Assays for detecting SARS-COV-2

Abstract

The present disclosure relates to methods for detecting a protein or fragment thereof derived from the SARS-CoV-2 virus in a sample obtained from a subject using a primary antibody or antigen-binding fragment thereof that binds to the protein or fragment thereof derived from the SARS-CoV-2 virus and a secondary antibody or antigen-binding fragment thereof that binds to the protein or fragment thereof derived from the SARS-CoV-2 virus.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 060,975, filed August 4, 2020, 63 / 065,898, filed August 14, 2020, 63 / 067,051, filed August 18, 2020, and 63 / 126,336, filed December 16, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing The text of the computer readable sequence listing filed herewith, entitled "38691-601_SEQUENCE_LISTING_ST25," created on August 4, 2021, and having a file size of 13,107 bytes, is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to components and methods for detecting SARS-CoV-2 antigens. [Background technology]

[0004] A novel coronavirus (SARS-CoV-2 (2019-nCoV)), which causes fever, severe respiratory illness, and pneumonia, emerged as a human pathogen in late 2019 in Hubei Province, China. The disease associated with SARS-CoV-2 (2019-nCoV) has been named COVID-19. The novel coronavirus is a member of the Betacoronavirus genus and is closely related to several bat coronaviruses and severe acute respiratory syndrome coronavirus (SARS-CoV). However, unlike SARS-CoV, SARS-CoV-2 (2019-nCoV) transmits rapidly between humans.

[0005] As of the end of July 2021, over 190 million cases of COVID-19 have been confirmed in over 200 countries, and COVID-19 complications have been the cause of over 4 million deaths. Given the health risks posed by SARS-CoV-2 infection, there is a need for methods and kits for assessing coronavirus infection in humans, including methods for determining the presence and / or quantity of SARS-CoV-2 proteins in one or more samples obtained from a subject. Summary of the Invention

[0006] The present disclosure provides methods, devices, and kits for detecting the presence or determining the amount of SARS-CoV-2 in a sample from a subject.

[0007] In some embodiments, the method includes contacting a sample obtained from the subject with a primary antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus, under conditions that allow binding of the protein or fragment thereof derived from the SARS-CoV-2 virus, if present in the sample, to the primary antibody or antigen-binding fragment thereof; contacting the sample with a conjugate comprising a secondary antibody that specifically binds to the protein or fragment thereof derived from the SARS-CoV-2 virus and a detectable label; and assessing the presence of a signal of the detectable label, wherein the presence of the signal of the detectable label indicates the presence of the protein or fragment thereof derived from the SARS-CoV-2 virus in the sample.

[0008] In some embodiments, the protein or fragment thereof derived from the SARS-CoV-2 virus is a nucleocapsid (N) protein. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may recognize different epitopes of a protein derived from the SARS-CoV-2 virus (e.g., a SARS-CoV-2 virus nucleocapsid (N) protein).

[0009] The primary antibody or antigen-binding fragment thereof may comprise (i) a heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO: 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 2, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID NO: 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 6. In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 8. In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 17 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 18.

[0010] The secondary antibody or antigen-binding fragment thereof may comprise (i) a heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO: 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 10, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID NO: 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 14. In some embodiments, the secondary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 16.

[0011] In some embodiments, the method is performed using an immunoassay, which may be an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay (LFA).

[0012] The sample can be any sample from a subject containing or suspected of containing SARS-CoV02. In some embodiments, the sample comprises a nasal or nasopharyngeal swab or brush, saliva, mucus, blood, serum, or plasma.

[0013] Also disclosed herein are lateral flow devices comprising a primary antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus, and a secondary antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus. In some embodiments, the primary antibody or antigen-binding fragment thereof is immobilized. In some embodiments, the test line comprises the primary antibody or antigen-binding fragment thereof. In some embodiments, the secondary antibody or antigen-binding fragment thereof comprises a detectable label. In some embodiments, the sample pad comprises the secondary antibody or antigen-binding fragment thereof.

[0014] In some embodiments, the protein or fragment thereof derived from the SARS-CoV-2 virus is a nucleocapsid (N) protein. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may recognize different epitopes of a protein derived from the SARS-CoV-2 virus (e.g., a SARS-CoV-2 virus nucleocapsid (N) protein).

[0015] The primary antibody or antigen-binding fragment thereof may comprise (i) a heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO: 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 2, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID NO: 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 6. In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 8. In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 17 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 18.

[0016] The secondary antibody or antigen-binding fragment thereof may comprise (i) a heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO: 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 10, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID NO: 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO: 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO: 14. In some embodiments, the secondary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO: 16.

[0017] Further disclosed are kits comprising the lateral flow devices described herein. The kits may further comprise at least one or both of an extraction buffer and a sampling device (e.g., a nasal swab).

[0018] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a graph of the dose response of an exemplary lateral flow assay to test the hook effect. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure is based, at least in part, on the development of a method using a pair of antibodies that allows for the detection of SARS-CoV-2 with increased specificity and a lower limit of detection.

[0021] As used herein, the words "comprise," "including," "having," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0022] When numerical ranges are recited herein, each intervening number is expressly contemplated, to the same degree of precision. For example, in the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0023] As used herein, the term "immunoglobulin" or "antibody" refers to a protein found in the blood or other body fluids of vertebrates, which is used by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein that contains at least one complementarity-determining region (CDR). The CDRs form the "hypervariable region" of the antibody, which is responsible for antigen binding (discussed further below). A whole immunoglobulin usually consists of four polypeptides: two identical copies of heavy (H) chain polypeptides and two identical copies of light (L) chain polypeptides. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H1 , C H2 , and C H3 ) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region. The light chain of an antibody can be assigned to one of two different types, kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. In a typical immunoglobulin, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0024] The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V LThe regions have the same general structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to the relatively conserved amino acid sequences within the variable region located between the CDRs. Each variable domain has four framework regions, designated FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides the structural framework for the variable region (see, e.g., CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)).

[0025] The framework regions are linked by three CDRs. As discussed above, the three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding. The CDRs form loops that connect, and in some cases include, a portion of the beta-sheet structure formed by the framework regions. The constant regions of the light and heavy chains are not directly involved in binding the antibody to the antigen, but the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular cytotoxicity through interactions with effector molecules and cells.

[0026] As used herein, when an antibody or other entity (e.g., an antigen-binding domain) "specifically recognizes" or "specifically binds" an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. In this regard, "substantially higher affinity" means an affinity that is high enough to allow detection of the antigen or epitope as distinct from the entity using a desired assay or measurement device. Typically, the affinity is at least 10 7 M -1 (e.g., >10 7 M -1 , >108 M -1 , >10 9 M -1 , >10 10 M -1 , >10 11 M -1 , >10 12 M -1 , >10 13 M -1 etc.) binding constant (K a ) means a binding affinity having a specific epitope. In certain such embodiments, the antibody is capable of binding to different antigens as long as the different antigens contain that particular epitope. In certain cases, for example, homologous proteins from different species may contain the same epitope.

[0027] As used herein, "antibody" and "antibodies" refer to monoclonal antibodies, monospecific antibodies (which may be, e.g., monoclonal or may be produced by other means that produce them from a common germ cell), multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, including, but not limited to, antibodies from birds (e.g., ducks or geese), sharks, whales, and non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.). and mammals, including humans, or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single-chain variable fragments ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFv"), and anti-idiotypic ("anti-Id") antibodies, dual-domain antibodies, dual-variable domain (DVD), or triple-variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods for making them are described in Wu, C., et al. al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application WO2001 / 058956, the contents of each of which are incorporated herein by reference), or domain antibodies (dAbs) (e.g., those described in Holt et al. (2014) Trends in Biotechnology 21:484-490, including naturally occurring single domain antibodies (sdAbs) from, for example, cartilaginous fish and camelids, or synthetic single domain antibodies (sdAbs) such as nanobodies, VHHs, or other domain structures), as well as functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain the analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.For simplicity, antibodies to an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies."

[0028] The terms "antibody fragment," "antibody fragment," and "antigen-binding fragment" of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally: Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Any antigen-binding fragment of an antibody described herein is within the scope of the present invention. An antibody fragment desirably includes, for example, one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or a combination thereof. In some embodiments, the portion does not include the constant heavy chain domains of the Fc region of an intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include: (i) V L , V H , C L , and C H1 (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V fragment, which is a single arm of an antibody. L and V H (iv) Fab' fragments obtained by cleaving the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); (vi) antibody single variable region domains (V) that specifically bind to an antigen. H or V L(xii) a single-chain polypeptide containing three CDRs of a light chain variable domain; (xiii) a single-chain polypeptide containing three CDRs of a light chain variable domain; (xiv) a single-chain polypeptide containing only one heavy chain variable domain; and (xv) a single-chain polypeptide containing three CDRs of a heavy chain variable domain.

[0029] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (which produces two identical Fab fragments) or pepsin (which produces the F(ab')2 fragment). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the variable and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is still capable of cross-linking antigen molecules like the parent IgG molecule.

[0030] As used herein, "fragment antigen-binding fragment" or "Fab fragment" refers to a fragment of an antibody that binds to an antigen and contains one antigen-binding site, one complete light chain, and part of one heavy chain. Fab is a fragment of an antibody that binds to an antigen and contains one antigen-binding site, one complete light chain, and part of one heavy chain. L , V H , C L , and C H1Fab is a monovalent fragment consisting of one constant domain and one variable domain from each of the heavy and light chains. Fab consists of one constant domain and one variable domain from each of the heavy and light chains. The variable domain contains the paratope (antigen-binding site), which comprises a series of complementarity-determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on the antigen. For example, Fab fragments can be produced as described in the art using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or they can be produced by recombinant means.

[0031] As used herein, "F(ab')2 fragment" refers to an antibody generated by pepsin digestion of a whole IgG antibody, removing most of the Fc region while leaving a portion of the hinge region intact. The F(ab')2 fragment has two antigen-binding F(ab) portions linked together by disulfide bonds and is therefore bivalent, with a molecular weight of approximately 110 kDa. Bivalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules, allowing for better tissue penetration and facilitating antigen recognition in immunohistochemistry. Furthermore, the use of F(ab')2 fragments avoids nonspecific binding to Fc receptors or protein A / G on live cells. The F(ab')2 fragment can bind to and precipitate antigens.

[0032] As used herein, "framework" (FR) or "framework sequence" can refer to the remaining sequence of the variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined in different systems, the meaning of a framework sequence is correspondingly subject to different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 in the light chain and CDR-H1, CDR-H2, and CDR-H3 in the heavy chain) also divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4), with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3, or FR4, framework regions referred to by others represent the combined FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that make up a framework region.

[0033] Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "acceptor" framework sequences (or simply "acceptor sequences") to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences listed in publicly available databases, such as V-base (hypertext transfer protocol: vbase(dot)mrc-cpe(dot)cam(dot)ac(dot))uk) or the International ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol: imgt(dot)cines(dot)fr / texts / IMGTrepertoire / LocusGenes / ).

[0034] "Recombinant antibody" and "recombinant antibody" refer to antibodies prepared in one or more steps, including recombinantly cloning nucleic acid sequences encoding all or part of one or more monoclonal antibodies into a suitable expression vector, followed by expression of the antibody in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate antibodies, DVD-Ig®, and other antibodies described herein (i). (Dual variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).

[0035] The terms "nucleic acid," "polynucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably herein and refer to polymers or oligomers of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The terms encompass any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of the bases. The polymer or oligomer can be heterogeneous or homogeneous in composition, isolated from natural sources, or artificially or synthetically produced. In addition, the nucleic acid can be DNA or RNA, or a mixture thereof, and can exist permanently or transiently in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence includes other types of nucleic acid structures, such as DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acids (LNAs; see, e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see, e.g., J. Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes. The terms "nucleic acid" and "nucleic acid sequence" can also encompass chains that include non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks (e.g., "nucleotide analogs") that can exhibit the same function as natural nucleotides.

[0036] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0037] The terms "immunogen" and "antigen" are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An "immune response" may involve, for example, antibody production and / or activation of immune effector cells. An antigen, in the context of the present disclosure, may include any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that elicits an immune response in a mammal. "Epitope" refers to the sequence of an antigen that is recognized by an antibody or antigen receptor. An epitope is also referred to in the art as an "antigenic determinant." In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope may comprise a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl, or a sulfonyl group. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a "conformational" epitope) and / or specific charge characteristics. The antigen may be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which elicits an immune response in a mammal, preferably resulting in protective immunity.

[0038] As used herein, the terms "detectable label" and "label" refer to a moiety capable of producing a signal detectable by visual or instrumental means. In some embodiments, the label is a direct label, e.g., an entity that is readily visible in its native state with the naked eye or with the aid of optical filters and / or applied stimuli, e.g., UV light, which promotes fluorescence. For example, colored microparticles, e.g., dye sols, metal sols (e.g., gold), and colored latex particles, are highly suitable. In some embodiments, the label is an indirect label, e.g., an enzyme, e.g., alkaline phosphatase and horseradish peroxidase. Indirect labels typically require the addition of one or more developing reagents, e.g., substrates, before a visible signal can be detected.

[0039] As used herein, "presence" or "absence" (or "present" or "absent") is used in a relative sense to describe the amount or level of a particular entity (e.g., an analyte). For example, when an analyte is said to be "present" in a test sample, it means that the level or amount of the analyte is above a predetermined threshold; conversely, when an analyte is said to be "absent" in a test sample, it means that the level or amount of the analyte is below a predetermined threshold. The predetermined threshold may be a detectability threshold associated with the particular test used to detect the analyte, or any other threshold. When an analyte is "detected" in a sample, it is "present" in the sample; when an analyte is "not detected," it is "absent" in the sample. Furthermore, a sample in which an analyte is "detected" or "present" is a sample that is "positive" for the analyte. A sample in which an analyte is "not detected" or "absent" is a sample that is "negative" for the analyte.

[0040] As used herein, the term "analyte" refers to a compound or composition to be detected and / or measured by specific binding to a ligand, receptor, or enzyme (e.g., an antibody or antigen). In some embodiments, the analyte is a protein or nucleic acid. In some embodiments, the analyte is an antigen. In some embodiments, the analyte is a fragment of an antigen. In some embodiments, the analyte is an analyte analog or analyte derivative (e.g., an analyte modified by chemical or biological methods). In some embodiments, the analyte is an epitope. In some embodiments, the term "analyte" refers to a protein and / or nucleic acid derived from the SARS-CoV-2 virus. In some embodiments, the analyte is a fragment and / or epitope of a protein and / or nucleic acid derived from the SARS-CoV-2 virus. In some embodiments, the analyte is the SARS-CoV-2 spike protein (the "S" protein provided at UniProtKB accession number P0DTC2) or the spike protein receptor-binding domain (see, e.g., Wrapp (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation" Science 367:1260-63; Walls (2020) "Structure, Function, and Antigenicity of the SARS CoV-2 Spike Glycoprotein" Cell 367:1260-63). 180:1-12, which are incorporated herein by reference. In some embodiments, the analyte is a viral transcription and / or replication protein (e.g., replicase polyprotein 1a (R1a) provided at UniProtKB Accession No. P0DTC1 or replicase polyprotein 1ab (R1ab) provided at UniProtKB Accession No. P0DTD1). In some embodiments, the analyte is a viral budding protein (e.g., protein 3a provided at UniProtKB Accession No. P0DTC3 or envelope small membrane protein (E) provided at UniProtKB Accession No. P0DTC4).In some embodiments, the analyte is a viral morphogenesis protein (e.g., membrane protein (M) provided in UniProtKB Accession No. P0DTC5). In some embodiments, the analyte is nonstructural protein 6 (e.g., provided in UniProtKB Accession No. P0DTC6), protein 7a (NS7A) (e.g., provided in UniProtKB Accession No. P0DTC7), protein 7b (NS7B) (e.g., provided in UniProtKB Accession No. P0DTD8), nonstructural protein 8 (NS8) (e.g., provided in UniProtKB Accession No. P0DTC8), or protein 9b (e.g., provided in UniProtKB Accession No. P0DTD2). In some embodiments, the analyte is a viral genome packaging protein (e.g., nucleoprotein (e.g., N), e.g., provided in UniProtKB Accession No. P0DTC9). In some embodiments, the analyte is an unidentified protein (eg, as provided in UniProtKB Accession No. P0DTD3 or A0A663DJA2).

[0041] As used herein, a "system" refers to multiple real and / or abstract components that work together for a common purpose. In some embodiments, a "system" is an integrated collection of hardware and / or software components. In some embodiments, each component of a system interacts with and / or is related to one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing a methodology.

[0042] As used herein, the term "sample" refers to any specimen that contains or potentially contains SARS-CoV-2 or a part or component thereof. Accordingly, the term "sample" refers to a material to be tested for the presence or quantity of an analyte, e.g., SARS-CoV-2 or a part or component thereof. Preferably, the sample is a fluid sample, preferably a liquid sample. For example, the sample may be a bodily fluid, such as blood (including, e.g., capillary blood, venous blood, dried blood spots, etc.), serum, plasma, ocular fluid, urine, mucus, semen, nasal or nasopharyngeal swab, throat swab, tears, sweat, or saliva. Viscous liquid, semi-solid, or solid specimens may be used to create solutions, eluates, suspensions, or extracts that can serve as samples. For example, a throat or genital swab may be suspended in a solution to create a sample.

[0043] "Point-of-care device" refers to a device used to provide medical diagnostic testing at or near the point of care (i.e., outside a laboratory) at the time and place of patient care (e.g., hospital, physician's office, emergency or other medical facility, patient's home, nursing home, and / or long-term care and / or hospice facility). Examples of point-of-care devices include those manufactured by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), Universal Biosensors (Rowville, Australia) (see US 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and Clinical Lab Products (Los Angeles, USA).

[0044] As used herein, the "sensitivity" of an assay refers to the ratio of subjects who are correctly identified as positive (e.g., correctly identifying subjects who suffer from the disease or condition for which they are being tested) to subjects who test positive. For example, this can include correctly distinguishing subjects who are infected with a coronavirus, such as a β-coronavirus, from subjects who are not infected with or have never been infected with a coronavirus, such as a β-coronavirus. In some embodiments, the sensitivity of an assay can be determined by assessing a change in the signal-to-noise (S / N) ratio of the assay. For example, in some embodiments, an increase in the S / N ratio can indicate improved sensitivity of the assay to a particular analyte (e.g., SARS-CoV-2 nucleocapsid protein).

[0045] As used herein, the "specificity" of an assay refers to the ratio of subjects who are correctly identified as negative (e.g., correctly identifying subjects who do not have the disease or condition for which they are being tested) to subjects who test negative. For example, this can include correctly distinguishing subjects who are infected with a coronavirus, such as a β-coronavirus, from subjects who have not been infected with a coronavirus, such as a β-coronavirus.

[0046] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms shall be clear. However, in the event of any latent ambiguity, the definitions provided herein shall take precedence over any dictionary or extrinsic definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0047] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0048] 1. Antibodies According to the disclosed methods, a sample is contacted with a primary antibody or antigen-binding fragment thereof (e.g., Fab) that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus, and a secondary antibody or antigen-binding fragment thereof (e.g., Fab) that specifically binds to a protein derived from the SARS-CoV-2 virus. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof recognize different epitopes of a protein derived from the SARS-CoV-2 virus or fragment thereof. In some embodiments, the protein derived from the SARS-CoV-2 virus is a viral genome packaging protein (e.g., the nucleocapsid (N) protein, e.g., provided in UniProtKB Accession No. P0DTC9).

[0049] The primary antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region comprising a CDR1 amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%) identical to SEQ ID NO: 1, a CDR2 amino acid sequence at least 70% identical to SEQ ID NO: 2, and a CDR3 amino acid sequence at least 70% identical to SEQ ID NO: 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence at least 70% identical to SEQ ID NO: 4, a CDR2 amino acid sequence at least 70% identical to SEQ ID NO: 5, and a CDR3 amino acid sequence at least 70% identical to SEQ ID NO: 6.

[0050] Alternatively, the primary antibody or antigen-binding fragment thereof may comprise heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences that are at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3, respectively, and / or light chain variable region CDR1, CDR2, and CDR3 amino acid sequences that are at least 90% identical to SEQ ID NO:4, SEQ ID NO:5, and / or SEQ ID NO:6, respectively.

[0051] In one embodiment, (i) each of the heavy chain variable region CDR1, CDR2, and / or CDR3 amino acid sequences comprises, consists essentially of, or consists of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3, respectively, and (ii) each of the light chain variable region CDR1, CDR2, and / or CDR3 amino acid sequences comprises, consists essentially of, or consists of SEQ ID NO:4, SEQ ID NO:5, and / or SEQ ID NO:6, respectively. When the CDR1, CDR2, and CDR3 of the heavy and / or light chains of the disclosed antibodies consist essentially of the above amino acid sequences, additional components may be included in the CDRs that do not substantially affect the antibody or antigen-binding fragment thereof (e.g., protein moieties such as biotin that facilitate purification or isolation). When the CDR1, CDR2, and CDR3 of the heavy and / or light chains of the disclosed antibodies consist of the above amino acid sequences, each CDR does not include any additional components (e.g., components that are not endogenous to the CDR). SEQ ID NO:1-CKASGYSFTSYWMHW SEQ ID NO:2-MIDPSDSETRLNQRFKDK SEQ ID NO:3-CARSLLRGVYAMDYW SEQ ID NO:4-CKASQSVSNDVAW SEQ ID NO:5-YYASNRYTGVPDR SEQ ID NO: 6-CQQDYSSPYTF

[0052] In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V) comprising, consisting essentially of, or consisting of SEQ ID NO:7. H ) amino acid sequence of SEQ ID NO: 8, and a light chain variable region (V L ) amino acid sequence. H The amino acid sequence consists essentially of SEQ ID NO:7, LWhen the amino acid sequence consists essentially of SEQ ID NO:8, additional components may be included in the heavy or light chain variable region that do not substantially affect the antibody or antigen-binding fragment thereof (e.g., protein moieties such as biotin or His tags that facilitate purification or isolation). H The amino acid sequence consists of SEQ ID NO: 7, and V L When the amino acid sequence consists of SEQ ID NO: 8, the heavy and light chain variable regions do not include any additional components (eg, components that are not endogenous to the heavy or light chain variable region).

[0053] In other embodiments, the primary antibody or antigen-binding fragment thereof may comprise a heavy chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:7 and a light chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:8. Sequence number 7-QVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIGMIDPSDSETRLNQRFKDKATLTVDRSSSTAYMQLSSPTSEDSAVYYCARSLLRGVYAMDYWGQGTSVTVSS SEQ ID NO:8- SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK

[0054] In some embodiments, the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V) comprising, consisting essentially of, or consisting of SEQ ID NO: 17. H ) amino acid sequence of SEQ ID NO: 18, and a light chain variable region (V L ) amino acid sequence. H The amino acid sequence consists essentially of SEQ ID NO: 17, LWhen the amino acid sequence consists essentially of SEQ ID NO: 18, additional components may be included in the heavy or light chain variable region that do not substantially affect the antibody or antigen-binding fragment thereof (e.g., protein moieties such as biotin or His tags that facilitate purification or isolation). H The amino acid sequence consists of SEQ ID NO: 17, L When the amino acid sequence consists of SEQ ID NO: 18, the heavy and light chain variable regions do not include any additional components (eg, components that are not endogenous to the heavy or light chain variable region).

[0055] In other embodiments, the primary antibody or antigen-binding fragment thereof may comprise a heavy chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:17 and a light chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:18. Sequence number 17-QVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIGMIDPSDSETRLNQRFKDKATLTVDRSSSTAYMQLSSPTSEDSAVYYCARSLLRGVYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC SEQ ID NO: 18- SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNES

[0056] The secondary antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region comprising a CDR1 amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%) identical to SEQ ID NO: 9, a CDR2 amino acid sequence at least 70% identical to SEQ ID NO: 10, and a CDR3 amino acid sequence at least 70% identical to SEQ ID NO: 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence at least 70% identical to SEQ ID NO: 12, a CDR2 amino acid sequence at least 70% identical to SEQ ID NO: 13, and a CDR3 amino acid sequence at least 70% identical to SEQ ID NO: 14.

[0057] Alternatively, the secondary antibody or antigen-binding fragment thereof may comprise heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences that are at least 90% identical to SEQ ID NO:9, SEQ ID NO:10, and / or SEQ ID NO:11, respectively, and / or light chain variable region CDR1, CDR2, and CDR3 amino acid sequences that are at least 90% identical to SEQ ID NO:12, SEQ ID NO:13, and / or SEQ ID NO:14, respectively.

[0058] In one embodiment, (i) each of the heavy chain variable region CDR1, CDR2, and / or CDR3 amino acid sequences comprises, consists essentially of, or consists of SEQ ID NO:9, SEQ ID NO:10, and / or SEQ ID NO:11, respectively, and (ii) each of the light chain variable region CDR1, CDR2, and / or CDR3 amino acid sequences comprises, consists essentially of, or consists of SEQ ID NO:12, SEQ ID NO:13, and / or SEQ ID NO:14, respectively. When the CDR1, CDR2, and CDR3 of the heavy and / or light chains of the disclosed antibodies consist essentially of the above amino acid sequences, additional components may be included in the CDRs that do not substantially affect the antibody or antigen-binding fragment thereof (e.g., protein moieties such as biotin that facilitate purification or isolation). When the CDR1, CDR2, and CDR3 of the heavy and / or light chains of the disclosed antibodies consist of the above amino acid sequences, each CDR does not include any additional components (e.g., components that are not endogenous to the CDR). SEQ ID NO:9-SYAIS SEQ ID NO: 10-GIIPIFGTANYAQKFQG SEQ ID NO: 11-GYWGSGYHYYGMDV SEQ ID NO: 12-GGNNIGSKSVH SEQ ID NO: 13-YDSDRPS SEQ ID NO: 14-QVWDRSSDLVV

[0059] In some embodiments, the secondary antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V) comprising, consisting essentially of, or consisting of SEQ ID NO: 15. H ) amino acid sequence of SEQ ID NO: 16, and a light chain variable region (V L ) amino acid sequence. H The amino acid sequence consists essentially of SEQ ID NO: 15, L When the amino acid sequence consists essentially of SEQ ID NO: 16, additional components may be included in the V or light chain variable region that do not substantially affect the antibody or antigen-binding fragment thereof (e.g., protein moieties such as biotin or His tags that facilitate purification or isolation). H The amino acid sequence consists of SEQ ID NO: 15, L When the amino acid sequence consists of SEQ ID NO: 16, the heavy and light chain variable regions do not include any additional components (eg, components that are not endogenous to the heavy or light chain variable region).

[0060] In other embodiments, the secondary antibody or antigen-binding fragment thereof may comprise a heavy chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:15 and a light chain variable region amino acid sequence that is at least 70% identical to SEQ ID NO:16. SEQ ID NO: 15-EVQLVESGGGVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGYWGSGYHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKC SEQ ID NO: 16-DIQMTQSPSSSVAPGKTARIPCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSSDLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS

[0061] Nucleic acid or amino acid sequence "identity" as described herein can be determined by comparing a nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. Percent identity is the number of nucleotides or amino acid residues that are the same (e.g., identical) between a sequence of interest and a reference sequence, divided by the length of the longest sequence (e.g., the length of either the sequence of interest or the reference sequence, whichever is longer). Numerous mathematical algorithms for obtaining optimal alignments and calculating identity between two or more sequences are known and are incorporated into many available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST2.1, BL2SEQ, and later versions thereof), and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also described, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge Disclosed in UK (1997).

[0062] One or more amino acids of the above-described antibodies or antigen fragments thereof can be replaced or substituted with a different amino acid. An amino acid "replacement" or "substitution" refers to the replacement of one amino acid at a given position or residue with another amino acid at the same position or residue within a polypeptide sequence.

[0063] Additionally, one or more amino acids can be inserted into an antibody or antigen-binding fragment thereof (e.g., insertion into the heavy and / or light chain variable region amino acid sequence). Any number of any suitable amino acids can be inserted into the amino acid sequence of an antibody or antigen-binding fragment thereof. In this regard, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or fewer, 15 or fewer, or 12 or fewer amino acids) can be inserted into the amino acid sequence of an antibody or antigen-binding fragment thereof. For example, 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can be inserted into the amino acid sequence of an antibody or antigen-binding fragment thereof. In this regard, an amino acid(s) can be inserted into any suitable position of an antibody or antigen-binding fragment thereof. Preferably, an amino acid(s) is inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of an antibody or antigen-binding fragment thereof.

[0064] The antibodies or antigen-binding fragments thereof used in the methods of the present invention are not limited to polypeptides containing the specific amino acid sequences described herein. Indeed, the antibodies or antigen-binding fragments thereof can include any heavy or light chain polypeptides that compete with the antibodies or antigen-binding fragments thereof of the present invention for binding to tenofovir or tenofovir derivatives. Antibody competition can be assayed using conventional peptide competition assays such as ELISA, Western blot, or immunohistochemistry (see, e.g., US Patents 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4:12 (2006)).

[0065] Antibodies can be produced by any of a number of techniques known in the art. For example, expression from host cells, where expression vector(s) encoding the heavy and light chains are transfected into the host cells using standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce endogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although antibodies can be expressed in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and expression of antibodies in mammalian host cells is most preferred, since such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0066] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovarian (CHO) cells (e.g., dhfr-CHO cells (described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)) used with the DHFR selection marker described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells). When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow for expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the medium using standard protein purification methods. In some embodiments, the antibody can be purified from CHO and / or HEK cells using routine techniques known in the art.

[0067] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations of the above procedures can be performed. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and / or heavy chains of an antibody. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibodies.

[0068] In a preferred system for recombinant expression of an antibody or its antigen-binding portion, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Furthermore, the present disclosure provides a method for synthesizing a recombinant antibody by culturing host cells in a suitable medium until the recombinant antibody is synthesized. The method can further include isolating the recombinant antibody from the culture medium.

[0069] A humanized antibody may be an antibody or variant, derivative, analog, or fragment or portion thereof that specifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody may be derived from a non-human species antibody that binds to the desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0070] As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to that of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and usually two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (e.g., a donor antibody) and all or substantially all of the framework regions are human immunoglobulin consensus sequences. According to one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. In some embodiments, a humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain, hi certain embodiments, a humanized antibody contains only humanized variable domains of the light and / or heavy chain.

[0071] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0072] The framework (FR) and CDR regions of a humanized antibody need not correspond exactly to the parental sequences. For example, the donor antibody CDR or consensus framework may be mutated with the substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently within a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0073] Humanized antibodies can be designed to minimize unwanted immune responses in rodents against anti-human antibodies, thereby limiting the duration and effectiveness of therapeutic use of such antibodies in human recipients. A humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human residues are often referred to as "import" residues, and are usually taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than intact human variable domains have been substituted by the corresponding sequence from a non-human species. See, e.g., U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. A humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. Humanization or modification of the antibodies of the present disclosure can be carried out using any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0074] Humanized antibodies may retain high affinity for SARS-CoV-2 antigens and other favorable biological properties. Humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, e.g., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences to achieve desired antibody characteristics, such as increased affinity for SARS-CoV-2. In general, hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

[0075] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be generated. For example, human antibodies can be isolated from libraries via PROfusion and / or yeast-related technologies. Transgenic animals (e.g., mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production) can also be generated. For example, the expression of antibody heavy chain joining regions (J) in chimeric and germ-line mutant mice can be improved. HHomozygous deletion of the .) gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690; 6,682,928; and 6,984,720, the contents of each of which are incorporated herein by reference.

[0076] 2. Method The disclosed methods include contacting a sample obtained from a subject with a primary antibody or antigen-binding fragment thereof that contains a detectable label and specifically binds to a protein or fragment or epitope from the SARS-CoV-2 virus, if present in the sample, under conditions that allow the protein or fragment or epitope from the SARS-CoV-2 virus, if present in the sample, to bind to the primary antibody or antigen-binding fragment to form a first complex, as described herein. In some embodiments, the protein from the SARS-CoV-2 virus is a viral genome packaging protein (e.g., the nucleocapsid (N) protein, e.g., as provided in UniProtKB Accession No. P0DTC9).

[0077] The sample may undergo one or more processing steps prior to contact with the primary antibody or antibody-binding fragment thereof. In some embodiments, such processing steps include the addition of one or more preservatives or stabilizers to facilitate storage or transport of the sample from the collection site to the testing site. In some embodiments, such processing steps include a purification step (e.g., using filters, centrifugation, etc.) to remove one or more components from the sample in order to enrich the sample for the analyte of interest.

[0078] The primary antibody or antigen-binding fragment thereof can be contacted with the sample using any suitable method known in the art. As used herein, the term "contacting" refers to any type of combined process that brings an antibody, particularly an antibody immobilized on a solid support, into sufficiently close proximity with an analyte of interest in a sample (e.g., a protein from the SARS-CoV-2 virus) so that a binding interaction occurs if the specific analyte of interest is present in the sample. Contacting can be achieved in a variety of different ways, including directly combining the antibody or antigen-binding fragment with the sample, or exposing the sample to a solid support containing the antibody or antigen-binding fragment by introducing the solid support in close proximity to the sample. Contacting can be repeated as many times or for as long as necessary for a binding interaction to occur.

[0079] The methods described herein are preferably carried out using an immunoassay. As used herein, the term "immunoassay" refers to a biochemical test that uses antibodies or antigens to measure the presence or concentration of large or small molecules in a solution. Any suitable immunoassay may be used, and a wide variety of immunoassay types, configurations, and formats are known in the art and are within the scope of the present disclosure. Suitable types of immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), lateral flow assays, competitive inhibition immunoassays (e.g., forward and reverse), radioimmunoassays (RIAs), fluoroimmunoassays (FIAs), chemiluminescent immunoassays (CLIAs), counting immunoassays (CIAs), enzyme-amplified immunoassay techniques (EMITs), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays, single-molecule detection assays, and the like. Such methods are described, for example, in U.S. Patent Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; 5,480,792; 11,022,598, International Patent Application Publication No. WO 2016 / 161400; and Adamczyk et al., Anal. Chim. Acta, 579(1):61-67 (2006).

[0080] Immunoassay formats can be "direct," "indirect," or "sandwich." The sandwich format involves the use of a capture antigen and a detection antigen to immobilize and detect an antigen in a sample. Specifically, the surface of a solid support (e.g., an ELISA plate, beads, etc.) is coated with a capture antibody or its antigen-binding fragment, and the capture antibody binds to and immobilizes the target antigen present in a sample applied to it. A detection antibody is then added to or contacts the complex. The detection antibody can be directly labeled with the antibody to allow detection and quantification of the antigen ("direct sandwich immunoassay"). Alternatively, if the detection antibody is unlabeled, a secondary enzyme-conjugated detection antibody may be used ("indirect sandwich assay").

[0081] Thus, the disclosed methods may further include contacting the sample with a conjugate comprising a second antibody, wherein the second antibody or antigen-binding fragment thereof, part of the conjugate, specifically binds to the target antigen (e.g., a protein from SARS-CoV-2 or a fragment or epitope thereof), resulting in binding of the conjugate to the captured analyte and formation of an immune sandwich (also referred to herein as an "immune sandwich complex"). It will be understood that in a sandwich immunoassay format, the primary and secondary antibodies recognize two different, non-overlapping epitopes on the target analyte / antigen.

[0082] In certain embodiments, the primary antibody or antigen-binding fragment thereof may be bound or immobilized to a solid support. The terms "solid phase" and "solid support" are used interchangeably herein and refer to any material that can be used to bind and / or attract and immobilize one or more antibodies. Any solid support known in the art can be used in the methods described herein. Examples of suitable solid supports include electrodes, test tubes, beads, microparticles, nanoparticles, wells of microwell or multiwell plates, gels, colloids, biological cells, sheets, strips, and chips.

[0083] In one embodiment, the solid support desirably comprises a plurality (e.g., 2 or more, 50 or more, 100 or more, 1,000 or more, or 5,000 or more) of antibodies or antigen-binding fragments thereof immobilized on its surface that bind to a protein or fragment or epitope derived from the SARS-CoV-2 virus. As used herein, the term "immobilized" refers to a stable association of the binding member with the surface of the solid support. As discussed herein, after a sufficient incubation time between the solid support and the sample, the protein or fragment or epitope derived from the SARS-CoV-2 virus, if present in the sample, is desirably captured on the surface of the solid support by the immobilized antibodies.

[0084] An antibody or antibody fragment can be bound to a solid support via a bond, which may include any portion, functionalization, or modification of the support and / or antibody that facilitates binding of the antibody to the support. The bond between the antibody and the support may include one or more chemical or physical bonds (e.g., nonspecific bonds via van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic / hydrophilic interactions, etc.) and / or a chemical spacer that provides such a bond(s). Many techniques can be used to bind antibodies to a wide variety of solid supports (see, e.g., U.S. Pat. No. 5,620,850; and Heller, Acc. Chem. Res., 23:128 (1990)).

[0085] In some embodiments, the binding affinity between a protein derived from the SARS-CoV-2 virus, or a fragment or epitope thereof, and a primary or secondary antibody or antibody fragment must be sufficient to maintain binding under assay conditions, including wash steps to remove nonspecifically bound molecules or particles. Contact is desirably maintained (e.g., incubated) for a time sufficient to allow binding interactions between the protein derived from the SARS-CoV-2 virus, or a fragment or epitope thereof, and the primary or secondary antibody or antibody fragment. In addition, incubation may be in a binding buffer that promotes specific binding interactions, such as, for example, albumin (e.g., BSA), non-ionic detergents (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). The binding affinity and / or specificity of a primary or secondary antibody or antibody fragment may be manipulated or altered in an assay by varying the binding buffer.

[0086] Any unbound antibody, antibody fragment, or conjugate components may be separated from the immuno-sandwich by any suitable means, such as, for example, droplet actuation, electrophoresis, electrowetting, dielectrophoresis, electrostatic actuation, electric field-mediated, electrode-mediated, capillary force, chromatography, centrifugation, aspiration, or surface acoustic wave (SAW)-based washing methods.

[0087] The method further includes assessing the presence of a signal from a detectable label conjugated to the second antibody, wherein the presence of a signal from the detectable label indicates the presence of a protein or a fragment or epitope thereof from the SARS-CoV-2 virus in the sample.

[0088] Suitable detectable labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). For example, detectable labels can be radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S, or 125 I), fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), or enzymes (e.g., alkaline phosphatase, β-galactosidase, or horseradish peroxidase). Any method known in the art for separately conjugating an antibody to a detectable label can be used in the context of the present disclosure (see, for example, Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982)). The signal generated from the detectable label bound to the antibody can be measured based on its spectroscopic properties.

[0089] It will be understood that different configurations of the above antigen capture and immunosandwich formation methods are within the scope of the present disclosure. Indeed, the various components of the above solid support, conjugate, and detectable label can be arranged or utilized in any suitable combination, configuration, or format. For example, the disclosed methods can be performed in a one-step, delayed one-step, or two-step format in which the sample is incubated with a primary antibody followed by a secondary antibody, or vice versa. Assay reagents (e.g., microparticles, conjugates, fluorophores, etc.) can be premixed or added sequentially, as needed.

[0090] In one embodiment, a lateral flow assay is used. Lateral flow assays provide a technique for qualitatively detecting and / or quantitatively measuring an analyte in a short time using antigen-antibody interactions (e.g., using immunochromatography). These tests typically use an assay device in the form of an assay test strip, or a device in which the assay strip is mounted within a plastic case. See, for example, International Patent Application Publication No. WO2011102563A1; U.S. Patent No. 8,828,739 (each of which is incorporated herein by reference).

[0091] Lateral flow assays are generally provided in a device that includes a lateral flow test strip (e.g., nitrocellulose or filter paper), a sample application area (e.g., a sample pad), a test result area (e.g., a test line), an optional control result area (e.g., a control line), and an analyte-specific binding reagent linked to a detectable label (e.g., a colored particle or an enzyme detection system). See, e.g., U.S. Patent Nos. 6,485,982; 6,187,598; 5,622,871; 6,565,808; 6,809,687; and 10,717,082 (each of which is incorporated herein by reference). In some embodiments, the technology relates to test devices that include reagent-impregnated test strips for providing specific binding assays, e.g., immunoassays.

[0092] In some embodiments, the present disclosure relates to lateral flow devices suitable for use in the home, clinic, or hospital, intended to provide rapid analytical results with minimal skill and user involvement. In some embodiments, use of the devices described herein involves a method in which the user performs a series of operations to provide an observable test result.

[0093] In some embodiments, the lateral flow device comprises a primary antibody that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus and a secondary antibody that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus. The antibody descriptions provided above pertain to the lateral flow devices described herein. In some embodiments, the test line of the device comprises the primary antibody. In some embodiments, the sample pad comprises the secondary antibody.

[0094] In some embodiments, a sample is applied to a portion of a test strip and allowed to permeate the strip material, typically using an elution solvent such as water and / or a suitable extraction buffer (e.g., optionally containing a surfactant). This causes the sample to move into or through a detection zone of the test strip, immobilizing a specific binding reagent (e.g., an antibody) for an analyte (e.g., a protein from the SARS-CoV-2 virus, or a fragment or epitope thereof) suspected to be present in the sample. Thus, analyte present in the sample can become bound in the detection zone. Labeling reagents, which may be incorporated into or subsequently applied to the test strip, can be used to determine the extent to which the analyte becomes bound in the zone.

[0095] In some embodiments, the analytical test device includes a hollow casing constructed of a moisture-impermeable solid material containing a dry porous carrier that is in direct or indirect communication with the exterior of the casing so that a liquid test sample can be applied to the porous carrier. In some embodiments, the device also includes a labeled specific binding reagent for the analyte, which is free to migrate within the porous carrier when wet. In some embodiments, the device includes an unlabeled specific binding reagent for the same analyte, which is permanently immobilized in a detection zone on the carrier material and therefore does not migrate when wet. The relative placement of the labeled reagent and the detection zone allows a liquid sample applied to the device to receive the labeled reagent and then penetrate the detection zone, and the device provides the extent to which the labeled reagent, if any, becomes observable within the detection zone.

[0096] In some embodiments, the device includes a porous solid phase material having a labeled reagent in a first zone. The labeled reagent is retained in the first zone while the porous material is in a dry state, but is free to migrate through the porous material when the porous material is wetted, for example, by application of an aqueous liquid sample suspected of containing the analyte. In some embodiments, the porous material includes an unlabeled specific binding reagent specific for the analyte in a second zone, spatially distinct from the first zone, which may engage with the labeled reagent in either a "sandwich" or "competitive" reaction. The unlabeled specific binding reagent is firmly immobilized on the porous material so that it does not migrate freely when the porous material is in a wet state.

[0097] In some embodiments, an aqueous liquid sample suspected of containing an analyte is contacted with a device described herein, such that the sample permeates through the porous solid phase material by capillary action through the first zone to the second zone, the labeled reagent migrates therewith from the first zone to the second zone, and the presence of the analyte in the sample is determined by observing the extent to which the labeled reagent, if any, becomes bound in the second zone.

[0098] In some embodiments, the labeled reagent is a specific binding partner for the analyte. The labeled reagent, the analyte (if present), and the immobilized unlabeled specific binding reagent cooperate in a "sandwich" reaction, such that if the analyte is present in the sample, the labeled reagent binds to the second zone. In the sandwich format, the two binding reagents have specificity for different epitopes of the analyte. In some embodiments, the primary antibody is immobilized. In some embodiments, the secondary antibody comprises a detectable label.

[0099] In some embodiments, the test strip (e.g., carrier material) comprises nitrocellulose, which has a significant advantage over other strip materials, such as paper, because it has a natural ability to bind proteins without the need for prior sensitization. Specific binding reagents, such as immunoglobulins, can be directly applied to and immobilized on the nitrocellulose. No chemical treatment is required, which could interfere with the reagent's intrinsic specific binding activity. Unused binding sites on the nitrocellulose can then be blocked using a simple material, such as polyvinyl alcohol. Furthermore, nitrocellulose is readily available in a wide range of pore sizes, facilitating the selection of a carrier material to meet specific requirements, such as sample flow rate.

[0100] In some embodiments, the porous solid phase material is bound to a porous receptive member to which a liquid sample can be applied, allowing the sample to penetrate the porous solid phase material. In some embodiments, the porous solid phase material is contained within a moisture-impermeable casing or housing, and the porous receptive member to which the porous solid phase material is bound extends outside the housing and can function as a means for allowing the liquid sample to enter the housing and penetrate the porous solid phase material. The housing should be provided with means, such as an appropriately positioned window, that allows a second zone of the porous solid phase material (carrying the immobilized unlabeled specific binding reagent) to be observed from outside the housing so that the results of the assay can be observed. Optionally, the housing can also be provided with additional means, allowing another zone of the porous solid phase material to be observed from outside the housing, allowing another zone 1 to take up a control reagent, thereby allowing an indicator to indicate whether the assay procedure is complete. In some embodiments, the housing is provided with a removable cap or shroud that can protect the protruding porous receiving member during storage before use. If desired, the cap or shroud can be replaced over the protruding porous receiving member after sample application, but the assay procedure has been performed; optionally, labeled reagents can be incorporated elsewhere within the device, for example, in the bibulous sample collection member, although this is not preferred.

[0101] In some embodiments, the devices are provided as a kit suitable for hospital, clinic, or home use, hi some embodiments, the kit includes multiple (e.g., two) devices individually packaged in moisture-impermeable packaging and packaged with appropriate instructions for the user.

[0102] In some embodiments, the device includes an optional "control zone." If present, the "control" zone can be designed to provide an independent signal to the user that the device is functioning. To confirm that the sample has penetrated the test strip, for example, the control zone can be loaded with an antibody (e.g., goat anti-rabbit IgG) that will bind to a labeled antibody, e.g., a labeled rabbit IgG, from the first zone. In some embodiments, the first zone contains an antigen and / or antibody that is unrelated to the analyte and specifically captured in the control zone. In some embodiments, the control zone can contain an anhydrous reagent, e.g., anhydrous copper sulfate, that undergoes a color change or development when wetted. This will turn blue when wetted with an aqueous sample. As a further alternative, the control zone can contain an immobilized analyte that reacts with excess labeled reagent from the first zone. Because the purpose of the control zone is to indicate to the user that the test is complete, the control zone should be located downstream of the second zone where the intended test result is recorded. Thus, a positive control indicator indicates to the user that the sample has penetrated the required distance through the test device.

[0103] Ideally, the results of the assay should be discernible by eye, and to facilitate this, the direct label should be concentrated in the detection zone. In some embodiments, the detectable label is a direct label, e.g., an entity that is easily visible in its natural state with the naked eye or with the aid of an optical filter and / or applied stimulus, e.g., UV light, which promotes fluorescence. For example, colored microparticles, e.g., dye sols, metal sols (e.g., gold), and colored latex particles, are highly suitable. Concentration of the label within a small zone or volume (e.g., a test line) produces an easily detectable signal, e.g., a strongly colored area. This can be assessed visually or, if necessary, by instrumentation.

[0104] In some embodiments, the technique involves the use of indirect labels. Indirect labels, such as enzymes, e.g., alkaline phosphatase and horseradish peroxidase, can be used, but these typically require the addition of one or more developing reagents, such as substrates, before a visible signal can be detected. They can be incorporated into the porous solid phase material or, if present, the sample receiving member, so that they dissolve or disperse in an aqueous liquid sample. Alternatively, the developing reagents can be added to the sample before contacting the porous material, or the porous material can be exposed to the developing reagents after the binding reaction has occurred.

[0105] In some embodiments, the sample flow continues beyond the detection zone, and sufficient sample is applied to the porous material so that any excess labeled reagent from the first zone that is not involved in any binding reaction in the second zone is washed away from the detection zone by this continued flow. Optionally, an absorbent "sink" can be provided at the distal end of the carrier material. The absorbent sink may be constructed, for example, from Whatman 3MM chromatography paper and should provide sufficient absorbent capacity to allow any unbound conjugate to be washed away from the detection zone. As an alternative to such a sink, it may be sufficient to have a length of porous solid phase material extending beyond the detection zone.

[0106] In some embodiments, the carrier material is in the form of a strip or sheet onto which reagents are applied in spatially distinct zones, allowing the liquid sample to permeate the sheet or strip from one side or end to another.

[0107] In some embodiments, the material comprising the porous solid phase is nitrocellulose, which has the advantage that the antibody in the second zone can be firmly immobilized without prior chemical treatment. For example, if the porous solid phase material comprises paper, the antibody in the second zone must be immobilized by chemical coupling, for example, using CNBr, carbonyldiimidazole, or tresyl chloride.

[0108] After application of the antibody to the detection zone, the remainder of the porous solid phase material is treated to block any remaining binding sites elsewhere. Blocking can be achieved by treatment with proteins (e.g., bovine serum albumin or milk proteins) or polyvinyl alcohol or ethanolamine, or any combination of these agents. The labeled reagent for the first zone can then be dispensed onto the dry carrier and, when wet, will be mobile within the carrier. Between each of these various process steps (sensitization, application of unlabeled reagent, blocking, and application of labeled reagent), the porous solid phase material is dried.

[0109] The disclosed methods may include a quality control component. "Quality control components" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., one or more, e.g., multiple) can be used to establish a calibration (standard) curve for interpolating the concentration of an analyte, such as an antigen. Alternatively, a single calibrator that approximates a reference or control level (e.g., a "low," "medium," or "high" level) can be used. Multiple calibrators (e.g., multiple calibrators or varying amounts of calibrator(s)) can be used in combination to form a "sensitivity panel." Optionally, a calibrator is part of a series of calibrators, each of which differs from the other calibrators in the series, for example, in concentration or detection method (e.g., colorimetric or fluorescent detection).

[0110] The disclosed methods may further include detecting one or more pathogens or antigens thereof in addition to SARS-CoV-2. A pathogen can be any infectious organism capable of causing an infection of interest or causing disease symptoms for which differential diagnosis for appropriate treatment is required. Infectious organisms of interest include bacteria (including, but not limited to, Escherichia species, Streptococcus species, Haemophilus species, Staphylococcus species, and Neisseria species), viruses (including, but not limited to, adenovirus, enterovirus, echovirus, human herpesvirus, mumps virus Ag, influenza, parainfluenza, respiratory syncytial virus (RSV), other human coronaviruses, rhinovirus, human metapneumovirus), or eukaryotic pathogens (including fungi and protozoa). In some embodiments, the method further includes detecting influenza, rhinovirus, RSV, and / or adenovirus.

[0111] Detection of other pathogens may use the same types of methods used to detect SARS-CoV-2 antigens, such as the immunoassays described above. Alternatively, detection of other pathogens may use non-immunoassays of detection, including, but not limited to, nucleic acid detection (e.g., microarrays), nucleic acid amplification (e.g., RT-PCR), and / or sequencing (e.g., next-generation sequencing), immunofluorescence assays, serological assays, and cell culture-based detection.

[0112] The detection of other pathogens can be performed simultaneously with, before, or after the detection of SARS-Cov-2 antigens. For example, if the method for detecting other pathogens is the same type of immunoassay, the sample can be incubated with the detection reagent for the other pathogens simultaneously with that for detecting SARS-Cov-2 antigens (e.g., a single sample pad for multiple test strips in a lateral flow assay).

[0113] 3. Kits and measuring instruments Also provided herein are kits for carrying out the above-described methods. The instructions included in the kits can be attached to packaging materials or included as a package insert. The instructions can be written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.

[0114] The kit may include a lateral flow device described herein. In some embodiments, the lateral flow device is in a sealed package (e.g., enclosed in moisture-tight wrapping). The lateral flow device may be disposable. In some embodiments, the device is provided as a kit suitable for use in a hospital, clinic, or home. In some embodiments, the kit includes multiple (e.g., two) devices individually packaged in moisture-tight packaging and packaged with appropriate user instructions.

[0115] The kit may include a sample container. The sample container may be a cuvette. The sample holder may be a cartridge including a microfluidics module. The sample holder may be an ELISA plate. The sample container may contain one or more reagents (e.g., binding buffer and antibodies) useful for performing the methods disclosed herein. The sample holder may also include other material(s) that may be desirable from a user's perspective, such as buffer(s), diluent(s), standard(s) (e.g., calibrators and controls), and / or any other material useful for sample processing, washing, or performing other steps of the assay.

[0116] The kit may further include a reference standard for detecting a protein or fragment thereof derived from the SARS-CoV-2 virus in a sample. The reference standard may be used to establish a standard curve for interpolating and / or extrapolating the protein from the concentration of the SARS-CoV-2 virus or fragment thereof. The kit may include reference standards with different concentration levels. For example, the kit may include one or more reference standards with high, medium, or low concentration levels. The concentration range of the reference standard may be optimized for each assay.

[0117] The kit may also include quality control components (e.g., sensitivity panels, calibrators, positive controls). Preparation of quality control reagents is well known in the art and is described on various immunodiagnostic product inserts. Sensitivity panel members are optionally used to verify assay performance characteristics and are a useful indicator of kit reagent integrity and assay standardization.

[0118] The kit may also optionally include other reagents necessary to detect additional pathogens in the sample, and any reference standards or quality control components thereof.

[0119] The kit may optionally include other reagents necessary to perform the assay or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components, such as buffers and solutions for isolating and / or processing the test sample (e.g., pretreatment reagents or extraction buffers), may also be included in the kit. The kit may further include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further include reagents suitable for reconstituting the lyophilized components. One or more of the components may be in liquid form.

[0120] The various components of the kit are optionally provided in suitable containers as needed. The kit may further include a container for holding or storing a sample (e.g., a sample container or cartridge). Optionally, the kit may optionally contain a reaction vessel, a mixing vessel, and reagents or other components that facilitate the preparation of a test sample. The kit may also include one or more sample collection / acquisition devices to assist in obtaining a test sample (e.g., a microsampling device, a microneedle, or other minimally invasive painless blood collection method for obtaining, storing, or aspirating a tissue sample; a blood collection tube(s); a lancet; a capillary blood collection tube; another single fingerstick blood collection method; an oral swab, a nasal / pharyngeal swab; a 16-gauge or other size needle, a surgical knife or a razor (e.g., particularly a handheld one), a syringe, a sterile container, or a cannula).

[0121] The concepts, kits, and methods described herein can be performed in any system or device, including any manual, automated, or semi-automated system for performing immunoassays. In certain embodiments, the assays, kits, and kit components described herein can be performed in a hospital, home, or clinic.

[0122] In certain embodiments, the assays, kits, and kit components described herein can be performed in a high-throughput immunoassay laboratory system, such as, for example, an Abbott™ ARCHITECT™ (Abbott Laboratories) immunoassay analyzer. Such devices and their components are described, for example, in U.S. Patent Nos. 5,468,646; 5,543,524; 5,545,739; 5,565,570; 5,669,819; and 5,783,699.

[0123] In certain embodiments, the assays, kits, and kit components described herein can be performed in electrochemical or other handheld or point-of-care assay systems, such as the Abbott Point-of-Care (I-STAT®, Abbott Laboratories) electrochemical assay system, which performs sandwich assays and Axis-Shield POC AS. Immunosensors and their methods of manufacture and operation in disposable test devices are described, for example, in U.S. Patent Nos. 5,063,081; 7,419,821; 7,682,833; and 7,723,099; and U.S. Patent Application Publication No. 2004 / 0018577. [Example]

[0124] Example 1 Lateral flow assay An exemplary lateral flow assay for detecting SARS-CoV-2 is provided, utilizing a sandwich-type assay. Specifically, the device includes a first zone (e.g., a reagent zone) containing a labeled antibody specific to a protein or fragment thereof derived from the SARS-CoV-2 virus, e.g., a monoclonal antibody labeled with a detectable label. The device includes a second zone (e.g., a detection zone) immobilized with a secondary antibody specific to a different epitope from the protein or fragment thereof derived from the SARS-CoV-2 virus. A positive test is indicated by the appearance of a visible line in the detection zone (e.g., a test line).

[0125] The initial testing of the device was performed with heat-inactivated SARS-CoV-2 virus diluted on a foam swab applied to the reagent zone. The LOD was 22.5 median tissue culture infectious dose (TCID 50 )(TCID 50 ) / test dilution or 1125 TCID 50 / mL.

[0126] No cross-reactivity was observed with many other viruses tested, including adenoviruses (types 1, 5, and 7), enteroviruses (EV68 and D68), echoviruses (types 1 and 2), human herpesviruses (types 1 and 2), mumps virus Ag, influenza virus A (strains H1N1 (A / Virginia / ATCC1 / 2009), H1N1 (A / WS / 33), and H3N2 (A / Hongkong / 8 / 68)), influenza B (strain B / Lee / 40), parainfluenza (types 1, 2, 3, and 4A), respiratory syncytial virus or RSV (types A and B), human coronaviruses (HKU1, NL63, OC43, and 229E), rhinovirus (type A16), MERs-CoV, and human metapneumovirus (type 16A1). Additionally, coinfection with non-SARS-CoV-2 viruses (influenza A, rhinovirus, RSV, or adenovirus) did not affect detection of SARS-CoV-2 near the limit of detection.

[0127] However, cross-reactivity to human SARS-CoV was observed when 25 ng / mL to 25 μg / mL of human SARS-CoV nucleoprotein was used. This is likely due to the 79.6% similarity between the genomes of SARS-CoV and SARS-CoV-2. No cross-reactivity occurred with samples containing 2.5 ng / mL of human SARS-CoV nucleoprotein.

[0128] No cross-reactivity was observed with other organisms, including Candida albicans, Chlamydia pneumoniae, Streptococcus pyogenes (Group A 19615), Staphylococcus aureus, Staphylococcus saprophyticus, Neisseria sp. (Neisseria lactamica), Escherichia coli, Staphylococcus haemolyticus, Streptococcus salivarius, Hemophilus parahaemolyticus, Proteus vulgaris, Moraxella catarrhalis, Klebsiella pneumoniae, Fusobacterium necrophorum, and Mycobacterium tuberculosis. Additionally, no cross-reactivity was observed in polled human nasal wash samples.

[0129] For example, endogenous substances such as mucin, hemoglobin, triglycerides, jaundice (bilirubin), rheumatoid factor, antinuclear antibodies, pregnancy, and whole blood, as well as expectorants such as guaiacol glyceryl ether; bronchodilators such as albuterol and ephedrine; antihistamines such as chlorpheniramine and diphenhydramine; nasal decongestants such as phenylephrine hydrochloride and oxymetazoline hydrochloride; antivirals such as Testing was performed to include a variety of known interfering substances, including ribavirin, oseltamivir, and zanamivir; antibiotics such as amoxicillin; common medications such as acetylsalicylic acid and ibuprofen; antihypertensives such as chlorothiazide and indapamide; antidiabetic drugs such as glimepiride (a sulfonylurea) and indapamide; and potential COVID-19 medications such as ivermectin, lopinavir, ritonavir, and chloroquine phosphate. Solutions of interfering substances were spiked onto foam swabs or sample repositories, and testing was completed as described above. No interference was observed at the concentrations tested.

[0130] Example 2 Lateral Flow Assay Kit An exemplary lateral flow assay kit includes at least one or all of the following, either separately or within a lateral flow device: a lateral flow device; a positive control swab containing recombinant SARS-CoV-2 N protein in 1X XTBE and 1% BSA solution; a nitrocellulose test strip with test and control lines; a patient swab (e.g., a foam-tipped applicator); and at least one or both of a primary antibody and a secondary antibody or fragment thereof. The lateral flow device includes test strip components including a bridge pad (e.g., Ahlstrom 1281 - Ahlstrom Filtration Inc., Mt. Holly Springs, Pa.), a conjugate pad (e.g., PNPK002123), a sample pad (e.g., Ahlstrom 1281 - Ahlstrom Filtration Inc., Mt. Holly Springs, Pa.), an Ab pad (e.g., Ahlstrom 904 - Ahlstrom Filtration Inc., Mt. Holly Springs, Pa.), and a housing. The extraction buffer contains 200 mM Tricine, 1.2% NaCl, 0.75% Zwittergent, 0.5% Tween 20, and 0.0125% azide (pH 8.8). Test lines were embedded or treated with a solution containing 2 mg / mL BSA-Fab, 1.5% trehalose, 50 mM Tris, 0.1% azide, and 0.02% Intrawhite (UV). Control lines were embedded or treated with a solution containing 1 mg / mL chicken IgY, 1% trehalose, 50 mM Tris, 0.1% azide, and 0.05% FD&C blue dye.

[0131] The secondary antibody or conjugated antibody or fragment thereof is provided in a resuspension buffer (e.g., 5 mM boric acid, 0.1% casein, 0.01% PEG compound, and 0.01% azide (pH 7.4)) and a drying buffer (e.g., 5 mM boric acid, 2% enzyme casein (NZ case), 0.1% Triton X-100, 2% Tween 20, 6% sucrose, and 0.02% azide (pH 8)). The secondary antibody or conjugated antibody or fragment thereof may include SEQ ID NOs: 15 and 16 or derivatives thereof, and BSA-free donkey anti-chicken.

[0132] Example 3 Lateral Flow Assay Kit Exemplary lateral flow devices containing the primary and secondary antibodies disclosed herein were tested using 243 nasopharyngeal specimens collected from individuals suspected of exposure to or exhibiting symptoms of COVID-19 within the past seven days (60 PCR positive and 183 PCR negative). The results show an overall agreement rate of 97.9% (Table 1). [Table 1]

[0133] Example 4 Comparison of lateral flow assays Exemplary lateral flow devices were tested for clinical sensitivity and specificity against a reference method, including real-time PCR (RT-PCR), for SARS-CoV-2 in samples derived from nasal swabs of patients suspected of COVID-19 infection. One exemplary lateral flow device contained full-length versions of the primary and secondary antibodies described herein, while the second lateral flow device contained Fab antibody fragments of the primary and secondary antibodies. Two nasal swabs were collected from each patient. One was for direct testing using the lateral flow assay, and the other was placed in virus transfer medium and cooled to 2–8°C for subsequent RT-PCR analysis. The order in which the nasal swabs were collected was randomized.

[0134] An exemplary lateral flow device containing full-length antibodies was found to have a positive agreement rate of approximately 68% across all samples from symptomatic patients only when compared with RT-PCR analysis (Table 2). The positive agreement rate was approximately 90% compared with culturable virus measurements (CT cutoff = 23), while the negative agreement rate was slightly reduced (Table 3). The majority of patients (100) developed symptoms within 7 days, and the results were similar to the summary results for all patients, as shown in Table 2. However, the smaller sample sizes for patients with symptoms for 8-10 days, 11-14 days, or more than 15 days resulted in larger 95% confidence intervals. [Table 2] [Table 3]

[0135] The exemplary lateral flow device containing Fab antibody fragments yielded significantly higher positive agreement rates than devices using full-length antibodies. The positive agreement rate for all patient samples (symptomatic and asymptomatic) was approximately 90%; for symptomatic patients, the positive agreement rate was 91.5% (Table 4). Similar results were found when comparing the Fab lateral flow device with culturable virus (CT cutoff = 23). Due to small sample sizes, the positive agreement rate for asymptomatic patients had a large margin of error, as shown by the 95% confidence interval (Table 4). [Table 4]

[0136] The majority of symptomatic patients (102 / 126) had symptoms for less than 7 days. For these samples, the positive agreement and overall agreement all increased to 97.1% and 98.0%, respectively; the negative agreement remained essentially the same. However, the small sample sizes of symptomatic patients with symptoms for 8 to 10 days (5 patients), 11 to 14 days (13 patients), and 15 or more days (6 patients), as well as the total number of asymptomatic patients, introduced a large amount of error into the positive agreement measurements of 100.0% (29.2, 100.0), 62.5% (24.5, 91.5), and 100.0% (2.5, 100.0). As shown in Table 5, samples from patients with symptoms for 10 days or less produced an overall agreement rate of approximately 98%. [Table 5]

[0137] The use of Fab antibody fragments instead of full-length antibodies in lateral flow devices increased the positive agreement rate from about 68% to about 90% and the overall agreement rate from about 88% to about 95% for patient samples tested, and increased the positive agreement rate, negative agreement rate, and overall agreement rate for samples from patients with symptoms for 10 days or less.

[0138] Example 5 Lateral flow assay Additional testing using a lateral flow device containing full-length versions of the primary and secondary antibodies described herein was performed on heat-inactivated SARS-CoV-2 isolated from a confirmed positive patient. The LOD was 79 median tissue culture infectious doses (TCID 50 ) / mL((TCID 50 ) / mL).

[0139] The samples were also used to determine the presence or absence of the hook effect (also known as the high-dose hook effect). The hook effect is caused by an excess of target protein reacting simultaneously and instantaneously with both the immobilized and labeled antibodies. Therefore, the hook effect refers to a false-negative result that can be seen when very high levels of target are present in the test sample. To overcome the hook effect, dilution of the specimen may be required. As shown in Figure 1, the hook effect can be as low as 1.0 x 10 5.8 (630,957)(TCID 50 ) / mL or less.

[0140] Example 6 Clinical evaluation of nasopharyngeal and nasal specimens using a lateral flow device An exemplary lateral flow device was used in a clinical evaluation of the sensitivity and specificity of SARS-CoV-2 versus a reference method, including real-time PCR (RT-PCR), in samples derived from nasopharyngeal specimens of patients suspected of having COVID-19 infection. The exemplary lateral flow device contained full-length versions of the primary and secondary antibodies described herein.

[0141] The exemplary lateral flow device containing full-length antibodies was found to have a sensitivity (also referred to as the positive agreement rate above) of approximately 91% across all samples when compared to RT-PCR analysis (Table 6). The specificity (also referred to as the negative agreement rate above) was greater than 99%.

[0142] Samples were also categorized based on the number of days since symptom onset. As shown in Table 7, sensitivity was greater than 90% for all positive subjects 0-7 days after symptom onset. Additionally, specificity was 100% for negative subjects 0-3 days after symptom onset and greater than 99% for negative subjects 4-7 days after symptom onset. [Table 6] [Table 7]

[0143] An exemplary lateral flow device was used to test the clinical sensitivity and specificity of SARS-CoV-2 against a reference method, including real-time PCR (RT-PCR), in samples derived from nasal swabs of patients suspected of having COVID-19 infection. The exemplary lateral flow device contained full-length versions of the primary and secondary antibodies described herein.

[0144] The exemplary lateral flow device containing full-length antibodies was found to have a sensitivity of approximately 91% across all samples when compared to RT-PCR analysis (Table 8). Specificity was greater than 99%.

[0145] Samples were also categorized based on the number of days since symptom onset. As shown in Table 9, sensitivity was greater than 90% for all positive subjects 0-7 days after symptom onset. Additionally, specificity was 100% for negative subjects 4-7 days after symptom onset and greater than 99% for negative subjects 4-7 days after symptom onset. [Table 8] [Table 9]

[0146] In addition, as described in Example 4 and used above, RT-PCR was completed using the remaining sample after some or most of the sample had been used in the lateral flow assay, rather than the second sample.

[0147] The exemplary lateral flow device containing full-length antibodies was found to have a sensitivity of approximately 98% for all samples compared to RT-PCR analysis of the remaining samples (Table 10). Specificity was greater than 99%. Overall concordance was greater than 99%.

[0148] Samples were also categorized by the number of days since symptom onset. As shown in Table 11, sensitivity was 100% for all positive subjects 0-3 days after symptom onset and greater than 96% for all positive subjects 4-7 days after symptom onset. Additionally, specificity was 100% for negative subjects 4-7 days after symptom onset and greater than 99% for negative subjects 4-7 days after symptom onset. [Table 10] [Table 11]

[0149] Example 7 SARS-CoV-2 variants To assess the impact of nucleocapsid mutations found in circulating SARS-CoV-2 strains, including delta and lambda strains, we prepared recombinant proteins bearing mutations identified in clinical specimens for testing. Mutations tested individually or in combination included D63G, R203K, R203M, G204R, R209I, A220V, Q229H, M234I, S235F, D348Y, P365S, E367Q, A376T, D377Y, and a wild-type (WT) Wuhan reference control. These mutations represent unique nucleocapsid sequence profiles of several circulating lineages: B.1.1.7, B.1.617.1, B.1.617.2, B.1.617.3, B.1.618, AY.1, AY.2, P.2, B.1.526, B.1.526.1, B.1.526.2, and a panel of Italian strains. Western blot and high-throughput immunoassays performed with the antibodies disclosed herein confirmed the detection of all mutant and WT recombinant antigens (rAgs) with sensitivity comparable to that of the WT control.

[0150] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be construed as limiting the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0151] Various changes and modifications to the disclosed embodiments, which will be apparent to those skilled in the art, can be made without departing from the spirit and scope thereof.

Claims

1. 1. A method for detecting SARS-CoV-2 virus in a sample obtained from a subject, comprising: contacting a sample obtained from a subject with a primary antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from SARS-CoV-2 virus under conditions that allow binding of the protein or fragment thereof derived from SARS-CoV-2 virus, if present in the sample, to the primary antibody or antigen-binding fragment thereof, wherein the primary antibody or antigen-binding fragment thereof: (i) a heavy chain variable region comprising a complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3; (ii) a light chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6; the steps of: contacting the sample with a conjugate comprising a second antibody that specifically binds to the protein or fragment thereof from the SARS-CoV-2 virus and a detectable label, wherein the second antibody or antigen-binding fragment thereof: (i) a heavy chain variable region comprising a complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 9, a CDR2 amino acid sequence of SEQ ID NO: 10, and a CDR3 amino acid sequence of SEQ ID NO: 11; (ii) a light chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14; and assessing the presence of a signal of the detectable label, wherein the presence of the signal of the detectable label indicates the presence of the SARS-CoV-2 virus-derived protein or fragment thereof in the sample; A method comprising:

2. The method of claim 1 , wherein the method is carried out using an immunoassay.

3. 3. The method of claim 2, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay (LFA).

4. The method of any of claims 1 to 3, wherein the sample comprises a nasal swab or brush, saliva, mucus, blood, serum, or plasma.

5. The method of any one of claims 1 to 4, wherein the primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof recognize different epitopes of a protein derived from the SARS-CoV-2 virus.

6. The method according to any one of claims 1 to 5, wherein the protein or fragment thereof derived from SARS-CoV-2 virus is nucleocapsid (N) protein.

7. 7. The method of claim 1, wherein the primary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 90% identity to SEQ ID NO:7 and a light chain variable region amino acid sequence having at least 90% identity to SEQ ID NO:

8.

8. 8. The method of any one of claims 1 to 7, wherein the secondary antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 90% identity to SEQ ID NO: 15 and a light chain variable region amino acid sequence having at least 90% identity to SEQ ID NO:

16.

9. A lateral flow device comprising: A primary antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus, (i) a heavy chain variable region comprising a complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3; (ii) a light chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6; a primary antibody or antigen-binding fragment thereof; and A second antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof derived from the SARS-CoV-2 virus, (i) a heavy chain variable region comprising a complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 9, a CDR2 amino acid sequence of SEQ ID NO: 10, and a CDR3 amino acid sequence of SEQ ID NO: 11; (ii) a light chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14; a secondary antibody or antigen-binding fragment thereof; 1. A lateral flow device comprising:

10. The lateral flow device of claim 9 , wherein the primary antibody or antigen-binding fragment thereof is immobilized.

11. The lateral flow device of claim 9 or 10, wherein the secondary antibody or antigen-binding fragment thereof comprises a detectable label.

12. The lateral flow device of any one of claims 9 to 11, wherein the primary antibody or antigen-binding fragment thereof and the secondary antibody or fragment thereof recognize different epitopes of a protein derived from the SARS-CoV-2 virus.

13. The lateral flow device according to any one of claims 9 to 12, wherein the sample pad comprises the secondary antibody or an antigen-binding fragment thereof.

14. The lateral flow device according to any one of claims 9 to 13, wherein the test line comprises the primary antibody or an antigen-binding fragment thereof.

15. A kit comprising the lateral flow device according to any one of claims 9 to 14 in a sealed package.

16. 16. The kit of claim 15, further comprising an extraction buffer.

17. 17. The kit of claim 15 or 16, further comprising a sampling device.

18. 18. The kit of claim 17, wherein the sampling device comprises a nasal swab.

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