Antigen detection method

Abstract

To provide an antigen detection method capable of detecting an antigen of a virus by using a single domain antibody with high sensitivity.SOLUTION: In an antigen detection method using a single domain antibody, a multimer of the single domain antibodies, in which the two or more single domain antibodies are serially connected through spacers, is used as a capture antibody, and the spacer is made of a peptide with an amino acid length of 8 to 30 and includes a rigid motif with the amino acid length of 2 to 8.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting an antigen using a single domain antibody. [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 about one-tenth that of IgG antibodies, and they are expected to bind to new epitopes that cannot be bound by conventional antibodies; (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 have a very high affinity 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. For these reasons, VHHs are expected to be used as antibodies to replace conventional immunoglobulins such as IgG antibodies and IgA antibodies that have been used in pharmaceuticals and diagnostic drugs.

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

[0005] A typical example of POCT is an antigen test kit based on the immunochromatography method. In the immunochromatography method, an antibody bound to gold colloid or latex particles prepared in advance (hereinafter referred to as labeled antibody) and an antigen contained in the sample form a complex and move on a nitrocellulose membrane by capillary action, and an antibody previously immobilized on the nitrocellulose membrane (hereinafter referred to as capture antibody) binds to the antigen-labeled antibody complex, resulting in coloration. Most of the immunochromatography kits currently available on the market use monoclonal or polyclonal IgG antibodies derived from mice, rabbits, etc.

[0006] The superiority of VHHs is also clear in the development of diagnostic drugs. In particular, the following characteristics of VHHs are considered to be superior to IgG antibodies: (1) VHHs can be densely immobilized on particles or nitrocellulose membranes, allowing more paratopes to be presented on the substrate, (2) VHHs have excellent protein stability, and products can be expected to have better storage stability, (3) VHHs can be mass-produced inexpensively using microorganisms, allowing for further reduction in manufacturing costs, and (4) VHHs do not have an Fc region that can cause nonspecific reactions in immunochromatography.

[0007] However, there are problems with using VHH in antigen testing methods such as immunochromatography. It is known that VHH consisting only of variable regions (1) have a low molecular weight compared to IgG antibodies, and therefore have low physical adsorption performance to substrates, (2) are prone to a decrease in binding activity due to protein denaturation effects during immobilization on substrates, and (3) unlike IgG antibodies, VHH do not have an Fc region, making it difficult to control the orientation in random adsorption to substrates. For these reasons, when VHH are used as capture antibodies, the binding activity is prone to decrease, which is a problem. In other words, it is known that it is difficult to detect antigens with high sensitivity when VHH 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, VHH and biotin-labeled VHH, are prepared in advance on a conjugation pad as labeled antibodies, and streptavidin, which exhibits binding activity to biotin, is immobilized on a nitrocellulose membrane. That is, a complex of labeled antibody-antigen-biotinylated VHH is formed on the nitrocellulose membrane, and the complex is captured by streptavidin (Prior Art 1).

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

[0010] In addition, Non-Patent Document 3 uses a single-chain variable region fragment in which a variable region composed of VH and VL, the minimum unit for an IgG antibody to recognize an antigen, is bound by a peptide spacer as a capture antibody in an immunochromatography method using a cellulose membrane. When a single-chain variable region fragment is used as a capture antibody, there is a problem with detection sensitivity, but this is solved by using a carbohydrate-binding module that binds to the cellulose membrane (Prior Art 3). That is, it is reported that by linking the single-chain variable region fragment to a carbohydrate-binding module, the amount and orientation of the single-chain variable region fragment immobilized on the cellulose membrane can be controlled, thereby improving the binding activity of the single-chain variable region fragment used as a capture antibody.

[0011] In the case of conventional techniques 1 and 2, it is necessary to modify VHH with biotin, and in many cases, chemical modification methods such as amine coupling are used. However, it is known that the amine coupling method often leads to a decrease in binding activity because it is difficult to site-specifically modify VHH. In addition, the use of biotin modification or streptavidin is not a preferable method in terms of work steps and costs. In particular, streptavidin also binds to vitamins other than avidin. When such vitamins are contained in the sample, the use of streptavidin as a part of the capture antibody poses the problem that the function of the capture antibody is reduced due to competitive inhibition.

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

[0013] In addition, there is an Enzyme-Linked Immuno Sorbent Assay (hereinafter referred to as ELISA), an antigen testing method that uses a capture antibody like the immunochromatography method. Non-Patent Document 4 reports that a method (conventional technique 4) using a single-domain multimer in which three single-domain antibodies are linked in series via a commonly used flexible linker [(GGGGS)3] as a capture antibody is effective in addressing the low detection sensitivity when a single-domain antibody is used as a capture antibody, which is also an issue in the ELISA method. [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 method capable of detecting antigens with high sensitivity using single domain antibodies. [Means for solving the problem]

[0016] The present inventors have investigated how to improve the antigen detection sensitivity of immunoassays using single domain antibodies such as VHHs, and have found that an immunoassay with high antigen detection sensitivity can be realized by using a VHH multimer in which VHHs are linked via a specific spacer as a capture antibody. They also found that a specific VHH with affinity for SARS-CoV-2 is suitable for detecting SARS-CoV-2 using the immunoassay.

[0017] That is, the present invention relates to the following 1) and 2). 1) A method for detecting an antigen using a single domain antibody, comprising using as a capture antibody a multimer of single domain antibodies in which two or more single domain antibodies are linked in series via a spacer, the spacer consisting of a peptide of 8 to 30 amino acids in length and including a rigid motif of 2 to 8 amino acids in length. 2) An antigen detection kit for carrying out the method of 1), comprising a capture antibody, a detection antibody and reagents necessary for detection. Effect of the Invention

[0018] According to the present invention, antigens can be detected with high sensitivity by immunoassay using single domain antibodies. In addition, by carrying out the immunoassay using a specific single domain antibody having affinity for SARS-CoV-2 of the present invention, it is possible to rapidly determine whether SARS-CoV-2 infection is caused by acute respiratory disease (COVID-19). [Brief description of the drawings]

[0019] [Figure 1] SDS-PAGE results of His-tagged VHH [Diagram 2]Evaluation of the binding activity of EN12, EN12-EN12-0 (without spacer), EN12-EN12-1 [(GGGGS)2 linkage], EN12-EN12-2 [(GGGGS)3 linkage], EN12-EN12-3 [(GGGGS)5], EN12-EN12-4 [(EAAAK)2], EN12-EN12-5 [(EAAAK)3], EN12-EN12-6 [(EAAAK)5], EN12-EN12-7 [(PQ)5], EN12-EN12-8 [(PQ)10], and EN12-EN12-9 [(PQ)15] to the N protein of SARS-CoV-2 by ELISA [Diagram 3] Criteria for determining the degree of coloration of the test line in the SARS-CoV-2 N protein lateral flow assay [Figure 4] SDS-PAGE results of His-tagged VHH [Diagram 5] Evaluation of binding activity of PN10, PN27, PN44, PN46 and PN63 to SARS-CoV-2 N protein by Sandwich ELISA [Figure 6] Evaluation of binding specificity of PN10, PN27, PN44, PN46 and PN63 to coronavirus N proteins by ELISA [Figure 7] SDS-PAGE results of His-tagged VHH dimers [Figure 8] Evaluation of the binding activity of PN27, PN27-PN27[(EAAAK)5], PN46, and PN46-PN46[(EAAAK)5] to the SARS-CoV-2 N protein by ELISA [Figure 9] SDS-PAGE results of GII.19 VLPs [Figure 10] SDS-PAGE results of His-tagged VHH dimers [Figure 11] Criteria for determining the degree of coloration of the test line in a lateral flow assay when using norovirus antigen [Figure 12] SDS-PAGE results of His-tagged VHH dimers DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The method of the present invention is an antigen detection method using a single domain antibody, in which a single domain antibody multimer in which two or more single domain antibodies are linked in series via a spacer is used as a capture antibody, and the spacer consists of a peptide of 8 to 30 amino acids in length and contains a rigid motif of 2 to 8 amino acids in length.

[0021] The antigen detection method of the present invention refers to a method for specifically detecting an antigen in a sample by utilizing the characteristics of an antibody that binds to a specific substance (called an antigen), and refers to an immunoassay that uses at least a capture antibody to immobilize the antibody on a solid phase. For example, a sandwich immunoassay is typically used, but also includes biolayer interference, surface plasmon resonance, field effect transistor, electrochemical impedance, fluorescence anisotropy measurement, quartz crystal microbalance, and other immunoassays that use only a capture antibody.

[0022] 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 labeled detection antibody and a substrate, and 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.

[0023] Antigens to be detected by the antigen detection method of the present invention are not particularly limited, but representative 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, 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 through a single variable region (antigen-binding domain). Single domain antibodies include antibodies whose variable region consists only of a heavy chain variable region (heavy chain single domain antibody) and antibodies whose variable region consists only of a light chain variable region (light chain single domain antibody). VHH, a heavy chain antibody identified in camelids (e.g., camel, llama, alpaca, etc.), and VNAR, a heavy chain antibody derived from cartilaginous fish (e.g., shark), are known as types of single domain antibodies, and VHH is preferred as the single domain antibody of the present invention. VHH has a molecular structure formed by cutting out the antigen-binding domain of an antibody heavy chain, and has three antigen-binding loops (antigen complementarity determining regions; CDRs) just like the heavy chain of a normal antibody.

[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 virus. The virus may be any type of virus, regardless of the type of nucleic acid (RNA, DNA) and whether it has an envelope or not, but is preferably an influenza virus, coronavirus, SARS coronavirus, SARS coronavirus-2, RS virus, or norovirus, which have RNA as nucleic acid, among the above-mentioned viruses, more preferably SARS coronavirus, SARS coronavirus-2, or norovirus, and more preferably SARS coronavirus-2 or norovirus. In the present invention, the use of VHH having affinity for SARS-CoV-2 as a single domain antibody makes it possible to detect 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, Patent Application No. 2022-065934 discloses EN2 (sequence number 7), EN3 (sequence number 8), EN4 (sequence number 9), EN5 (sequence number 10), EN6 (sequence number 11), EN11 (sequence number 12), and EN12 (sequence number 13) as VHHs that exhibit binding activity against the N protein of SARS-CoV-2, and International Publication No. 2022 / 181550 discloses CoVHH-N1 (sequence number 14) and CoVHH-N2 (sequence number 15). In addition, examples of VHHs that have affinity for SARS-CoV-2 and can detect SARS-CoV-2 with high sensitivity in a lateral flow assay include anti-SARS-CoV-2 antibodies having a structural domain containing CDR1 to 3 shown in (a), (b), (c), (d), or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GFTFSRYDMS (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by AISWNGGSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence represented by LDWLQWDWAY (SEQ ID NO: 18). (b) CDR1 consisting of the amino acid sequence represented by GFTFSSYAMT (SEQ ID NO: 19), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 20), and CDR3 consisting of the amino acid sequence represented by GQHHQELQHWYAWDY (SEQ ID NO: 21). (c) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 22), CDR2 consisting of the amino acid sequence represented by AITWNGGSTY (SEQ ID NO: 23), and CDR3 consisting of the amino acid sequence represented by LQDHNSVLADAY (SEQ ID NO: 24). (d) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 22), CDR2 consisting of the amino acid sequence represented by AISWSGDSTH (SEQ ID NO: 25), and CDR3 consisting of the amino acid sequence represented by LGEIDGLEENDY (SEQ ID NO: 26) (e) CDR1 consisting of the amino acid sequence represented by GFTFSDYAMG (SEQ ID NO: 27), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 20), and CDR3 consisting of the amino acid sequence represented by LGQNKEHVGKRIEDY (SEQ ID NO: 28).

[0027] SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory disease (COVID-19), and its viral genome is a single-stranded positive-strand RNA virus with approximately 29,903 bases. The antibodies (a) to (e) above are antibodies that bind to SARS-CoV-2, and more specifically, they are antibodies that bind 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 interference, surface plasmon resonance, and the equilibrium dissociation constant KD.

[0028] The structural domain of a single domain antibody (VHH) shown in (a) to (e) above has three CDRs, namely, 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. The single domain antibodies described above in (a) to (e) may have framework regions on both ends of CDR1, CDR2, and CDR3. The framework region is a region in the variable region of an antibody molecule excluding the complementarity determining regions, and refers to a highly conserved region. That is, in one embodiment, the structural domain of the single domain antibody has, in this order, a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3 and a fourth framework region (FR4). Examples of amino acid sequences of framework regions in structural domains include the amino acid sequences shown below or amino acid sequences having 80% or more identity to said amino acid sequences. FR1: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 29) FR2: WFRQAPGKGREFVA (SEQ ID NO:30) WFRQAPGKGREGVA (SEQ ID NO:31) FR3: YAESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 32) YADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAV (SEQ ID NO: 33) YADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 34) YAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 35) FR4: WGQGTLVTVSS (SEQ ID NO: 36)

[0029] Examples of framework regions consisting of amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 29 to 36 include framework regions consisting of amino acid sequences having preferably 85% or more, more preferably 90% 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 identity.

[0030] Here, the identity of amino acid sequences refers to the percentage (%) of the number of positions at which identical amino acid residues exist in two amino acid sequences when the two sequences are aligned relative to the total number of amino acid residues. The identity of sequences can be calculated, for example, by performing an analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

[0031] Among the antibodies of the present invention, an antibody having a structural domain (SEQ ID NO: 37) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 being the amino acid sequence shown in SEQ ID NO: 16, CDR2 being the amino acid sequence shown in SEQ ID NO: 17, CDR3 being the amino acid sequence shown in SEQ ID NO: 18, FR1 being the amino acid sequence shown in SEQ ID NO: 29, FR2 being the amino acid sequence shown in SEQ ID NO: 30, FR3 being the amino acid sequence shown in SEQ ID NO: 32, and FR4 being the amino acid sequence shown in SEQ ID NO: 36, and an antibody having a structural domain (SEQ ID NO: 37) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 19, an antibody having a structural domain (SEQ ID NO: 38) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 22, CDR2 is the amino acid sequence shown in SEQ ID NO: 23, CDR3 is the amino acid sequence shown in SEQ ID NO: 24, and FR1 is the amino acid sequence shown in SEQ ID NO: 25; An antibody having a structural domain (SEQ ID NO:39) in which CDR1 is the amino acid sequence shown in SEQ ID NO:22, CDR2 is the amino acid sequence shown in SEQ ID NO:25, CDR3 is the amino acid sequence shown in SEQ ID NO:26, FR1 is the amino acid sequence shown in SEQ ID NO:29, FR2 is the amino acid sequence shown in SEQ ID NO:31, FR3 is the amino acid sequence shown in SEQ ID NO:34, and FR4 is the amino acid sequence shown in SEQ ID NO:36; an antibody having a structural domain (SEQ ID NO:40) in which CDR1 is the amino acid sequence shown in SEQ ID NO:22, CDR2 is the amino acid sequence shown in SEQ ID NO:25, CDR3 is the amino acid sequence shown in SEQ ID NO:26, FR1 is the amino acid sequence shown in SEQ ID NO:29, FR2 is the amino acid sequence shown in SEQ ID NO:31, FR3 is the amino acid sequence shown in SEQ ID NO:34, and FR4 is the amino acid sequence shown in SEQ ID NO:36; and an antibody having a structural domain (SEQ ID NO:41) in which CDR1 is the amino acid sequence shown in SEQ ID NO:27, CDR2 is the amino acid sequence shown in SEQ ID NO:20, CDR3 is the amino acid sequence shown in SEQ ID NO:28, FR1 is the amino acid sequence shown in SEQ ID NO:29, FR2 is the amino acid sequence shown in SEQ ID NO:31, FR3 is the amino acid sequence shown in SEQ ID NO:35, and FR4 is the amino acid sequence shown in SEQ ID NO:36,The clones (SEQ ID NO: 37 is PN10, SEQ ID NO: 38 is PN27, SEQ ID NO: 39 is PN44, SEQ ID NO: 40 is PN46, and SEQ ID NO: 41 is PN63) that are highly reactive to SARS-CoV-2 were obtained by screening using the cDNA display method, and are suitable anti-SARS-CoV-2 antibodies.

[0032] In the method of the present invention, single domain antibodies can be used as both capture and detection antibodies in immunoassays, and in such cases, in addition to being used as single domain antibodies, they can also be used as multimers in which single chain antibodies, heavy chain antibodies, and variable region fragments are linked via peptide spacers or the like, but at least for the capture antibody, a multimer of single domain antibodies is used in which two or more identical or different single domain antibodies are linked in series via a spacer consisting of a peptide of 8 to 30 amino acids in length and containing a rigid motif of 2 to 8 amino acids in length, which dramatically improves the antigen detection sensitivity of the immunoassay.

[0033] In the present invention, the term "rigid motif" refers to a partial peptide or its amino acid sequence that extends linearly. The term "rigidity" is used to indicate the degree to which peptides with an alpha-helical structure and polymers with a helical structure, such as deoxyribonucleic acid, maintain their linearity (also expressed as "persistence length" or "long-range regularity"). In the present invention, from the viewpoint of improving the antigen detection sensitivity of immunoassays, the proportion of the rigid motif in the spacer is preferably 30% or more and 100% or less, more preferably 70% or more and 100% or less, and even more preferably 90% or more and 100% or less.

[0034] Known rigid motifs include peptides that have an alpha-helical structure, such as EAAR (sequence number 1), EAAK (sequence number 2), EAAAR (sequence number 3), EAAAK (sequence number 4), EEEERRRR (sequence number 5), and EEEEKKKK (sequence number 6), as well as proline-rich peptides whose amino acid molecules themselves exhibit a rigid structure.

[0035] In the present invention, as one embodiment, the rigid motif is a peptide that forms a helix structure, specifically, an amino acid sequence in which EAAR (SEQ ID NO: 1) is repeated 3 to 7 times [(EAAR) n [n is an integer from 3 to 7.]], an amino acid sequence in which EAAK (SEQ ID NO: 2) is repeated 3 to 7 times [(EAAK) n [wherein n is an integer from 3 to 7.]], an amino acid sequence in which EAAAR (SEQ ID NO: 3) is repeated 2 to 6 times [(EAAAR) n [n is an integer from 2 to 6.]], an amino acid sequence in which EAAAK (SEQ ID NO: 4) is repeated 2 to 6 times [(EAAAK) n [n represents an integer of 2 to 6.]], an amino acid sequence in which EEEERRRR (SEQ ID NO:5) is repeated two or three times [(EEEERRRR) n [n represents an integer of 2 or 3.]], an amino acid sequence in which EEEEKKKK (SEQ ID NO: 6) is repeated two or three times [(EEEEKKKK) n (n is an integer of 2 or 3).

[0036] In another embodiment, the peptide is a peptide containing a large amount of proline, preferably in which 50% or more of the amino acids constituting the motif are proline, or in which 50% or more of the amino acids constituting the motif are XP or PX (X represents any amino acid residue), specifically, an amino acid sequence having three or more XP or PX (X represents any amino acid residue) [e.g., EPKTPKPQSGS (SEQ ID NO: 132), EPKTPKPQS (SEQ ID NO: 139), etc.], an amino acid sequence in which P, XP or PX (X represents any amino acid residue) is repeated 5 to 15 times, or a combination thereof [(P) l , (XP) m , (PX) nor a combination thereof [l, m, and n each independently represent an integer of 6 to 15, and X represents any amino acid residue.]. Note that X represents any amino acid residue, and preferably includes 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), proline (P), or threonine (T) residues.

[0037] In addition, it is preferable that the glycine content of the amino acids in the portion other than the rigid motif of the spacer containing such a rigid motif is 50% or more and 100% or less. Specifically, the amino acid sequence in the portion other than the rigid motif may contain, for example, (G) n , (GX) n , (GX) n , (GGX) n , (GXG) n , (XGG) n , (GGGX) n , (GGXG) n , (GXGG) n , (XGGG) n , (GGGGX (SEQ ID NO: 135)) n , (GGGXG (SEQ ID NO: 136)) n , (GXGGG (SEQ ID NO: 137)) n , and (XGGGG (SEQ ID NO: 138)) n (wherein n is an integer of 1 to 5, and X is any amino acid residue.)

[0038] Furthermore, the single domain antibody and single domain antibody multimer of the present invention may further comprise 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: 44)), FLAG tag (SEQ ID NO: 45), Strep tag (SEQ ID NO: 46), and HA tag (SEQ ID NO: 47). The tag can be linked, for example, to the N-terminus or C-terminus of the single domain antibody or single domain antibody multimer of the present invention, directly or via a peptide linker (e.g., GS).

[0039] In the method of the present invention, the multimer of single domain antibodies linked via the spacer used as a capture antibody may be either a linkage between identical single domain antibodies or a linkage between 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 binding 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 identical or different single domain antibody to the C-terminus of the linker.

[0040] In the method of the present invention, when the single domain antibodies (VHH) having affinity for SARS-CoV-2 shown in (a) to (e) above are used, suitable examples of combinations of the multimer used as the capture antibody and the detection antibody are shown in Table 1. In the table, the single domain antibodies shown in (a) to (e) are represented as (a), (b), (c), (d), and (e), respectively, and the spacers are represented as Y1 and Y2.

[0041] [Table 1]

[0042] The method for producing the single domain antibody and the antibody multimer of the present invention is not particularly limited, and they can be easily produced by known techniques in the art. For example, they can be produced by combining solid-phase peptide synthesis and native chemical ligation (NCL) methods, or by genetic engineering, but a preferred method is to design an artificial gene optimized for the expression of the target antibody in a host cell by subjecting a nucleic acid encoding the single domain antibody or the antibody multimer of the present invention to a process such as codon optimization, incorporate the artificial gene into an appropriate vector, and introduce the vector into a host cell to produce the recombinant antibody.

[0043] Examples of host cells used in the production of recombinant antibodies include Escherichia coli, Bacillus subtilis, mold, animal cells, plant cells, baculovirus / insect cells, and yeast cells. The expression vector for expressing the antibody can be a vector suitable for various host cells. Examples of expression vectors that can be used include vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common between Escherichia coli and Bacillus subtilis such as pHY300PLK; vectors derived from yeast 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, a selection marker, and a promoter suitable for each vector, and may have an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, in order to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a base sequence for expressing a fused tag such as a FLAG tag, a His tag, an HA tag, or a GST tag.

[0044] When extracting the expressed single domain antibody or antibody multimer of the present invention from cultured bacteria or cells, the bacteria or cultured cells are collected after cultivation by a known method, suspended in an appropriate buffer, and disrupted by ultrasound, lysozyme and / or freeze-thawing, etc., and then a soluble extract is obtained by centrifugation or filtration. The desired single domain antibody or antibody multimer can be obtained from the obtained extract by an appropriate combination of known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that utilize mainly 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 reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0045] According to the antigen detection method of the present invention, the presence or absence of an antigen of interest (e.g., SARS-CoV-2) in a test sample can be confirmed by contacting the sample with the antigen, the sample containing or potentially containing the antigen. Specifically, in the antigen detection method of the present invention, the sample is contacted with a single domain antibody, an antigen (e.g., SARS-CoV-2) in the sample is captured by the single domain antibody multimer, a detection antibody is reacted with the bound product, and the reactant (antigen-antibody bound product) is measured to detect the presence of the antigen in the sample.

[0046] When detecting SARS-CoV-2 in a sample, examples of samples include biological samples such as tracheal swabs, nasal swabs, throat swabs, nasal washings, nasal aspirates, nasal mucus, nasal mucus, saliva, sputum, blood, serum, urine, feces, tissues, cells, and tissue or cell fragments, as well as samples taken from solid surfaces on which the virus may be attached, such as doorknobs and toilets.

[0047] The contact between the single domain antibody and the sample may be any amount that allows sufficient antigen-antibody reaction, and the concentration, contact amount, and contact time of the single domain antibody may be appropriately set.

[0048] The antigen detection method of the present invention is preferably carried out in the form of a lateral flow assay, which is an immunoassay that operates along a single axial direction of a lateral flow strip. The strip in a lateral flow immunoassay consists of 1) a sample pad, 2) a conjugate pad or a reagent pad, 3) a reaction membrane, and 4) an absorbent reservoir or a waste reservoir. The components are immobilized on an inert backing material and are manufactured as a kit in a convenient dipstick format or in a plastic case. Here, 1) the sample pad is an adsorption pad on which the sample is applied. 2) the conjugate pad or reagent pad contains a single domain antibody (detection antibody) specific to the analyte antigen (e.g., SARS-CoV-2) labeled with colored particles (usually colloidal gold nanoparticles, latex microparticles, etc.). 3) the reaction membrane is generally a nitrocellulose membrane or a cellulose acetate membrane, on which a single domain antibody multimer (capture antibody) specific to the analyte antigen is immobilized in a line shape, and the control line contains an antibody that captures the antibody labeled to the particle. 4) the water absorption reservoir or waste liquid reservoir is an absorption pad designed to suck up the sample that has migrated through the reaction membrane by capillary action.

[0049] 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 and 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, the antigen detection kit or SARS-CoV-2 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, and 4) water absorption reservoir or waste liquid reservoir. EXAMPLES

[0050] (Production Example 1) Production of VHH using protease-deficient recombinant Bacillus subtilis (1) Construction of plasmid for VHH production Regarding EN12 (SEQ ID NO: 13) described in Japanese Patent Application No. 2022-065934, EN12-EN12-0 (SEQ ID NO: 59) in which two of the EN12 fragments are directly linked, EN12-EN12-1 (SEQ ID NO: 61) in which the fragments are linked with (GGGGS)2 (SEQ ID NO: 60), EN12-EN12-2 (SEQ ID NO: 63) in which the fragments are linked with (GGGGS)3 (SEQ ID NO: 62), or EN12-EN12-3 (SEQ ID NO: 64) in which the fragments are linked with (GGGGS)5 (SEQ ID NO: 65) are also available. 65), or EN12-EN12-4 (SEQ ID NO: 67) linked by (EAAAK)2 (SEQ ID NO: 66), or EN12-EN12-5 (SEQ ID NO: 69) linked by (EAAAK)3 (SEQ ID NO: 68), or EN12-EN12-6 (SEQ ID NO: 71) linked by (EAAAK)5 (SEQ ID NO: 70), or EN12-EN12-7 (SEQ ID NO: 73) linked by (PQ)5 (SEQ ID NO: 72), or (PQ) 10 EN12-EN12-8 (SEQ ID NO: 75) linked with (SEQ ID NO: 74), or (PQ) 15(SEQ ID NO: 76) was linked to give EN12-EN12-9 (SEQ ID NO: 77). The amino acid sequence of each VHH synthesized in this production example was provided with a His tag sequence (SEQ ID NO: 44) on the C-terminus via a linker sequence (SEQ ID NO: 53). The VHHs synthesized in this manner are designated His-tagged EN12 (SEQ ID NO: 78), His-tagged EN12-EN12-0 (SEQ ID NO: 79), His-tagged EN12-EN12-1 (SEQ ID NO: 80), His-tagged EN12-EN12-2 (SEQ ID NO: 81), His-tagged EN12-EN12-3 (SEQ ID NO: 82), His-tagged EN12-EN12-4 (SEQ ID NO: 83), His-tagged EN12-EN12-5 (SEQ ID NO: 84), His-tagged EN12-EN12-6 (SEQ ID NO: 85), His-tagged EN12-EN12-7 (SEQ ID NO: 86), His-tagged EN12-EN12-8 (SEQ ID NO: 87), and His-tagged EN12-EN12-9 (SEQ ID NO: 88).

[0051] To synthesize these His-tagged VHHs, the recombinant plasmid pHY-S237 (JP Patent Publication No. 2014-158430) constructed based on pHY300PLK was used as a template, and the plasmid sequence was amplified by PCR using the primer set 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 89) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 90) and PrimeSTAR Max DNA polymerase (TaKaRa). The genome of Bacillus subtilis 168 strain was used as a template, and the promoter DNA derived from the spoVG gene was amplified by PCR using the primer set 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 91) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 92). The obtained promoter DNA was incorporated into a plasmid sequence using 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 by incorporating a base sequence encoding a His-tagged VHH between SEQ ID NO: 93 and SEQ ID NO: 94 of the previously constructed VHH expression plasmid using GenPlus cloning by Genescript. The constructed plasmid was introduced into a strain (Dpr8ΔsigF) obtained by deleting eight extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE) from Bacillus subtilis 874 strain according to the method described in JP-A-2006-174707 and further deleting the sigF gene involved in sporulation according to the method described in JP-A-4336082, according to the procedure shown in (2) below.

[0052] (2) Construction of recombinant Bacillus subtilis The above-mentioned plasmid was introduced into the Bacillus subtilis strain by the protoplast method described below. Bacillus subtilis was cultured in a 96-well deep well plate. Bacillus subtilis stocked in a solution containing glycerol was inoculated into a well containing 0.8 mL of LB liquid medium, and cultured overnight with shaking at 30°C and 1,500 r / min. Next, 8 μL of the culture solution was inoculated into fresh 0.8 mL of LB liquid medium, and cultured with shaking at 30°C and 1,500 r / min for about 2 hours. After the culture was completed, the culture solution was collected in a 2 mL tube, 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 obtained pellet, and the tube was incubated at 37°C for 1 hour.

[0053] After the 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 obtain a suspension. 33 μL of the resulting suspension was added to another tube, and the plasmids prepared as above were added and mixed thereto, and 100 μL of 40% PEG was further added and vortexed to obtain 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. Then, 200 μL of the culture solution after incubation was applied onto the agar medium of a plate containing DM3 agar medium that had been prepared in advance, and incubated at 30°C for 2 to 3 days to obtain recombinant Bacillus subtilis.

[0054] (3) Production of VHH The recombinant Bacillus 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 at 1% into 1 mL of 2×L-mal medium placed in each well of a 96-well deep-well plate and cultured at 30°C with shaking for 72 hours. At the end of the culture, all the cultures were collected in 15 mL tubes 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 attached to the kit. PBS containing 30 mM imidazole was used as the eluent during purification.

[0055] (4) Confirmation by SDS-PAGE 2 μL of each His-tagged VHH aqueous solution (both 1 mg / mL), 2.5 μL of NuPAGE™ LDS Sample Buffer (4×) (Thermo Fisher Scientific), 1 μL of NuPAGE™ Sample Reducing Agent (10×) (Thermo Fisher Scientific), and 4.5 μL of H2O were mixed and heated at 100°C. Then, 10 μL of the sample solution was applied to the wells of the gel. As a molecular weight marker, 5 μL of Novex™ Sharp Pre-stained Protein Standard (Thermo Fisher Scientific) was also applied to the wells of the gel. The gel used was NuPAGE™ 10%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well (Thermo Fisher Scientific). The electrophoresis buffer used was prepared by diluting NuPAGE™ MES SDS Running Buffer (20x) (Thermo Fisher Scientific) 20 times with water. The XCell SureLock™ Mini Cell Electrophoresis System (Thermo Fisher Scientific) was used as the electrophoresis tank, and electrophoresis was performed for 40 minutes at a voltage of 200V by connecting it to a PowerEase™ 90W Power Supply (Thermo Fisher Scientific). The gel was then stained with GelCode™ Blue Stain Reagent (Thermo Fisher Scientific) to confirm the presence or absence of a band for the target protein. The results are shown in Figure 1. It was confirmed that the desired His-tagged VHH was produced.

[0056] (Test Example 1) Evaluation of binding activity of VHH by Sandwich ELISA method Pierce TM100 μL of each His-tagged VHH, prepared at 10 μg / mL in PBS, was added to each well of Nickle Coated Plates, Clear, 96-well (Thermo Fisher Scientific), and incubated at room temperature for 1 hour for immobilization. After that, the VHH solution was carefully removed using a pipette, and then 200 μL of PBST was added and carefully removed using a pipette (washing) three times. Next, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added to each well, and the plates were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and then the washing operation was repeated three times. SARS-CoV-2 Nucleocapsid-Fc fusion protein (Fc-tagged N protein, InvivoGen) was prepared at 0, 10, 100, and 1000ng / mL in dilution buffer (20mM Tris-HCl, 500mM NaCl, 0.05% Tween20). 100μL of prepared N protein was added to each well and incubated at room temperature for 1 hour. After that, the target protein solution was carefully removed using a pipette, and the washing operation was repeated three times. Goat pAb to Hu IgG(HRP)(Abcam) was diluted 1 / 5,000 with PBST and added to each well at a concentration of 100μL as a detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the washing operation was repeated three times. As a luminescent substrate, OPD tablets (Thermo Fisher Scientific) were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific) and 100 μL was added to each well. After incubation for 30 minutes at room temperature and in the dark, the absorbance at 450 nm was measured using the GloMax (registered trademark) Explorer System (Promega). The results are shown in Figure 2, and the binding activity of His-tagged EN12 and His-tagged EN12 dimer to N protein was confirmed.

[0057] (Test Example 2) Lateral flow assay (1) (1) Sensitization of labeled antibodies to pigment particles Colored cellulose particles NanoAct (chemically bonded type, Asahi Kasei) were used as the dye particles. Sensitization of the labeled antibody was performed according to the attached protocol. The outline is as follows. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, 540 μL of 100 mM MES (pH 6), 7.5 μL of 4 wt% EDC, and 15 μL of 4 wt% NHS were added thereto, and 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 to remove the supernatant, 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 wt% 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 x g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. The mixture was centrifuged again at 13,000 x g for 15 minutes to remove the supernatant, and 1.3 mL of storage solution (33 mM Boric Acid, 0.2 wt% Casein, 15 wt% Sucrose, pH 9.2) was added, followed by sonication, and stored at 4°C until use.

[0058] (2) Assembling the Half Strip A nitrocellulose membrane was prepared by cutting Hi-Flow Plus HF120 (Merck) to 5 mm × 40 mm. A 5 mm × 20 mm cut CM5 (Cytiva) was used as an absorbent pad. The nitrocellulose membrane and absorbent pad were laminated together with a 10 mm overlap and attached with a backing sheet (GL-57888, Lohmann) to assemble a half strip.

[0059] (3) Immobilization of capture antibodies onto nitrocellulose membrane The capture antibody, His-tagged EN12 or His-tagged EN12 dimer, was prepared in advance at 1 mg / mL in a PBS solution containing 30 mM imidazole. 1 μL of the antibody was spotted with a pipette at a point 2 cm from the bottom end of the strip assembled in (2) above. Then, the antibody was immobilized by drying at 37°C for 20 minutes. Hereinafter, the site where the capture antibody was immobilized is referred to as the test line.

[0060] (4) Preparation of developing solution 20 mM HEPES, pH 7 was prepared. NaCl and Tween 20 were added to give final concentrations of 500 mM and 0.5% to prepare an HBST solution, which was used as a developing solution.

[0061] (5) Performance evaluation of the constructed immunochromatography SARS-CoV-2 Nucleocapsid-Fc fusion protein (InvivoGen) was added to the developing solution to be evaluated, and a positive sample was prepared by diluting it stepwise 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 dye particles prepared in (1) were added to one well of a 96-well plate and mixed well by pipetting. Then, the capture antibody-immobilized membrane prepared in (3) was immersed in the well and left to stand until the liquid had completely flowed away. As shown in Figure 3, a blue dot that could be clearly confirmed on the test line was marked as "++", a dot that could be confirmed visually was marked as "+", a dot that could be confirmed but was very faint was marked as "±", and a dot that could not be confirmed was marked as "-".

[0062] The results of immunochromatographic performance evaluation are shown in Table 2. Compared to when EN12 monomer was used as the capture antibody, when any EN12 dimer was used as the capture antibody, the detection sensitivity was improved, but different results were obtained depending on the characteristics of the peptide used as the linker. When (GGGGS)2, (GGGGS)3 or (GGGGS)5 was used as a highly flexible spacer, a visual judgment of ± was obtained for an antigen amount of 0.5 ng (see Table 2, EN12-EN12-1, EN12-EN12-2, EN12-EN12-3). This result supported the conventional technique 4. On the other hand, when a spacer containing a rigid motif consisting of EAAAK or PQ was used, a visual judgment of ± was obtained even for an antigen amount of 0.05 ng (see Table 2, EN12-EN12-3 to EN12-EN12-9). These results indicate that the physical properties of the spacer used in the VHH dimer used as the capture antibody strongly affect the detection sensitivity, and demonstrate that a spacer containing a rigid motif is suitable for linking the VHH dimer used as the capture antibody.

[0063] [Table 2]

[0064] (Production Example 2) Screening of VHHs that bind to the novel coronavirus N protein 1. Screening target molecule The target molecule used was SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein, Sino Biological).

[0065] 2. Synthesis of Display cDNA (1) Synthesis of initial DNA library PCR amplification was performed using the synthetic VHH library PharmaLogical library (PL library), PL_Upright6-Y, PL_Upright12-Y, PL_Upright6-V, PL_Upright12-V, PL_Roll_12, and PL_Roll_15, as template DNA. The PCR solution was prepared for a 500μL reaction scale. For each test tube, 150μL of each VHH library was added as template DNA, 7.5μL of 20μM Newleft primer (5'-GATCCCGCGAAATTAATACGACTCACTATAGGG-3', sequence number 48), 7.5μL of 20μM NewYtag_for_polyA_cnvk-linker primer (5'-TTTCCACGCCGCCCCCCGTCCT-3', sequence number 49), 250μL of 2xPrimeSTAR MAX (Takara Bio), and 85μL of H2O were added. PCR was performed at 98°C for 1 min, followed by 5 cycles of 98°C for 10 s, 60°C for 5 s, 72°C for 10 s, and 72°C for 1 min. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. An equal amount of beads was added per 1 μL of PCR product, mixed by pipetting, and left to stand for 2 min, after which the supernatant was removed and the beads were washed three times with 400 μL of 70% ethanol. After air drying the beads, 500 μL of Nuclease Free Water (NFW) was added to suspend the beads, and the supernatant was collected. The DNA concentration was then quantified by absorbance measurement (A260 / A280).

[0066] (2) In vitro transcription T7 RiboMAX Express Large Scale RNA Production System (Promega) was used. 25 μL of RiboMAX (trademark) Express T7 2×Buffer, 10 pmol of PCR product, and 5 μL of Enzyme Mix were mixed and incubated at 37°C for 30 minutes. 5 μL of RQ1 RNase-Free DNase was then added and incubated at 37°C for 15 minutes. RNAClean XP (Beckman Coulter) was used to purify the transcription product. 1.8 μL of beads were added per 1 μL of transcription product, mixed by pipetting, and left to stand for 5 minutes. After standing on a magnetic plate until it became transparent, the supernatant was removed. 200 μL of 70% ethanol was added, left to stand for 30 seconds, and the supernatant was removed. This ethanol washing was performed three times. After removing the supernatant, the beads were left to stand on a magnetic plate for 10 minutes and air-dried. The tube was removed from the magnetic plate, 300 μL of NFW was added, the beads were suspended by pipetting, and the tube was left to stand for 5 minutes. The tube was left to stand on the magnetic plate for 1 minute, and the supernatant was collected. After purification, RNA was quantified using NanoPad DS-11FX (DeNovix).

[0067] (3) Ligation of mRNA with puromycin linker 20 μL of 0.25 M Tris-HCl (pH 7.5), 20 μL of 1 M NaCl, 100 pmol of the mRNA prepared in (2), and 100 pmol of cnvK riboG linker were mixed to prepare a reaction solution of 100 μL with NFW. Annealing was performed using the ProFlex PCR System (Life technologies) under the conditions of 90°C for 2 minutes, 70°C for 1 minute, 25°C for 30 seconds, and incubation at 4°C. The ramp rate was set to 0.1°C / second. Next, the sample was irradiated with ultraviolet light at a wavelength of 365 nm for 5 minutes using a Handheld UV Lamp, 6W, UVGL-58, 254 / 365 nm, 100V (Analytik jena US, An Endress+Hauser Company). The mRNA-linker conjugate was protected from light and cooled on ice until use.

[0068] (4) Cell-free translation PUREfrex (registered trademark) 1.0 (Gene Frontier) was used to synthesize the mRNA-VHH conjugate from the mRNA-linker conjugate. 12 μL of Nuclease Free Water, 37.5 μL of Purefrex Solution 1 (Gene Frontier), 3.75 μL of Purefrex Solution 2 (Gene Frontier), 3.75 μL of Purefrex Solution 3 (Gene Frontier), and 18 μL of the mRNA-linker conjugate were mixed. After incubating this reaction solution at 37° C. for 15 minutes, 36 μL of IVV formation buffer (3M KCl, 1M MgCl2) mixture was added, and the mixture was further reacted at 37° C. for 20 minutes to synthesize the mRNA-VHH conjugate.

[0069] (5) Immobilization of streptavidin onto magnetic beads 60 μL of 2×Binding buffer (20 mM Tris-HCl, 2 M NaCl, 0.2% Tween 20, 2 mM EDTA, pH 8) was added to 60 μL of Dynabeads My One Streptavidin C1 (Thermo Fisher Scientific), and suspended by pipetting for 1 minute. The mixture was left on a magnetic plate for 1 minute, and the supernatant was discarded. This washing was performed twice. 75 μL of mRNA-VHH conjugate, 75 μL of 2×Binding buffer, and washed streptavidin magnetic beads were mixed and incubated at room temperature for 30 minutes. The mixture was left on a magnetic plate for 1 minute, and the supernatant was removed. 200 μL of Binding buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% Tween 20, 1 mM EDTA, pH 8) was added, and the mixture was pipetted for 1 minute, and the supernatant was removed. This washing was performed twice.

[0070] (6) Reverse transcription reaction 55.5 μL of Nuclease Free Water, 15 μL of 5×RT Buffer (Nippon Gene), and 1.5 μL of GeneAce Reverse Transcriptase (200 U / μL) (Nippon Gene) were mixed. The mRNA-VHH conjugate immobilized on streptavidin magnetic beads was added to the reaction solution, and the mixture was incubated at 42° C. for 30 minutes to carry out reverse transcription, thereby synthesizing a cDNA-VHH conjugate.

[0071] (7) Cutting out beads His-tag wash buffer (20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, 0.05% Tween 20, pH 7.4) was added to the cDNA-VHH conjugates immobilized on streptavidin magnetic beads, and suspended by pipetting for 1 minute. The mixture was left to stand on a magnetic plate for 1 minute, and the supernatant was discarded. Next, 30 μL of His-tag wash buffer containing 10 U RNase T1 (Thermo Fisher Scientific) was added, suspended by pipetting for 1 minute, and then left to stand at 37°C for 15 minutes to elute the cDNA-VHH conjugates.

[0072] (8) Refining 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed on a magnetic plate for 1 minute, the supernatant was discarded, and the beads were resuspended in His-tag wash buffer. This washing procedure was performed twice. The cDNA-VHH conjugate eluate and the His Mag Sepharose Ni Beads suspension were mixed and incubated at room temperature for 30 minutes, then placed on a magnetic plate for 1 minute, and the supernatant was discarded. 200 μL of His-tag wash buffer was added, and suspended by pipetting for 1 minute. The beads were placed on a magnetic plate for 1 minute, and the supernatant was discarded. This washing procedure was performed twice. 10 μL of His-tag elution buffer (20 mM Sodium phosphate, 500 mM NaCl, 250 mM Imidazole, 0.05% Tween 20, pH 7.4) was added, and the beads were incubated at room temperature for 15 minutes to elute the cDNA-VHH conjugate.

[0073] 3. Selection (1) Immobilization of target molecules and blocking In this study, SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein, Sino Biological) was used as the target molecule. The target molecule, which had been adjusted in concentration with PBS, was immobilized in each well of a Nunc-ImmunoTM Plate II (Thermo Fisher Scientific). Specifically, 100 μL of the target factor, which had been adjusted to 50 μg / mL in selection round 1 (R1), 10 μg / mL in R2 (R2), and 1 μg / mL in rounds R3 and after, was added and incubated overnight at 4 °C. After removing the target factor using a pipette, 5% skim milk / PBST was added and incubated at room temperature for 1 hour. Then, the skim milk / PBST was carefully removed. Finally, the operation of adding and removing 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween 20) (washing) was repeated three times.

[0074] (2) Screening procedures for selection round 1 (R1) In R1, 100 μL of library solution was used, which was prepared by adding 35 μL of HBT (20 mM HEPES, 0.02% Tween20) and 40 μL of HBST to 200 pmol of the synthesized cDNA-VHH linked library. The library solution was added to the wells where the immobilization of the target molecule was completed, and incubated at room temperature for 1 hour. The library solution was then carefully removed. Next, the operation of adding and removing 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween20) (washing) was repeated six times. Finally, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) was added, carefully pipetted 10 times, and incubated at room temperature for 10 minutes. After that, the wells were carefully pipetted 10 times again, and the eluate was collected in a new tube.

[0075] (3) Screening procedures for R2 and later The synthesized cDNA-VHH linked library was adjusted to 100 μL by adding 35 μL of HBT (20 mM HEPES, 0.02% Tween 20) and 40 μL of HBST. 9 pmol of library solution was used in R2, and 18 pmol in R3 and later. In R2 and later, 100 μL of 5% skim milk / PBST was added to each well in advance, incubated at room temperature for 1 hour, and then washed three times with 200 μL of HBST to block the wells. The library solution was added to the blocked wells and incubated at room temperature for 30 minutes. This operation removed VHH clones with affinity to the blocking agent. After that, the library solution was added to the wells where the target molecule had been solid-phased, and incubated at room temperature for 1 hour. The library solution was then carefully removed. Next, 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween 20) was added and removed (washing) six times. Finally, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) was added and carefully pipetted 10 times, and then incubated at room temperature for 10 minutes. After that, the mixture was carefully pipetted again 10 times, and the eluate was collected in a new tube.

[0076] (4) PCR amplification The entire amount of the eluate after selection was used for PCR in R1, half the amount for R2, and 1 / 4 the amount for R3 and after. PCR was prepared by mixing 25 μL of KAPA Hifi Hotstart Ready Mix (×2) (KAPA biosystems), 1.5 μL of 10 μM forward primer (5'-GATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC-3', sequence number 50), 1.5 μL of 10 μM reverse primer (5'-TTTCCACGCCGCCCCCCGTCCT-3', sequence number 49), 12.5 μL of cDNA-VHH conjugate, and 9.5 μL of NFW per PCR tube. Using a ProFlex PCR system (Life technologies), PCR was performed under the following conditions: 2 minutes at 95°C, 22 cycles (R1) or 28 cycles (R2 and later) of [20 seconds at 98°C, 15 seconds at 68°C, 20 seconds at 72°C], and 5 minutes at 72°C. Next, purification of the PCR product was performed using Agencourt AMPue XP (Beckman Coulter). 1.8 μL of beads were added per 1 μL of PCR product, and the mixture was mixed by pipetting and left to stand for 5 minutes. After leaving the mixture on a magnetic plate until it became transparent, the supernatant was removed. Next, 1 mL of 70% ethanol was added, left to stand for 30 seconds, and the supernatant was removed. This operation was repeated twice. After removing the supernatant, the mixture was left to stand on a magnetic plate for 3 minutes, and the beads were air-dried. The tube was removed from the magnetic plate, thoroughly suspended in 45 μL of NFW, and then left to stand for 5 minutes. Finally, the mixture was placed on a magnetic plate for 1 minute, and the supernatant was collected. For the next round, the purified DNA was used for in vitro transcription, followed by synthesis, selection, and manipulation of cDNA display.

[0077] 4. Extraction of VHH candidate sequences (1) Quantification and concentration adjustment of PCR products The PCR products were quantified using a PicoGreen™ dsDNA reagent kit (Thermo Fisher Scientific). The concentration of each sample was adjusted to 100 ng / mL according to the quantification value.

[0078] (2) Preparation of sequencing library 1 st PCR was performed. The reaction mixture was prepared by mixing 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 2.5 μL of the PCR product described in the previous paragraph, and 9 μL of NFW per sample. The primers used were 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNATGGAAGTACAATTAGTTGAATCTGGTGGTGGGCTTG-3' (SEQ ID NO: 51) and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNTGAAGAGACTGTCACCAACGTGCCTTG-3' (SEQ ID NO: 52). PCR was performed at 95°C for 3 minutes, followed by 16 cycles of 98°C for 20 seconds, 62°C for 15 seconds, and 72°C for 20 seconds, and then 72°C for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. 1.8 μL of beads were added per 1 μL of PCR product, mixed by pipetting, and left to stand for 5 minutes. The tube was left to stand on a magnetic plate until it became clear, and then the supernatant was removed. 200 μL of 70% ethanol was added, left to stand for 30 seconds, and then the supernatant was removed. This ethanol washing was performed twice. After removing the supernatant, the tube was left to stand on a magnetic plate for 5 minutes, and the beads were air-dried. The tube was removed from the magnetic plate, 20 μL of NFW was added, and the beads were suspended by pipetting and left to stand for 5 minutes. The tube was left to stand on a magnetic plate for 1 minute, and the supernatant was collected. Next, index PCR was performed. The reaction mixture was prepared by mixing 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1 μL of 10 μM forward index primer (Nextera XT Index Kit v2, Illumina), 1 μL of 10 μM reverse index primer (Nextera XT Index Kit v2, Illumina), 2.5 μL of template DNA, and 8 μL of NFW per sample. PCR was performed at 95°C for 3 minutes, followed by 8 cycles of 98°C for 20 seconds, 55°C for 15 seconds, and 72°C for 30 seconds, and then 72°C for 5 minutes. PCR products were purified using Agencourt AMPure XP (Beckman Coulter). After confirming the size of the PCR product by polyacrylamide gel electrophoresis, the PCR product was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and the molar concentration was calculated according to the following formula (1). Based on the obtained quantitative value, the product was diluted with NFW to a concentration of 4 nM. DNA concentration [ng / μL] / (660[g / mol]×550[bp])×10 6 =DNA concentration [nM] (1)

[0079] (3) Sequence The library was prepared according to the recommended protocol for use with 4 nM libraries. The final concentration of the denatured and diluted sample library was adjusted to 7 pM. The PhiX control in the sample library was adjusted to 5%. Sequencing was performed using MiSeq (Illumina) and MiSeq Reagent Nano Kit v2 500 cycle (Illumina).

[0080] (4) Extraction of VHH amino acid sequences from sequence data and measurement of overlaps The sequence data demultiplexed using the MiSeq Controller was subjected to analysis. The base sequence region encoding VHH was extracted from the sequence data and translated into amino acid sequence. The number of amino acid sequences that perfectly matched each amino acid sequence was then counted. As a result, PN10 (SEQ ID NO: 37), PN27 (SEQ ID NO: 38), PN44 (SEQ ID NO: 39), PN46 (SEQ ID NO: 40), and PN63 (SEQ ID NO: 41) appeared frequently, and were selected as antibodies with high affinity for N protein.

[0081] In the amino acid sequence of PN10 (sequence number 37), positions 1 to 25 are FR1 (sequence number 29), positions 26 to 35 are CDR1 (sequence number 16), positions 36 to 49 are FR2 (sequence number 30), positions 50 to 59 are CDR2 (sequence number 17), positions 60 to 98 are FR3 (sequence number 32), positions 99 to 108 are CDR3 (sequence number 18), and positions 109 to 119 are FR4 (sequence number 36). In the amino acid sequence of PN27 (sequence number 38), positions 1 to 25 are FR1 (sequence number 29), positions 26 to 35 are CDR1 (sequence number 19), positions 36 to 49 are FR2 (sequence number 30), positions 50 to 59 are CDR2 (sequence number 20), positions 60 to 98 are FR3 (sequence number 33), positions 99 to 113 are CDR3 (sequence number 21), and positions 109 to 124 are FR4 (sequence number 36). In the amino acid sequence of PN44 (sequence number 39), positions 1 to 25 are FR1 (sequence number 29), positions 26 to 35 are CDR1 (sequence number 22), positions 36 to 49 are FR2 (sequence number 31), positions 50 to 59 are CDR2 (sequence number 23), positions 60 to 98 are FR3 (sequence number 34), positions 99 to 110 are CDR3 (sequence number 24), and positions 109 to 121 are FR4 (sequence number 36). In the amino acid sequence of PN46 (sequence number 40), positions 1 to 25 are FR1 (sequence number 29), positions 26 to 35 are CDR1 (sequence number 22), positions 36 to 49 are FR2 (sequence number 31), positions 50 to 59 are CDR2 (sequence number 25), positions 60 to 98 are FR3 (sequence number 34), positions 99 to 110 are CDR3 (sequence number 26), and positions 109 to 121 are FR4 (sequence number 36). In the amino acid sequence of PN63 (sequence number 41), positions 1 to 25 are FR1 (sequence number 29), positions 26 to 35 are CDR1 (sequence number 27), positions 36 to 49 are FR2 (sequence number 31), positions 50 to 59 are CDR2 (sequence number 20), positions 60 to 98 are FR3 (sequence number 35), positions 99 to 113 are CDR3 (sequence number 28), and positions 109 to 124 are FR4 (sequence number 36).

[0082] (Production Example 3) Production of VHH using protease-deficient recombinant Bacillus subtilis (2) PN10 (SEQ ID NO: 37), PN27 (SEQ ID NO: 38), PN44 (SEQ ID NO: 39), PN46 (SEQ ID NO: 40), and PN63 (SEQ ID NO: 41) obtained by Production Example 2 were produced. The amino acid sequence of each VHH synthesized in this Production Example was given a His tag sequence (SEQ ID NO: 44) at its C-terminus via a linker sequence (SEQ ID NO: 53). The VHHs synthesized in this manner are called His-tagged PN10 (SEQ ID NO: 54), His-tagged PN27 (SEQ ID NO: 55), His-tagged PN44 (SEQ ID NO: 56), His-tagged PN46 (SEQ ID NO: 57), and His-tagged PN63 (SEQ ID NO: 58). These VHHs represented by SEQ ID NOs: 54 to 58 are collectively referred to as His-tagged VHHs. His-tagged VHHs were produced according to Production Example 1. The results are shown in FIG. 4. Bands were confirmed at around 15 kDa for all samples, and it was revealed that each His-tagged VHH was produced.

[0083] (Test Example 3) Evaluation of binding activity of VHH by Sandwich ELISA method Nunc-Immuno(trademark) 100 μL of each His-tagged VHH, prepared at 20 μg / mL in PBS, was added to each well of Plate II (Thermo Fisher Scientific) and incubated at room temperature for 1 hour for immobilization. After that, the VHH solution was carefully removed using a pipette, and then 200 μL of PBST was added and carefully removed using a pipette (washing) three times. Next, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added to each well, and the wells were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and then the washing operation was repeated three times. SARS-CoV-2 Nucleocapsid-Fc fusion protein (Fc-tagged N protein, InvivoGen) was prepared at 0, 10, 100, and 1000ng / mL in dilution buffer (20mM Tris-HCl, 500mM NaCl, 0.05% Tween20). 100μL of prepared N protein was added to each well and incubated at room temperature for 1 hour. After that, the target protein solution was carefully removed using a pipette, and the washing operation was repeated three times. Goat pAb to Hu IgG(HRP)(Abcam) was diluted 1 / 5,000 with PBST and added to each well at a concentration of 100μL as a detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the washing operation was repeated three times. As a luminescent substrate, OPD tablets (Thermo Fisher Scientific) were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific) and added to each well at 100 μL. After incubation at room temperature for 30 minutes in the dark, the absorbance at 450 nm was measured using the GloMax® Explorer System (Promega).

[0084] The ELISA results are shown in Figure 5, which revealed that His-tagged PN10, His-tagged PN27, His-tagged PN44, His-tagged PN46, and His-tagged PN63 all showed clear binding activity to the SARS-CoV-2 N protein.

[0085] (Test Example 4) Evaluation of binding specificity of VHH by Direct ELISA method Nunc-Immuno(trademark) 50 μL of N protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, prepared at 10 μg / mL in PBS, was added to each well of Plate II (Thermo Fisher Scientific), and incubated overnight at 4 °C for immobilization. The N proteins used are shown in Table 3. After that, the N protein solution was carefully removed using a pipette, and then 200 μL of PBST was added and the operation of carefully removing the solution with a pipette (washing) was repeated three times. Next, 200 μL of 5% skim milk / PBST was added to each well, and the wells were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and the washing operation was repeated three times. Each His-tagged VHH was prepared at 1 μg / mL in PBST, and 100 μL was added to each well and incubated at room temperature for 1 hour. After that, the target protein solution was carefully removed using a pipette, and the washing operation was repeated three times. Peroxidase AffiniPure Goat Anti-Alpaca IgG, VHH domain (Jackson ImmunoResearch) was diluted 1 / 5,000 with PBST and added to each well at 100 μL as a detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the washing operation was repeated three times. As a luminescence substrate, OPD tablets were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific) and 100 μL was added to each well. After that, the plate was incubated at room temperature and in the dark for 30 minutes, and the absorbance at 450 nm was measured using the GloMax (registered trademark) Explorer System.

[0086] [Table 3]

[0087] The results of the Direct ELISA are shown in Figure 6. His-tagged PN10 and His-tagged PN27 were found to bind specifically to the N protein of SARS-CoV-2. In addition, His-tagged PN44, His-tagged PN46, and His-tagged PN63 were found to show specific and clear binding activity to the N protein of SARS-CoV-2, although they also showed some binding activity to the N protein of SARS-CoV.

[0088] (Production Example 4) Production of VHH using protease-deficient recombinant Bacillus subtilis (3) For each VHH of PN27 (SEQ ID NO: 38) and PN46 (SEQ ID NO: 40), VHH dimers were produced by linking the same VHH with (EAAAK)5 (SEQ ID NO: 70). The amino acid sequences of each VHH synthesized in this production example were His-tagged PN27-PN27 (SEQ ID NO: 95) and His-tagged PN46-PN46 (SEQ ID NO: 96). These His-tagged VHH dimers were produced using Bacillus subtilis according to the method shown in Production Example 1. Hereinafter, the monomer of VHH X will be represented as "X" and the dimer as "XX". SDS-PAGE was performed on the produced samples according to Production Example 3, and it was found that all His-tagged VHH dimers were produced as shown in Figure 7.

[0089] (Test Example 5) Evaluation of binding activity of VHH dimers by ELISA method The binding activity of the VHH dimers prepared in Production Example 4 was evaluated by ELISA according to Test Example 4. The binding activity of His-tagged PN27, His-tagged PN27-PN27, His-tagged PN46, and His-tagged PN46-PN46 was compared to evaluate the change in binding activity due to dimerization. The results are shown in FIG. 8, and all VHHs showed good binding activity to N protein. Furthermore, it was revealed that the binding activity of both His-tagged PN27 and His-tagged PN46 was improved by dimerization. Compared with each His-tagged VHH monomer, all of the provided His-tagged VHH dimers had improved binding activity to N protein. From these results, it was found that the binding activity of VHH was enhanced by dimerization, and therefore all of the linked VHH domains contributed to binding to the antigen.

[0090] (Test Example 6) Lateral flow assay (2) Immunochromatography was constructed according to Test Example 2 using His-tagged PN10 as the labeled antibody, His-tagged PN27 or PN27-PN27 as the capture antibody, or His-tagged PN27 as the labeled antibody and His-tagged PN46 or PN46-PN46 as the capture antibody. His-tagged N protein was used as the antigen. The results are shown in Table 4. When PN10 was used as the labeled antibody, detection was possible up to 5 ng when the capture antibody was PN27, and up to 0.05 ng when the capture antibody was PN27-PN27. This means that the detection sensitivity was improved 100-fold by dimerizing PN27 using (EAAAK)5. In addition, when PN27 was used as the labeled antibody, detection was possible up to 50 ng when the capture antibody was PN46, and up to 0.05 ng when the capture antibody was PN46-PN46. This means that dimerization of PN46 using (EAAAK)5 improved the detection sensitivity by 1000-fold. As described above, it was confirmed that when both the labeled antibody and the capture antibody are VHHs, dimerization of the capture antibody improves the detection sensitivity by 100 to 1000-fold.

[0091] [Table 4]

[0092] (Test Example 7) Lateral flow assay (3) Immunochromatography was performed according to Test Example 3 using His-tagged PN10, His-tagged PN63, His-tagged PN44, and His-tagged PN27 as labeled antibodies, and His-tagged PN27-PN27 and His-tagged PN46-PN46 as capture antibodies. However, His-tagged N protein was used as the antigen. The results are shown in Table 8. When PN10 or PN63 was used as the labeled antibody and His-tagged PN27 was used as the capture antibody, up to 32 pg was detected. When His-tagged PN44 was used as the labeled antibody, His-tagged PN27-PN27 was used as the capture antibody, or His-tagged PN277 was used as the labeled antibody and PN46-PN46 was used as the capture antibody, up to 125 pg was detected.

[0093] [Table 5]

[0094] (Test Example 8) Lateral flow assay (4) An immunochromatography was constructed according to Test Example 2 using His-tagged PN10 as the labeling antibody and His-tagged PN27-PN27 as the capture antibody. As antigens, N proteins derived from SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, which are classified in the same coronavirus genus as SARS-CoV-2, were prepared at 100 ng / mL and used. The results are shown in Table 6, and the N protein derived from SARS-CoV-2 could be clearly detected. On the other hand, the N proteins derived from other coronavirus genera could not be detected. In other words, it was confirmed that the immunochromatography constructed in this example can specifically detect only the new coronavirus.

[0095] [Table 6]

[0096] (Production Example 5) 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 7. LB medium (polypeptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7-7.4, Wako Pure Chemical Industries, Ltd.) was used for culturing DH5α and DH10bac. Agar medium contained 15 g / L agarose (Wako Pure Chemical Industries, Ltd.). Cultivation was carried out at 37°C, with shaking (200 rpm) as necessary. Antibiotics, etc. were added during transformation as necessary. Sf-900III SFM (Thermo Fisher Scientific) was used for culturing Sf9. 10 mL of 10000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) was added per 1 L of medium (hereinafter referred to as Sf900 medium). Express Five SFM (Thermo Fisher Scientific) was used for culturing H5. 10 mL of 10000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) and 100 mL of 200 mM L-Glutamine (Thermo Fisher Scientific) were added per 1 L of medium (hereinafter referred to as H5 medium). The culture temperature for both Sf9 and H5 was 27°C. Subculture of cells was performed in a stationary culture system using T flasks (Becton Dickinson). Amplification of baculovirus and VLP production were performed in a suspension culture system using Erlenmeyer flasks.

[0097] [Table 7]

[0098] (2) Artificial synthesis of genes The sequences of ORF2 and ORF3 encoding the norovirus capsid protein were obtained from the public gene database NoroNet (https: / / www.rivm.nl / en / noronet) or NCBI (https: / / www.ncbi.nlm.nih.gov / ) (GII.19, virus strain name: swine / GII / OH-QW170 / 03 / US, accession number: AY823306, SEQ ID NO: 97). For the genotypes for which the sequences were available, the 4 bases (GTGA) upstream of ORF2 were added to the 5' side of the ORF2 / ORF3 sequence, and the 3'-untranslated region of GII.19 (SEQ ID NO: 98) was added to the 3' side to design the desired gene construct. Synthesis was requested from FASMAC, and a pUCFa vector into which the desired construct was inserted was obtained.

[0099] (3) Insertion of NoV gene into pDEST8 vector Using a plasmid vector containing an artificially synthesized construct as a template, each NoV gene fragment was amplified by PCR using primers for amplifying NoV gene fragments (SEQ ID NO: 99, SEQ ID NO: 100). In addition, a linear pDEST8 vector was obtained using inverse PCR primers (SEQ ID NO: 101, SEQ ID NO: 102) with a pDEST8 vector (Thermo Fisher Scientific) as a template. KOD Plus Neo (TOYOBO) was used as the PCR enzyme. Each NoV gene fragment was inserted into the pDEST8 vector using In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio). The NoV gene fragment, linear pDEST8 vector, and reaction reagents were mixed as shown in Table 8 and incubated at 50°C for 15 minutes to obtain an In-Fusion reaction solution.

[0100] [Table 8]

[0101] (4) Transformation of DH5α 2.5 μL of the infusion reaction solution was added to 100 μL of DH5α competent cells melted 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 culture at 37°C, the grown colonies were picked up and cultured overnight in LB medium containing 100 μg / mL ampicillin. Plasmids were extracted from the culture using a QIAprep Spin Miniprep Kit (QIAGEN), and the pDEST8 vector into which the target construct was inserted was obtained.

[0102] (5) Construction of recombinant Bacmid (rBacmid) DH10Bac competent cells were thawed on ice, and 1 μL (100 ng) of pDEST8 vector solution was added to 50 μL of competent cells. pDEST8 vector was introduced by heat shock in the same manner as in 1-4. 900 μL of SOC medium was added to the heat-shocked cells, and recovery culture was performed at 37 °C and 200 rpm for 4 hours. Appropriately diluted bacterial solution 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. Static culture was performed at 37 °C, and white colonies were picked up 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.

[0103] (6) Introduction of rBacmid into insect cells To obtain recombinant baculovirus (rBV) carrying the target construct, rBacmid was introduced into Sf9 cells by transfection. 6Sf9 cells were seeded at 1000 cells / well and allowed to settle on the bottom of the plate. While the cells were settling, the 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 at 25 μg / mL in Grace's insect medium, unsupplemented was added to this solution, and the mixture was gently mixed by pipetting and then incubated at room temperature for 15 minutes. The cells were confirmed to have settled under a microscope, and the medium was removed. Plating medium (1.5 mL Grace's insect medium Supplemented with 10% FBS, 8.5 mL Grace's insect medium Unsupplemented (Thermo Fisher Scientific)) was added at 2.5 mL / well. The prepared transfection reagent was added to the wells in its entirety 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, protected from light with aluminum foil, and cultured at 27°C for 1 week. The collected culture medium was centrifuged at 8000 rpm, 10 min, and 4°C, and the supernatant was used as P0 rBV seed.

[0104] (7) Amplification of rBV seeds 4x10 in a 250mL Erlenmeyer flask (Nalgene) 5 30 mL of Sf9 cell solution prepared at 1000 cells / mL was added. 1 mL of P0 rBV seed solution was added and cultured at 27℃ and 125 rpm for 1 week under rotating conditions. The culture was centrifuged at 8000 rpm for 10 min at 4℃, 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℃. If the infectious titer of the rBV seed was insufficient, further amplification was performed using the same procedure as necessary.

[0105] (8) Plaque Assay-1 2.4x10 in a 6-well plate 6 Sf9 was seeded at 100 cells / well. After leaving the plate at room temperature to allow the cells to settle, the supernatant was removed and 900 μL of fresh Sf900 medium was added. 100 μL of rBV seed solution diluted 10-fold with Sf900 medium was added to each well. The plate was shielded from light with aluminum foil and incubated at 27°C for 1 hour with gentle stirring every 15 minutes. The supernatant was removed and 3 mL of Overlay1 medium prepared as shown in Table 9 was added to each well. After leaving the plate at room temperature until solidified, the plate was wrapped in plastic wrap and aluminum foil, turned upside down, and cultured at 27°C for 4 days.

[0106] [Table 9]

[0107] (9) Plaque Assay-2 The reagents were mixed as shown in Table 10 to prepare Overlay 2 medium. 2 mL of Overlay 2 medium was layered on each well and allowed to stand at room temperature to solidify. The plate was wrapped in plastic wrap and aluminum foil, turned upside down, and allowed to stand at 27°C for 2 days. When plaques could be visually confirmed, white light was applied to the bottom of the plate, and the plaques in the wells with the appropriate dilution ratio were counted. The average number of plaques in two wells was multiplied by the dilution ratio, and then multiplied by 10 to obtain the infectious titer (pfu / mL) of the rBV seed.

[0108] [Table 10]

[0109] (10) Production of VLPs VLP production was carried out in a suspension culture system using a 250 mL Erlenmeyer flask. 6The H5 cell solution prepared in H5 medium to give 100 cells / mL and rBV seeds were added to a 250 mL Erlenmeyer flask, and VLP production was carried out in a culture volume of 40 mL. The rBV seeds were added to the culture medium so that the MOI (Multiplicity of Infection) was 0.01 or more. The flask was shielded from light with aluminum foil, and cultured with shaking at 27°C and 125 rpm 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.

[0110] (11) Purification of VLPs The entire culture was centrifuged at 8000 rpm, 10 min, and 4°C to roughly remove the cells. The mixture was then centrifuged at 11000 rpm, 1 h, and 4°C, and the supernatant was collected in a new centrifuge tube. The supernatant was transferred to a centrifuge tube (Beckman Coulter) and ultracentrifuged at 32000 rpm, 2 h, and 4°C using an Optima XPN-100 (Beckman Coulter) and an SW32-Ti rotor to precipitate proteins including VLPs. The supernatant was discarded, 1 mL of fresh H5 medium was added, and the mixture was left to stand at 4°C for several hours to swell the pellet. The swollen pellet was well suspended by pipetting until no precipitate remained. 1.8 g of CsCl (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out 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, 20 h, and 4°C using an SW55-Ti rotor. After centrifugation, white light was applied from above the tube, and only the band portion derived from VLP was collected with a pipette. The collected solution was transferred to a centrifuge tube for pelleting. The solution was made up with 1xPBS (Thermo Fisher Scientific), and ultracentrifuged at 32000 rpm, 2 h, and 4°C to precipitate the VLPs. The supernatant was discarded, and fresh H5 medium was added and left to stand overnight at 4°C to swell the pellet. The swollen pellet was well suspended by pipetting to obtain a purified VLP solution.

[0111] (12) Quantification of VLPs The VLP concentration was quantified by the Bradford method. BSA diluted 2-fold from 0.5 mg / mL to 0.067 mg / mL was used as the standard substance. 200 μL of staining solution (Bio-Rad) was added to 800 μL of ion-exchanged water, and 20 μL of appropriately diluted sample was added and mixed well. The mixture was left to stand at room temperature for 5 minutes, and the absorbance at 600 nm was measured using a spectrophotometer.

[0112] (13) SDS-PAGE analysis of VLPs The acquisition of VLPs was confirmed by detection of VP1 protein by SDS-PAGE. 6.5 μL of sample prepared so that 1 μg was electrophoresed was mixed with 2.5 μL of 4xLDS sample buffer (Thermo Fisher Scientific), and 1 μL of 10xSample Reducing Agent (Thermo Fisher Scientific), and heat-treated at 100°C for 5 minutes. Electrophoresis was performed using NuPAGE 10% Bis-Tris Protein Gels (Thermo Fisher Scientific) at a constant pressure of 200 V for 40 minutes. After electrophoresis, the gel was stained with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific). After decolorization with tap water, the gel image was captured with a scanner (EPSON, GT-9300UF) (Figure 9). As a result, synthesis of the target norovirus VP1 protein was confirmed.

[0113] (Production Example 6) Screening of VHHs that bind to GII.19 norovirus (1) Preparation of cDNA for VHH display Construction of a DNA library encoding the single variable domain of the heavy chain of a heavy-chain antibody (VHH) of a full-length alpaca-derived antibody Using two types of alpaca-derived naive VHH library genes provided by RePHAGEN, S-hinge and L-hinge, as templates, the genes were amplified by PCR using an S-hinge VHH-specific primer pair (sequence numbers 103 and 104) and an L-hinge VHH-specific primer pair (sequence numbers 103 and 105). The genes were then amplified by extension PCR using overlapping PCR primers (sequence numbers 106 and 107) to prepare DNA fragments consisting of a T7 promoter, an omega (ω) enhancer, a Kozak consensus sequence, a VHH gene, a His tag, and a linker hybridization region (Y tag), thereby preparing a full-length VHH-encoding DNA library.

[0114] (2) Preparation of cDNA display Each cDNA display was prepared according to the following procedures (3) to (7). The buffers used are shown in Table 11.

[0115] [Table 11]

[0116] (3) Transcription The full-length VHH-encoding DNA library prepared in Example 1 was transcribed using the T7 RiboMAX Express Large Scale RNA Production System (Promega) according to the attached manual. The amount of DNA used was 6.6 μg. The amount of DNA for each subsequent selection was 0.1 to 1 μg from the second round onwards. The obtained transcription product was purified using RNAClean XP (Beckman Coulter) according to the attached manual. The concentration of the purified product was quantified using NanoPad DS-11FX (DeNovix).

[0117] (4) Ligation NaCl (final concentration 0.2 M) and Tris-HCl (pH 7.5, final concentration 0.05 M) were added to 20 pmol of purified mRNA and 20 pmol of puromycin linker, cnvK riboG linker (Epsilon Molecular Engineering), and incubated at 90°C for 1 minute. The temperature was then lowered to 70°C at a rate of 0.1°C / sec, and incubated at 70°C for 1 minute. The temperature was then lowered to 25°C at a rate of 0.1°C / sec, and then lowered to 10°C at a rate of 2°C / sec, to hybridize the cnvK linker to the 3' end of the mRNA. Then, a UVP CrossLinker (CL-3000), 365 nm, 100-115 V (Analytik Jena) was used to irradiate 365 nm UV at 4060 mJ / cm. 2 By irradiation, the cnvK linker and mRNA were photocrosslinked to obtain an mRNA-linker complex.

[0118] (5) Preparation of mRNA display 6 pmol of the mRNA-linker complex was incubated at 30°C for 30 minutes using a 50 μL-scale cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), manufactured by Promega). Next, MgCL2 and KCL were added to final concentrations of 75 mM and 900 mM, respectively, and incubated at 37°C for 1 hour to display a peptide corresponding to the mRNA on the puromycin on the mRNA-linker. Next, EDTA (pH 8.0) was added to a final concentration of 70 mM, and incubated at 4°C for 5 minutes to prepare the mRNA display.

[0119] (6) Preparation of cDNA display 60 μL of Dynabeads Myone streptavidin C1 (manufactured by Thermo Fisher Scientific) was placed in a Protein Lobind Tube, washed with 200 μL of binding buffer, and the mRNA display prepared by the above method (1)-(4) was added and stirred at 25 ° C for 30 minutes. After washing with 200 μL of binding buffer, the reverse transcription reaction was carried out by incubating at 42 ° C for 30 minutes in a reaction solution having the composition shown in Table 12 to prepare an mRNA / cDNA - VHH conjugate. After washing with 200 μL of binding buffer, 39 μL of His tag binding / washing buffer and 1 μL of 1,000 U / μL RNase T1 were added and stirred at 37 ° C for 15 minutes to elute the mRNA / cDNA - VHH conjugate (hereinafter referred to as cDNA display) from Dynabeads Myone streptavidin C1 (manufactured by Veritas).

[0120] [Table 12]

[0121] (7) Purification of cDNA display 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed in an Eppendorf tube and washed with 200 μL of His tag binding / washing buffer, after which the cDNA display prepared by methods 2-1 to 2-6 was added and stirred for 30 minutes at 25° C. After washing with 200 μL of His tag binding / washing buffer, 30 μL of His tag elution buffer was added and stirred at 37° C. for 15 minutes to elute the cDNA display.

[0122] (8) In vitro selection experiments Selection was performed using cDNA display prepared from the library obtained in 2-7. The synthesis scale of cDNA display used in each selection round is shown in Table 13.

[0123] [Table 13]

[0124] (9) Screening procedure for selection cycle 1 (R1) 100 μL of 10 μg / mL GII.19 type VLP prepared using PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4°C. After removing the norovirus VLP dispersion that was not adsorbed to the well, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added and incubated at room temperature for 1 hour, and then the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.05% Tween20, pH 7.2) was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs and incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37 ° C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 108) and T7 omeganew (sequence number 109) as primers. The reaction solution was prepared by mixing 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5 μL of 10 μM cnvK NewYtag for poly A primer, 1.5 μL of 10 μM T7 omeganew primer, 12.5 μL of VHH-presenting cDNA display, and 9.5 μL of nuclease free water per sample. PCR was performed at 95 ° C for 2 minutes, followed by 22 cycles of 98 ° C for 20 seconds, 68 ° C for 15 seconds, and 72 ° C for 20 seconds, and then 72 ° C for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. The obtained purified DNA library (hereinafter referred to as R1 library) was subjected to R2 selection.

[0125] (10) Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library by the same method as above, at the synthesis scale shown in Table 3. 100 μL of 1 μg / mL GII.19 type VLP prepared with PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4 ° C. After removing the norovirus VLP dispersion that was not adsorbed to the well, 200 μL of PBST was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added, and the plate was incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs, and the mixture was incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37 ° C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 108) and T7 omeganew (sequence number 109) as primers. The reaction solution was prepared by mixing 25μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5μL of 10μM cnvK NewYtag for poly A primer, 1.5μL of 10μM T7 omeganew primer, 12.5μL of VHH-presenting cDNA display, and 9.5μL of nuclease free water per sample. PCR was performed at 95℃ for 2 minutes, followed by 24 cycles of 98℃ for 20 seconds, 68℃ for 15 seconds, and 72℃ for 20 seconds, and then 72℃ for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. The obtained purified DNA library (hereinafter referred to as R2 library) was subjected to R3 selection.

[0126] (11) Screening procedure for selection cycles (R3 and R4) A cDNA display library was prepared from the R2 library using the same method as above, with the synthesis scale shown in Table 3. Screening was then carried out up to R4 according to the method in 2-10. The resulting purified DNA library (hereinafter referred to as the R4 library) was subjected to R5 selection.

[0127] (12) Screening procedure for selection cycle 5 (R5) A cDNA display library was prepared from the R4 library by the same method as above, at the synthesis scale shown in Table 3. 100 μL of 0.1 μg / mL GII.19 type VLP prepared with PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno™ Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4°C. After removing the norovirus VLP dispersion that had not been adsorbed to the well, 200 μL of PBST was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added, and the plate was incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs and incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37 ° C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 108) and T7 omeganew (sequence number 109) as primers. The reaction mixture was prepared by mixing 25μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5μL of 10μM cnvK NewYtag for poly A primer, 1.5μL of 10μM T7 omeganew primer, 12.5μL of VHH-presenting cDNA display, and 9.5μL of nuclease free water per sample. PCR was performed at 95℃ for 2 minutes, followed by 24 cycles of 98℃ for 20 seconds, 68℃ for 15 seconds, and 72℃ for 20 seconds, and then 72℃ for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. This resulted in the R5 library.

[0128] (13) Next-generation sequence analysis To confirm the degree of convergence of the R4 library or the R5 library in detail, sequence analysis was performed using a next-generation sequencer. The preparation of the sequence samples was performed with reference to the 2-step PCR Amplicon Library Preparation method provided by Illumina. First, Amplicon PCR was performed using the PCR products after each selection as templates and NGS Fw 1st PCR primer (SEQ ID NO: 110) and NGS Rv 1st PCR primer (SEQ ID NO: 111) as primers. The PCR conditions were 98°C for 1 minute, followed by 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, and 72°C for 35 seconds, and then 72°C for 1 minute. The obtained PCR products were purified according to the instructions for Agencourt AMPure XP (Beckman Coulter), and then index PCR was performed according to the instructions for Illumina. The resulting PCR product was purified according to the Agencourt AMPure XP instructions, and then its concentration was quantified using a NanoPad DS-11FX (DeNovix). Sequence analysis was then performed using MiSeq (Illumina) and MiSeq Reagent Nano kit v2 (500 cycles) according to the Illumina instructions.

[0129] (14) Sequence data analysis The sequence data demultiplexed using the MiSeq Controller was subjected to analysis. The base sequence region encoding VHH was extracted from the sequence data and translated into amino acid sequence. The number of amino acid sequences that perfectly matched each amino acid sequence was then counted. As a result, NoVHH53 (sequence number 112) and NoVHH54 (sequence number 113) appeared frequently, and were selected as antibodies with high affinity to the antigen.

[0130] In the amino acid sequence of NoVHH53 (sequence number 112), positions 1 to 26 are FR1 (sequence number 114), positions 27 to 36 are CDR1 (sequence number 115), positions 37 to 50 are FR2 (sequence number 116), positions 51 to 60 are CDR2 (sequence number 117), positions 61 to 99 are FR3 (sequence number 118), positions 100 to 104 are CDR3 (sequence number 119), and positions 105 to 115 are FR4 (sequence number 120).

[0131] In the amino acid sequence of NoVHH54 (sequence number 113), positions 1 to 26 are FR1 (sequence number 121), positions 27 to 36 are CDR1 (sequence number 122), positions 37 to 50 are FR2 (sequence number 123), positions 51 to 59 are CDR2 (sequence number 124), positions 60 to 98 are FR3 (sequence number 125), positions 99 to 116 are CDR3 (sequence number 126), and positions 109 to 127 are FR4 (sequence number 120).

[0132] (Production Example 7) Production of VHH using protease-deficient recombinant Bacillus subtilis (1) Artificial synthesis of genes The amino acid sequence of the synthesized VHH was provided with a His tag (SEQ ID NO: 129) at the C-terminus of the amino acid sequence of the VHH of NoVHH53 (SEQ ID NO: 127) or NoVHH54 (SEQ ID NO: 128) via a linker sequence. The VHHs synthesized in this manner are called His-tagged NoVHH53 (SEQ ID NO: 130) and His-tagged NoVHH54 (SEQ ID NO: 131). In addition, a His-tagged NoVHH53 dimer (SEQ ID NO: 133) and a His-tagged NoVHH54 dimer (SEQ ID NO: 134) were designed in which two VHHs were linked in series via a peptide linker (EPKTPKPQSGS: SEQ ID NO: 132). These VHHs represented by SEQ ID NOs: 127 to 130 are collectively referred to as His-tagged VHHs. These His-tagged VHH dimers were produced using Bacillus subtilis by the method shown in Production Example 3. The produced samples were subjected to SDS-PAGE according to Production Example 3, and it was found that all His-tagged VHH dimers were produced (FIG. 10).

[0133] (Test Example 11) Lateral flow assay (7) (1) Sensitization of labeled antibodies to pigment particles Colored cellulose particles NanoAct (chemically bonded type, manufactured by Asahi Kasei Corporation) were used as the dye particles. Sensitization with the detection antibody was performed according to the attached protocol. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, 540 μL of 100 mM MES (pH 6), 7.5 μL of 4 wt% EDC (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide), and 15 μL of 4 wt% NHS were added thereto, and the tube was left to stand at room temperature for 15 minutes to perform esterification of the carboxyl group. The tube was centrifuged at 13,000 × g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. 60 μg of His-tagged NoVHH53 or His-tagged NoVHH54 was added as the detection antibody, followed by vortexing and incubation at 37 ° C for 120 minutes. Next, 7.2 mL of blocking buffer (1 wt% 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 to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. The mixture was centrifuged again at 13,000×g for 15 minutes to remove the supernatant, and 1.3 mL of storage solution (33 mM Boric Acid, 0.2 wt% Casein, 15 wt% Sucrose, pH 9.2) was added, followed by sonication, and stored at 4°C until use (hereinafter referred to as sensitized particles).

[0134] (2) Assembling the Half Strip FF120HP Plus or FF80HP Plus (Cytiva) cut to 5 mm x 40 mm was used as the nitrocellulose membrane. CM5 (Cytiva) cut to 5 mm x 20 mm was used as the absorbent pad. The nitrocellulose membrane and absorbent pad were laminated together with a 10 mm overlap and adhered with a backing sheet (GL-57888, Lohmann) to assemble a half strip.

[0135] (3) Immobilization of capture antibodies onto nitrocellulose membrane The capture antibodies used were His-tagged NoVHH53, His-tagged NoVHH54, His-tagged NoVHH53 dimer, and His-tagged NoVHH54 dimer (spacer: EPKTPKPQSGS (SEQ ID NO: 132)). 2 μg of His-tagged VHH was spotted with a pipette at a point 2 cm from the bottom end of the strip assembled in 6-2. Then, immobilization was performed by drying at 37 ° C for 30 minutes. Hereinafter, the site where the capture antibody was immobilized is referred to as the test spot.

[0136] (4) Evaluation by dipstick-type lateral flow assay A borate buffer (100 mM boric acid, 1% casein, 150 mM NaCl, pH 8.2) was used as the developing solution. A 1 to 1000 ng / mL GII.19 type VLP dispersion was prepared using the developing solution (hereinafter referred to as a positive sample). For conditions that did not contain GII.19 type VLP, a borate buffer was used as the developing solution (hereinafter referred to as a negative sample). 100 μL of a positive sample or a negative sample and 20 μL of the sensitized particles prepared in 6-1 were added to one well of a 96-well plate and mixed well by pipetting. Then, the half strip prepared in 6-3 was immersed by standing it up against the well and left to stand until the developing solution had completely flowed out. Then, the presence or absence of coloring in the test spot was visually judged. The criteria for judging the presence or absence of coloring are as shown in FIG. 11. The results of the lateral flow assay are shown in Table 14. When His-tagged NoVHH53 or His-tagged NoVHH54 was used as the capture antibody, no coloration was observed within the range of antigen concentrations tested. On the other hand, when a His-tagged NoVHH53 dimer was used as a capture antibody, 500 ng of GII.19 VLP was detected, and when a His-tagged NoVHH54 dimer was used, 50 ng of GII.19 VLP was detected. The capture antibody spacer (EPKTPKPQSGS (SEQ ID NO: 132)) used here consists of a total of 11 amino acids. This spacer has three XP or PX (EP or PK, TP or PK, KP or PQ) as rigid motifs, and is composed of a heterogeneous amino acid sequence containing one GS that exhibits non-rigid properties. When a VHH dimer linked by such a spacer was used as a capture antibody, the detection sensitivity was improved by 100 times or more compared to the monomer. Therefore, it can be said that a spacer used to link dimers can function as the spacer of the present invention as long as it contains at least a rigid motif consisting of 2 to 8 amino acids in length.

[0137] [Table 14]

[0138] (Production Example 8) Production of VHH using protease-deficient recombinant Bacillus subtilis EN12-EN12-10 (SEQ ID NO: 140), in which two EN12 (SEQ ID NO: 13) described in Japanese Patent Application No. 2022-065934 are linked with EPKTPKPQS (SEQ ID NO: 139), 15The following VHHs were designed: EN12-EN12-11 (SEQ ID NO: 142) linked by (SEQ ID NO: 141), EN12-EN12-12 (SEQ ID NO: 144) linked by (PQ)8 (SEQ ID NO: 143), EN12-EN12-13 (SEQ ID NO: 145) linked by EEEEKKKK (SEQ ID NO: 6), and EN12-EN12-14 (SEQ ID NO: 147) linked by GGGGSEAAAK (SEQ ID NO: 146). The amino acid sequence of each VHH synthesized in this production example was given a His tag sequence (SEQ ID NO: 44) on the C-terminal side via a linker sequence (SEQ ID NO: 53). The VHHs synthesized in this manner are called His-tagged EN12-EN12-10 (SEQ ID NO: 148), His-tagged EN12-EN12-11 (SEQ ID NO: 149), His-tagged EN12-EN12-12 (SEQ ID NO: 150), His-tagged EN12-EN12-13 (SEQ ID NO: 151), and His-tagged EN12-EN12-14 (SEQ ID NO: 152). These His-tagged VHH dimers were produced using Bacillus subtilis according to the method shown in Production Example 3. When the produced samples were subjected to SDS-PAGE according to Production Example 3, it was found that all of the His-tagged VHH dimers were produced (Figure 12).

[0139] (Test Example 12) Lateral flow assay (8) Following the example 2, immunochromatography was constructed using COVID-19 Nucleocapsid Monoclonal Antibody, A03100 (BiosPacific) as the labeled antibody and His-tagged EN12-EN12-10 to 14 as the capture antibody. Fc-tagged N protein was used as the antigen. The results are shown in Table 15. When linked with the highly flexible (GGGGS)3, which is a comparative example, a visual judgment of ± was obtained for an antigen amount of 0.5 ng (EN12-EN12-2). Next, when linked with EPKTPKPQS (sequence number 139), in which the rigid motif composed of EP, TP, and KP (all of which are XP type, where X is any amino acid) accounts for 67%, a visual judgment of ± was obtained even for an antigen amount of 0.05 ng (EN12-EN12-10). P composed only of proline 15When the ligation was performed at 0.05ng, a visual judgment of ± was obtained even for an antigen amount of 0.05ng (EN12-EN12-11). (XP) n When the rigid motif (PQ)8 of the type (X is any amino acid, n is an integer) was linked, a visual judgment of ± was obtained even for an antigen amount of 0.05 ng (EN12-EN12-12). When the rigid motif EEEEKKKK (SEQ ID NO: 6) was linked, a visual judgment of + was obtained for an antigen amount of 0.5 ng (EN12-EN12-13). When the rigid motif GGGGSEAAAK (SEQ ID NO: 146), in which the rigid motif EAAAK accounts for 50%, was linked, a visual judgment of + was obtained for an antigen amount of 0.5 ng (EN12-EN12-14). From the above, it was confirmed that the detection sensitivity was improved when any of the highly rigid spacers was used compared to when a highly flexible spacer was used as a comparative example. These results strongly suggest that a spacer containing a rigid motif is suitable for linking VHH dimers used as capture antibodies.

[0140] [Table 15]

Claims

1. A method for detecting an antigen using a single domain antibody, comprising using a multimer of single domain antibodies in which two or more single domain antibodies are linked in series via a spacer as a capture antibody, the spacer consisting of a peptide of 8 to 30 amino acids in length and including a rigid motif consisting of 2 to 8 amino acids in length.

2. The method according to claim 1 , wherein the proportion of rigid motifs in the spacer is 30% or more and 100% or less.

3. The method of claim 1 or 2, wherein the rigid motif is a motif that forms a helical structure.

4. The amino acid sequence of the rigid motif is (EAAR). ｎ [n represents an integer of 3 to 7.], (EAAK) ｎ [n is an integer from 3 to 7.], (EAAAR) ｎ [n is an integer from 2 to 6.], (EAAAK) ｎ [n is an integer from 2 to 6.] (EEEERRRR) ｎ [n is an integer of 2 or 3.] or (EEEEKKKKK). ｎ The method according to claim 1 or 2, wherein n is an integer of 2 or 3.

5. 3. The method according to claim 1 or 2, wherein 50% or more of the amino acids constituting the rigid motif are XP or PX (X represents any amino acid residue).

6. The amino acid sequence of the rigid motif is (P) ｌ , (XP) ｍ , (PX) ｎ or a combination thereof, wherein l, m, and n independently represent an integer of 6 to 15, and X represents any amino acid residue.

7. The method according to claim 1 or 2, wherein in the spacer, the glycine content of amino acids in a portion other than the rigid motif is 50% or more and 100% or less.

8. The spacer is provided in the amino acid sequence other than the rigid motif, ｎ , (GX) ｎ , (GX) ｎ , (GGX) ｎ , (GXG) ｎ , (XGG) ｎ , (GGGX) ｎ , (GGXG) ｎ , (GXGG) ｎ , (XGGG) ｎ , (GGGGX) ｎ , (GGGGXG) ｎ , (GXGGG) ｎ , and (XGGGG) ｎ (wherein n is an integer of 1 to 5, and X is any amino acid residue).

9. The method according to any one of claims 1 to 8, wherein the single domain antibody multimer is composed of two or more types of single domain antibodies that exhibit binding activity to the same antigen.

10. The method according to any one of claims 1 to 8, wherein the single domain antibody multimer consists of two or more identical single domain antibodies.

11. 11. The method of any one of claims 1 to 10, wherein the antigen detection is detection of SARS-CoV-2 in a sample, and the single domain antibody comprises CDR1-3 selected from the following: (a), (b), (c), (d), (e) and (f). (a) CDR1 consisting of the amino acid sequence represented by GFTFSRYDMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by AISWNGGSTY (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by LDWLQWDWAY (SEQ ID NO: 6). (b) CDR1 consisting of the amino acid sequence represented by GFTFSSYAMT (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GQHHQELQHWYAWDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AITWNGGSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by LQDHNSVLADAY (SEQ ID NO: 12). (d) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AISWSGDSTH (SEQ ID NO: 13), and CDR3 consisting of the amino acid sequence represented by LGEIDGLEENDY (SEQ ID NO: 14). (e) CDR1 consisting of the amino acid sequence represented by GFTFSDYAMG (SEQ ID NO: 15), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by LGQNKEHVGKRIEDY (SEQ ID NO: 16).

12. The method according to any one of claims 1 to 11, which is a lateral flow assay.

13. An antigen detection kit for carrying out the method according to any one of claims 1 to 12, comprising a capture antibody, a detection antibody and reagents necessary for detection.

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