン、ポリプロピレンなどの多孔質材料が挙げられる。この中でもセルロース製の濾紙が好適である。 In the present invention, 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.
　さらに、イムノクロマト試験片は、サンプルパッド２、コントロールライン５とテストライン６の位置に、それぞれ生体試料を注入するための開口部、捕捉された検出粒子量を定量するための開口部を有する適当なプラスチック製のハウジングケースに収容しても良い。 Furthermore, 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.
（イムノクロマト試験片の製造方法）
　本発明のイムノクロマト試験片の製造方法は特に限定されない。例えば、コンジュゲーションパッドにおいては、帯状のガラスファイバーに一定量の検出試薬を均一に塗布した後、恒温槽内で適当な温度で一定時間乾燥することで作製することが出来る。捕捉抗体と抗ビオチン抗体は、多孔性メンブレンパッド上にそれぞれ一定量をライン状に塗布すればよい。多孔性メンブレンパッド上に塗布する方法は、特に限定されないが、市販のイムノクロマトディスペンサーを使用することができる。捕捉抗体と抗ビオチン抗体の塗布量は、好ましくはライン長１０ｃｍあたり１０μｌ以下、より好ましくは８μｌ以下、さらに好ましくは６μｌ以下である。また、好ましくは４μｌ以上である。捕捉抗体と抗ビオチン抗体の濃度は、好ましくは０．１～２．０ｍｇ／ｍｌ、より好ましくは０．２～１．８ｍｇ／ｍｌ、さらに好ましくは０．３～１．６ｍｇ／ｍｌである。 (Method for producing immunochromatographic test piece)
The method for producing the immunochromatographic test piece of the present invention is not particularly limited. For example, 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. Although the method of apply | 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 μl or less per 10 cm line length, more preferably 8 μl or less, and even more preferably 6 μl or less. Moreover, it is preferably 4 μl 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.
　多孔性メンブレンパッド上に塗布した捕捉抗体と抗ビオチン抗体は、乾燥するのみで極めて容易に固定化することが出来る。乾燥温度は特に限定されないが、好ましくは２０℃～８０℃、より好ましくは３０～７０℃、さらに好ましくは４０～６０℃である。乾燥時間は、乾燥温度によって異なるが、５～１２０分間、好ましくは１０～６０分間である。 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.
（イムノクロマト試験片を使用した検出対象物質の測定方法）
　上記のようなイムノクロマト試験片を用いて測定を行う方法は特に限定されない。例えば、以下の方法が例示される。まず、生体試料を必要に応じて適当な展開溶媒（検体希釈液）と混合して展開可能な混合液を得た後、前記混合液をサンプルパッド２上に注入（滴下）する。前記混合液は、サンプルパッド２を通過してコンジュゲーションパッド３に展開する。前記混合液は、コンジュゲーションパッド３に含浸された検出試薬とコントロールライン検出試薬を溶解しながら、生体試料中の検出対象物質と検出試薬が免疫複合体を形成し、多孔性メンブレンパッドに展開する。その後、毛細管現象によって吸収パッド４側に流れる。前記混合液が多孔性メンブレンパッド上のコントロールライン５に到達すると、前記コントロールライン検出試薬は、抗ビオチン抗体で捕捉され集積し、コントロールライン５が発色する。さらに、前記混合液は、コントロールライン５を通過してテストライン６に到達する。ここで、前記混合液中の免疫複合体は捕捉抗体で捕捉され集積し、テストライン６が発色する。残りの混合液は、最終的に吸収パッドで吸収される。コントロールライン５とテストライン６の発色強度をイムノクロマトリーダー等を使用して測定することで検出対象物質の濃度を測定（定量）することができる。 (Measurement method of detection target substance using immunochromatographic test strip)
A method for performing measurement using the immunochromatographic test piece as described above is not particularly limited. For example, the following method is exemplified. First, 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. . Then, it flows to the absorption pad 4 side by capillary action. When the mixed solution reaches the control line 5 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. By measuring the color intensity of the control line 5 and the test line 6 using an immunochromatography reader or the like, the concentration of the detection target substance can be measured (quantified).
（コントロールラインの発色強度）
　コントロールラインの発色強度は特に制限されないが、目視あるいはイムノクロマトリーダーで検出することが出来る範囲の発色強度であれば良い。好ましくは１０ｍＡｂｓ～５００ｍＡｂｓ、より好ましくは５０ｍＡｂｓ～４５０ｍＡｂｓ、さらに好ましくは１００ｍＡｂｓ～４００ｍＡｂｓである。 (Color intensity of control line)
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.
（イムノクロマト分析キット）
　本発明のイムノクロマト分析キットは、イムノクロマト試験片に加えて、生体試料の前処理や希釈を目的とした検体希釈液および分析装置を含む。なお、イムノクロマト試験片は、生体試料を滴下するための検体滴下部と検出ラインの発色強度を測定するための観測窓を有するプラスチック製のハウジングケースに収納された状態でも良い。 (Immunochromatography kit)
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.
　以下、本発明を実施例に基づいて詳細に説明する。本発明はこれらの実施例に制限されるものではない。 Hereinafter, the present invention will be described in detail based on examples. The present invention is not limited to these examples.
［実施例１］
（ｈＣＧ検出試薬の調製）
　１２５μｌの金コロイド液（ＯＤ５２０＝１２）（ＷＲＧＨ１、株式会社ワインレッドケミカル社製）に５０μｌの１０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ９．２を加え、ボルテックスで撹拌した。市販の抗ｈＣＧモノクローナル抗体（製品名：Ａｎｔｉ ＣＧＢ／ｈＣＧ β，Ｈｕｍａｎ （Ｍｏｕｓｅ)、品番：ＬＳ－Ｃ１９６９０２－１００、ＬｉｆｅＳｐａｎ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ．製）を１０ｍＭ　Ｔｒｉｓ－ＨＣｌで０．１ｍｇ／ｍｌに調製した。１００μｌの抗ｈＣＧモノクローナル抗体溶液を金コロイド液に加え、ボルテックスで撹拌した後、室温で１５分間静置した。２００μｌの０．３ｗｔ％Ｂｌｏｃｋｉｎｇ Ｐｅｐｔｉｄｅ Ｆｒａｇｍｅｎｔ（東洋紡株式会社製）（以下、ＢＰＦと表記する場合がある）＋１．０ｗｔ％ＰＥＧ２００００水溶液（Ｓａｎｔａ　Ｃｒｕｚ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｃ製）（以下、ＰＥＧと表記する場合がある）を加え、ボルテックスで撹拌した後、１５分間室温で静置した。その後、７０００ｒｐｍで１０分間遠心分離し、上清を除去した。２００μｌの０．３ｗｔ％ＢＰＦ＋１．０ｗｔ％ＰＥＧ水溶液を加え、ボルテックスで撹拌した後、１５分間室温で静置した。７０００ｒｐｍで１０分間遠心分離し、上清を除去した。１ｍｌの０．３ｗｔ％ＢＰＦ＋５．０ｗｔ％Ｄ（＋）-トレハロース（ナカライテスク株式会社製）水溶液を加え、ボルテックスで撹拌した後、１５分間室温で静置した。 [Example 1]
(Preparation of hCG detection reagent)
To 125 μl of colloidal gold solution (OD 520 = 12) (WRGH1, manufactured by Wine Red Chemical Co., Ltd.), 50 μl of 10 mM Tris-HCl pH 9.2 was added and vortexed. A commercially available anti-hCG monoclonal antibody (product name: Anti CGB / hCG β, Human (Mouse), product number: LS-C196902-100, manufactured by LifeSpan Biosciences, Inc.) was prepared to 0.1 mg / ml with 10 mM Tris-HCl. . 100 μl of the anti-hCG monoclonal antibody solution was added to the gold colloid solution, vortexed, and allowed to stand at room temperature for 15 minutes. 200 μl of 0.3 wt% Blocking Peptide Fragment (manufactured by Toyobo Co., Ltd.) (hereinafter may be referred to as BPF) +1.0 wt% PEG 20000 aqueous solution (manufactured by Santa Cruz Biotechnology Inc) (hereinafter may be referred to as PEG) Was added and stirred by vortexing, and then allowed to stand at room temperature for 15 minutes. Thereafter, the mixture was centrifuged at 7000 rpm for 10 minutes, and the supernatant was removed. After adding 200 μl of 0.3 wt% BPF + 1.0 wt% PEG aqueous solution and stirring by vortex, it was allowed to stand at room temperature for 15 minutes. Centrifugation was performed at 7000 rpm for 10 minutes, and the supernatant was removed. 1 ml of 0.3 wt% BPF + 5.0 wt% D (+)-trehalose (manufactured by Nacalai Tesque) was added and stirred by vortexing, and then allowed to stand at room temperature for 15 minutes.
（コントロールライン検出試薬の調製）
　３．１５ｍｇのＤ－ビオチン（ナカライテスク株式会社製）を７２μｌの蒸留水で溶解した。また、１０ｍｇの１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（ナカライテスク株式会社製）と１０ｍｇのＮ－ヒドロキシスクシンイミド（ナカライテスク株式会社製）を１００μｌの蒸留水に溶解した。これに前記Ｄ－ビオチン溶液を加え、室温で１時間緩やかに攪拌した。得られた溶液に８０μｌの３ｗｔ％ＢＰＦ溶液を加えて３０分間室温で静置し、ビオチン標識ＢＰＦ溶液を調製した。次に、５０μｌの金コロイド液（ＯＤ５２０＝１２）に４５０μｌの１０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ９．２を加え、ボルテックスで撹拌した。得られた溶液に１５μｌのビオチン標識ＢＰＦ溶液を加え、ボルテックスで撹拌した後、１５分間室温で静置した。２００μｌの０．３ｗｔ％ＢＰＦ＋１．０ｗｔ％ＰＥＧ水溶液（Ｓａｎｔａ　Ｃｒｕｚ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｃ製）を加え、ボルテックスで撹拌した後、１５分間室温で静置した。その後、７０００ｒｐｍで１０分間遠心分離し、上清を除去した。２００μｌの０．３ｗｔ％ＢＳＡ＋１．０ｗｔ％ＰＥＧ水溶液を加え、ボルテックスで撹拌した後、１５分間室温で静置した。７０００ｒｐｍで１０分間遠心分離し、上清を除去した。１ｍｌの０．３ｗｔ％ＢＰＦ＋５ｗｔ％Ｄ（＋）－トレハロース（ナカライテスク株式会社製）水溶液を加え、ボルテックスで撹拌した後、１５分間室温で静置した。 (Preparation of control line detection reagent)
3.15 mg of D-biotin (manufactured by Nacalai Tesque) was dissolved in 72 μl of distilled water. Further, 10 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (manufactured by Nacalai Tesque) and 10 mg of N-hydroxysuccinimide (manufactured by Nacalai Tesque) were dissolved in 100 μl of distilled water. The D-biotin solution was added thereto, and the mixture was gently stirred at room temperature for 1 hour. 80 μl of 3 wt% BPF solution was added to the resulting solution and allowed to stand at room temperature for 30 minutes to prepare a biotin-labeled BPF solution. Next, 450 μl of 10 mM Tris-HCl pH 9.2 was added to 50 μl of colloidal gold solution (OD 520 = 12) and vortexed. 15 μl of a biotin-labeled BPF solution was added to the resulting solution, vortexed, and allowed to stand at room temperature for 15 minutes. 200 μl of 0.3 wt% BPF + 1.0 wt% PEG aqueous solution (manufactured by Santa Cruz Biotechnology Inc) was added, stirred by vortexing, and allowed to stand at room temperature for 15 minutes. Thereafter, the mixture was centrifuged at 7000 rpm for 10 minutes, and the supernatant was removed. After adding 200 μl of 0.3 wt% BSA + 1.0 wt% PEG aqueous solution and stirring by vortex, it was allowed to stand at room temperature for 15 minutes. Centrifugation was performed at 7000 rpm for 10 minutes, and the supernatant was removed. 1 ml of 0.3 wt% BPF + 5 wt% D (+)-trehalose (manufactured by Nacalai Tesque) solution was added, stirred by vortexing, and allowed to stand at room temperature for 15 minutes.
（ｈＣＧ検出用多孔性メンブレンパッドの作製）
　２５ｍｍ×１５０ｍｍのニトロセルロースメンブレン（商品名：Ｈｉ－Ｆｌｏｗ　ＰｌｕｓタイプＨＦ１２０（メルクミリポア株式会社製））の一端（上流側）から約１０ｍｍの位置にライン幅約１ｍｍのコントロールラインとして、１．０ｍｇ／ｍｌの抗ビオチンモノクローナル抗体（製品名：Ａｎｔｉ－Ｂｉｏｔｉｎ ａｎｔｉｂｏｄｙ、品番：ＧＴＸ４４３４４、ＧＥＮＥＴＥＸ，ｉｎｃ．製）溶液（５０ｍＭ ＫＨ２ＰＯ４，ｐＨ７．０＋５ｗｔ％スクロース）をイムノクロマトディスペンサーを用いて約１．０μｌ／ｃｍの塗布量で塗布した。次に、ニトロセルロースメンブレンの一端（上流側）から約１５ｍｍの位置にライン幅約１ｍｍのテストラインとして、０．５ｍｇ／ｍｌの抗ｈＣＧモノクローナル抗体（製品名：Ａｎｔｉ Ｃｈｏｒｉｏｎｉｃ Ｇｏｎａｄｏｔｒｏｐｉｎ，Ｈｕｍａｎ(Ｒａｂｂｉｔ)、品番：ＡＴ－２６０１－２、ＥＹ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ．製）溶液（５０ｍＭ ＫＨ２ＰＯ４，ｐＨ７．０＋５ｗｔ％スクロース）をイムノクロマトディスペンサーを用いて約１．０μｌ／ｃｍの塗布量で塗布した。その後、５０℃で３０分乾燥した。 (Preparation of porous membrane pad for hCG detection)
As a control line with a line width of about 1 mm at a position about 10 mm from one end (upstream side) of a 25 mm × 150 mm nitrocellulose membrane (trade name: Hi-Flow Plus type HF120 (manufactured by Merck Millipore)), 1.0 mg / ml of an anti-biotin monoclonal antibody (product name: Anti-Biotin antibody, product number: GTX44344, GENETEX, Inc.) solution (50 mM KH 2 PO 4 , pH 7.0 + 5 wt% sucrose) was added to about 1.0 μl / ml using an immunochromatographic dispenser. It was applied at a coating amount of cm. Next, as a 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) was applied at an application amount of about 1.0 μl / cm using an immunochromatographic dispenser. Then, it dried at 50 degreeC for 30 minutes.
（ｈＣＧ測定用コンジュゲーションパッドの作製）
　１０ｍｍ×１５０ｍｍのコンジュゲーションパッド（商品名：シュアウィック（メルクミリポア株式会社製））に前記ｈＣＧ検出試薬とコントロールライン検出試薬の混合液（混合比１：１）１．０ｍｌを均一に塗布した。その後、デシケータ内で一晩、室温で減圧乾燥し、ｈＣＧ検出試薬とコントロールライン検出試薬を含浸したｈＣＧ測定用コンジュゲーションパッドを作製した。 (Preparation of conjugation pad for hCG measurement)
1.0 ml of a mixed solution (mixing ratio 1: 1) of the hCG detection reagent and the control line detection reagent was uniformly applied to a 10 mm × 150 mm conjugation pad (trade name: Surewick (manufactured by Merck Millipore)). Thereafter, the hCG measurement conjugation pad impregnated with the hCG detection reagent and the control line detection reagent was produced by drying in a desiccator overnight at room temperature.
（ｈＣＧ測定用試験片の作製）
　ｈＣＧ測定用試験片は、イムノクロマトグラフで一般的に使用されるスライド構成で作製した。具体的には、サンプルパッド（商品名：シュアウィック（メルクミリポア株式会社製））、コンジュゲーションパッド（商品名：シュアウィック（メルクミリポア株式会社製））、ニトロセルロースメンブレン（商品名：Ｈｉ－Ｆｌｏｗ　ＰｌｕｓタイプＨＦ１２０（メルクミリポア株式会社製））、吸収パッド（商品名：シュアウィック（メルクミリポア株式会社製））を直列連結し作製した。次に、横幅約４ｍｍ、縦幅約６０ｍｍの試験片状に裁断機を用いてカットした。 (Preparation of hCG measurement specimen)
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.
（ｈＣＧ試料の調製）
　市販のｈＣＧ抗原を５０ｍＭ ＫＨ２ＰＯ４，ｐＨ７．２＋１．０ｗｔ％ＢＳＡ＋０．１ｗｔ％ＮａＮ３で希釈し、１００ＩＵ／ＬのｈＣＧ試料を調製した。 (Preparation of hCG sample)
A commercially available hCG antigen was diluted with 50 mM KH2PO4, pH 7.2 + 1.0 wt% BSA + 0.1 wt% NaN 3 to prepare a 100 IU / L hCG sample.
（ｈＣＧの測定）
　作製したｈＣＧ測定用試験片を水平な台に設置した。次に、調製した１００ＩＵ／ＬのｈＣＧ試料を４０μｌピペットで取り、サンプルパッドに滴下した。検体を滴下してから２分毎にイムノクロマトリーダー（Ｃ１００６０－１０（浜松ホトニクス社製））を用いて発色強度を測定した。結果を図２および表１に示す。コントロールラインの発色強度は、測定開始から１２分後に３１０ｍＡｂｓに到達し、１４分後に３０８ｍＡｂｓであったことから、測定開始から１４分後に安定化したと判断した。次に、１４分後のテストラインの発色強度は３０３ｍＡｂｓであった。コントロールラインとテストラインを測定するために要した時間は約１４分であった。 (Measurement of hCG)
The prepared hCG measurement specimen was placed on a horizontal base. Next, the prepared 100 IU / L hCG sample was taken with a 40 μl 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
［比較例１］
（ｈＣＧ測定用試験片の作製）
　実施例１と同様の試薬および材料を用いて、コントロールラインとテストラインの配置を入れ替えたｈＣＧ検出用多孔性メンブレンパッドを作製した。得られた多孔性メンブレンパッドを用いて、実施例１と同様にしてｈＣＧ試験片を作製した。 [Comparative Example 1]
(Preparation of hCG measurement specimen)
Using the same reagents and materials as in Example 1, a porous membrane pad for hCG detection in which the arrangement of the control line and the test line was exchanged was produced. An hCG test piece was prepared in the same manner as in Example 1 using the obtained porous membrane pad.
（ｈＣＧの測定）
　前記作製したｈＣＧ試験片を用いて、実施例１と同様の方法でｈＣＧを測定した。結果を図３および表２に示す。コントロールラインの発色強度は測定開始から１８分後に３１２ｍＡｂｓに到達し、２０分後に３１４ｍＡｂｓであったことから、測定開始から２０分後に安定化したと判断した。次に、２０分後のテストラインの発色強度は３１３ｍＡｂｓであった。コントロールラインとテストラインを測定するために要した時間は約２０分であった。 (Measurement of hCG)
HCG was measured in the same manner as in Example 1 using the hCG test piece prepared above. The results are shown in FIG. The color intensity of the control line reached 312 mAbs 18 minutes after the start of measurement and was 314 mAbs after 20 minutes, so it was judged that the control line had stabilized 20 minutes after the start of measurement. Next, the color intensity of the test line after 20 minutes was 313 mAbs. The time required to measure the control line and the test line was about 20 minutes.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　以上、説明した通り、検出対象物質と特異的に結合する検出試薬とコントロールライン検出試薬がコンジュゲーションパッドに共に含浸されたイムノクロマト試験片とすることにより、テストラインの上流側にコントロールラインを配設しても迅速かつ簡便で測定精度の高い定量分析が可能となった。 As described above, the 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.
　本発明により、迅速かつ簡便で測定精度の高いイムノクロマト試験片を提供することができる。 According to the present invention, it is possible to provide an immunochromatographic test piece which is quick, simple and highly accurate.
１．多孔性メンブレンパッド
２．サンプルパッド
３．コンジュゲーションパッド
４．吸収パッド
５．コントロールライン
６．テストライン
７．粘着シート
 
  1. 1. Porous membrane pad 2. Sample pad Conjugation pad 4. 4. Absorption pad Control line 6. 6. Test line Adhesive sheet

Claims (9)

  1. 　生体試料中の検出対象物質を定量するためのイムノクロマト試験片であって、
    a)前記検出対象物質と特異的に結合する検出試薬及びコントロールライン検出試薬が含浸されたコンジュゲーションパッド、および
    b)上流側にコントロールライン検出試薬を特異的に捕捉するための抗体が固定化されており、かつ下流側に前記検出対象物質を捕捉するための抗体が固定化されている多孔性メンブレンパッド
    を有することを特徴とするイムノクロマト試験片。     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. 　前記コントロールライン検出試薬は、ビオチン標識タンパク質が結合した検出粒子を含むことを特徴とする請求項１に記載のイムノクロマト試験片。 2. The immunochromatographic test strip according to claim 1, wherein the control line detection reagent contains detection particles to which a biotin-labeled protein is bound.
  3. 　前記タンパク質は、微生物由来または動物由来のタンパク質であること特徴とする請求項２に記載のイムノクロマト試験片。 The immunochromatographic test piece according to claim 2, wherein the protein is a protein derived from a microorganism or an animal.
  4. 　前記微生物由来のタンパク質は、Ｂｌｏｃｋｉｎｇ Ｐｅｐｔｉｄｅ Ｆｒａｇｍｅｎｔであることを特徴とする請求項３に記載のイムノクロマト試験片。 The immunochromatographic test piece according to claim 3, wherein the protein derived from the microorganism is Blocking Peptide Fragment.
  5. 　前記動物由来のタンパク質は、ウシ血清アルブミンまたはカゼインであることを特徴とする請求項３に記載のイムノクロマト試験片。 The immunochromatographic test piece according to claim 3, wherein the animal-derived protein is bovine serum albumin or casein.
  6. 　前記コントロールライン検出試薬を特異的に捕捉するための抗体は、抗ビオチン抗体であることを特徴とする請求項１～５のいずれかに記載のイムノクロマト試験片。 The immunochromatographic test strip according to any one of claims 1 to 5, wherein the antibody for specifically capturing the control line detection reagent is an anti-biotin antibody.
  7. 　請求項１～６のいずれかに記載のイムノクロマト試験片を用いる、生体試料中の検出対象物質を定量する方法。 A method for quantifying a substance to be detected in a biological sample, using the immunochromatographic test strip according to any one of claims 1 to 6.
  8. 　請求項１～６のいずれかに記載のイムノクロマト試験片、検体希釈液および分析装置を含むイムノクロマト分析キット。 An immunochromatographic analysis kit comprising the immunochromatographic test strip according to any one of claims 1 to 6, a specimen diluent, and an analyzer.
  9. 　請求項１～６のいずれかに記載のイムノクロマト試験片の製造方法。
     
          The method for producing an immunochromatographic test piece according to any one of claims 1 to 6.

PCT/JP2016/085690 2015-12-02 2016-12-01 Immunochromatographic test piece WO2017094825A1 (en)

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