WO2020166698A1 - Une éprouvette immunochromatographique et procédé de mesure l'utilisant - Google Patents

Une éprouvette immunochromatographique et procédé de mesure l'utilisant Download PDF

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WO2020166698A1
WO2020166698A1 PCT/JP2020/005776 JP2020005776W WO2020166698A1 WO 2020166698 A1 WO2020166698 A1 WO 2020166698A1 JP 2020005776 W JP2020005776 W JP 2020005776W WO 2020166698 A1 WO2020166698 A1 WO 2020166698A1
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particles
control line
detection
measurement
immunochromatographic test
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PCT/JP2020/005776
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English (en)
Japanese (ja)
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岡本　淳
美穂 松尾
圭三 米田
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東洋紡株式会社
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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 an immunochromatographic test strip for measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. Specifically, it relates to an immunochromatographic test strip having high measurement accuracy and a measurement method using the same.
  • POCT Point Of Care Testing
  • a clinical test performed by a medical staff beside a subject Unlike the clinical test performed in the central laboratory of a large-scale hospital, etc., POCT is capable of instantly obtaining test results on the spot, so POCT is spreading in a wide range of test items.
  • a typical example of POCT is immunochromatography.
  • the immunochromatography method is that from one end of a porous body on which an antibody that specifically binds to a measurement target substance contained in a measurement sample is immobilized, the measurement sample develops in the porous body by a capillary phenomenon and is labeled in the process. It is an immunoassay method for determining the presence or absence of a measurement target substance in a measurement sample by causing the measurement target substance and an antibody to bind with each other by an antigen-antibody reaction and accumulate over time and develop color locally.
  • Advantages of the immunochromatography method include quick results, easy operation, and low cost.
  • In vitro diagnostic agents such as pregnancy test agents and influenza diagnostic agents, which take advantage of these advantages, have become widespread worldwide.
  • visual judgment quantitative evaluation
  • the amount of the substance to be measured contained in the measurement sample was quantified using an analyzer such as an immunochromatographic reader that measures the color development intensity. Technology to do so is being developed.
  • One of the methods for quantifying the amount of a substance to be measured using the immunochromatography method is a sandwich method using an antigen-antibody reaction.
  • the sandwich method uses two types of antibodies having different epitopes for the substance to be measured.
  • One of the antibodies forms a test line as a capture antibody linearly immobilized on the surface of the porous body.
  • the other antibody is used as a detection antibody (hereinafter may be referred to as a test line detection reagent) sensitized with detection particles such as gold colloid, colored latex particles, and fluorescent particles in order to detect the test line.
  • an antibody that specifically captures the test line detection reagent is linearly immobilized at a position different from the test line on the surface of the porous body to form a control line.
  • the substance to be measured in the measurement sample develops from one end (upstream side) of the porous body, moves while forming an immune complex with the test line detection reagent, and contacts the capture antibody on the test line to be captured and develop color. To do.
  • the free test line detection reagent that is not captured on the test line is captured by the capture antibody on the control line and develops color.
  • the amount of the substance to be measured can be quantified by using a device such as an immunochromatographic reader for these color development intensities.
  • the mechanism that captures the free test line detection reagent, which was not captured on the test line, by the capture antibody on the control line affects the concentration of the measurement target substance and changes the color intensity of the control line. It was a problem to do. For example, when a high concentration of the substance to be measured is present in the measurement sample, a large amount of the test line detection reagent forms an immune complex with the substance to be measured and is captured by the test line on the upstream side. Will be higher. In other words, the amount of free test line detection reagent is small, so that the color intensity of the control line is low.
  • the amount of test line detection reagent that forms an immune complex with the substance to be measured will also be small, so a large amount of free test line detection reagent will be captured by the control line As a result, there is a problem that the color development intensity of the control line is increased.
  • An immunochromatographic test strip in which the color intensity of the control line reflects the amount of the test sample (or test line detection reagent) developed on the porous body in order to accurately measure the measurement target substance contained in the measurement sample by the immunochromatographic method.
  • the color development intensity of the control line may be used to correct the variation in the spread amount of the measurement sample on the porous body due to the individual difference of the immunochromatographic test strip. In that case, when a certain amount of measurement sample (or test line detection reagent) is spread on the porous body, it is desired that the color intensity of the control line is always constant.
  • a detection particle labeled with a low molecular weight compound (hereinafter sometimes referred to as a control line detection reagent) and an antibody that specifically captures the low molecular weight compound are lined on the surface of a porous body.
  • a control line detection reagent a low molecular weight compound
  • an antibody that specifically captures the low molecular weight compound are lined on the surface of a porous body.
  • the control line reflects the inflow amount of the measurement sample (or the test line detection reagent) into the porous body to some extent
  • the color intensity of the test line is corrected by the color intensity of the control line to obtain the measurement sample.
  • the substance to be measured contained in the substance can be measured with a certain degree of accuracy.
  • the test line and control line are usually immobilized at different positions on the porous body, the time until the test line detection reagent is captured by the test line and the time until the control line detection reagent is captured by the control line are However, the above correction was insufficient.
  • An object of the present invention is to provide an immunochromatographic test strip for measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. Specifically, it is an object to provide an immunochromatographic test strip with high measurement accuracy and a measurement method using the same.
  • the present inventor has found that the test line is located upstream of the control line (on the sample pad side) and is detected in the test line detection reagent composed of the detection antibody and the detection particles A.
  • the measurement accuracy is improved as compared with the conventional technique. Have been found to improve.
  • the average particle size of the detection particles A in the test line detection reagent captured by the test line located on the upstream side is the average particle size of the detection particles B in the control line detection reagent captured by the control line located on the downstream side.
  • the difference between the time until the test line detection reagent is captured in the test line and the retention time until the control line detection reagent is captured in the control line is reduced, and more accurate correction is possible. It is supposed to be because it became.
  • the present inventor has found that the immunochromatographic reader can be miniaturized by making the detection particles A and the detection particles B have the same color, and completed the present invention.
  • the representative present invention is as follows.
  • An immunochromatographic test strip for quantifying a substance to be measured contained in a measurement sample wherein the immunochromatographic test strip has a structure in which a sample pad, a conjugation pad, a membrane, and an absorption pad are arranged in sequence.
  • the membrane is provided with a test line and a control line that develop color by an antigen-antibody reaction, the test line is located upstream of the control line, and the average particle size of the detection particles A captured by the test line and the An immunochromatographic test strip in which the ratio of the average particle diameters of the detection particles B captured on the control line is 3:1 to 100:1.
  • the immunochromatographic test strip of the present invention and the measuring method using the same are such that the color intensity of the control line is stabilized because the independent antigen-antibody reaction proceeds between the test line and the control line, and the test line detection reagent is captured by the test line.
  • the test line detection reagent is captured by the test line.
  • the measurement sample is not particularly limited, but examples thereof include biological samples such as blood, lymph, spinal fluid, sweat, urine, lacrimal fluid, saliva, skin, mucous membrane, and hair.
  • biological samples such as blood, lymph, spinal fluid, sweat, urine, lacrimal fluid, saliva, skin, mucous membrane, and hair.
  • serum, blood cells or plasma obtained by centrifugation of blood can be used as a sample.
  • the measurement sample is not limited to a human-derived sample, and a biological sample derived from a mammal such as a dog, a cat, or a cow is also targeted.
  • the measurement items are not particularly limited, but for example, HbA1c, C-peptide, Insulin, m-Alumin, Cys-C, NGAL, TriponinI, D-dimer, NT-proBNP, CK-MB, Myoglobin, h.
  • -FABP hs-CRP
  • PSA AFP
  • CEA CEA
  • FOB Ferritin
  • PCT CRP
  • SAA ASO
  • hCG LH
  • TSH FSH
  • T3, T4 VitaminD
  • influenzaA/B Dengue, HBV, HCV, HIV, Syphilis, Malaria, H.
  • Infectious disease items such as pylori, Rota, Chlamydia, StrepA, Adeno, Zika, Chikungunya, RSV and the like can be mentioned.
  • the structure of the immunochromatographic test piece is such that the addition portion (dropping portion) of the measurement sample solution of the immunochromatographic test piece is on the upstream side, and the sample pad having the addition portion, the conjugation pad, the membrane, and the absorption pad are connected in this order. It is preferable to have the above structure.
  • an example of the immunochromatographic test strip of the present invention will be described with reference to the drawings, but the present invention is not limited thereto. 1 and 2, 1 is a sample pad, 2 is a conjugation pad, 3 is a membrane, 4 is an absorbent pad, 5 is a backing sheet, 6 is a test line, 7 is a control line, and 8 is an adhesive sheet.
  • the immunochromatographic test piece has an elongated strip shape having a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm).
  • a test line 6 in which a capture antibody that specifically binds to the substance to be measured is linearly immobilized is formed at a position about 10 mm from the upstream end of the membrane 3 of the immunochromatographic test strip.
  • a control line 7 is formed at a position of about 15 mm from the end portion, in which a capture antibody that specifically binds to a low molecular weight compound described later is linearly immobilized.
  • the conjugation pad 2 of the immunochromatographic test strip is composed of a test line detection reagent composed of a detection antibody that specifically binds to the substance to be measured and detection particles A, and a detection particle B labeled with a low molecular weight compound.
  • the control line detection reagent is loaded.
  • the average particle size of the detection particles A in the test line detection reagent and the average particle size of the detection particles B in the control line detection reagent are not particularly limited, but are preferably 10 to 1000 nm, more preferably 10 to 500 nm.
  • the average particle size of the detection particles is large, the downstream development may be delayed and the measurement time may be long. In addition, it is likely to be captured on the membrane, and the background itself may develop color, which may make the color development on the test line and control line unclear.
  • the average particle size of the detection particles is small, in the case of the test line detection reagent, the amount of detection antibody that can be physically adsorbed or chemically bound decreases, and the measurement sensitivity may decrease. Further, in the case of the control line detection reagent, the amount of the low molecular weight compound that can be labeled may decrease, and the correction may be insufficient.
  • the ratio of the average particle size of the detection particles A to the average particle size of the detection particles B is preferably 3:1 to 100:1, more preferably 4:1 to 50:1, and more preferably 5:1 to 20:. 1 is more preferred.
  • the test line detection reagent that is, the detection particles A
  • the detection particles B is a test line on the upstream side. The difference between the time to reach the control line and the time to reach the control line on the downstream side of the control line reagent (that is, the detection particles B) becomes small.
  • the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is less than 3:1, the retention time until the test line detection reagent is captured in the test line and the control line detection reagent is captured in the control line Since there is a large difference in the retention time before the adjustment, there is a possibility that the correction of the color intensity of the control line will be insufficient.
  • the detection particle A is larger than 1000 nm or the detection particle B is smaller than 10 nm, which is not preferable as described above.
  • the detection particles A and the detection particles B are not particularly limited, but colored particles or fluorescent particles can be used.
  • the colored particles include metal particles, latex particles, cellulose particles and the like.
  • metal particles include gold colloid, silver colloid, platinum colloid, palladium colloid, gold nanorods, gold nanoplates, and silver nanoplates.
  • the latex particles include particles made of materials such as polystyrene, polymethylmethacrylate, and acrylic acid polymer.
  • the fluorescent particles include polystyrene, polymethylmethacrylate, polyvinyltoluene, silica and the like, and examples of the fluorescent dye include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives and the like. be able to.
  • both the detection particles A and the detection particles B are colored particles, and it is more preferable that the detection particles A and the detection particles B have the same color.
  • the same color means that the maximum absorption wavelength is within ⁇ 50 nm.
  • the immunochromatographic reader becomes large and expensive for detecting fluorescence, which is not preferable from the viewpoint of POCT.
  • the detection particles A and the detection particles B have different colors, for example, in a general light emitting diode (hereinafter sometimes abbreviated as LED)-photodiode (hereinafter sometimes abbreviated as PD) immunochromatographic reader. In the case of measurement, it is necessary to combine a plurality of LED-PDs corresponding to each color of the test line and the control line, which makes the device large and expensive, which is not preferable from the viewpoint of POCT.
  • LED general light emitting diode
  • PD photodiode
  • the detection particles A are organic particles and the detection particles B are Are preferably inorganic particles.
  • the detection particles A are cellulose particles or latex particles
  • the detection particles B are a combination of spherical gold fine particles. From the viewpoint of highly sensitive measurement, it is more preferable that the detection particles A are cellulose particles and the detection particles B are spherical gold fine particles.
  • the detection particles A and B are both blue, the detection particles A are cellulose particles or latex particles, and the detection particles B are a combination of plate-shaped gold fine particles. From the viewpoint of highly sensitive measurement, it is more preferable that the detection particles A are cellulose particles and the detection particles B are plate-shaped gold fine particles. It should be noted that the fine gold particles have a red color in a spherical shape and a blue color in a plate shape.
  • the detection antibody in the test line detection reagent is not particularly limited as long as it can specifically bind to the substance to be measured.
  • an anti-hCG antibody can be used
  • an anti-Hb antibody or an anti-HbA1c antibody can be used.
  • the detection antibody may be a commercially available product or may be separately produced by a known method. Further, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the method of binding the detection antibody and the detection particles in the test line detection reagent is not particularly limited, but it is preferable to sensitize by physical adsorption by a hydrophobic bond or chemical bond by a covalent bond, and the operation is simple and the cost is low. Physical adsorption is more preferred.
  • a reactive active group may be introduced into the detection particles.
  • the reactive active group is not particularly limited, but examples thereof include a carboxyl group, an amino group, an aldehyde group, a thiol group, an epoxy group, and a hydroxyl group. Among these, a carboxyl group and an amino group are preferable. In the case of a carboxyl group, carbodiimide can be used to form a covalent bond with the amino group of the ligand.
  • the low molecular weight compound in the control line detection reagent is not particularly limited, but biotin or digoxigenin is preferable, and biotin is more preferable, because it is relatively inexpensive, easily available, and has a proven record in the field of protein labeling.
  • the test line detection reagent and the control line detection reagent are blocked with a blocking protein.
  • the blocking protein is not particularly limited, and Blocking Peptide Fragment (hereinafter sometimes abbreviated as BPF) of a microorganism-derived protein, bovine serum albumin (hereinafter sometimes abbreviated as BSA) of an animal-derived protein, and casein are preferable. ..
  • BPF Blocking Peptide Fragment
  • BSA bovine serum albumin
  • the blocking protein may be a commercially available product, or may be separately produced by a known method. BPF is commercially available from Toyobo Co., Ltd. and is easily available.
  • the linearly immobilized capture antibody forming the test line 6 on the membrane 3 used in the present invention is not particularly limited as long as it can specifically bind to the substance to be measured.
  • an anti-hCG antibody can be used
  • an anti-Hb antibody or an anti-HbA1c antibody can be used.
  • the detection antibody and the capture antibody have different epitopes for the substance to be measured.
  • the capture antibody may be a commercially available product or may be separately produced by a known method. Further, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the linearly immobilized capture antibody forming the control line 7 on the membrane 3 used in the present invention is not particularly limited as long as it can specifically bind to the low molecular weight compound of the control line detection reagent.
  • the low molecular weight compound is biotin
  • an anti-biotin antibody can be used
  • digoxigenin an anti-digoxigenin antibody can be used.
  • the capture antibody may be a commercially available product or may be separately produced by a known method. Further, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the material of the sample pad 1 is not particularly limited as long as it can quickly absorb the measurement sample and then spread to the downstream conjugation pad, membrane, or absorbent pad.
  • cellulose filter paper or nonwoven fabric glass. Filter paper or non-woven fabric, polyester filter paper or non-woven fabric, polyethylene filter paper or non-woven fabric. Of these, cellulose filter paper is preferable.
  • the thickness of the sample pad 1 is preferably 0.1 to 2.0 mm, more preferably 0.2 to 1.0 mm. When the thickness is small, the flow of the measurement sample on the downstream side becomes non-uniform, and the measurement accuracy may decrease. On the other hand, if the thickness is large, the downstream expansion may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream development increases.
  • the material of the conjugation pad 2 should be one that can hold the test line detection reagent and the control line detection reagent in a dry state, and can rapidly release both of the detection reagents as the measurement sample is developed downstream.
  • the thickness of the conjugation pad 2 is preferably 0.1 to 2.0 mm, more preferably 0.2 to 1.0 mm. If the thickness is small, it may not be possible to keep the target amount of the test line detection reagent and the control line detection reagent in a dry state. If the thickness is large, the downstream expansion may be slow and the measurement time may be long. In addition, the amount of measurement sample required for downstream development increases.
  • the material of the membrane 3 is not particularly limited as long as it can accurately and uniformly spread the measurement sample, and examples thereof include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride. , Or a nylon membrane. Among these, a nitrocellulose membrane is preferable.
  • the material of the absorbent pad 4 is not particularly limited as long as it can quickly absorb the measurement sample that has been developed from the upstream and then hold it so that it does not backflow.
  • Polyester filter paper or non-woven fabric polyethylene filter paper or non-woven fabric. Of these, cellulose filter paper is preferable.
  • the thickness of the absorbent pad 4 is preferably 0.2 to 5.0 mm, more preferably 0.5 to 2.0 mm. When the thickness is small, the measurement sample once absorbed by the absorption pad may flow back to the membrane side depending on the amount of the measurement sample dropped. If the thickness is large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also becomes large, which is not preferable from the viewpoint of POCT.
  • the method of supporting the test line detection reagent and the control line detection reagent on the conjugation pad 2 is not particularly limited, and for example, the test line detection reagent and the control line detection reagent are mixed at a constant ratio, and then the mixed solution is mixed.
  • the coating amount of the mixed solution is not particularly limited, but is preferably 5 ⁇ L to 50 ⁇ L per 1 cm line length. Then, it is preferable to dry after the application.
  • the drying temperature is not particularly limited, but is preferably 20°C to 80°C, more preferably 20°C to 60°C.
  • the drying time varies depending on the drying temperature, but is usually 5 minutes to 120 minutes.
  • the method of linearly immobilizing the capture antibody forming the test line and the capture antibody forming the control line on the membrane 3 is not particularly limited.
  • the capture antibody forming the test line and the control line are formed. It can be prepared by applying a fixed amount of each of the capture antibodies to different positions on the line and then drying at a suitable temperature for a fixed time in a constant temperature bath.
  • the coating amount of both capture antibodies is not particularly limited, but 0.1 ⁇ L to 2 ⁇ L per 1 cm line length is preferable. Then, it is preferable to dry after the application.
  • the drying temperature is not particularly limited, but is preferably 20°C to 80°C, more preferably 20°C to 60°C.
  • the drying time varies depending on the drying temperature, but is usually 5 minutes to 120 minutes.
  • the above-prepared membrane 3 is attached to the vicinity of the center of the adhesive sheet 8, and then the conjugation pad 2 is attached to one end of the membrane 3 so as to partially overlap with it, and then the sample pad 1 is partially overlapped and pasted on the end opposite to the overlapping of the conjugation pad 2 with the membrane 3, and then the absorbent pad 4 is partially overlapped and pasted on the other end of the membrane 3, It can be manufactured by cutting it into a strip having a constant width.
  • the test line 6 and the control line 7 may be prepared after producing the test piece, or may be prepared before producing the test piece.
  • the immunochromatographic test piece has at least a first opening for dropping a measurement sample on the sample pad 1 and a second opening for measuring the test line 6 and the control line 7 on the membrane 3 It may be housed in a plastic housing case.
  • the concentration of the substance to be measured is more preferably measured from a correction value obtained by dividing the color development intensity of the test line by the color development intensity of the control line in consideration of the development unevenness of the measurement sample.
  • the measuring method of the test line and control line of the immunochromatographic test piece is not particularly limited, and a commercially available immunochromatographic reader may be used, or the immunochromatographic reader may be manufactured by a separately known method.
  • the detection system is not particularly limited, but for example, LED-FD, LED-CMOS, LED-CCD can be used.
  • Shape of detection particles Each of the detection particles was observed with a scanning electron microscope (SEM, S-4800 manufactured by Hitachi Ltd.) and classified into a [1] sphere, a spheroid, and a plate that is a spherical [2] prism that is an ellipsoid.
  • Average particle size of detected particles Each particle was observed with a scanning electron microscope (SEM), and when the shape was [1] spherical, the diameter of 100 particles was measured and the average diameter (average particle diameter) was calculated. When the shape was a [2] plate shape, the maximum length of the plane portion of 100 particles was measured, and the average length (average particle diameter) was calculated.
  • test line detection reagent for hCG measurement 5.0 mg/mL anti-hCG- ⁇ monoclonal antibody (5014, manufactured by Medix Biochemica) was prepared to 1.0 mg/mL with distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.). did.
  • 1.0 wt% cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red, average particle size 340 nm, manufactured by Asahi Kasei) 100 ⁇ L, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) (900 ⁇ L) and the above 1.0 mg/mL (0.1 wt%) of anti-hCG- ⁇ monoclonal antibody (100 ⁇ L) were added to a 15 mL centrifuge tube and vortexed. Then, it was placed in a low temperature incubator (IN604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37° C. and left standing for 120 minutes.
  • a low temperature incubator I604, manufactured by Yamato Scientific Co., Ltd.
  • a blocking solution consisting of 1.0 wt% casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) was added. In addition, it was further left to stand for 60 minutes in a low temperature incubator adjusted to 37°C. Then, using a centrifuge (MX-307, manufactured by Tommy Seiko), a rack in rotor (TMA-300, manufactured by Tommy Seiko), and a rack (AR510-04, manufactured by Tommy Seiko), centrifuge at 13,000 G.
  • MX-307 manufactured by Tommy Seiko
  • TMA-300 rack in rotor
  • AR510-04 manufactured by Tommy Seiko
  • the reaction was carried out at 25°C for 15 minutes to precipitate the antibody-sensitized cellulose particles, and then the supernatant was removed. Then, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer was added and treated with an ultrasonic disperser (UH-50, SMT) for 10 seconds. Then, using a centrifuge, a rack in rotor and a rack, centrifugation at 13,000 G was performed at 25° C. for 15 minutes to precipitate the antibody-sensitized cellulose particles, and then the supernatant was removed.
  • a washing solution pH 10.0
  • UH-50, SMT ultrasonic disperser
  • a coating solution (pH 9.2) consisting of 15 wt% sucrose (196-00001, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 wt% casein, and 62 mM borate buffer was added, and ultrasonic dispersion was performed.
  • the sample was treated with a machine for 10 seconds to obtain a test line detection reagent 1 for hCG measurement.
  • EDC 15022-86, manufactured by Nacalai Tesque
  • NHS 18948-02, manufactured by Nacalai Tesque
  • 1,000 ⁇ L of distilled water was added to a 5 mL microtube and stirred by vortex to obtain an EDC/NHS solution.
  • 1,000 ⁇ L of the D biotin solution and 1,000 ⁇ L of the EDC/NHS solution were mixed and then placed in a low temperature incubator adjusted to 25° C. and left standing for 15 minutes to obtain a D biotin/EDC/NHS solution.
  • Bovine serum album:BSA (A7906, manufactured by Sigma-Aldrich) and 1,000 ⁇ L of distilled water were added to a 5 mL microtube, and the mixture was vortexed to obtain a BSA solution. Then, 1,000 ⁇ L of the D biotin/EDC/NHS solution and 1,000 ⁇ L of the BSA solution were mixed and then placed in a low temperature incubator adjusted to 25° C. and left standing for 30 minutes to obtain a D biotin-BSA solution.
  • BSA Bovine serum album:BSA
  • a colloidal gold preservation solution consisting of 0.05 wt% PEG20,000, 150 mM sodium chloride (198-01675, manufactured by Wako Pure Chemical Industries, Ltd.), 1.0 wt% BSA, and 20 mM Tris buffer. ) 30 mL was added and the mixture was treated with an ultrasonic disperser for 10 seconds. Then, using a centrifuge, a rack in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed.
  • a gold colloid coating solution (pH 8.2) consisting of 0.05 wt% PEG20,000, 37.5 mM sodium chloride, 0.25 wt% BSA, 2.5 wt% sucrose, and 20 mM Tris buffer was OD. 520 was added to 3.75 and the mixture was treated with an ultrasonic disperser for 10 seconds to obtain a control line detection reagent 1.
  • hCG Measurement Membrane Card 5.0 mg/mL of anti-hCG- ⁇ monoclonal antibody (6601, manufactured by MedixBiochemica) was prepared to 0.5 mg/mL with distilled water. Then, 1.0 mg/mL of anti-biotin polyclonal antibody (A150-111A, manufactured by BETHYL) was prepared to 0.5 mg/mL with distilled water. Next, a 60 mm x 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards) consisting of a 20 mm x 300 mm adhesive tape part on the upstream side, a 25 mm x 300 mm membrane part in the center, and a 15 mm x 300 mm adhesive tape part on the downstream side.
  • Hi-Flow Plus 120 Membrane Cards consisting of a 20 mm x 300 mm adhesive tape part on the upstream side, a 25 mm x 300 mm membrane part in the center, and a 15 mm x 300 mm adhesive tape part on the downstream side.
  • the hCG measurement membrane card 1 was obtained by forming a control line having a line width of about 1 mm by applying a coating amount of 3 to 30° C. and drying it with a dryer adjusted to 45° C. for 30 minutes.
  • a 20 mm ⁇ 300 mm absorption pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the 15 mm ⁇ 300 mm adhesive tape portion on the downstream side of the hCG measurement membrane card so as to overlap with the membrane portion by 5 mm.
  • a guillotine-type cutting module (CM5000, manufactured by BIODOT) was used to cut into strips having a width of 4 mm and a length of 60 mm to obtain an immunochromatographic test piece 1 for hCG measurement.
  • hCG antigen (30-1132, manufactured by Fitzgerald) was diluted with a diluent (pH 7.0) consisting of 50 mM potassium dihydrogen phosphate and 1.0 wt% BSA, 1 IU/L and 100 IU/L diluted hCG samples were prepared.
  • pH 7.0 a diluent
  • Example 8 (1) Preparation of Test Line Detection Reagent for hCG Measurement Instead of cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red, average particle size 340 nm, manufactured by Asahi Kasei Co., Ltd.) as detection particles in the test line detection reagent, cellulose was used.
  • a test line detection reagent 8 for measuring hCG was prepared in the same manner as in Example 1 except that particles (NanoAct (registered trademark), BL2: Dark Navy, blue, average particle size 365 nm, manufactured by Asahi Kasei Corporation) were used.
  • a gold colloid storage solution (pH 8.2) consisting of 0.05 wt% PEG 6,000, 150 mM sodium chloride, 1.0 wt% BSA, and 20 mM Tris buffer was added, and the mixture was ultrasonically dispersed for 10 seconds. Processed. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 10 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed.
  • a gold colloid storage solution pH 8.2
  • a rack-in rotor and a rack centrifugation at 8,000 G was performed at 25° C. for 10 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed.
  • a gold colloid coating solution (pH 8.2) consisting of 0.05 wt% PEG 6,000, 37.5 mM sodium chloride, 0.25 wt% BSA, 2.5 wt% sucrose, and 20 mM Tris buffer was OD. 610 was added to be 3.75, and the mixture was treated with an ultrasonic disperser for 10 seconds to obtain a control line detection reagent 8.
  • Immunochromatography for hCG measurement was performed in the same manner as in Example 1 except that the hCG measurement test line detection reagent 8 was used in place of the hCG measurement test line detection reagent 1, and the control line detection reagent 8 was used in place of the control line detection reagent 1. Test piece 8 was prepared and evaluated. Table 1 shows the obtained evaluation results. In addition, with the change of detection particles from red to blue, the measurement mode of the immunochromatographic reader was also changed from Gold Colloid to Latex.
  • test line detection reagent for hCG measurement 5.0 mg/mL of anti-hCG- ⁇ monoclonal antibody was added to 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.0).
  • 50 mM potassium dihydrogen phosphate 166-04255, manufactured by Wako Pure Chemical Industries, Ltd.
  • pH 7.0 50 mM potassium dihydrogen phosphate
  • a cleaning solution consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and the mixture was treated with an ultrasonic disperser for 10 seconds. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate antibody-sensitized latex particles, and then the supernatant was removed.
  • a coating solution consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and treated with an ultrasonic disperser for 10 seconds, and a test line detection reagent for hCG measurement.
  • a coating solution pH 7.0
  • BSA Bovine serum albumin
  • 50 mM potassium dihydrogen phosphate was added, and treated with an ultrasonic disperser for 10 seconds, and a test line detection reagent for hCG measurement.
  • An hCG measurement immunochromatographic test piece 10 was prepared and evaluated in the same manner as in Example 1 except that the hCG measurement test line detection reagent 10 was used in place of the hCG measurement test line detection reagent 1. Table 1 shows the obtained evaluation results.
  • Example 11 Instead of the membrane card (Hi-Flow Plus 120 Membrane Cards, water absorption rate 120 seconds/4 cm, HF120, manufactured by Millipore), the membrane card (Hi-Flow Plus 75 Membrane Cards, water absorption rate 75 seconds/4 cm, HF75, Millipore) was used. (Example 11), a membrane card (Hi-Flow Plus 180 Membrane Cards, water absorption speed 180 seconds/4 cm, HF180, manufactured by Millipore) (Example 12) (Example 12) (Example 12) (Example 12) The immunochromatographic test pieces 11 and 12 for measurement were produced and evaluated. Table 1 shows the obtained evaluation results.
  • Example 13 Instead of 5.0 mg/mL anti-hCG- ⁇ monoclonal antibody as the detection antibody in the test line detection reagent, 8.57 mg/mL anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY) was used as a test line.
  • HbA1c Antibody OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY
  • Example except that a 3.6 mg/mL anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) was used as the capture antibody forming the antibody instead of 5.0 mg/mL anti-hCG- ⁇ monoclonal antibody.
  • An immunochromatographic test strip 13 for HbA1c measurement was prepared in the same manner as in 1.
  • the immunochromatographic test piece 13 for HbA1c measurement was evaluated in the same manner as in Example 1 except that the diluted HbA1c samples (L1, L5) were used instead of the diluted hCG samples (1 IU/L, 100 IU/L). Table 1 shows the obtained evaluation results.
  • a blocking solution consisting of 1.0 wt% Bovine serum albumin:BSA was added, and the mixture was allowed to stand for 60 minutes in a low temperature incubator adjusted to 25°C. Then, using a centrifuge, a rack-in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate D-biotin-sensitized latex particles, and then the supernatant was removed.
  • 12 mL of a cleaning solution (pH 7.0) consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and the mixture was treated with an ultrasonic disperser for 10 seconds.
  • An immunochromatographic test strip 16 for hCG measurement was prepared and evaluated in the same manner as in Example 1 except that the control line detection reagent 16 was used in place of the control line detection reagent 1. Table 2 shows the obtained evaluation results.
  • Comparative example 4 An immunochromatographic test strip 17 for hCG measurement was prepared and evaluated in the same manner as in Comparative Example 3 except that the hCG measurement test line detection reagent 1 was used in place of the hCG measurement test line detection reagent 1. Table 2 shows the obtained evaluation results.
  • Comparative Example 2 when the ratio of the average particle size of the detection particles A captured on the test line to the average particle size of the detection particles B captured on the control line is larger than 100:1, the test is performed.
  • the retention time until the line detection reagent is captured on the test line is longer than the retention time until the control line detection reagent is captured on the control line, resulting in insufficient correction by the color intensity of the control line.
  • the measurement accuracy deteriorated.
  • the CV of the reflection absorbance of the test line itself was about 6%, whereas the CV after the correction was 7.2%, and the measurement accuracy by the correction deteriorated.
  • the average particle size of the detection particles B was as small as 2 nm, the amount of the low molecular weight compound that could be labeled decreased, and as a result, the control line was unclear.
  • the substance to be measured contained in the measurement sample can be measured quickly, simply, inexpensively and with high measurement accuracy, which greatly contributes to the industry.

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Abstract

Le problème décrit par la présente invention est de fournir une éprouvette immunochromatographique permettant de mesurer avec précision une substance à mesurer dans un échantillon à mesurer en utilisant une immunochromatographie, et un procédé de mesure utilisant l'éprouvette immunochromatographique. Selon l'invention, la solution porte sur une éprouvette immunochromatographique pour quantifier une substance à mesurer dans un échantillon de mesure dans laquelle : l'éprouvette immunochromatographique est formée en connectant dans l'ordre suivant un tampon d'échantillon, un tampon de conjugaison, une membrane et un tampon absorbant ; la membrane est pourvue d'une ligne de commande et d'une ligne d'essai qui acquièrent une couleur en réponse à une réaction antigène-anticorps ; la ligne d'essai est positionnée en amont de la ligne de commande ; et le rapport de la taille moyenne des particules de détection A capturées sur la ligne d'essai et de la taille moyenne des particules de détection B capturées sur la ligne de commande est de 3:1 à 100:1.
PCT/JP2020/005776 2019-02-14 2020-02-14 Une éprouvette immunochromatographique et procédé de mesure l'utilisant WO2020166698A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115951072A (zh) * 2022-12-06 2023-04-11 北京鸿宇泰生物科技有限公司 一种糖化血红蛋白-c肽联合检测试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020045195A1 (en) * 2000-10-13 2002-04-18 Hubscher Thomas T. Method for the visual detection of specific antibodies in human serum by the use of lateral flow assays
JP2008164403A (ja) * 2006-12-28 2008-07-17 Sysmex Corp 呼吸器感染症の検査方法
WO2011062157A1 (fr) * 2009-11-17 2011-05-26 旭化成せんい株式会社 Microparticules organiques colorées, kit de réactifs de diagnostic contenant ces microparticules, et procédé de diagnostic in vitro
JP2013053869A (ja) * 2011-09-01 2013-03-21 Furukawa Electric Co Ltd:The イムノクロマトグラフィー用試験キット、これを用いた検出方法及びこれに用いられる標識試薬
WO2017065213A1 (fr) * 2015-10-16 2017-04-20 東洋紡株式会社 Pièce de test immunochromatographique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020045195A1 (en) * 2000-10-13 2002-04-18 Hubscher Thomas T. Method for the visual detection of specific antibodies in human serum by the use of lateral flow assays
JP2008164403A (ja) * 2006-12-28 2008-07-17 Sysmex Corp 呼吸器感染症の検査方法
WO2011062157A1 (fr) * 2009-11-17 2011-05-26 旭化成せんい株式会社 Microparticules organiques colorées, kit de réactifs de diagnostic contenant ces microparticules, et procédé de diagnostic in vitro
JP2013053869A (ja) * 2011-09-01 2013-03-21 Furukawa Electric Co Ltd:The イムノクロマトグラフィー用試験キット、これを用いた検出方法及びこれに用いられる標識試薬
WO2017065213A1 (fr) * 2015-10-16 2017-04-20 東洋紡株式会社 Pièce de test immunochromatographique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115951072A (zh) * 2022-12-06 2023-04-11 北京鸿宇泰生物科技有限公司 一种糖化血红蛋白-c肽联合检测试剂盒
CN115951072B (zh) * 2022-12-06 2024-05-14 北京鸿宇泰生物科技有限公司 一种糖化血红蛋白-c肽联合检测试剂盒

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