WO2022265106A1 - Immunochromatographic test piece - Google Patents

Immunochromatographic test piece Download PDF

Info

Publication number
WO2022265106A1
WO2022265106A1 PCT/JP2022/024381 JP2022024381W WO2022265106A1 WO 2022265106 A1 WO2022265106 A1 WO 2022265106A1 JP 2022024381 W JP2022024381 W JP 2022024381W WO 2022265106 A1 WO2022265106 A1 WO 2022265106A1
Authority
WO
WIPO (PCT)
Prior art keywords
sars
cov
protein
antibody
manufactured
Prior art date
Application number
PCT/JP2022/024381
Other languages
French (fr)
Japanese (ja)
Inventor
美穂 松尾
淳 岡本
研吾 西村
Original Assignee
東洋紡株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 東洋紡株式会社 filed Critical 東洋紡株式会社
Priority to JP2023530439A priority Critical patent/JPWO2022265106A1/ja
Publication of WO2022265106A1 publication Critical patent/WO2022265106A1/en

Links

Images

Classifications

    • 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 provides an immunochromatographic test strip 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 high specificity, and an immunochromatographic test strip. relating to kits containing
  • 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 immunocomplexes with the substance to be measured and the sensitized detection antibody pass over the test line and are captured by the control line antibody to develop color.
  • the presence or absence of the substance to be measured can be determined by visually confirming these color development intensities (Patent Document 1).
  • An object of the present invention is to provide an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and high specificity, and a kit containing the immunochromatographic test strip.
  • the present inventors have found that the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein as a detection antibody or capture antibody is an antibody with a weighted reaction ratio of 40 or more.
  • an immunochromatographic specimen that does not react with the SARS-CoV N protein but reacts with the SARS-CoV-2 N protein can be obtained.
  • 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) A conjugate carrying a complex of antibody A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample and cellulose-based colored fine particles gaming pad and (3) A membrane in which antibody B that specifically binds to the SARS-CoV-2 N protein in the measurement sample and antibody C that specifically binds to the antibody A are linearly immobilized at different positions. , (4) an absorbent pad; An immunochromatographic test strip, wherein the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A or antibody B is 40 or more. 2.
  • the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A and antibody B is 40 or more; Immunochromatographic test piece according to. 3. 1. or 2.
  • 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 high specificity.
  • 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 of the present invention 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 may be a biological sample or a non-biological sample.
  • the measurement sample includes not only the sample as it is collected but also the sample subjected to pretreatment such as removal of contaminants and the like.
  • biological sample 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, Bronchial lavage, pleural effusion, ascites, amniotic fluid, intestinal lavage, urine, feces, cell extract, tissue extract, organ extract and the like.
  • non-biological samples are not particularly limited, but examples thereof include samples collected from environmental surfaces such as faucets, handles, handrails, straps, switches, walls, floors, desks, chairs, and toilet seats.
  • the N protein of SARS-CoV-2 refers to one having the amino acid sequence disclosed in GenBank Accession No. (MN908947).
  • the N protein of SARS-CoV refers to one having 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.
  • the immunochromatographic test piece has a long and narrow rectangular shape with a width of 3 mm to 5 mm (preferably about 4 mm) and a length of 40 mm to 100 mm (preferably about 60 mm).
  • the conjugation pad 2 of the immunochromatographic test strip carries a complex of antibody A and cellulose-based colored fine particles for specifically capturing the N protein of SARS-CoV-2 in the measurement sample.
  • a test line in which an antibody B for specifically capturing the SARS-CoV-2 N protein in the measurement sample is linearly fixed at a position about 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip. 6 is formed.
  • a control line 7 is formed at a position about 20 mm from the end, on which an antibody C that specifically binds to the antibody A is linearly fixed.
  • the material of the sample pad 1 is not particularly limited as long as it can rapidly absorb the sample to be measured and then spread to downstream conjugation pads, membranes, and absorbent pads.
  • cellulose filter paper For example, cellulose filter paper.
  • the thickness of the sample pad 1 is preferably 0.1 mm to 2 mm, more preferably 0.2 mm to 1 mm. If the thickness is too small, the flow of the sample to be measured downstream becomes non-uniform, and the accuracy of measurement may deteriorate. 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 material of the conjugation pad 2 can hold in a dry state a complex of the antibody A that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the cellulose-based colored fine particles, and
  • the material is not particularly limited as long as it can rapidly release the complex as the sample develops downstream, 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 mm to 2 mm, more preferably 0.2 mm 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 material of the membrane 3 is not particularly limited as long as it can spread the measurement sample uniformly with high accuracy.
  • examples include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, and polyvinyl chloride. , polyvinylidene fluoride, or nylon membranes. Among these, nitrocellulose membranes are preferred.
  • the material of the absorbent pad 4 is not particularly limited as long as it can quickly absorb the measurement sample developed from upstream and then hold it so that it does not flow back.
  • Filter paper or non-woven fabric made of glass, filter paper or non-woven fabric made of polyester, filter paper or non-woven fabric made of polyethylene can be mentioned. Among these, cellulose filter paper is preferable.
  • the thickness of the absorbent pad 4 is preferably 0.2 mm to 5 mm, more preferably 0.5 mm 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.
  • antibodies 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.
  • the antibody A used as a detection antibody is required to specifically bind to the SARS-CoV-2 N protein, and the weighted reaction of the SARS-CoV-2 N protein to the SARS-CoV N protein
  • the ratio is preferably 40 or more, more preferably 100 or more, even more preferably 200 or more.
  • a low weighted reaction ratio may cause the immunochromatographic strip to react with both SARS-CoV and SARS-CoV-2. It may also have poor reactivity with SARS-CoV-2.
  • a weighted reaction ratio as a mixture may be adopted.
  • the weighted reaction ratio ⁇ (reflection absorbance of SARS-CoV-2) 2 /(reflection absorbance of SARS-CoV) ⁇ is the reaction ratio ⁇ ( Reflection absorbance of SARS-CoV-2) / (Reflection absorbance of SARS-CoV) ⁇ is an indicator that can detect the N protein of SARS-CoV-2 with high sensitivity (Reflection absorbance of SARS-CoV-2). It is the multiplied value. It can be said that the higher the weighted reaction ratio, the more sensitive and highly specific the antibody can detect the N protein of SARS-CoV-2.
  • 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% by mass of the cellulose-based colored fine particles is preferably derived from cellulose, more preferably 20 to 80% by mass, and even more preferably 20 to 70% by mass.
  • the average particle size of the cellulose-based colored fine particles is not particularly limited, and is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle size is large, the downstream development may be delayed or 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, and examples thereof 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 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 A, and is not particularly limited. 1:0.01 to 1:1 is preferred, 1:0.02 to 1:0.5 is more preferred, and 1:0.02 to 1:0.2 is even more preferred. If the mass ratio is outside the above range, the binding amount of antibody A to the cellulose-based colored microparticles becomes insufficient, or the binding amount of antibody A to the cellulose-based colored microparticles increases excessively, resulting in antibody A that does not contribute to the antigen-antibody reaction. increases, which can reduce measurement sensitivity.
  • the method of binding the antibody A and the cellulose-based colored fine particles is not particularly limited, but sensitization is preferably performed by physical adsorption by hydrophobic bonding or chemical bonding by covalent bonding, and physical adsorption is preferred because of simple operation and low cost. preferable.
  • 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 A that specifically binds to the SARS-CoV-2 N protein in the measurement sample and the cellulose-based colored fine particles is not particularly limited.
  • the conjugation pad can be prepared by uniformly applying, spraying or impregnating the 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 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 antibody B used as the capture antibody forming the test line 6 is required to specifically bind to the SARS-CoV-2 N protein, and the SARS-CoV-2 against the SARS-CoV N protein is preferably 40 or more, more preferably 100 or more, even more preferably 200 or more.
  • a low weighted reaction ratio may cause the immunochromatographic strip to react with both SARS-CoV and SARS-CoV-2. It may also have poor reactivity with SARS-CoV-2.
  • antibody B is a mixture of a plurality of antibodies, a weighted reaction ratio as a mixture may be adopted.
  • antibody C used as a capture antibody forming control line 7 is an antibody that specifically binds to antibody A.
  • 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 the above to different positions on the line, and then drying for a given period of time at an appropriate temperature in a constant temperature bath.
  • 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 the cost is increased. Then, it is preferable to dry after the coating.
  • 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. may contain filters for
  • the measurement sample diluent preferably contains a nonionic surfactant that improves the spreadability of the measurement sample and does not affect immune reactions.
  • the nonionic surfactant is not particularly limited, and polyoxyethylene alkylphenyl ether (Triton (registered trademark) surfactant, etc.), polyoxyethylene alkyl ether (Brij (registered trademark) surfactant, etc.) , polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactant, etc.), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester, and the like.
  • the said surfactant may be used individually, or may be used in combination of 2 or more types.
  • the concentration of the nonionic surfactant is preferably 0.01 to 5% by mass, more preferably 0.05 to 4% by mass, even more preferably 0.1 to 3% by mass. 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 separate, resulting in failure to obtain a measurement value.
  • 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 sufficient buffering capacity in the target pH range.
  • 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 complex of antibody A and cellulose-based colored fine particles 1.0 wt% 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 solution (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 1 (Anti- SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo Co., Ltd.) 100 ⁇ L was added to a 15 mL centrifuge tube, stirred with a vortex, and allowed to stand at 37° C.
  • NaAct registered trademark
  • BL2 Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation
  • a blocking solution 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 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. for 15 minutes to precipitate the antibody-sensitized cellulose-based colored fine particles, and then the supernatant was removed.
  • a centrifuge MX-307, manufactured by Tomy Seiko Co., Ltd.
  • 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 centrifuge MX-307, manufactured by Tomy Seiko Co., Ltd.
  • 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 dispersing machine (UH-50, manufactured by SMTE). A composite 1 with colored fine particles was obtained.
  • SARS-CoV-2 immobilized membrane card Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, manufactured by RayBiotech), which is the N protein of commercially available SARS-CoV-2, was added to PBS (-) ( 166-23555, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a SARS-CoV-2 solution.
  • PBS -
  • a 60 mm ⁇ 300 mm membrane card Hi-Flow Plus 120 Membrane Cards
  • 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.
  • the SARS-CoV -2 After applying the solution at a coating amount of 1 ⁇ L / cm, dry it for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45 ° C. to form a test line with a line width of about 1 mm.
  • a SARS-CoV-2 immobilized membrane card 1 was obtained.
  • SARS-CoV nucleocapsid recombinant protein (ARG70215, manufactured by Arigo Biolaboratories), which is a commercially available SARS-CoV N protein instead of SARS-CoV-2 solution, was added to PBS (- ) (166-23555, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) except for using a SARS-CoV solution diluted to 100 ⁇ g/mL in the same manner as in (2) to obtain a SARS-CoV immobilized membrane card. . Then, SARS-CoV immobilized half strip 1 was obtained in the same manner as in (3)
  • Example 2 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 2 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-109, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles got 2. The resulting SARS-CoV-2 N protein to SARS-CoV N protein complex 2 reaction ratios and weighted reaction ratios are shown in Table 1.
  • Example 3 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. A complex of antibody A and cellulose-based colored fine particles got 3. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 3 are shown in Table 1.
  • Example 4 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 4 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles Got 4. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 4 are shown in Table 1.
  • Example 5 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 5 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles Got 5. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 5 are shown in Table 1.
  • Example 6 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 6 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-103, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles got 6. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 6 are shown in Table 1.
  • Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex ⁇ of antibody A and cellulose-based colored fine particles was obtained in the same manner as in Example 1 except that N protein monoclonal antibody ⁇ (COVID-19 NP Antibody, HM1147, manufactured by EastCoastBio) was used.
  • the resulting reaction ratios and weighted reaction ratios of SARS-CoV-2 N protein to SARS-CoV N protein of complex ⁇ are shown in Table 1.
  • Example 2 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex ⁇ of antibody A and cellulose-based colored microparticles was obtained in the same manner as in Example 1 except that N protein monoclonal antibody ⁇ (COVID-19 NP Antibody, HM1145, manufactured by EastCoastBio) was used. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios of complex ⁇ are shown in Table 1.
  • Table 1 shows the evaluation in each example.
  • the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein is ⁇ (bad) when it is 40 or less, ⁇ (average) when it is 40 or more and 100 or less, ⁇ (good) when it is 100 or more and 200 or less, 200 or more is indicated by ⁇ (excellent). Since the weighted response ratio of SARS-CoV-2 N protein to SARS-CoV N protein in Examples 1-6 is greater than 40, Antibodies 1-6 used in each device are not reactive to SARS-CoV-2. It can be seen that the reactivity of SARS-CoV is high and/or significantly lower than that of SARS-CoV-2.
  • the antibodies 1-4 used in each device are antibodies capable of detecting the SARS-CoV-2 N protein with high sensitivity and high specificity.
  • the weighted response ratio of SARS-CoV-2 N protein to SARS-CoV N protein in Comparative Examples 1-4 is less than 40, the antibodies ⁇ to ⁇ used in each device react to SARS-CoV-2. It is clear that the reactivity of SARS-CoV is significantly lower and/or the reactivity of SARS-CoV has not been kept lower than that of SARS-CoV-2.
  • Example 7 Preparation of membrane card for detecting SARS-CoV-2 N protein
  • antibody B 2.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo Co., Ltd.) was prepared.
  • Antibody C a 1.0 mg/mL anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) was prepared.
  • 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.
  • HF120 manufactured by Millipore
  • a dispensing platform XYZ3060, manufactured by BIODOT
  • BIODOT Bio Jet nozzle
  • a 20 mm ⁇ 300 mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, CFSP002000, (manufactured by Millipore) was pasted together. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), a strip of 4 mm wide and 60 m long was cut to obtain an immunochromatographic test strip 1 for detecting N protein of SARS-CoV-2.
  • the immunochromatographic test piece 1 for detecting N protein of SARS-CoV-2 thus obtained 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.
  • SARS-CoV-2 N protein detection immunochromatography test piece evaluation of specificity
  • the SARS-CoV-2 N protein detection immunochromatography device was placed on a horizontal table. Then, 100 ⁇ L of the SARS-CoV sample was dispensed with a micropipette, gently dropped onto the sample pad, and left standing at 25° C. for 15 minutes. Then, the reflectance absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). Table 2 shows the results obtained.
  • Example 10 Instead of the complex 2 of the antibody A and the cellulose-based colored fine particles, the complex 6 of the antibody A and the cellulose-based colored fine particles is used, and the mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 is used as the antibody B.
  • Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 (Anti-SARS-CoV-2-NP Monoclonal (11) non-specific adsorption, (12) sensitivity, and (13) specificity were evaluated in the same manner as in Example 7, except that antibody, SCV-106, manufactured by Toyobo Co., Ltd.) was used. Table 2 shows the results obtained.
  • Example 11 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
  • Example 12 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
  • Example 14 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
  • Example 15 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
  • Example 16 Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
  • Example 17 Instead of the complex 2 of the antibody A and the cellulose-based colored fine particles, the complex 1 of the antibody A and the cellulose-based colored fine particles is used, and the mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 is used as the antibody B.
  • Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody ⁇ (COVID-19 NP Antibody, HM1147, EastCoastBio (11) non-specific adsorption, (12) sensitivity, and (13) specificity were evaluated in the same manner as in Example 7, except that the product was used. Table 2 shows the results obtained.
  • Table 2 shows the evaluation in each example.
  • Non-specific adsorption is indicated by ⁇ (bad) for 50 mAbs or more, ⁇ (average) for 30 mAbs or more and 50 mAbs or less, ⁇ (good) for 10 mAbs or more and 30 mAbs or less, and ⁇ (excellent) for 10 mAbs or less.
  • Sensitivity of 50 mAbs or less was indicated by ⁇ (bad), 50 mAbs to 150 mAbs by ⁇ (average), 150 mAbs to 250 mAbs by O (good), and 250 mAbs or more by ⁇ (excellent).
  • the immunochromatographic test strips of Examples 7 to 17 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of 40 or more as a capture antibody and / or a detection antibody in combination. Therefore, it was confirmed that the N protein of SARS-CoV-2 can be detected with high sensitivity and high specificity.
  • the immunochromatographic test strips of Examples 7 to 12 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of 40 or more as a capture antibody and a detection antibody in combination. Therefore, it was confirmed that the N protein of SARS-CoV-2 can be detected with extremely high sensitivity and high specificity.
  • the immunochromatographic test strips of Comparative Examples 5 to 7 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of less than 40 in combination as a capture antibody and a detection antibody. Therefore, the result was low sensitivity and low specificity.
  • the present invention can provide an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and high specificity.
  • sample pad 2 conjugation pad 3: membrane 4: absorbent pad 5: backing sheet 6: test line 7: control line 8: adhesive sheet

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

[Problem] To provide an immunochromatographic test piece that can detect N proteins of SARS-CoV-2 with high sensitivity and high specificity. [Solution] The present invention relates to an immunochromatographic test piece characterized by being configured from (1) a sample pad, (2) a conjugation pad on which is supported a complex of cellulose-based coloring microparticles and antibodies A that specifically bind to nucleocapsid proteins (N proteins) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement specimen, (3) a membrane to which antibodies B that specifically bind to N proteins of SARS-CoV-2 in a measurement specimen and antibodies C that specifically bind to the antibodies A are linearly secured at mutually different positions, and (4) an absorption pad, the weighting reaction ratio of the N proteins of SARS-CoV-2 to the N proteins of SARS-CoV in the antibodies A or antibodies B being 40 or greater.

Description

イムノクロマト試験片Immunochromatographic test piece
　本発明は、高感度かつ特異性高く重症急性呼吸器症候群（Ｓｅｖｅｒｅ　Ａｃｕｔｅ　Ｒｅｓｐｉｒａｔｏｒｙ　Ｓｙｎｄｒｏｍｅ）コロナウイルス２（ＳＡＲＳ－ＣｏＶ－２）のヌクレオカプシドタンパク質（Ｎタンパク質）を検出し得るイムノクロマト試験片、およびイムノクロマト試験片を含むキットに関する。 The present invention provides an immunochromatographic test strip 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 high specificity, and an immunochromatographic test strip. relating to kits containing
　ＳＡＲＳ－ＣｏＶ－２は、新型コロナウイルス感染症（ＣＯＶＩＤ－１９）の原因ウイルスであり、２０２０年初より世界中で急速に流行している。ＳＡＲＳ－ＣｏＶ－２は、一般的なコロナウイルスと同様に、ヌクレオカプシドおよび当該ヌクレオカプシドを取り囲むエンベロープで構成され、前記ヌクレオカプシドには、ウイルスゲノム（ＲＮＡ）および当該ウイルスゲノムに結合するヌクレオカプシドタンパク質（Ｎタンパク質）が含まれ、前記エンベロープには、脂質および当該脂質に結合するスパイクタンパク質（Ｓタンパク質）、膜タンパク質（Ｍタンパク質）、およびエンベロープタンパク質（Ｅタンパク質）が含まれる。 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. and 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).
　Ｎタンパク質は、ウイルスコアの形成、並びに、ウイルスゲノムのパッケージングおよび転写等に関与するタンパク質であり、セリン（Ｓ）／アルギニン（Ｒ）リッチな領域を有するリンカーを介してＮ末端ドメイン（ＮＴＤ）およびＣ末端ドメイン（ＣＴＤ）が結合した構造を有する。非特許文献1には、Ｎタンパク質が多量にリン酸化されることが記載され、リン酸化部位のマッピングが示されている。Ｎタンパク質のアミノ酸配列は株間で保存されているため、Ｎタンパク質は診断マーカー等として使用される。 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. 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.
　複数ある感染症診断法の中でも迅速・簡便・安価であるイムノクロマト法は最も普及した技術の１つである。イムノクロマト法とは毛細管現象を利用した免疫測定法であり、インフルエンザ検査などにおいて世界的に普及している。イムノクロマト法を用いて測定対象物質を検出する手法の一つとしては、抗原抗体反応を利用したサンドイッチ法が挙げられる。サンドイッチ法では測定対象物質に対してエピトープの異なる２種類の抗体を利用する。一方の抗体は、金コロイド、着色ラテックス粒子、蛍光粒子等の検出粒子と感作した検出抗体として使用する。他方の抗体は、多孔質支持体の表面に線状に固定した捕捉抗体としてテストラインを形成する。加えて、前記検出抗体を特異的に捕捉する抗体を多孔質支持体の表面の、前記テストラインとは異なる位置に線状に固定しコントロールラインを形成する。測定試料中に含まれる測定対象物質は、多孔質支持体の一端（上流側）から展開し、検出抗体と免疫複合体を形成しながら移動し、テストライン上で捕捉抗体と接触して捕捉され発色する。測定対象物質と免疫複合体を形成しなかった遊離の検出粒子と感作した検出抗体はテストライン上を通過し、コントロールラインの抗体に捕捉され発色する。これらの発色強度を目視で確認することで測定対象物質の有無を判定することができる（特許文献１）。 Among the multiple diagnostic methods for infectious diseases, the immunochromatographic method, which is quick, simple, and inexpensive, is one of the most popular techniques. 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. In the 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. The other antibody forms the test line as a capture antibody linearly immobilized on the surface of the porous support. In addition, 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 immunocomplexes with the substance to be measured and the sensitized detection antibody pass over the test line and are captured by the control line antibody to develop color. The presence or absence of the substance to be measured can be determined by visually confirming these color development intensities (Patent Document 1).
特開２００６－６７９７９号公報JP-A-2006-67979
　本発明は、高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得るイムノクロマト試験片、イムノクロマト試験片を含むキットを提供することを課題とするものである。 An object of the present invention is to provide an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and high specificity, and a kit containing the immunochromatographic test strip.
　本発明者は、上記課題を解決するために鋭意研究した結果、検出抗体または捕捉抗体としてＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上の抗体を使用することで、ＳＡＲＳ－ＣｏＶのＮタンパク質とは反応せず、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質とは反応するイムノクロマト試験片を得られることを見出した。また、検出粒子としてセルロース系着色微粒子を使用することにより、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質をさらに高感度で検出できることを見出し、本発明を完成させた。 As a result of intensive research to solve the above problems, the present inventors have found that the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein as a detection antibody or capture antibody is an antibody with a weighted reaction ratio of 40 or more. Thus, it was found that an immunochromatographic specimen that does not react with the SARS-CoV N protein but reacts with the SARS-CoV-2 N protein can be obtained. 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.
　すなわち、代表的な本発明は以下の通りである。
１．　（１）サンプルパッドと、
（２）測定試料中の重症急性呼吸器症候群コロナウイルス２（ＳＡＲＳ－ＣｏＶ－２）のヌクレオカプシドタンパク質（Ｎタンパク質）と特異的に結合する抗体Ａとセルロース系着色微粒子との複合体を担持したコンジュゲーションパッドと、
（３）測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合する抗体Ｂと、前記抗体Ａと特異的に結合する抗体Ｃとを、それぞれ異なる位置に線状に固定したメンブレンと、
（４）吸収パッドと、から構成され、
前記抗体Ａまたは抗体ＢのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であることを特徴とするイムノクロマト試験片。
２．　前記抗体Ａおよび抗体ＢのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であることを特徴とする１．に記載のイムノクロマト試験片。
３．　１．または２．のいずれかに記載のイムノクロマト試験片、測定試料採取具、フィルター、測定試料希釈液からなることを特徴とするイムノクロマトキット。 That is, the representative present invention is as follows.
1. (1) a sample pad;
(2) A conjugate carrying a complex of antibody A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample and cellulose-based colored fine particles gaming pad and
(3) A membrane in which antibody B that specifically binds to the SARS-CoV-2 N protein in the measurement sample and antibody C that specifically binds to the antibody A are linearly immobilized at different positions. ,
(4) an absorbent pad;
An immunochromatographic test strip, wherein the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A or antibody B is 40 or more.
2. 1. The weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A and antibody B is 40 or more; Immunochromatographic test piece according to.
3. 1. or 2. 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.
　本発明のイムノクロマト試験片は、特定の抗体、および検出粒子を、特定の配置で担持させているため、高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出することができる。 Since 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 high specificity.
本発明のイムノクロマト試験片の一例を示す図（上面図）である。1 is a diagram (top view) showing an example of an immunochromatographic test strip of the present invention. FIG. 本発明のイムノクロマト試験片の一例を示す図（側面図）である。1 is a diagram (side view) showing an example of an immunochromatographic test strip of the present invention. FIG.
　本発明のイムノクロマト試験片は測定試料中の重症急性呼吸器症候群（Ｓｅｖｅｒｅ　Ａｃｕｔｅ　Ｒｅｓｐｉｒａｔｏｒｙ　Ｓｙｎｄｒｏｍｅ）コロナウイルス２（ＳＡＲＳ－ＣｏＶ－２）のヌクレオカプシドタンパク質（Ｎタンパク質）を検出するための試験片である。本発明に用いる測定試料は、生物試料であってもよく非生物試料であってもよい。また、測定試料には、採取したままの試料のみならず、当該試料に対して夾雑物等の除去等の前処理を施したものも含まれる。 The immunochromatographic test strip of the present invention 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 may be a biological sample or a non-biological sample. Moreover, the measurement sample includes not only the sample as it is collected but also the sample subjected to pretreatment such as removal of contaminants and the like.
　前記生物試料は、特に限定されないが、例えば血液、血清、血漿、骨髄液、リンパ液、涙、鼻汁、鼻腔洗浄液、鼻腔拭い液、唾液、うがい液、喀痰、咽頭拭い液、汗、気管吸引液、気管支洗浄液、胸水、腹水、羊水、腸管洗浄液、尿、糞便、細胞抽出液、組織抽出液、臓器抽出液等が挙げられる。また、非生物試料は、特に限定されないが、例えば蛇口、取っ手、手すり、つり革、スイッチ、壁、床、机、椅子、便座等の環境表面から採取した試料等が挙げられる。 Examples of the biological sample 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, Bronchial lavage, pleural effusion, ascites, amniotic fluid, intestinal lavage, urine, feces, cell extract, tissue extract, organ extract and the like. In addition, non-biological samples are not particularly limited, but examples thereof include samples collected from environmental surfaces such as faucets, handles, handrails, straps, switches, walls, floors, desks, chairs, and toilet seats.
　本発明において、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質は、ＧｅｎＢａｎｋアクセッション番号（ＭＮ９０８９４７）に開示されたアミノ酸配列を有するものを指す。なお、ＳＡＲＳ－ＣｏＶのＮタンパク質は、ＧｅｎＢａｎｋアクセッション番号（ＡＹ２７８７４１）に開示されたアミノ酸配列を有するものを指す。 In the present invention, the N protein of SARS-CoV-2 refers to one having the amino acid sequence disclosed in GenBank Accession No. (MN908947). The N protein of SARS-CoV refers to one having the amino acid sequence disclosed in GenBank Accession No. (AY278741).
　本発明において、イムノクロマト試験片の構成は、イムノクロマト試験片の測定試料溶液の添加部（滴下部）を上流側として、前記添加部を有するサンプルパッド、コンジュゲーションパッド、メンブレン、吸収パッドの順に連接配置されている。次いで、本発明のイムノクロマト試験片の一例を、図面を参照して説明する。図１および図２において、１はサンプルパッド、２はコンジュゲーションパッド、３はメンブレン、４は吸収パッド、５はバッキングシート、６はテストライン、７はコントロールライン、８は粘着シートを示す。 In the present invention, 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. It is Next, an example of the immunochromatographic test strip of the present invention will be described with reference to the drawings. 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.
　図１および図２の例では、イムノクロマト試験片は、幅３ｍｍ～５ｍｍ（好ましくは、４ｍｍ程度）、長さ４０ｍｍ～１００ｍｍ（好ましくは６０ｍｍ程度）の細長い短冊状の形態をしている。なお、イムノクロマト試験片のコンジュゲーションパッド２には測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質を特異的に捕捉するための抗体Ａとセルロース系着色微粒子との複合体が担持される。また、イムノクロマト試験片のメンブレン３の上流側の端部から約１５ｍｍの位置に測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質を特異的に捕捉するための抗体Ｂを線状に固定したテストライン６が形成される。また、前記端部から約２０ｍｍの位置に抗体Ａと特異的に結合する抗体Ｃを線状に固定したコントロールライン７が形成される。 　In the examples of Figures 1 and 2, the immunochromatographic test piece has a long and narrow rectangular shape with a width of 3 mm to 5 mm (preferably about 4 mm) and a length of 40 mm to 100 mm (preferably about 60 mm). The conjugation pad 2 of the immunochromatographic test strip carries a complex of antibody A and cellulose-based colored fine particles for specifically capturing the N protein of SARS-CoV-2 in the measurement sample. In addition, a test line in which an antibody B for specifically capturing the SARS-CoV-2 N protein in the measurement sample is linearly fixed at a position about 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip. 6 is formed. Further, a control line 7 is formed at a position about 20 mm from the end, on which an antibody C that specifically binds to the antibody A is linearly fixed.
　本発明において、サンプルパッド１の材質は、測定試料を速やかに吸収した後、下流のコンジュゲーションパッド、メンブレン、吸収パッドへ展開できる材質のものであれば、特に限定されず、例えばセルロース製のろ紙または不織布、ガラス製のろ紙または不織布、ポリエステル製のろ紙または不織布、ポリエチレン製のろ紙または不織布が挙げられる。これらの中でも、セルロース製のろ紙が好ましい。また、前記サンプルパッド１の厚みは、０．１ｍｍ～２ｍｍが好ましく、０．２ｍｍ～１ｍｍがより好ましい。厚みが小さいと、下流での測定試料の流れが不均一となり測定精度が低下することがある。一方、厚みが大きいと、下流への展開が遅くなり測定時間が長くなることがある。また、下流への展開に必要となる測定試料量が多くなる。 In the present invention, the material of the sample pad 1 is not particularly limited as long as it can rapidly absorb the sample to be measured and then spread to downstream conjugation pads, membranes, and absorbent pads. For example, cellulose filter paper. Alternatively, non-woven fabric, filter paper or non-woven fabric made of glass, filter paper or non-woven fabric made of polyester, filter paper or non-woven fabric made of polyethylene can be used. Among these, cellulose filter paper is preferable. The thickness of the sample pad 1 is preferably 0.1 mm to 2 mm, more preferably 0.2 mm to 1 mm. If the thickness is too small, the flow of the sample to be measured downstream becomes non-uniform, and the accuracy of measurement may deteriorate. 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.
　本発明において、コンジュゲーションパッド２の材質は、測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合する抗体Ａとセルロース系着色微粒子との複合体を乾燥状態で保持でき、かつ測定試料の下流への展開と共に前記複合体を速やかに放出することができる材質のものであれば、特に限定されず、例えばセルロース製のろ紙または不織布、ガラス製のろ紙または不織布、ポリエステル製のろ紙または不織布、ポリエチレン製のろ紙または不織布が挙げられる。これらの中でも、ガラス製のろ紙が好ましい。また、前記コンジュゲーションパッド２の厚みは、０．１ｍｍ～２ｍｍが好ましく、０．２ｍｍ～１ｍｍがより好ましい。厚みが小さいと、目的量の前記複合体を乾燥状態で保持できないことがある。一方、厚みが大きいと、下流への展開が遅くなり測定時間が長くなることがある。また、下流への展開に必要となる測定試料量が多くなる。 In the present invention, the material of the conjugation pad 2 can hold in a dry state a complex of the antibody A that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the cellulose-based colored fine particles, and The material is not particularly limited as long as it can rapidly release the complex as the sample develops downstream, 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 mm to 2 mm, more preferably 0.2 mm 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.
　本発明において、メンブレン３の材質は、測定試料を精度よく均一に展開できるものであれば、特に限定されず、例えばセルロース、セルロース誘導体、ニトロセルロース、酢酸セルロース、ポリウレタン、ポリエステル、ポリエチレン、ポリ塩化ビニル、ポリフッ化ビニリデン、またはナイロン製のメンブレンが挙げられる。これらの中でも、ニトロセルロース製のメンブレンが好ましい。 In the present invention, the material of the membrane 3 is not particularly limited as long as it can spread the measurement sample uniformly with high accuracy. Examples include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, and polyvinyl chloride. , polyvinylidene fluoride, or nylon membranes. Among these, nitrocellulose membranes are preferred.
　本発明において、吸収パッド４の材質は、上流より展開してきた測定試料を速やかに吸収した後、逆流しないよう保持できる材質のものであれば、特に限定されず、例えばセルロース製のろ紙または不織布、ガラス製のろ紙または不織布、ポリエステル製のろ紙または不織布、ポリエチレン製のろ紙または不織布が挙げられる。これらの中でも、セルロース製のろ紙が好ましい。また、前記吸収パッド４の厚みは、０．２ｍｍ～５ｍｍが好ましく、０．５ｍｍ～２ｍｍがより好ましい。厚みが小さいと、測定試料の滴下量によっては一度吸収パッドに吸収された測定試料がメンブレン側に逆流することがある。一方、厚みが大きいと、イムノクロマト試験片およびイムノクロマト試験片を覆うハウジングケースのサイズも大きくなり、ＰＯＣＴの観点から好ましくない。 In the present invention, the material of the absorbent pad 4 is not particularly limited as long as it can quickly absorb the measurement sample developed from upstream and then hold it so that it does not flow back. Filter paper or non-woven fabric made of glass, filter paper or non-woven fabric made of polyester, filter paper or non-woven fabric made of polyethylene can be mentioned. Among these, cellulose filter paper is preferable. The thickness of the absorbent pad 4 is preferably 0.2 mm to 5 mm, more preferably 0.5 mm 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.
　本発明において、抗体はモノクローナル抗体であってもポリクローナル抗体であってもよいが、モノクローナル抗体が好ましい。抗体は、任意のアイソタイプ、例えばＩｇＧ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＭ等であることができるが、ＩｇＧが好ましい。また、抗体は、市販品を用いてもよいし、別途公知の方法で製造してもよい。 In the present invention, antibodies 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. In addition, the antibody may be a commercially available product, or may be separately produced by a known method.
　本発明において、検出抗体として用いる抗体Ａは、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合することが必要であり、ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であるのが好ましく、１００以上がより好ましく、２００以上がさらに好ましい。加重反応比が小さいと、イムノクロマト試験片がＳＡＲＳ－ＣｏＶおよびＳＡＲＳ－ＣｏＶ－２の両方と反応することがある。また、ＳＡＲＳ－ＣｏＶ－２との反応性に乏しいこともある。なお、抗体Ａが複数の抗体の混合物の場合は、混合物としての加重反応比を採用してもよい。 In the present invention, the antibody A used as a detection antibody is required to specifically bind to the SARS-CoV-2 N protein, and the weighted reaction of the SARS-CoV-2 N protein to the SARS-CoV N protein The ratio is preferably 40 or more, more preferably 100 or more, even more preferably 200 or more. A low weighted reaction ratio may cause the immunochromatographic strip to react with both SARS-CoV and SARS-CoV-2. It may also have poor reactivity with SARS-CoV-2. When antibody A is a mixture of multiple antibodies, a weighted reaction ratio as a mixture may be adopted.
　加重反応比｛（ＳＡＲＳ－ＣｏＶ－２の反射吸光度）２／（ＳＡＲＳ－ＣｏＶの反射吸光度）｝とは、特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得る指標である反応比｛（ＳＡＲＳ－ＣｏＶ－２の反射吸光度）／（ＳＡＲＳ－ＣｏＶの反射吸光度）｝に、高感度にＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得る指標である（ＳＡＲＳ－ＣｏＶ－２の反射吸光度）を掛けた数値である。加重反応比が大きいほど、高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得る抗体であると言える。 The weighted reaction ratio {(reflection absorbance of SARS-CoV-2) 2 /(reflection absorbance of SARS-CoV)} is the reaction ratio {( Reflection absorbance of SARS-CoV-2) / (Reflection absorbance of SARS-CoV)} is an indicator that can detect the N protein of SARS-CoV-2 with high sensitivity (Reflection absorbance of SARS-CoV-2). It is the multiplied value. It can be said that the higher the weighted reaction ratio, the more sensitive and highly specific the antibody can detect the N protein of SARS-CoV-2.
　セルロース系着色微粒子は、大量の水酸基を有するため、多くの反応性染料を共有結合により保持することができるだけでなく、濃染化した後も水などへの安定分散性を保持することができる。セルロース系着色微粒子としては、再生セルロース、精製セルロース、天然セルロース等を用いることができ、一部誘導体化されたセルロースを用いてもよい。前記セルロース系着色微粒子の２０～９０質量％はセルロース由来であることが好ましく、２０～８０質量％がより好ましく、２０～７０質量％がさらに好ましい。 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% by mass of the cellulose-based colored fine particles is preferably derived from cellulose, more preferably 20 to 80% by mass, and even more preferably 20 to 70% by mass.
　セルロース系着色微粒子の平均粒子径は、特に限定されず、１００ｎｍ～１０００ｎｍが好ましく、２００ｎｍ～８００ｎｍがより好ましい。平均粒子径が大きいと、下流への展開が遅くなるとか、測定時間が長くなることがある。また、メンブレン上に捕捉されやすくなり、バックグラウンド自体が発色してしまうことでテストラインおよびコントロールラインでの発色が不明瞭になることがある。一方、平均粒子径が小さいと、物理吸着または化学結合できる抗体量が低下し、測定感度が低下することがある。 The average particle size of the cellulose-based colored fine particles is not particularly limited, and is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle size is large, the downstream development may be delayed or 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.
　セルロース系着色微粒子の色は、特に限定されず、例えば赤色、青色、黄色、緑色、黒色、白色、蛍光色が挙げられる。これらの中でも、視認性のよい赤色、青色、黒色が好ましい。このようなセルロース系着色微粒子としては、旭化成社製の着色セルロースナノビーズ（ＮａｎｏＡｃｔ（登録商標））が挙げられる。 The color of the cellulose-based colored fine particles is not particularly limited, and examples thereof 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.
　セルロース系着色微粒子への抗体Ａの結合量は、セルロース系着色微粒子と抗体Ａの仕込み質量比を調整することで制御でき、特に限定されないが、セルロース系着色微粒子と抗体Ａとの仕込み質量比は１：０．０１～１：１が好ましく、１：０．０２～１：０．５がより好ましく、１：０．０２～１：０．２がさらに好ましい。質量比が前記範囲を外れると、セルロース系着色微粒子への抗体Ａの結合量が不十分となるとか、セルロース系着色微粒子への抗体Ａの結合量が増えすぎ、抗原抗体反応に寄与しない抗体Ａが増えるため、測定感度が低下することがある。 The binding amount of the antibody 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 A, and is not particularly limited. 1:0.01 to 1:1 is preferred, 1:0.02 to 1:0.5 is more preferred, and 1:0.02 to 1:0.2 is even more preferred. If the mass ratio is outside the above range, the binding amount of antibody A to the cellulose-based colored microparticles becomes insufficient, or the binding amount of antibody A to the cellulose-based colored microparticles increases excessively, resulting in antibody A that does not contribute to the antigen-antibody reaction. increases, which can reduce measurement sensitivity.
　抗体Ａとセルロース系着色微粒子との結合方法は、特に限定されないが、疎水結合による物理吸着または共有結合による化学結合で感作するのが好ましく、操作が簡便でありかつコストも安い物理吸着がより好ましい。なお、感作効率を向上させるため、セルロース系着色微粒子に反応性活性基を導入してもよい。反応性活性基としては、特に限定されないが、例えばカルボキシル基、アミノ基、アルデヒド基、チオール基、エポキシ基、水酸基が挙げられる。これらの中でも、カルボキシル基、アミノ基が好ましい。カルボキシル基の場合は、カルボジイミドを用いてリガンドのアミノ基と共有結合を形成することができる。 The method of binding the antibody A and the cellulose-based colored fine particles is not particularly limited, but sensitization is preferably performed by physical adsorption by hydrophobic bonding or chemical bonding by covalent bonding, and physical adsorption is preferred because of simple operation and low cost. preferable. In addition, in order to improve the sensitization efficiency, a reactive active group may be introduced into the cellulose-based colored fine particles. Examples of 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.
　コンジュゲーションパッド２に測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合する抗体Ａとセルロース系着色微粒子との複合体を担持させる方法は、特に限定されないが、例えば前記複合体の溶液をコンジュゲーションパッドに均一に塗布、噴霧または含浸した後、恒温槽内で適当な温度で一定時間乾燥することで作製することができる。前記複合体の溶液の塗布量は、特に限定されないが、ライン長１ｃｍ辺り５μＬ～５０μＬが好ましい。また、前記複合体の溶液におけるセルロース系着色微粒子濃度は、特に限定されないが、０．０１～０．５質量％が好ましく、０．０２～０．２質量％がより好ましく、０．０２～０．１質量％がさらに好ましい。濃度が低いと、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質を十分捕捉・検出することができず、測定感度が低下することがある。一方、濃度が高くても、測定感度の向上は見られず、コストだけが高くなる。次いで、前記塗布後に乾燥するのが好ましい。乾燥温度は、特に限定されないが、２０℃～８０℃が好ましく、２０℃～６０℃がより好ましい。乾燥時間は乾燥温度によって異なるが、通常は５～１２０分間である。 The method of causing the conjugation pad 2 to support the complex of the antibody A that specifically binds to the SARS-CoV-2 N protein in the measurement sample and the cellulose-based colored fine particles is not particularly limited. The conjugation pad can be prepared by uniformly applying, spraying or impregnating the 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 μL to 50 μL per 1 cm line length. Further, 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. .1% by weight 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. Then, it is preferable to dry after the coating. 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.
　本発明において、テストライン６を形成する捕捉抗体として用いる抗体Ｂは、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合することが必要であり、ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であるのが好ましく、１００以上がより好ましく、２００以上がさらに好ましい。加重反応比が小さいと、イムノクロマト試験片がＳＡＲＳ－ＣｏＶおよびＳＡＲＳ－ＣｏＶ－２の両方と反応することがある。また、ＳＡＲＳ－ＣｏＶ－２との反応性に乏しいこともある。なお、抗体Ｂが複数の抗体の混合物の場合は、混合物としての加重反応比を採用してもよい。 In the present invention, the antibody B used as the capture antibody forming the test line 6 is required to specifically bind to the SARS-CoV-2 N protein, and the SARS-CoV-2 against the SARS-CoV N protein is preferably 40 or more, more preferably 100 or more, even more preferably 200 or more. A low weighted reaction ratio may cause the immunochromatographic strip to react with both SARS-CoV and SARS-CoV-2. It may also have poor reactivity with SARS-CoV-2. In addition, when antibody B is a mixture of a plurality of antibodies, a weighted reaction ratio as a mixture may be adopted.
　本発明において、コントロールライン７を形成する捕捉抗体として用いる抗体Ｃは、抗体Ａと特異的に結合する抗体である。 In the present invention, antibody C used as a capture antibody forming control line 7 is an antibody that specifically binds to antibody A.
　メンブレン３にテストライン６を形成する捕捉抗体とコントロールライン７を形成する捕捉抗体を線状に固定する方法は、特に限定されないが、例えばテストラインを形成する捕捉抗体とコントロールラインを形成する捕捉抗体を、それぞれ線上に一定量を異なる位置に塗布した後、恒温槽内で適当な温度で一定時間乾燥することで作製することができる。前記両捕捉抗体の塗布量は、特に限定されないが、ライン長１ｃｍ辺り０．１μＬ～２μＬが好ましい。また、前記両捕捉抗体の塗布濃度は、特に限定されないが、０．１ｍｇ／ｍＬ～１０ｍｇ／ｍＬが好ましく、０．２ｍｇ／ｍＬ～８ｍｇ／ｍＬがより好ましく、０．５ｍｇ／ｍＬ～５ｍｇ／ｍＬがさらに好ましい。濃度が低いと、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質を十分捕捉・検出することができず、測定感度が低下することがある。一方、濃度が高くても、測定感度の向上は見られず、コストが高くなる。次いで、前記塗布後に乾燥するのが好ましい。乾燥温度は、特に限定されないが、２０℃～８０℃が好ましく、２０℃～６０℃がより好ましい。乾燥時間は乾燥温度によって異なるが、通常は５分間～１２０分間である。 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 the above to different positions on the line, and then drying for a given period of time at an appropriate temperature in a constant temperature bath. The amount of the two capture antibodies to be applied is not particularly limited, but is preferably 0.1 μL to 2 μL per 1 cm of line length. In addition, 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 the cost is increased. Then, it is preferable to dry after the coating. 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.
　本発明のイムノクロマト試験片は、前記作製したメンブレン３を粘着シート８の中央付近に貼り付け、次いでコンジュゲーションパッド２をメンブレン３の一方の末端上に一部重ね合わせて貼り付け、次いでサンプルパッド１をコンジュゲーションパッド２のメンブレン３との重なりとは逆の末端上に一部重ね合わせて貼り付け、次いで吸収パッド４をメンブレン３の他方の末端上に一部重ね合わせて貼り付けた後、一定幅の短冊状に切断することで作製することができる。なお、テストライン６およびコントロールライン７は試験片を作製した後に調製してもよいし、試験片を作製する前に調製してもよい。 In the immunochromatographic test piece of the present invention, 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. Note that 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.
　イムノクロマト試験片は、少なくともサンプルパッド１上に測定試料を滴下するための第一の開口部、メンブレン３上にテストライン６とコントロールライン７を目視で確認するための第二の開口部を有する適当なプラスチック製のハウジングケースに収容されていても良い。 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.
　本発明において、イムノクロマト測定キットは、イムノクロマト試験片に加え、測定試料を採取するための測定試料採取具、測定試料を前処理および／または希釈するための測定試料希釈液、測定試料をろ過するためのフィルターを含むことがある。 In the present invention, 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. may contain filters for
　前記測定試料希釈液は、測定試料の展開性を向上させ、かつ免疫反応に影響しないノニオン性界面活性剤を含むことが好ましい。前記ノニオン性界面活性剤としては、特に限定されず、ポリオキシエチレンアルキルフェニルエーテル（Ｔｒｉｔｏｎ（登録商標）系界面活性剤等）、ポリオキシエチレンアルキルエーテル（Ｂｒｉｊ（登録商標）系界面活性剤等）、ポリオキシエチレンソルビタン脂肪酸エステル（Ｔｗｅｅｎ（登録商標）系界面活性剤等）、ポリオキシエチレン脂肪酸エステル、ソルビタン脂肪酸エステル、アルキルグルコシド、ショ糖脂肪酸エステル等が挙げられる。また、前記界面活性剤は単独で用いても、二種以上を組み合わせて用いてもよい。ノニオン性界面活性剤の濃度としては、０．０１～５質量％が好ましく、０．０５～４質量％がより好ましく、０．１～３質量％がさらに好ましい。濃度が低いと、下流への展開が困難となることがある。また、展開が不均一となり測定精度が低下することがある。一方、濃度が高いと、物理的に吸着している、検出粒子と抗体、および／またはメンブレンと抗体が乖離し、測定値が得られないことがある。 The measurement sample diluent preferably contains a nonionic surfactant that improves the spreadability of the measurement sample and does not affect immune reactions. The nonionic surfactant is not particularly limited, and polyoxyethylene alkylphenyl ether (Triton (registered trademark) surfactant, etc.), polyoxyethylene alkyl ether (Brij (registered trademark) surfactant, etc.) , polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactant, etc.), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester, and the like. Moreover, the said surfactant may be used individually, or may be used in combination of 2 or more types. The concentration of the nonionic surfactant is preferably 0.01 to 5% by mass, more preferably 0.05 to 4% by mass, even more preferably 0.1 to 3% by mass. 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 separate, resulting in failure to obtain a measurement value.
　前記測定試料希釈液は、無機塩類やｐＨ調整に用いる緩衝剤を添加しても良い。前記緩衝剤としては、目的とするｐＨ範囲において充分な緩衝能力を有していれば、いかなる種類の緩衝剤を用いてもよく、例えば、トリス、リン酸、フタル酸、クエン酸、マレイン酸、コハク酸、シュウ酸、ホウ酸、酒石酸、酢酸、炭酸、グッドバッファー（ＭＥＳ、ＡＤＡ、ＰＩＰＥＳ、ＡＣＥＳ、コラミン塩酸、ＢＥＳ、ＴＥＳ、ＨＥＰＥＳ、アセトアミドグリシン、トリシン、グリシンアミド、ビシン）が挙げられる。これらの中でも、本発明に用いる抗体の至適ｐＨ範囲である７．０付近において充分な緩衝能力を有する等の理由から、トリス、リン酸、ＭＥＳ、ＰＩＰＥＳ、ＴＥＳ、ＨＥＰＥＳが好ましく、トリス、リン酸、ＰＩＰＥＳがより好ましい。 Inorganic salts and buffers used for pH adjustment may be added to the measurement sample diluent. As the buffering agent, any type of buffering agent may be used as long as it has sufficient buffering capacity in the target pH range. succinic 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). Among these, 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.
　以下、本発明を実施例に基づいて詳細に説明する。本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples. The invention is not limited to these examples.
（実施例１）
（１）抗体Ａとセルロース系着色微粒子との複合体の調製
　１．０質量％のセルロース系着色微粒子（ＮａｎｏＡｃｔ（登録商標）、ＢＬ２：Ｄａｒｋ　Ｎａｖｙ、平均粒子径３６５ｎｍ、旭化成社製）１００μＬ、１０ｍＭのトリス緩衝液（２０４－０７８８５、富士フィルム和光純薬社製）（ｐＨ８．０）９００μＬ、抗体Ａとして１．０ｍｇ／ｍＬのマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）１００μＬを１５ｍＬの遠沈管に加え、ボルテックスで撹拌した後、３７℃で１２０分間静置した。次いで、１．０質量％のカゼイン（０３０－０１５０５、富士フィルム和光純薬社製）、１００ｍＭのホウ酸緩衝液（０２１－０２１９５、富士フィルム和光純薬社製）からなるブロッキング液（ｐＨ８．０）１２ｍＬを加え、３７℃で６０分間静置した。次いで、遠心分離機（ＭＸ－３０７、トミー精工社製）を用い、１３，０００×ｇの遠心を２５℃で１５分間行い、抗体感作セルロース系着色微粒子を沈降させた後に上澄みを除去した。次いで、５０ｍＭのホウ酸緩衝液（０２１－０２１９５、富士フィルム和光純薬社製）からなる洗浄液（ｐＨ１０．０）１２ｍＬを加え、超音波分散機（ＵＨ－５０、エスエムテー社製）で１０秒間処理した。次いで、遠心分離機（ＭＸ－３０７、トミー精工社製）を用い、１３，０００×ｇの遠心を２５℃で１５分間行い、抗体感作セルロース系着色微粒子を沈降させた後に上澄みを除去した。次いで、１５質量％のスクロース（１９６－０００１５、富士フィルム和光純薬社製）、０．２質量％のカゼイン（０３０－０１５０５、富士フィルム和光純薬社製）、６２ｍＭのホウ酸緩衝液（０２１－０２１９５、富士フィルム和光純薬社製）からなる塗布液（ｐＨ９．２）２．０ｍＬを加え、超音波分散機（ＵＨ－５０、エスエムテー社製）で１０秒間処理し、抗体Ａとセルロース系着色微粒子との複合体１を得た。 (Example 1)
(1) Preparation of complex of antibody A and cellulose-based colored fine particles 1.0 wt% cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) 100 µL, 10 mM Tris buffer solution (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.0) 900 μL, 1.0 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti- SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo Co., Ltd.) 100 μL was added to a 15 mL centrifuge tube, stirred with a vortex, and 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 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. for 15 minutes to precipitate the antibody-sensitized cellulose-based colored fine particles, and then the supernatant was removed. Next, 12 mL of a cleaning solution (pH 10.0) 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. Next, 15% by mass of 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 dispersing machine (UH-50, manufactured by SMTE). A composite 1 with colored fine particles was obtained.
（２）ＳＡＲＳ－ＣｏＶ－２固定化メンブレンカードの作製
　市販のＳＡＲＳ－ＣｏＶ－２のＮタンパク質であるＲｅｃｏｍｂｉｎａｎｔ　ＳＡＲＳ－ＣｏＶ－２　Ｎｕｃｌｅｏｃａｐｓｉｄ　ｐｒｏｔｅｉｎ（２３０－３０１６４、ＲａｙＢｉｏｔｅｃｈ社製）を、ＰＢＳ（－）（１６６－２３５５５、富士フィルム和光純薬社製）にて１μｇ／ｍＬに希釈し、ＳＡＲＳ－ＣｏＶ―２溶液を準備した。次いで、上流側に２０ｍｍ×３００ｍｍの粘着テープ部、中央に２５ｍｍ×３００ｍｍのメンブレン部、下流側に１５ｍｍ×３００ｍｍの粘着テープ部から構成される６０ｍｍ×３００ｍｍのメンブレンカード（Ｈｉ－Ｆｌｏｗ　Ｐｌｕｓ　１２０　Ｍｅｍｂｒａｎｅ　Ｃａｒｄｓ、ＨＦ１２０、Ｍｉｌｌｉｐｏｒｅ社製）のメンブレン部の上流側から８ｍｍの位置に、分注プラットフォーム（ＸＹＺ３０６０、ＢＩＯＤＯＴ社製）と、Ｂｉｏ Ｊｅｔノズル（ＢＨＱＨＲ－ＸＹＺ、ＢＩＯＤＯＴ社製）を用い、前記ＳＡＲＳ－ＣｏＶ―２溶液を１μＬ／ｃｍの塗布量で塗布した後、４５℃に調整した乾燥機（ＷＦＯ－５１０、東京理化器械社製）で３０分間乾燥し、ライン幅約１ｍｍのテストラインを形成することで、ＳＡＲＳ－ＣｏＶ－２固定化メンブレンカード１を得た。 (2) Preparation of SARS-CoV-2 immobilized membrane card Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, manufactured by RayBiotech), which is the N protein of commercially available SARS-CoV-2, was added to PBS (-) ( 166-23555, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a SARS-CoV-2 solution. Next, 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. , HF120, manufactured by Millipore), using a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT) at a position 8 mm from the upstream side of the membrane part, the SARS-CoV -2 After applying the solution at a coating amount of 1 μL / cm, dry it for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45 ° C. to form a test line with a line width of about 1 mm. A SARS-CoV-2 immobilized membrane card 1 was obtained.
（３）ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップの作製
　前記ＳＡＲＳ－ＣｏＶ－２固定化メンブレンカード１の上流側の２０ｍｍ×３００ｍｍの粘着テープ部を切断し除去した。次いで、前記ＳＡＲＳ－ＣｏＶ－２固定化メンブレンカード１の下流側の１５ｍｍ×３００ｍｍの粘着テープ部に、メンブレン部と約５ｍｍ重なるよう２０ｍｍ×３００ｍｍの吸収パッド（ＣＥＬＬＵＬＯＳＥ　ＦＩＢＥＲ　ＳＡＭＰＬＥ　ＰＡＤＳ、ＣＦＳＰ００２０００、Ｍｉｌｌｉｐｏｒｅ社製）を貼り合わせた。次いで、ギロチン式カッティングモジュール（ＣＭ５０００、ＢＩＯＤＯＴ社製）を用い、幅３ｍｍ、長さ６０ｍｍの短冊状にカットすることで、ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップ１を得た。 (3) Production of SARS-CoV-2-immobilized half strip A 20 mm × 300 mm adhesive tape portion on the upstream side of the SARS-CoV-2-immobilized membrane card 1 was cut and removed. Then, an absorbent pad of 20 mm x 300 mm (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) is attached to the adhesive tape portion of 15 mm x 300 mm on the downstream side of the SARS-CoV-2 immobilized membrane card 1 so that it overlaps the membrane portion by about 5 mm. ) were pasted together. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), the strips were cut into strips with a width of 3 mm and a length of 60 mm to obtain SARS-CoV-2 immobilized half strips 1.
（４）ＳＡＲＳ－ＣｏＶ固定化ハーフストリップの作製
　ＳＡＲＳ－ＣｏＶ―２溶液の代わりに市販のＳＡＲＳ－ＣｏＶのＮタンパク質であるＳＡＲＳ－ＣｏＶ ｎｕｃｌｅｏｃａｐｓｉｄ ｒｅｃｏｍｂｉｎａｎｔ ｐｒｏｔｅｉｎ（ＡＲＧ７０２１５、Ａｒｉｇｏ Ｂｉｏｌａｂｏｒａｔｏｒｉｅｓ社製）を、ＰＢＳ（－）（１６６－２３５５５、富士フィルム和光純薬社製）にて１００μｇ／ｍＬに希釈したＳＡＲＳ－ＣｏＶ溶液を用いた以外は（２）と同様の方法にてＳＡＲＳ－ＣｏＶ固定化メンブレンカードを得た。次いで、（３）と同様の方法にてＳＡＲＳ－ＣｏＶ固定化ハーフストリップ１を得た (4) Preparation of SARS-CoV immobilized half strip SARS-CoV nucleocapsid recombinant protein (ARG70215, manufactured by Arigo Biolaboratories), which is a commercially available SARS-CoV N protein instead of SARS-CoV-2 solution, was added to PBS (- ) (166-23555, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) except for using a SARS-CoV solution diluted to 100 μg/mL in the same manner as in (2) to obtain a SARS-CoV immobilized membrane card. . Then, SARS-CoV immobilized half strip 1 was obtained in the same manner as in (3)
（５）測定試料希釈液の調製
　１００ｍＭのトリス緩衝液（２０４－０７８８５、富士フィルム和光純薬社製）（ｐＨ８．５）１Ｌ、塩化ナトリウム（１９１－０１６６５、富士フィルム和光純薬社製）８．７７ｇ、ポリオキシエチレン（２０）ソルビタンモノラウレート：Ｔｗｅｅｎ（登録商標）２０（１６６－２１２１３、富士フィルム和光純薬社製）２ｇ、ポリオキシエチレン（１０）オクチルフェニルエーテル：ＴｒｉｔｏｎＸ（登録商標）－１００（１６０－２４７５１、富士フィルム和光純薬社製）９ｇをガラス瓶に加え、溶解することで測定試料希釈液を調製した。 (5) Preparation of measurement sample dilution solution 100 mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.5) 1 L, sodium chloride (191-01665, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 8 .77 g, polyoxyethylene (20) sorbitan monolaurate: Tween (registered trademark) 20 (166-21213, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 2 g, polyoxyethylene (10) octylphenyl ether: TritonX (registered trademark) -100 (160-24751, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) (9 g) was added to a glass bottle and dissolved to prepare a diluted measurement sample solution.
（６）ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比の評価
　９６ｗｅｌｌプレートの１つのｗｅｌｌに前記抗体Ａとセルロース系着色微粒子との複合体１を３μＬ、前記検体希釈液を２０μＬ添加し、ピペッティングにてよく混合した後、前記ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップ１を差し込み、２５℃で１０分間静置した。次いで、メンブレン上のテストラインの反射吸光度（ｍＡｂｓ）をイムノクロマトリーダー（Ｃ１００６０－１０、測定モード：Ｌａｔｅｘ、Ｌｉｎｅ、浜松ホトニクス社製）を用いて測定した。また、前記ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップ１の代わりに前記ＳＡＲＳ－ＣｏＶ固定化ハーフストリップ１を使用する以外は同様にして、メンブレン上のテストラインの反射吸光度（ｍＡｂｓ）を測定した。得られた２つの反射吸光度（ｍＡｂｓ）から下記（式１）および（式２）にてＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を算出した。 (6) Evaluation of reaction ratio and weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein . After adding 20 μL of the sample diluent and mixing well by pipetting, the SARS-CoV-2 immobilized half strip 1 was inserted and allowed to stand at 25° C. for 10 minutes. Next, the reflectance absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). In addition, the reflection absorbance (mAbs) of the test line on the membrane was measured in the same manner except that the SARS-CoV-immobilized half strip 1 was used instead of the SARS-CoV-2 immobilized half strip 1. From the two reflected absorbances (mAbs) obtained, the reaction ratio and weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein were calculated using the following (Formula 1) and (Formula 2).
（ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップ１の反射吸光度）／（ＳＡＲＳ－ＣｏＶ固定化ハーフストリップ１の反射吸光度）　　　　　　…　（式１） (Reflection absorbance of SARS-CoV-2 immobilized half strip 1) / (Reflection absorbance of SARS-CoV immobilized half strip 1) (Formula 1)
（ＳＡＲＳ－ＣｏＶ－２固定化ハーフストリップ１の反射吸光度）２／（ＳＡＲＳ－ＣｏＶ固定化ハーフストリップ１の反射吸光度）　　　　　　…　（式２） (Reflection absorbance of SARS-CoV-2 immobilized half strip 1) 2 /(Reflection absorbance of SARS-CoV immobilized half strip 1) … (Equation 2)
（実施例２）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体２（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０９、東洋紡社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体２を得た。得られた複合体２のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Example 2)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 2 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-109, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles got 2. The resulting SARS-CoV-2 N protein to SARS-CoV N protein complex 2 reaction ratios and weighted reaction ratios are shown in Table 1.
（実施例３）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体３（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０６、東洋紡社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体３を得た。得られた複合体３のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Example 3)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. A complex of antibody A and cellulose-based colored fine particles got 3. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 3 are shown in Table 1.
（実施例４）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体４（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０１、東洋紡社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体４を得た。得られた複合体４のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Example 4)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 4 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles Got 4. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 4 are shown in Table 1.
（実施例５）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体５（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１００、東洋紡社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体５を得た。得られた複合体５のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Example 5)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 5 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles Got 5. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 5 are shown in Table 1.
（実施例６）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体６（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０３、東洋紡社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体６を得た。得られた複合体６のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Example 6)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 6 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-103, manufactured by Toyobo Co., Ltd.) was used in the same manner as in Example 1. Complexes of antibody A and cellulose-based colored fine particles got 6. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios for Complex 6 are shown in Table 1.
（比較例１）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体α（ＣＯＶＩＤ－１９　ＮＰ　Ａｎｔｉｂｏｄｙ、ＨＭ１１４７、ＥａｓｔＣｏａｓｔＢｉｏ社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体αを得た。得られた複合体αのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Comparative example 1)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex α of antibody A and cellulose-based colored fine particles was obtained in the same manner as in Example 1 except that N protein monoclonal antibody α (COVID-19 NP Antibody, HM1147, manufactured by EastCoastBio) was used. The resulting reaction ratios and weighted reaction ratios of SARS-CoV-2 N protein to SARS-CoV N protein of complex α are shown in Table 1.
（比較例２）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体β（ＣＯＶＩＤ－１９　ＮＰ　Ａｎｔｉｂｏｄｙ、ＨＭ１１４５、ＥａｓｔＣｏａｓｔＢｉｏ社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体βを得た。得られた複合体βのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Comparative example 2)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex β of antibody A and cellulose-based colored microparticles was obtained in the same manner as in Example 1 except that N protein monoclonal antibody β (COVID-19 NP Antibody, HM1145, manufactured by EastCoastBio) was used. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios of complex β are shown in Table 1.
（比較例３）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体γ（ＣＯＶＩＤ－１９　ＮＰ　Ａｎｔｉｂｏｄｙ、ＨＭ１１４４、ＥａｓｔＣｏａｓｔＢｉｏ社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体γを得た。得られた複合体γのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Comparative Example 3)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex γ of antibody A and cellulose-based colored fine particles was obtained in the same manner as in Example 1, except that N protein monoclonal antibody γ (COVID-19 NP Antibody, HM1144, manufactured by EastCoastBio) was used. The resulting SARS-CoV-2 N protein to SARS-CoV N protein response ratios and weighted response ratios of complex γ are shown in Table 1.
（比較例４）
　抗体Ａとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりにマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体δ（ＣＯＶＩＤ－１９　ＮＰ　Ａｎｔｉｂｏｄｙ、ＨＭ１１４３、ＥａｓｔＣｏａｓｔＢｉｏ社製）を用いた以外は実施例１と同様の方法で抗体Ａとセルロース系着色微粒子との複合体δを得た。得られた複合体δのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の反応比および加重反応比を表１に示す。 (Comparative Example 4)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as antibody A (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 A complex δ of antibody A and cellulose-based colored microparticles was obtained in the same manner as in Example 1 except that N protein monoclonal antibody δ (COVID-19 NP Antibody, HM1143, manufactured by EastCoastBio) was used. The resulting SARS-CoV-2 N protein to SARS-CoV N protein reaction ratios and weighted reaction ratios of complex δ are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に各実施例での評価を示す。ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以下を×（ｂａｄ）、４０以上１００以下を△（ａｖｅｒａｇｅ）、１００以上２００以下を○（ｇｏｏｄ）、２００以上を◎（ｅｘｃｅｌｌｅｎｔ）で示した。実施例１～６のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比は４０より大きいため、各デバイスで使用した抗体１～６はＳＡＲＳ－ＣｏＶ－２に対する反応性が高い、および／またはＳＡＲＳ－ＣｏＶ－２に比べてＳＡＲＳ－ＣｏＶの反応性が大幅に低いことが理解できる。特に、実施例１～４の加重反応比は２００より大きいため、各デバイスで使用した抗体１～４は高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得る抗体であると言える。一方、比較例１～４のＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比は４０より小さいため、各デバイスで使用した抗体α～δはＳＡＲＳ－ＣｏＶ－２に対する反応性が著しく低い、および／またはＳＡＲＳ－ＣｏＶ－２に比べてＳＡＲＳ－ＣｏＶの反応性を低く抑えることができていないことが明らかである。 Table 1 shows the evaluation in each example. The weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein is × (bad) when it is 40 or less, △ (average) when it is 40 or more and 100 or less, ○ (good) when it is 100 or more and 200 or less, 200 or more is indicated by ⊚ (excellent). Since the weighted response ratio of SARS-CoV-2 N protein to SARS-CoV N protein in Examples 1-6 is greater than 40, Antibodies 1-6 used in each device are not reactive to SARS-CoV-2. It can be seen that the reactivity of SARS-CoV is high and/or significantly lower than that of SARS-CoV-2. In particular, since the weighted reaction ratio of Examples 1-4 is greater than 200, it can be said that the antibodies 1-4 used in each device are antibodies capable of detecting the SARS-CoV-2 N protein with high sensitivity and high specificity. . On the other hand, since the weighted response ratio of SARS-CoV-2 N protein to SARS-CoV N protein in Comparative Examples 1-4 is less than 40, the antibodies α to δ used in each device react to SARS-CoV-2. It is clear that the reactivity of SARS-CoV is significantly lower and/or the reactivity of SARS-CoV has not been kept lower than that of SARS-CoV-2.
(実施例７)
（７）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用メンブレンカードの作製
　抗体Ｂとして２．０ｍｇ／ｍＬのマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）を準備した。次いで、抗体Ｃとして１．０ｍｇ／ｍＬの抗マウスＩｇＧポリクローナル抗体（Ｍｏｕｓｅ　ＩｇＧ－ｈｅａｖｙ　ａｎｄ　ｌｉｇｈｔ　ｃｈａｉｎ　ａｎｔｉｂｏｄｙ、Ａ９０－１１７Ａ、ＢＥＴＨＹＬ社製）を準備した。次いで、上流側に２０ｍｍ×３００ｍｍの粘着テープ部、中央に２５ｍｍ×３００ｍｍのメンブレン部、下流側に１５ｍｍ×３００ｍｍの粘着テープ部から構成される６０ｍｍ×３００ｍｍのメンブレンカード（Ｈｉ－Ｆｌｏｗ　Ｐｌｕｓ　１２０　Ｍｅｍｂｒａｎｅ　Ｃａｒｄｓ、ＨＦ１２０、Ｍｉｌｌｉｐｏｒｅ社製）のメンブレンカードの上流側から８ｍｍの位置に、分注プラットフォーム（ＸＹＺ３０６０、ＢＩＯＤＯＴ社製）と、Ｂｉｏ Ｊｅｔノズル（ＢＨＱＨＲ－ＸＹＺ、ＢＩＯＤＯＴ社製）を用い、前記抗体Ｂを１．０μＬ／ｃｍの塗布量で塗布した後、４５℃に調整した乾燥機（ＷＦＯ－５１０、東京理化器械社製）で３０分間乾燥し、ライン幅約１ｍｍのテストラインを形成した。さらに、前記テストラインを形成したメンブレンカードのメンブレン部の上流側から１５ｍｍの位置に、分注プラットフォーム（ＸＹＺ３０６０、ＢＩＯＤＯＴ社製）と、Ｂｉｏ Ｊｅｔノズル（ＢＨＱＨＲ－ＸＹＺ、ＢＩＯＤＯＴ社製）を用い、前記抗体Ｃを１．０μＬ／ｃｍの塗布量で塗布した後、４５℃に調整した乾燥機（ＷＦＯ－５１０、東京理化器械社製）で３０分間乾燥し、ライン幅約１ｍｍのコントロールラインを形成することで、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用メンブレンカード１を得た。 (Example 7)
(7) Preparation of membrane card for detecting SARS-CoV-2 N protein As antibody B, 2.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo Co., Ltd.) was prepared. Next, as Antibody C, a 1.0 mg/mL anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) was prepared. Next, 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. , HF120, manufactured by Millipore) at a position 8 mm from the upstream side of the membrane card, using a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT), the antibody B is added. After coating with a coating amount of 1.0 μL/cm, it was dried for 30 minutes in a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45° C. to form a test line with a line width of about 1 mm. Furthermore, using a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT) at a position 15 mm from the upstream side of the membrane part of the membrane card on which the test line is formed, Antibody C is applied at a coating amount of 1.0 μL/cm, and dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45° C. to form a control line with a line width of about 1 mm. Thus, membrane card 1 for detecting N protein of SARS-CoV-2 was obtained.
（８）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用コンジュゲーションパッドの作製
　１０ｍｍ×３００ｍｍのコンジュゲーションパッド（ＧＬＡＳＳＦＩＢＥＲ　ＤＩＡＧＮＯＳＴＩＣ　ＰＡＤ、ＧＦＤＸ００１０５０、Ｍｉｌｌｉｐｏｒｅ社製）の全面に、分注プラットフォーム（ＸＹＺ３０６０、ＢＩＯＤＯＴ社製）と、Ａｉｒ Ｊｅｔノズル（ＡＪＱＨＲ－ＸＹＺ、ＢＩＯＤＯＴ社製）を用い、実施例２の抗体Ａとセルロース系着色微粒子との複合体２を１５μＬ／ｃｍの塗布量で均一に塗布した後、４５℃に調整した乾燥機（ＷＦＯ－５１０、東京理化器械社製）で３０分間乾燥し、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用コンジュゲーションパッド１を得た。 (8) Preparation of SARS-CoV-2 N protein detection conjugation pad On the entire surface of a 10 mm × 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore), a dispensing platform (XYZ3060, manufactured by BIODOT) Then, using an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the composite 2 of antibody A and cellulose-based colored fine particles of Example 2 was uniformly applied at a coating amount of 15 μL / cm, and then heated to 45 ° C. It was dried for 30 minutes with an adjusted dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) to obtain a conjugation pad 1 for detecting N protein of SARS-CoV-2.
（９）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片およびデバイスの作製
　前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用メンブレンカード１の上流側の２０ｍｍ×３００ｍｍの粘着テープ部に、メンブレン部と２ｍｍ重なるよう前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用コンジュゲーションパッド１を貼り合わせた。次いで、さらに上流側に前記コンジュゲーションパッドと３ｍｍ重なるように１５ｍｍ×３００ｍｍのサンプルパッド（ＣＥＬＬＵＬＯＳＥ　ＦＩＢＥＲ　ＳＡＭＰＬＥ　ＰＡＤＳ、ＣＦＳＰ００２０００、Ｍｉｌｌｉｐｏｒｅ社製）を貼り合わせた。次いで、前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用メンブレンカード１の下流側の１５ｍｍ×３００ｍｍの粘着テープ部に、メンブレン部と５ｍｍ重なるように２０ｍｍ×３００ｍｍの吸収パッド（ＣＥＬＬＵＬＯＳＥ　ＦＩＢＥＲ　ＳＡＭＰＬＥ　ＰＡＤＳ、ＣＦＳＰ００２０００、Ｍｉｌｌｉｐｏｒｅ社製）を貼り合わせた。次いで、ギロチン式カッティングモジュール（ＣＭ５０００、ＢＩＯＤＯＴ社製）を用い、幅４ｍｍ、長さ６０ｍの短冊状にカットすることで、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片１を得た。得られたＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片１をハウジングケース（Ｋ００７、Ｓｈｅｎｇｆｅｎｇ　Ｐｌａｓｔｉｃ社製）に収納することでＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマトデバイス１を得た。 (9) Preparation of immunochromatographic test strip and device for detecting N protein of SARS-CoV-2 In the adhesive tape part of 20 mm × 300 mm on the upstream side of the membrane card 1 for detecting N protein of SARS-CoV-2, the membrane part and The SARS-CoV-2 N protein detection conjugation pad 1 was adhered so as to overlap by 2 mm. Next, a 15 mm×300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was pasted on the upstream side so as to overlap the conjugation pad by 3 mm. Then, a 20 mm × 300 mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, CFSP002000, (manufactured by Millipore) was pasted together. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), a strip of 4 mm wide and 60 m long was cut to obtain an immunochromatographic test strip 1 for detecting N protein of SARS-CoV-2. The immunochromatographic test piece 1 for detecting N protein of SARS-CoV-2 thus obtained 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.
（１０）ＳＡＲＳ－ＣｏＶ２試料およびＳＡＲＳ－ＣｏＶ試料の調製
　市販のＳＡＲＳ－ＣｏＶ－２のＮタンパク質であるＲｅｃｏｍｂｉｎａｎｔ　ＳＡＲＳ－ＣｏＶ－２　Ｎｕｃｌｅｏｃａｐｓｉｄ　ｐｒｏｔｅｉｎ（２３０－３０１６４、ＲａｙＢｉｏｔｅｃｈ社製）を、前記測定試料希釈液にて１ｎｇ／ｍＬに希釈することでＳＡＲＳ－ＣｏＶ－２試料を得た。他方、市販のＳＡＲＳ－ＣｏＶのＮタンパク質であるＳＡＲＳ－ＣｏＶ ｎｕｃｌｅｏｃａｐｓｉｄ ｒｅｃｏｍｂｉｎａｎｔ ｐｒｏｔｅｉｎ（ＡＲＧ７０２１５、Ａｒｉｇｏ Ｂｉｏｌａｂｏｒａｔｏｒｉｅｓ社製）を、前記測定試料希釈液にて１００ｎｇ／ｍＬに希釈することでＳＡＲＳ－ＣｏＶ試料を得た。 (10) Preparation of SARS-CoV2 sample and SARS-CoV sample Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, manufactured by RayBiotech), which is a commercially available N protein of SARS-CoV-2, was added to the measurement sample diluent. SARS-CoV-2 samples were obtained by diluting to 1 ng/mL at . On the other hand, a commercially available SARS-CoV nucleocapsid recombinant protein (ARG70215, manufactured by Arigo Biolaboratories), which is the N protein of SARS-CoV, was diluted to 100 ng/mL with the measurement sample diluent to obtain a SARS-CoV sample. .
（１１）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片の評価：非特異吸着の評価
　前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマトデバイスを水平な台に設置した。次いで、前記測定試料希釈液１００μＬを、マイクロピペットで分取し、サンプルパッドに緩やかに滴下し、２５℃で１０分間静置した。次いで、メンブレン上のテストラインの反射吸光度（ｍＡｂｓ）をイムノクロマトリーダー（Ｃ１００６０－１０、測定モード：Ｌａｔｅｘ、Ｌｉｎｅ、浜松ホトニクス社製）を用いて測定した。得られた結果を表２に示す。 (11) Evaluation of immunochromatographic test piece for detecting N protein of SARS-CoV-2: evaluation of non-specific adsorption The immunochromatographic device for detecting N protein of SARS-CoV-2 was placed on a horizontal table. Next, 100 μL of the diluted measurement sample solution was dispensed with a micropipette, gently dropped onto the sample pad, and allowed to stand at 25° C. for 10 minutes. Next, the reflectance absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). Table 2 shows the results obtained.
（１２）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片の評価：感度の評価
　前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマトデバイスを水平な台に設置した。次いで、前記ＳＡＲＳ－ＣｏＶ－２試料１００μＬを、マイクロピペットで分取し、サンプルパッドに緩やかに滴下し、２５℃で１５分間静置した。次いで、メンブレン上のコントロールラインおよびテストラインの反射吸光度（ｍＡｂｓ）をイムノクロマトリーダー（Ｃ１００６０－１０、測定モード：Ｌａｔｅｘ、Ｌｉｎｅ、浜松ホトニクス社製）を用いて測定した。得られた結果を表２に示す。 (12) Evaluation of SARS-CoV-2 N protein detection immunochromatography test piece: Evaluation of sensitivity The SARS-CoV-2 N protein detection immunochromatography device was placed on a horizontal table. Then, 100 μL of the SARS-CoV-2 sample was dispensed with a micropipette, gently dropped onto the sample pad, and left standing at 25° C. for 15 minutes. Then, the reflectance absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). Table 2 shows the results obtained.
（１３）ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマト試験片の評価：特異性の評価
　前記ＳＡＲＳ－ＣｏＶ－２のＮタンパク質検出用イムノクロマトデバイスを水平な台に設置した。次いで、前記ＳＡＲＳ－ＣｏＶ試料１００μＬを、マイクロピペットで分取し、サンプルパッドに緩やかに滴下し、２５℃で１５分間静置した。次いで、メンブレン上のコントロールラインおよびテストラインの反射吸光度（ｍＡｂｓ）をイムノクロマトリーダー（Ｃ１００６０－１０、測定モード：Ｌａｔｅｘ、Ｌｉｎｅ、浜松ホトニクス社製）を用いて測定した。得られた結果を表２に示す。 (13) Evaluation of SARS-CoV-2 N protein detection immunochromatography test piece: evaluation of specificity The SARS-CoV-2 N protein detection immunochromatography device was placed on a horizontal table. Then, 100 μL of the SARS-CoV sample was dispensed with a micropipette, gently dropped onto the sample pad, and left standing at 25° C. for 15 minutes. Then, the reflectance absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). Table 2 shows the results obtained.
(実施例８)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体４を用いた以外は実施例７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 8)
(11) non-specific adsorption, (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例９)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体５を用いた以外は実施例７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 9)
(11) non-specific adsorption, (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１０)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体６を用い、前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体３（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０６、東洋紡社製）を用いた以外は実施例７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 10)
Instead of the complex 2 of the antibody A and the cellulose-based colored fine particles, the complex 6 of the antibody A and the cellulose-based colored fine particles is used, and the mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 is used as the antibody B. (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 (Anti-SARS-CoV-2-NP Monoclonal (11) non-specific adsorption, (12) sensitivity, and (13) specificity were evaluated in the same manner as in Example 7, except that antibody, SCV-106, manufactured by Toyobo Co., Ltd.) was used. Table 2 shows the results obtained.
(実施例１１)
　前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体３（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０６、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体４（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０１、東洋紡社製）を用いた以外は実施例１０と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 11)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１２)
　前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体３（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０６、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体５（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１００、東洋紡社製）を用いた以外は実施例１０と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 12)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 3 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-106, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１３)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体αを用いた以外は実施例７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 13)
(11) non-specific adsorption, (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１４)
　前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体３（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０６、東洋紡社製）を用いた以外は実施例１３と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 14)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１５)
　前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体４（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０１、東洋紡社製）を用いた以外は実施例１３と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 15)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１６)
　前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体６（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０３、東洋紡社製）を用いた以外は実施例１３と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 16)
Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 as the antibody B (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV- (11) non-specific adsorption, ( 12) sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(実施例１７)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体１を用い、前記抗体Ｂとしてマウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体１（Ａｎｔｉ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ　Ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ、ＳＣＶ－１０８、東洋紡社製）の代わりに、マウス由来抗ＳＡＲＳ－ＣｏＶ－２のＮタンパク質モノクローナル抗体α（ＣＯＶＩＤ－１９　ＮＰ　Ａｎｔｉｂｏｄｙ、ＨＭ１１４７、ＥａｓｔＣｏａｓｔＢｉｏ社製）を用いた以外は実施例７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Example 17)
Instead of the complex 2 of the antibody A and the cellulose-based colored fine particles, the complex 1 of the antibody A and the cellulose-based colored fine particles is used, and the mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 is used as the antibody B. (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo) instead of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody α (COVID-19 NP Antibody, HM1147, EastCoastBio (11) non-specific adsorption, (12) sensitivity, and (13) specificity were evaluated in the same manner as in Example 7, except that the product was used. Table 2 shows the results obtained.
(比較例５)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体βを用いた以外は実施例１７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Comparative Example 5)
(11) non-specific adsorption, (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(比較例６)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体γを用いた以外は実施例１７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Comparative Example 6)
(11) Non-specific adsorption, (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
(比較例７)
　前記抗体Ａとセルロース系着色微粒子との複合体２の代わりに前記抗体Ａとセルロース系着色微粒子との複合体δを用いた以外は実施例１７と同様の方法にて（１１）非特異吸着、（１２）感度、（１３）特異性を評価した。得られた結果を表２に示す。 (Comparative Example 7)
(11) Non-specific adsorption in the same manner as in Example 17 except that the complex δ of the antibody A and the cellulose-based colored fine particles was used instead of the complex 2 of the antibody A and the cellulose-based colored fine particles. (12) Sensitivity and (13) specificity were evaluated. Table 2 shows the results obtained.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に各実施例での評価を示す。（１１）非特異吸着が５０ｍＡｂｓ以上を×（ｂａｄ）、３０ｍＡｂｓ以上５０ｍＡｂｓ以下を△（ａｖｅｒａｇｅ）、１０ｍＡｂｓ以上３０ｍＡｂｓ以下を○（ｇｏｏｄ）、１０ｍＡｂｓ以下を◎（ｅｘｃｅｌｌｅｎｔ）で示した。また、（１２）感度が５０ｍＡｂｓ以下を×（ｂａｄ）、５０ｍＡｂｓ以上１５０ｍＡｂｓ以下を△（ａｖｅｒａｇｅ）、１５０ｍＡｂｓ以上２５０ｍＡｂｓ以下を○（ｇｏｏｄ）、２５０ｍＡｂｓ以上を◎（ｅｘｃｅｌｌｅｎｔ）で示した。さらに、（１３）特異性が３０ｍＡｂｓ以上を×（ｂａｄ）、２０ｍＡｂｓ以上３０ｍＡｂｓ以下を△（ａｖｅｒａｇｅ）、１０ｍＡｂｓ以上２０ｍＡｂｓ以下を○（ｇｏｏｄ）、１０ｍＡｂｓ以下を◎（ｅｘｃｅｌｌｅｎｔ）で示した。表２より実施例７～１７のイムノクロマト試験片は、ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上の抗体を捕捉抗体および／または検出抗体として組み合わせて使用しているため、高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得ることを確認できた。特に、実施例７～１２のイムノクロマト試験片は、ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上の抗体を捕捉抗体および検出抗体として組み合わせて使用しているため、非常に高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得ることを確認できた。一方、比較例５～７のイムノクロマト試験片は、ＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０より小さい抗体を捕捉抗体および検出抗体として組み合わせて使用しているため、低感度かつ特異性も低い結果となった。 Table 2 shows the evaluation in each example. (11) Non-specific adsorption is indicated by × (bad) for 50 mAbs or more, Δ (average) for 30 mAbs or more and 50 mAbs or less, ○ (good) for 10 mAbs or more and 30 mAbs or less, and ◎ (excellent) for 10 mAbs or less. (12) Sensitivity of 50 mAbs or less was indicated by × (bad), 50 mAbs to 150 mAbs by Δ (average), 150 mAbs to 250 mAbs by O (good), and 250 mAbs or more by ⊚ (excellent). Furthermore, (13) specificity of 30 mAbs or more is indicated by × (bad), 20 mAbs to 30 mAbs by Δ (average), 10 mAbs to 20 mAbs by ○ (good), and 10 mAbs or less by ◎ (excellent). From Table 2, the immunochromatographic test strips of Examples 7 to 17 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of 40 or more as a capture antibody and / or a detection antibody in combination. Therefore, it was confirmed that the N protein of SARS-CoV-2 can be detected with high sensitivity and high specificity. In particular, the immunochromatographic test strips of Examples 7 to 12 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of 40 or more as a capture antibody and a detection antibody in combination. Therefore, it was confirmed that the N protein of SARS-CoV-2 can be detected with extremely high sensitivity and high specificity. On the other hand, the immunochromatographic test strips of Comparative Examples 5 to 7 use an antibody with a weighted reaction ratio of SARS-CoV-2 N protein to SARS-CoV N protein of less than 40 in combination as a capture antibody and a detection antibody. Therefore, the result was low sensitivity and low specificity.
　本発明により、高感度かつ特異性高くＳＡＲＳ－ＣｏＶ－２のＮタンパク質を検出し得るイムノクロマト試験片を提供することができる。 The present invention can provide an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and high specificity.
１：サンプルパッド
２：コンジュゲーションパッド
３：メンブレン
４：吸収パッド
５：バッキングシート
６：テストライン
７：コントロールライン
８：粘着シート 1: sample pad 2: conjugation pad 3: membrane 4: absorbent pad 5: backing sheet 6: test line 7: control line 8: adhesive sheet

Claims (3)

  1. 　（１）サンプルパッドと、
    （２）測定試料中の重症急性呼吸器症候群コロナウイルス２（ＳＡＲＳ－ＣｏＶ－２）のヌクレオカプシドタンパク質（Ｎタンパク質）と特異的に結合する抗体Ａとセルロース系着色微粒子との複合体を担持したコンジュゲーションパッドと、
    （３）測定試料中のＳＡＲＳ－ＣｏＶ－２のＮタンパク質と特異的に結合する抗体Ｂと、前記抗体Ａと特異的に結合する抗体Ｃとを、それぞれ異なる位置に線状に固定したメンブレンと、
    （４）吸収パッドと、から構成され、
    前記抗体Ａまたは抗体ＢのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であることを特徴とするイムノクロマト試験片。     (1) a sample pad;
    (2) A conjugate carrying a complex of antibody A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample and cellulose-based colored fine particles gaming pad and
    (3) A membrane in which antibody B that specifically binds to the SARS-CoV-2 N protein in the measurement sample and antibody C that specifically binds to the antibody A are linearly immobilized at different positions. ,
    (4) an absorbent pad;
    An immunochromatographic test strip, wherein the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A or antibody B is 40 or more.
  2. 　前記抗体Ａおよび抗体ＢのＳＡＲＳ－ＣｏＶのＮタンパク質に対するＳＡＲＳ－ＣｏＶ－２のＮタンパク質の加重反応比が４０以上であることを特徴とする請求項１に記載のイムノクロマト試験片。 The immunochromatographic test strip according to claim 1, wherein the weighted reaction ratio of the SARS-CoV-2 N protein to the SARS-CoV N protein of the antibody A and antibody B is 40 or more.
  3. 　請求項１または２に記載のイムノクロマト試験片、測定試料採取具、フィルター、測定試料希釈液からなることを特徴とするイムノクロマトキット。
          3. An immunochromatographic kit comprising the immunochromatographic test piece according to claim 1 or 2, a measurement sample collecting tool, a filter, and a measurement sample diluent.
PCT/JP2022/024381 2021-06-17 2022-06-17 Immunochromatographic test piece WO2022265106A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023530439A JPWO2022265106A1 (en) 2021-06-17 2022-06-17

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021-101042 2021-06-17
JP2021101042 2021-06-17

Publications (1)

Publication Number Publication Date
WO2022265106A1 true WO2022265106A1 (en) 2022-12-22

Family

ID=84526529

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/024381 WO2022265106A1 (en) 2021-06-17 2022-06-17 Immunochromatographic test piece

Country Status (2)

Country Link
JP (1) JPWO2022265106A1 (en)
WO (1) WO2022265106A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008014751A (en) * 2006-07-05 2008-01-24 Denka Seiken Co Ltd Membrane assay method using colored latex particle, and kit
WO2019138898A1 (en) * 2018-01-09 2019-07-18 東洋紡株式会社 Immunochromatography test piece, measurement kit, and measurement method
CN111733141A (en) * 2020-06-19 2020-10-02 清华大学深圳国际研究生院 Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008014751A (en) * 2006-07-05 2008-01-24 Denka Seiken Co Ltd Membrane assay method using colored latex particle, and kit
WO2019138898A1 (en) * 2018-01-09 2019-07-18 東洋紡株式会社 Immunochromatography test piece, measurement kit, and measurement method
CN111733141A (en) * 2020-06-19 2020-10-02 清华大学深圳国际研究生院 Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "SARS-CoV-2 {COVID-19) Research", GENE TEX., 1 January 2020 (2020-01-01), XP093015721, Retrieved from the Internet <URL:https://www.lubio.ch/assets/PDFs/GeneTex_SARS-CoV-2_brochure_2020_original.pdf> [retrieved on 20230119] *
LI MAOHUA, JIN RONGHUA, PENG YA, WANG CUIYAN, REN WENLIN, LV FUDONG, GONG SITAO, FANG FENG, WANG QIANYUN, LI JIANLI, SHEN TONG, SU: "Generation of antibodies against COVID-19 virus for development of diagnostic tools", MEDRXIV, 27 February 2020 (2020-02-27), pages 1 - 17, XP009531584, DOI: 10.1101/2020.02.20.20025999 *
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, AMSTERDAM, NL, vol. 300, 1 July 2021 (2021-07-01), NL , pages 1 - 7, XP093014610, ISSN: 0168-1702, DOI: 10.1016/j.virusres.2021.198445 *
YAMAKAWA, KENTARO ET AL.: "Development of Rapid Immunochromatographic Enzyme Immunoassay for SARS CoV-2 nucleocapsid antigen", JAPANESE JOURNAL OF MEDICINE AND PHARMACEUTICAL SCIENCE, vol. 77, no. 6, 27 May 2020 (2020-05-27), pages 937 - 944, XP009536599 *
YAMAOKA YUTARO, MIYAKAWA KEI, JEREMIAH SUNDARARAJ STANLEYRAJ, FUNABASHI RIKAKO, OKUDELA KOJI, KIKUCHI SAYAKA, KATADA JUNICHI, WADA: "Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests", CELL REPORTS MEDICINE, vol. 2, 1 June 2021 (2021-06-01), pages 1 - 18, XP055896515, ISSN: 2666-3791, DOI: 10.1016/j.xcrm.2021.100311 *

Also Published As

Publication number Publication date
JPWO2022265106A1 (en) 2022-12-22

Similar Documents

Publication Publication Date Title
JP6143818B2 (en) Immunochromatographic analyzer for detection of Mycoplasma pneumoniae
JP2020530567A (en) Assay Methods for Improved Analyze Detection
JP4918627B2 (en) Sample extract that suppresses false positive reaction
JP6348240B2 (en) Detection method using immunochromatography
EP3076177B1 (en) Immunochromatography-assisted detection method
WO2019138898A1 (en) Immunochromatography test piece, measurement kit, and measurement method
EP3339862A1 (en) Immunological detection method and test strip used therefor
US20230341398A1 (en) SARS-CoV-2 DETECTION KIT AND SARS-CoV-2 DETECTION METHOD
WO2012133615A1 (en) Detection method using immunochromatography capable of determining sample without addition of specimen as operation-failure sample, and test strip for use in same
WO2020196296A1 (en) Immunochromatographic analysis method, and test strip used in said immunochromatographic analysis method
WO2023025074A1 (en) Immunoassay kit having multiple test scales, and application thereof
JP2008275511A (en) Method for measuring immunity of influenza virus antigen and device used for the same
WO2023112859A1 (en) Immunochromatography test strip and immunochromatography kit, immunoassay method using same, and sample filtration method
WO2022265106A1 (en) Immunochromatographic test piece
WO2023017817A1 (en) Immunoassay method, specimen diluent, and immunochromatography kit
JP2023016530A (en) Immunochromatographic test piece
JP2023016529A (en) Immunochromatographic test piece
WO2022265105A1 (en) Immunochromatographic test strip and immunochromatographic kit
JP2009002822A (en) Immuno-chromatography measuring method and kit
JP4426122B2 (en) Blood antigen detection method and apparatus
WO2024090518A1 (en) Immunochromatographic test piece
WO2024005055A1 (en) Test method, test reagent, and test kit
WO2020196295A1 (en) Immunological analysis method for respiratory tract infection virus and detection kit
WO2021193792A1 (en) Immunochromatography kit and immunochromatography method
WO2016194986A1 (en) Immunochromatographic analyzer for mycoplasma pneumoniae detection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22825094

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023530439

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE