WO2022265105A1

WO2022265105A1 - Immunochromatographic test strip and immunochromatographic kit

Info

Publication number: WO2022265105A1
Application number: PCT/JP2022/024380
Authority: WO
Inventors: 美穂松尾; 淳岡本; 研吾西村
Original assignee: 東洋紡株式会社
Priority date: 2021-06-17
Filing date: 2022-06-17
Publication date: 2022-12-22
Also published as: JPWO2022265105A1

Abstract

[Problem] To provide an immunochromatographic test strip that can detect an N protein of SARS-CoV-2 at high sensitivity while suppressing false positives. [Solution] An immunochromatographic test strip according to the present invention comprises (1) a sample pad, (2) a conjugation pad which supports a composite of fine colored cellulose particles and an antibody composition A specifically binding to a nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample, (3) a membrane to which an antibody composition B specifically binding to the N protein of SARS-CoV-2 in the measurement sample and an antibody composition C specifically binding to the antibody composition A are linearly fixed at respective positions that differ from each other, and (4) an absorption pad, wherein the antibody composition A is a mixture of an antibody A1 and an antibody A2 that are from different origins, and the antibody composition B is a mixture of an antibody B1 and an antibody B2 that are from different origins.

Description

Immunochromatographic test strips and immunochromatographic kits

The present invention is an immunochromatography test piece that can detect the nucleocapsid protein (N protein) of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome) coronavirus 2 (SARS-CoV-2) with high sensitivity and suppressing false positives, and immunochromatography It relates to a kit containing test strips.

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).

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.

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 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.

Generally, immunoassay methods using mouse monoclonal antibodies are used to measure analytes (measurement targets) contained in samples such as blood and urine. However, in immunoassays based on the specific binding of antigen-antibody reactions, non-specific reactions other than the intended specific antigen-antibody reactions often impair the reliability of the measured values. It recognized.

This phenomenon is caused by the reaction of components other than the antigen contained in the specimen with the labeled antibody. Heterophilic antibodies, such as human anti-mouse antibodies (HAMA) contained in the specimen to be tested, allow binding to the solid phase, for example in a typical sandwich ELISA assay, even though the analyte is not present. Non-specific cross-linking between the labeled antibody and the labeled antibody to be detected occurs, resulting in a false positive signal. While automation of testing has progressed and rapid measurement has become possible, false reactions such as HAMA have increased, and these non-specific reactions are often overlooked. In this way, a non-specific reaction is observed in the immunoassay system, and by reacting with a sample that does not contain the target analyte, a sample that is originally negative may be determined to be positive (Non-Patent Document 2, See Non-Patent Document 3). This phenomenon also applies to immunochromatography.

One of the reasons why HAMA is present in human-derived specimens is that it is caused by the administration of large doses of mouse monoclonal antibodies to patients as a treatment. In vivo administration of a mouse antibody produces HAMA, which poses a problem of eliciting an immune response to a heterologous antigen. Recent antibody drugs include chimeric antibodies fusing a mouse-derived antigen-binding site and a human-derived constant region, and advances in humanized antibody production technology. is increasing and the problem cannot be completely ignored.

Heterophilic antibodies are known not only for mice, but also for animals such as goats, sheep, and rabbits. (Goat: HAGA, Sheep: HASA, Rabbit: HARA).

If the binding between the monoclonal antibody used for measurement and the heterophile antibody is not prevented, serious problems such as diagnostic errors may occur due to inaccurate measurement results. Even if a monoclonal antibody with excellent specificity is used in an actual measurement system, it is a serious problem that its performance cannot be fully utilized (see Patent Document 1).

Various attempts have been made in the past to obtain correct measurement values that avoid the above-mentioned non-specific reactions. For example, it is common practice to heat the sample to be measured or pretreat it with an appropriate reagent, or add various animal sera, immunoglobulin fractions, albumin, skimmed milk, surfactants, etc. to the measurement system. It's been broken In addition, antibody fragments such as Fab and F(ab')2 are used for specific reactions to avoid non-specific reactions caused by the Fc portion of antibodies (Patent Documents 2 and 3). reference). In addition, a monoclonal antibody that has a different reaction specificity from that of the monoclonal antibody used in the measurement system and that does not inhibit the reaction related to the measurement system is also added to the measurement system. For example, the use of aggregates derived from monoclonal or polyclonal antibodies has been suggested. This aggregate may be a homopolymer of the antibody, an antibody fragment, or a heteropolymer with a protein such as albumin or a polysaccharide macromolecule such as dextran.

In addition, in order to suppress non-specific reaction, the monoclonal antibody used for specific reaction was prepared by heat treatment etc. Although the specific activity of the original antibody is lost, the non-specific reaction suppressing activity is retained. A substance derived from a monoclonal antibody has been disclosed (see Patent Document 4). However, although these methods are effective in suppressing non-specific reactions to some extent, they are still insufficient for some specimens and may partially inhibit the target antigen-antibody reaction. It was not always satisfactory.

Japanese Patent No. 2109086 JP-A-54-119292 JP-A-04-221762 Japanese Patent No. 2561134

An object of the present invention is to provide an immunochromatographic test strip that can detect the SARS-CoV-2 N protein with high sensitivity and reduced false positives, and a kit containing the immunochromatographic test strip.

As a result of intensive research to solve the above problems, the present inventors have found that by using a mixture of two types of antibodies of different origins as a capture antibody and a detection antibody, SARS-1 can be detected with high sensitivity and reduced false positives. It was found that the N protein of CoV-2 could be detected. In addition, the inventors have found that the N protein of SARS-CoV-2 can be detected with higher sensitivity by using cellulose-based colored microparticles as detection particles, and have completed the present invention.

That is, the representative present invention is as follows.
1. (1) a sample pad;
(2) Carrying a complex of an antibody composition A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample and cellulose-based colored fine particles a conjugation pad with
(3) Line the antibody composition B that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the antibody composition C that specifically binds to the antibody composition A at different positions. a membrane fixed in a shape;
(4) an absorbent pad;
The antibody composition A is a mixture of antibody A1 and antibody A2 derived from different origins,
An immunochromatographic test strip, wherein the antibody composition B is a mixture of antibody B1 and antibody B2 derived from different origins.
2. 1. Any one of the antibody A1 and the antibody A2 is a mouse-derived antibody. Immunochromatographic test piece according to.
3. 1. The antibody A1 and the antibody A2 are supported on the conjugation pad at a mixing ratio (mass ratio) of 10:1 to 1:10. or 2. Immunochromatographic test piece according to.
4. 1. Any one of the antibody B1 and the antibody B2 is a mouse-derived antibody. to 3. Immunochromatographic test strip according to any one of.
5. 1. The antibody B1 and the antibody B2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. to 4. Immunochromatographic test strip according to any one of.
6. 1. The antibody composition C is a mixture of an antibody C1 that binds to the antibody A1 and an antibody C2 that binds to the antibody A2. to 3. Immunochromatographic test strip according to any one of.
7. 6. The antibody C1 and the antibody C2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. Immunochromatographic test piece according to.
8. 1. to 7. 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, it is possible to detect the SARS-CoV-2 N protein with high sensitivity and reduced false positives.

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.

In the present invention, the immunochromatographic test strip is a test strip for detecting the nucleocapsid protein (N protein) of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample. The measurement sample used in the present invention includes not only the sample as collected but also the sample subjected to pretreatment such as removal of contaminants.

Examples of the sample to be measured include, but are not limited to, blood, serum, plasma, bone marrow fluid, lymph, tears, nasal discharge, nasal wash, nasal swab, saliva, gargle, sputum, pharyngeal swab, sweat, tracheal aspirate, bronchi. Lavage, pleural fluid, ascites, amniotic fluid, intestinal lavage, urine, feces, cell extract, tissue extract, organ extract and the like.

In the present invention, the N protein of SARS-CoV-2 has the amino acid sequence disclosed in GenBank Accession No. (MN908947). The SARS-CoV N protein has the amino acid sequence disclosed in GenBank Accession No. (AY278741).

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, respectively.

　In the examples of Figures 1 and 2, the immunochromatographic test piece has a long and narrow rectangular shape with a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm). The conjugation pad 2 of the immunochromatography test strip carries a complex of antibody composition A and cellulose-based colored fine particles for specifically capturing the N protein of SARS-CoV-2 in the measurement sample. . In addition, the antibody composition B for specifically capturing the SARS-CoV-2 N protein in the measurement sample was linearly immobilized at a position about 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip. A test line 6 is formed. Further, a control line 7 is formed in which the antibody composition C that specifically binds to the antibody composition A is linearly fixed at a position about 20 mm from the end.

In the present invention, the sample pad 1 is not particularly limited as long as it is made of a material that can rapidly absorb the measurement sample and then spread to downstream conjugation pads, membranes, and absorbent pads. For example, cellulose filter paper or non-woven fabric. , glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, polyethylene filter paper or non-woven fabric. Among these, cellulose filter paper is preferable. Moreover, the thickness of the sample pad 1 is preferably 0.1 to 2 mm, more preferably 0.2 to 1 mm. If the thickness is too small, the flow of the sample to be measured downstream may become non-uniform, resulting in a decrease in measurement accuracy. On the other hand, if the thickness is large, the downstream deployment may be delayed and the measurement time may be lengthened. In addition, the amount of measurement sample required for downstream development is increased.

In the present invention, the conjugation pad 2 can hold in a dry state a complex of the antibody composition A that specifically binds to the SARS-CoV-2 N protein in the measurement sample and the cellulose-based colored fine particles, and the measurement The material is not particularly limited as long as it can rapidly release the complex as the sample is developed downstream, but for example, cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or Non-woven fabrics, polyethylene filter paper or non-woven fabrics can be mentioned. Among these, glass filter paper is preferable. Moreover, the thickness of the conjugation pad 2 is preferably 0.1 to 2 mm, more preferably 0.2 to 1 mm. If the thickness is too small, it may not be possible to retain the desired amount of the composite in a dry state. On the other hand, if the thickness is large, the downstream deployment may be delayed and the measurement time may be lengthened. In addition, the amount of measurement sample required for downstream development is increased.

In the present invention, the membrane 3 is not particularly limited as long as it can accurately and uniformly develop the measurement sample. Membranes made of vinylidene chloride or nylon can be mentioned. Among these, nitrocellulose membranes are preferred.

In the present invention, the absorbent pad 4 is not particularly limited as long as it is made of a material that can quickly absorb the measurement sample developed from upstream and then hold it so that it does not flow back. 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. Moreover, the thickness of the absorbent pad 4 is preferably 0.2 to 5 mm, more preferably 0.5 to 2 mm. If the thickness is small, the measurement sample once absorbed by the absorbent pad may flow back to the membrane side depending on the amount of the measurement sample dropped. On the other hand, if the thickness is large, the sizes of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also become large, which is not preferable from the point of view of POCT.

The antibodies used in the present invention may be monoclonal antibodies or polyclonal antibodies, but monoclonal antibodies are preferred. Antibodies can be of any isotype, eg, IgG, IgA, IgD, IgE, IgM, etc., but IgG is preferred. In addition, the antibody may be a commercially available product, or may be separately produced by a known method.

Antibody composition A used as a detection antibody in the present invention must be a mixture of antibody A1 and antibody A2 that specifically binds to the N protein of SARS-CoV-2 and has different origins. Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. Therefore, since they have different reactivities with respect to antigens, the degree of binding of antibodies to antigens increases compared to the case of antibodies alone, resulting in higher sensitivity. In particular, mouse-derived antibodies are presumed to have a structure with a higher degree of binding to antigens than other antibodies. Therefore, it is preferable that either the antibody A1 or the antibody A2 is a mouse-derived antibody. If the antibody composition A is not a mixture of antibodies A1 and A2 derived from different origins, the detection sensitivity may be reduced. Also, false positives may occur.

The mixing ratio (mass ratio) of antibody A1 and antibody A2 having different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease. Also, false positives may occur.

The reflection absorbance of the test line is preferably 40 mAbs or more, more preferably 60 mAbs or more, and even more preferably 80 mAbs or more, because the visibility at the time of completion of measurement is good and the line can be visually recognized at an early point from the start of measurement.

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

The average particle size of the cellulose-based colored fine particles is not particularly limited, but is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle size is large, downstream development may be delayed and the measurement time may be lengthened. In addition, it tends to be captured on the membrane, and the background itself develops color, which may obscure the color development on the test line and the control line. On the other hand, when the average particle size is small, the amount of antibody that can be physically adsorbed or chemically bonded decreases, and the measurement sensitivity may decrease.

The color of the cellulose-based colored fine particles is not particularly limited, but examples include red, blue, yellow, green, black, white, and fluorescent colors. Among these, red, blue, and black, which are highly visible, are preferable. Examples of such colored cellulose-based fine particles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation.

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

The method of binding the antibody composition A and the cellulose-based colored microparticles is not particularly limited, but sensitization is preferably performed by physical adsorption by hydrophobic bonding or chemical bonding by covalent bonding. Adsorption is more preferred. 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.

The method of causing the conjugation pad 2 to support the complex of the antibody composition A that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the cellulose-based colored fine particles is not particularly limited. It can be produced by uniformly applying, spraying or impregnating the body solution onto the conjugation pad and then drying it in a constant temperature bath at an appropriate temperature for a certain period of time. The amount of the composite solution to be applied is not particularly limited, but is preferably 5 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. The applied conjugation pad is then dried. The drying temperature is not particularly limited, but is preferably 20°C to 80°C, more preferably 20°C to 60°C. The drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.

In the present invention, antibody composition B used as a capture antibody forming test line 6 specifically binds to the N protein of SARS-CoV-2 and is a mixture of antibody B1 and antibody B2 of different origins. is necessary. Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. As a result, they have different reactivities to the antigen, and the degree of binding of the antibody to the antigen increases, resulting in higher sensitivity. In particular, it is presumed that the mouse-derived antibody has a structure with a higher degree of binding to an antigen than other antibodies due to the attachment of sugar chains. Therefore, it is preferable that either the antibody B1 or the antibody B2 is a mouse-derived antibody. If antibody composition B is not a mixture of antibody B1 and antibody B2 derived from different origins, the detection sensitivity may be reduced. Also, false positives may occur.

The mixing ratio (mass ratio) of antibody B1 and antibody B2 having different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease. Also, false positives may occur.

In the present invention, the antibody composition C used as the capture antibody forming the control line 7 must be an antibody that specifically binds to the antibody composition A. Moreover, the antibody composition C is preferably a mixture of the antibody C1 that binds to the antibody A1 and the antibody C2 that binds to the antibody A2. If antibody composition C is not a mixture of antibody C1 and antibody C2, the control line will be faint and may be judged as re-measurement.

The mixing ratio (mass ratio) of the antibodies C1 and C2 is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the control line becomes thin and may be judged as re-measurement.

The method of linearly immobilizing the capturing antibody forming the test line 6 and the capturing antibody forming the control line 7 on the membrane 3 is not particularly limited, but for example, the capturing antibody forming the test line and the capturing antibody forming the control line are fixed. can be produced by applying a given amount of each of and to different positions on the line, and then drying at an appropriate temperature in a constant temperature bath for a given period of time. The amount of the two capture antibodies to be applied is not particularly limited, but is preferably 0.1 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 only the cost is increased. Next, the membrane 3 after the application is dried. The drying temperature is not particularly limited, but is preferably 20°C to 80°C, more preferably 20°C to 60°C. The drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.

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.

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.

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 the immune reaction. Examples of nonionic surfactants include, but are not limited to, polyoxyethylene alkylphenyl ethers (Triton (registered trademark) surfactants, etc.), polyoxyethylene alkyl ethers (Brij (registered trademark) surfactants, etc.), Examples include polyoxyethylene sorbitan fatty acid esters (Tween (registered trademark) surfactants, etc.), polyoxyethylene fatty acid esters, sorbitan fatty acid esters, alkylglucosides, sucrose fatty acid esters, and the like. Moreover, surfactants may be used alone or in combination of two or more. The concentration of the nonionic surfactant is preferably 0.01% by mass to 5.0% by mass, more preferably 0.05% by mass to 4.0% by mass, and 0.1% by mass to 3.0% by mass. More preferred. Low concentrations can make downstream deployment difficult. In addition, the deployment may become uneven and the measurement accuracy may be lowered. On the other hand, if the concentration is high, the physically adsorbed detection particles and the antibody, and/or the membrane and the antibody, may deviate from each other, resulting in failure to obtain measurement values.

Inorganic salts and buffers used for pH adjustment may be added to the measurement sample diluent. As the buffering agent, any type of buffering agent may be used as long as it has a sufficient buffering capacity in the target pH range. acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good's buffer (MES, ADA, PIPES, ACES, colamin hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine). 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.

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

(Example 1)
(1) Preparation of complexes of antibody A1, antibody A2 and cellulose-based colored fine particles 1.0% by mass of cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) 100 μL, 10 mM Tris buffer (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 as antibody A1 ( Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, manufactured by Toyobo) 50 μL, and 1.0 mg / mL rabbit-derived anti-SARS-CoV-2 N protein monoclonal antibody (SARS-CoV- 2 (COVID-19) nucleocapsid antibody, [HL5511], Cat No. GTX635689, manufactured by GeneTex) (50 μL) 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). 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 disperser (UH-50, manufactured by SMTE). A composite with cellulose-based colored fine particles was obtained.

(2) Preparation of membrane card for detecting SARS-CoV-2 N protein 2.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo), and 2.0 mg/mL rabbit-derived anti-SARS-CoV-2 N protein monoclonal antibody as antibody B2 (SARS-CoV-2 (COVID-19) nucleocapsid antibody, [HL146 ], Cat No. GTX635680, manufactured by GeneTex) were mixed at a mixing ratio (mass ratio) of 1:1. Then, 1.0 mg/mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) as antibody C1, and 1.0 mg/mL goat-derived anti-antibody as antibody C2. A rabbit IgG polyclonal antibody (Rabbit IgG-heavy and light chain antibody, A120-101A, manufactured by BETHYL) was mixed at a mixing ratio (mass ratio) of 1:1. 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, antibody B1 and antibody After applying the mixture with B2 at a coating amount of 1.0 μL / cm, it was dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45 ° C., and a test line with a line width of about 1 mm was formed. formed. 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 was formed, antibody C1 was applied. and C2 at a coating amount of 1.0 μL / cm, dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) adjusted to 45 ° C., and a control line with a line width of about 1 mm. By forming, a membrane card for detecting N protein of SARS-CoV-2 was obtained.

(3) Preparation of conjugation pad for detecting N protein of SARS-CoV-2 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 complexes of the antibody A1, antibody A2 and cellulose-based colored fine particles were uniformly applied at a coating amount of 15 μL / cm, and then adjusted to 45 ° C. It was dried for 30 minutes with a drier (WFO-510, manufactured by Tokyo Rikakikai Co., Ltd.) to obtain a conjugation pad for detecting N protein of SARS-CoV-2.

(4) Preparation of immunochromatographic test strip and device for detecting N protein of SARS-CoV-2 A 20 mm × 300 mm adhesive tape part on the upstream side of the membrane card for detecting N protein of SARS-CoV-2 overlaps the membrane part by 2 mm. A conjugation pad for detecting the N protein of SARS-CoV-2 was attached as shown. 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, Millipore Co., Ltd. ) were pasted together. Then, using a guillotine cutting module (CM5000, manufactured by BIODOT), a strip of 4 mm in width and 60 mm in length was cut to obtain an immunochromatographic test strip for detecting SARS-CoV-2 N protein. The obtained immunochromatographic test piece for detecting N protein of SARS-CoV-2 was housed in a housing case (K007, manufactured by Shengfeng Plastic) to obtain an immunochromatographic device 1 for detecting N protein of SARS-CoV-2.

(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) Preparation of positive 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 1 ng/mL with the measurement sample diluent. A positive sample was obtained by

(7) Preparation of Negative Sample A negative sample was obtained by diluting commercially available HAMA (3PH490, manufactured by Scantibodies Laboratory) to 100 ng/mL with the measurement sample diluent.

(8) 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 15 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 1 shows the evaluation results.
Non-specific adsorption of the immunochromatographic device for detecting N protein of SARS-CoV-2 in Example 1 was confirmed to have no problem with the reflectance absorbance of the test line <10 mAbs.

(9) 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. Next, 100 μL of the positive sample was dispensed with a micropipette, gently dropped onto the sample pad, and allowed to stand 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 evaluation results.
The immunochromatographic device for detecting N protein of SARS-CoV-2 of Example 1 was capable of highly sensitive measurement.

(10) Evaluation of immunochromatographic test strip for detecting SARS-CoV-2 N protein: evaluation of false positives The immunochromatographic device for detecting SARS-CoV-2 N protein was placed on a horizontal table. Next, 100 μL of the negative sample was dispensed with a micropipette, gently dropped onto the sample pad, and allowed to stand at 25° C. for 15 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 3 shows the evaluation results.
The immunochromatographic device for detecting N protein of SARS-CoV-2 of Example 1 suppressed false positives and enabled highly specific measurement.

(Example 2)
Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by East Coast Bio) was used as antibody A2, and a goat-derived anti-rat IgG polyclonal antibody (Rat An immunochromatographic device 2 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that IgG-heavy and light chain antibody, A110-105A, manufactured by BETHYL) was used. Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

(Example 3)
SARS-CoV in the same manner as in Example 1 except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by EastCoast Bio) was used as antibody B2. An immunochromatographic device 3 for N protein detection of -2 was obtained. Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

(Example 4)
Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A2, and rat-derived anti-SARS-CoV-2 was used as antibody B2. N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by East Coast Bio) was used, and antibody C2 was a goat-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-105A, An immunochromatographic device 4 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except for using BETHYL). Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

(Example 5)
Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A1, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat An immunochromatographic device 5 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that IgG-heavy and light chain antibody, A110-122A, manufactured by BETHYL) was used. Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

(Example 6)
SARS-CoV in the same manner as in Example 1 except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by EastCoast Bio) was used as antibody B1. An immunochromatographic device 6 for N protein detection of -2 was obtained. Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

(Example 7)
Rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, manufactured by EastCoast Bio) was used as antibody A1, and rat-derived anti-SARS-CoV-2 was used as antibody B1. N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, manufactured by East Coast Bio) was used, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-122A, A110-122A, An immunochromatographic device 7 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that BETHYL) was used. Table 1 shows the evaluation results of (8) nonspecific adsorption, Table 2 shows the evaluation results of (9) sensitivity, and Table 3 shows the evaluation results of (10) false positives of the obtained device.

From the results shown in Table 1, it was confirmed that the non-specific adsorption of the immunochromatographic devices for detecting the N protein of SARS-CoV-2 in Examples 2 to 7 had no problem with the test line reflection absorbance <10 mAbs. Further, from the results shown in Table 2, it was confirmed that the sensitivity of the immunochromatographic devices for detecting the N protein of SARS-CoV-2 in Examples 2 to 4 was satisfactory with a test line reflection absorbance >40 mAbs. Furthermore, from the results shown in Table 3, it was confirmed that the false positives of the immunochromatographic devices for detecting the N protein of SARS-CoV-2 in Examples 2 to 4 were no problem with the reflectance absorbance of the test line <10 mAbs.

(Example 8)
An immunochromatographic device 8 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that the concentration of antibody A1 was 0.222 mg/mL and the concentration of antibody A2 was 1.778 mg/mL. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 9)
An immunochromatographic device 9 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was 1.818 mg/mL and the concentration of antibody A2 was 0.182 mg/mL. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 10)
An immunochromatographic device 10 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was 1.905 mg/mL and the concentration of antibody A2 was 0.095 mg/mL. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 11)
An immunochromatographic device 11 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 1:8. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 12)
An immunochromatographic device 12 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 10:1. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 13)
An immunochromatographic device 13 for detecting SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed at a mass ratio of 20:1. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 14)
An immunochromatographic device 14 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 1:0. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 15)
An immunochromatographic device 15 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 1:8. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 16)
An immunochromatographic device 16 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 10:1. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

(Example 17)
An immunochromatographic device 17 for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed at a mass ratio of 20:1. Table 4 shows the evaluation results of (8) nonspecific adsorption, Table 5 shows the evaluation results of (9) sensitivity, and Table 6 shows the evaluation results of (10) false positive of the obtained device.

From the results shown in Table 4, it was confirmed that the nonspecific adsorption of the immunochromatographic devices for detecting N protein of SARS-CoV-2 in Examples 5 to 17 was satisfactory, with the test line reflectance absorbance <10 mAbs. Also, in the control line, the closer the C1 and C2 antibody concentrations, the higher the reflection absorbance. Further, from the results shown in Table 5, it was confirmed that the sensitivity of the immunochromatographic devices for detecting the N protein of SARS-CoV-2 in Examples 5 to 14 was satisfactory, with the test line reflectance absorbance >40 mAbs. Furthermore, from the results shown in Table 6, it was confirmed that the false positives of the immunochromatographic devices for detecting N protein of SARS-CoV-2 in Examples 5 to 14 were no problem with the test line reflection absorbance <10 mAbs.

(Comparative example 1)
0.5 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, Toyobo Co., Ltd.) 100 μL was used as antibody A1, and antibody A2 was Without using, 1.0 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo) as antibody B1, antibody B2 was not used, 0.5 mg / mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) was used as antibody C1, except that antibody C2 was not used. An immunochromatographic device A for detecting N protein of SARS-CoV-2 was obtained in the same manner as in Example 1. Table 7 shows the evaluation results of (8) nonspecific adsorption, Table 8 shows the evaluation results of (9) sensitivity, and Table 9 shows the evaluation results of (10) false positives of the obtained device.

(Comparative example 2)
0.5 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, Toyobo Co., Ltd.) 100 μL was used as antibody A1, and antibody A2 was 0.5 mg/mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) was used as antibody C1, except that antibody C2 was not used. An immunochromatographic device B for detecting N protein of SARS-CoV-2 was obtained in the same manner as in 1. Table 7 shows the evaluation results of (8) nonspecific adsorption, Table 8 shows the evaluation results of (9) sensitivity, and Table 9 shows the evaluation results of (10) false positives of the obtained device.

(Comparative Example 3)
1.0 mg/mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo) was used as antibody B1, and antibody B2 was used. An immunochromatographic device C for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1 except that it was not used. Table 7 shows the evaluation results of (8) nonspecific adsorption, Table 8 shows the evaluation results of (9) sensitivity, and Table 9 shows the evaluation results of (10) false positives of the obtained device.

From the results shown in Table 7, it was confirmed that the non-specific adsorption of the immunochromatographic devices for detecting N protein of SARS-CoV-2 in Comparative Examples 1 to 3 had no problem with the reflectance absorbance of the test line <10 mAbs. Further, from the results shown in Table 8, it was confirmed that the immunochromatographic devices for detecting N protein of SARS-CoV-2 of Comparative Examples 1 to 3 have clearly lower sensitivity than Example 1. Furthermore, from the results shown in Table 9, it was confirmed that the immunochromatographic devices for detecting N protein of SARS-CoV-2 of Comparative Examples 1 to 3 generated false positives and had low specificity.

According to the present invention, it is possible to provide an immunochromatographic test strip that can detect the N protein of SARS-CoV-2 with high sensitivity and suppressed false positives.

1: sample pad 2: conjugation pad 3: membrane 4: absorbent pad 5: backing sheet 6: test line 7: control line 8: adhesive sheet

Claims

(1) a sample pad;
(2) Carrying a complex of an antibody composition A that specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample and cellulose-based colored fine particles a conjugation pad with
(3) Line the antibody composition B that specifically binds to the N protein of SARS-CoV-2 in the measurement sample and the antibody composition C that specifically binds to the antibody composition A at different positions. a membrane fixed in a shape;
(4) an absorbent pad;
The antibody composition A is a mixture of antibody A1 and antibody A2 derived from different origins,
An immunochromatographic test strip, wherein the antibody composition B is a mixture of antibody B1 and antibody B2 derived from different origins.
The immunochromatographic test strip according to claim 1, wherein either the antibody A1 or the antibody A2 is a mouse-derived antibody.
The antibody A1 and antibody A2 are in a mixing ratio (mass ratio) of 10: 1 to 1: 10, and the immunochromatographic test strip according to claim 1 or 2, wherein the conjugation pad is supported. .
The immunochromatographic test strip according to any one of claims 1 to 3, wherein either the antibody B1 or the antibody B2 is a mouse-derived antibody.
5. The antibody B1 and the antibody B2 according to any one of claims 1 to 4, wherein the antibody B1 and the antibody B2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. of the immunochromatographic specimen.
The immunochromatographic test strip according to any one of claims 1 to 3, wherein said antibody composition C is a mixture of antibody C1 that binds to said antibody A1 and antibody C2 that binds to said antibody A2.
The immunochromatographic test strip according to claim 6, wherein the antibody C1 and the antibody C2 are linearly immobilized on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10.
An immunochromatographic kit comprising the immunochromatographic test piece according to any one of claims 1 to 7, a measurement sample collecting tool, a filter, and a measurement sample diluent.