WO2020166699A1 - Pièce de test immunochromatographique et procédé d'immunochromatographie l'utilisant - Google Patents
Pièce de test immunochromatographique et procédé d'immunochromatographie l'utilisant Download PDFInfo
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- WO2020166699A1 WO2020166699A1 PCT/JP2020/005780 JP2020005780W WO2020166699A1 WO 2020166699 A1 WO2020166699 A1 WO 2020166699A1 JP 2020005780 W JP2020005780 W JP 2020005780W WO 2020166699 A1 WO2020166699 A1 WO 2020166699A1
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- immunochromatographic test
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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
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- the present invention relates to an immunochromatographic test strip for rapidly measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. More specifically, the present invention relates to an immunochromatographic test strip that is fast and has high measurement sensitivity, 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 test line is placed upstream of the control line, and the color of the control line on the downstream side ensures that the sample has normally passed the test line. It may be placed upstream. The latter contributes to the shortening of the measurement time because the control line takes less time to develop color than the former.
- 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 the test line detection reagent that forms an immune complex with the measurement target substance also becomes small, so a large amount of free test line detection reagent is 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 since 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.
- Patent Document 1 since the test line is arranged on the upstream side of the control line, there is a problem that it takes time for the control line to develop color, that is, the measurement time becomes long. In addition, since it takes a short time for the test line detection reagent to reach the test line, the measurement sensitivity is slightly lowered.
- Patent Document 2 since the control line is arranged on the upstream side of the test line, there is a certain effect in improving the measurement sensitivity, but the shortening of the measurement time is insufficient.
- An object of the present invention is to provide an immunochromatographic test strip for rapidly measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. More specifically, it is an object of the present invention to provide an immunochromatographic test strip that is rapid and has high measurement sensitivity, and a measurement method using the same.
- the present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, the detection in the test line detection reagent in which the control line is located upstream of the test line (on the sample pad side) and which is composed of the detection antibody and the detection particles A
- the detection in the test line detection reagent in which the control line is located upstream of the test line (on the sample pad side) and which is composed of the detection antibody and the detection particles A By using an immunochromatographic test strip in which the average particle size of the particles A is larger than the average particle size of the detection particles B in the control line detection reagent composed of the detection particles B labeled with a low molecular weight compound, it is faster and faster than the prior art. It was found that the measurement sensitivity was improved.
- the average particle size of the detection particles A in the test line detection reagent captured by the test line located on the downstream 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 upstream side.
- the diameter By making it larger than the diameter, it takes less time for the control line detection reagent to be captured on the control line, and more time for the test line detection reagent to be captured on the test line, resulting in quick and high It is speculated that sensitive measurements have become possible.
- 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 configuration in which a sample pad, a conjugation pad, a membrane, and an absorption pad are arranged in order.
- the membrane is provided with a test line and a control line that develop color by an antigen-antibody reaction, the control line is located upstream of the test line, and the average particle diameter of the detection particles A captured by the test line is large. And an average particle size ratio of the detection particles B captured by the control line is 3:1 to 100:1.
- the immunochromatographic test strip of the present invention and the measuring method using the same the color development intensity of the control line is stabilized because the independent antigen-antibody reaction proceeds in the test line and the control line, and the control line detection reagent is captured by the control line. It is possible to measure the substance to be measured contained in the measurement sample quickly and with high sensitivity by shortening the time until the measurement is performed and increasing the time until the test line detection reagent is captured in the test line. it can.
- 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 constitution 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 arranged in this order.
- 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 control line, 7 is a test line, and 8 is an adhesive sheet.
- the immunochromatographic test piece is in the form of an elongated strip 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 7 in which a capture antibody that specifically binds to the substance to be measured is linearly immobilized is formed at a position approximately 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip.
- a control line 6 in which a capture antibody that specifically binds to a low molecular weight compound described later is linearly immobilized is formed at a position of about 10 mm from the end.
- 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.
- a control line detection reagent is carried.
- 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. If the average particle size of the detection particles is large, the downstream development becomes slow and the measurement time becomes long. In addition, it is easy to be captured on the membrane and the background itself is colored, so that the coloring on the test line and the control line becomes unclear. On the other hand, when 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 decreases. Further, in the case of the control line detection reagent, the amount of the low molecular weight compound that can be labeled decreases, and the color development on the line becomes unclear.
- 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 A is located on the downstream side of the test line. It takes a longer time to reach, that is, the time for the antigen-antibody reaction becomes longer, so that highly sensitive measurement becomes possible.
- the time required for the control line detection reagent (that is, the detection particles B) to reach the control line on the upstream side becomes shorter, that is, the time until the control line develops color becomes shorter, which enables quick measurement.
- the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is small, the time until the test line detection reagent is captured in the test line is shortened, or the control line detection reagent is captured in the control line. Since it takes a long time until the measurement is completed, it becomes difficult to achieve both high-sensitivity measurement and quick measurement.
- the test line detection reagent undergoes an antigen-antibody reaction with the substance to be measured during development, a longer residence time contributes to higher sensitivity, but the control line detection reagent does not react during development. Therefore, the shorter the retention time, the faster it contributes to speeding up.
- the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is large, for example, the detection particles A are larger than 1000 nm or the detection particles B are 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, and the detection particles B are spherical gold fine particles. Therefore, 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 the detection particles B are blue, the detection particles A are preferably cellulose particles or latex particles, and the detection particles B are preferably plate-shaped gold fine particles.
- the detection particles A are cellulose particles and the detection particles B are plate-shaped gold fine particles. It should be noted that the gold fine particles exhibit 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 7 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 6 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. 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 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 mm to 5.0 mm, more preferably 0.5 mm 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. On the other hand, when 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 7 and the control line 6 may be prepared after producing the test piece, or may be prepared before producing the test piece.
- the immunochromatographic test strip 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 7 and the control line 6 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 1 and then drying for 30 minutes with a dryer adjusted to 45°C.
- 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
- the measurement time at the time when the reflection absorbance ⁇ 50 mAbs at which the control line is visible is calculated: t (second) is calculated, and t ⁇ 60 seconds is ⁇ (excellent), 60 seconds ⁇ t ⁇ 90 seconds Was evaluated as ⁇ (good), and 90 seconds ⁇ t was evaluated as ⁇ (bad).
- the reflection absorbance (mAbs) of the test line was measured over time from 0 seconds to 10 minutes using an immunochromatographic reader, and the measurement time at the time when the reflection absorbance of the control line exceeded 50 mAbs: 5 minutes after t (seconds)
- the reflection absorbance of the control line and the test line of was used as the measured value.
- 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 and the average particle size of the detection particles B captured on the control line is larger than 100:1, detection is performed. Since the average particle size of the particle B is as small as 2 nm, the amount of low-molecular compounds that can be labeled is reduced, and as a result, the control line becomes unclear, and the reflectance is ⁇ 50 mAbs, which is the level at which the control line, which is an index of rapidity, can be visually recognized. Until: It was difficult to determine t (seconds). Therefore, evaluation of sensitivity has not been carried out.
- the substance to be measured contained in the measurement sample can be measured quickly, easily, inexpensively and with high measurement sensitivity, which greatly contributes to the industry.
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Abstract
Le problème décrit par la présente invention est de fournir une pièce de test immunochromatographique pour mesurer rapidement et avec une sensibilité élevée, une substance à mesurer dans un échantillon de mesure par immunochromatographie, et un procédé de mesure utilisant la pièce de test immunochromatographique. La solution selon l'invention porte sur une pièce de test immunochromatographique pour quantifier une substance à mesurer dans un échantillon de mesure: la pièce de test immunochromatographique est formée en disposant de manière connectée et 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 de test qui développent une couleur en réponse à une réaction antigène-anticorps; la ligne de commande est positionnée en amont de la ligne de test; et le rapport de la taille de particule moyenne des particules de détection A capturées sur la ligne de test et de la taille de particule moyenne des particules de détection B capturées sur la ligne de commande est de 3:1-100:1.
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CN113848330A (zh) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | 一种同型半胱氨酸和维生素b12的检测方法、检测试纸条及其应用 |
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