Capture antibody and method for detecting antigen using same

Abstract

To provide a capture antibody that enables highly sensitive antigen detection by using single domain antibodies and a method of antigen detection using the same.SOLUTION: Provided is a capture antibody to be used in an antigen detection method using single domain antibodies. The capture antibody comprises a multimer of two or more single domain antibodies tandemly linked to each other through spacers, where each of the spacers is a peptide of 5-30 amino acid residues in length. The CV value of the end-to-end distances between the peptides as calculated by molecular dynamics simulation, that is, (standard deviation of the end-to-end distances between the peptides) / (cumulative average value of the end-to-end distances between the peptides), is 0.3 or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a capture antibody, including a single domain antibody, and a method for detecting an antigen using the same. [Background technology]

[0002] VHH (variable domain of heavy chain of heavy chain antibody) is a single-domain antibody consisting only of the variable domain of a heavy chain antibody found in camelids. Other single-domain antibodies include VNAR (variable domain of new antigen receptor), which is the variable domain of a heavy chain antibody found in sharks. Single-domain antibodies also include variable domains consisting of amino acid sequences artificially modified based on the amino acid sequence information of VHH or VNAR.

[0003] While VHHs exhibit binding activity equivalent to that of IgG antibodies, they have the following characteristics: (1) their molecular weight is approximately one-tenth that of IgG antibodies, and they are expected to target novel epitopes that conventional antibodies cannot bind to; (2) unlike IgG antibodies, they have a highly reversible protein structure and are highly resistant to heat and pressure; and (3) unlike IgG antibodies, they can be produced using microorganisms such as yeast and bacteria. Furthermore, (4) VHHs are highly compatible with in vitro antibody selection techniques such as cDNA display and phage display, and can be developed in a shorter period of time than IgG antibodies obtained by immunizing mice or rabbits. Therefore, VHHs are expected to be used as alternatives to conventional immunoglobulins such as IgG and IgA antibodies, which have been used in pharmaceuticals and diagnostic reagents.

[0004] In response to the recent global spread of COVID-19 caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), POCT (Point of Care Testing) The need for g) is greater than ever before.

[0005] A typical POCT is an antigen test kit based on the immunochromatography method. In immunochromatography, an antibody bound to pre-prepared gold colloids or latex particles (hereinafter referred to as labeled antibody) and an antigen contained in the test sample form a complex and move on a nitrocellulose membrane by capillary action, and an antibody (hereinafter referred to as capture antibody) that has been immobilized on the nitrocellulose membrane in advance binds to the antigen-labeled antibody complex, resulting in color development. Currently, most commercially available immunochromatography kits use monoclonal or polyclonal IgG antibodies derived from mice, rabbits, etc.

[0006] VHHs also have clear advantages in the development of diagnostic reagents. In particular, VHHs have the following advantages over IgG antibodies: (1) they can be densely immobilized on particles or nitrocellulose membranes, allowing for the presentation of more paratopes on the substrate; (2) they are highly stable as proteins, leading to the expectation of superior shelf life; (3) they can be mass-produced inexpensively by microorganisms, further reducing manufacturing costs; and (4) they lack the Fc region, which can cause nonspecific reactions in immunochromatography.

[0007] However, there are challenges in using VHHs in antigen testing methods such as immunochromatography. VHHs, which consist only of variable regions, have the following known characteristics: (1) their molecular weight is lower than that of IgG antibodies, resulting in poor physical adsorption to substrates; (2) their binding activity is easily reduced due to protein denaturation during immobilization on a substrate; and (3) unlike IgG antibodies, VHHs lack an Fc region, making it difficult to control the orientation of random adsorption to a substrate. For these reasons, when VHHs are used as capture antibodies, their binding activity is easily reduced. In other words, it is known that highly sensitive antigen detection is difficult when VHHs are used as capture antibodies in immunochromatography.

[0008] To address this issue, Non-Patent Document 1 uses a method in which two types of antibodies, a VHH and a biotin-labeled VHH, are pre-prepared as labeled antibodies on a conjugation pad, and streptavidin, which exhibits binding activity to biotin, is immobilized on a nitrocellulose membrane. That is, the method forms a complex of labeled antibody-antigen-biotinylated VHH on the nitrocellulose membrane, and then captures the complex with streptavidin (Prior Art 1).

[0009] Furthermore, Non-Patent Document 2 uses VHH as a labeled antibody, and a pre-prepared biotinylated VHH-streptavidin complex is immobilized on a nitrocellulose membrane as a capture antibody, which is a method in which immobilization of VHH on the nitrocellulose membrane depends on the interaction between streptavidin and the nitrocellulose membrane (Prior Art 2).

[0010] Furthermore, Non-Patent Document 3 reports that single-chain variable region fragments, in which variable regions consisting of VH and VL, the minimum unit for IgG antibodies to recognize antigens, are linked via a peptide spacer, are used as capture antibodies in immunochromatography using a cellulose membrane. Although using a single-chain variable region fragment as a capture antibody poses a problem in detection sensitivity, this issue is resolved by using a carbohydrate-binding module that binds to the cellulose membrane (Prior Art 3). Specifically, the authors report that linking the single-chain variable region fragment to the carbohydrate-binding module controls the amount and orientation of the single-chain variable region fragment immobilized on the cellulose membrane, thereby improving the binding activity of the single-chain variable region fragment used as a capture antibody.

[0011] In Prior Art 1 and Prior Art 2, VHH must be biotin-modified, and chemical modification methods such as amine coupling are often used. However, it is known that the site-specific modification of VHH is difficult with amine coupling, which often results in a decrease in binding activity. Furthermore, biotin modification and the use of streptavidin are not preferable methods in terms of work steps and cost. In particular, streptavidin also binds to vitamins other than biotin. When such vitamins are present in the analyte, using streptavidin as part of the capture antibody poses a problem: it is subject to competitive inhibition, resulting in a decrease in the function of the capture antibody.

[0012] Regarding Prior Art 3, the method of linking an adsorption module to a substrate to a VHH is an excellent method in terms of the amount immobilized on the substrate, orientation, and avoidance of protein denaturation during adsorption. However, it is necessary to search for an adsorption module for each substrate used, making it difficult to find one that is versatile. Another issue is that, depending on the type of adsorption module linked to a VHH, production using microorganisms may be difficult. Furthermore, it has been noted that the carbohydrate-binding module described in Non-Patent Document 4 does not exhibit binding activity to nitrocellulose membranes. Therefore, Prior Art 3 is a method of immobilizing antibodies that is limited to cellulose membranes as a substrate for immobilizing capture antibodies.

[0013] Similarly to immunochromatography, there is also an antigen testing method that uses a capture antibody, called Enzyme-Linked Immunosorbent Assay (hereinafter referred to as ELISA). Non-Patent Document 4 reports that a method using a single-domain multimer, in which three single-domain antibodies are linked in series via a commonly used flexible linker [(GGGGS)3 (SEQ ID NO: 1)], as a capture antibody, is effective in addressing the low detection sensitivity that occurs when a single-domain antibody is used as a capture antibody, which is also an issue with ELISA (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus. mSphere. 2016;1:e00219-16. [Non-patent document 2] Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections. Sci Rep. 2018;8:9019. [Non-patent document 3] Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays. Sci Rep. 2021;11:7880. [Non-patent document 4] Multivalent nanobody as capture antibody-based enzyme linked immunosorbent assay for detection of 3-phenoxybenzoic acid in urine.Anal Biochem. 2021;632:114390. Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention relates to providing a capture antibody that enables highly sensitive detection of an antigen using a single domain antibody, and a method for detecting an antigen using the same. [Means for solving the problem]

[0016] The present inventors have investigated ways to improve the antigen detection sensitivity of immunoassays using single domain antibodies such as VHH, and have found that immunoassays with high antigen detection sensitivity can be achieved by using a multimer of single domain antibodies containing VHH, in which single domain antibodies containing VHH are linked via a specific spacer, as a capture antibody.

[0017] That is, the present invention relates to the following 1) to 4). 1) A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is a peptide of 5 to 30 amino acids in length, and the CV value of the distance between the two ends of the peptide calculated by molecular dynamics simulation, i.e., the standard deviation of the distance between the two ends of the peptide / the integrated average value of the distance between the two ends of the peptide, is 0.3 or less. 2) A method for detecting an antigen in a sample, comprising the step of contacting the sample with the capture antibody of 1). 3) An antigen detection kit containing the capture antibody of 1). 4) A method for producing the capture antibody of 1) immobilized on a solid phase, which comprises applying a solution of the capture antibody to the surface of a solid phase and then drying the solution. [Effects of the Invention]

[0018] According to the present invention, antigens can be detected with high sensitivity by immunoassay using single domain antibodies. [Brief explanation of the drawings]

[0019] [Figure 1] Graph showing the time course of the cumulative mean and standard deviation of the distance between both ends of a peptide analyzed by molecular dynamics simulation. [Figure 2] Criteria for determining the degree of color development in lateral flow assays. [Figure 3] Lateral flow assay results. DETAILED DESCRIPTION OF THE INVENTION

[0020] In the present invention, the term "antigen detection method" refers to a method for specifically detecting an antigen in a sample by utilizing the properties of an antibody that binds to a specific substance (referred to as an antigen), and refers to an immunoassay that uses at least a capture antibody to immobilize the antigen on a solid phase. Representative examples include sandwich immunoassays, but also include immunoassays that use only a capture antibody, such as biolayer interference, surface plasmon resonance, field-effect transistor, electrochemical impedance spectroscopy, fluorescence anisotropy measurement, and quartz crystal microbalance.

[0021] A sandwich immunoassay is a method in which two types of antibodies (a capture antibody and a detection antibody) that recognize different epitopes on the antigen to be analyzed are prepared, and the antigen is detected by sandwiching it between the capture antibody and the detection antibody. Sandwich immunoassays include a method in which an antigen captured by a capture antibody is detected using a labeled detection antibody, and a method in which an antigen captured by a capture antibody is detected by adding a detection antibody and a substrate, but either method is acceptable. Specifically, sandwich immunoassays are carried out in the form of ELISA (enzyme-linked immunosorbent assay) or lateral flow assay (also called immunochromatographic assay), but in the present invention, it is preferable to carry out the assay as a lateral flow assay.

[0022] Examples of solid phases for immobilizing capture antibodies include polystyrene reaction plates used in ELISA, nitrocellulose or cellulose acetate reaction membranes used in lateral flow assays, biosensors used in biolayer interferometry, gold-coated biochips used in surface plasmon resonance, and electrodes in electrochemical impedance spectroscopy. Methods for immobilizing a capture antibody on a solid phase are not limited as long as they do not impair the function of the capture antibody, and include physical adsorption, specific substance interaction, and covalent bonding. Physical adsorption methods include applying a capture antibody solution to a solid phase. Another example of physical adsorption is applying a capture antibody solution to a solid phase and then drying it. A specific substance interaction method involves attaching a tag molecule that adsorbs to a specific substance to the capture antibody, and then reacting the capture antibody with a solid phase bearing a molecule on its surface that the tag molecule adsorbs. When a histidine tag is used as the tag molecule, an example of this is the reaction with a solid phase bearing nickel ions on its surface, such as by coating it with Ni-NTA. A covalent bonding method includes amine coupling. A carboxyl group on the solid phase can be activated and esterified to immobilize the capture antibody via the amino group. Among these, immobilization by physical adsorption is preferred in sandwich immunoassays, specifically, a method in which a capture antibody solution is applied to a solid phase is preferred in ELISA, and a method in which the capture antibody solution is applied to a solid phase and then dried is preferred in lateral flow assays. In surface plasmon resonance, a method based on the interaction between specific substances is preferred, and a method in which a tag molecule is added to the capture antibody is preferred.

[0023] Antigens to be detected by the above antigen detection method are not particularly limited, but typical antigens include viral antigens, tumor-associated antigens, metabolic substances, antibodies, drugs, enzymes, etc., with viral antigens being preferred. Examples of viruses that can be used as viral antigens include influenza virus, coronavirus, SARS coronavirus, SARS coronavirus-2, respiratory syncytial virus, mumps virus, Lassa virus, dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, rubella virus, human immunodeficiency virus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, rotavirus, coxsackievirus, enterovirus, sapovirus, human herpesvirus, vaccinia virus, hepatitis B virus, adenovirus, B19 virus, papovavirus, and human papillomavirus.

[0024] In the present invention, a single-domain antibody refers to an antibody that has the property of specifically binding to an antigen via a single variable region (antigen-binding domain). Single-domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy-chain single-domain antibodies) and antibodies whose variable region consists only of the variable region of a light chain (light-chain single-domain antibodies). VHHs, which are heavy-chain antibodies identified in camelids (e.g., camels, llamas, alpacas, etc.), and VNARs, which are heavy-chain antibodies derived from cartilaginous fish (e.g., sharks), are known as types of single-domain antibodies, and VHHs are preferred as the single-domain antibodies of the present invention. VHHs have a molecular structure in which the antigen-binding domain of a heavy-chain antibody is excised, and, like the heavy chains of conventional antibodies, have three antigen-binding loops (antigen complementarity-determining regions; CDRs).

[0025] In the present invention, the single domain antibody may be an antibody having affinity for any antigen, but is preferably a VHH having affinity for a protein. The protein antigen may be derived from a virus, and the virus may be any type of virus, regardless of the type of nucleic acid (RNA or DNA) or whether it is enveloped or not. However, among the above-mentioned viruses, influenza virus, SARS-CoV, SARS-CoV-2, respiratory syncytial virus, norovirus, dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, and tick-borne encephalitis virus, which have RNA as nucleic acid, are preferred, and SARS-CoV, SARS-CoV-2, and norovirus are more preferred, with SARS-CoV-2 and norovirus being even more preferred. In the present invention, the use of a VHH having affinity for SARS-CoV-2 as the single domain antibody enables detection of SARS-CoV-2 in a sample.

[0026] Examples of VHHs with affinity for SARS-CoV-2 include antibodies or peptides described in International Publication No. 2021 / 221136, International Publication No. 2022 / 071581, Patent Application No. 2022-065934, International Publication No. 2022 / 181550, etc. For example, Japanese Patent Application No. 2022-065934 discloses EN2 (sequence number 2), EN3 (sequence number 3), EN4 (sequence number 4), EN5 (sequence number 5), EN6 (sequence number 6), EN11 (sequence number 7), and EN12 (sequence number 8) as VHHs that exhibit binding activity against the N protein of SARS-CoV-2; Japanese Patent Application No. 2023-064507 discloses PN4 (sequence number 9), PN10 (sequence number 10), PN27 (sequence number 11), PN44 (sequence number 12), PN46 (sequence number 13), and PN63 (sequence number 14); and International Publication No. 2022 / 181550 discloses CoVHH-N1 (sequence number 15) and CoVHH-N2 (sequence number 16).

[0027] SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory disease (COVID-19). Its viral genome is a single-stranded, positive-sense RNA virus with approximately 29,903 base pairs. The above-mentioned antibodies bind to SARS-CoV-2, specifically, to the N protein of SARS-CoV-2. The binding ability to SARS-CoV-2 can be evaluated by methods known to those skilled in the art, such as ELISA, immunochromatography, isothermal titration calorimetry, biolayer interferometry, surface plasmon resonance, and the equilibrium dissociation constant (KD).

[0028] The structural domain of a single domain antibody, including a VHH, typically has three CDRs: CDR1, CDR2, and CDR3. A CDR (Complementarity Determining Region) contains a sequence-variable antigen recognition site or a random sequence region, and is also called a hypervariable region. In the structural domain, the three CDRs are present in the order of CDR1, CDR2, and CDR3 from the N-terminus. A framework region is a region in the variable region of an antibody molecule excluding the CDRs, and refers to a highly conserved region. That is, one embodiment of the structural domain of the single domain antibody includes a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4), in this order.

[0029] In the present invention, single-domain antibodies can be used as both capture and detection antibodies in immunoassays. In such cases, they can be used as single-domain antibodies or as multimers in which single-chain antibodies, heavy-chain antibodies, or variable region fragments are linked via peptide spacers or the like. However, at least the capture antibody is a single-domain antibody multimer, preferably a dimer, in which two or more different single-domain antibodies that exhibit binding activity to the same or the same antigen are linked in series via peptides that are 5 to 30 amino acids long and have a CV value (standard deviation of the distance between the ends of the peptides / integrated average of the distance between the ends of the peptides) of 0.3 or less, as calculated by molecular dynamics simulation. This improves the antigen detection sensitivity of the immunoassay.

[0030] The present inventors hypothesized that when single-domain antibodies are linked via peptide spacers to form multimers, one end of the multimeric molecule is adsorbed to a substrate, and then the multimer undergoes a morphological change over time, and if the other end also adsorbs to the substrate (double-end adsorption), the exposure of the antibody exhibiting binding activity decreases, leading to its susceptibility to inactivation. The inventors hypothesized that such morphological changes of multimers over time are significantly affected by the degree of spacer flexibility, and that in the case of multimers linked via a flexible spacer with high flexibility, both ends of the antibody are more likely to be adsorbed to the substrate, whereas in the case of multimers linked via a rigid spacer with low flexibility, the morphological change is suppressed, and only one end of the antibody is adsorbed to the substrate, leaving the other end open to the surface without being adsorbed, thereby increasing the exposure of the antibody. As will be described in the Examples below, the inventors have found that when peptides 5 to 30 amino acids in length and having a CV value of the distance between the two ends of the peptide calculated by molecular dynamics simulation, i.e., the standard deviation of the distance between the two ends of the peptide divided by the cumulative average value of the distance between the two ends of the peptide, are selected as peptide spacers and used to link single domain antibodies, they can produce capture antibodies with excellent antigen detection sensitivity without reducing binding activity.

[0031] Peptides generally form a fixed tertiary structure in solution, but the atoms that make up a peptide do not remain stationary in solution, but gradually change position. Molecular Dynamics (MD) simulations are used to reproduce such atomic movements in a computer. That is, in the present invention, molecular dynamics simulation refers to a method for simulating the movements of individual atoms in biomolecules such as proteins and peptides on a computer. In MD simulation, steps (1) to (3) are repeated on a computer: (1) determining the arrangement of atoms, (2) calculating the forces acting on the atoms, and (3) calculating the movement of the atoms. Physical quantities and three-dimensional structures that change over time are arbitrarily extracted, and these are statistically processed and displayed as images to analyze the structure and physical properties of biomolecules and compounds. To perform a stable simulation, the calculation system must be relaxed. Therefore, it is often necessary to first perform MD simulations under constant particle number, volume, and temperature conditions (called the NVT ensemble) with positional constraints on the protein's main chain atoms to allow for structural relaxation of the solvent molecules. Then, MD simulations under constant particle number, pressure, and temperature conditions (called the NPT ensemble) are performed to equilibrate the entire calculation system. By using the final structure obtained in the equilibration calculations to perform further MD simulations under the NPT ensemble, stable molecular simulations that reproduce the phenomena that occur at actual temperature and pressure can be continued.

[0032] Examples of molecular dynamics calculation programs for peptides include GROMACS (http: / / www.gromacs.org / ), LAMMPS (https: / / www.lammps.org / ), NAMD (https: / / www.ks.uiuc.edu / Research / namd), CHARMM (https: / / www.charmm.org / ), and AMBER (https: / / ambermd.org / ). For calculations, a conventional constant-temperature, constant-particle-number-pressure (NPT) ensemble calculation is preferred, but is not limited to this.

[0033] In the present invention, the distance between both ends of a peptide is generally calculated by the following procedures 1) and 2), and specifically, it can be calculated by the method shown in the test examples described later. 1) An NPT ensemble MD simulation is performed on a computational system in which one peptide molecule of 5 to 30 amino acids in length is placed in water, and all atomic coordinate data is obtained over time. 2) The distance between the two ends is defined as the norm of the difference in position vectors between the N-terminal nitrogen atom and the C-terminal carbon atom of the peptide, and the average value and standard deviation of the distance between the two ends over the entire simulation time are calculated.

[0034] The cumulative average value of the distance (Å) between both ends of the spacer peptide thus calculated is preferably 10 Å or more, more preferably 20 Å or more, and is preferably 70 Å or less, more preferably 45 Å or less. The coefficient of variation (CV value), i.e., the standard deviation of the distance between the two peptide termini / the cumulative mean value of the distance between the two peptide termini, is then calculated from the integrated mean and standard deviation of the distance (Å) between the two peptide termini. Those with a CV value of 0.3 or less, preferably 0.28 or less, more preferably 0.26 or less, more preferably 0.13 or less, and more preferably 0.12 or less, can serve as spacers linking single domain antibodies in the single domain antibody multimer used as the capture antibody of the present invention. Furthermore, the smaller the CV value, the more preferable. The lower limit may be any value greater than 0, and examples include 0.01, 0.02, and 0.04. In one embodiment, the CV value is a CV value calculated by a 100 ns molecular dynamics simulation employing the CHARMM36 force field and the TIP3P water model at a temperature of 290 K and a pressure of 0.1 MPa, and the CV value is 0.3 or less, preferably 0.28 or less, more preferably 0.26 or less, more preferably 0.13 or less, and more preferably 0.12 or less.

[0035] The fluctuation distance between the two termini (N-terminus and C-terminus) of a peptide calculated by molecular dynamics simulation is known to be an index of the susceptibility of the peptide to fluctuation (Construction of a linker library with widely controllable flexibility for fusion protein design. Appl Microbiol Biotechnol. 2016;100:215-25.). Therefore, the peptide spacer defined in the present invention can also be said to be an amino acid sequence that is easily elongated linearly and exhibits little structural fluctuation. Known structures that are easily elongated linearly and exhibit little structural fluctuation include peptides with an alpha-helical structure and polymers with a helical structure, such as deoxyribonucleic acid.

[0036] The spacer of the present invention can be obtained by screening any peptide having a length of 5 to 30 amino acids for peptides for which the CV value of the distance between both ends of the peptide calculated by the above-mentioned molecular dynamics simulation is 0.3 or less.

[0037] Examples of peptides with a CV value of 0.3 or less include peptides with an alpha-helical structure, such as peptides consisting of the amino acid sequences shown in EAAR (SEQ ID NO: 17), EAAK (SEQ ID NO: 18), EAAAR (SEQ ID NO: 19), EAAAK (SEQ ID NO: 20), EEEERRRR (SEQ ID NO: 21), and EEEEKKKK (SEQ ID NO: 22), as well as peptides containing a large amount of proline, which means that the amino acid molecules themselves have a rigid structure.

[0038] Furthermore, as a peptide having a CV value of 0.3 or less, a peptide that forms a helix structure, an amino acid sequence in which EAAR (SEQ ID NO: 17) is repeated 2 to 7 times [(EAAR) n [n represents an integer of 2 to 7.]], an amino acid sequence in which EAAK (SEQ ID NO: 18) is repeated 2 to 7 times [(EAAK) n[n represents an integer of 2 to 7.]], an amino acid sequence in which EAAAR (SEQ ID NO: 19) is repeated 2 to 6 times [(EAAAR) n [n represents an integer of 2 to 6.]], an amino acid sequence in which EAAAK (SEQ ID NO: 20) is repeated 1 to 6 times [(EAAAK) n [n represents an integer of 1 to 6.]], an amino acid sequence in which EEEERRRR (SEQ ID NO: 21) is repeated 1 to 3 times [(EEEERRRR) n [n represents an integer of 1 to 3.]], an amino acid sequence in which EEEEKKKK (SEQ ID NO: 22) is repeated two or three times [(EEEEKKKK) n [n represents an integer of 1 to 3], or a peptide consisting of a combination thereof.

[0039] In another embodiment, the peptide having a CV value of 0.3 or less is a peptide containing a large amount of proline, preferably a peptide in which 25% or more, preferably 50% or more of the constituent amino acids are proline, specifically an amino acid sequence in which P is repeated 5 to 30 times [P n [n is an integer of 5 to 30], XP or PX [X is any amino acid residue] repeated 5 to 15 times [(X m , (PX) n [m and n independently represent an integer of 5 to 15, and X represents any amino acid residue], or a combination thereof. X represents any amino acid residue, preferably glutamine (Q), glutamic acid (E), lysine (K), proline (P), threonine (T), glycine (G), alanine (A), arginine (R), etc., and more preferably glutamine (Q), glutamic acid (E), lysine (K), or threonine (T) residues. Further, examples of peptides containing a large amount of proline include peptides consisting of the amino acid sequences shown by EPKTPKPQS (SEQ ID NO: 87) and EPKTPKPQSGS (SEQ ID NO: 64).

[0040] Other peptides with a CV value of 0.3 or less include peptides consisting of the amino acid sequences GGGGX (where X represents any amino acid residue) (SEQ ID NO: 23) and GGGGSEAAAK (SEQ ID NO: 88).

[0041] Furthermore, single domain antibody multimers used as capture antibodies of the present invention may contain a peptide tag consisting of a specific amino acid sequence. Examples of peptide tags include peptide tags for protein isolation / purification, such as histidine tag (His tag (SEQ ID NO: 24)), FLAG tag (SEQ ID NO: 25), Myc tag (SEQ ID NO: 26), HA tag (SEQ ID NO: 27), V5 tag (SEQ ID NO: 28), Strep-tag II (SEQ ID NO: 29), CBD (Chitin Binding Domain) tag (SEQ ID NO: 30), and CBP (Calmodulin Binding Peptide) tag (SEQ ID NO: 31). The tag can be linked, for example, directly or via a peptide linker (e.g., GS) to the N-terminus or C-terminus of the single domain antibody multimer used as the capture antibody of the present invention.

[0042] The single domain antibody multimer used as the capture antibody of the present invention may be a conjugate of identical single domain antibodies, or a conjugate of different single domain antibodies, for example, heterologous single domain antibodies that exhibit binding activity to the same antigen. The number of linked antibodies may be 2 to 4, preferably 2 to 3, more preferably 2 (dimer), and more preferably a dimer in which two identical single domain antibodies are linked. For example, a dimer is formed by linking the N-terminus of a peptide linker to the C-terminus of a single domain antibody via a peptide bond, and then binding the N-terminus of another single domain antibody, either the same or different, to the C-terminus of the linker.

[0043] The method for producing the single domain antibody multimers used as the capture antibodies of the present invention is not particularly limited, and they can be easily produced using techniques known in the art. For example, they can be produced by combining solid-phase peptide synthesis with native chemical ligation (NCL) or by genetic engineering. That is, a preferred method involves designing an artificial gene optimized for expression of the target antibody in host cells by subjecting nucleic acids encoding the single domain antibody multimers used as the capture antibodies of the present invention to processes such as codon optimization, incorporating the artificial gene into an appropriate vector, and introducing the vector into host cells to produce the recombinant antibody.

[0044] Host cells used to produce recombinant antibodies include, for example, Escherichia coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus / insect cells, and yeast cells. Expression vectors suitable for various host cells can be used to express antibodies. Examples of expression vectors that can be used include E. coli-derived vectors such as pBR322, pBR325, pUC12, and pUC13; Bacillus subtilis-derived vectors such as pUB110, pTP5, and pC194; shuttle vectors compatible with both E. coli and Bacillus subtilis, such as pHY300PLK; yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified from these. These expression vectors have a replication origin, selection marker, and promoter appropriate for each vector, and may also have, as necessary, an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a nucleotide sequence for expressing a fused tag such as a FLAG tag, His tag, HA tag, or GST tag.

[0045] When expressing single domain antibody multimers from cultured bacterial cells or cells, the bacterial cells or cultured cells are collected after cultivation using known methods, suspended in an appropriate buffer, and disrupted using ultrasound, lysozyme, and / or freeze-thawing, followed by centrifugation or filtration to obtain a soluble extract. The desired single domain antibody multimers can be obtained from the resulting extract by appropriately combining known separation and purification methods. Alternatively, bacteria that secrete and express proteins outside the cell, such as Bacillus subtilis, can also be used to express single domain antibody multimers. In this case, a culture medium separated from the bacterial cells or containing the bacterial cells can be obtained using known methods such as centrifugation or filtration, and the desired single domain antibody multimers can be obtained by appropriately combining known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that primarily utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; methods that utilize differences in isoelectric point, such as isoelectric focusing; and methods that utilize interactions between peptide tags consisting of specific amino acid sequences and substances that bind to them. Examples of peptide tags include peptide tags for protein isolation / purification, such as histidine tag (His tag (SEQ ID NO: 24)), FLAG tag (SEQ ID NO: 25), Myc tag (SEQ ID NO: 26), HA tag (SEQ ID NO: 27), V5 tag (SEQ ID NO: 28), Strep-tag II (SEQ ID NO: 29), CBD (Chitin Binding Domain) tag (SEQ ID NO: 30), and CBP (Calmodulin Binding Peptide) tag (SEQ ID NO: 31).

[0046] The method for detecting an antigen in a sample of the present invention uses a single-domain antibody multimer as a capture antibody, in which two or more of the above-mentioned single-domain antibodies are linked in series via a specific spacer. Specifically, the capture antibody is brought into contact with the sample to be tested, and the antigen (e.g., SARS-CoV-2) in the sample is captured by the capture antibody. The presence of the antigen is confirmed using a detection system that uses a labeling substance such as an enzyme, colored particle, fluorescent dye, luminescent dye, or radioactive substance as a marker. That is, the method for detecting an antigen in a sample of the present invention includes a step of contacting the sample with a capture antibody, and further includes a step of detecting a signal derived from a labeling substance incorporated into the reaction system.

[0047] When detecting SARS-CoV-2 in a sample, examples of samples include biological samples such as tracheal swabs, nasal swabs, throat swabs, nasal washes, nasal aspirates, nasal mucus / nasal blows, saliva, sputum, blood, serum, urine, feces, tissues, cells, and tissue or cell fragments, as well as samples collected from solid surfaces on which the virus may be attached, such as doorknobs and toilets.

[0048] The contact between the capture antibody and the sample may be any as long as it allows a sufficient antigen-antibody reaction, and the concentration, contact amount, and contact time of the single domain antibody may be appropriately set.

[0049] Examples of methods for incorporating a labeling substance into a reaction system include labeling the antigen through an antigen-antibody reaction using a method in which an antibody (primary antibody) that recognizes the target antigen is linked to a labeling substance in advance, or a method in which an antibody (secondary antibody) that binds to the antibody that recognizes the target antigen is linked to a labeling substance in advance; labeling with a substance having a structure that adsorbs to part of the antigen; and labeling the antigen by providing an affinity site to the antigen and labeling it with a substance that has affinity for the affinity site. As the labeling substance, enzymes, colored particles, fluorescent dyes, luminescent dyes, radioactive substances, magnetic substances, conductive substances, quantum dots, etc. are usually used. For example, antigens can be labeled with enzyme labels (e.g., peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, etc.), fluorescent labels (e.g., fluorescamine, fluorescein isothiocyanate (FITC), Oregon Green, carboxyfluorescein, etc.), radioactive labels (e.g., tritium, iodine-125, iodine-131, carbon-14, etc.), luminescent labels (e.g., luminol, luminol derivatives, luciferin, lucigenin, etc.), etc.

[0050] The form of immunoassay to which the antigen detection method of the present invention is applied is not limited, but any method using a capture antibody is sufficient, and it is preferably carried out in the form of an ELISA method or a lateral flow assay, more preferably a lateral flow assay. In the present invention, the lateral flow assay is an immunoassay that operates along a single axis of the lateral flow strip. It is a method for detecting antigens using a two-antibody sandwich method in which a test substance (antigen) is reacted with a labeled antibody bound to a labeled substance that serves as a marker, the resulting antigen-antibody complex is moved across a membrane, and is captured by a capture antibody that has been immobilized in advance on a solid phase such as a membrane. That is, the method for detecting an antigen in a sample of the present invention, which is carried out by a lateral flow assay, includes the steps of forming a complex between a test substance contained in the sample and a labeled antibody, and capturing and detecting the complex with the capture antibody of the present invention. Specifically, the lateral flow assay works as follows. 1) The sample is dropped or applied to the sample pad and accepted by it. 2) The sample received in the sample pad passes through the sample pad by capillary action and moves to the conjugate pad containing the labeled antibody. In the conjugate pad, if an analyte is present in the sample, a complex between the analyte and the labeled antibody is formed. 3) The complex then migrates downstream by capillary action, and is captured at the test line by the immobilized capture antibody of the present invention, whereupon a signal derived from the labeled substance in the complex is detected. That is, if the test substance is present in the sample, a signal derived from the labeled substance is detected at the test line, whereas if the test substance is not present in the sample, no signal is detected at the test line.

[0051] The strip used in lateral flow immunoassays consists of 1) a sample pad, 2) a conjugate or reagent pad, 3) a reaction membrane, 4) an absorbent or waste reservoir, and 5) a backing sheet. It is typically sold as a kit in a convenient dipstick format or in a plastic case. A simpler version is available: a half-strip consisting of 3) a reaction membrane, 4) an absorbent or waste reservoir, and 5) a backing sheet. In this case, the labeled antibody in the conjugate or reagent pad can be premixed with the antigen before use in the half-strip. Here, 1) the sample pad is an adsorption pad into which a liquid sample is introduced. 2) The conjugate pad or reagent pad contains a labeled antibody, such as an antibody labeled with colored particles (usually colloidal gold nanoparticles, latex microparticles, etc.), that specifically binds to the antigen to be analyzed (e.g., SARS-CoV-2 or norovirus). 3) The reaction membrane is generally a nitrocellulose membrane or a cellulose acetate membrane, and is immobilized by applying a solution containing the capture antibody of the present invention specific to the antigen to be analyzed in a line or spot pattern and drying. The control line contains an antibody that captures the labeled antibody. The capture antibody is immobilized on the reaction membrane by applying a predetermined amount of capture antibody solution to the reaction membrane and then drying. 4) The water absorption reservoir or waste reservoir is an absorbent pad designed to absorb the sample that has completely migrated through the reaction membrane by capillary action. 5) The backing sheet serves to support components 1 to 4 or 4 to 5.

[0052] The ELISA method is a method for detecting a target antigen by 1) binding between the capture antibody and the target antigen, followed by 2) binding between the target antigen and a labeled antibody, such as an antibody labeled with an enzyme or fluorescent dye, in a reaction plate made of polypropylene or polystyrene on the surface of which the capture antibody is immobilized. The antigen detection kit of the present invention is a kit used for carrying out the antigen detection method of the present invention, and can include, in addition to the above-mentioned capture antibody of the present invention, a detection antibody, reagents and instruments necessary for detection, such as a solid phase carrier, a buffer solution, an enzyme reaction stopping solution, a microplate reader, etc. When the antigen detection method of the present invention is carried out as a lateral flow immunoassay, an antigen detection kit for carrying out the method may be composed of the above-mentioned 1) sample pad, 2) conjugate pad or reagent pad, 3) reaction membrane, 4) water-absorbing reservoir or waste liquid reservoir, or 3) reaction membrane, 4) water-absorbing reservoir or waste liquid reservoir, and 5) backing sheet.

[0053] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is a peptide 5 to 30 amino acids in length, and the CV value of the distance between the two ends of the peptide calculated by molecular dynamics simulation, i.e., the standard deviation of the distance between the two ends of the peptide / the integrated average value of the distance between the two ends of the peptide, is 0.3 or less. <2> The CV value is 0.3 or less, preferably 0.28 or less, more preferably 0.26 or less, more preferably 0.13 or less, more preferably 0.12 or less; <1> The capture antibody according to claim 1. <3> The CV values ​​are calculated by a 100 ns molecular dynamics simulation using the CHARMM36 force field and the TIP3P water model at a temperature of 290 K and a pressure of 0.1 MPa. <1> or <2> The capture antibody according to claim 1. <4> The CV value is 0.01 or more, 0.02 or more, or 0.04 or more. <1> ~ <3> 1. A capture antibody according to any one of the preceding claims. <5> the CV value is 0.3 or less and 0.01 or more, 0.3 or less and 0.02 or more, 0.3 or less and 0.04 or more, 0.28 or less and 0.01 or more, 0.28 or less and 0.02 or more, 0.28 or less and 0.04 or more, 0.26 or less and 0.01 or more, 0.26 or less and 0.02 or more, 0.26 or less and 0.04 or more, 0.13 or less and 0.02 or more, 0.13 or less and 0.04 or more, 0.12 or less and 0.01 or more, 0.12 or less and 0.02 or more, or 0.12 or less and 0.04 or more; <1> ~ <3> 1. A capture antibody according to any one of the preceding claims. <6> the integrated average distance between both ends of the peptide calculated by molecular dynamics simulation is 10 Å or more and 70 Å or less; <1> ~ <5> 1. A capture antibody according to any one of the preceding claims. <7> the integrated average distance between both ends of the peptide calculated by molecular dynamics simulation is 20 Å or more and 70 Å or less, preferably 20 Å or more and 45 Å or less; <1> ~ <5> 1. A capture antibody according to any one of the preceding claims. <8> the spacer is a peptide that takes a helix structure; <1> ~ <7> 1. A capture antibody according to any one of the preceding claims. <9> Peptides that take on a helical structure (EAAR) n [n represents an integer of 2 to 7.], (EAAK) n [n represents an integer of 2 to 7.], (EAAAR) n [n represents an integer of 1 to 6.], (EAAAK) n [n represents an integer of 1 to 6.], (EEEERRRR) n [n represents an integer of 1 to 3], or (EEEEKKKK) n [n represents an integer of 1 to 3]. <8> The capture antibody according to claim 1. <10> The spacer is a peptide containing 25% or more proline among its constituent amino acids. <1> ~ <7> 1. A capture antibody according to any one of the preceding claims. <11> Peptides containing 25% or more proline among their constituent amino acids are P n (n is an integer of 5 to 30); (XP) mor (PX) n [m and n independently represent an integer of 5 to 15, and X represents any amino acid residue] or a combination thereof; a peptide having an amino acid sequence represented by EPKTPKPQS or EPKTPKPQSGS, <10> The capture antibody according to claim 1. <12> The spacer is a peptide consisting of an amino acid sequence represented by GGGGS or GGGGSEAAAK. <1> ~ <7> 1. A capture antibody according to any one of the preceding claims.

[0054] <13> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is a peptide that has a helix structure. <14> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is (EAAR) n [n represents an integer of 2 to 7.], (EAAK) n [n represents an integer of 2 to 7.], (EAAAR) n [n represents an integer of 1 to 6.], (EAAAK) n [n represents an integer of 1 to 6.], (EEEERRRR) n [n represents an integer of 1 to 3], or (EEEEKKKK) n [n is an integer of 1 to 3]. <15> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is a peptide containing 25% or more proline among its constituent amino acids. <16> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is P n (n is an integer of 5 to 30); (XP)m or (PX) n [m and n independently represent an integer of 5 to 15, and X represents any amino acid residue] or a combination thereof; a capture antibody which is a peptide having the amino acid sequence represented by EPKTPKPQS or EPKTPKPQSGS. <17> A capture antibody used in an antigen detection method using a single domain antibody, which is a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, and the spacer is a peptide consisting of the amino acid sequence represented by GGGGS or GGGGSEAAAK.

[0055] <18> The single domain antibody is preferably a heavy chain single domain antibody or a light chain single domain antibody, more preferably a heavy chain single domain antibody, even more preferably a VHH or VNAR, even more preferably a VHH; <1> ~ <17> 1. A capture antibody according to any one of the preceding claims. <19> A single domain antibody multimer is a multimer of one or more types of single domain antibodies that exhibit binding activity to the same antigen. <1> ~ <18> 1. A capture antibody according to any one of the preceding claims. <20> A single domain antibody multimer is a multimer of two or more types of single domain antibodies that exhibit binding activity to the same antigen. <19> The capture antibody according to claim 1. <21> The single domain antibody multimer is a multimer, preferably a dimer, of two or more identical single domain antibodies. <1> ~ <18> 1. A capture antibody according to any one of the preceding claims. <22> A single domain antibody multimer is a dimer of two identical single domain antibodies linked together. <1> ~ <18> 1. A capture antibody according to any one of the preceding claims. <23> The antigen is preferably a viral antigen, a tumor-associated antigen, a metabolite, an antibody, a drug, or an enzyme, more preferably a viral antigen, and the virus that serves as the viral antigen is preferably influenza virus, coronavirus, SARS coronavirus, SARS coronavirus-2, respiratory syncytial virus, mumps virus, Lassa virus, dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, rubella virus, human immunodeficiency virus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, rotavirus, coxsackievirus, enterovirus, sapovirus, human herpesvirus, vaccinia virus, hepatitis B virus, adenovirus, B19 virus, papovavirus, or human papillomavirus, more preferably SARS coronavirus, SARS coronavirus-2, or norovirus, and more preferably SARS coronavirus-2 or norovirus. <1> ~ <22> 1. A capture antibody according to any one of the preceding claims. <24> Immobilized on a solid phase <1> ~ <23> 1. A capture antibody according to any one of the preceding claims. <25> the solid phase is a reaction membrane in a lateral flow immunoassay, a reaction plate in an enzyme-linked immunosorbent assay, a biosensor used in biolayer interferometry, a gold-coated biochip used in surface plasmon resonance, or an electrode in electrochemical impedance spectroscopy; <24> The capture antibody according to claim 1. <26> The solid phase is a reaction membrane made of nitrocellulose or cellulose acetate or a reaction plate made of polystyrene; <25> The capture antibody according to claim 1.

[0056] <27> A method for detecting an antigen in a sample, comprising: <1> ~ <26> A method comprising contacting a sample with a capture antibody according to any one of the preceding claims. <28> Preferably, it is a sandwich immunoassay, more preferably an enzyme-linked immunosorbent assay, more preferably a lateral flow assay, and even more preferably a lateral flow assay. <27> The method described below.

[0057] <29> <1> ~ <26> An antigen detection kit comprising the capture antibody according to any one of the above items. <30> further comprising a detection antibody, <29> The antigen detection kit according to claim 1. <31> Further, reagents necessary for detection are included. <30> The antigen detection kit according to claim 1.

[0058] <32> Applying the solution of the capture antibody to a solid surface, or applying the solution of the capture antibody to a solid surface and then drying; <24> ~ <26> A method for producing a capture antibody according to any one of the above. [Example]

[0059] (Test Example 1) Prediction of peptide conformation by molecular dynamics simulation Three-dimensional atomic coordinate data for the amino acid sequences of the 13 peptides shown in Table 1 was obtained using the UniProt web service operated by EMBL-EBI (https: / / www.uniprot.org / (UniProt: the Universal Protein Knowledgebase in 2025. Nucleic Acids Res. 2025;53:D609-D617.)). The atomic coordinate data for the target peptides was obtained by removing unnecessary amino acid residue information from the obtained protein atomic coordinate data. A single molecule of the target peptide was placed so that its center of mass coincided with the center of a cubic periodic boundary cell measuring 5 nm x 5 nm x 5 nm for peptides with 16 residues or less, and 8 nm x 8 nm x 8 nm for peptides with more than 16 residues. The density of the system was approximately 1 g / cm. 3 The surrounding area was filled with water molecules so that

[0060] (1) Energy minimization using the steepest descent method for up to 50,000 steps, (2) Equilibration for 100 ps under constant volume conditions at T = 300 K, (3) Equilibration for 100 ps under constant pressure conditions at T = 300 K and P = 0.1 MPa, (4) Equilibration for 10 ns under constant pressure conditions at T = 290 K and P = 0.1 MPa, and (5) Equilibration for 300 ns under constant pressure conditions at T = 290 K and P = 0.1 MPa. The time step per step from (2) onwards was set to 2 fs. In addition, in the equilibration processes of (1)-(4), in order to avoid the collapse of the three-dimensional structure of the peptide due to thermal fluctuations and changes in the volume of the system, the spring constant k is set according to the conditions shown in equation (1). pr = 1,000 kJ mol -1 nm -2 and the position was constrained.

[0061] [Number 1] V pr (r i )=1 / 2k pr |r i -R i | 2 ···(1) [V pr : position constraint potential, k pr : spring constant, r i :Atomic position, R i :Reference atom position〕

[0062] CHARMM36 was selected as the force field parameters. TIP3P was selected as the water model. The cutoff distance for the Lennard-Jones interaction was set to 1.0 nm, and the Coulomb interaction was treated using the Particle Mesh Ewald method. The bond distance involving hydrogen atoms was constrained using the LINCS method. (2) Nose was used for subsequent temperature control. (3) The pressure control from (4) onwards was performed using the Parrinello-Rahman method, with uniform pressure control in all x, y, and z directions of the cubic cell. GROMACS-2022.5 was used for the series of procedures from creating the initial configuration to running the MD simulation.

[0063] The distance between the peptide ends was determined at each time point, and the cumulative mean, standard deviation, and coefficient of variation (CV) were calculated. The time course of the cumulative mean and standard deviation of the distance between the two ends of the peptide analyzed is shown in Figure 1. It was confirmed that the peptide structure was stable after 100 nanoseconds.

[0064] <Evaluation of peptide chain fluctuation> The distance between the peptide ends was determined from the coordinate data obtained from the 100 ns MD simulation, and the cumulative average, standard deviation, and coefficient of variation (CV value) were calculated. The coefficient of variation calculated from the cumulative average and standard deviation of the distance between both ends of the peptides used in the analysis is shown in Table 1. The larger the coefficient of variation, the greater the fluctuation of the peptide used, and conversely, the smaller the coefficient of variation, the greater the fluctuation of the peptide used. The smaller the peptide, the smaller the fluctuations in the physical properties of the peptide tested. n (n is an integer between 2 and 5) has a large CV value and is composed of a helix structure and a proline-rich structure (EAAAK). n (n is an integer between 1 and 5) showed small CV values. From these results, it was concluded that the CV values ​​obtained by this MD simulation can be used as an index of the structural fluctuation of peptides.

[0065] [Table 1]

[0066] (Production Example 1) Production of VHH using protease-deficient recombinant Bacillus subtilis (1) Construction of plasmid for VHH production For EN12 (SEQ ID NO: 8), an anti-SARS-CoV-2 N protein VHH antibody described in Japanese Patent Application No. 2022-065934, VHH dimers were produced using VHH monomers and various spacer types as linkers between VHHs. That is, a VHH dimer was designed that contains, from the N-terminus to the C-terminus of the amino acid sequence, the structure N-terminus-EN12-(spacer)-EN12-C-terminus. Specifically, EN12-EN12-11 (SEQ ID NO: 32) linked without a spacer, EN12-EN12-3 (SEQ ID NO: 34) linked with GGGGS (SEQ ID NO: 33), EN12-EN12-5 (SEQ ID NO: 36) linked by (GGGGS)2 (SEQ ID NO: 35); EN12-EN12-4 (SEQ ID NO: 37) linked by (GGGGS)3 (SEQ ID NO: 1); EN12-EN12-6 (SEQ ID NO: 39) linked by (GGGGS)5 (SEQ ID NO: 38); EN12-EN12-17 (SEQ ID NO: 41) linked with (GPGGA)3 (SEQ ID NO: 40), EN12-EN12-7 (SEQ ID NO: 42) linked with EAAAK (SEQ ID NO: 20); EN12-EN12-8 (SEQ ID NO: 44) linked with (EAAAK)2 (SEQ ID NO: 43), EN12-EN12-9 (SEQ ID NO: 46) linked with (EAAAK)3 (SEQ ID NO: 45), EN12-EN12-10 (SEQ ID NO: 48) linked with (EAAAK)5 (SEQ ID NO: 47), EN12-EN12-12 (SEQ ID NO: 50) linked with (PQ)5 (SEQ ID NO: 49), EN12-EN12-19 (SEQ ID NO: 52) linked with (PQ)8 (SEQ ID NO: 51), (PQ) 10 EN12-EN12-13 (SEQ ID NO: 54) linked with (SEQ ID NO: 53), (PQ) 15 EN12-EN12-14 (SEQ ID NO: 56) linked with (SEQ ID NO: 55), P 15 EN12-EN12-15 (SEQ ID NO: 58) linked with (SEQ ID NO: 57), EN12-EN12-20 (SEQ ID NO: 59) linked with EEEEKKKK (SEQ ID NO: 22), The resulting fragment was designated EN12-EN12-18 (SEQ ID NO: 61) linked by GGGGSEAAAK (SEQ ID NO: 60).

[0067] For PN27 (SEQ ID NO: 11) described in Patent Application No. 2023-064507, an anti-SARS-CoV-2 N protein VHH antibody different from EN12, VHH dimers were produced using VHH monomers and various spacer types as linkers between VHHs. That is, a VHH dimer was designed that contains, from the N-terminus to the C-terminus of the amino acid sequence, the structure N-terminus-PN27-(spacer)-PN27-C-terminus. Specifically, PN27-PN27-1 (SEQ ID NO: 62) linked by (GGGGS)5 (SEQ ID NO: 38) and PN27-PN27-2 (SEQ ID NO: 63) linked by (EAAAK)5 (SEQ ID NO: 47) were used as VHH dimers.

[0068] A VHH monomer was produced for PN10 (SEQ ID NO: 10) described in Japanese Patent Application No. 2023-064507, which is an anti-SARS-CoV-2 N protein VHH antibody different from EN12 and PN27.

[0069] The amino acid sequence of each VHH synthesized in this production example was conferred a His tag sequence (SEQ ID NO: 24) on the C-terminal side via a linker sequence (EPKTPKPQS: SEQ ID NO: 64).

[0070] To synthesize these His-tagged VHHs, the recombinant plasmid pHY-S237 (JP 2014-158430 A) constructed based on pHY300PLK was used as a template. The plasmid sequence was amplified by PCR using the primer set 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 65) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 66) and PrimeSTAR Max DNA polymerase (TaKaRa). The promoter DNA derived from the spoVG gene was amplified by PCR using the Bacillus subtilis 168 genome as a template and the primer set 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 67) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 68). The resulting promoter DNA was integrated into a plasmid sequence using the In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter. A His-tagged VHH expression plasmid was synthesized using Genescript's GenPlus cloning system, with the nucleotide sequence encoding the His-tagged VHH inserted between SEQ ID NO: 69 and SEQ ID NO: 70 of the previously constructed VHH expression plasmid. The constructed plasmid was then introduced into a strain (Dpr8ΔsigF) obtained by deleting eight extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprE) from Bacillus subtilis 874 according to the method described in Japanese Patent Publication No. 2006-174707, and further deleting the sigF gene involved in sporulation according to the method described in Japanese Patent No. 4336082, according to the procedure described in (2) below.

[0071] (2) Construction of recombinant Bacillus subtilis The above plasmids were introduced into Bacillus subtilis strains using the protoplast method described below. Bacillus subtilis was cultured in a 96-well deep-well plate. Bacillus subtilis stocked in a glycerol-containing solution was inoculated into a well containing 0.8 mL of LB liquid medium and cultured overnight at 30°C and 1,500 rpm with shaking. Next, 8 μL of the culture was inoculated into 0.8 mL of fresh LB liquid medium and cultured at 30°C and 1,500 rpm for approximately 2 hours with shaking. After incubation, the culture was collected in a 2 mL tube and centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. 500 μL of SMMP containing 4 mg / mL lysozyme (SIGMA) was added to suspend the resulting pellet, and the tube was incubated at 37°C for 1 hour.

[0072] After incubation, the tube was centrifuged at 3,500 rpm for 10 minutes and the supernatant was removed. 400 μL of SMMP was added to the tube, and the resulting pellet was suspended to form a suspension. 33 μL of the resulting suspension was added to another tube, and each of the plasmids prepared as described above was added and mixed. 100 μL of 40% (w / w) PEG was then added and mixed using a test tube mixer to form a mixture. 350 μL of SMMP was added to this mixture, which was then mixed by inversion and incubated at 30°C for 2 hours. 200 μL of the incubated culture medium was then spread onto agar medium on a plate containing DM3 agar medium prepared in advance and incubated at 30°C for 2-3 days to obtain recombinant Bacillus subtilis.

[0073] (3) Production of VHH The recombinant B. subtilis prepared in (2) was inoculated into 1 mL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C with shaking to obtain a preculture solution. The preculture solution was inoculated into 1 mL of 2xL-mal medium placed in each well of a 96-well deep-well plate at 1% concentration and cultured at 30°C with shaking for 72 hours. At the end of the culture, the entire culture was collected into a 15 mL tube and centrifuged at 4°C and 7,500 rpm for 5 minutes to collect the supernatant. Each VHH contained in the collected supernatant was purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries) according to the protocol provided with the kit. PBS containing 30 mM imidazole was used as the eluent for purification.

[0074] (Test Example 2) Lateral flow assay (1) Sensitization of antibodies to pigment particles NanoAct colored cellulose particles (chemically bonded, Asahi Kasei) were used as the dye particles. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, to which 540 μL of 100 mM MES (pH 6), 7.5 μL of 4% (w / w) EDC, and 15 μL of 4% (w / w) NHS were added. The tube was left to stand at room temperature for 15 minutes to esterify the carboxyl groups. The tube was centrifuged at 13,000 × g for 20 minutes, the supernatant was removed, and 600 μL of 100 mM MES (pH 6) was added and sonicated. 60 μg of COVID-19 Nucleocapsid Monoclonal Antibody, A03100 (BiosPacific) was added as the labeled antibody, followed by vortexing and incubation at 37°C for 120 minutes. Next, 7.2 mL of blocking buffer (1% (w / w) casein, 100 mM boric acid, pH 8.5) was added, followed by vortexing and incubation at 37°C for 60 minutes. The mixture was centrifuged at 13,000 × g for 20 minutes, the supernatant was removed, 600 μL of 100 mM MES (pH 6) was added, and the mixture was sonicated. The mixture was centrifuged again at 13,000 × g for 15 minutes, the supernatant was removed, and 1.3 mL of storage solution (33 mM boric acid, 0.2% (w / w) casein, 15% (w / w) sucrose, pH 9.2) was added, the mixture was sonicated, and the mixture was stored at 4°C until use.

[0075] (2) Assembling the half strip A 5mm x 40mm piece of Hi-Flow Plus HF120 (Merck) was used as the nitrocellulose membrane. A 5mm x 20mm piece of CM5 (Cytiva) was used as the absorbent pad. The nitrocellulose membrane and absorbent pad were overlapped by 10mm and attached with a backing sheet (GL-57888, Lohmann) to form half strips.

[0076] (3) Immobilization of capture antibodies onto nitrocellulose membrane EN12 monomer or EN12 dimer was prepared in advance at 1 mg / mL in PBS containing 30 mM imidazole. 1 μL of antibody was spotted with a pipette at a point 2 cm from the bottom of the strip assembled in (2) above. The antibody was then immobilized by drying at 37°C for 20 minutes.

[0077] (4) Preparation of developing solution 20 mM HEPES, pH 7 was prepared. NaCl and 0.5% (w / w) Tween 20 were added to give final concentrations of 500 mM and 0.5% (w / w), respectively, to prepare an HBST solution, which was used as a developing solution.

[0078] (5) Performance evaluation of the constructed immunochromatography A positive sample was prepared by adding SARS-CoV-2 Nucleocapsid-Fc fusion protein (InvivoGen) to the developer solution to be evaluated and serially diluting it to 1,000 ng / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of the positive sample solution and 20 μL of the sensitized dye particles prepared in (1) were added to one well of a 96-well plate and mixed thoroughly by pipetting. The capture antibody-immobilized membrane prepared in (3) was then immersed in the well and allowed to stand until the solution had completely drained away. As shown in Figure 3, a clearly defined, deep blue spot was designated "++," a fully visible spot was designated "+," a partially visible spot was designated "±," and no visible spot was designated "-."

[0079] The performance evaluation results of each immunochromatography are summarized in Table 2. Note that "±*" indicates stronger color development than "±." Comparative Examples 3, 4, and 5 use EN12 dimers with (GGGGS)5 (SEQ ID NO: 38), (GGGGS)3 (SEQ ID NO: 1), and (GPGGA)3 (SEQ ID NO: 40) as spacers, respectively, and were judged as "±" when evaluated with an antigen amount of 0.5 ng / test. This indicates that the sensitivity is higher than when the EN12 monomer shown in Comparative Example 1 is used as the capture antibody. Of these comparative examples, Comparative Example 4 uses the conventional technology 4 described in Non-Patent Document 4, and this study also demonstrated that higher sensitivity is possible compared to when a VHH monomer is used.

[0080] On the other hand, Examples 1 to 9 each involve an EN12 dimer in which a peptide spacer has a CV value of 0.3 or less for the distance between the two ends of the peptide (standard deviation of the distance between the two ends of the peptide / integrated average value of the distance between the two ends of the peptide) calculated by molecular dynamics simulation. In all Examples, a "+" result was obtained when the antigen amount was evaluated at 0.5 ng / test. This indicates that higher sensitivity was achieved than in Comparative Examples 3 to 5, in which an EN12 dimer in which a peptide spacer has a CV value of 0.37 or more was used. Furthermore, Examples 6 to 9 involve an EN12 dimer in which a peptide spacer has a CV value of 0.12 or less, calculated from the average and standard deviation of the distance between the two ends of the spacer by molecular simulation. In all Examples, a "±" result was obtained when the antigen amount was evaluated at 0.05 ng / test.

[0081] [Table 2]

[0082] (Test Example 3) Prediction of peptide conformation by molecular dynamics simulation According to the method of Test Example 1, however, for EPKTPKPQS (SEQ ID NO: 87) and EPKTPKPQSGS (SEQ ID NO: 64), since there was no corresponding 3D atomic coordinate data registered in UniProt, 3D atomic coordinate data was generated using the web service AlphaFold2 (https: / / alphafold.com / (Highly accurate protein structure prediction with AlphaFold. Nature, 2021;596:583-589.)) provided by EMBL-EBI. In addition, (PQ) 15 Since this peptide could not be accommodated in the periodic boundary cell employed in the method of Test Example 1, a 12 nm x 12 nm x 12 nm cubic periodic boundary cell was employed.

[0083] [Table 3]

[0084] (Production Example 2) Production of VHH using protease-deficient recombinant Bacillus subtilis Variants of VHH monomers were prepared using EN12 (SEQ ID NO: 8). These variants differ in the sequence of the connecting peptide connecting the VHH body (N-terminus) and the His tag. The EN12 monomer was constructed by attaching a His tag (SEQ ID NO: 24) to the C-terminus of EN12 (SEQ ID NO: 8) via the connecting peptide EPKTPKPQS (SEQ ID NO: 87). Similarly, the following monomers were designed: EN12 monomer-2 (SEQ ID NO: 71) incorporating EAAAK (SEQ ID NO: 20) as the connecting peptide; EN12 monomer-3 (SEQ ID NO: 72) incorporating (EAAAK) (SEQ ID NO: 45) as the connecting peptide; EN12 monomer-4 (SEQ ID NO: 73) incorporating (EAAAK) (SEQ ID NO: 47) as the connecting peptide; and EN12 monomer-5 (SEQ ID NO: 74) incorporating SEGEWQQQQHQWAHQE (SEQ ID NO: 75) as the connecting peptide. EN12 monomers—2, 3, 4, and 5—were all prepared according to Preparation Example 1.

[0085] (Test Example 4) Lateral flow assay (2) Following Test Example 2, performance evaluation was performed when EN12 monomer, EN12 monomer-2, EN12 monomer-3, EN12 monomer-4, and EN12 monomer-5 were each used as capture antibodies. The results are shown in Table 4. Comparative Examples 7, 8, and 10 were only able to detect 5 ng / test, as in Comparative Example 1. Comparative Example 9 was only able to detect 0.5 ng / test.

[0086] [Table 4]

[0087] (Test Example 5) Lateral flow assay (3) (1) Sensitization of labeled antibodies to pigment particles Following the procedure of Test Example 2, PN10 was sensitized to the dye particles.

[0088] (2) Assembling the half strip Following the procedure of Test Example 2, half strips were assembled.

[0089] (3) Immobilization of capture antibodies onto nitrocellulose membrane PN27, PN27-PN27-1, or PN27-PN27-2 was prepared in advance at 1 mg / mL in PBS containing 30 mM imidazole. The antibody was applied to the strip assembled in (2) above, 1.5 cm from the bottom, using an antibody applicator (CyberJet2, Musashi Engineering) at a rate of 1 μL / cm. The strip was then dried at 50°C for 24 hours for immobilization.

[0090] (4) Preparation of developing solution A developing solution was prepared to have a pH of 8, containing 200 mM Tris-HCl, 200 mM NaCl, 1.5% Triton X-100, and 0.5% Tween 20.

[0091] (5) Performance evaluation of the constructed immunochromatography SARS-CoV-2 Nucleocapsid-His Recombinant Protein (Sino Biologicals) was added to the developer solution to be evaluated, and serial dilutions of 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL were used to prepare positive samples. 50 μL of the positive sample solution and 20 μL of the dye particles prepared in (1) were added to one well of a 96-well plate and mixed thoroughly by pipetting. The capture antibody-immobilized membrane prepared in (3) was then immersed in the well and allowed to stand until the solution had completely drained. The results are shown in Figure 3. The lower limit of detection when PN27 was used as the capture antibody was 100 ng / mL, 10 ng / mL for PN27-PN27-1, and 1 ng / mL for PN27-PN27-2.

[0092] (Production Example 3) Production of VHH using protease-deficient recombinant Bacillus subtilis Based on the amino acid sequences of the anti-norovirus VHH antibodies (NoVHH53 (sequence number 76) and NoVHH54 (sequence number 77)) described in JP 2024-143793, NoVHH53 dimer (sequence number 78), NoVHH53 monomer (sequence number 79), and NoVHH54 monomer (sequence number 80) were produced according to Production Example 1.

[0093] (Production Example 4) Preparation of virus-like hollow particles of norovirus (1) Cell lines and culture media used The bacterial strains and cell lines used in this study are shown in Table 5. DH5α and DH10bac were cultured in LB medium (polypeptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7-7.4, Wako Pure Chemical Industries). Agar medium contained 15 g / L agarose (Wako Pure Chemical Industries). Culture was performed at 37°C, with shaking (200 rpm) as needed. Antibiotics were added as needed during transformation. Sf9 was cultured in Sf-900III SFM (Thermo Fisher Scientific). 10 mL of 10,000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) was added per liter of medium (hereafter referred to as Sf900 medium). H5 was cultured in Express Five SFM (Thermo Fisher Scientific). 10 mL of 10,000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) and 100 mL of 200 mM L-Glutamine (Thermo Fisher Scientific) were added per liter of medium (hereafter referred to as H5 medium). Both Sf9 and H5 were cultured at 27°C. Subculture of cells was performed in a static culture system using T flasks (Becton Dickinson). Baculovirus amplification and VLP production were performed in a suspension culture system using Erlenmeyer flasks.

[0094] [Table 5]

[0095] (2) Artificial synthesis of genes The sequences of ORF2 and ORF3 encoding the norovirus capsid protein were obtained from the public gene databases NoroNet (https: / / www.rivm.nl / en / noronet) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) (GII.19, virus strain name: swine / GII / OH-QW170 / 03 / US, accession number: AY823306, SEQ ID NO: 81). For genotypes for which sequences were available, the target gene construct was designed by adding four bases (GTGA) upstream of ORF2 to the 5' end of the ORF2 / ORF3 sequence and the 3'-untranslated region of GII.19 (SEQ ID NO: 82) to the 3' end. Synthesis was requested from FASMAC, and the pUCFa vector containing the target construct was obtained.

[0096] (3) Insertion of the NoV gene into the pDEST8 vector Using a plasmid vector containing the artificially synthesized construct as a template, each NoV gene fragment was amplified by PCR using NoV gene fragment amplification primers (SEQ ID NO: 83, SEQ ID NO: 84). Additionally, a linearized pDEST8 vector was obtained using pDEST8 vector (Thermo Fisher Scientific) as a template and inverse PCR primers (SEQ ID NO: 85, SEQ ID NO: 86). KOD Plus Neo (TOYOBO) was used as the PCR enzyme. Each NoV gene fragment was inserted into the pDEST8 vector using the In-Fusion® HD Cloning Kit (Takara Bio Inc.). The NoV gene fragment, linearized pDEST8 vector, and reaction reagents listed in Table 6 were mixed and incubated at 50°C for 15 minutes to obtain the In-Fusion reaction mixture.

[0097] [Table 6]

[0098] (4) Transformation of DH5α 2.5 μL of the infusion reaction solution was added to 100 μL of DH5α competent cells thawed on ice and mixed gently. After standing on ice for 5 minutes, the cells were incubated at 42°C for 45 seconds and then left on ice for 2 minutes. 1 mL of LB medium was added, and the appropriately diluted solution was plated on LB agar medium containing 100 μg / mL ampicillin. After overnight incubation at 37°C, grown colonies were picked and cultured overnight in LB medium containing 100 μg / mL ampicillin. Plasmids were extracted from the culture medium using a QIAprep Spin Miniprep Kit (QIAGEN), and the pDEST8 vector containing the target construct was obtained.

[0099] (5) Construction of recombinant Bacmid (rBacmid) DH10Bac competent cells were thawed on ice, and 1 μL (100 ng) of the pDEST8 vector solution was added to 50 μL of competent cells. After 5 minutes on ice, the cells were incubated at 42°C for 45 seconds and then left on ice for 2 minutes. 900 μL of SOC medium was added, and the cells were allowed to recover for 4 hours at 37°C and 200 rpm. Appropriately diluted bacterial suspension was plated on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. After static cultivation at 37°C, white colonies were picked on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. Transformants containing the target sequence were selected by PCR. The transformant of interest was cultured in LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline, and then rBacmid was extracted from the culture medium using a QIAprep Spin Miniprep Kit.

[0100] (6) Introduction of rBacmid into insect cells To obtain recombinant baculovirus (rBV) carrying the target construct, rBacmid was transfected into Sf9 cells. Sf9 cells were seeded at 8.5 x 106 cells / well in a 6-well plate (Becton Dickinson) and allowed to settle on the bottom of the plate. While the cells were settling, a transfection reagent was prepared as follows: 8 μL of Cellfectin Reagent (Thermo Fisher Scientific) was mixed with 100 μL of Grace's insect medium, unsupplemented (Thermo Fisher Scientific). 100 μL of rBacmid solution (prepared in Grace's insect medium, unsupplemented) at 25 μg / mL was added to this solution, mixed gently by pipetting, and then incubated at room temperature for 15 minutes. Cell settling was confirmed under a microscope, and the medium was removed. Plating medium (1.5 mL Grace's insect medium supplemented with 10% (w / w) FBS, 8.5 mL Grace's insect medium unsupplemented (Thermo Fisher Scientific)) was added at 2.5 mL per well. The entire volume of the prepared transfection reagent was added to the wells and incubated at 27°C for 3 to 5 hours. The supernatant was removed, and 2 mL of Sf900 medium was added. To prevent evaporation of the culture medium, the wells were wrapped in plastic wrap and protected from light with aluminum foil. The wells were then cultured statically at 27°C for 1 week. The collected culture medium was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was used as P0 rBV seed.

[0101] (7) Amplification of rBV seeds 30 mL of Sf9 cell solution prepared at 4 x 105 cells / mL was added to a 250 mL Erlenmeyer flask (Nalgene). 1 mL of P0 rBV seed solution was added, and the cells were cultured at 27°C and 125 rpm for 1 week with shaking. The culture was centrifuged at 8000 rpm for 10 minutes at 4°C, and the collected supernatant was used as P1 rBV seed. The infectious titer of the rBV seed was measured by plaque assay. The obtained rBV seed was dispensed into 1 mL aliquots and frozen and stored at -80°C. If the infectious titer of the rBV seed was insufficient, further amplification was performed using the same procedure as above, if necessary.

[0102] (8) Plaque Assay-1 Sf9 cells were seeded at 2.4 x 106 cells / well in a 6-well plate. After allowing the cells to settle at room temperature, the supernatant was removed and 900 μL of fresh Sf900 medium was added. 100 μL of rBV seed solution diluted 1:10 with Sf900 medium was added to each well. The plates were shielded from light with aluminum foil and incubated at 27°C for 1 hour with gentle agitation every 15 minutes. The supernatant was removed and 3 mL of Overlay 1 medium, prepared as described in Table 7, was added to each well. After allowing the medium to solidify at room temperature, the plates were wrapped in plastic wrap and aluminum foil, inverted, and incubated at 27°C for 4 days.

[0103] [Table 7]

[0104] (9) Plaque Assay-2 Reagents were mixed as shown in Table 8 to prepare Overlay 2 medium. 2 mL of Overlay 2 medium was layered onto each well and allowed to solidify at room temperature. The plate was wrapped in plastic wrap and aluminum foil, inverted, and incubated at 27°C for 2 days. Once plaques were visible, white light was shone onto the bottom of the plate, and the plaques in the wells with the appropriate dilution ratio were counted. The average plaque counts from two wells were multiplied by the dilution ratio, and then multiplied by 10 to obtain the infectious titer (pfu / mL) of the rBV seed.

[0105] [Table 8]

[0106] (10) Production of VLPs VLP production was carried out in a suspension culture system using a 250 mL Erlenmeyer flask. The H5 cell suspension, prepared in H5 medium to a final concentration of 1 x 106 cells / mL, and rBV seeds were added to a 250 mL Erlenmeyer flask, and VLP production was carried out in a 40 mL culture volume. The rBV seeds were added to the culture medium so that the MOI (multiplicity of infection) was 0.01 or greater. The flask was shielded from light with aluminum foil and cultured at 27°C and 125 rpm with shaking for one week. Two days after the start of culture, protease inhibitor (Roche) was added. One tablet of protease inhibitor was dissolved in 1 mL of H5 medium, and 100 μL was added per 10 mL of medium.

[0107] (11) Purification of VLPs The entire culture medium was centrifuged at 8,000 rpm for 10 minutes at 4°C to roughly remove the cells. This was followed by centrifugation at 11,000 rpm for 1 hour at 4°C, and the supernatant was collected in a new centrifuge tube. This supernatant was transferred to a centrifuge tube (Beckman Coulter) and ultracentrifuged at 32,000 rpm for 2 hours at 4°C using an Optima XPN-100 (Beckman Coulter) with an SW32-Ti rotor to precipitate proteins, including VLPs. The supernatant was discarded, and 1 mL of fresh H5 medium was added. The mixture was left to stand at 4°C for several hours to allow the pellet to swell. The swollen pellet was resuspended by pipetting until no precipitate remained. 1.8 g of CsCl (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed per tube, dissolved in 2 mL of H5 medium, and mixed with the pellet suspension. The mixture was placed in a centrifuge tube (Beckman Coulter) and ultracentrifuged at 40,000 rpm for 20 hours at 4°C using an SW55-Ti rotor. After centrifugation, white light was shone from above the tube, and only the VLP-derived band was collected using a pipette. The collected solution was transferred to a centrifuge tube for pelleting. The solution was made up to 1x PBS (Thermo Fisher Scientific) and ultracentrifuged at 32,000 rpm for 2 hours at 4°C to precipitate the VLPs. The supernatant was discarded, and fresh H5 medium was added. The mixture was left to stand overnight at 4°C to swell the pellet. The swollen pellet was suspended well by pipetting to obtain a purified VLP solution.

[0108] (Test Example 6) Lateral flow assay (4) (1) Sensitization of labeled antibodies to pigment particles In accordance with Test Example 2, the NoVHH54 monomer was sensitized to the dye particles.

[0109] (2) Assembling the half strip The nitrocellulose membrane was made of FF120HP Plus (Cytiva) cut to 5 mm x 40 mm. The absorbent pad was made of CM5 (Cytiva) cut to 5 mm x 20 mm. The nitrocellulose membrane and absorbent pad were attached together with a 10 mm overlap and a backing sheet (GL-57888, Lohmann) was used to assemble the half strip.

[0110] (3) Immobilization of capture antibodies onto nitrocellulose membrane The capture antibody used was a NoVHH53 monomer or a NoVHH53 dimer (spacer: EPKTPKPQSGS (SEQ ID NO: 64)). 2 μg of the capture antibody was spotted with a pipette 2 cm from the bottom of the half strip assembled in (2). The antibody was then immobilized by drying at 37°C for 30 minutes. Hereinafter, the site where the capture antibody was immobilized is referred to as the test spot.

[0111] (4) Performance evaluation of the constructed immunochromatography The developing solution was borate buffer (100 mM boric acid, 1% (w / w) casein, 150 mM NaCl, pH 8.2). A GII.19 VLP dispersion was prepared using the developing solution (hereafter referred to as the positive sample). For conditions that did not contain GII.19 VLP, borate buffer was used as the developing solution (hereafter referred to as the negative sample). 100 μL of a positive or negative sample and 20 μL of the sensitized particles prepared in (1) were added to one well of a 96-well plate and mixed thoroughly by pipetting. The half strip prepared in (3) was then placed against the well to immerse it and allowed to stand until the developing solution had completely drained away. The presence or absence of color development in the test spot was then visually determined. The results of the lateral flow assay are shown in Table 9. When NoVHH53 monomer was used as the capture antibody, no color development was observed within the range of antigen concentrations tested. On the other hand, when NoVHH53 dimer was used as the capture antibody, 50 ng of GII.19 VLPs was detected.

[0112] [Table 9]

Claims

1. A capture antibody used in an antigen detection method using a single domain antibody, the capture antibody being a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer, the spacer being a peptide 5 to 30 amino acids in length, and the CV value of the distance between the two ends of the peptide calculated by molecular dynamics simulation, i.e., the standard deviation of the distance between the two ends of the peptide / the integrated average value of the distance between the two ends of the peptide, is 0.3 or less.

2. The capture antibody of claim 1, having a CV value of 0.13 or less.

3. 2. The capture antibody according to claim 1, wherein the cumulative average distance between the two ends of the peptide calculated by molecular dynamics simulation is 10 Å or more and 70 Å or less.

4. 2. The capture antibody according to claim 1, wherein the cumulative average distance between both ends of the peptide calculated by molecular dynamics simulation is 20 Å or more and 70 Å or less.

5. The capture antibody of claim 1 , wherein the spacer is a peptide that forms a helix structure.

6. Peptides that take on a helix structure (EARR) ｎ [n represents an integer of 2 to 7.], (EAAK) ｎ [n represents an integer of 2 to 7.], (EAAAR) ｎ [n represents an integer of 1 to 6.], (EAAAK) ｎ [n represents an integer of 1 to 6.], (EEEERRRR) ｎ [n represents an integer of 1 to 3], or (EEEEKKKK) ｎ The capture antibody according to claim 5, which is a peptide consisting of an amino acid sequence represented by the formula: [n is an integer of 1 to 3].

7. The capture antibody according to claim 1, wherein the spacer is a peptide containing 25% or more proline amino acids among its constituent amino acids.

8. Peptides containing 25% or more proline among their constituent amino acids are called P ｎ [n represents an integer of 5 to 30]; (XP) ｍ Or (PX) ｎ [m and n independently represent an integer of 5 to 15, and X represents any amino acid residue] or a combination thereof; or a peptide consisting of an amino acid sequence represented by EPKTPKPQS or EPKTPKPQSGS.

9. The capture antibody of claim 1 , wherein the spacer is a peptide consisting of the amino acid sequence represented by GGGGS or GGGGS EAAAK.

10. The capture antibody of claim 1, wherein the single domain antibody multimer is a multimer of one or more types of single domain antibodies that exhibit binding activity to the same antigen.

11. The capture antibody according to claim 10, wherein the single domain antibody multimer is a multimer of two or more types of single domain antibodies that exhibit binding activity to the same antigen.

12. The capture antibody of claim 1 , wherein the single domain antibody multimer is a multimer of two or more identical single domain antibodies.

13. The capture antibody of claim 1 , which is immobilized on a solid phase.

14. The capture antibody according to claim 13, wherein the solid phase is a reaction membrane in a lateral flow immunoassay or a reaction plate in an enzyme-linked immunosorbent assay.

15. A method for detecting an antigen in a sample, comprising contacting the sample with a capture antibody according to any one of claims 1 to 14.

16. 16. The method of claim 15, which is a lateral flow assay.

17. An antigen detection kit comprising the capture antibody according to any one of claims 1 to 14.

18. The method for producing a capture antibody according to claim 13, wherein the solution of the capture antibody is applied to a solid surface and then dried.

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