WO2022265105A1 - Éprouvette d'immunochromatographie, et kit d'immunochromatographie - Google Patents

Éprouvette d'immunochromatographie, et kit d'immunochromatographie Download PDF

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Publication number
WO2022265105A1
WO2022265105A1 PCT/JP2022/024380 JP2022024380W WO2022265105A1 WO 2022265105 A1 WO2022265105 A1 WO 2022265105A1 JP 2022024380 W JP2022024380 W JP 2022024380W WO 2022265105 A1 WO2022265105 A1 WO 2022265105A1
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antibody
protein
cov
sars
immunochromatographic
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PCT/JP2022/024380
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English (en)
Japanese (ja)
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美穂 松尾
淳 岡本
研吾 西村
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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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention is an immunochromatography test piece that can detect the nucleocapsid protein (N protein) of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome) coronavirus 2 (SARS-CoV-2) with high sensitivity and suppressing false positives, and immunochromatography It relates to a kit containing test strips.
  • N protein nucleocapsid protein
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 is the causative virus of the new coronavirus infection (COVID-19), and has spread rapidly around the world since the beginning of 2020.
  • SARS-CoV-2 like common coronaviruses, is composed of a nucleocapsid and an envelope surrounding the nucleocapsid.
  • the nucleocapsid contains a viral genome (RNA) and a nucleocapsid protein (N protein) that binds to the viral genome.
  • the envelope includes a lipid and a spike protein (S protein) that binds to the lipid, a membrane protein (M protein), and an envelope protein (E protein).
  • the N protein is a protein involved in the formation of the viral core, packaging of the viral genome, transcription, etc., and is linked to the N-terminal domain (NTD) via a linker having a serine (S)/arginine (R)-rich region. and a C-terminal domain (CTD) bound together.
  • NTD N-terminal domain
  • S serine
  • R arginine
  • Non-Patent Document 1 describes that the N protein is extensively phosphorylated and shows mapping of phosphorylation sites. Since the amino acid sequence of the N protein is conserved among strains, the N protein is used as a diagnostic marker and the like.
  • Immunochromatography is an immunoassay method that utilizes capillary action, and is widely used worldwide in influenza testing and the like.
  • One technique for detecting a substance to be measured using immunochromatography is a sandwich method that utilizes an antigen-antibody reaction.
  • sandwich method two types of antibodies with different epitopes are used for the substance to be measured.
  • One antibody is used as a detection antibody sensitized with detection particles such as colloidal gold, colored latex particles, fluorescent particles and the like.
  • detection particles such as colloidal gold, colored latex particles, fluorescent particles and the like.
  • the other antibody forms the test line as a capture antibody linearly immobilized on the surface of the porous support.
  • an antibody that specifically captures the detection antibody is linearly immobilized on the surface of the porous support at a position different from the test line to form a control line.
  • the substance to be measured contained in the measurement sample develops from one end (upstream side) of the porous support, moves while forming an immune complex with the detection antibody, and is captured by coming into contact with the capture antibody on the test line. develop color.
  • Free detection particles that did not form an immune complex with the substance to be measured and the sensitized detection antibody pass through the test line are captured by the control line antibody, and develop color. The presence or absence of the substance to be measured can be determined by visually confirming these color development intensities.
  • immunoassay methods using mouse monoclonal antibodies are used to measure analytes (measurement targets) contained in samples such as blood and urine.
  • analytes measured targets
  • samples such as blood and urine.
  • non-specific reactions other than the intended specific antigen-antibody reactions often impair the reliability of the measured values. It recognized.
  • Heterophilic antibodies such as human anti-mouse antibodies (HAMA) contained in the specimen to be tested, allow binding to the solid phase, for example in a typical sandwich ELISA assay, even though the analyte is not present.
  • HAMA human anti-mouse antibodies
  • Non-specific cross-linking between the labeled antibody and the labeled antibody to be detected occurs, resulting in a false positive signal. While automation of testing has progressed and rapid measurement has become possible, false reactions such as HAMA have increased, and these non-specific reactions are often overlooked.
  • Non-Patent Document 2 See Non-Patent Document 3. This phenomenon also applies to immunochromatography.
  • HAMA human-derived specimens
  • In vivo administration of a mouse antibody produces HAMA, which poses a problem of eliciting an immune response to a heterologous antigen.
  • Recent antibody drugs include chimeric antibodies fusing a mouse-derived antigen-binding site and a human-derived constant region, and advances in humanized antibody production technology. is increasing and the problem cannot be completely ignored.
  • Heterophilic antibodies are known not only for mice, but also for animals such as goats, sheep, and rabbits. (Goat: HAGA, Sheep: HASA, Rabbit: HARA).
  • aggregates derived from monoclonal or polyclonal antibodies has been suggested.
  • This aggregate may be a homopolymer of the antibody, an antibody fragment, or a heteropolymer with a protein such as albumin or a polysaccharide macromolecule such as dextran.
  • An object of the present invention is to provide an immunochromatographic test strip that can detect the SARS-CoV-2 N protein with high sensitivity and reduced false positives, and a kit containing the immunochromatographic test strip.
  • the present inventors have found that by using a mixture of two types of antibodies of different origins as a capture antibody and a detection antibody, SARS-1 can be detected with high sensitivity and reduced false positives. It was found that the N protein of CoV-2 could be detected. In addition, the inventors have found that the N protein of SARS-CoV-2 can be detected with higher sensitivity by using cellulose-based colored microparticles as detection particles, and have completed the present invention.
  • the representative present invention is as follows. 1. (1) a sample pad; (2) Carrying a complex of an antibody composition A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample and cellulose-based colored fine particles a conjugation pad with (3) Line the antibody composition B that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the antibody composition C that specifically binds to the antibody composition A at different positions.
  • N protein nucleocapsid protein
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the antibody composition A is a mixture of antibody A1 and antibody A2 derived from different origins, An immunochromatographic test strip, wherein the antibody composition B is a mixture of antibody B1 and antibody B2 derived from different origins.
  • Any one of the antibody A1 and the antibody A2 is a mouse-derived antibody.
  • the antibody A1 and the antibody A2 are supported on the conjugation pad at a mixing ratio (mass ratio) of 10:1 to 1:10. or 2.
  • 4. 1. Any one of the antibody B1 and the antibody B2 is a mouse-derived antibody. to 3. Immunochromatographic test strip according to any one of. 5. 1.
  • the antibody B1 and the antibody B2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. to 4.
  • Immunochromatographic test strip according to any one of. 6.
  • the antibody composition C is a mixture of an antibody C1 that binds to the antibody A1 and an antibody C2 that binds to the antibody A2. to 3.
  • the antibody C1 and the antibody C2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10.
  • An immunochromatographic kit comprising the immunochromatographic test piece according to any one of 1, a measurement sample collecting tool, a filter, and a measurement sample diluent.
  • the immunochromatographic test strip of the present invention carries specific antibodies and detection particles in a specific arrangement, it is possible to detect the SARS-CoV-2 N protein with high sensitivity and reduced false positives.
  • FIG. 1 is a diagram (top view) showing an example of an immunochromatographic test strip of the present invention.
  • FIG. 1 is a diagram (side view) showing an example of an immunochromatographic test strip of the present invention.
  • FIG. 1 is a diagram (side view) showing an example of an immunochromatographic test strip of the present invention.
  • the immunochromatographic test strip is a test strip for detecting the nucleocapsid protein (N protein) of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample.
  • the measurement sample used in the present invention includes not only the sample as collected but also the sample subjected to pretreatment such as removal of contaminants.
  • sample to be measured examples include, but are not limited to, blood, serum, plasma, bone marrow fluid, lymph, tears, nasal discharge, nasal wash, nasal swab, saliva, gargle, sputum, pharyngeal swab, sweat, tracheal aspirate, bronchi. Lavage, pleural fluid, ascites, amniotic fluid, intestinal lavage, urine, feces, cell extract, tissue extract, organ extract and the like.
  • the N protein of SARS-CoV-2 has the amino acid sequence disclosed in GenBank Accession No. (MN908947).
  • the SARS-CoV N protein has the amino acid sequence disclosed in GenBank Accession No. (AY278741).
  • the configuration of the immunochromatography test piece is such that the addition portion (dropping portion) of the measurement sample solution of the immunochromatography 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.
  • 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
  • 8 is an adhesive sheet, respectively.
  • the immunochromatographic test piece has a long and narrow rectangular shape with a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm).
  • the conjugation pad 2 of the immunochromatography test strip carries a complex of antibody composition A and cellulose-based colored fine particles for specifically capturing the N protein of SARS-CoV-2 in the measurement sample.
  • the antibody composition B for specifically capturing the SARS-CoV-2 N protein in the measurement sample was linearly immobilized at a position about 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip.
  • a test line 6 is formed.
  • a control line 7 is formed in which the antibody composition C that specifically binds to the antibody composition A is linearly fixed at a position about 20 mm from the end.
  • the sample pad 1 is not particularly limited as long as it is made of a material that can rapidly absorb the measurement sample and then spread to downstream conjugation pads, membranes, and absorbent pads.
  • cellulose filter paper or non-woven fabric. glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, polyethylene filter paper or non-woven fabric.
  • the thickness of the sample pad 1 is preferably 0.1 to 2 mm, more preferably 0.2 to 1 mm. If the thickness is too small, the flow of the sample to be measured downstream may become non-uniform, resulting in a decrease in measurement accuracy. On the other hand, if the thickness is large, the downstream deployment may be delayed and the measurement time may be lengthened. In addition, the amount of measurement sample required for downstream development is increased.
  • the conjugation pad 2 can hold in a dry state a complex of the antibody composition A that specifically binds to the SARS-CoV-2 N protein in the measurement sample and the cellulose-based colored fine particles, and the measurement
  • the material is not particularly limited as long as it can rapidly release the complex as the sample is developed downstream, but for example, cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or Non-woven fabrics, polyethylene filter paper or non-woven fabrics can be mentioned. Among these, glass filter paper is preferable.
  • the thickness of the conjugation pad 2 is preferably 0.1 to 2 mm, more preferably 0.2 to 1 mm. If the thickness is too small, it may not be possible to retain the desired amount of the composite in a dry state. On the other hand, if the thickness is large, the downstream deployment may be delayed and the measurement time may be lengthened. In addition, the amount of measurement sample required for downstream development is increased.
  • the membrane 3 is not particularly limited as long as it can accurately and uniformly develop the measurement sample.
  • Membranes made of vinylidene chloride or nylon can be mentioned. Among these, nitrocellulose membranes are preferred.
  • the absorbent pad 4 is not particularly limited as long as it is made of a material that can quickly absorb the measurement sample developed from upstream and then hold it so that it does not flow back.
  • cellulose filter paper or nonwoven fabric glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, and polyethylene filter paper or non-woven fabric.
  • the thickness of the absorbent pad 4 is preferably 0.2 to 5 mm, more preferably 0.5 to 2 mm. If the thickness is small, the measurement sample once absorbed by the absorbent pad may flow back to the membrane side depending on the amount of the measurement sample dropped. On the other hand, if the thickness is large, the sizes of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also become large, which is not preferable from the point of view of POCT.
  • the antibodies used in the present invention may be monoclonal antibodies or polyclonal antibodies, but monoclonal antibodies are preferred.
  • Antibodies can be of any isotype, eg, IgG, IgA, IgD, IgE, IgM, etc., but IgG is preferred.
  • the antibody may be a commercially available product, or may be separately produced by a known method.
  • Antibody composition A used as a detection antibody in the present invention must be a mixture of antibody A1 and antibody A2 that specifically binds to the N protein of SARS-CoV-2 and has different origins. Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. Therefore, since they have different reactivities with respect to antigens, the degree of binding of antibodies to antigens increases compared to the case of antibodies alone, resulting in higher sensitivity. In particular, mouse-derived antibodies are presumed to have a structure with a higher degree of binding to antigens than other antibodies. Therefore, it is preferable that either the antibody A1 or the antibody A2 is a mouse-derived antibody. If the antibody composition A is not a mixture of antibodies A1 and A2 derived from different origins, the detection sensitivity may be reduced. Also, false positives may occur.
  • the mixing ratio (mass ratio) of antibody A1 and antibody A2 having different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease. Also, false positives may occur.
  • the reflection absorbance of the test line is preferably 40 mAbs or more, more preferably 60 mAbs or more, and even more preferably 80 mAbs or more, because the visibility at the time of completion of measurement is good and the line can be visually recognized at an early point from the start of measurement.
  • Cellulose-based colored fine particles have a large amount of hydroxyl groups, so they can not only hold many reactive dyes through covalent bonds, but also maintain stable dispersibility in water even after deep dyeing.
  • As the cellulose-based colored fine particles regenerated cellulose, purified cellulose, natural cellulose, etc. can be used, and partially derivatized cellulose may also be used.
  • 20 to 90 mass % of the mass of the cellulose-based colored fine particles is preferably derived from cellulose, more preferably 20 to 80 mass %, even more preferably 20 to 70 mass %.
  • the average particle size of the cellulose-based colored fine particles is not particularly limited, but is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle size is large, downstream development may be delayed and the measurement time may be lengthened. In addition, it tends to be captured on the membrane, and the background itself develops color, which may obscure the color development on the test line and the control line. On the other hand, when the average particle size is small, the amount of antibody that can be physically adsorbed or chemically bonded decreases, and the measurement sensitivity may decrease.
  • the color of the cellulose-based colored fine particles is not particularly limited, but examples include red, blue, yellow, green, black, white, and fluorescent colors. Among these, red, blue, and black, which are highly visible, are preferable.
  • Examples of such colored cellulose-based fine particles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation.
  • the binding amount of the antibody composition A to the cellulose-based colored fine particles can be controlled by adjusting the charged mass ratio of the cellulose-based colored fine particles and the antibody composition A, and is not particularly limited.
  • the charged mass ratio with substance A is preferably 1:0.01 to 1:1, more preferably 1:0.02 to 1:0.5, and even more preferably 1:0.02 to 1:0.2. If the mass ratio is outside the above range, the binding amount of the antibody composition A to the cellulose-based colored fine particles becomes insufficient, or the binding amount of the antibody composition A to the cellulose-based colored fine particles increases excessively, resulting in an antigen-antibody reaction. Since the amount of antibody composition A that does not contribute increases, the measurement sensitivity may decrease.
  • the method of binding the antibody composition A and the cellulose-based colored microparticles is not particularly limited, but sensitization is preferably performed by physical adsorption by hydrophobic bonding or chemical bonding by covalent bonding. Adsorption is more preferred.
  • a reactive active group may be introduced into the cellulose-based colored fine particles.
  • reactive active groups include, but are not limited to, carboxyl groups, amino groups, aldehyde groups, thiol groups, epoxy groups, and hydroxyl groups. Among these, a carboxyl group and an amino group are preferred. In the case of carboxyl groups, carbodiimides can be used to form covalent bonds with amino groups of ligands.
  • the method of causing the conjugation pad 2 to support the complex of the antibody composition A that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the cellulose-based colored fine particles is not particularly limited. It can be produced by uniformly applying, spraying or impregnating the body solution onto the conjugation pad and then drying it in a constant temperature bath at an appropriate temperature for a certain period of time.
  • the amount of the composite solution to be applied is not particularly limited, but is preferably 5 ⁇ L to 50 ⁇ L per 1 cm line length.
  • the concentration of the cellulose-based colored fine particles in the solution of the composite is not particularly limited, but is preferably 0.01 to 0.5% by mass, more preferably 0.02 to 0.2% by mass, and 0.02 to 0.2% by mass.
  • the applied conjugation pad is then dried.
  • 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 to 120 minutes.
  • antibody composition B used as a capture antibody forming test line 6 specifically binds to the N protein of SARS-CoV-2 and is a mixture of antibody B1 and antibody B2 of different origins. is necessary.
  • Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. As a result, they have different reactivities to the antigen, and the degree of binding of the antibody to the antigen increases, resulting in higher sensitivity.
  • the mouse-derived antibody has a structure with a higher degree of binding to an antigen than other antibodies due to the attachment of sugar chains. Therefore, it is preferable that either the antibody B1 or the antibody B2 is a mouse-derived antibody. If antibody composition B is not a mixture of antibody B1 and antibody B2 derived from different origins, the detection sensitivity may be reduced. Also, false positives may occur.
  • the mixing ratio (mass ratio) of antibody B1 and antibody B2 having different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease. Also, false positives may occur.
  • the antibody composition C used as the capture antibody forming the control line 7 must be an antibody that specifically binds to the antibody composition A. Moreover, the antibody composition C is preferably a mixture of the antibody C1 that binds to the antibody A1 and the antibody C2 that binds to the antibody A2. If antibody composition C is not a mixture of antibody C1 and antibody C2, the control line will be faint and may be judged as re-measurement.
  • the mixing ratio (mass ratio) of the antibodies C1 and C2 is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the control line becomes thin and may be judged as re-measurement.
  • the method of linearly immobilizing the capturing antibody forming the test line 6 and the capturing antibody forming the control line 7 on the membrane 3 is not particularly limited, but for example, the capturing antibody forming the test line and the capturing antibody forming the control line are fixed. can be produced by applying a given amount of each of and to different positions on the line, and then drying at an appropriate temperature in a constant temperature bath for a given period of time.
  • the amount of the two capture antibodies to be applied is not particularly limited, but is preferably 0.1 ⁇ L to 2 ⁇ L per 1 cm of line length.
  • the application concentration of both the capturing antibodies is not particularly limited, but is preferably 0.1 mg/mL to 10 mg/mL, more preferably 0.2 mg/mL to 8 mg/mL, and 0.5 mg/mL to 5 mg/mL. is more preferred. If the concentration is too low, the N protein of SARS-CoV-2 cannot be sufficiently captured and detected, and the measurement sensitivity may decrease. On the other hand, even if the concentration is high, the measurement sensitivity is not improved, and only the cost is increased.
  • 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 to 120 minutes.
  • the membrane 3 prepared above is attached near the center of the adhesive sheet 8, and then the conjugation pad 2 is partially overlapped on one end of the membrane 3 and attached. is partially overlapped on the end of the conjugation pad 2 opposite to the overlap with the membrane 3, then the absorbent pad 4 is partially overlapped on the other end of the membrane 3, and then fixed It can be produced by cutting it into width strips.
  • the test line 6 and the control line 7 may be prepared after preparing the test piece, or may be prepared before preparing the test piece.
  • the immunochromatographic test piece has at least a first opening for dropping the measurement sample onto the sample pad 1 and a second opening for visually confirming the test line 6 and the control line 7 on the membrane 3. may be housed in a plastic housing case.
  • the immunochromatographic measurement kit includes, in addition to the immunochromatographic test strip, a measurement sample collecting tool for collecting a measurement sample, a measurement sample diluent for pretreating and / or diluting the measurement sample, and a measurement sample for filtering. preferably contains a filter of
  • the measurement sample diluent preferably contains a nonionic surfactant that improves the spreadability of the measurement sample and does not affect the immune reaction.
  • nonionic surfactants include, but are not limited to, polyoxyethylene alkylphenyl ethers (Triton (registered trademark) surfactants, etc.), polyoxyethylene alkyl ethers (Brij (registered trademark) surfactants, etc.), Examples include polyoxyethylene sorbitan fatty acid esters (Tween (registered trademark) surfactants, etc.), polyoxyethylene fatty acid esters, sorbitan fatty acid esters, alkylglucosides, sucrose fatty acid esters, and the like.
  • surfactants may be used alone or in combination of two or more.
  • concentration of the nonionic surfactant is preferably 0.01% by mass to 5.0% by mass, more preferably 0.05% by mass to 4.0% by mass, and 0.1% by mass to 3.0% by mass. More preferred. Low concentrations can make downstream deployment difficult. In addition, the deployment may become uneven and the measurement accuracy may be lowered. On the other hand, if the concentration is high, the physically adsorbed detection particles and the antibody, and/or the membrane and the antibody, may deviate from each other, resulting in failure to obtain measurement values.
  • Inorganic salts and buffers used for pH adjustment may be added to the measurement sample diluent.
  • the buffering agent any type of buffering agent may be used as long as it has a sufficient buffering capacity in the target pH range. acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good's buffer (MES, ADA, PIPES, ACES, colamin hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine).
  • tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferable, because they have sufficient buffering capacity around 7.0, which is the optimum pH range of the antibody used in the present invention.
  • Acid, PIPES is more preferred.
  • Example 1 Preparation of complexes of antibody A1, antibody A2 and cellulose-based colored fine particles 1.0% by mass of cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) 100 ⁇ L, 10 mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.0) 900 ⁇ L, 1.0 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody as antibody A1 ( Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, manufactured by Toyobo) 50 ⁇ L, and 1.0 mg / mL rabbit-derived anti-SARS-CoV-2 N protein monoclonal antibody (SARS-CoV- 2 (COVID-19) nucleocapsid antibody, [HL5511], Cat No.
  • NaAct registered trademark
  • BL2 Dark Navy,
  • GTX635689, manufactured by GeneTex 50 ⁇ L was added to a 15 mL centrifuge tube and vortexed. Then, it was allowed to stand at 37°C for 120 minutes. Next, a blocking solution (pH 8.0) consisting of 1.0% by mass of casein (030-01505, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ) was added, and the mixture was allowed to stand at 37° C. for 60 minutes. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), centrifugation was performed at 13,000 ⁇ g at 25° C.
  • a cleaning solution consisting of 50 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added, and treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMTE). did. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), centrifugation was performed at 13,000 ⁇ g at 25° C. for 15 minutes to precipitate the antibody-sensitized cellulose-based colored fine particles, and then the supernatant was removed.
  • a cleaning solution consisting of 50 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added, and treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMTE). did. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), centrifugation was performed at 13,000 ⁇ g at 25° C. for 15 minutes to precipitate the antibody
  • sucrose (196-00015, manufactured by Fujifilm Wako Pure Chemical Industries), 0.2% by mass of casein (030-01505, manufactured by Fujifilm Wako Pure Chemical Industries), 62 mM borate buffer (021 -02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added with 2.0 mL of a coating solution (pH 9.2), and treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMTE). A composite with cellulose-based colored fine particles was obtained.
  • 1.0 mg/mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) as antibody C1
  • 1.0 mg/mL goat-derived anti-antibody as antibody C2.
  • a rabbit IgG polyclonal antibody (Rabbit IgG-heavy and light chain antibody, A120-101A, manufactured by BETHYL) was mixed at a mixing ratio (mass ratio) of 1:1.
  • a 60 mm ⁇ 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards) composed of an adhesive tape portion of 20 mm ⁇ 300 mm on the upstream side, a membrane portion of 25 mm ⁇ 300 mm in the center, and an adhesive tape portion of 15 mm ⁇ 300 mm on the downstream side.
  • a 20 mm ⁇ 300 mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore Co., Ltd. ) were pasted together. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), a strip of 4 mm in width and 60 mm in length was cut to obtain an immunochromatographic test strip for detecting SARS-CoV-2 N protein. The obtained immunochromatographic test piece for detecting N protein of SARS-CoV-2 was housed in a housing case (K007, manufactured by Shengfeng Plastic) to obtain an immunochromatographic device 1 for detecting N protein of SARS-CoV-2.
  • CM5000 manufactured by BIODOT
  • Example 2 Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by East Coast Bio) was used as antibody A2, and a goat-derived anti-rat IgG polyclonal antibody (Rat An immunochromatographic device 2 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that IgG-heavy and light chain antibody, A110-105A, manufactured by BETHYL) was used.
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Example 3 SARS-CoV in the same manner as in Example 1 except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by EastCoast Bio) was used as antibody B2.
  • An immunochromatographic device 3 for N protein detection of -2 was obtained.
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A2, and rat-derived anti-SARS-CoV-2 was used as antibody B2.
  • N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by East Coast Bio) was used, and antibody C2 was a goat-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-105A,
  • An immunochromatographic device 4 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except for using BETHYL).
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A1, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat An immunochromatographic device 5 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that IgG-heavy and light chain antibody, A110-122A, manufactured by BETHYL) was used.
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Example 6 SARS-CoV in the same manner as in Example 1 except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by EastCoast Bio) was used as antibody B1.
  • An immunochromatographic device 6 for N protein detection of -2 was obtained.
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A1, and rat-derived anti-SARS-CoV-2 was used as antibody B1.
  • N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by East Coast Bio) was used, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-122A, A110-122A, An immunochromatographic device 7 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that BETHYL) was used.
  • Table 1 shows the evaluation results of (8) nonspecific adsorption
  • Table 2 shows the evaluation results of (9) sensitivity
  • Table 3 shows the evaluation results of (10) false positives of the obtained device.
  • Example 8 An immunochromatographic device 8 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that the concentration of antibody A1 was 0.222 mg/mL and the concentration of antibody A2 was 1.778 mg/mL.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 9 An immunochromatographic device 9 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was 1.818 mg/mL and the concentration of antibody A2 was 0.182 mg/mL.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 10 An immunochromatographic device 10 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was 1.905 mg/mL and the concentration of antibody A2 was 0.095 mg/mL.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 11 An immunochromatographic device 11 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 1:8.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 12 An immunochromatographic device 12 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 10:1.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 13 An immunochromatographic device 13 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 20:1.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 14 An immunochromatographic device 14 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 1:0.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 15 An immunochromatographic device 15 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 1:8.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 16 An immunochromatographic device 16 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 10:1.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • Example 17 An immunochromatographic device 17 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 20:1.
  • Table 4 shows the evaluation results of (8) nonspecific adsorption
  • Table 5 shows the evaluation results of (9) sensitivity
  • Table 6 shows the evaluation results of (10) false positive of the obtained device.
  • An immunochromatographic device A for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1.
  • Table 7 shows the evaluation results of (8) nonspecific adsorption
  • Table 8 shows the evaluation results of (9) sensitivity
  • Table 9 shows the evaluation results of (10) false positives of the obtained device.
  • Example 3 1.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo) was used as antibody B1, and antibody B2 was used.
  • An immunochromatographic device C for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that it was not used.
  • Table 7 shows the evaluation results of (8) nonspecific adsorption
  • Table 8 shows the evaluation results of (9) sensitivity
  • Table 9 shows the evaluation results of (10) false positives of the obtained device.
  • an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and suppressed false positives.
  • sample pad 2 conjugation pad 3: membrane 4: absorbent pad 5: backing sheet 6: test line 7: control line 8: adhesive sheet

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Abstract

L'invention fournit une éprouvette d'immunochromatographie permettant de détecter la protéine N du ＳＡＲＳ－ＣｏＶ－２ selon une sensibilité élevée et en réduisant les faux positifs. Plus précisément, l'invention concerne une éprouvette d'immunochromatographie qui est configurée par : (1) un tampon d'échantillon ; (2) un tampon de conjugaison sur lequel est supporté un composite de microparticules colorées de cellulose et d'une composition d'anticorps (A) s'unissant spécifiquement à la protéine de nucléocapside (protéine N) du coronavirus du syndrome respiratoire aigu sévère 2 (ＳＡＲＳ－ＣｏＶ－２) dans un échantillon mesuré ; (3) une membrane sur laquelle une composition d'anticorps (B) s'unissant spécifiquement à la N protéine du ＳＡＲＳ－ＣｏＶ－２ dans l'échantillon mesuré, et une composition d'anticorps (C) s'unissant spécifiquement à ladite composition d'anticorps (A), se fixent de manière linéaire en des positions réciproquement différentes ; et (4) un tampon absorbant. Ladite composition d'anticorps (A) consiste en un mélange d'un anticorps (A1) et d'un anticorps (A2) d'origines différentes, et ladite composition d'anticorps (B) consiste en un mélange d'un anticorps (B1) et d'un anticorps (B2) d'origines différentes.
PCT/JP2022/024380 2021-06-17 2022-06-17 Éprouvette d'immunochromatographie, et kit d'immunochromatographie WO2022265105A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019187271A1 (fr) * 2018-03-30 2019-10-03 株式会社バイオメディカル研究所 Ensemble d'essai et procédé d'essai
CN111733141A (zh) * 2020-06-19 2020-10-02 清华大学深圳国际研究生院 一种可分泌抗新型冠状病毒n蛋白单克隆抗体的杂交瘤细胞、单克隆抗体及应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019187271A1 (fr) * 2018-03-30 2019-10-03 株式会社バイオメディカル研究所 Ensemble d'essai et procédé d'essai
CN111733141A (zh) * 2020-06-19 2020-10-02 清华大学深圳国际研究生院 一种可分泌抗新型冠状病毒n蛋白单克隆抗体的杂交瘤细胞、单克隆抗体及应用

Non-Patent Citations (2)

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Title
TIAN XINGUI, MO CHUNCONG, ZHOU LILING, YANG YUJIE, ZHOU ZHICHAO, YOU AIPING, FAN YE, LIU WENKUAN, LI XIAO, ZHOU RONG: "Epitope mapping of severe acute respiratory syndrome-related coronavirus nucleocapsid protein with a rabbit monoclonal antibody", VIRUS RESEARCH, vol. 300, 1 July 2021 (2021-07-01), NL , pages 1 - 7, XP093014610, ISSN: 0168-1702, DOI: 10.1016/j.virusres.2021.198445 *
YAMAKATUA KENTARO, FUJIMOTO AKIRA, MIYATNOTO KAZUYOSHI, OSHIMA TAKUMA, SUZUKI TADAKI, NAGATA NORIYO, SHIRATO KAZUYA, SATO HIDEO, H: "Development of Rapid Immunochromatographic Enzyme Immunoassay for SARS CoV-2 nucleocapsid antigen.", IKAGAKU TO YAKUGAKU = JAPANESE JOURNAL OF MEDICINE AND PHARMACEUTICAL SCIENCE, vol. 77, no. 6, 27 May 2020 (2020-05-27), JP , pages 937 - 944, XP009536599, ISSN: 0389-3898 *

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