Anti-SARS-COV-2 monoclonal antibody, and SARS-COV-2 immunoassay method and immunoassay device using same

Abstract

Disclosed are: a monoclonal antibody which enables the rapid, simple, and highly sensitive detection and measurement of SARS-COV-2 contained in a test sample; and an SARS-COV-2 immunoassay method and immunoassay instrument using the monoclonal antibody. A monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain CDR1-CDR3 having a specific amino acid sequence, and a light chain CDR1-CDR3 having a specific amino acid sequence. The immunoassay method for the SARS-COV-2 comprises the step of performing immunoassay on the SARS-COV-2 by utilizing the monoclonal antibody or the antigen binding fragment thereof to react with the antigen-antibody of the SARS-COV-2 in a test sample.

Description

Technical Field

[0001] The present invention relates to an anti-SARS-CoV-2 monoclonal antibody and a SARS-CoV-2 immunoassay method and immunoassay device using the same. Background Art

[0002] SARS-CoV-2 is a virus belonging to the Coronaviridae family, with a single-stranded, positive-sense RNA genome. In March 2020, the infectious disease (COVID-19) caused by SARS-CoV-2 reached pandemic proportions, with a large number of infections reported worldwide. As of 2022, countries are developing vaccines and promoting vaccination globally, but the emergence of variants suggests that the pandemic shows no sign of ending.

[0003] In the case of COVID-19, SARS-CoV-2 infection is diagnosed using PCR and antigen testing. An immunochromatographic method using anti-SARS-CoV-2 antibodies has been developed as a rapid and convenient method for detecting SARS-CoV-2. However, with no sign of an end to the pandemic, there is a demand for more accurate testing.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: WO2021 / 181994 Summary of the Invention

[0007] Problems to be solved by the invention

[0008] Currently produced monoclonal antibodies against SARS-CoV-2 have insufficient sensitivity for detecting SARS-CoV-2, and there is a demand for monoclonal antibodies that react with higher sensitivity.

[0009] The object of the present invention is to provide a monoclonal antibody capable of rapidly, simply, and highly sensitively detecting and measuring SARS-CoV-2 contained in a test sample, and a SARS-CoV-2 immunoassay method and immunoassay device using the same.

[0010] Means for solving problems

[0011] The inventors of the present application conducted intensive research on the above-mentioned problems and, as a result, discovered a specific monoclonal antibody that reacts with SARS-COV-2 with high sensitivity, thereby completing the present invention.

[0012] That is, the present invention provides the following solutions.

[0013] (1) A monoclonal antibody or an antigen-binding fragment thereof comprising the following heavy chain CDR1 to CDR3 (a-1) to (c-1) and the following light chain CDR1 to CDR3 (d-1) to (f-1).

[0014] (a-1) heavy chain CDR1 consisting of the amino acid sequence of GYTFTSYWMH (SEQ ID NO: 1),

[0015] (b-1) a heavy chain CDR2 consisting of the amino acid sequence of YINPTTGYTDYNQKFKD (SEQ ID NO: 2),

[0016] (c-1) a heavy chain CDR3 consisting of the amino acid sequence of EIYFGDVKTWFAY (SEQ ID NO: 3),

[0017] (d-1) light chain CDR1 consisting of the amino acid sequence of STSQDISNYLN (SEQ ID NO: 4),

[0018] (e-1) light chain CDR2 consisting of the amino acid sequence of TSNLQSG (SEQ ID NO: 5),

[0019] (f-1) Light chain CDR3 consisting of the amino acid sequence of QQYSKFPYT (SEQ ID NO: 6)

[0020] (2) A monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-2) to (c-2) and the light chain CDR1 to CDR3 of the following (d-2) to (f-2).

[0021] (a-2) heavy chain CDR1 consisting of the amino acid sequence of GYTFSNYWIE (SEQ ID NO: 7),

[0022] (b-2) a heavy chain CDR2 consisting of the amino acid sequence of EILPGSDITNYNEKFKD (SEQ ID NO: 8),

[0023] (c-2) a heavy chain CDR3 consisting of the amino acid sequence VYYYPGSLAWFAY (SEQ ID NO: 9),

[0024] (d-2) light chain CDR1 consisting of the amino acid sequence of SASQDISNYLN (SEQ ID NO: 10),

[0025] (e-2) light chain CDR2 consisting of the amino acid sequence of YTSSLHS (SEQ ID NO: 11),

[0026] (f-2) Light chain CDR3 consisting of the amino acid sequence of QQYSKLPYT (SEQ ID NO: 12)

[0027] (3) A monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-3) to (c-3) and the light chain CDR1 to CDR3 of the following (d-3) to (f-3).

[0028] (a-3) heavy chain CDR1 consisting of the amino acid sequence of GYTFSSYWIE (SEQ ID NO: 13),

[0029] (b-3) heavy chain CDR2 consisting of the amino acid sequence of EILPGSGNTYYNEKFKG (SEQ ID NO: 14),

[0030] (c-3) a heavy chain CDR3 consisting of the amino acid sequence of WEWLLRLYGLDY (SEQ ID NO: 15),

[0031] (d-3) light chain CDR1 consisting of the amino acid sequence of KASQSVDYDGESYMN (SEQ ID NO: 16),

[0032] (e-3) light chain CDR2 consisting of the amino acid sequence of AASNLES (SEQ ID NO: 17),

[0033] (f-3) Light chain CDR3 consisting of the amino acid sequence of QQSNEDPYT (SEQ ID NO: 18)

[0034] (4) A monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-4) to (c-4) and the light chain CDR1 to CDR3 of the following (d-4) to (f-4).

[0035] (a-4) heavy chain CDR1 consisting of the amino acid sequence of GFSFSDYFMY (SEQ ID NO: 19),

[0036] (b-4) a heavy chain CDR2 consisting of the amino acid sequence of TISDGGIYTYYPDSVKG (SEQ ID NO: 20),

[0037] (c-4) a heavy chain CDR3 consisting of the amino acid sequence of DLDYFGSTIAY (SEQ ID NO: 21),

[0038] (d-4) light chain CDR1 consisting of the amino acid sequence of RSSAGALTTRNYAN (SEQ ID NO: 22),

[0039] (e-4) light chain CDR2 consisting of the amino acid sequence of GTSNRAP (SEQ ID NO: 23),

[0040] (f-4) Light chain CDR3 consisting of the amino acid sequence of ALWYSNHWV (SEQ ID NO: 24)

[0041] (5) A monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, each of which has a sequence identity of 60% or more to the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 shown in any one of (1) to (4), and which undergoes an antigen-antibody reaction with SARS-COV-2.

[0042] (6) A method for immunoassaying SARS-COV-2, comprising immunoassaying SARS-COV-2 using the monoclonal antibody or antigen-binding fragment thereof described in any one of (1) to (5) and the antigen-antibody reaction of SARS-COV-2 in a sample.

[0043] (7) The method according to (6), wherein the immunoassay method is a sandwich method, and the monoclonal antibody or antigen-binding fragment thereof is used as at least one of a label or a solid phase.

[0044] (8) A SARS-CoV-2 immunoassay device comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (5).

[0045] Effects of the Invention

[0046] The present invention provides a monoclonal antibody capable of rapidly, simply, and highly sensitively detecting and measuring SARS-CoV-2 contained in a test sample, and a SARS-CoV-2 immunoassay method and immunoassay device using the monoclonal antibody. DETAILED DESCRIPTION

[0047] Hereinafter, embodiments of the present invention will be described in detail.

[0048] 1. Monoclonal antibodies or their antigen-binding fragments

[0049] The monoclonal antibody of the present invention is a monoclonal antibody having each of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 specified in (1) to (4) below. (1)

[0051] (a-1) heavy chain CDR1 consisting of the amino acid sequence of GYTFTSYWMH (SEQ ID NO: 1),

[0052] (b-1) a heavy chain CDR2 consisting of the amino acid sequence of YINPTTGYTDYNQKFKD (SEQ ID NO: 2),

[0053] (c-1) a heavy chain CDR3 consisting of the amino acid sequence of EIYFGDVKTWFAY (SEQ ID NO: 3),

[0054] (d-1) light chain CDR1 consisting of the amino acid sequence of STSQDISNYLN (SEQ ID NO: 4),

[0055] (e-1) light chain CDR2 consisting of the amino acid sequence of TSNLQSG (SEQ ID NO: 5),

[0056] (f-1) Light chain CDR3 consisting of the amino acid sequence of QQYSKFPYT (SEQ ID NO: 6) (2)

[0058] (a-2) heavy chain CDR1 consisting of the amino acid sequence of GYTFSNYWIE (SEQ ID NO: 7),

[0059] (b-2) a heavy chain CDR2 consisting of the amino acid sequence of EILPGSDITNYNEKFKD (SEQ ID NO: 8),

[0060] (c-2) a heavy chain CDR3 consisting of the amino acid sequence VYYYPGSLAWFAY (SEQ ID NO: 9),

[0061] (d-2) light chain CDR1 consisting of the amino acid sequence of SASQDISNYLN (SEQ ID NO: 10),

[0062] (e-2) light chain CDR2 consisting of the amino acid sequence of YTSSLHS (SEQ ID NO: 11),

[0063] (f-2) Light chain CDR3 consisting of the amino acid sequence of QQYSKLPYT (SEQ ID NO: 12) (3)

[0065] (a-3) heavy chain CDR1 consisting of the amino acid sequence of GYTFSSYWIE (SEQ ID NO: 13),

[0066] (b-3) heavy chain CDR2 consisting of the amino acid sequence of EILPGSGNTYYNEKFKG (SEQ ID NO: 14),

[0067] (c-3) a heavy chain CDR3 consisting of the amino acid sequence of WEWLLRLYGLDY (SEQ ID NO: 15),

[0068] (d-3) light chain CDR1 consisting of the amino acid sequence of KASQSVDYDGESYMN (SEQ ID NO: 16),

[0069] (e-3) light chain CDR2 consisting of the amino acid sequence of AASNLES (SEQ ID NO: 17),

[0070] (f-3) Light chain CDR3 consisting of the amino acid sequence of QQSNEDPYT (SEQ ID NO: 18) (4)

[0072] (a-4) heavy chain CDR1 consisting of the amino acid sequence of GFSFSDYFMY (SEQ ID NO: 19),

[0073] (b-4) a heavy chain CDR2 consisting of the amino acid sequence of TISDGGIYTYYPDSVKG (SEQ ID NO: 20),

[0074] (c-4) a heavy chain CDR3 consisting of the amino acid sequence of DLDYFGSTIAY (SEQ ID NO: 21),

[0075] (d-4) light chain CDR1 consisting of the amino acid sequence of RSSAGALTTRNYAN (SEQ ID NO: 22),

[0076] (e-4) light chain CDR2 consisting of the amino acid sequence of GTSNRAP (SEQ ID NO: 23),

[0077] (f-4) Light chain CDR3 consisting of the amino acid sequence of ALWYSNHWV (SEQ ID NO: 24)

[0078] The monoclonal antibody or antigen-binding fragment thereof of the present invention has a basic structure consisting of a heavy chain and a light chain, each of which has a variable region that can specifically bind to an antigen. VH refers to the variable region of the heavy chain, and VL refers to the variable region of the light chain. The variable regions of the heavy chain and the light chain each comprise an amino acid sequence of a complementarity determining region (CDR), i.e., CDR1, CDR2, and CDR3, and a framework region (FR). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) together with three CDRs.

[0079] The monoclonal antibodies of the present invention also include four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, and bispecific antibodies). Furthermore, the type of monoclonal antibody is not limited to IgG and may also be IgM or IgY.

[0080] In the present invention, the antigen-binding fragment of a monoclonal antibody refers to a fragment obtained by isolating only the antigen-binding site of a monoclonal antibody. Examples thereof include fragments having specific antigen-binding properties such as Fab, Fab', F(ab')2, and single-chain antibodies (scFv) produced by known methods.

[0081] When preparing such antigen-binding fragments, preparation methods known to those skilled in the art can be used, for example, methods in which the antibody is digested using a protease (e.g., pepsin, papain, etc.) using conventional methods and then purified using known protein separation and purification methods; preparation methods based on genetic recombination, etc.

[0082] As the monoclonal antibodies of the present invention, monoclonal antibodies having the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of Antibodies 1 to 4 obtained in the following Examples are preferred from the perspective of being able to detect SARS-CoV-2 with higher sensitivity. The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of Antibodies 1 to 4 are shown in Table 2 below.

[0083] It should be noted that the following monoclonal antibodies are also monoclonal antibodies of the present invention, which comprise heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having a sequence identity of 60% or more, preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more with each of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of each of the above-mentioned antibodies 1 to 4, and undergo an antigen-antibody reaction with SARS-COV-2. It should be noted that, here, "sequence identity" is obtained by aligning two amino acid sequences in a manner such that the number of identical amino acids is the largest (inserting gaps if necessary), dividing the number of identical amino acids by the number of all amino acids (in the case where the number of all amino acids is different, the longer number of amino acids), and expressing the result as a percentage. This can be easily calculated using publicly known software such as BLAST.

[0084] 2. Method for producing monoclonal antibodies

[0085] The monoclonal antibodies of the present invention can be obtained by immunizing an animal with a complex or extract containing SARS-CoV-2 as the target antigen, or with SARS-CoV-2 or a partial peptide thereof using known immunological methods, and then using cells from the immunized animal to generate hybridomas. The length of the peptide used for immunization is not particularly limited; however, peptides of 5 or more amino acids, and more preferably 10 or more amino acids, can be used as immunogens.

[0086] Immunogens can also be obtained from culture fluid, or by integrating DNA encoding any SARS-COV-2 antigen into a plasmid vector and introducing it into a host cell for expression. Any SARS-COV-2 antigen or its partial peptide as an immunogen can also be expressed in the form of a fusion protein with the protein exemplified below, and used as an immunogen after purification or in an unpurified state. In the preparation of fusion proteins, those skilled in the art can utilize glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), Nus tags, S tags, HSV tags, FRAG tags, polyhistidine tags, etc., which are commonly used as "protein expression purification tags". The fusion protein formed with them is preferably used as an immunogen after using a digestive enzyme to cut off any SARS-COV-2 antigen or its partial peptide portion and the tag portion in addition thereto and is isolated and purified.

[0087] Monoclonal antibodies can be easily prepared from immunized animals by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as spleen cells and lymphocytes are recovered from the immunized animal, and these antibody-producing cells are fused with mouse myeloma cells using conventional methods to produce hybridomas. The resulting hybridomas are cloned using limiting dilution methods, and monoclonal antibodies produced by each cloned hybridoma are selected for those that react with the antigen used for animal immunization.

[0088] The purification of monoclonal antibodies from ascites and culture supernatants can utilize known immunoglobulin purification methods. For example, fractionation based on salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, etc. can be cited. In addition, depending on the type of immunized animal and the type of monoclonal antibody, purification may also be performed by affinity chromatography using a carrier bound to any one of protein A, protein G, and protein L.

[0089] Furthermore, the monoclonal antibodies used in the present invention can be produced by genetically modifying plants and can be produced using a plant transient expression system.

[0090] In addition, the monoclonal antibody used in the present invention can also be obtained using mammalian cells as the form of the gene recombinant of expression host.As mammalian cells in this case, CHO (Chinese hamster ovary cell, Chinese Hamster Ovary) cell, HEK293 (human embryonic kidney cell, Human Embryonic Kidney cells293) cell etc. can be enumerated, but are not limited to this.In addition, about the method obtained with the form of gene recombinant, the transient (transient) expression system of the plasmid vector lacking autonomous replication ability, viral vector, the semi-stable (semi-stable) expression system of the episomal vector (episomal vector) that uses to give nuclear localization signal and has autonomous replication ability and the stable (stable) expression system of the genome of target gene inserted into expression host etc. can be enumerated, are not particularly limited.

[0091] It should be noted that, for example, in the above-mentioned antibodies 1 to 4, since the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 have been determined, it is possible to produce monoclonal antibodies having the same six CDR regions as these monoclonal antibodies, i.e., having the same binding affinity, by expressing genes incorporating polynucleotides encoding these six CDRs using genetic engineering methods. Such methods are well known and are described, for example, in Japanese Patent No. 6614523.

[0092] 3. Immunoassay Method

[0093] In addition to using the above-described monoclonal antibody of the present invention or an antigen-binding fragment thereof, any known immunoassay method may be employed, and each immunoassay method itself can be performed in the same manner as conventional methods.

[0094] In the present invention, by using the aforementioned monoclonal antibodies or antigen-binding fragments thereof, SARS-CoV-2 can be detected with very high sensitivity. In the following descriptions before the Examples, "monoclonal antibody" means "monoclonal antibody or antigen-binding fragment thereof," unless the context clearly indicates otherwise.

[0095] In the present invention, SARS-CoV-2 detection is performed by immunoassay using the aforementioned monoclonal antibodies to react with SARS-CoV-2 antigens and antibodies in a test sample. The so-called antigen-antibody reaction between the monoclonal antibodies and SARS-CoV-2 refers to a specific reaction between the monoclonal antibodies and SARS-CoV-2. "Specificity" means that, in a system where the antigen protein and the monoclonal antibody are mixed, the antibody does not undergo a detectable antigen-antibody reaction with proteins other than the target protein component of the antigen; or, even if coexisting substances other than the antibody and antigen undergo some binding or association reaction with the antibody and antigen, the reaction is significantly weaker than the antigen-antibody reaction between the antibody and the antigen.

[0096] In the present invention, any method known to those skilled in the art, such as a competitive method, an agglutination method, a Western blotting method, an immunostaining method, and a sandwich method, can be used as the immunoassay method.

[0097] As the immunoassay method of the present invention, the sandwich method is preferred. In the sandwich method, a complex is formed by sandwiching an antigen between two antibodies, and the complex is detected. The sandwich method itself is well known in the field of immunoassays and can be performed, for example, using immunochromatography or ELISA. These sandwich methods are well known in themselves, and the method of the present invention can be performed using the aforementioned monoclonal antibodies or antigen-binding fragments thereof, in addition to using known sandwich methods.

[0098] In the sandwich method, one or more antibodies (antibodies immobilized on a solid phase and labeled antibodies) that recognize the antigen are used. When two or more antibodies are used, at least one of the two antibodies is the monoclonal antibody described above. Alternatively, the same two antibodies may be used to sandwich the antigen to form a complex. Preferably, one or two antibodies that recognize the peptide are used.

[0099] In the immunoassay based on the sandwich method as the detection principle, as the solid phase for antibody immobilization, any solid phase that can utilize known technology to immobilize the antibody can be used, for example, any known article such as a porous film (membrane), a particulate material, a test tube, a resin plate with capillary action can be selected. In addition, as the material for labeling the antibody, enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, etc. can be used. In the immunoassay utilizing the aforementioned various materials, particularly from the viewpoint of simplicity and rapidity of clinical examination, it is preferred to use an immunochromatographic method as a lateral flow immunoassay utilizing a membrane.

[0100] In the present invention, when using monoclonal antibodies to quantify or semi-quantitate SARS-CoV-2, since quantification and semi-quantification necessarily involve "assay," they are also included in the "assay" of the present invention. That is, in the present invention, "assay" in immunoassay includes any of quantification, semi-quantification, and detection.

[0101] The present invention also provides an immunoassay device that can perform immunoassays in a lateral flow manner using the above-mentioned monoclonal antibodies. The immunoassay device provided by the present invention includes: a support having a detection area on which an antibody (antibody 1) that captures the assay object (antigen) is immobilized; a labeling area having a removable labeled antibody (antibody 2); a sample pad for dripping the sample; an absorption belt that absorbs the developed sample liquid; and a backing sheet for bonding these components together, wherein at least one of the antibody 1 and the antibody 2 is the above-mentioned monoclonal antibody of the present invention. The immunoassay device is also referred to as an immunochromatographic test piece. It should be noted that the support having a detection area immobilized with an antibody (antibody 1) that captures the assay object (antigen) and the removable labeled antibody (antibody 2) can use a variety of monoclonal antibodies. In addition, polyclonal antibodies can also be combined.

[0102] The support is a material with the performance of the antibody immobilization for capturing the detected substance (antigen), and has the performance that does not hinder the passage of liquid in the horizontal direction. It is preferably a porous film with capillary action, which is a material that can transport liquid and the components dispersed therein by absorption. The material forming the support is not particularly limited, and for example, cellulose, nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), glass fiber, nylon, polyketone, etc. can be cited. Wherein, nitrocellulose is more preferably used to make a film. The film immobilized with the antibody is referred to as an antibody immobilized membrane.

[0103] The labeled region is formed from a porous substrate containing the labeled antibody. Commonly used materials for the substrate include glass fiber and nonwoven fabrics. To allow for the impregnation of a large amount of labeled antibody, the substrate is preferably in the form of a pad with a thickness of approximately 0.3 mm to 0.6 mm. A porous substrate impregnated with labeled antibody and dried is also referred to as a dry pad.

[0104] The labeling of labeled antibodies mostly uses enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloids, silica particles, cellulose particles, colored polystyrene particles and colored latex particles. When using colored particles such as metal colloid particles, colored polystyrene particles and colored latex particles, since these labeling reagents produce coloration due to aggregation, the coloration is measured. The particles immobilized with the antibody are referred to as antibody-immobilized particles. The immobilized amount of the antibody is not particularly limited, and the presence of several ng to tens of μg in the labeled region is sufficient.

[0105] The detection region refers to a portion of the support where antibodies that capture the substance to be detected (antigen) are immobilized. The detection region is provided with at least one region where antibodies that capture the antigen are immobilized. The detection region only needs to be contained within the support, and the antibodies can be immobilized on the support. The amount of immobilized antibody is not particularly limited, and a few ng to tens of μg of antibody can be immobilized within the detection region.

[0106] The sample pad is a porous material used to drip the sample. It is located at the uppermost part of the immunoassay instrument. Typical materials used for this pad include filter paper, fiberglass, and non-woven fabric. To accommodate large quantities of sample for immunoassays, a pad with a thickness of approximately 0.3 mm to 1 mm is preferred. Samples prepared using the sample are also included, such as those obtained by suspending the sample in another solution.

[0107] The absorbent tape is used to absorb components supplied to the support but not involved in the reaction in the detection area. This material can be made of commonly used natural or synthetic polymers, such as filter paper or sponge with high water retention. However, to facilitate sample development, highly absorbent materials are preferred.

[0108] The base plate is a component to which all the aforementioned materials, namely the support, specimen pad, marker area, absorbent tape, etc., are partially overlapped and fixed. As long as these materials are arranged and fixed at optimal intervals, a base plate is not essential, but it is generally preferred for ease of manufacture and use.

[0109] The immunoassay device of the present invention may further include a control display area (component). The control display area is a portion that indicates that the test has been correctly performed. For example, the control display area is located downstream of the detection area, and when the test sample passes through the detection area and reaches the control display area, a signal is emitted by coloring, etc. The control display area may be pre-immobilized with a substance that binds to an antibody bound to a labeled carrier, or may be pre-immobilized with a pH indicator or other reagent that changes color when the test sample arrives. In the case where the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0110] The size of the immunoassay instrument is not limited, and may be, for example, several centimeters to several dozen centimeters in length and several millimeters to several centimeters in width.

[0111] The immunoassay device of the present invention can be stored in a storage container, which can prevent degradation caused by, for example, ultraviolet light or atmospheric moisture. Furthermore, when using contaminating or infectious samples, the storage container can prevent contamination or infection of the test subject. For example, a resin housing of an appropriately sized storage container can be used as the storage container, and the device of the present invention can be housed within this housing. The storage container and the immunoassay device housed therein are sometimes collectively referred to as an immunoassay device.

[0112] The present invention can provide a kit comprising a SARS-CoV-2 immunoassay device containing the above-mentioned monoclonal antibody. The kit may also include a brochure, a sample collection device, etc.

[0113] In the method of the present invention, capillary action is utilized to allow a complex of an antibody 2, which is capable of binding to a target substance (labeling reagent) labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid, and the target substance to develop and migrate within a solid support immobilized with antibody 1. As a result, a complex of the immobilized substance, target substance, and labeling reagent forms on the solid support. The target substance can be detected by detecting the labeling reagent signal emitted by this complex (in the case of gold colloid, the portion of the solid support immobilized with the target substance-binding substance turns red). This immunoassay method can be performed at 5 to 30°C, preferably at room temperature.

[0114] The number of detection regions and the type of labeled antibodies contained in the marker region are not limited to one. By using a plurality of antibodies corresponding to the analyte, two or more antigens can be detected using the same immunoassay instrument.

[0115] The method of the present invention can detect whether a person is infected with SARS-CoV-2. If a SARS-CoV-2 antigen is detected in a test sample, it can be determined that the person is infected with SARS-CoV-2.

[0116] When the above-mentioned antibodies of the present invention are used, SARS-CoV-2 can be specifically identified. The above-mentioned monoclonal antibodies do not recognize other viruses, such as adenovirus, coxsackievirus, echovirus, herpes simplex virus, human metapneumovirus, influenza virus, measles virus, mumps virus, parainfluenza virus, RS virus (respiratory syncytial virus infection), etc., and will not cause false detection of these viruses.

[0117] Regarding the sample to be tested, as a sample, for example, there can be mentioned: body fluids such as blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites or amniotic fluid of humans or animals; mucus; feces; organs such as blood vessels or liver; tissues; cells or their extracts, etc., which may contain SARS-CoV-2 proteins, preferably cells and secretions from the oral cavity, tonsils, nasal cavity, pharynx, larynx, trachea, bronchi or lungs, etc., which are easy to collect, nasal swabs, throat swabs, mouthwashes, sputum, tracheal aspirates, bronchoalveolar lavage fluid, saliva, etc. The method for collecting these samples is not particularly limited, and known methods can be used. Specifically, a method using a cotton swab can be mentioned.

[0118] Example

[0119] The present invention will be described in more detail below based on examples. However, the present invention is not limited to the following examples.

[0120] Example 1 Preparation of Monoclonal Antibodies Recognizing the N Protein of SARS-CoV-2

[0121] 1. Preparation of SARS-CoV-2 N protein antigen

[0122] The DNA encoding the SARS-CoV-2N protein was expressed in Escherichia coli using an expression vector, and the protein was purified after several days of culture, and the resulting product was used.

[0123] 2. Preparation of monoclonal antibodies against SARS-CoV-2N protein

[0124] BALB / c mice were immunized with the SARS-CoV-2 N protein antigen described in 1. After a period of feeding, the iliac lymph nodes were removed from the mice, and the "mouse iliac lymph node method" (Sado Y et al., Acta Histochem. Cytochem. 39: 89-94 (2006)) was used to obtain multiple hybridoma cell lines that produced anti-SARS-CoV-2 N protein antibodies.

[0125] The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice. Approximately two weeks later, ascites containing antibodies was collected. IgG was purified from the resulting ascites using affinity chromatography using a Protein A column to obtain several purified anti-SARS-CoV-2 N protein monoclonal antibodies (hereinafter sometimes referred to as "anti-N protein antibodies").

[0126] In the following examples, four antibodies 1 to 4 were used, which were selected based on reactivity and specificity from a plurality of obtained anti-SARS-CoV-2 N protein monoclonal antibodies.

[0127] Example 2 Immunoassay Device for SARS-CoV-2

[0128] 1. Immobilization of anti-SARS-CoV-2N protein antibodies on nitrocellulose membranes

[0129] A solution of the anti-N protein antibodies classified in Example 1 diluted with a buffer and anti-mouse IgG antibodies were prepared. The anti-N protein antibodies were applied in a line pattern to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibodies were applied in a line pattern to the absorbent side. The nitrocellulose membrane was then thoroughly dried with hot air to obtain an anti-N protein antibody-immobilized membrane.

[0130] 2. Immobilization of Anti-SARS-CoV-2N Protein Antibodies on Colored Polystyrene Particles

[0131] The anti-N protein antibody prepared in Example 1 was bound to colored polystyrene particles, suspended in a buffer solution, and fully dispersed by ultrasonic treatment to obtain anti-N protein antibody-bound colored polystyrene particles. These are referred to herein as anti-N protein antibody-immobilized particles.

[0132] 3. Coating and Drying of Anti-SARS-CoV-2N Protein Antibody-Conjugated Colored Polystyrene Particles

[0133] The anti-N protein antibody-immobilized particles prepared in step 2 were applied to a glass fiber nonwoven fabric in a predetermined amount and dried thoroughly under hot air. In this specification, this is referred to as a marker pad.

[0134] 4. Fabrication of SARS-CoV-2 Testing Equipment

[0135] The anti-N protein antibody-immobilized membrane prepared in step 1 and the marker pad prepared in steps 2 and 3 were bonded to other components (base plate, absorption belt, sample pad), and cut into 5 mm widths to prepare a SARS-CoV-2 inspection device.

[0136] 5. Confirmation of the specificity and accuracy of SARS-CoV-2 detection equipment

[0137] 50 μL of a buffer solution containing viruses that cause respiratory infections (10 mM Tris (pH 7.0), 1% (w / v) polyoxyethylene octylphenyl ether, 3% (w / v) arginine, 3% (w / v) BSA) was added to the SARS-CoV-2 inspection device prepared in 4 and allowed to stand for 8 minutes.

[0138] A positive result was determined when both the anti-mouse IgG antibody and the anti-N protein antibody were visually detected at the application site. A negative result was determined when only the anti-mouse IgG antibody was visually detected, and no color was detected at the application site of the anti-N protein antibody. Table 1 shows several items from the determination results for each monoclonal antibody combination. Positive results were determined as +++, ++, +, and ±, in order of color intensity, and negative results were determined as -.

[0139] [Table 1]

[0140] Kit 1 Kit 2 No antigen - - SARS-CoV-2 N protein antigen +++ +++ SARS-CoV-2 inactivated antigen ++ ++ 229E inactivated antigen - ± OC43 inactivated antigen - - HKU1 inactivated antigen - - NL63 inactivated antigen - - MARS inactivated antigen - - SARS-CoV-1 inactivated antigen ± ±

[0141] Note) In kit 1, antibody 3 is bound to the membrane, and antibody 1 is bound to the colored particles.

[0142] In the case of kit 2, antibody 2 is bound to the membrane, and antibody 4 is bound to the colored particles.

[0143] Example 3 Variable Region Analysis of Anti-SARS-CoV-2N Protein Monoclonal Antibodies

[0144] 1. Sequence analysis of the variable regions of anti-SARS-CoV-2 N protein monoclonal antibodies

[0145] The hybridoma cell lines producing anti-SARS-CoV-2 N protein monoclonal antibodies 1 to 4 prepared in Example 1 were sent to BioPeak Inc. for base sequence analysis of the variable regions of the antibodies. BioPeak Inc. extracted RNA from the hybridoma cell lines, and the base sequences were obtained from the amplified products of the anti-SARS-CoV-2 N protein monoclonal antibodies variable regions obtained by PCR.

[0146] 2. Amino acid sequence prediction of the variable region of anti-SARS-CoV-2N protein monoclonal antibodies

[0147] Based on the base sequence information obtained in 1, the amino acid sequence of the variable region was predicted using the Kabat numbering system. The results are shown in Table 2.

[0148] [Table 2]

[0149]

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-1) to (c-1) and the light chain CDR1 to CDR3 of the following (d-1) to (f-1), (a-1) heavy chain CDR1 consisting of the amino acid sequence of GYTFTSYWMH, (b-1) a heavy chain CDR2 consisting of the amino acid sequence of YINPTTGYTDYNQKFKD, (c-1) heavy chain CDR3 consisting of the amino acid sequence of EIYFGDVKTWFAY, (d-1) light chain CDR1 consisting of the amino acid sequence of STSQDISNYLN, (e-1) light chain CDR2 consisting of the amino acid sequence of TSNLQSG, (f-1) Light chain CDR3 consisting of the amino acid sequence of QQYSKFPYT.

2. A monoclonal antibody or antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-2) to (c-2) and the light chain CDR1 to CDR3 of the following (d-2) to (f-2), (a-2) heavy chain CDR1 consisting of the amino acid sequence of GYTFSNYWIE, (b-2) heavy chain CDR2 consisting of the amino acid sequence of EILPGSDITNYNEKFKD, (c-2) heavy chain CDR3 consisting of the amino acid sequence of VYYYPGSLAWFAY, (d-2) light chain CDR1 consisting of the amino acid sequence of SASQDISNYLN, (e-2) light chain CDR2 consisting of the amino acid sequence of YTSSLHS, (f-2) Light chain CDR3 consisting of the amino acid sequence of QQYSKLPYT.

3. A monoclonal antibody or antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-3) to (c-3) and the light chain CDR1 to CDR3 of the following (d-3) to (f-3), (a-3) heavy chain CDR1 consisting of the amino acid sequence of GYTFSSYWIE, (b-3) a heavy chain CDR2 consisting of the amino acid sequence of EILPGSGNTYYNEKFKG, (c-3) heavy chain CDR3 consisting of the amino acid sequence of WEWLLRLYGLDY, (d-3) light chain CDR1 consisting of the amino acid sequence of KASQSVDYDGESYMN, (e-3) light chain CDR2 consisting of the amino acid sequence of AASNLES, (f-3) Light chain CDR3 consisting of the amino acid sequence of QQSNEDPYT.

4. A monoclonal antibody or antigen-binding fragment thereof comprising the heavy chain CDR1 to CDR3 of the following (a-4) to (c-4) and the light chain CDR1 to CDR3 of the following (d-4) to (f-4), (a-4) heavy chain CDR1 consisting of the amino acid sequence of GFSFSDYFMY, (b-4) a heavy chain CDR2 consisting of the amino acid sequence of TISDGGIYTYYPDSVKG, (c-4) heavy chain CDR3 consisting of the amino acid sequence of DLDYFGSTIAY, (d-4) light chain CDR1 consisting of the amino acid sequence of RSSAGALTTRNYAN, (e-4) light chain CDR2 consisting of the amino acid sequence of GTSNRAP, (f-4) Light chain CDR3 consisting of the amino acid sequence of ALWYSNHWV.

5. A monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 that each have a sequence identity of 60% or more to the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 shown in any one of claims 1 to 4, and having an antigen-antibody reaction with SARS-COV-2.

6. A method for immunoassaying SARS-COV-2, comprising immunoassaying SARS-COV-2 using the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 to react with an antigen-antibody of SARS-COV-2 in a sample.

7. The method according to claim 6, wherein: The immunoassay method is a sandwich method, and the monoclonal antibody or antigen-binding fragment thereof is used for at least one of a label or a solid phase.

8. A SARS-CoV-2 immunoassay device comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

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