JP2023016530A - Immunochromatographic test piece - Google Patents

Abstract

To provide an immunochromatographic test piece that can detect an influenza virus A, an influenza virus B, and a severe acute respiratory syndrome coronavirus 2 simultaneously with high sensitivity.SOLUTION: The present invention is an immunochromatographic test piece comprising: a conjugation pad 1 carrying complexes of antibodies A1, B1 that specifically bind to a nucleocapsid protein (N protein) of influenza viruses A, B (Flu-A, B) in a measurement sample and cellulose colored fine particles A, B; a conjugation pad 2 carrying a complex of an antibody C1 that specifically binds to a N protein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellulose colored fine particles C; a membrane 1 provided with test lines A, B immobilizing antibodies A2, B2 that specifically bind to the N protein of the Flu-A, B; and a membrane 2 provided with a test line C immobilizing an antibody C2 that specifically binds to the N protein of the SARS-CoV-2.SELECTED DRAWING: None

Description

The present invention provides the nucleocapsid protein (N protein) of influenza virus A (Flu-A), the N protein of influenza virus B (Flu-B), and Severe Acute Respiratory Syndrome coronavirus 2 (SARS- The present invention relates to an immunochromatographic test strip capable of simultaneously and highly sensitively detecting the N protein of CoV-2), and a kit containing the immunochromatographic test strip.

Influenza is an infectious disease that is prevalent in winter and presents with clinical symptoms such as high fever, upper respiratory tract inflammation, and malaise. It is not easy to distinguish between them. Here, SARS-CoV-2 is the causative virus of the novel coronavirus infection (COVID-19), and has been rapidly prevalent all over the world since the beginning of 2020.

Influenza virus is a virus whose genomic gene is a segmented negative-strand RNA, and consists of eight segments: HA, NA, PA, PB1, PB2, M, NP, and NS. Influenza viruses are classified into types A, B and C according to the difference in antigenicity between the matrix protein (M1) and the N protein (NP) among the constituent proteins. Since the proteins differ in their antigenicity, they do not show cross-reactivity between different types.

Oseltamivir phosphate, zanamivir hydrate, peramivir hydrate, laninamivir octanoate hydrate, and amantadine hydrochloride are used as therapeutic agents for influenza, but amantadine hydrochloride, which is an M2 inhibitor, It is used only for the treatment of influenza A because it does not inhibit the infection and proliferation of influenza B virus. Furthermore, although the former four are therapeutic drugs that show the same action point as neuraminidase (NA) inhibitors, it has been pointed out that there are differences in efficacy, such as fever-reducing time and virus survival rate, depending on the type of influenza (non Patent document 1). Therefore, influenza type discrimination is important for the subsequent selection of appropriate therapeutic agents.

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). 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 2 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, which is rapid, simple, and inexpensive, is one of the most widely used diagnostic methods for infectious diseases. 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 an immune complex with the substance to be measured and the sensitized detection antibody pass through the test line, are captured by the control line antibody, and develop color. The presence or absence of the substance to be measured can be determined by visually confirming these color development intensities. Anti-NP antibodies (Patent Documents 1 and 2) and the like have been particularly used for detection of conventional influenza.

Currently, influenza and SARS-CoV-2 testing is performed using individual test kits for each item. For this reason, for example, if a patient who shows clinical symptoms such as high fever, upper respiratory tract inflammation, and malaise tests negative for SARS-CoV-2, the patient should be re-tested for influenza. must be taken. A specimen is collected from the nasal cavity or pharynx using a cotton swab or the like, which is painful and burdensome for the patient.

JP 2007-285749 A WO2005/007698

J Infect. 2008 Jan;56(1):51-57. Epub 2007 Oct 15. Klann et al. , Molecular Cell, 80(1), 2020, pp. 164-174

The present invention provides an immunochromatographic test strip that can detect Flu-A N protein, Flu-B N protein, and SARS-CoV-2 N protein simultaneously and with high sensitivity, and a kit containing the immunochromatographic test strip. is the subject.

As a result of intensive research to solve the above problems, the present inventors have found that complexes of antibody A1 and detection particles that specifically bind to N protein of Flu-A, and N protein of Flu-B specifically A conjugation pad 1 carrying a complex of binding antibody B1 and detection particles, and a conjugation pad 2 carrying a complex of antibody C1 specifically binding to the N protein of SARS-CoV-2 and detection particles. , a test line A linearly immobilized antibody A2 that specifically binds to the N protein of Flu-A, a test line B linearly immobilized antibody B2 that specifically binds to the N protein of Flu-B, And the membrane 1 equipped with a control line 1 in which the antibody D1 that specifically binds to the antibody A2 or B2 is linearly immobilized, and the antibody C2 that specifically binds to the SARS-CoV-2 N protein is linearly By using a membrane 2 equipped with an immobilized test line C and a control line 2 linearly immobilized with an antibody D2 that specifically binds to the antibody C2, N protein of Flu-A, N protein of Flu-B It was found that an immunochromatographic test strip capable of simultaneously and highly sensitively detecting the protein and the N protein of SARS-CoV-2 was obtained. In addition, by using cellulose-based colored fine particles as detection particles, it was found that Flu-A N protein, Flu-B N protein, and SARS-CoV-2 N protein can be detected with higher sensitivity. completed.

That is, the representative present invention is as follows.
1. (1) an absorbent pad 1;
(2) Test line A in which antibody A2 that specifically binds to the nucleocapsid protein (N protein) of influenza virus A (Flu-A) in the measurement sample is linearly immobilized, N of influenza virus B (Flu-B) A membrane 1 having a test line B linearly immobilized with an antibody B2 that specifically binds to a protein and a control line 1 linearly immobilized with an antibody D1 that specifically binds to the antibody A2 or B2;
(3) A complex of antibody A1 that specifically binds to Flu-A N protein and cellulose-based colored fine particle A, and antibody B1 that specifically binds to Flu-B N protein and cellulose-based colored fine particle B A conjugation pad 1 carrying a complex of
(4) a sample pad;
(5) Conjugation pad 2 carrying a complex of antibody C1 that specifically binds to the N protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample and cellulose-based colored fine particles C and,
(6) Test line C linearly immobilized antibody C2 that specifically binds to the N protein of SARS-CoV-2, and control line 2 linearly immobilized antibody D2 that specifically binds to the antibody C2 a membrane 2 comprising
(7) an absorbent pad 2;
An immunochromatographic test strip characterized by comprising:
2. 1. The absorbent pad 1, the membrane 1, the conjugation pad 1, the sample pad, the conjugation pad 2, the membrane 2, and the absorbent pad 2 are arranged in sequence. Immunochromatographic test piece according to.
3. 1. The test line A and the test line B have different coloring, and the coloring of the test line A or the test line B and the coloring of the test line C are the same. or 2. Immunochromatographic test piece according to.
4. 1, wherein the color development of the test line A/test line B/test line C is red/blue/red, red/blue/blue, blue/red/red, or blue/red/blue, respectively; . ~3. Immunochromatographic test strip according to any one of.
5. 1. The test line A and the test line B have the same coloring, and the coloring of the test line C is different. or 2. Immunochromatographic test piece according to.
6. 1. The color development of the test line A/test line B/test line C is red/red/blue or blue/blue/red, respectively. 2. or 5. Immunochromatographic test strip according to any one of.
7. 1. ~6. 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.

Since the immunochromatographic test strip of the present invention carries specific antibodies and detection particles in a specific arrangement, Flu-A N protein, Flu-B N protein, and SARS-CoV can be detected simultaneously and with high sensitivity. -2 N proteins can be detected.

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.

The immunochromatographic test strip of the present invention includes influenza virus A (Flu-A) nucleocapsid protein (N protein), influenza virus B (Flu-B) N protein, and severe acute respiratory syndrome (Severe Acute Respiratory Syndrome) in the measurement sample. ) is a test strip for detecting the N protein of coronavirus 2 (SARS-CoV-2).

In the present invention, Flu-A is H1N1, H1N2, H2N2, H2N3, H3N2, H3N8, H4N6, H5N1, H5N2, H6N2, H7N1, H7N7, H7N9, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8 , and H16N3 subtypes.

In the present invention, the Flu-B type is classified into two groups that are antigenically and genetically different, namely, the Yamagata lineage and the Victoria lineage. Target stocks.

In the present invention, SARS-CoV-2 N protein refers to that 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).

In the present invention, the measurement sample 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.

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. Examples of non-biological samples include, but are not limited to, samples collected from environmental surfaces such as faucets, handles, handrails, straps, switches, walls, floors, desks, chairs, and toilet seats.

The configuration of the immunochromatographic test strip of the present invention is such that, with the addition portion (dropping portion) of the measurement sample solution of the immunochromatographic test strip as the upstream side, the sample pad having the addition portion, the conjugation pad, the membrane, and the absorbent pad are connected in this order. ing. 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, 21 is a conjugation pad 1, 22 is a conjugation pad 2, 31 is a membrane 1, 32 is a membrane 2, 41 is an absorbent pad 1, 42 is an absorbent pad 2, and 5 is a backing. 6 to 7 are test lines A to B; 8 is test line C; 91 is control line 1; 92 is control line 2;

In the examples of FIGS. 1 and 2, the immunochromatographic test piece has a long strip shape with a width of 3-5 mm (preferably about 4 mm) and a length of 80-160 mm (preferably about 100 mm). In the conjugation pad 1 of the immunochromatographic test strip, a complex of antibody A1 and cellulose-based colored fine particles A that specifically binds to Flu-A N protein in the measurement sample, and Flu-B N protein and specific A complex of the antibody B1 and the cellulose-based colored fine particle B that specifically binds to each other, and a complex of the antibody C1 that specifically binds to the N protein of SARS-CoV-2 and the cellulose-based colored fine particle C on the conjugation pad 2. are carried respectively. In addition, a test line A in which an antibody A2 that specifically binds to the N protein of Flu-A is linearly fixed at a position about 8 mm from the upstream end of the membrane 1 of the immunochromatographic test strip, about 10 mm from the end A test line B in which an antibody B2 that specifically binds to the N protein of Flu-B is linearly fixed at the position of , and an antibody D1 that specifically binds to the antibody A2 or B2 at a position about 14 mm from the end. A linearly fixed control line 1 is formed. In addition, a test line C in which an antibody C2 that specifically binds to the N protein of SARS-CoV-2 is linearly fixed at a position about 8 mm from the upstream end of the membrane 2 of the immunochromatographic test strip, from the end A control line 2 is formed at a position of about 14 mm, in which the antibody D2 that specifically binds to the antibody C2 is linearly immobilized.

The sample pad used in the present invention is not particularly limited as long as it is made of a material that can rapidly absorb the measurement sample and then spread to downstream conjugation pads, membranes, and absorbent pads. For example, cellulose filter paper or nonwoven 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 mentioned. Among these, cellulose filter paper is preferable. Moreover, the thickness of the sample pad is preferably 0.1 to 2.0 mm, more preferably 0.2 to 1.0 mm. If the thickness is too small, the flow of the sample to be measured downstream may become non-uniform, resulting in a decrease in measurement accuracy. On the other hand, if the thickness is large, the downstream deployment may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream development is increased.

Conjugation pads 1 and 2 used in the present invention are complexes of antibody A1 and cellulose-based colored fine particles A that specifically bind to Flu-A N protein in the measurement sample, and are specific to Flu-B N protein. A complex of the antibody B1 and the cellulose-based colored fine particle B that binds to and a complex of the antibody C1 that specifically binds to the N protein of SARS-CoV-2 and the cellulose-based colored fine particle C can be kept in a dry state, The material is not particularly limited as long as it can rapidly release the composite together with the development of the measurement sample downstream. For example, cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper, etc. Filter paper or non-woven fabric, filter paper or non-woven fabric made of polyethylene can be mentioned. Among these, glass filter paper is preferable. The thickness of the conjugation pads 1 and 2 is preferably 0.1 mm to 2.0 mm, more preferably 0.2 mm to 1.0 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 long. In addition, the amount of measurement sample required for downstream development is increased.

The membranes 1 and 2 used in the present invention are not particularly limited as long as they can spread the measurement sample uniformly with high accuracy. For example, cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride. , polyvinylidene fluoride, or nylon membranes. Among these, nitrocellulose membranes are preferred.

The absorbent pads 1 and 2 used in the present invention are not particularly limited as long as they can quickly absorb the measurement sample developed from upstream and then hold it so that it does not flow back. For example, cellulose filter paper or nonwoven fabric, glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, and polyethylene filter paper or non-woven fabric. Among these, cellulose filter paper is preferable. The thickness of the absorbent pads 1 and 2 is preferably 0.2 mm to 5.0 mm, more preferably 0.5 mm to 2.0 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, when 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 A1 to C1, antibodies A2 to C2, and antibodies D1 to D2 used in the present invention may be monoclonal antibodies or polyclonal antibodies, but monoclonal antibodies are preferred. Antibodies can be of any isotype, eg, IgG, IgA, IgD, IgE, IgM, etc., but IgG is preferred. In addition, the antibody may be a commercially available product, or may be separately produced by a known method.

Since the cellulose-based colored fine particles A to C have a large amount of hydroxyl groups, they can not only hold many reactive dyes by covalent bonds, but also maintain stable dispersibility in water etc. even after deep dyeing. be able to. As the cellulose-based colored fine particles, regenerated cellulose, purified cellulose, natural cellulose, etc. can be used, and partially derivatized cellulose may also be used. 20 to 90 mass % of the mass of the cellulose-based colored fine particles is preferably derived from cellulose, more preferably 20 to 80 mass %, even more preferably 20 to 70 mass %.

The average particle size of the cellulose-based colored fine particles A to C is not particularly limited, but is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle size is large, the development to the downstream becomes slow and the measurement time becomes long. In addition, they are likely to be trapped on the membrane, and the background itself develops color, which makes the color development on the test line and control line unclear. On the other hand, when the average particle size is small, the amount of antibody that can be physically adsorbed or chemically bonded is reduced, resulting in decreased measurement sensitivity.

Examples of the cellulose-based colored fine particles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation. The color of the cellulose-based colored fine particles is not particularly limited, but examples thereof include red, orange, yellow, green, blue, dark blue, purple, black, and fluorescence. Specifically, NanoAct® Red (RE2) has an OD of 240 at 1% by weight particle concentration, NanoAct® Blue (BL2) has an OD of 265 at 1% by weight of particle concentration, and NanoAct® Green Red and blue are preferred because (GR1) has an OD of 160 at a particle concentration of 1% by weight and NanoAct® Black (KR1) has an OD of 150 at a particle concentration of 1% by weight.

The binding amount of the antibodies A1 to C1 to the cellulose-based colored fine particles A to C can be controlled by adjusting the charged mass ratio of the cellulose-based colored fine particles and the antibody, and is not particularly limited. The charged mass ratio is preferably 1:0.01 to 1:1, more preferably 1:0.02 to 1:0.5, and even more preferably 1:0.02 to 1:0.2. If the mass ratio is out of the above range, the amount of antibody binding to the cellulose-based colored fine particles becomes insufficient, or the amount of antibody binding to the cellulose-based colored fine particles increases excessively, resulting in an increase in the number of antibodies that do not contribute to the antigen-antibody reaction. , the measurement sensitivity may decrease.

The method of binding the antibodies A1 to C1 and the cellulose-based colored microparticles A to C is not particularly limited, but sensitization is preferably performed by physical adsorption by hydrophobic bonding or chemical bonding by covalent bonding, and the operation is simple and cost effective. Physical adsorption, which is also cheaper, is more 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.

On the conjugation pad 1, a complex of the antibody A1 that specifically binds to the Flu-A N protein in the measurement sample and the cellulose-based colored microparticles A, and an antibody that specifically binds to the Flu-B N protein. A method for supporting a complex of B1 and cellulose-based colored fine particles B, and an antibody C1 that specifically binds to the N protein of SARS-CoV-2 in a measurement sample on the conjugation pad 2 and cellulose-based colored fine particles C. Although the method for supporting the composite is not particularly limited, for example, the solution of the composite is uniformly applied, sprayed or impregnated on the conjugation pad, and then dried at an appropriate temperature in a constant temperature bath for a certain period of time. be able to. 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. At low concentrations, Flu-A, Flu-B, and SARS-CoV-2 N proteins may not be sufficiently captured and detected, resulting in reduced measurement sensitivity. 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.

The method for linearly immobilizing the capturing antibodies A2 to C2 forming the test lines A to C and the capturing antibodies D1 and D2 forming the control lines 1 and 2 on the membranes 1 and 2 is not particularly limited. Capture antibodies A2 to C2 that form test lines A to C and capture antibodies D1 and D2 that form the control lines 1 and 2 are respectively applied to different positions on the lines, and then placed in a constant temperature bath at an appropriate temperature. It can be produced by drying for a certain period of time. 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. At low concentrations, the Flu-A, Flu-B, and SARS-CoV-2 N proteins cannot be sufficiently captured and detected, resulting in decreased measurement sensitivity. 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.

In the immunochromatographic test piece of the present invention, the membranes 1 and 2 prepared above are attached to two locations on the left and right sides of the adhesive sheet 10, and then the conjugation pads 1 and 2 are partially overlapped on the upstream ends of the membranes 1 and 2. Then, the sample pad was attached to the central portion of the adhesive sheet 10, and then the absorbent pads 1 and 2 were partially overlapped on the downstream ends of the membranes 1 and 2, and then a strip of constant width was attached. It can be produced by cutting into a shape. Test lines A to C and control lines 1 and 2 may be prepared after preparing the test piece, or may be prepared before preparing the test piece.

The immunochromatographic test strip has at least a first opening for dropping a measurement sample on the sample pad, a second opening for visually checking the test lines A and B and the control line 1 on the membrane 1, It may be housed in a suitable plastic housing case with a third opening for visual confirmation of the test line C and the control line 2 above the membrane 2 .

In the immunochromatographic test piece of the present invention, from the viewpoint of visibility, in test lines A to C, the test line A and test line B have different colors, and the color of test line A or test line B and the color of test line C are preferably the same, and the colors of the test lines A to C are preferably red/blue/red, red/blue/blue, blue/red/red, or blue/red/blue, respectively. . Since the colors of the adjacent test lines A and B are different, it is possible to determine at a glance which of Flu-A and Flu-B was positive from the color and position of the line when the line appeared. . In addition, SARS-CoV-2 has a line at a distant position, so there is no problem in visibility even if the same coloring as Flu-A or Flu-B is used. In particular, in NanoAct (registered trademark), as described above, red and blue are particularly highly visible, so a combination of red and blue is suitable. With the color development, if a line appears, it is possible to reduce the risk of misidentifying Flu-A or Flu-B as SARS-CoV-2. The control line is a mechanism that captures the detection particles that were not captured by the test line and develops color.

Further, in the immunochromatographic test piece of the present invention, in the test lines A to C, the color development of the test line A and the test line B is the same, and the color development of the test line C is preferably different, More preferably, the test lines A to C are colored red/red/blue or blue/blue/red, respectively. Since the color development has the same color of Flu-A and Flu-B and is different from the color of SARS-CoV-2, if a line appears, from the position of the line Flu-A and Flu-B, the line From the color of , it can be judged at a glance whether Flu-A or Flu-B and SARS-CoV-2 were positive. In particular, in NanoAct (registered trademark), as described above, red and blue are particularly highly visible, so a combination of red and blue is suitable. With the color development, if a line appears, it is possible to reduce the risk of misidentifying Flu-A or Flu-B as SARS-CoV-2.

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. preferably contains a filter of

The measurement sample diluent preferably contains a nonionic surfactant that improves the spreadability of the measurement sample and does not affect immune reactions. The nonionic surfactant is not particularly limited, but 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 surfactants may be used alone, or two or more of them may be used in combination. The concentration of the nonionic surfactant is preferably 0.01 to 5.0% by mass, more preferably 0.05 to 4.0% by mass, even more preferably 0.1 to 3.0% by mass. If the concentration is lower than 0.01% by mass, downstream development may become difficult. In addition, the deployment may become uneven and the measurement accuracy may be lowered. On the other hand, if the concentration is higher than 5.0% by mass, the physically adsorbed detection particles and the antibody, and/or the membrane and the antibody may deviate, resulting in failure to obtain a measurement value.

An inorganic salt or a buffer 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.

Hereinafter, the present invention will be described in detail based on examples. The invention is not limited to these examples.

(Example 1)
(1) Preparation of complex of antibody A1 and cellulose-based colored fine particles A
1.0% by mass of blue cellulose-based colored fine particles A (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei) 100 μL, 10 mM Tris buffer (204-07885, Fujifilm Wako Pure) Pharmaceutical company) (pH 8.0) 900 μL, 1.0 mg / mL mouse-derived anti-Flu-A N protein monoclonal antibody 1 (Influenza A nucleoprotein, antibody, Cat: 3IN5, MAbs: InA245, HyTest Co., Ltd.) as antibody A1 ) was added to a 15 mL centrifuge tube and vortexed. Then, it was allowed to stand at 37°C for 120 minutes. Next, a blocking solution (pH 8.0) consisting of 1.0% by mass of casein (030-01505, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ) was added, and the mixture was allowed to stand at 37° C. for 60 minutes. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), centrifugation was performed at 13,000×g at 25° C. 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). bottom. 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 with an ultrasonic disperser (UH-50, manufactured by SMTE) for 10 seconds. A composite 1 with colored fine particles A was obtained.

(2) Preparation of a complex of antibody B1 and cellulose-based colored fine particles B Mouse-derived anti-Flu-B N protein monoclonal antibody 1 (Influenza B virus Monoclonal Antibody, Cat: 1131, ViroStat) as antibody B1 instead of antibody A1 ) instead of red cellulose-based colored fine particles A, cellulose-based colored fine particles B (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Co., Ltd.) (1) A complex 1 of antibody B1 and cellulose-based colored fine particles B was obtained in the same manner as the preparation of the complex of antibody A1 and cellulose-based colored fine particles A.

(3) Preparation of complex of antibody C1 and cellulose-based colored fine particle C Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti-SARS-CoV-2-NP Monoclonal) as antibody C1 instead of antibody A1 Antibody, SCV-108, manufactured by Toyobo Co., Ltd.), in the same manner as in (1) Preparation of complex of antibody A1 and cellulose-based colored fine particles A, antibody C1 and blue cellulose-based colored fine particles C (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation).

(4) Production of Flu-A, Flu-B N protein detection membranes, and SARS-CoV-2 N protein detection membranes 2.0 mg/mL mouse-derived anti-Flu-A N protein monoclonal as antibody A2 Antibody 2 (Influenza A Virus (NP) Antibody, Cat: BMRia017, Clone: FLA-859, Bio Matrix Research Inc.), 2.0 mg / mL mouse-derived anti-Flu-B N protein monoclonal antibody as antibody B2 2 (Influenza B nucleoprotein, antibody, Cat: 3IF18/RIF17, MAbs: R2/3, manufactured by HyTest), 2.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 2 (Anti -SARS-CoV-2-NP Monoclonal antibody, SCV-109, manufactured by Toyobo), 1.0 mg / mL rabbit-derived anti-mouse IgG polyclonal antibody as antibodies D1 and D2 (Mouse IgG-heavy and light chain antibody, A90- 117A, manufactured by BETHYL) was prepared. Next, using a dispensing platform (XYZ3060, manufactured by BIODOT) and a BioJet nozzle (BHQHR-XYZ, manufactured by BIODOT), a position 8 mm from the upstream side of a 25 mm × 300 mm immunochromatography membrane (IAB090, manufactured by Advantec) Then, the antibody A2 was applied at a coating amount of 1.0 μL/cm (test line A), and the antibody B2 was applied at a coating amount of 1.0 μL/cm at a position 10 mm from the upstream side of the membrane (test line A). Line B), after applying the antibody D1 at a position of 14 mm from the upstream side of the membrane at a coating amount of 1.0 μL / cm (control line 1), a dryer adjusted to 45 ° C. (WFO-510, Tokyo Rika (manufactured by Kikai Co., Ltd.) for 30 minutes to form test lines A and B and a control line 1 with a line width of about 1 mm to obtain a membrane 1 for N protein detection of Flu-A and Flu-B. Next, using a dispensing platform (XYZ3060, manufactured by BIODOT) and a BioJet nozzle (BHQHR-XYZ, manufactured by BIODOT), a 25 mm × 300 mm immunochromatography membrane (IAB090, manufactured by Advantec) different from the membrane 1 was applied. At a position 8 mm from the upstream side, the antibody C2 is applied at an application amount of 1.0 μL / cm (test line C), and at a position 14 mm from the upstream side of the membrane, the antibody D2 is applied at 1.0 μL / cm. (control line 2), dried for 30 minutes in a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45 ° C. to form a test line C and a control line 2 with a line width of about 1 mm. Thus, SARS-CoV-2 N protein detection membrane 2 was obtained.

(5) Preparation of Flu-A, Flu-B N protein detection conjugation pads, and SARS-CoV-2 N protein detection conjugation pads 10 mm × 300 mm conjugation pads (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, Millipore (manufactured by BIODOT), using a dispensing platform (XYZ3060, manufactured by BIODOT) and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), 15 μL / cm, and the composite 1 of the antibody B1 and the cellulose-based colored fine particles B was uniformly applied at a coating amount of 15 μL/cm, and then dried in a dryer (WFO-510, Tokyo) adjusted to 45 ° C. Rikakigi Co., Ltd.) and dried for 30 minutes to obtain a conjugation pad 1 for N protein detection of Flu-A and Flu-B. Next, a dispensing platform (XYZ3060, manufactured by BIODOT) and an Air Jet nozzle (AJQHR- XYZ, manufactured by BIODOT), the composite 1 of the antibody C1 and the cellulose-based colored fine particles C was uniformly applied at a coating amount of 15 μL / cm, and then a dryer adjusted to 45 ° C. (WFO-510, Tokyo Rika (manufactured by Kikai Co., Ltd.) for 30 minutes to obtain a conjugation pad 2 for detecting the N protein of SARS-CoV-2.

(6) Flu-A, Flu-B and SARS-CoV-2 preparation of immunochromatographic test strips for detecting N protein The Flu-A and Flu-B N protein detection membranes 1 and the SARS-CoV-2 N protein detection membrane 2 were laminated at positions 10 mm to 35 mm from the other end of the backing sheet. Then, on the upstream side of the Flu-A and Flu-B N protein detection membrane 1, the Flu-A and Flu-B N protein detection conjugation pad 1 is placed so that it overlaps the membrane by 5 mm. A conjugation pad 2 for detecting the N protein of SARS-CoV-2 was attached to the upstream side of the membrane 2 for detecting the N protein of 2 so as to overlap the membrane by 5 mm. Next, a 25 mm×300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the center of the backing sheet. Next, on the downstream side of the N protein detection membrane 1 of Flu-A and Flu-B, an absorbent pad 1 of 20 mm × 300 mm (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) so as to overlap the membrane by 5 mm. - Absorbent pads 2 (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) of 20 mm × 300 mm were attached to the downstream side of the membrane 2 for detecting N protein of CoV-2 so as to overlap the membrane by 5 mm. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), by cutting into strips with a width of 4 mm and a length of 90 mm, Flu-A, Flu-B, immunochromatography for N protein detection of SARS-CoV-2 A test piece 1 was obtained.

(7) 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 Fujifilm Wako Pure Chemical Industries, Ltd.) (9 g) was added to a 500 mL glass bottle and dissolved to prepare a diluted measurement sample solution.

(8) Preparation of Flu-A sample, Flu-B sample and SARS-CoV2 sample Purified Influenza A protein (H1N1 strain) (30-AI50, manufactured by Fitzgerald), which is the N protein of commercially available Flu-A, was measured as described above. Flu-A samples were obtained by diluting to 10 μg/mL with sample diluent. Purified Influenza B protein (30-AI75, manufactured by Fitzgerald), which is the N protein of Flu-B on the market, was diluted to 10 μg/mL with the measurement sample diluent to obtain a Flu-B sample. Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, manufactured by RayBiotech), which is a commercially available SARS-CoV-2 N protein, is diluted to 10 ng/mL with the measurement sample diluent to obtain SARS-CoV- Two samples were obtained.

(9) Flu-A, Flu-B, evaluation of immunochromatographic test strip for N protein detection of SARS-CoV-2: Evaluation of non-specific adsorption Flu-A, Flu-B, N protein detection of SARS-CoV-2 An immunochromatographic test piece for 2D was placed on a horizontal table. Next, 150 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 15 minutes. Then, the reflection absorbance (mAbs) of the test lines A to C on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line (when measuring the red line) or Latex, Line (when measuring the blue or green line), (manufactured by Hamamatsu Photonics K.K.). Table 1 shows the results obtained. It should be noted that non-specific adsorption can be judged to be good if the reflection absorbance is 10 mAbs or less.

(10) Flu-A, Flu-B, evaluation of immunochromatographic test strip for N protein detection of SARS-CoV-2: evaluation of sensitivity Flu-A, Flu-B, immunochromatography for N protein detection of SARS-CoV-2 The specimen was placed on a horizontal table. Then, 150 μL of the Flu-A sample, Flu-B sample, or SARS-CoV-2 sample was dispensed with a micropipette, gently dropped onto the sample pad, and left at 25° C. for 15 minutes. Then, the reflected absorbance (mAbs) of the control line and the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line (when measuring the red line) or Latex, Line (when measuring the blue or green line), (manufactured by Hamamatsu Photonics K.K.). Table 1 shows the results obtained. The sensitivity was judged to be poor when the reflection absorbance was 50 mAbs or less, and good when it exceeded 50 mAbs.

(11) Flu-A, Flu-B, Evaluation of immunochromatographic test strip for detecting N protein of SARS-CoV-2: Evaluation of visibility Flu-A, Flu-B, for detecting N protein of SARS-CoV-2 An immunochromatographic test piece was placed on a horizontal table. Then, 150 μL of the Flu-A sample (test line A and control line develop color), Flu-B sample 150 μL (test line B and control line develop color), SARS-CoV-2 sample 150 μL (test line C and control line develop Coloring), or 150 μL of a mixture of 50 μL of Flu-A sample, 50 μL of Flu-B sample, and 50 μL of SARS-CoV-2 sample (coloring of test lines A to C and control line) is dispensed with a micropipette and sample It was gently dropped onto the pad and allowed to stand at 25°C for 15 minutes. Next, the control line and the test line on the membrane were visually observed by 10 people, and 10 out of 10 people judged that the item was easy to judge ◎, 8 to 9 out of 10 people judged that the item was easy ◯ when 6 to 7 out of 10 panelists determined that the item was easy to determine; Here, item determination was made by visually observing the control line and the test line on the membrane, and evaluating whether or not it was possible to immediately determine to which item each of the four lines that appeared were lines. Table 1 shows the results obtained.

(Example 2)
Red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Corporation) are added to cellulose-based colored fine particles A, and blue particles (NanoAct (registered trademark), BL2: Dark Navy are added to cellulose-based colored fine particles B. , average particle size 365 nm, manufactured by Asahi Kasei), and red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei) for the cellulose-based colored fine particles C). Immunochromatographic test strip 2 for N protein detection of Flu-A, Flu-B, SARS-CoV-2 was prepared in the same manner as in 1, (9) nonspecific adsorption, (10) sensitivity, (11) visibility evaluated. Table 1 shows the results obtained.

(Example 3)
Red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Corporation) are added to cellulose-based colored fine particles A, and blue particles (NanoAct (registered trademark), BL2: Dark Navy are added to cellulose-based colored fine particles B. , average particle size 365 nm, manufactured by Asahi Kasei) in the same manner as in Example 1 except that each is used. Immunochromatographic test strip 3 for N protein detection of Flu-A, Flu-B, and SARS-CoV-2 was prepared. (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 4)
Flu-A and Flu- were prepared in the same manner as in Example 1, except that red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Corporation) were used as the cellulose-based colored fine particles C. B, immunochromatographic test strip 4 for N protein detection of SARS-CoV-2 was prepared, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 5)
Flu-A and Flu- were obtained in the same manner as in Example 1, except that red particles (NanoAct (registered trademark), RE2: Dark Red, average particle diameter 340 nm, manufactured by Asahi Kasei Corporation) were used as the colored cellulose-based fine particles A. B, immunochromatographic test strip 5 for N protein detection of SARS-CoV-2 was prepared, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 6)
Blue particles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) are added to cellulose-based colored fine particles B, and red particles (NanoAct (registered trademark), RE2: Dark Red are added to cellulose-based colored fine particles C. , average particle size 340 nm, manufactured by Asahi Kasei) in the same manner as in Example 1 except that each is used. Immunochromatographic test strip 6 for N protein detection of Flu-A, Flu-B, and SARS-CoV-2 was prepared. (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 7)
Green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei Corporation) are added to the cellulose-based colored fine particles A, and blue particles (NanoAct (registered trademark), BL2: Dark Navy, are added to the cellulose-based colored fine particles B. Average particle size 365 nm, manufactured by Asahi Kasei), and green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei) for the cellulose-based colored fine particles C). Flu-A, Flu-B, immunochromatographic test strip 7 for N protein detection of SARS-CoV-2 was prepared in the same manner, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. bottom. Table 1 shows the results obtained.

(Example 8)
Flu-A and Flu-B are prepared in the same manner as in Example 1, except that green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei Corporation) are used as the cellulose-based colored fine particles B. , SARS-CoV-2 N protein detection immunochromatographic test strip 8 was prepared, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 9)
Green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei Corporation) are added to the cellulose-based colored fine particles A, and green particles (NanoAct (registered trademark), GR1: Green, average Flu-A, Flu-B, and SARS-CoV-2 in the same manner as in Example 1 except that the particle size is 335 nm, manufactured by Asahi Kasei). (9) non-specific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 10)
Blue particles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) are added to the colored cellulose-based fine particles B, and green particles (NanoAct (registered trademark), GR1: Green, Flu-A, Flu-B, and SARS-CoV-2 in the same manner as in Example 1 except that each of them uses an average particle size of 335 nm, manufactured by Asahi Kasei Co., Ltd. An immunochromatographic test strip 10 for N protein detection was prepared. , (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 11)
Red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Corporation) are added to cellulose-based colored fine particles A, and blue particles (NanoAct (registered trademark), BL2: Dark Navy are added to cellulose-based colored fine particles B. , average particle size 365 nm, manufactured by Asahi Kasei), and green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei) in cellulose-based colored fine particles C. Example 1 Immunochromatographic test strip 11 for N protein detection of Flu-A, Flu-B, and SARS-CoV-2 was prepared in the same manner as, (9) nonspecific adsorption, (10) sensitivity, and (11) visibility. evaluated. Table 1 shows the results obtained.

(Example 12)
Green particles (NanoAct (registered trademark), GR1: Green, average particle size 335 nm, manufactured by Asahi Kasei Corporation) are added to the cellulose-based colored fine particles A, and blue particles (NanoAct (registered trademark), BL2: Dark Navy, are added to the cellulose-based colored fine particles B. Example 1 except that red particles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340 nm, Asahi Kasei Co., Ltd.) are used for the cellulose-based colored fine particles C, respectively. Immunochromatographic test strip 12 for N protein detection of Flu-A, Flu-B, and SARS-CoV-2 is prepared in the same manner as, (9) nonspecific adsorption, (10) sensitivity, and (11) visibility. evaluated. Table 1 shows the results obtained.

(Example 13)
1.0 mg / mL mouse-derived anti-Flu-A N protein monoclonal antibody 1 as antibody A1 (Influenza A nucleoprotein, antibody, Cat: 3IN5, MAbs: InA245, HyTest) instead of 1.0 mg / mL Mouse-derived anti-Flu-A N protein monoclonal antibody 3 (Monoclonal Antibody to Influenza A (Nucleoprotein), C01732M, manufactured by Meridian) was used, and 2.0 mg/mL mouse-derived anti-Flu-A N protein was used as antibody A2. Instead of monoclonal antibody 2 (Influenza A Virus (NP) Antibody, Cat: BMRia017, Clone: FLA-859, Bio Matrix Research Inc.), 2.0 mg / mL mouse-derived anti-Flu-A N protein monoclonal Flu-A, Flu-B, N protein detection of SARS-CoV-2 in the same manner as in Example 1 except for using antibody 4 (Monoclonal Antibody to Influenza A (Nucleoprotein), C01731M, manufactured by Meridian) Immunochromatographic test strip 13 was prepared, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 1 shows the results obtained.

(Example 14)
Mouse-derived anti-Flu-B N protein monoclonal antibody 1 as antibody B1 (Influenza B virus Monoclonal Antibody, Cat: 1131, manufactured by ViroStat) instead of 1.0 mg / mL mouse-derived anti-Flu-B N protein monoclonal Antibody 3 (Monoclonal Antibody to Influenza B (Nucleoprotein), C01744M, manufactured by Meridian) was used, and 2.0 mg/mL mouse-derived anti-Flu-B N protein monoclonal antibody 2 (Influenza B nucleoprotein, antibody, Cat: 3IF18/RIF17, MAbs: R2/3, manufactured by HyTest), 2.0 mg / mL mouse-derived anti-Flu-B N protein monoclonal antibody 4 (Monoclonal Antibody, Influenza B, MAB7042P, manufactured by BBI ) to prepare an immunochromatographic test strip 14 for N protein detection of Flu-A, Flu-B, and SARS-CoV-2 in the same manner as in Example 1, (9) non-specific adsorption, (10 ) sensitivity and (11) visibility were evaluated. Table 1 shows the results obtained.

(Comparative example 1)
(12) Preparation of complex of antibody A1 and colloidal gold
OD520 = 1.0 colloidal gold particles (EMGC40: Red, average particle size 40 nm, manufactured by BBI Solutions) 13.5 mL, 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ( pH 7.0) 1.5 mL, and 0.05 mg / mL mouse-derived anti-Flu-A N protein monoclonal antibody 1 (Influenza A nucleoprotein, antibody, Cat: 3IN5, MAbs: InA245, manufactured by HyTest) 1 as antibody A1 0.5 mL was added to a 50 mL centrifuge tube, vortexed gently, and allowed to stand at 25° C. for 10 minutes. Next, 1 mL of a blocking solution consisting of 10% by mass of Bovine serum albumin:BSA (A7906, manufactured by Sigma-Aldrich) was added and gently stirred. Next, using a centrifuge (MX-307, manufactured by Tomy Seiko) and a rack-in rotor (TMA-300, manufactured by Tomy Seiko), centrifugation was performed at 8,000 × g for 15 minutes at 25 ° C. to perform antibody sensitization. After allowing the colloidal gold particles to settle, the supernatant was removed. Next, 1.0% by mass of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich), 150 mM sodium chloride (192-13925, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 20 mM Tris buffer (204- 07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.2) was added, and treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMTE). Then, using a centrifuge (MX-307, Tomy Seiko Co., Ltd.), centrifugation was performed at 8,000×g for 15 minutes at 25° C. to settle the antibody-sensitized colloidal gold particles, and then the supernatant was removed. Then, 2.5% by mass of sucrose (196-00015, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.25% by mass of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich), 37.5 mM chloride A coating solution (pH 8.2) containing sodium (192-13925, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 20 mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, followed by ultrasonic dispersion. A complex X of antibody A1 and colloidal gold was obtained by treating for 10 seconds with a machine (UH-50, manufactured by SMTE) and adjusting the amount of coating liquid so that OD520 was 4.0.

(13) Preparation of complex of antibody B1 and colloidal gold Instead of antibody A1, mouse-derived anti-Flu-B N protein monoclonal antibody 1 (Influenza B virus Monoclonal Antibody, Cat: 1131, manufactured by ViroStat) was used as antibody B1. Complex Y of antibody B1 and colloidal gold was obtained in the same manner as in (12) Preparation of complex of antibody A1 and colloidal gold, except that it was used.

(14) Preparation of complex of antibody C1 and colloidal gold Mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV -108, manufactured by Toyobo), a complex Z of antibody C1 and colloidal gold was obtained in the same manner as in (12) Preparation of complex of antibody A1 and colloidal gold.

(15) Preparation of Flu-A and Flu-B N protein detection membranes and SARS-CoV-2 N protein detection membranes (4) Flu-A and Flu-B N protein detection membranes, and Flu-A, Flu-B N protein detection membrane 1, and SARS-CoV-2 N protein detection membrane 2 were obtained in the same manner as the preparation of the SARS-CoV-2 N protein detection membrane. .

(16) Flu-A, Flu-B N protein detection conjugation pad, and SARS-CoV-2 N protein detection conjugation pad Substitute for complex 1 of antibody A1 and cellulose-based colored fine particle A complex X of antibody A1 and colloidal gold, complex Y of antibody B1 and colloidal gold instead of complex 1 of antibody B1 and cellulose-based colored fine particle B, antibody C1 and cellulose-based colored fine particle C, and (5) Flu-A, Flu-B N protein detection conjugation pad, and SARS-CoV-2 Conjugation pad X for detecting N protein of Flu-A and Flu-B, and conjugation pad Y for detecting N protein of SARS-CoV-2 were obtained in the same manner as the conjugation pad for N protein detection. .

(17) Preparation of Flu-A, Flu-B, N protein detection immunochromatographic test strip and device for SARS-CoV-2 Flu-A instead of conjugation pad 1 for N protein detection of Flu-A, Flu-B , Flu-B N protein detection conjugation pad X, SARS-CoV-2 N protein detection conjugation pad Y instead of SARS-CoV-2 N protein detection conjugation pad 1, respectively. Except for (6) Flu-A, Flu-B, SARS-CoV-2 in the same manner as the preparation of immunochromatographic test strips for N protein detection, Flu-A, Flu-B, N of SARS-CoV-2 An immunochromatographic test strip X for protein detection was prepared, and (9) nonspecific adsorption, (10) sensitivity, and (11) visibility were evaluated. Table 2 shows the results obtained.

In the test pieces of Examples 1 to 14, the test lines A to C and the control line were darkly colored and had good visibility. On the other hand, in the device of Comparative Example 1, the test lines A to C and the control line are thin, and the four lines are all the same color.

INDUSTRIAL APPLICABILITY The present invention can provide an immunochromatographic test strip that can detect Flu-A N protein, Flu-B N protein, and SARS-CoV-2 N protein simultaneously and with high sensitivity.

1: sample pad 21: conjugation pad 1
22: Conjugation pad 2
31: Membrane 1
32: Membrane 2
41: Absorbent pad 1
42: Absorbent pad 2
5: Backing sheet 6: Test line A or B
7: Test line B or A
8: Test line C
91: Control line 1
92: Control line 2
10: Adhesive sheet

Claims (7)

(1) an absorbent pad 1;
(2) Test line A in which antibody A2 that specifically binds to the nucleocapsid protein (N protein) of influenza virus A (Flu-A) in the measurement sample is linearly immobilized, N of influenza virus B (Flu-B) A membrane 1 having a test line B linearly immobilized with an antibody B2 that specifically binds to a protein and a control line 1 linearly immobilized with an antibody D1 that specifically binds to the antibody A2 or B2;
(3) A complex of antibody A1 that specifically binds to Flu-A N protein and cellulose-based colored fine particle A, and antibody B1 that specifically binds to Flu-B N protein and cellulose-based colored fine particle B A conjugation pad 1 carrying a complex of
(4) a sample pad;
(5) Conjugation pad 2 carrying a complex of antibody C1 that specifically binds to the N protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample and cellulose-based colored fine particles C and,
(6) Test line C linearly immobilized antibody C2 that specifically binds to the N protein of SARS-CoV-2, and control line 2 linearly immobilized antibody D2 that specifically binds to the antibody C2 a membrane 2 comprising
(7) an absorbent pad 2;
An immunochromatographic test strip characterized by comprising: 2. The method according to claim 1, wherein said absorbent pad 1, said membrane 1, said conjugation pad 1, said sample pad, said conjugation pad 2, said membrane 2, and said absorbent pad 2 are successively arranged in a continuous manner. Immunochromatographic test strip as described. The immunochromatographic test strip according to claim 1 or 2, wherein the test line A and the test line B have different colors, and the test line A or the test line B has the same color as the test line C. . The color development of the test line A/test line B/test line C is red/blue/red, red/blue/blue, blue/red/red, or blue/red/blue, respectively. Item 4. The immunochromatographic test strip according to any one of items 1 to 3. 3. The immunochromatographic test strip according to claim 1, wherein the test line A and the test line B have the same color development and are different from the test line C color development. The immunochromatographic test according to any one of claims 1, 2 or 5, wherein the test line A/test line B/test line C are colored red/red/blue or blue/blue/red, respectively. piece. An immunochromatographic kit comprising the immunochromatographic test piece according to any one of claims 1 to 6, a measurement sample collecting tool, a filter, and a measurement sample diluent.

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