Novel peptide, antibody comprising same or antigen-binding fragment thereof, and substance for detecting SARS-cov-2, comprising same

Abstract

Disclosed are: a novel peptide; an antibody comprising the peptide, or an antigen-binding fragment thereof; a nucleic acid coding the peptide, antibody or antigen-binding fragment thereof; a recombinant expression vector comprising the nucleic acid; a cell transformed using the recombinant expression vector; a substance for detecting SARS-CoV-2 comprising the antibody or antigen-binding fragment; a one-step SARS-CoV-2 diagnostic kit comprising the substance for detecting SARS-CoV-2; and a one-step SARS-CoV-2 diagnosis method using the one-step SARS-CoV-2 diagnostic kit.

Description

Novel peptide, antibody or antigen-binding fragment thereof containing the same, and substance for detecting SARS-COV-2 containing the same

[0001] It relates to a novel peptide, an antibody or antigen-binding fragment thereof containing the same, a substance for detecting SARS-CoV-2 containing the same, etc.

[0002] More specifically, the present invention relates to a novel peptide, an antibody comprising the peptide or an antigen-binding fragment thereof, a nucleic acid encoding the peptide or the antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid, a cell transformed with the recombinant expression vector, a substance for detecting SARS-CoV-2 comprising the antibody or antigen-binding fragment, a one-step SARS-CoV-2 diagnostic kit comprising the substance for detecting SARS-CoV-2, and a one-step SARS-CoV-2 diagnostic method using the one-step SARS-CoV-2 diagnostic kit.

[0003] The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered the global COVID-19 pandemic. For over 20 years, three zoonotic coronaviruses—SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2—have infected humans, and these viruses can cause acute respiratory distress syndrome (ARDS). CoVs are known to produce four main types of proteins: envelope proteins, membrane proteins, nucleocapsid proteins, and spike proteins. Among these proteins, the spike protein has been extensively utilized in the development of vaccines and diagnostic assays. In particular, SARS-CoV-2 is known to infect host cells through the binding of the spike protein (SP) on the viral surface to the angiotensin-converting enzyme 2 (ACE2) on the host envelope. The interaction between SP and ACE2 has been reported to occur at the receptor binding domain (RBD) of SP, which is located in the S1 domain of SP.

[0004] In the present invention, an Fv antibody library was used to screen for Fv antibodies with binding affinity to the RBD, and these Fv antibodies were used to create a one-step immunoassay based on a switching peptide for the detection of SARS-CoV-2 by mixing samples without a washing step. An Fv antibody library for expression of Fv antibodies in the outer membrane of E. coli was constructed using autodisplay technology. Fv antibodies are the corresponding VH region of IgG, which consists of three complementarity determining regions (CDRs) and four framework regions (FRs). The Fv antibody library was generated by randomizing the amino acid sequence of CDR3, which consists of 11 amino acid residues, by site-directed mutagenesis. Expression of the Fv antibody library in the outer membrane was performed using autodisplay technology. In autodisplay technology, the target protein (Fv antibody) is expressed by transport of Fv antibodies through the beta-barrel of AIDA-1. Over 105 using auto display technology High expression yields of more than 90% were achieved in protein / E. coli and the entire E. coli population. The Fv-antibody library prepared in this way was 10 5It was reported to have high clone / library diversity. The high expression yield of the Fv-antibody library enabled effective screening of target Fv-antibodies from the library without repeated biopanning processes. The screened Fv-antibodies were expressed for a one-step immunoassay for SARS-CoV-2. A one-step immunoassay based on switching peptides was used to detect SARS-CoV-2 using the expressed Fv-antibodies. Switching peptides were synthesized from the FR regions located on the heavy and light chains of IgG. These FRs from different IgG chains are known to self-assemble to form the binding pocket of IgG. Since the expressed Fv-antibody is composed of the CDRs and FRs of the IgG heavy chain, the switching peptide of the IgG light chain is thought to bind to the expressed Fv-antibody. These switching peptides were used in a one-step immunoassay through the release of fluorescently labeled switching peptides upon binding to the target antigen. For the construction of the one-step immunoassay, switching peptides with higher binding affinity constants (KD) for Fv antibodies compared to the target antigen were selected and optimized for quantitative assays. The one-step assay for SARS-CoV-2 based on the switching peptides was validated using expressed Fv antibodies. The binding affinity of the Fv antibodies was measured using a modified RBD antigen and then using pseudovirus particles expressing SP on the envelope. Finally, heat-inactivated SARS-CoV-2 was used as a real sample. In the present invention, Fv antibodies against the SARS-CoV-2 spike protein were screened from an autodisplayed Fv antibody library expressed on the outer membrane of E. coli. Four screened Fv-antibodies were expressed as soluble proteins and their binding affinities (K) to RBD (immobilized and soluble) were determined. D) were measured. The selectivity of Fv antibodies against SARS-CoV-2 pseudovirus particles with different spike proteins (original, delta, and two omicrons) was measured. A one-step immunoassay based on switching peptides was validated for the detection of SARS-CoV-2 virus.

[0005] One object of the present invention is to provide a peptide capable of binding to the SARS-CoV-2 virus.

[0006] Another object of the present invention is to provide an antibody or an antigen-binding fragment thereof capable of binding to the SARS-CoV-2 virus.

[0007] Another object of the present invention is to provide a nucleic acid encoding the peptide or the antibody or an antigen-binding fragment thereof, which can be used for the synthesis of the peptide or the antibody or an antigen-binding fragment thereof.

[0008] Another object of the present invention is to provide a recombinant expression vector capable of expressing the peptide or the antibody or an antigen-binding fragment thereof, including the nucleic acid.

[0009] Another object of the present invention is to provide a cell that can be transformed with the recombinant expression vector and used for the synthesis of the peptide or the antibody or antigen-binding fragment thereof.

[0010] Another object of the present invention is to provide a substance for detecting SARS-CoV-2, which exhibits fluorescence depending on whether or not the SARS-CoV-2 virus is bound, including the peptide or the antibody or an antigen-binding fragment thereof.

[0011] Another object of the present invention is to provide a one-step SARS-CoV-2 diagnostic kit comprising the substance for detecting SARS-CoV-2.

[0012] Another object of the present invention is to provide a one-step SARS-CoV-2 diagnostic method using the one-step SARS-CoV-2 diagnostic kit.

[0013] In one aspect, the present invention provides a peptide that specifically binds to SARS-CoV-2, the peptide comprising at least one peptide sequence selected from the group consisting of the peptide sequence of SEQ ID NO: 3, the peptide sequence of SEQ ID NO: 4, the peptide sequence of SEQ ID NO: 5, and the peptide sequence of SEQ ID NO: 6.

[0014] By selecting the sequence of the peptide as described above, the formed peptide can have the property of specifically binding to SARS-CoV-2, and this effect will be made clear by the following examples.

[0015] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2, comprising a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6.

[0016] By having a variable region as described above, the antibody or antigen-binding fragment thereof according to an embodiment of the present invention may have the property of specifically binding to SARS-CoV-2, and this effect will be made clear by the following examples.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; and a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6.

[0018] In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single domain antibody.

[0019] In another aspect, the present invention provides a nucleic acid encoding the peptide or the antibody or an antigen-binding fragment thereof.

[0020] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0021] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0022] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0023] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0024] In another aspect, the present invention provides a substance for detecting SARS-CoV-2, comprising: the antibody or an antigen-binding fragment thereof; a switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy chain or light chain of the antibody or an antigen-binding fragment thereof; and a marker bound to the switching peptide.

[0025] By being configured as described above, the SARS-CoV-2 detection material according to an embodiment of the present invention can provide rapid and accurate qualitative and quantitative analysis of the SARS-CoV-2 virus by dissociating the switching peptide bound to the fluorescent marker from the antibody or its antigen-binding fragment by exposure to the detection target substance.

[0026] In one embodiment, the antibody or antigen-binding fragment thereof may be single-stranded.

[0027] In one embodiment, the marker may be a fluorescent marker.

[0028] As described above, by including a single-strand fragment rather than a whole antibody, detection of SARS-CoV-2 using the substance for detecting SARS-CoV-2 can be achieved more quickly and accurately, and in particular, detection can be possible without a washing process.

[0029] In another aspect, the present invention provides a one-step SARS-CoV-2 diagnostic kit comprising the substance for detecting SARS-CoV-2.

[0030] In another aspect, the present invention provides a one-step SARS-CoV-2 diagnostic method, comprising the step of mixing a substance for detecting SARS-CoV-2 in a one-step SARS-CoV-2 diagnostic kit and a sample to be detected, and then identifying a marker.

[0031] In one embodiment, the marker may be a fluorescent marker.

[0032] The peptide according to an embodiment of the present invention is a novel peptide that can bind to the SARS-CoV-2 virus, particularly the RBD region of the SARS-CoV-2 virus.

[0033] An antibody or antigen-binding fragment thereof according to an embodiment of the present invention can bind to a SARS-CoV-2 virus by including the peptide, particularly by including the peptide in the CDR3 region.

[0034] The nucleic acid according to an embodiment of the present invention can be used for expression by encoding the peptide and / or the antibody or antigen-binding fragment thereof.

[0035] A recombinant expression vector according to an embodiment of the present invention can be used to express the peptide and / or the antibody or antigen-binding fragment thereof, including the nucleic acid.

[0036] A cell according to an embodiment of the present invention can be transformed with the recombinant expression vector to express the peptide and / or the antibody or antigen-binding fragment thereof.

[0037] A substance for detecting SARS-CoV-2 according to an embodiment of the present invention can detect all or part of the SARS-CoV-2 virus and its analogs without a separate washing process.

[0038] SARS-CoV-2 can be diagnosed quickly and accurately through a one-step SARS-CoV-2 diagnostic kit according to an embodiment of the present invention.

[0039] A one-step SARS-CoV-2 diagnostic method according to an embodiment of the present invention can diagnose SARS-CoV-2 in one step using the one-step SARS-CoV-2 diagnostic kit.

[0040] FIG. 1 is a schematic diagram illustrating a material for detecting SARS-CoV-2 according to an embodiment of the present invention.

[0041] FIG. 2 is a flowchart illustrating a one-step SARS-CoV-2 diagnostic method according to an embodiment of the present invention.

[0042] Figures 3 to 18 are drawings according to experimental examples of the present invention.

[0043] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0044] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" should be understood to indicate the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.

[0045] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0046] A peptide according to an embodiment of the present invention is a peptide that specifically binds to SARS-CoV-2 and may include one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 3, the peptide sequence of SEQ ID NO: 4, the peptide sequence of SEQ ID NO: 5, and the peptide sequence of SEQ ID NO: 6.

[0047] The peptide sequences of SEQ ID NOs: 3 to 6 may be made clear elsewhere in this specification, but SEQ ID NO: 3 may be DISGEKHPADF, SEQ ID NO: 4 may be DGTPKGPRNDF, SEQ ID NO: 5 may be DPPTGTMVTDF, and SEQ ID NO: 6 may be YGSLQAQAPDD. In the context of this specification, all sequences should be interpreted as including identical or similar sequences that would be obvious to a person skilled in the art. For example, unless otherwise specified herein, all sequences should be considered identical or similar sequences to their reverse sequences and sequences with minor modifications. Furthermore, unless otherwise specified herein, sequences that perform the same function, for example, nucleic acid sequences that encode the same amino acid, should be considered identical or similar sequences.

[0048] By selecting the sequence of the peptide as described above, the formed peptide can have the property of specifically binding to SARS-CoV-2, and this effect will be made clear by the following examples.

[0049] An antibody or antigen-binding fragment thereof according to an embodiment of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2, and may include a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5; and a variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6. By having the variable region as described above, the antibody or antigen-binding fragment thereof according to an embodiment of the present invention may have a property of specifically binding to SARS-CoV-2, and this effect will be made clear by the following examples.

[0050] In the context of this specification, "CDR" may refer to complementarity determining regions. In the context of this specification, the term "antibody or antigen-binding fragment thereof" may refer to a region capable of binding to an antigen in an antigen-antibody reaction, or a fragment comprising the region, or an antibody comprising the region. In addition, since the present invention relates to the detection of SARS-CoV-2, unless expressly excluded herein, the term "antigen" may refer to all or part of the SARS-CoV-2 virus or a particle substantially identical thereto.

[0051] In one embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; and a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6.

[0052] As long as the antibody or antigen-binding fragment thereof according to an embodiment of the present invention has the above sequence and / or performs the above function, the form of the antibody or antigen-binding fragment thereof is not particularly limited. In one embodiment, the antibody or antigen-binding fragment thereof may be in the form of a single-domain antibody.

[0053] Meanwhile, the nucleic acid according to an embodiment of the present invention may encode the peptide or the antibody or an antigen-binding fragment thereof.

[0054] In the context of this specification, unless specifically defined otherwise, "coding" is defined based on the relationship between a nucleic acid sequence and a peptide sequence defined by transcription and translation mechanisms known to those skilled in the art. Therefore, as described above, the nucleic acid or nucleic acid sequence referred to herein may be a term encompassing nucleic acids or nucleic acid sequences that encode identical or similar peptide sequences, taking into account cases where there is one or more nucleic acids corresponding to the amino acids contained in the peptide sequence. Such nucleic acids may be used to derive the peptides or antibodies or antigen-binding fragments thereof discovered in the present invention.

[0055] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of the present specification, a "recombinant expression vector" or "recombinant expression vector" may refer to a vector that uses genetic engineering technology to bring in a DNA fragment from outside and promote the expression of a gene in a specific host cell or tissue. This vector may insert an external gene into a host cell and include a control region and sequence necessary for gene expression.

[0056] Therefore, the recombinant expression vector can be used to express the peptide or the antibody or antigen-binding fragment thereof by including the nucleic acid according to the embodiment of the present invention.

[0057] Meanwhile, the cell according to an embodiment of the present invention may be a cell transformed with the recombinant expression vector. In the context of this specification, the term "transformation" or "transformation" may refer to a form of genetic engineering technology, a process of introducing a gene into a cell or tissue that receives an external gene from outside, and may be used primarily to induce gene expression by delivering a recombinant expression vector created externally using recombinant DNA technology into a cell.

[0058] Accordingly, the cell according to the embodiment of the present invention can express the peptide or the antigen or an antigen-binding fragment thereof from the recombinant expression vector using any system related to intracellular gene expression.

[0059] As long as the above function is performed, the type of the cell is not particularly limited. In one embodiment, the cell may include one or more cells selected from the group including animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0060] FIG. 1 is a schematic diagram illustrating a material for detecting SARS-CoV-2 according to an embodiment of the present invention.

[0061] Referring to FIG. 1, a material (100) for detecting SARS-CoV-2 according to an embodiment of the present invention may include: an antibody or an antigen-binding fragment thereof (110); a switching peptide (120) capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3, and FR4) of a heavy chain or a light chain of the antibody or an antigen-binding fragment thereof (110); and a marker (130) bound to the switching peptide (120).

[0062] The description of the substance (100) for detecting SARS-CoV-2 according to the embodiment of the present invention may be applied identically or similarly to the terminology or configuration in the description of the antibody or antigen-binding fragment thereof according to the embodiment of the present invention described above. Therefore, the antibody or antigen-binding fragment thereof (110) may be the antibody or antigen-binding fragment thereof according to the embodiment of the present invention described above, or an analog thereof. Accordingly, the antibody or antigen-binding fragment thereof (110) may include a variable region including CDR1 (110-1), CDR2 (110-2), and CDR3 (110-3), as illustrated in FIG. 1.

[0063] The antibody or antigen-binding fragment thereof (110) can bind to all or part of the SARS-CoV-2 virus or an analog thereof. As described above, as long as the antibody or antigen-binding fragment thereof (110) exhibits the above-described function by including the sequence discovered in the present invention, particularly by including it in CDR3 (110-3), other sequences and structures of the antibody or antigen-binding fragment thereof (110) are not particularly limited.

[0064] In one embodiment, the antibody or antigen-binding fragment thereof (110) may include a variable region comprising CDR1 (110-1) of SEQ ID NO: 1, CDR2 (110-2) of SEQ ID NO: 2, and CDR3 (110-3) of SEQ ID NO: 3; a variable region comprising CDR1 (110-1) of SEQ ID NO: 1, CDR2 (110-2) of SEQ ID NO: 2, and CDR3 (110-3) of SEQ ID NO: 4; a variable region comprising CDR1 (110-1) of SEQ ID NO: 1, CDR2 (110-2) of SEQ ID NO: 2, and CDR3 (110-3) of SEQ ID NO: 5; and a variable region comprising CDR1 (110-1) of SEQ ID NO: 1, CDR2 (110-2) of SEQ ID NO: 2, and CDR3 (110-3) of SEQ ID NO: 6.

[0065] The above switching peptide (120) is a peptide that can specifically and reversibly bind to one or more of the first to fourth framework regions (FR1, FR2, FR3, and FR4) of the heavy chain or light chain of the antibody or antigen-binding fragment thereof (110). Since the switching peptide (120) has the above properties, the switching peptide (120) can bind to the antibody or antigen-binding fragment thereof (110) to which an antigen is not bound, and can be released when an antigen is bound to the antibody or antigen-binding fragment thereof (110).

[0066] In one embodiment, the switching peptide (120) may be an L2-switching peptide.

[0067] The above marker (130) may have a signal according to the binding state of the switching peptide (120), and is a sub-substance that allows the practitioner to confirm the binding state of the switching peptide (120). The type of the marker (130) is not particularly limited as long as it has a signal that the practitioner can recognize. In one embodiment, the marker (130) may be a fluorescent marker.

[0068] By being configured as described above, the SARS-CoV-2 detection material (100) according to an embodiment of the present invention can provide rapid and accurate qualitative and quantitative analysis of the SARS-CoV-2 virus by causing the switching peptide (120) to which the fluorescent marker is bound to dissociate from the antibody or its antigen-binding fragment (110) upon exposure to the detection target material.

[0069] In one embodiment, the antibody or antigen-binding fragment thereof (110) may be single-stranded. As described above, by the antibody or antigen-binding fragment thereof (110) being composed of a single strand, a technical advantage can be obtained in the detection of SARS-CoV-2 using the switching peptide (120), which will be made clear by the following examples. As described above, by including a single-stranded fragment rather than a whole antibody, the detection of SARS-CoV-2 using the SARS-CoV-2 detection substance (100) can be achieved more quickly and accurately, and in particular, detection can be possible without a washing process.

[0070] A one-step SARS-CoV-2 diagnostic kit according to an embodiment of the present invention may include a substance for detecting SARS-CoV-2. The substance for detecting SARS-CoV-2 may be a substance for detecting SARS-CoV-2 according to an embodiment of the present invention described above.

[0071] FIG. 2 is a flowchart illustrating a one-step SARS-CoV-2 diagnostic method according to an embodiment of the present invention.

[0072] Referring to FIG. 2, a one-step SARS-CoV-2 diagnostic method (200) according to an embodiment of the present invention may include a step (S210) of mixing a SARS-CoV-2 detection substance and a detection target sample in a one-step SARS-CoV-2 diagnostic kit and then confirming the marker. The one-step SARS-CoV-2 diagnostic kit may be the one-step SARS-CoV-2 diagnostic kit according to the embodiment of the present invention described above. In addition, the SARS-CoV-2 detection substance may be the SARS-CoV-2 detection substance according to the embodiment of the present invention described above.

[0073] The above marker confirmation step (S210) is a step of confirming the binding status of the switching peptide of the SARS-CoV-2 detection substance as a signal. As described above, the marker may have a signal according to the binding status of the switching peptide, etc., and is a sub-substance that allows the practitioner to confirm the binding status of the switching peptide. Therefore, by confirming the marker, the practitioner can confirm whether all or part of the SARS-CoV-2 virus or its analogue is bound to the SARS-CoV-2 detection substance in the one-step SARS-CoV-2 diagnostic kit.

[0074] As described above, in one embodiment, the marker may be a fluorescent marker. If the marker is a fluorescent marker, the practitioner can determine by fluorescence whether all or part of the SARS-CoV-2 virus or its analogue has bound to the SARS-CoV-2 detection substance in the one-step SARS-CoV-2 diagnostic kit.

[0075] At this time, as described above, when using a material for detecting SARS-CoV-2 according to an embodiment of the present invention, SARS-CoV-2 can be detected quickly and accurately without a separate washing process, which provides a technical advantage to the present invention and will be made clear by the following examples.

[0076] Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0077] ingredient

[0078] The SARS-CoV-2 RBD antigen (amino acid residues 319–541, 52.1 kDa) and Fv antibody (41 kDa) were custom synthesized as fusion proteins with superfolder GFP by Cosmogenetech (Seoul, Korea). The Fv library plasmid construction was described in previous reports (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). The L2-switching peptide was synthesized by Peptron (Daejeon, Korea). Microplate (Maxisorp, 96-well), Dulbecco's modified Eagle's medium (DMEM, Gibco #11995065), Opti-MEM (Gibco, #31985-070), and penicillin-streptomycin (Gibco, #15140122) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Korea Seoul, Korea. NATtrol™ SARS-CoV-2 reagent (10 6copies / mL, Ct value=27.5) and NATtrol™ negative control reagent were purchased from Zeptometrix (Buffalo, NY, USA). Lenti-X™ HEK293T cells (#632180) and Lenti-X™ concentrators (#631231) were purchased from Clontech (Palo Alto, CA, USA). Fetal bovine serum (FBS, Cytiva, #SH-30919.03) and FuGENE (#E5911) were purchased from Promega (Southampton, UK). psPAX2 (#12260) was purchased from Addgene. Spike Pseudotyping Vectors (Original D614, Delta, Omicron BA.2, Omicron BA.4 / BA.5) were purchased from Invivogen (San Diego, CA, USA).

[0079] Screening of Fv-antibody libraries

[0080] Preparation of Fv-antibody library and autodisplay of Fv-variants were performed using conventional techniques (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). Referring to Fig. 3, target E. coli with binding activity to SARS-CoV-2 RBD from the autodisplayed Fv-antibody library were screened according to the following steps: (1) SARS-CoV-2 RBD antigen (2 μM) was added to the autodisplayed Fv-antibody library (100 μL, OD 600nm=0.5) and cultured (37°C, 10 rpm, 1 hour); (2) After washing with 0.01% PBST and PBS, target E. coli cells (n=1,000) were analyzed using a FACSCalibur™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) using the FL1 fluorescence channel (λ) for GFP fusion antigen. Ex =488nm,λ Em =530 nm) (see Fig. 4); (3) E. coli screened for SARS-CoV-2 RBD were spread on an agar plate to obtain screened E. coli clones; (4) To confirm binding activity, the screened E. coli clones (100 μL, OD600=0.5, n=30) were mixed with SARS-CoV-2 RBD (2 μM), and after washing, flow cytometry analysis was performed using a FACSCalibur™ flow cytometer as shown in Fig. 5; (5) The binding constant (K) of the screened E. coli clones D ) was measured using flow cytometry as shown in Fig. 6. Screened E. coli clones (100 μL, OD 600 =0.5) was mixed with SARS-CoV-2 RBD at concentrations ranging from 62 to 4,000 nM for 1 hour.

[0081] Expression and purification of Fv antibodies

[0082] Plasmids (pJY005, pJY006, pJY007, and pJY008) were synthesized by Cosmogenetech (Seoul, Korea) to contain the open reading frame (ORF) of an Fv antibody with binding activity to SARS-CoV-2 RBD (see Fig. 7 ). The amino acid sequences and oligonucleotide sequences of the CDR3 region are summarized in Table 1.

[0083] <h2 style=";text-align:left;direction:ltr">Clone-#Oligonucleotide sequence(33 bp)CDR3 sequence(11 mer)서열목록 번호83-GAT ATA AGC GGA GAG AAA CAC CCC GCG GAT TTC-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> DISGE<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> KHPAD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F3113-GAC GGT ACA CCG AAA GGC CCT AGA AAT GAT TTC-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> DGTPK<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> GPRND<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F4153-GAC CCT CCA ACT GGT ACA ATG GTA ACA GAT TTT-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> DPPTG<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> TMVTD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F5293-TAC GGA AGT CTA CAA GCA CAA GCA CCT GAT GAT-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> YGSLQ<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> AQAPD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> D6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] To express Fv antibodies intracellularly in E. coli, competent cells were transformed with a custom plasmid, and the transformed E. coli were cultured in 20 mL of luria bertani (LB) medium with 1 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) and 30 μg / mL carbenicillin at 30°C for 16 h. The E. coli pellet was collected by centrifugation (2,000 × g for 2 min) and resuspended in 20 mL of binding buffer (5 mM Tris-HCl and 0.5 M NaCl) containing 3 M urea. The resuspended E. E. coli cells were sonicated using a sonic reactor (Vibra cell VCX-130, Sonics, USA), and the lysate was centrifuged at 25,000 × g for 10 min. The Fv antibody in the supernatant was purified using a His-tag purification column from Roche (Bassel, Switzerland) and eluted using elution buffer (binding buffer containing 3 M urea and 100 mM imidazole) (Spriestersbach et al., 2015). Finally, the purified Fv antibody was dialyzed at 150 rpm and 4°C for 16 h to remove imidazole and urea.

[0085] Production of pseudovirus particles

[0086] Pseudo-viral particles were produced by co-transfecting Lenti-X™ HEK293T cells with the SARS-CoV-2 spike pseudotyping vector (Original D614, Delta, Omicron BA.2, Omicron BA.4 / BA.5) and the helper plasmid psPAX2 using Fugene Transfection Reagent. The production of pseudo-viral particles followed the following steps: (1) Lenti-X™ HEK 293T cells were cultured in DMEM 14 supplemented with 10% FBS, and the cells were seeded at a density of 3 × 10 before transfection. 6 Cells were plated in 100-pi dishes; (2) Fugene (25 μL) and plasmids (7.5 μg of psPAX2 and 5 μg of SARS-CoV-2 spike pseudotyping vector) were added to Opti-MEM (3.5 mL) and incubated at 37°C for 10 min; (3) the medium was replaced with 10.5 mL of fresh DMEM containing 10% FBS; (4) the Fugene-plasmid mixture in Opti-MEM was added dropwise to the cells; (5) After 72 h, the supernatant was centrifuged at 500 g for 10 min to remove debris, and the supernatant was then mixed into 3 volumes of Lenti-X™ concentrator and incubated at 4 °C for 3 h; (6) the supernatant was centrifuged at 1500 g at 4 °C for 45 min; (7) After removing the supernatant, the pellet was resuspended in DMEM; (8) Finally, the pseudo-virus particles were stored at -70 °C in disposable aliquots; (9) The pseudo-virus particles were used in a viral titer assay using the Lenti-X™ qRT-PCR titration kit (#631235, Takara Bio, USA). The obtained viral titer was 10 8-9 It was in the range of copies / mL.

[0087] Measurement of Fv-antibody binding using surface plasmon resonance

[0088] Binding constant of Fv-antibody (K D ) was measured using an SPR biosensor from Iclubio (Seongnam, Korea). The SPR chip was prepared using the following steps: BK-7 glass (1 × 1 cm 2 ) was sputter-coated with a 2 nm titanium layer and a 48 nm gold layer as an adhesive. SPR measurements were prepared using the expressed Fv-antibody or SARS-CoV-2 RBD immobilized on the gold surface as shown in Figures 8 and 9. The SPR chip was incubated with SARS-CoV-2 RBD or Fv-antibody (20 μg / mL each) at 37°C for 1 h. After washing with PBS, the blocking process was performed by incubating with BSA (1 mg / mL) for 1 h. After washing, solution samples of SARS-CoV-2 RBD (6.2 to 50.0 nM) or Fv-antibody (25 to 200 nM) were injected into the flow cell at a flow rate of 15 μL / min for 10 min as a binding step. As shown in Figures 10 and 11, PBS was injected into the flow cell at a flow rate of 15 μL / min for 10 min as a dissociation step. The binding properties of the pseudo-virus particles of the wild type (Wuhan-1 and three variants) and the expressed Fv-antibody to NATtrol™ SARS-CoV-2 were measured using the Fv-antibody immobilized on the gold surface of the SPR biosensor (see Figures 12 and 13). 2.5 × 10 6 1.5×10 5Pseudovirus particles at a dilution range of 100 copies / mL and NATtrol™ SARS-CoV-2 solutions at a dilution range of 100-fold (Ct value = 34.1) to 800-fold (Ct value = 37.1) were analyzed using the SPR biosensor in the same manner. The SPR signal was calculated by fitting the dose-response curve of the sample containing the solution protein or virus particles to the isotherm model:

[0089] R = (Rmax·[Ag]) / (Kd+[Ag])

[0090] Here, R represents the SPR signal, Rmax represents the maximum SPR signal, [Ag] represents the antigen concentration, and Kd represents the binding constant (Bong et al., 2020; Bong et al., 2021a). The SPR signal results were fitted to Hill's equation.

[0091] y = (ad) / [1+(x / c)^b]+d

[0092] Here, a and d represent the maximum and minimum signals, respectively; c represents the concentration of solution protein; and b represents the Hill's slope of the curve (Oshannessy et al., 1993; Saleviter et al., 2019; Lee et al., 2022).

[0093] One-Step Immuno Essay

[0094] As shown in Figure 14, Fv antibody (10 μg / mL, 100 μL / well) was incubated in a microplate (Maxisorp, 96-well) at 4 °C for 16 h. After blocking with BSA (1 mg / mL, 150 μL), L2 switching peptide (FAM-labeled, 100 nM, 100 μL / well) was added to each well and incubated for 1 h. After washing with PBS, NATtrol™ SARS-CoV-2 reagent (100 μL / well) diluted 100-fold (Ct value = 34.1) to 24,300-fold (Ct value = 42.1) was incubated for 1 h. The supernatant from each well was transferred to another microplate. The fluorescence intensity of the supernatant was measured using a PerkinElmer Inc. Measurements were made using a Victor X5 fluorescence spectrometer (Waltham, MA, USA).

[0095] Screening of Fv antibodies against the SARS-CoV-2 spike protein (RBD)

[0096] The Fv-antibody library comprises the variable region (V) of immunoglobulin G as described in the prior art. H ) is prepared using autodisplay (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). The Fv library has three CDR regions and four frame regions, and the amino acid sequence of the CDR3 region was randomized by site-directed mutagenesis as shown in Figure 3.

[0097] The Fv-antibody library prepared in this way was expressed on the outer membrane of E. coli using autodisplay technology, and it was expressed up to 10 for more than 90% of the total E. coli population of the Fv-antibody library. 5High expression yields of Fv-antibodies / E. coli can be achieved (Jose and Meyer, 2007; Jung et al., 2021c; Jung et al., 2021d).

[0098] SARS-CoV-2 is known to bind to the ACE2 receptor on host cells using the spike protein on the viral surface, and the core region of the binding has been reported to be the receptor binding domain (RBD) of the spike protein (Lan et al., 2020; Sun et al., 2020). The amino acid sequence of RBD is known to be residues 319-541 (222 amino acids, 24.9 kDa), and it was used as an antigen for screening a Fv-antibody library. As shown in Figure 15, the RBD antigen was produced as a fusion protein with GFP, consisting of the RBD region (amino acid residues 319-541) and GFP. As shown in SDS-PAGE, the RBD antigen was expressed as a soluble protein with a molecular weight of 52.1 kDa. The RBD antigen was used to screen a Fv-antibody library, and target E bound to the RBD antigen was identified using FACS. E. coli cells were observed to have a fluorescent signal. FACS analysis of the Fv-antibody library revealed a high fluorescent region, indicating target E. coli cells with binding affinity for the RBD antigen. These target E. coli cells were isolated using FACS and cultured on agar plates. In the case of the control E. coli, this high fluorescent region was not observed in the FACS analysis, indicating that the RBD antigen did not bind to the control E. coli. Therefore, it can be confirmed that the binding of the RBD antigen to E. coli (Fv-antibody library) occurred through specific binding between the RBD antigen and the Fv-antibody on the outer membrane. Among the screened and cultured clones, 30 clones were selected and tested for binding to the RBD antigen using FACS. As shown in Figure 5, the control E. coli cells with only the CDR1 and CDR2 regions were tested.Compared to E. coli, several clones were observed to have significantly higher binding to the RBD antigen, and eight clones (clone numbers 8, 9, 11, 15, 25, 26, 29, and 30) were observed. These clones were then reacted with different concentrations of the RBD antigen, and quantitative binding characteristics were observed for four clones (clone numbers 8, 11, 15, and 29). As shown in Fig. 6, the binding constant (K) of each clone was determined from the dose response curve. D) was measured from 100 to 140 μM, and the response from the control E. coli was measured at the baseline level over the entire concentration range of the RBD antigen. These results demonstrate that the Fv antibody on the autodisplayed E. coli outer membrane has a specific binding affinity for the RBD antigen. The oligonucleotide sequences of the CDR3 region were analyzed for four clones selected from gene sequencing, and the corresponding amino acid sequences were correlated as listed in Table 1. Using the genetic information, the Fv antibody was expressed as a fusion protein with GFP from the four selected clones. As previously reported, the fusion protein with GFP has the following advantages: (1) Fv antibody has limited solubility, and the fusion protein with GFP has much improved solubility (Pedelacq et al., 2006; Liu et al., 2019); (2) GFP can be effectively used for immobilization on the metal surface of the biosensor. As shown in Fig. 7, autodisplayed Fv antibodies were expressed using an expression vector (pJB030). The expression of four Fv antibodies was confirmed to have a molecular weight of 41 kDa by SDS-PAGE, and the Fv antibodies were named Anti-RBD1 for clone number 8, Anti-RBD2 for clone number 11, Anti-RBD3 for clone number 15, and Anti-RBD4 for clone number 29 as listed in Table 1. The binding properties of the expressed Fv antibodies were analyzed using an SPR biosensor. First, the binding properties of the Fv antibodies were analyzed for the Fv antibodies immobilized on the SPR biosensor. As shown in Fig. 8, the Fv antibodies were immobilized on the gold surface of the SPR biosensor, and the RBD antigen was treated in a concentration range of 25-200 nM to obtain the binding and dissociation profiles. As shown in Fig. 10, the binding constant (K D) was measured as 81 nM for Anti-RBD1, 85 nM for Anti-RBD2, 71 nM for Anti-RBD3, and 70 nM for Anti-RBD4. As shown in Fig. 9, the RBD antigen was immobilized on the gold surface of the SPR biosensor, and four types of Fv-antibodies were treated in the concentration range of 6.2 to 50.0 nM to obtain the binding and dissociation profiles. As shown in Fig. 11, the binding constant (K D ) was measured as 20 nM for Anti-RBD1, 24 nM for Anti-RBD2, 13 nM for Anti-RBD3, and 24 nM for Anti-RBD4. In the next step, the binding properties of Fv-antibodies to RBD antigens in solution were analyzed using SPR biosensor. In previous studies (Lan et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Zahradnik et al., 2021), the binding constant (K) of SARS-CoV-1 spike protein to the ACE2 receptor of host cells D ) was reported to be 31 to 100 nM and the binding constant (K) of SARS-CoV-2 D ) was reported to be 4.7 to 10.0 nM, which confirms that about 10 times stronger binding to the ACE2 receptor occurred in SARS-CoV-2 compared to SARS-CoV-1. Therefore, the binding constant (K) between Fv-antibody and RBD antigen D ) was measured to have relatively strong binding to SARS-CoV and the ACE2 receptor of host cells. These results demonstrate that the expressed Fv-antibody can be applied to detect SARS-CoV.

[0099] Virus detection using Fv antibodies against RBD antigens

[0100] The expressed Fv antibodies were used for the detection of pseudovirus particles expressing the SARS-CoV-2 spike protein of wild-type (Wuhan-1) and variants (Delta, Omicron BA.2, Omicron BA.4 / 5) in lentivirus envelopes. The amino acid sequences of the SARS-CoV-2 SP variants in pseudoviruses are summarized in Table 2.

[0101] SARS-CoV-2 SP variantsAmino acid mutations in SARS-CoV-2 SP variantsCountsOriginal (D614)D614G1Delta (B.1.617.2)T19R, T95I, G142D, E156G, F157-R158,L452R, T478K, D614G, P681R, D950N10Omicron (BA.2)T19I, L24-P26, A27S, G142D, V213G,G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K29Omicron (BA.4 / BA.5)T19I, L24-P26, A27S, H69+V70, G142D, V213G,G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K31

[0102] The concentration of pseudo-virus particles was determined in copies / mL using the Lenti-X™ qRT-PCR Titration kit (#631235, Takara Bio, USA) in 10 8-9The binding properties of the expressed Fv-antibody to pseudo-virus particles were measured using the Fv-antibody immobilized on the gold surface of the SPR biosensor, as shown in Figure 12. The dose response curve was 1.5 × 10 5 2.5×10 6 copies / mL were treated with wild-type (Wuhan-1 and three mutants) pseudo-virus particles. In the case of Anti-RBD1, as shown in Fig. 16, four types of pseudo-virus particles showed a quantitatively increasing response depending on the concentration of the pseudo-virus particles. Among the four types of pseudo-virus particles, the wild-type and Delta-mutant showed similar levels of response, and the two Omicron-mutants showed relatively lower responses compared to the other two. In addition, the limits of detection (LOD) were measured as 168 copies / μL for the wild-type, 189 copies / μL for the Delta-mutant, 187 copies / μL for Omicron BA.2, and 206 copies / μL for Omicron BA.4 / 5. These results indicate that (1) Anti-RBD1 was not sensitive enough to recognize amino acid sequence variations of the wild-type RBD antigen, and (2) Anti-RBD1 can be used to detect wild-type and mutants. For anti-RBD3, the wild-type and Delta mutant showed similar levels of response, while the two Omicron mutants showed much lower responses than the other two mutants, as in the case of anti-RBD2. The LOD was measured as 206 copies / μL for the wild-type, 230 copies / μL for the Delta mutant, 408 copies / μL for Omicron BA.2, and 553 copies / μL for Omicron BA.4 / 5. These results indicate that (1) anti-RBD3 was much more sensitive to the wild-type and Delta mutant compared to the two Omicron mutants, and (2) anti-RBD2 can also be used to selectively detect the wild-type among the two Omicron mutants. For anti-RBD4, a quantitative increase in four types of pseudovirus particles was observed depending on the concentration of pseudovirus particles. Among the four pseudovirus particles, the wild type showed relatively higher sensitivity than the other variants.And the LOD was measured as 158 copies / μL for wild type, 165 copies / μL for Delta-mutant, 267 copies / μL for Omicron BA.2, and 362 copies / μL for Omicron BA.4 / 5. These results indicate that Anti-RBD4 can be used for the sensitive detection of wild-type RBD antigen and Delta-mutant. The expressed Fv-antibody was used for the detection of real SARS-CoV-2 (wild type, Wuhan-1), which was manufactured in a medical sample matrix (called NATtrol™ SARS-CoV-2) through head inactivation by Zeptometrix Co. (Buffalo, NY, USA) (Wei et al., 2021; Park et al., 2022a). According to the manufacturer, the virus particle concentration was 10. 6 copies / mL. The binding properties of Fv-antibodies were measured using Fv-antibodies immobilized on the gold surface of an SPR biosensor, as shown in Figure 13. Then, SARS-CoV-2 samples in the concentration range of 34.1 to 37.1 (Ct) were treated to obtain binding and dissociation profiles. As shown in Figure 17, the binding constant (K D) was measured as 35.7 (Ct) for Anti-RBD1, 35.6 (Ct) for Anti-RBD2, 36.2 (Ct) for Anti-RBD3, and 35.4 (Ct) for Anti-RBD4. And LOD was measured as 0.83 copies / μL (Ct=37.7) for Anti-RBD1, 0.67 copies / μL (Ct=38.0) for Anti-RBD2, 0.71 copies / μL (Ct=37.9) for Anti-RBD3, and 0.70 copies / μL (Ct=37.9) for Anti-RBD4. Since the conventional cutoff value for a positive COVID-19 test was usually reported to be 34-35 (Ct) using real-time PCR (Engelmann et al., 2021; Rabaan et al., 2021), the LOD for detecting SARS-CoV-2 using the screened Fv antibody can be considered feasible for the medical diagnosis of COVID-19. A one-step immunoassay based on switching peptides was constructed for the detection of real SARS-CoV-2 (wild type, Wuhan-1) using the expressed Fv antibody. As previously reported (Bong et al., 2021a; Lee et al., 2022; Park et al., 2022b), the switching peptides were developed using amino acid sequences of the framework regions (FRs) of the heavy and light chains of immunoglobulin G (IgG). This region is known to form the binding pocket of IgG through self-assembly and preserves its amino acid sequence across all types of IgG. Switching peptides are created using amino acids from the FR and can bind to all types of IgG. These switching peptides can be released from IgG when target antigen(s) bind to the binding pocket of IgG through specific interactions with the CDR regions. Therefore, a one-step immunoassay based on fluorescently labeled switching peptides was developed to detect target antigens by mixing samples without a washing step.In the present invention, the switching peptide was conjugated to an expressed Fv antibody for the construction of a one-step immunoassay. Since the expressed Fv antibody has an IgG VH framework with four FR and three CDR regions, a switching peptide (L2 switching peptide) made from the IgG light chain can bind to the Fv antibody, and this switching peptide can quantitatively release the concentration of SARS-CoV-2 bound to the Fv antibody. The one-step immunoassay for SARS-CoV-2 detection was constructed by conjugating a fluorescently labeled switching peptide (L2 switching peptide) to an immobilized Fv antibody, as shown in Figure 18. The actual SARS-CoV-2 sample was used after serial dilution of the previously used NATtrol™ SARS-CoV-2 from Zeptometrix Co. (Buffalo, NY, USA). Four types of expressed Fv antibodies were immobilized on a microplate, and fluorescently labeled L2-switching peptides were incubated to bind them. The one-step immunoassay plate prepared in this way was used to detect SARS-CoV-2 samples in the concentration range of 34.1–42.1 (Ct). As shown in Figure 14, the binding constant (K. D ) was measured as 37.1 (Ct) for Anti-RBD1, 36.4 (Ct) for Anti-RBD2, 36.2 (Ct) for Anti-RBD3, and 37.7 (Ct) for Anti-RBD4. And LOD was measured as 0.22 copies / μL (Ct=39.6) for Anti-RBD1, 0.14 copies / μL (Ct=40.3) for Anti-RBD2, 0.47 copies / μL (Ct=38.5) for Anti-RBD3, and 0.16 copies / μL (Ct=40.1) for Anti-RBD4. Therefore, the LOD for detecting SARS-CoV-2 using the one-step immunoassay method based on switching peptide is confirmed to be feasible for the medical diagnosis of COVID-19.

[0103] The sequence list used in this specification is as follows.

[0104] SequenceIDNumberSequence NumberSequenceNameSequence NameMoleculeTypeMoleculeTypeOrganismSequenceSequence1CDR1AAsynthetic constructTYGIQ2CDR2AAsynthetic constructWIHAGTGGTKYSRKFQG3CDR3_8AAsynthetic constructDISGEKHPADF4CDR3_11AAsynthetic constructDGTPKGPRNDF5CDR3_15AAsynthetic constructDPPTTGTMVTDF6CDR3_29AAsynthetic constructYGSLQAQAPDD

[0105] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0106] [Explanation of symbols]

[0107] 100: Substance for detecting SARS-CoV-2

[0108] 110: Antibody or antigen-binding fragment thereof

[0109] 110-1: CDR1

[0110] 110-2: CDR2

[0111] 110-3: CDR3

[0112] 120: Switching Peptide

[0113] 130: Marker

[0114] 200: One-step SARS-CoV-2 diagnostic method

Claims

1. As a peptide that specifically binds to SARS-CoV-2, Comprising one or more peptide sequences selected from the group comprising the peptide sequence of SEQ ID NO: 3, the peptide sequence of SEQ ID NO: 4, the peptide sequence of SEQ ID NO: 5, and the peptide sequence of SEQ ID NO:

6. Peptide.

2. An antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2, A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4; A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5; and A variable region comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6; Including those selected from the group consisting of, An antibody or antigen-binding fragment thereof.

3. In paragraph 2, The above antibody or antigen-binding fragment thereof, A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 5; and A heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 6; Including those selected from the group consisting of, An antibody or antigen-binding fragment thereof.

4. In paragraph 2, The above antibody or antigen-binding fragment thereof is in the form of a single domain antibody, An antibody or antigen-binding fragment thereof.

5. Coding a peptide according to paragraph 1 or an antibody or antigen-binding fragment thereof according to paragraph 2; Nucleic acid.

6. Containing nucleic acid according to Article 5, Recombinant expression vector.

7. Transformed with a recombinant expression vector according to Article 6, cell.

8. In paragraph 7, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli, and insect cells. cell.

9. In paragraph 8, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp.) and Neurospora crassa (Neurospora crassa), comprising one or more cells selected from the group consisting of cell.

10. Antibody or antigen-binding fragment thereof according to paragraph 2; A switching peptide capable of specifically and reversibly binding to one or more of the first to fourth framework regions (FR1, FR2, FR3 and FR4) of the heavy or light chain of the antibody or antigen-binding fragment thereof; and A marker bound to the above switching peptide; comprising; Substance for detecting SARS-CoV-2.

11. In paragraph 10, The above antibody or antigen-binding fragment thereof is single-stranded, Substance for detecting SARS-CoV-2.

12. In paragraph 10, The above marker is a fluorescent marker, Substance for detecting SARS-CoV-2.

13. Containing a substance for detecting SARS-CoV-2 according to Article 10; One-step SARS-CoV-2 diagnostic kit.

14. A step of mixing a substance for detecting SARS-CoV-2 in a one-step SARS-CoV-2 diagnostic kit according to Article 13 and a sample to be detected, and then confirming the marker; A one-step SARS-CoV-2 diagnostic method.

15. In paragraph 14, The above marker is prepared as a fluorescent marker, A one-step SARS-CoV-2 diagnostic method.

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