WO2022103793A1

WO2022103793A1 - Engineered proteins that bind the sars-cov-2 nucleocapsid protein

Info

Publication number: WO2022103793A1
Application number: PCT/US2021/058722
Authority: WO
Inventors: Hadley SIKES JOHNSON; Eric Alexander MILLER
Original assignee: Massachusetts Institute Of Technology
Priority date: 2020-11-10
Filing date: 2021-11-10
Publication date: 2022-05-19

Abstract

The present disclosure relates to proteins comprising a SARS-CoV-2 nucleoprotein- binding domain for detection of SARS-CoV-2, methods, compositions and kits thereof.

Description

ENGINEERED PROTEINS THAT BIND THE SARS-CQV-2 NUCLEQCAPSID PROTEIN

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional application number 63/111,769, filed November 10, 2020, the content of which is incorporated herein by reference in its entirety.

FIELD

Methods and compositions for detecting targets of interest are disclosed herein.

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus and the causative agent of coronavirus disease 2019 (Covid- 19). As an emerging threat to humans and other mammals, SARS-CoV-2 has presented an urgent challenge to doctors and public health officials worldwide.

SUMMARY

Containing and minimizing the effects of SARS-CoV-2 requires generation of new diagnostic methods to specifically and efficiently detect SARS-CoV-2. Rapid, specific, economical testing for SARS-CoV-2 is needed to combat a global health crisis. Herein we describe a paper-based diagnostic using recombinant proteins that bind the SARS-Co-V nucleoprotein (N protein) linked to cellulose binding domains. The instant disclosure relates to recombinant scaffold proteins, in particular engineered reduced-charge Sso7d (rcSso7d), having the ability to bind SARS-CoV-2 nucleoprotein/nucleocapsid protein (N protein). These recombinant scaffold proteins may be used alone or in combination with domains capable of binding to paper, such as cellulose binding domains (CBD), to generate inexpensive, scalable diagnostics for rapid, specific detection of SARS-CoV-2.

In an aspect, the instant disclosure relates to an antigen-binding protein comprising an engineered reduced-charge Sso7d (rcSso7d) antigen-binding protein that binds a SARS-CoV- 2 nucleoprotein/nucleocapsid protein (N protein), wherein the antigen-binding protein comprises a variable region, said variable region comprising the sequence of SEQ ID NO: 31-40, 42-50, and 52-60. In some embodiments, the rcSso7d antigen-binding protein comprises a scaffold, said scaffold comprising the sequence of SEQ ID NO: 3.

In some embodiments, the antigen-binding protein further comprises a cellulose binding domain (CBD); the antigen-binding protein is linked to the CBD through a linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the C-terminus of the antigen-binding protein is linked to the N-terminus of the CBD. In some embodiments, the CBD is a type 3a CBD, or the type 1 dimerized CBD (dCBD). In some embodiments, the type 3a CBD is a domain of the protein CipA from Clostridium thermocellum.

In another aspect, the instant disclosure also relates to a method of detecting SARS- CoV-2 nucleoprotein (N protein). In some embodiments, the method comprises: (a) contacting the antigen-binding protein with a cellulose-containing substrate for a time sufficient for the antigen-binding protein to bind the cellulose-containing substrate; (b) contacting the antigen-binding protein bound to the cellulose-containing substrate with a sample comprising or suspected to comprise a SARS-CoV-2 N protein; and (c) detecting the SARS-CoV-2 N protein, if present, bound by the antigen-binding protein. In some embodiments, the method comprises (a) contacting an antigen-binding protein disclosed herein with a sample comprising or suspected to comprise a SARS-CoV-2 N protein, wherein the SARS-CoV-2 N protein binds to the antigen-binding protein and forms a complex; (b) contacting the complex with a cellulose-containing substrate for a time sufficient for the complex to bind to the cellulose-containing substrate; and (c) detecting the SARS-CoV-2 N protein, if present, bound by the antigen-binding protein. In some embodiments, the disclosure relates to a method for assessing a presence or amount of a SARS-CoV-2 nucleoprotein (N protein) in a sample, comprising contacting a sample with one of the antigen-binding proteins disclosed herein and measuring the presence or amount of the SARS-CoV-2 N protein in the sample.

In some embodiments, the sample is a biological sample from a subject. In some embodiments, the subject is a mammal, and, in some embodiments, the subject is a human.

In some embodiments, detecting comprises addition of a detectably-labeled protein which binds to SARS-CoV-2 N protein. In some embodiments, the detectably-labeled protein is an enzyme-labeled protein. In some embodiments, the enzyme-labeled protein is an engineered rcSso7d antigen-binding protein that binds a SARS-CoV-2 N protein. In some embodiments, the enzyme-labeled protein is an antibody that binds SARS-CoV-2 N protein. In some embodiments, the enzyme-labeled antibody is labeled with horseradish peroxidase (HRP).

In some embodiments, the antigen-binding protein is in molar excess of the SARS- CoV-2 N protein. In some embodiments, the antigen-binding protein is in at least 10-fold molar excess of the anti-SARS-CoV-2 N protein. In some embodiments, at least 50% of the SARS-CoV-2 N protein is bound by the antigen-binding protein.

In some embodiments, the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder. In some embodiments, the cellulose-containing substrate is chromatography paper. In some embodiments, the chromatography paper is unmodified.

In some embodiments, the method further comprises rinsing the cellulose-containing substrate with a buffer solution before detecting the SARS-CoV-2 N protein bound by the antigen-binding protein. In some embodiments, the method further comprises treating the subject if SARS-CoV-2 N protein is detected.

In another aspect, the disclosure relates to a kit comprising a container containing one of the antigen-binding proteins disclosed herein. In some embodiments, the kit further comprising a cellulose-containing substrate. In some embodiments, the antigen-binding protein is bound to the cellulose-containing substrate; in some embodiments, the antigenbinding protein is not bound to the cellulose-containing substrate. In some embodiments, the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder. In some embodiments, the cellulose-containing substrate is chromatography paper. In some embodiments, the chromatography paper is unmodified.

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Each of the above embodiments and aspects may be linked to any other embodiment or aspect. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIG. 1 depicts a schematic demonstrating selection of SARS-CoV-2 N protein binders.

FIG. 2 depicts the function of binders in sandwich assays. The data in the column on the left (Full sandwich complex) show binding signals only in the presence of the SARS- CoV-2 N protein. Controls in the right column (Empty sandwich complex) show that binding signals are not present in the absence of the SARS-CoV-2 N protein.

FIG. 3 depicts a subset of N protein binders that show strong binding signals when this SARS-CoV-2 protein was present in low concentrations, e.g., 100 pM. The bottom row of data shows the specificity of the binding reagents. A 1000-fold higher concentration of a non-target viral protein (DENV2 NS1) did not lead any spurious binding signals.

DETAILED DESCRIPTION

Rapid, specific, economical testing for SARS-CoV-2 is needed to combat a global health crisis. A paper-based diagnostic using recombinant proteins that bind the SARS-Co-V nucleoprotein (N protein) linked to cellulose binding domains is described herein.

Aspects of the present disclosure relate to the development of compositions and methods for capture of a SARS-CoV-2 N protein, using a fusion protein which includes a SARS-CoV-2 N protein-binding protein or SARS-CoV-2 N protein-binding domain and a substrate-anchoring domain, such as a cellulose binding domain (CBD) or a carbohydrate binding module (CBM).

SARS-CoV-2 Detection Proteins

The instant disclosure relates to proteins for the detection of SARS-CoV-2. These proteins comprise a domain that binds a SARS-CoV-2 N protein. In some aspects, the domain that binds a SARS-CoV-2 N protein is linked to a substrate-anchoring domain, which anchors the SARS-CoV-2 N protein-binding domain to a carbohydrate, paper or paper-like substrate. In some aspects, the SARS-CoV-2 N protein-binding domain and the substrate-anchoring domain comprise a fusion protein.

Domain that binds SARS-CoV-2 N protein

According to some aspects, the instant disclosure relates to a domain that binds to SARS-CoV-2 N protein. In some embodiments, the SARS-CoV-2 N protein-binding protein is an engineered Sso7d SARS-CoV-2 N protein-binding protein. The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is a small protein (7 kDa) with high thermal stability (T_m of 98°C), which is highly positively charged since it is a DNA-binding protein. The high positive charges in Sso7d introduce a strong specificity constraint for binding epitopes and leads to nonspecific interaction with mammalian cell membranes. Charge-neutralized variants of Sso7d that maintain high thermal stability have been reported (Traxlmayr et al., J Biol Chem (2016) 291(43):22496-508).

In some embodiments, the Sso7d SARS-CoV-2 N protein-binding protein comprises the amino acid sequence of SEQ ID NO: 12, corresponding to the amino acid sequence of Sso7d from Sulfolobus solfataricus (UniProtKb: P39476; European Nucleotide Archive: AAK42212.1)

Amino acid sequence of Sso7d from Sulfolobus solfataricus (SEQ ID NO: 12):

MATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDA

PKELLQMLEKQKK

(SEQ ID NO: 12)

Orthologs of Sso7d have been described in various species, including Sulfolobus islandicus (NCBI Reference Sequence: WP_012713334.1), Sulfolobus tokodaii (NCBI Reference Sequence: WP_010978621.1), Sulfolobus sp. A20 (Sequence ID: WP_069284107.1), Acidianus hospitalis (NCBI Reference Sequence: WP_013777046.1), and Acidianus manzaensis (GenBank: ARM76167.1).

In some embodiments, the Sso7d SARS-CoV-2 N protein-binding protein is a reduced-charge variant of Sso7d (rcSso7d). In some embodiments, the rcSso7d SARS-CoV- 2 N protein-binding protein comprises the amino acid sequence of SEQ ID NO: 3.

Amino acid sequence of rcSso7d from Sulfolobus solfataricus (SEQ ID NO: 3):

MATVKFTYQGEEKQVDISKIKKVWRVGQMISFTYDEGGGATGRGAVSEKDA PKELLQMLEKQ (SEQ ID NO: 3) In some embodiments, the engineered SARS-CoV-2 N protein-binding protein is Sso7a. In some embodiments, the Sso7a SARS-CoV-2 N protein-binding protein is from Sulfolobus solfataricus (UniProtKB: P61991; European Nucleotide Archive: AAK42090.1).

Amino acid sequence of Sso7a from Sulfolobus solfataricus (SEQ ID NO: 11): MATVKFKYKG EEKQVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK (SEQ ID NO: 11)

In some embodiments, a reduced charge variant of Sso7a is contemplated herein.

In some embodiments, the SARS-CoV-2 N protein-binding protein is Sac7d from Sulfolobus acidocaldarius (UniProtKB: P13123). In some embodiments, the SARS-CoV-2 N protein-binding protein is a reduced-charge variant of Sac7d (rcSac7d).

Amino acid sequence of Sac7d from Sulfolobus acidocaldarius (SEQ ID NO: 13): MVKVKFKYKG EEKEVDTSKI KKVWRVGKMV SFTYDDNGKT GRGAVSEKDA PKELLDMLAR AEREKK (SEQ ID NO: 13)

In some embodiments, the Sso7 SARS-CoV-2 N protein-binding protein is a variant that is at least or about 50% identical, at least or about 60% identical, at least or about 70% identical, at least or about 80% identical, at least or about 85% identical, at least or about 90% identical, at least or about 95% identical, at least or about 96% identical, at least or about 97% identical, at least or about 98% identical, at least or about 99% identical, at least or about 99.5% identical, at least or about 99.9% identical, or about 100% identical to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

In some embodiments, the Sso7 SARS-CoV-2 N protein-binding protein includes variants which are shorter or longer than amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, or more.

Any orthologs of the sequences described herein may be identified conducting a BLAST search of the sequence of interest.

As disclosed herein, the SARS-CoV-2 N protein-binding protein is an engineered rcSso7d SARS-CoV-2 N protein-binding protein, which binds to a SARS-CoV-2 N protein. As described herein, an “antigen” or “SARS-CoV-2 N protein” refers to any full length SARS-CoV-2 N protein or fragment thereof that can bind to the rcSso7d SARS-CoV-2 N protein-binding protein. In some embodiments, a SARS-CoV-2 N protein is a molecule capable of inducing an immune response (to produce an antibody) in a host organism.

A SARS-CoV-2 Nucleoprotein (N protein) is a protein produced by the SARS-CoV-2 virus and present in a SARS-CoV-2 virion, such that its presence in a sample indicates presence of SARS-CoV-2. As described herein, the engineered rcSso7d SARS-CoV-2 N protein-binding protein includes a motif which recognizes and/or binds to a specific SARS- CoV-2 N protein. The SARS-CoV-2 N protein has the amino acid sequence: MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALT QHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYL GTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKG FYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRL NQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQT QGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKL DDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLL PAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 4).

Substrate anchoring domain

In some aspects, provided herein are substrate-anchoring domains that bind a carbohydrate or paper substrate. In some embodiments, the substrate-anchoring domain is a CBM or CBD. In some embodiments the CBM has carbohydrate-binding activity. In some embodiments, the CBM is CBM1, CBM2, CBM3, CBM4, CBM5, CBM6, CBM9, CBM10, CBM11, CBM12, CBM14, CBM15, CBM17, CBM18, CBM19, CBM20, CBM21, CBM25, CBM27, CBM28, CBM32, CBM33, CBM48, or CBM49. The nucleic acid and amino acid sequences of CBMs contemplated herein have been described, such as those disclosed in www.cazypedia.org/index.php/Carbohydrate-binding_modules, and can be readily identified by one of ordinary skill in the art using a BLAST search.

In some embodiments, the substrate-anchoring domain is a CBD. Orthologs of CBDs have been described in various species, including, but not limited to Micromonospora mirobrigensis (GenBank ID: SCF42127.1), Mycobacterium tuberculosis (GenBank ID: CNE10097.1), Micromonospora nigra (GenBank ID: SCL15442.1), Micromonospora mirobrigensis (GenBank ID: SCF04121.1), Cellulomonas Fimi (PDB: 1EXH_A), Mycobacterium kansasii 732 (GenBank: EUA13076.1), Ruminococcus albus 8 (GenBank: EGC02462.1), Leifsonia aquatic (NCBI Reference Sequence: WP_021763186.1), Schizosaccharomyces pombe (NCBI Reference Sequence: NP_593986.1), Desulfitobacterium hafniense (GenBank: CDX04743.1). CBDs expressed in other species that are known to one of ordinary skill in the art, such as CBDs of families I, II, III and IV disclosed, for instance, in Tomme et al., J Chromatogr B Biomed Sci Appl (1998) 715(1 ):283-96, are also contemplated herein.

Different types of CBDs are also contemplated herein. In some embodiments, a type 1 CBD is contemplated herein and serves as the substrate-anchoring domain of a fusion protein described herein. In some embodiments, the type 1 CBD is identified by SEQ ID NO: 10.

Amino acid sequence of type 1 CBD (SEQ ID NO: 10)

AGPGANPPGTTTTSRPATTTGSSPGPQACSSVWGQCGGQNWSGPTCCASGST CVYSNDYYSQCLPGANPPGTTTTSRPATTTGSSPGPTQSHYGQCGGIGYSGPT VCASGTTCQVLNPYYSQCL (SEQ ID NO: 10)

Orthologs of type 1 CBDs have been described in various species, including, but not limited to Trichoderma reesei QM6a (NCBI Reference Sequence: XP_006969224.1); Rhizopus oryzae (GenBank: BAC53988.1); Schizosaccharomyces japonicus yFS275 (NCBI Reference Sequence: XP_002172247.1); Trichoderma virens Gv29-8 (NCBI Reference Sequence: XP_013954979.1); Trichoderma viride (GenBank: CAA37878.1) are also contemplated herein. Type 1 CBDs or orthologs thereof in other species known to one of ordinary skill in the art are also contemplated herein.

In some embodiments, a type 3a CBD is contemplated herein and serves as the substrate-anchoring domain of a fusion protein described herein. In some embodiments, the type 3a CBD is a domain of the CipA protein from Clostridium thermocellum (Genbank: HF912725.1; UniProtKB/TrEMBL: N1JW75).

Amino acid sequence of CipA protein from Clostridium thermocellum (SEQ ID NO: 1)

MRKVISMLLV VAMLTTIFAA MIPQTVSAAT MTVEIGKVTA AVGSKVEIPI TLKGVPSKGM ANCDFVLGYD PNVLEVTEVK PGSIIKDPDP SKSFDSAIYP DRKMIVFLFA EDSGRGTYAI TQDGVFATIV ATVKSAAAAP ITLLEVGAFA DNDLVEISTT FVAGGVNLGS SVPTTQPNVP SDGVVVEIGK VTGSVGTTVE IPVYFRGVPS KGIANCDFVF RYDPNVLEII GIDPGDIIVD PNPTKSFDTA IYPDRKIIVF LFAEDSGTGA YAITKDGVFA KIRATVKSSA PGYITFDEVG GFADNDLVEQ KVSFIDGGVN VGNATPTKGA TPTNTATPTK SATATPTRPS VPTNTPTNTP ANTPVSGNLK VEFYNSNPSD TTNSINPQFK VTNTGSSAID LSKLTLRYYY TVDGQKDQTF WCDHAAIIGS NGSYNGVTSN VKGTFVKMSS STNNADTYLE ISFTGGTLEP GAHVQIQGRF AKNDWSNYTQ SNDYSFKSAS QFVEWDQVTA YLNGVLVWGK EPGGSVVPST QPVTTPPATT KPPATTIPPS DDPNAIKIKV DTVNAKPGDT VNIPVRFSGI PSKGIANCDF VYSYDPNVLE IIEIKPGELI VDPNPDKSFD TAVYPDRKII VFLFAEDSGT GAYAITKDGV FATIVAKVKS GAPNGLSVIK FVEVGGFANN DLVEQKTQFS DGGVNVGGTT VPTTPPASTT PTDDPNAIKI KVDTVNAKPG DTVNIPVRFS GIPSKGIANC DFVYSYDPNV LEIIEIKPGE LIVDPNPDKS FDTAVYPDRK IIVFLLTEDS GTGAYAITKD GVFATIVAKV KSGAPNGLSV IKFVEVGGFA NNDLVEQKTQ FFDGGVNVGD TTVPTTPTTP VTTPTDDPNA VRIKVDTVNA KTGDTVRIPV RFSGIPSKGI ANCDFVYSYD PNVLEIIEIE PGDIIVDPNP DKSFDTAVYP DRKIIVFLFA EDSGTGAYAI TKDGVFATIV AKVKSGAPNG LSVIKFVEVG GFANNDLVEQ KTQFFDGGVN VGDTTEPATP TTPVTTPTTT DGLDAVRIKV DTVNAKPGDT VRIPVRFSGI PSKGIANCDF VYSYDPNVLE IIEIEPGDII VDPNPDKSFD TAVYPDRKII VFLFAEDSGT GAYAITKDGV FATIVAKVKS GAPNGLSVIK FVEVGGFANN DLVEQRTQFF DGGVNVGDTT VPTTPTTPVT TPTDDSNAVR IKVDTVNAKP GDTVRIPVRF SGIPSKGIAN CDFVYSYDPN VLEIIEIEPG DIIVDPNPDK SFDTAVYPDR KIIVFLFAED SGTGAYAITK DGVFATIVAK VKSGAPNGLS VIKFVEVGGF ANNDLVEQKT QFFDGGVNVG DTTVPTTSPT TTPPEPTIAP NKLTLKIGRA EGRPGDTVEI PVNLYGVPQK GIASGDFVVS YDPNVLEIIE IEPGELIVDP NPTKSFDTAV YPDRKMIVFL FAEDSGTGAY AITEDGVFAT IVAKVKEGAP EGFSAIEISE FGAFADNDLV EVETDLINGG VLVTNKTVIE GYKVSGYILP DFSFDATVAP LVKAGFKVEI VGTELYAVTD ANGYFEITGV PANASGYTLK ISRATYLDRV IANVVVTGDT SVSTSQAPIM MWVGDIVKDN SINLLDVAEV IRCFNATKGS ANYVEELDIN RNGAINMQDI MIVHKHFGAT SSDY (SEQ ID NO: 1)

In some embodiments, the underlined valine (V) residue of SEQ ID NO: 1 is an isoleucine (I), which corresponds to SEQ ID NO: 15. Amino acid sequence of CipA protein from Clostridium thermocellum with an isoleucine in place of a valine (SEQ ID NO: 15)

MRKVISMLLV VAMLTTIFAA MIPQTVSAAT MTVEIGKVTA AVGSKVEIPI TLKGVPSKGM ANCDFVLGYD PNVEEVTEVK PGSIIKDPDP SKSFDSAIYP DRKMIVFLFA EDSGRGTYAI TQDGVFATIV ATVKSAAAAP ITLLEVGAFA DNDLVEISTT FVAGGVNLGS SVPTTQPNVP SDGVVVEIGK VTGSVGTTVE IPVYFRGVPS KGIANCDFVF RYDPNVLEII GIDPGDIIVD PNPTKSFDTA IYPDRKIIVF LFAEDSGTGA YAITKDGVFA KIRATVKSSA PGYITFDEVG GFADNDLVEQ KVSFIDGGVN VGNATPTKGA TPTNTATPTK SATATPTRPS VPTNTPTNTP ANTPVSGNLK VEFYNSNPSD TTNSINPQFK VTNTGSSAID LSKLTLRYYY TVDGQKDQTF WCDHAAIIGS NGSYNGITSN VKGTFVKMSS STNNADTYLE ISFTGGTLEP GAHVQIQGRF AKNDWSNYTQ SNDYSFKSAS QFVEWDQVTA YLNGVLVWGK EPGGSVVPST QPVTTPPATT KPPATTIPPS DDPNAIKIKV DTVNAKPGDT VNIPVRFSGI PSKGIANCDF VYSYDPNVLE IIEIKPGELI VDPNPDKSFD TAVYPDRKII VFLFAEDSGT GAYAITKDGV FATIVAKVKS GAPNGLSVIK FVEVGGFANN DLVEQKTQFS DGGVNVGGTT VPTTPPASTT PTDDPNAIKI KVDTVNAKPG DTVNIPVRFS GIPSKGIANC DFVYSYDPNV LEIIEIKPGE LIVDPNPDKS FDTAVYPDRK IIVFLLTEDS GTGAYAITKD GVFATIVAKV KSGAPNGLSV IKFVEVGGFA NNDLVEQKTQ FFDGGVNVGD TTVPTTPTTP VTTPTDDPNA VRIKVDTVNA KTGDTVRIPV RFSGIPSKGI ANCDFVYSYD PNVLEIIEIE PGDIIVDPNP DKSFDTAVYP DRKIIVFLFA EDSGTGAYAI TKDGVFATIV AKVKSGAPNG LSVIKFVEVG GFANNDLVEQ KTQFFDGGVN VGDTTEPATP TTPVTTPTTT DGLDAVRIKV DTVNAKPGDT VRIPVRFSGI PSKGIANCDF VYSYDPNVLE IIEIEPGDII VDPNPDKSFD TAVYPDRKII VFLFAEDSGT GAYAITKDGV FATIVAKVKS GAPNGLSVIK FVEVGGFANN DLVEQRTQFF DGGVNVGDTT VPTTPTTPVT TPTDDSNAVR IKVDTVNAKP GDTVRIPVRF SGIPSKGIAN CDFVYSYDPN VLEIIEIEPG DIIVDPNPDK SFDTAVYPDR KIIVFLFAED SGTGAYAITK DGVFATIVAK VKSGAPNGLS VIKFVEVGGF ANNDLVEQKT QFFDGGVNVG DTTVPTTSPT TTPPEPTIAP NKLTLKIGRA EGRPGDTVEI PVNLYGVPQK GIASGDFVVS YDPNVLEIIE IEPGELIVDP NPTKSFDTAV YPDRKMIVFL FAEDSGTGAY AITEDGVFAT IVAKVKEGAP EGFSAIEISE FGAFADNDLV EVETDLINGG VLVTNKTVIE GYKVSGYILP DFSFDATVAP LVKAGFKVEI VGTELYAVTD ANGYFEITGV PANASGYTLK ISRATYLDRV IANVVVTGDT SVSTSQAPIM MWVGDIVKDN SINLLDVAEV IRCFNATKGS ANYVEELDIN RNGAINMQDI MIVHKHFGAT SSDY (SEQ ID NO: 15)

The amino acid sequence of the type 3 a CBD of CipA protein from Clostridium thermocellum, which corresponds to amino acids 364-522 of the CipA protein from Clostridium thermocellum corresponds to SEQ ID NO: 2.

PVSGNLK VEFYNSNPSD TTNSINPQFK VTNTGSSAID LSKLTLRYYY TVDGQKDQTF WCDHAAIIGS NGSYNGVTSN VKGTFVKMSS STNNADTYLE ISFTGGTLEP GAHVQIQGRF AKNDWSNYTQ SNDYSFKSAS QFVEWDQVTA YLNGVLVWGK EP (SEQ ID NO: 2)

In some embodiments, the underlined valine (V) residue of SEQ ID NO: 2 is an isoleucine (I), which corresponds to SEQ ID NO: 16.

PVSGNLK VEFYNSNPSD TTNSINPQFK VTNTGSSAID LSKLTLRYYY

TVDGQKDQTF WCDHAAIIGS NGSYNGITSN VKGTFVKMSS STNNADTYLE ISFTGGTLEP GAHVQIQGRF AKNDWSNYTQ SNDYSFKSAS QFVEWDQVTA YLNGVLVWGK EP (SEQ ID NO: 16)

Orthologs of type 3a CBDs have been described in various species, including, but not limited to Ruminiclostridium thermocellum AD2 (GenBank: ALX08828.1), Caldicellulosiruptor lactoaceticus 6A (GenBank: AEM74847.1), Niastella koreensis GR20- 10 (GenBank: AEV99440.1), Actinobacteria bacterium OV450 (GenBank: KPH97519), Spirosoma linguale DSM 74 (GenBank: ADB37689.1). Type 3 CBDs, including type 3a CBDs, from other species known to one of ordinary skill in the art are also contemplated herein.

In some embodiments, the CBD includes a variant that is at least or about 50% identical, at least or about 60% identical, at least or about 70% identical, at least or about 80% identical, at least or about 85% identical, at least or about 90% identical, at least or about 95% identical, at least or about 96% identical, at least or about 97% identical, at least or about 98% identical, at least or about 99% identical, at least or about 99.5% identical, at least or about 99.9% identical, or about 100% identical to the amino acid sequence of SEQ ID

NO: 1.

In some embodiments, the type 1 CBD includes a variant that is at least or about 50% identical, at least or about 60% identical, at least or about 70% identical, at least or about 80% identical, at least or about 85% identical, at least or about 90% identical, at least or about 95% identical, at least or about 96% identical, at least or about 97% identical, at least or about 98% identical, at least or about 99% identical, at least or about 99.5% identical, at least or about 99.9% identical, or about 100% identical to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the type 3a CBD includes a variant that is at least or about 50% identical, at least or about 60% identical, at least or about 70% identical, at least or about 80% identical, at least or about 85% identical, at least or about 90% identical, at least or about 95% identical, at least or about 96% identical, at least or about 97% identical, at least or about 98% identical, at least or about 99% identical, at least or about 99.5% identical, at least or about 99.9% identical, or about 100% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 16.

In some embodiments, the CBD includes a variant which is shorter or longer than the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, by 200 amino acids, by 300 amino acids, by 400 amino acids, by 500 amino acids, 800 amino acids, 1000 amino acids, 1200 amino acids, 1400 amino acids or more.

In some embodiments, the type 1 CBD includes a variant which is shorter or longer than the amino acid sequence of a type 1 CBD of SEQ ID NO: 10 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more.

In some embodiments, the type 3a CBD includes a variant which is shorter or longer than the amino acid sequence of a CBD of SEQ ID NO: 2 or SEQ ID NO: 16 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more.

Fusion proteins

In some aspects, provided herein is a fusion protein that incorporates a substrateanchoring domain and a domain that binds SARS-CoV-2 N protein.

The fusion protein described herein can be exemplified by the use of a SARS-CoV-2 N protein binding fusion protein bound to a cellulose-containing substrate, such as a chromatography paper (e.g., Whatman® Grade 1 Qualitative Filtration Paper). The fusion protein bound to the cellulose-containing substrate can be contacted with a sample, such as a biological sample (e.g., blood), obtained from a subject, that contains, or is suspected to contain, a SARS-CoV-2 N protein. The SARS-CoV-2 N protein can be a blood-based biomarker of active SARS-CoV-2 obtained from a subject that has or is suspected of having SARS-CoV-2, which, in some instances, may be used to determine whether the subject has SARS-CoV-2.

In some embodiments, the fusion protein incorporates a substrate-anchoring domain and a SARS-CoV-2 N protein-binding domain, in which the SARS-CoV-2 N protein-binding domain is expressed as a genetic fusion to the substrate-anchoring domain. In some embodiments, the SARS-CoV-2 N protein-binding domain is not expressed as a genetic fusion to the substrate-anchoring domain. In some embodiments, the SARS-CoV-2 N protein-binding domain interacts with the substrate-anchoring domain.

The amino acid sequence of an exemplary fusion protein (rcSso7d.SARS-CoV- 2.NP.MBS2.1-CBD) construct described herein can be represented as follows:

MGSSHHHHHHSSGLVPRGSHMATVKFTYQGEEKQVDISKIKNVKRYGQIIAFI YDEGGGAYGAGGVSEKDAPKEELOMLEKQGGGGSGGGGSGGGGS.PySGNL

DHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQI

NO: 14).

The single underlined amino acids correspond to a histidine tag-thrombin site for purification. The double underlined amino acids correspond to the rcSso7d.SARS-CoV- 2.NP.MBS2.1 (i.e., rcSso7d antigen binding protein variant that binds to SARS-CoV-2 N protein). The dash underlined amino acids correspond to the (G+Sja linker (SEQ ID NO: 17). The zig-zag underlined amino acids correspond to the CBD. In some embodiments, any of the fusion protein constructs described herein have a similar arrangement, consisting of a purification tag and cleavage site, followed by the amino acid sequence of a SARS-CoV-2 N protein-binding protein contemplated herein, followed by a linker, and followed by the amino acid sequence of a CBD domain contemplated herein.

In some embodiments, the fusion protein comprises more than one rcSso7d SARS- CoV-2 N protein-binding protein. In some embodiments, the fusion protein comprises at least or 2, at least or 3, at least or 4, at least or 5, at least or 6, at least or 7, at least or 8, at least or 9, at least or 10, at least or 12, at least or 14, at least or 16, at least or 18, at least or 20, at least or 25, at least or 30, at least or 35, at least or 40, at least or 45, at least or 50, at least or 55, at least or 60, at least or 65, at least or 70, at least or 75, at least or 80, at least or 85, at least or 90, at least or 95, or at least or 100 SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein.

In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are genetically fused together. The more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are genetically fused together by using an expression vector that includes more than one copy of a nucleic acid sequence that encodes the SARS-CoV-2 N protein-binding protein or domain. In some embodiments, the nucleic acid sequence that encodes one SARS-CoV-2 N protein-binding protein or domain is separated from another nucleic acid sequence that encodes one SARS-CoV-2 N proteinbinding protein or domain by a nucleic acid encoding a linker. Non-limiting examples of linkers encoded by a nucleic acid contemplated herein include a protein linker or a peptide linker, such as a Gly-Ser linker (e.g., a linker that includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 17), known as (G₄S)₃). The Gly-Ser linker can be replicated n number of times, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30, for example. Additional non-limiting examples of linkers disclosed herein and/or known to one of ordinary skill in the art are also contemplated herein. In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are not genetically fused together. In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are chemically fused. In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are chemically fused together. The more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are chemically fused by a chemical reagent after the proteins have been expressed from a nucleic acid sequence. In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein are chemically fused after SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein is expressed, for instance, from an expression vector. In some embodiments, the more than one rcSso7d SARS-CoV-2 N protein-binding proteins are chemically fused by a linker, such as a bifunctional linker, or using other methods known to one of ordinary skill in the art. In some embodiments, the more than one SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein, are chemically fused by a fusion via disulfide linkages between cysteine residues at the N- and C-termini, or via dual-maleimide chemical reagents. In some embodiments, in vivo ligation tags such as HALO or SPY tags to attach orthogonal reactive moieties to the SARS-CoV-2 N protein-binding proteins or domains, such as any of the rcSso7d or its variants disclosed herein, allowing separate molecules to react together, are contemplated. In some embodiments, residues of SARS-CoV-2 N proteinbinding proteins or domains, such as any of the rcSso7d or its variants disclosed herein, could be chemically altered to feature aldehyde moieties, which can be reacted with primary amines to form covalent imine linkages. (See e.g., Tuley et al., Chemical communications (2014) 50(56):7424-7426. doi:10.1039/c4cc02000f). In some embodiments, a sortase-based method could be used for in vitro fusion of a SARS-CoV-2 N protein-binding protein or domain, such as any of the rcSso7d or its variants disclosed herein.

In some embodiments, the SARS-CoV-2 N protein-binding protein is bound to a detection reagent. In some embodiments, the detection reagent is a fluorophore. In some embodiments, the detection agent is a fluorophore, such as Alexa Fluor 647 (AF647). In some embodiments, the fluorophore is hydroxycoumarin, methoxycoumarin, aminocoumarin, Cy2, FAM, Alexa Fluor 405 (AF405), Alexa Fluor 488 (AF488), Fluorescein FITC, Alexa Fluor 430 (AF430), Alexa Fluor 532 (AF532), HEX, Cy3, TRITC, Alexa Fluor 546 (AF546), Alexa Fluor 555 (AF555), R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa Fluor 568 (AF568), Red 613, Texas Red, Alexa Fluor 594 (AF594), Alexa Fluor 633 (AF633), Allophycocyanin, Cy5, Alexa Fluor 660 (AF660), Cy5.5, TruRed, Alexa Fluor 680 (AF680), Cy7, Cy7.5 or any other fluorophores known to one of ordinary skill in the art (see e.g., www.biosyn.com/Images/ArticleImages/Comprehensive%20fluorophore%201ist.pdf). In some embodiments, the fluorophore is a fluorescent protein or a chromophore, such as green fluorescent protein (GFP), chromoprotein from the coral Acropora millepora (amilCP), a chromoprotein from the coral Acropora millepora (amilGFP), a fluorescent protein from Acropora millepora (amilRFP), etc., or other species chemically linked to a detection reagent known to one of ordinary skill in the art. In some embodiments, one or more fluorophores could be used for the purification of chemically-labeled molecules to ensure 100% or near 100% labeling efficiency. In some embodiments, the SARS-CoV-2 N protein-binding domain is coupled to a molecule that emits a detectable signal. In some embodiments, the molecule is horseradish peroxidase or phycoerythrin. In some embodiments, the molecule that emits a detectable signal is a color-producing enzyme (e.g., beta-galactosidase), APEX2 for metal sequestration and high contrast electron microscopy (EM), or a chemiluminescent species. In some embodiments, any of the SARS-CoV-2 N protein-binding proteins disclosed herein, such as a multimeric rcSso7d binding protein associated or not associated with a substrate-anchoring domain includes a binding face that binds an analyte, antigen or SARS-CoV-2 N protein and a second binding face that binds one or more of the detection reagents disclosed herein. Other detection reagents, fluorophores or molecules that emit a detectable signal known to one of ordinary skill in the art are also contemplated herein. In some embodiments, the detection reagent, fluorophore or molecule that emits a detectable signal is directly or indirectly linked to one or more of streptavidin, to IgG antibody (polyclonal or monoclonal), any of the biomarkers disclosed herein, any of the SARS-CoV-2 N protein-binding proteins disclosed herein [e.g., rcSso7d, rcSso7d-based detection reagents (e.g., BA-MBP-rcSso7d)], a nucleic acid (e.g., DNA, RNA, etc.), or an organic or inorganic nanoparticle (e.g., a nanoparticle comprising gold, carbon, latex, cellulose, etc.)

In some embodiments, two or more SARS-CoV-2 N protein binding proteins are used in an assay. In some embodiments, the two or more SARS-CoV-2 N protein-binding proteins bind to different sites on a SARS-CoV-2 N protein, allowing more than one SARS-CoV-2 N protein-binding protein to bind to a single SARS-CoV-2 N protein. In some embodiments, one SARS-CoV-2 N protein binding protein is paired with a CBD, allowing it to bind a cellulose-containing substrate and immobilize bound SARS-CoV-2 N protein for detection. A second SARS-CoV-2 N protein binding protein is bound to a detectable domain, such as those described above. In some embodiments, SARS-CoV-2 N protein binds a SARS-CoV-2 N protein-binding protein bound to CBD and immobilized on a cellulose-containing substrate, then exposed to a second SARS-CoV-2 N protein binding protein bound to a detectable domain. In some embodiments, SARS-CoV N protein is bound by a SARS-CoV-2 N protein-binding protein bound to a detectable domain, and then exposed to a SARS-CoV-2 N protein-binding protein bound to CBD and immobilized on a cellulose-containing substrate.

In some embodiments, a substrate-anchoring domain, such as a CBD, and a SARS- CoV-2 N protein-binding domain, are directly attached. The substrate-anchoring domain, such as a CBD, can be directly attached to the SARS-CoV-2 N protein-binding protein or a SARS-CoV-2 N protein-binding domain (e.g., an engineered rcSso7d SARS-CoV-2 N protein-binding protein) through a peptide bond between the substrate-anchoring domain and the SARS-CoV-2 N protein-binding protein or SARS-CoV-2 N protein-binding domain. In some embodiments, a substrate-anchoring domain, such as a CBD, and a SARS-CoV-2 N protein-binding domain (e.g., engineered rcSso7d SARS-CoV-2 N protein-binding protein) are indirectly attached. In some embodiments, the engineered Sso7d SARS-CoV-2 N protein-binding protein (e.g., rcSso7d) is indirectly attached to the CBD through a linker (i.e., is linked). Non-limiting examples of linkers contemplated herein include a protein linker; a peptide linker, such as a Gly-Ser linker (e.g., a linker that includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 17), known as (G₄S)₃). The Gly-Ser linker can be replicated n number of times, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30, for example. Additional non-limiting examples of linkers known to one of ordinary skill in the art, such as chemical linkers (e.g., crosslinkers, bifunctional linkers, trifunctional trilinkers), such as Bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl] sulfone, O,O'-Bis[2-(N-Succinimidyl- succinylamino)ethyl]polyethylene glycol 2,000, O,O'-Bis[2-(N-Succinimidyl- succinylamino)ethyl]polyethylene glycol 3,000, O,O'-Bis[2-(N-Succinimidyl- succinylamino)ethyl]polyethylene glycol 10,000, BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), 4,4 '-Diisothiocyanatostilbene-2, 2 '-disulfonic acid disodium salt hydrate, bromoacetic acid N-hydroxy succinimide ester, maleimide-PEG2-succinimidyl ester, SBAP (succinimidyl 3-(bromoacetamido)propionate), 5-Azido-2-nitrobenzoic acid N- hydroxysuccinimide ester, etc.; flexible linkers (e.g., (Gly)e (SEQ ID NO: 18), (Gly)s (SEQ ID NO: 19), etc.), rigid linkers (e.g., (EAAAK)₃ (SEQ ID NO: 20), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 21), PAPAP (SEQ ID NO: 22), etc.) and cleavable linkers (e.g., disulfide, VSQTSKLTRJ.AETVFPDV (SEQ ID NO: 23), RVLJ.AEA (SEQ ID NO: 24); EDVVCC^SMSY (SEQ ID NO: 25); GGIEGRJ.GS (SEQ ID NO: 26); GFLGj, (SEQ ID NO: 27), etc.) naturally-occurring or synthetic, such as those disclosed in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-69, are also contemplated herein.

In some embodiments, the C-terminus of the engineered rcSso7d SARS-CoV-2 N protein-binding protein is either directly or indirectly attached to the N-terminus of the CBD. In some embodiments, the C-terminus of the engineered rcSso7d SARS-CoV-2 N proteinbinding protein is directly attached to the N-terminus of the CBD. In some embodiments, the C-terminus of the engineered rcSso7d SARS-CoV-2 N protein-binding protein is indirectly attached to the N-terminus of the CBD through a linker. In some embodiments, the N- terminus of the engineered rcSso7d SARS-CoV-2 N protein-binding protein is either directly or indirectly attached to the C-terminus of the CBD. In some embodiments, the N-terminus of the engineered rcSso7d SARS-CoV-2 N protein-binding protein is directly attached to the C-terminus of the CBD. In some embodiments, the N-terminus of the engineered rcSso7d SARS-CoV-2 N protein-binding protein is indirectly attached to the C-terminus of the CBD through a linker.

Also disclosed herein are nucleic acids that encode for any of the fusion proteins described herein, libraries that contain any of the nucleic acids and/or fusion proteins described herein, and compositions that contain any of the nucleic acids and/or fusion proteins described herein. It should be appreciated that libraries containing nucleic acids or proteins can be generated using methods known in the art. A library containing nucleic acids can contain fragments of genes and/or full-length genes and can contain wild-type sequences and mutated sequences. A library containing proteins can contain fragments of proteins and/or full length proteins and can contain wild-type sequences and mutated sequences.

The development and selection of a SARS-CoV-2 N protein-binding protein described herein, such as the rcSso7d. SARS-CoV-2. NP.MBS2.1 or the rcSso7d.SARS-CoV- 2.NP.PF1.B.4 can be produced by methods disclosed in Miller et al., 2016. Briefly, a SARS- CoV-2 N protein-binding protein, such as rcSso7d. SARS-CoV-2. NP.MBS2.1 or the rcSso7d.SARS-CoV-2.NP.PFl.B.4 is selected from a yeast surface display library based on the reduced-charge Sso7d scaffold (rcSso7d). The yeast library can be generated using trinucleotide oligo synthesis and in vivo homologous recombination with a linearized plasmid, such as the pCTcon2 plasmid (Traxlmayr et al., 2016). Methods of isolation, such as the highly-avid magnetic bead sorting (Ackerman et al., 2009) (MBS) and fluorescence- activated cell sorting (FACS) (Chao et al., 2006) can be employed to select binders against a SARS-CoV-2 N protein and stringency increased over rounds of FACS-based library screening, after which a sub-library can be sequenced and the SARS-CoV-2 N proteinbinding protein that binds the SARS-CoV-2 N protein (e.g., rcSso7d.SARS-CoV- 2.NP.PF1.B.4) can be selected for further characterization, such as robust expression in a system, such as a bacterial system, for downstream applications. Additional methods for creating a yeast surface display library include methods known to one of ordinary skill in the art.

In some embodiments, one or more of the SARS-CoV-2 N protein-binding domains or proteins disclosed herein are expressed in a recombinant expression vector. As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.

A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA).

A nucleic acid molecule that encodes a fusion protein or antigen or any other molecule disclosed herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc.

Any type of cell that can be engineered to recombinantly express genes can be used in the methods described herein, including prokaryotic and eukaryotic cells. In some embodiments the cell is a bacterial cell, such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. The bacterial cell can be a Gram-negative cell such as an Escherichia coli (E. coll) cell, or a Gram-positive cell such as a species of Bacillus. In other embodiments, the cell is a fungal cell such as a yeast cell, e.g., Saccharomyces spp. (e.g., S. cerevisiae), Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. Other examples of fungi include Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp. In other embodiments, the cell is an algal cell, or a plant cell. Compositions

In some aspects, compositions of the fusion proteins described herein are also provided. In some embodiments, the composition includes any of the fusion proteins described herein bound to a cellulose-containing substrate. In some embodiments, the cellulose-containing substrate is paper (e.g., chromatography paper) or nitrocellulose. In certain embodiments, the cellulose-containing substrate is modified in an oxidizing chemical bath to yield covalent chemical linkage of the protein to the substrate, passivated with a blocking agent to reduce non-specific protein adsorption to the substrate, or pre-incubated with a stabilizing species such as trehalose in order to improve assay functionality and stability. In certain embodiments, the cellulose-containing substrate is not modified (unmodified). In some embodiments, the cellulose-containing substrate is an unmodified chromatography paper, such as unmodified Whatman® Grade 1 Qualitative Filtration Paper. Additional non-limiting examples of cellulose-containing substrates also contemplated herein include cellulose powder, cellulose microbeads, or cellulosic fabrics/yarns.

In some embodiments, at least or about 0.1 micromole, at least or about 0.2 micromoles, at least or about 0.3 micromoles, at least or about 0.4 micromoles, at least or about 0.5 micromoles, at least or about 0.6 micromoles, at least or about 0.7 micromoles, at least or about 0.8 micromoles, at least or about 0.9 micromoles, at least or about 1 micromole, at least or about 1.1 micromoles, at least or about 1.2 micromoles, at least or about 1.3 micromoles, at least or about 1.4 micromoles, at least or about 1.5 micromoles, at least or about 1.6 micromoles, at least or about 1.7 micromoles, at least or about 1.8 micromoles, at least or about 1.9 micromoles, at least or about 2 micromoles, at least or about 2.1 micromoles, at least or about 2.2 micromoles, at least or about 2.3 micromoles, at least or about 2.4 micromoles, at least or about 2.5 micromoles, at least or about 2.6 micromoles, at least or about 2.7 micromoles, at least or about 2.8 micromoles, at least or about 2.9 micromoles, at least or about 3 micromoles, at least or about 3.5 micromoles, at least or about 4 micromoles, at least or about 4.5 micromoles, or at least or about 5 micromoles of any of the fusion proteins described herein are attached to a cellulose-containing substrate per gram of cellulose of the cellulose-containing substrate.

In some embodiments, at least or about 1 pM, at least or about 25 pM, at least or about 50 pM, at least or about 60 pM, at least or about 70 pM, at least or about 80 pM, at least or about 90 pM, at least or about 100 pM, at least or about 150 pM, at least or about 200 pM, at least or about 250 pM, at least or about 300 pM, at least or about 350 pM, at least or about 400 pM, at least or about 500 pM, at least or about 550 pM, at least or about 600 pM, at least or about 650 pM, at least or about 700 pM, at least or about 750 pM, at least or about 800 pM, at least or about 850 pM, at least or about 900 pM, at least or about 950 pM, at least or about 1 mM, at least or about 1.5 mM, at least or about 2 mM, at least or about 2.5 mM, at least or about 3 mM, at least or about 3.5 mM, at least or about 4 mM, at least or about 4.5 mM, at least or about 5 mM of volume- average concentration of any of the fusion proteins described herein are attached to a cellulose-containing substrate.

SARS-CoV-2 Detection Methods

Methods for detecting SARS-CoV-2 are provided herein. In some embodiments, the method includes contacting any of the fusion proteins described herein with a cellulose- containing substrate for a time sufficient for the fusion protein to bind to the cellulose- containing substrate; contacting the fusion protein bound to the cellulose-containing substrate with a sample comprising a SARS-CoV-2 N protein; and detecting the SARS-CoV-2 N protein bound by the engineered reduced charge Sso7d SARS-CoV-2 N protein-binding protein (e.g., rcSso7d. SARS-CoV-2. NP.PF1.B.4).

In some embodiments, the method includes contacting any of the fusion proteins described herein with a sample comprising a SARS-CoV-2 N protein, wherein the SARS- CoV-2 N protein binds to the fusion protein and forms a complex; contacting the complex with a cellulose-containing substrate for a time sufficient for the complex to bind to the cellulose-containing substrate; and detecting the SARS-CoV-2 N protein bound by the engineered Sso7d SARS-CoV-2 N protein-binding protein.

In some embodiments, the method includes contacting any of the fusion proteins described herein, such as rcSso7d-CBD, with a cellulose-containing substrate for a time sufficient for fusion protein to bind to the cellulose-containing substrate; contacting a sample, such as a biological sample, comprising or suspected to comprise SARS-CoV-2, or SARS- CoV-2 N protein, for a time sufficient to allow the SARS-CoV-2 or SARS-CoV-2 N protein to bind to the fusion protein and form a complex; contacting the complex with an antibody that recognizes the SARS-CoV-2 N protein; and detecting the antibody. In some embodiments, the antibody is directly or indirectly linked to a fluorophore or a molecule that emits a detectable signal to detect the antigen or SARS-CoV-2 N protein. In some embodiments, the antibody is biotinylated. In some embodiments, the biotinylated antibody is contacted with a streptavidin molecule that is directly or indirectly linked to a fluorophore or a molecule that emits a detectable signal to detect the antigen or SARS-CoV-2 N protein.

In some embodiments, the fusion protein or the complex is in solution. In some embodiments, the solution includes a buffer, such as a buffer known to one of ordinary skill in the art. The bifunctional protein may be in solution at a desired concentration. In some embodiments, the fusion protein is at a desired concentration of or about 5 pM, of or about 10 pM, of or about 15 pM, of or about 20 pM, of or about 25 pM, of or about 30 pM, of or about 35 pM, of or about 40 pM, of or about 45 pM, of or about 50 pM, of or about 60 pM, of or about 70 pM, of or about 80 pM, of or about 90 pM, of or about 100 pM, of or about 200 pM, of or about 300 pM, or of or about 400 pM.

In some embodiments, the fusion protein described herein is contacted with the cellulose-containing substrate for about 5 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 1 hour.

In some embodiments, the fusion protein bound to the cellulose-containing substrate is contacted with a sample that contains SARS-CoV-2 or SARS-CoV-2 N protein. In some embodiments, the fusion protein described herein is contacted with a sample comprising a SARS-CoV-2 N protein, wherein the SARS-CoV-2 N protein binds to the fusion protein and forms a complex; the complex is then contacted with a cellulose-containing substrate for a time sufficient for the complex to bind to the cellulose-containing substrate.

In some embodiments, the sample is a biological sample. The biological sample may be obtained from a subject. As described herein, the term “biological sample” is used to generally refer to any biological material obtained from a subject. The biological sample typically is a fluid sample. Solid tissues may be made into fluid samples using routine methods in the art. In some embodiments, the biological sample is tissue, feces, or a cell obtained from a subject. In some embodiments, the biological sample comprises a bodily fluid from a subject. The bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid or combinations thereof.

In some embodiments, the cellulose-containing substrate is paper (e.g., chromatography paper) or nitrocellulose. In certain embodiments, the cellulose-containing substrate is modified in an oxidizing chemical bath to yield covalent chemical linkage of the protein to the substrate, passivated with a blocking agent (See e.g., Y. Zhu, et al., Anal Chem. (2014) 86:2871-5; M. Vuoriluoto, et al., ACS Appl. Mater. Interfaces (2016) 8, 5668-78) to reduce non-specific protein adsorption to the substrate, or pre-incubated with a stabilizing species such as trehalose in order to improve assay functionality and stability. In certain embodiments, the cellulose-containing substrate is not modified (unmodified). In some embodiments, the cellulose-containing substrate is an unmodified chromatography paper, such as unmodified Whatman® Grade 1 Qualitative Filtration Paper. Additional non-limiting examples of cellulose-containing substrates also contemplated herein include cellulose powder, cellulose microbeads, cellulosic fabrics/yams, etc.

In some embodiments, the cellulose-containing substrate is oxidized. In some embodiments, the cellulose-containing substrate is oxidized with sodium metaperiodate to functionalize the cellulose surfaces with aldehyde groups or other methods to oxidize cellulose known to one of ordinary skill in the art. (See e.g., Badu-Tawiah, et al., Lab Chip, (2015) 15:655-9).

For instance, a non-limiting example is the use of rcSso7d.SARS-CoV-2.NP.MBS2.1 -CBD fusion protein bound to a cellulose-containing substrate, such as a chromatography paper (e.g., Whatman® Grade 1 Qualitative Filtration Paper), which is contacted with a sample that contains a SARS-CoV-2 N protein, for example a blood sample obtained from a subject that has or is suspected of having SARS-CoV-2, which, in some instances, may be used to determine whether the subject has SARS-CoV-2.

In some embodiments, at least or about 0.1 micromole, at least or about 0.2 micromoles, at least or about 0.3 micromoles, at least or about 0.4 micromoles, at least or about 0.5 micromoles, at least or about 0.6 micromoles, at least or about 0.7 micromoles, at least or about 0.8 micromoles, at least or about 0.9 micromoles, at least or about 1 micromole, at least or about 1.1 micromoles, at least or about 1.2 micromoles, at least or about 1.3 micromoles, at least or about 1.4 micromoles, at least or about 1.5 micromoles, at least or about 1.6 micromoles, at least or about 1.7 micromoles, at least or about 1.8 micromoles, at least or about 1.9 micromoles, at least or about 2 micromoles, at least or about 2.1 micromoles, at least or about 2.2 micromoles, at least or about 2.3 micromoles, at least or about 2.4 micromoles, at least or about 2.5 micromoles, at least or about 2.6 micromoles, at least or about 2.7 micromoles, at least or about 2.8 micromoles, at least or about 2.9 micromoles, at least or about 3 micromoles, at least or about 3.5, at least or about 4 micromoles, at least or about 4.5 micromoles, or at least or about 5 micromoles of any of the fusion proteins described herein are attached to a cellulose-containing substrate per gram of cellulose of the cellulose-containing substrate.

In some embodiments, at least or about 1 pM, at least or about 25 pM, at least or about 50 pM, at least or about 60 pM, at least or about 70 pM, at least or about 80 pM, at least or about 90 pM, at least or about 100 pM, at least or about 150 pM, at least or about 200 pM, at least or about 250 pM, at least or about 300 pM, at least or about 350 pM, at least or about 400 pM, at least or about 500 pM, at least or about 550 pM, at least or about 600 pM, at least or about 650 pM, at least or about 700 pM, at least or about 750 pM, at least or about 800 pM, at least or about 850 pM, at least or about 900 pM, at least or about 950 pM, at least or about 1 mM, at least or about 1.5 mM, at least or about 2 mM, at least or about 2.5 mM, at least or about 3 mM, at least or about 3.5 mM, at least or about 4 mM, at least or about 4.5 mM, at least or about 5 mM of volume- average concentrations any of the fusion proteins described herein are attached to a cellulose-containing substrate.

In some aspects, the molar abundance or molar excess of the SARS-CoV-2 N proteinbinding protein in the fusion protein, such as a rcSso7d linked to a CBD, relative to the SARS-CoV-2 N protein allows the rapid capture and, in some embodiments, efficient and complete depletion of the SARS-CoV-2 N protein from a sample.

In some embodiments, at least or about a 10-fold molar excess of fusion protein or SARS-CoV-2 N protein-binding protein completely depletes a SARS-CoV-2 N protein from a sample or solution. In some embodiments, at least or about a 10-fold volume-average concentration excess leads to rapid capture and/or immobilization of a fusion protein or SARS-CoV-2 N protein-binding protein.

In some embodiments, the fusion protein is in molar excess of the SARS-CoV-2 N protein. In some embodiments, the fusion protein is in at least or about 2-fold molar excess, at least or about 3-fold molar excess, at least or about 4-molar excess, at least or about 5-fold molar excess, at least or about 6-fold molar excess, at least or about 7-fold molar excess, at least or about 8-fold molar excess, at least or about 9-fold molar excess, at least or about 10- fold molar excess, at least or about 15-fold molar excess, at least or about 20-fold molar excess, at least or about 25-fold molar excess, at least or about 30-fold molar excess, at least or about 35-fold molar excess, at least or about 40-fold molar excess, at least or about 45-fold molar excess, at least or about 50-fold molar excess, at least or about 60-fold molar excess, at least or about 65-fold molar excess, at least or about 70-fold molar excess, at least or about 80-fold molar excess, at least or about 90-fold molar excess, at least or about 100-fold molar excess, at least or about 200-fold molar excess, at least or about 300-fold molar excess, at least or about 400-fold molar excess, at least or about 500-fold molar excess, at least or about 600-fold molar excess, at least or about 700-fold molar excess, at least or about 800-fold molar excess, at least or about 900-fold molar excess, at least or about 1000-fold molar excess, at least or about 1500-fold molar excess, or at least or about 2000-fold molar excess relative to the SARS-CoV-2 N protein in the sample.

In some embodiments, the fusion protein is in such excess that the SARS-CoV-2 N protein is depleted from the sample. In some embodiments, about or at least 10%, about or at least 20%, about or at least 30%, about or at least 40%, about or at least 50%, about or at least 55%, about or at least 60%, about or at least 65%, about or at least 70%, about or at least 75%, about or at least 80%, about or at least 81%, about or at least 82%, about or at least 83%, about or at least 84%, about or at least 85%, about or at least 86%, about or at least 87%, about or at least 88%, about or at least 89%, about or at least 90%, about or at least 91%, about or at least 92%, about or at least 93%, about or at least 94%, about or at least 95%, about or at least 95.5%, about or at least 96%, about or at least 96.5%, about or at least 97%, about or at least 97.5%, about or at least 98%, about or at least 98.5%, about or at least 99%, about or at least 99.5%, or about 100% of the SARS-CoV-2 N protein is depleted from the sample, such as a biological sample.

In some aspects, standard curves can be prepared given the advantageous properties of the disclosure in which complete or near-complete depletion of a SARS-CoV-2 N protein can be achieved from a sample or solution. The abundance of the captured antigen can be detected and measured or determined using a readout, such as a fluorescent readout or a colorimetric readout.

In some embodiments, the surface-immobilized concentration of the SARS-CoV-2 N protein-binding protein (e.g., rcSso7d.SARS-CoV-2.NP.MBS2.1-CBD) is quantified using a protein assay, such as a micro bicinchoninic acid (BCA) assay. A standard curve can be prepared by evaporating known quantities of protein onto cellulose test zones, depositing these test zones into the wells of a micro BCA assay, and quantifying the signal development in this format. The same procedure is followed for the experimental samples (following the substrate washing step), and the associated signal for each sample is then mapped to this standard curve in order to determine the mass of immobilized rcSso7d.SARS-CoV- 2.NP.MBS2.1-CBD.

In some embodiments, the sample is a biological sample from a subject. A subject includes, but is not limited to, any mammal, such as a human, a primate, a mouse, a rat, a dog, a cat, a horse, or agricultural stocks (e.g., fish, pigs, cows, sheep, and birds - particularly chickens). In certain embodiments, the subject is a human. In some embodiments, the sample is a solution, such as a buffer solution.

In some embodiments, the cellulose-containing substrate is rinsed with a buffer solution before detecting the SARS-CoV-2 N protein bound to the engineered reduced charge Sso7d SARS-CoV-2 N protein-binding protein (e.g., rcSso7d). In some embodiments, the buffer is phosphate buffered saline (PBS) or another buffer known to one of ordinary skill in the art that provides a stable environment for a macromolecule, such as a protein, protein complex, antigen, etc.

In some embodiments, the method further includes detecting the SARS-CoV-2 N protein bound by the engineered reduced charge Sso7d SARS-CoV-2 N protein-binding protein (e.g., rcSso7d) in the fusion protein. In some embodiments, the SARS-CoV-2 N protein bound to the fusion protein is contacted with a cellulose-containing substrate in which the CBD of the fusion protein binds the cellulose-containing substrate (e.g., chromatography paper such as Whatman® Grade 1 Qualitative Filtration Paper). The method allows for the separation or isolation of the SARS-CoV-2 N protein from any other molecules that may be present in a sample, such as a biological sample (e.g., blood). In some embodiments, the presence or amount of the SARS-CoV-2 N protein is determined or measured using a signalgenerating reagent that specifically recognizes the SARS-CoV-2 N protein and generates a signal.

In some embodiments, the fusion protein (e.g., rcSso7d-CBD) is immobilized on a cellulose substrate (e.g., chromatography paper, cellulose powder, etc.), and then is brought into contact with the solution/biological sample bearing the SARS-CoV-2 N protein (either forced convection to draw the fluid across or through the test zone, or soluble co-incubation of the CBD/substrate and antigen). This immobilized complex then is contacted with a second, epitope- specific variant of rcSso7d (not fused to CBD, but fused instead to a biotin acceptor sequence, or modified with a fluorophore). The second species (e.g., rcSso7d) binds to a second epitope of the captured antigen. This second species is conjugated to a means of transducing this binding reaction; several examples are outlined below. All of these steps can be done directly on the cellulose-containing substrate.

Non-limiting examples of signal-generating molecules that can be fused to the SARS- CoV-2 N protein-binding protein (e.g., rcSso7d) include, without limitation, gold nanoparticles, enzymes (expressed as fusion partners or indirectly bound to rcSso7d) which yield a colorimetric response, enzymes which yield an amperometric or impedometric signal (e.g., glucose oxidase), a macrophotoinitiator which can initiate a polymerization reaction, cellulose nanobeads, other metallic nanoparticles, dye-filled liposomes, DNA which can be amplified enzymatically, RNA which can be expressed for the production of a colorproducing enzyme, etc. The presence or amount of the signal-generating reagent can be detected using an imaging device, such as a digital imager. Additional non-limiting examples of detecting the signal-generating reagent include gold nanoparticles, which can be used in a point-of-care setting, and are the reagents used in traditional pregnancy tests. The spatial localization of gold nanoparticles, mediated by the SARS-CoV-2 N protein-binding interaction, concentrates the optical signal (which is also amplified by the occurrence of surface plasmon resonance). This can be detected by the naked eye. Polymerization-based amplification use the localization of a macrophotoinitiator in order to yield a rapid, durable polymerization response following incubation with a monomer solution and irradiation with the appropriate wavelength of light. Entrained phenolphthalein yields a high-contrast colorimetric readout following the application of a basic solution, which can be detected with the naked eye. An amperometric method, such as fusing glucose oxidase to the second rcSso7d species and contacting the tests with gold probes and a glucose solution, allow for smart phone based detection. Enzymatic methods can also be used, and rely upon a fusion of the second species (e.g., rcSso7d) to an enzyme and contacting the tests with a labile substrate which becomes colored following enzymatic cleavage. Impedometric means of detecting the signal generating reagent are also possible, and can be achieved using smartphone-compatible adaptors.

In some aspects, provided herein are also methods for enhancing the sensitivity of an assay. The method includes binding of a target to a target-binding species, which includes fusing a target-binding species that binds to a target of interest to a cellulose binding domain (CBD). Any SARS-CoV-2 N protein-binding protein that can be attached to a cellulose- binding domain can benefit from its favorable properties; the high immobilized abundance of fusion protein with a CBD results in high molar abundance of the binding species, thereby allowing, in some instances, depletion of a SARS-CoV-2 N protein and a high local concentration of this species, thereby allowing, in some instances, rapid capture of a SARS- CoV-2 N protein. In some embodiments, the SARS-CoV-2 N protein is in solution. In contrast to traditional immunoassays in which the immobilized binding partner is the limiting reagent and the SARS-CoV-2 N protein is captured slowly and incompletely, the present disclosure allows for the antigen capture/detection to rapidly proceed to completion. Additionally, because the fusion protein, and thus the SARS-CoV-2 N protein-binding domain, is at a high local abundance, this allows the use of higher sample volumes containing higher amounts of antigen, which are captured and depleted, in some instances, to provide high signal over a method previously available in the art in which the SARS-CoV-2 N protein-binding species is actually the limiting reagent, reducing the amount of antigen that can be captured and detected at a given point. This could be applied to any binding scaffold by expressing the binding scaffold as a fusion partner to the CBD.

Kits

In some aspects, the fusion protein and compositions described herein are provided in a kit. In some embodiments, the kit is used to assess the presence or amount of a molecule, such as an antigen or a SARS-CoV-2 N protein and includes a container containing any of the fusion proteins described herein.

In some embodiments, the kit further comprises a cellulose-containing substrate. In some embodiment, the fusion protein is not bond to the cellulose-containing substrate. In some embodiments, the fusion protein is bound to the cellulose-containing substrate. In some embodiments, at least or about 0.1 micromole, at least or about 0.2 micromoles, at least or about 0.3 micromoles, at least or about 0.4 micromoles, at least or about 0.5 micromoles, at least or about 0.6 micromoles, at least or about 0.7 micromoles, at least or about 0.8 micromoles, at least or about 0.9 micromoles, at least or about 1 micromole, at least or about

1.1 micromoles, at least or about 1.2 micromoles, at least or about 1.3 micromoles, at least or about 1.4 micromoles, at least or about 1.5 micromoles, at least or about 1.6 micromoles, at least or about 1.7 micromoles, at least or about 1.8 micromoles, at least or about 1.9 micromoles, at least or about 2 micromoles, at least or about 2.1 micromoles, at least or about

2.2 micromoles, at least or about 2.3 micromoles, at least or about 2.4 micromoles, at least or about 2.5 micromoles, at least or about 2.6 micromoles, at least or about 2.7 micromoles, at least or about 2.8 micromoles, at least or about 2.9 micromoles, at least or about 3 micromoles, at least or about 3.5, at least or about 4 micromoles, at least or about 4.5 micromoles, or at least or about 5 micromoles of any of the fusion proteins described herein are attached to the cellulose-containing substrate per gram of cellulose of the cellulose- containing substrate.

In some embodiments, at least or about 1 pM, at least or about 25 pM, at least or about 50 pM, at least or about 60 pM, at least or about 70 pM, at least or about 80 pM, at least or about 90 pM, at least or about 100 pM, at least or about 150 pM, at least or about 200 pM, at least or about 250 pM, at least or about 300 pM, at least or about 350 pM, at least or about 400 pM, at least or about 500 pM, at least or about 550 pM, at least or about 600 pM, at least or about 650 pM, at least or about 700 pM, at least or about 750 pM, at least or about 800 pM, at least or about 850 pM, at least or about 900 pM, at least or about 950 pM, at least or about 1 mM, at least or about 1.5 mM, at least or about 2 mM, at least or about 2.5 mM, at least or about 3 mM, at least or about 3.5 mM, at least or about 4 mM, at least or about 4.5 mM, at least or about 5 mM of volume-concentration of any of the fusion proteins described herein are attached to the cellulose-containing.

In some embodiments, the fusion protein is bound to the cellulose-containing substrate. In some embodiments, the cellulose-containing substrate is paper (e.g., chromatography paper), nitrocellulose or cellulose powder. In certain embodiments, the cellulose-containing substrate is modified in an oxidizing chemical bath to yield covalent chemical linkage of the protein to the substrate, passivated with a blocking agent to reduce non-specific protein adsorption to the substrate, or pre-incubated with a stabilizing species such as trehalose in order to improve assay functionality and stability. In certain embodiments, the cellulose-containing substrate is not modified (unmodified). In some embodiments, the cellulose-containing substrate is an unmodified chromatography paper, such as unmodified Whatman® Grade 1 Qualitative Filtration Paper. Additional non-limiting examples of cellulose-containing substrates also contemplated herein include cellulose powder, or cellulose microbeads, cellulosic fabrics/yarns.

EXAMPLES

Example 1. Selection and Characterization of rcSso7d Binding Variants that Bind to SARS- CoV-2 Nucleoprotein

Moderate binding proteins were developed that bind to SARS-CoV-2 Nucleoprotein (N protein). SARS-CoV-2 N protein was recombinantly expressed and purified with an N- terminal hexahistidine tag. A biotinylated version of SARS-CoV-2 N protein was cloned, expressed, and purified with an additional biotin acceptor sequence tag on the C-terminus.

The amino acid sequence of the selected binding variants can be seen below (Table 1). Flow cytometry data indicating the specific binding activity of selected rcSso7d clones, in the presence or absence of SARS-CoV-2 N protein, is shown in the FACS plots in FIG. 2.

Flow cytometry data was collected using the yeast-surface display platform, in which the particular rcSso7d variant is displayed on the surface of a clonal population of yeast. The target- specific binding activity of each particular rcSso7d variant was assessed using fluorescent reagents specific to epitope fusion tags associated with the target biomarker (either a biotin acceptor tag or a hexahistidine tag).

Flow cytometry data was also collected in the presence of an off-target viral protein (DENV2) at lOOuM, compared to activity in the presence of SARS-CoV-2 N protein at lOOpM. FIG. 3 shows specificity of selected rcSso7d.NP proteins for SARS-CoV-2 N protein in comparison to a much higher concentration of DENV2.

Datasets include both secondary controls and experimental samples demonstrating baseline binding in an idealized 0.1% BSA/PBS buffer. Secondary controls indicate the extent of off-target binding to the fluorescent reagents used to detect binding activity, and are thus a proximate measure of the binding specificity of the rcSso7d variant. The experimental samples indicate the activity of the surface-displayed rcSso7d variant against the purified SARS-CoV-2 N biomarkers, at a concentration denoted in the corresponding figure. The x- axis signifies rcSso7d expression level on the surface of the yeast (using the cMyc or HA tags on the yeast-surface displayed rcSso7d with a biotinylated antibody). The y-axis signifies binding to SARS-CoV-2 N protein. Specific binding variants are observed to exhibit an increase in fluorescence signal on the y-axis of the flow cytometry plots.

Table 1. Primary protein structure of selected rcSso7d binding variants that bind to SARS-

CoV-2 N protein.

Example 2. Paper-Based Diagnostics In The Antigen-Depletion Regime: High-Density

Immobilization Of rcSso7d-Cellulose-Binding Domain Fusion Proteins For Efficient Target

Capture

Materials and Methods

Materials

Unless otherwise stated, all chemical reagents, biological materials, and consumables were procured from the same source as outlined in the supplementary information of Miller et al., 2016, SI. All DNA cloning enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Streptavidin-eosin conjugate was prepared as previously described (Miller et al., 2016, SI).

Yeast surface display selection and characterization of rcSso7d-based binding variants Development and selection of rcSso7d that binds streptavidin (rcSso7d.SA) was described in previous work (Miller et al., 2016). The SARS-CoV-2 N protein-binding variant of rcSso7d was selected in similar fashion, from a yeast surface display library based on the reduced-charge Sso7d scaffold (rcSso7d). This yeast library was generated using trinucleotide oligo synthesis and in vivo homologous recombination with the linearized pCTcon2 plasmid (Traxlmayr et al., 2016).

Both highly-avid magnetic bead sorting (Ackerman et al., 2009) (MBS) and fluorescence-activated cell sorting (FACS) (Chao et al., 2006) were used to select binders against a biotinylated SARS-CoV-2 N protein target (FIG. 1). The sorting stringency was increased over five rounds of FACS-based library screening, after which a sub-library was sequenced and 30 candidates were selected for further characterization (Table 2). The affinity of this species was assessed in a yeast surface display format, via a soluble titration of biotinylated SARS-CoV-2 N protein against the displayed rcSso7d variant.

Recombinant protein expression, purification, and characterization

The genes for the selected sub-library candidates were both cloned from the pCTcon2 yeast display plasmid into the pET28b(+) bacterial expression plasmid as previously described (Miller et al., 2016). CBD fusion gene products were generated by Integrated DNA Technologies (IDT; Coralville, IA, USA) via gene synthesis, and traditional PCR cloning was used to integrate the individual sub-library candidate modules into separate rcSso7d-CBD fusion constructs. All gene products were modified with an N-terminal hexahistidine tag for purification via immobilized metal affinity chromatography (IMAC). The pET14b- NP plasmid was provided by the lab of Dr. Antonio Campos-Neto at the Forsyth Institute (Napolitano et al., 2008).

The heterologous expression of all protein species was conducted in a BE21(DE3) strain of E. coli, and induced via the addition of 0.5 mM isopropyl P-D-l- thiogalactopyranoside (IPTG). Induced cells were lysed by ultrasonification, and the recombinant product was purified from the clarified lysate via IMAC. A 3-kDa Amicon Ultracentrifuge Filter cassette was used to buffer exchange the 9.24-kDa rcSso7d monomer 1,000-fold into the resuspension buffer (40 mM sodium acetate, pH 5.5). Products featuring a CBD fusion partner were buffer-exchanged using a 3.5kDa MWCO Slide- A-Lyzer Dialysis Cassette (Thermo Fisher Scientific, Waltham, MA, USA), in order to prevent the adsorption of the CBD fusion products to the cellulose acetate membrane of the spin filters.

SARS-CoV-2 N protein was expressed in similar fashion using BL21(DE3) E. coli, and was resuspended in 50 mM HEPES buffer (pH 8.0) using a lOkDa MWCO Slide-A- Lyzer Dialysis Cassette. Purified SARS-CoV-2 N protein was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin No-Weigh Format labeling kit from Thermo Fisher Scientific, and desalted using Micro G-25 Spin Columns from Santa Cruz Biotech (Dallas, TX, USA).

The concentrations of all purified proteins were assessed using a bicinchoninic acid (BCA) assay, and all standards and purified samples were tested in triplicate for greater accuracy. Protein purity was assessed using a freshly cast 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, stained using Coomassie Brilliant Blue G-250.

Fabrication and testing of biofunctional cellulose test zones

Unmodified Whatman No. 1 chromatography paper are used as shipped for the immobilization of rcSso7d-CBD fusion proteins. In order to enable the covalent immobilization of rcSso7d variants lacking a CBD fusion partner, Whatman No. 1 chromatography paper is functionalized in 30 mM sodium metaperiodate solution as previously described (Miller et al., 2016). This oxidized, aldehyde-functionalized cellulose is stored under vacuum in a desiccator until use, whereas non-functionalized paper is stored under ambient conditions. As previously described, a solid ink printer is used to produce test zone arrays, and this printed wax is melted through the paper thickness (0.18 mm) to yield test zones with an average area of 2.5 ± 0.1 mm² (unless otherwise noted).

Stock solutions of purified rcSso7d and rcSso7d-CBD variants are diluted to the desired concentrations in resuspension buffer. For bare rcSso7d species, glycerol is also added to the solution at a final volumetric concentration of 10% in order to prevent evaporation during the extended initial incubation. Unless otherwise stated, all binding protein solutions are prepared at a final concentration of 30 pM. Negative controls for functionalized paper samples consisted of test zones contacted with 1 mg/mL bovine serum albumin (BSA). Bare paper test zones are used as the negative control for unmodified paper samples.

Functionalized test zones are modified with the bare rcSso7d variants, washed, and neutralized in Tris-buffered saline as described in previous work. Both rcSso7d-CBD variants are contacted with unmodified paper in 6 pL aliquots for at least thirty seconds, and then washed twice in 20 pL of lx phosphate-buffered saline (PBS; pH 7.4).

Protein-coated test zones are then contacted with 10 pL of the relevant antigen, diluted to the desired concentration in sterile-filtered lx PBS/1% w/v BSA. rcSso7d.Cov2- CBD candidates are contacted with biotinylated recombinant SARS-CoV-2 N protein. All test zones are incubated with antigen solution for 30 minutes at room temperature, after which they are washed twice with PBS. Negative controls are incubated in PBS in the absence of soluble antigen during this period.

Assay test zones are then subjected to an additional 30-minute incubation with SA- E/SA-AF647 at a concentration of 256 nM. SA-E samples are prepared in a citric acid- sodium phosphate buffer system (50mM citric acid, 90mM Na2HPO4, pH 4.5) containing 1% BSA, and washed in the same acidic buffer lacking BSA, in order to reduce non-specific binding to the detection reagent . Developed samples are blotted dry and stored in the dark in a freezer box until needed for fluorescence microscopy imaging.

Fluorescence microscopy

All samples are imaged as previously described (Miller et al., 2016), using an Olympus 1X81 Microscope. Unless otherwise noted, all samples developed with SA-E are exposed for 1000 ms using a Semrock TxRed-4040C filter set. Samples developed with SA- AF647 are exposed for either 80 ms or 100 ms (as noted) using a Semrock Cy5-4040C filter set. The ImageJ Auto Threshold function (Default algorithm) is used to identify the bounds of each sample zone, and the mean fluorescence intensity (MFI) of each sample was calculated by averaging the brightness of all pixels within the thresholded area. Four technical replicates are prepared for all experimental conditions, and the resultant MFI values are averaged for all replicates. Error bars represent one standard deviation from this mean intensity value. Quantification of surface-immobilized CBD fusion proteins

A micro BCA assay (Thermo Fisher Scientific) are used to determine the immobilized surface density of the engineered rcSso7d-CBD fusion protein on non-functionalized Whatman No. 1 chromatography paper. A series of standards is prepared by contacting test zones with known masses of rcSso7d-CBD and allowing these solutions to evaporate in a vacuum chamber at room temperature for 30 minutes, yielding complete protein adsorption to the cellulosic substrate. Experimental samples are generated by applying a series of known soluble rcSso7d-CBD concentrations to the test zones, followed by a PBS wash step.

All samples are excised from the test strips and deposited into the wells of a 96-well plate pre-filled with 150 pL of 40mM sodium acetate (pH 5.5). These test zones are vigorously stirred with clean pipette tips, and 150 pL of Working Reagent is then added to each sample well. The plate is incubated at 37°C for two hours, and after removing the paper test zones (wringing any entrained fluid back into the sample well), the absorbance at 562 nm is quantified for all samples.

The response curve for the evaporated standards is fit to a second-order polynomial, and this standard curve is used to determine the effective quantity of rcSso7d-CBD immobilized on the washed samples. Proportional rcSso7d-CBD retention is calculated by comparing these experimentally determined quantities to the known protein masses applied to the surface. In order to quantify the binding capacity of the cellulose substrate under these processing conditions, the density of Whatman No. 1 chromatography paper is measured in triplicate, and is found to be 0.088 ± 0.00016 mg/mm² The area of the test zones is measured by determining the pixel density at 40x magnification (0.287 megapixels/mm²), and measuring the thresholded test zone area in ImageJ. For this micro BCA experiment, the average area of the test zones is found to be 3.65 ± 0.25 mm², corresponding to a cellulose mass of 0.32 ± 0.021 mg.

Combinatorial Library Screening

The pCTcon2-encoded library of rcSso7d variants is expressed and exported to the exterior of the yeast membrane as a C-terminal fusion to the yeast Aga2p mating protein. This permits the selection of yeast carrier cells based on the binding activity of the displayed protein, allowing the population genetics to be biased towards plasmids encoding for functional rcSso7d variants. In order to select binding variants against the recombinant SARS-CoV-2 N protein antigen, two rounds of target positive MBS are used to reduce the library diversity. This sub-library is then screened via five rounds of FACS, sequentially increasing the sorting stringency by decreasing the concentration of available antigen and the captured proportion of the library population.

A sub-population of yeast is sequenced following the final FACS round, and 30 rcSso7d.NP (rcSso7d that binds SARS-CoV-N protein) proteins are selected based on their superior binding properties. The binding affinity of the rcSso7d.NP species is assessed in a yeast-surface display format, via a titration of the soluble, biotinylated SARS-CoV-2 N protein against the displayed rcSso7d binding species. The antigen concentration is varied from 256 nM to 0.25 nM, and at every concentration of SARS-CoV-2 N protein the yeast cells are resuspended in sufficient volume such that the antigen is present in ten-fold molar excess of the displayed binding species (assuming 50,000 displayed copies per cell, and efficient display in 60% of the population). Samples are incubated with continuous mixing for sufficient time to achieve greater than 99% of theoretical equilibrium binding. Following fluorescent labeling with streptavidin Alexa Fluor 647, the cell surface fluorescence is analyzed using a BD FACS LSR Fortessa II flow cytometer and the FACSDiva software package. All samples are analyzed using the 488 nm and 640 nm lasers, set to a voltage of 300V. The total geometric mean fluorescence intensity of all rcSso7d-displaying cells is quantified, and a sigmoidal function is fit to these data points to determine the affinity of the rcSso7d.NP binding species.

Production of Gene Constructs rcSso7d-NP constructs are cloned from the pCTcon2 yeast display plasmid into the pET28b(+) bacterial expression plasmid as previously described (Miller et al., 2016). Briefly, polymerase chain reaction (PCR) amplification of the desired gene is conducted using the primers rcSso7d-for and rcSso7d-rev (Table 1), at an annealing temperature of 58.3 °C. This PCR amplicon is subjected to an Ndel/Xhol double digest at 37°C for three hours (adding the Ndel enzyme after two hours to prevent aberrant cleavage), and this cleaved product is subsequently ligated into the digested pET-28b( +) plasmid backbone at room temperature in order to generate stable rcSso7d.NP constructs. All ligation mixtures are purified using the DNA Clean and Concentrator-5 Kit from Zyrno Research (Irvine, CA, USA), and eluted in 12 pL of PCR-grade water. 4 pL of this ligation product is transformed into DH5a E. coli (F- (p801acZAM15A(lacZYA-argF) U169 recAl end Al hsdR17 (rk-, mk+) gal- phoA supE44 k- thi-1 gyrA96 relAl) via electroporation. The entirety of this transformation mixture is plated on LB-kan plates and incubated overnight at 37°C. Positive clones are verified via both N- and C-terminal sequencing, using the T7 promoter and T7 terminator sequencing primers.

This general workflow is used for all cloning projects, and all relevant primers can be found in Table 2.

Table 2. Oligonucleotide sequences of primers used in sequencing reactions and plasmid cloning of selected binders.

REFERENCES

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Chao, G., Lau, W.L., Hackel, B.J., Sazinsky, S.L., Lippow, S.M., Wittrup, K.D., 2006. Nat. Protoc. 1, 755-68.

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Napolitano, D.R., Pollock, N., Kashino, S.S., Rodrigues, V., Campos-Neto, A., 2008. Clin. Vaccine Immunol. 15, 638-43.

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OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims

What is claimed is:

1. An antigen-binding protein comprising an engineered reduced-charge Sso7d (rcSso7d) antigen-binding protein that binds a SARS-CoV-2 nucleoprotein (N protein), wherein the antigen-binding protein comprises a variable region, said variable region comprising the sequence of any one of SEQ ID NO: 31-40, 42-50, and 52-60.

2. The antigen-binding protein of claim 1, wherein the rcSso7d antigen-binding protein comprises a scaffold, said scaffold comprising the sequence of SEQ ID NO: 3.

3. The antigen-binding protein of claim 1 or claim 2, wherein the SARS-CoV-2 nucleoprotein comprises the sequence of SEQ ID NO: 4.

4. The antigen-binding protein of any one of claims 1-3 further comprising a cellulose binding domain (CBD).

5. The antigen-binding protein of claim 4, wherein the antigen-binding protein is linked to the CBD through a linker.

6. The antigen-binding protein of claim 5, wherein the linker is a Gly-Ser linker.

7. The antigen-binding protein of any one of claims 4-6, wherein the C-terminus of the antigen-binding protein is linked to the N-terminus of the CBD.

8. The antigen-binding protein of any one of claims 4-7, wherein the CBD is a type 3a CBD, or the type 1 dimerized CBD (dCBD).

9. The antigen-binding protein of claim 8, wherein the type 3a CBD is a domain of the protein CipA from Clostridium thermocellum.

10. The antigen-binding protein of any one of claims 4-9, comprising the sequence of SEQ ID NO: 14.

11. A method of detecting SARS-CoV-2 nucleoprotein (N protein), comprising:

47 (a) contacting the antigen-binding protein of any one of claims 4-10 with a cellulose- containing substrate for a time sufficient for the antigen-binding protein to bind the cellulose-containing substrate;

(b) contacting the antigen-binding protein bound to the cellulose-containing substrate with a sample comprising or suspected to comprise a SARS-CoV-2 N protein; and

(c) detecting the SARS-CoV-2 N protein, if present, bound by the antigen-binding protein.

12. The method of claim 11, wherein the sample is a biological sample from a subject.

13. The method of claim 12, wherein the subject is a mammal.

14. The method of claim 13, wherein the subject is a human.

15. The method of any one of claims 11-14, wherein detecting comprises addition of a detectably-labeled protein which binds to SARS-CoV-2 N protein.

16. The method of claim 15, wherein the detectably-labeled protein is an enzyme-labeled protein.

17. The method of claim 16, wherein the enzyme-labeled protein is an engineered rcSso7d antigen-binding protein that binds a SARS-CoV-2 N protein.

18. The method of claim 16, wherein the enzyme-labeled protein is an antibody that binds SARS-CoV-2 N protein.

19. The method of any one of claims 16-18, wherein the enzyme-labeled antibody is labeled with horseradish peroxidase (HRP).

20. The method of any one of claims 11-19, wherein the antigen-binding protein is in molar excess of the SARS-CoV-2 N protein.

21. The method of claim 20, wherein the antigen-binding protein is in at least 10-fold molar excess of the anti-SARS-CoV-2 N protein.

22. The method of any one of claims 11-21, wherein at least 50% of the SARS-CoV-2 N protein is bound by the antigen-binding protein.

48

23. The method of any one of claims 11-22, wherein the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder.

24. The method of any one of claims 11-23, wherein the cellulose-containing substrate is chromatography paper.

25. The method of claim 24, wherein the chromatography paper is unmodified.

26. The method of any one of claims 11-25, further comprising rinsing the cellulose- containing substrate with a buffer solution before detecting the SARS-CoV-2 N protein bound by the antigen-binding protein.

27. The method of any one of claims 12-26, further comprising treating the subject if SARS- CoV-2 N protein is detected.

28. A method for detecting a SARS-CoV-2 nucleoprotein (N protein), the method comprising:

(a) contacting the antigen-binding protein of any one of claims 4-10 with a sample comprising or suspected to comprise a SARS-CoV-2 N protein, wherein the SARS-CoV-

2 N protein binds to the antigen-binding protein and forms a complex;

(b) contacting the complex with a cellulose-containing substrate for a time sufficient for the complex to bind to the cellulose-containing substrate; and

29. The method of claim 28 wherein the sample is a biological sample from a subject.

30. The method of claim 29, wherein the subject is a mammal.

31. The method of claim 30, wherein the subject is a human.

32. The method of any one of claims 28-31, wherein detecting comprises addition of a detectably-labeled protein which binds to SARS-CoV-2 N protein.

33. The method of claim 32, wherein the detectably-labeled protein is an enzyme-labeled protein.

49

34. The method of claim 33, wherein the enzyme-labeled protein is an engineered rcSso7d antigen-binding protein that binds a SARS-CoV-2 N protein.

35. The method of claim 33, wherein the enzyme-labeled protein is an antibody that binds SARS-CoV-2 N protein.

36. The method of any one of claims 33-35, wherein the enzyme-labeled antibody is labeled with horseradish peroxidase (HRP).

37. The method of any one of claims 28-36, wherein the antigen-binding protein is in molar excess of the SARS-CoV-2 N protein.

38. The method of claim 36, wherein the antigen-binding protein is in at least 10-fold molar excess of the anti-SARS-CoV-2 N protein.

39. The method of any one of claims 28-38, wherein at least 50% of the SARS-CoV-2 N protein is bound by the antigen-binding protein.

40. The method of any one of claims 28-39, wherein the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder.

41. The method of any one of claims 28-39, wherein the cellulose-containing substrate is chromatography paper.

42. The method of claim 41, wherein the chromatography paper is unmodified.

43. The method of any one of claims 28-42, further comprising rinsing the cellulose- containing substrate with a buffer solution before detecting the SARS-CoV-2 N protein bound by the antigen-binding protein.

44. The method of any one of claims 29-43, further comprising treating the subject if SARS- CoV-2 N protein is detected.

45. A method for assessing a presence or amount of a SARS-CoV-2 nucleoprotein (N protein) in a sample, the method comprising contacting the sample with the antigenbinding protein of any one of claims 4-10 and measuring the presence or amount of the SARS-CoV-2 N protein in the sample.

50 The method of claim 45, wherein the sample is a biological sample from a subject. The method of claim 46, wherein the subject is a mammal. The method of claim 47, wherein the subject is a human. The method of any one of claims 45-48, wherein measuring comprises addition of a detectably-labeled protein which binds to SARS-CoV-2 N protein. The method of claim 49, wherein the detectably-labeled protein is an enzyme-labeled protein. The method of claim 50, wherein the enzyme-labeled protein is an engineered rcSso7d antigen-binding protein that binds a SARS-CoV-2 N protein. The method of claim 50, wherein the enzyme-labeled protein is an antibody that binds SARS-CoV-2 N protein. The method of any one of claims 50-52, wherein the enzyme-labeled antibody is labeled with horseradish peroxidase (HRP). The method of any one of claims 45-53, wherein the antigen-binding protein is in molar excess of the SARS-CoV-2 N protein. The method of claim 54, wherein the antigen-binding protein is in at least 10-fold molar excess of the anti-SARS-CoV-2 N protein. The method of any one of claims 45-55, wherein at least 50% of the SARS-CoV-2 N protein is bound by the antigen-binding protein. The method of any one of claims 45-56, wherein the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder. The method of any one of claims 45-56, wherein the cellulose-containing substrate is chromatography paper. The method of claim 58, wherein the chromatography paper is unmodified.

60. The method of any one of claims 45-59, further comprising rinsing the cellulose- containing substrate with a buffer solution before detecting the SARS-CoV-2 N protein bound by the antigen-binding protein.

61. The method of any one of claims 46-60, further comprising treating the subject if SARS- CoV-2 N protein is detected.

62. A kit comprising a container containing the antigen-binding protein of any one of claims 4-10.

63. The kit of claim 62, further comprising a cellulose-containing substrate.

64. The kit of claim 63, wherein the antigen-binding protein is bound to the cellulose- containing substrate.

65. The kit of claim 63, wherein the antigen-binding protein is not bound to the cellulose- containing substrate.

66. The kit of any one of claims 62-65, wherein the cellulose-containing substrate is paper, nitrocellulose, or cellulose powder. 67. The kit of any one of claims 62-65, wherein the cellulose-containing substrate is chromatography paper.

68. The kit of claim 67, wherein the chromatography paper is unmodified.