Biosensors for calprotectin

Abstract

Provided herein are compositions, methods, and kits suitable for detection and / or quantification of calprotectin in a sample (including extracts thereof). In some aspects, compositions, methods, and / or kits comprise a Fab-Enabled Split Luciferase Calprotectin Assay (FESCA) or components thereof, a novel quantitative method for calprotectin measurement. In some aspects, provided herein are two new fragment antigen binding proteins (Fabs), CP16 and CP17, which bind to different epitopes of the calprotectin complex. These Fabs can be fused, directly or indirectly, with a reporter system, such as split NanoLuciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. Technologies provided herein can be utilized to detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, whole blood, and stool extracts, and can be utilized in methods of treating a subject.

Description

BIOSENSORS FOR CALPROTECTINCLAIM OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 646,094, filed on May 13, 2024, which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM119840 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on January 4, 2024, is named ARCD_P0826USPl_Sequence_Listing.xml and is 20,548 bytes in size.FIELD AND BACKGROUND1. Field

[0004] The present disclosure relates generally to the fields of chemistry and medicine. More particularly, it concerns compositions, methods, and kits for detecting and / or measuring a biological agent and methods for therapeutically intervening according thereto.2. Background

[0005] Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various inflammatory diseases, such as inflammatory bowel disease (IBD). Currently, clinical testing for calprotectin levels, especially fecal calprotectin levels, relies on enzyme-linked immunosorbent assays (ELISAs), which require trained personnel and expensive equipment, and are therefore limited to laboratory settings. In addition, data collection of fecal calprotectin levels is usually impeded by low patient compliance to the generally inconvenient fecal immunochemical testing requirements (see e.g., PMID 30684199), and specifically among patients with IBD (see e.g., PMID 36776647), with the necessity of sample transportation to the clinic often acting as a primary deterrent (see e.g., PMID: 36776647, and 28570431), making monitoring the disease progression and guiding management challenging for both the physicians and the patients.

[0006] There is a pressing need for improved compositions and / or methods for detecting and / or quantifying calprotectin levels in various sample types in an efficient, reliable, mobile, and / or convenient manner.SUMMARY

[0007] This disclosure describes solutions to at least some of the problems associated with accurate, rapid, and / or convenient detection of calprotectin levels in a sample. In some aspects, the solution resides in engineered polypeptides comprising binding specificity to calprotectin complexes. In some aspects, the polypeptides can be linked to a reporter system that can be fragmented limiting production of reporter signal to instances when the reporter fragments are in sufficient proximity. In some aspects, the polypeptides are linked to a fragmented luciferase enzyme that becomes functional when the polypeptides bind a calprotectin complex. The signal produced by polypeptides of the disclosure can be proportionate to the levels of calprotectin in a sample thereby providing at least quantitative measurements.

[0008] As disclosed herein, among other things, the inventors have created a Fab-Enabled Split Luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA can utilize fragment antigen binding proteins (Fabs), namely the newly created CP16 (SEQ ID NOs: 1 and 2) and / or CP17 (SEQ ID NOs: 7 and 8) Fabs, which bind to different epitopes of the calprotectin complex. These Fabs can be fused with reporter systems (e.g., detectable and / or can produce a detectable signal), such as but not limited to luciferase fragments (e.g., NANOLUCIFERASE™, PROMEGA®), enabling the reconstitution of a reporter system (e.g., an active luciferase enzyme) upon binding to calprotectin either in solution or in varied immobilized assay formats. As disclosed herein, in certain aspects, FESCA can detect physiologically relevant calprotectin levels across various sample types, including but not limited to, serum, plasma, whole blood, and stool extracts. Notably, in certain aspects, FESCA's output luminescence can be quantitatively measured with standard laboratory equipment and / or consumer-grade cell phone cameras (e.g., IPHONE™ 13 PRO dark mode, APPLE ®).

[0009] Provided herein are compositions, methods of making, methods of use, uses in the manufacture of a medicament, and kits (“technologies”) comprising reagents that can be utilized to detect calprotectin in a sample, such as through FESCA methods. In some aspects, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples. In some aspects, FESCA compositions / methods can beutilized to improve the diagnosis and / or monitoring of inflammatory diseases (such as but not limited to IBD), especially in at-home and / or point-of-care settings.

[0010] In some aspects, provided herein are antibody or antigen binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 of, of at least, or of at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to an HCDR1, HCDR2, HCDR3 from a heavy chain variable region of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 of, of at least, or of at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to an LCDR1 from a light chain variable region of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0011] In some aspects, a heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acid sequence of a HCDR1, HCDR2, and HCDR3 of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence of the LCDR1 from the light chain variable region of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7). In some aspects, a HCDR1, HCDR2, HCDR3, and LCDR1 each comprise an amino acid sequence of, of at least, or of at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to an HCDR1, HCDR2, HCDR3, and LCDR1 of CP 16 or CP 17 heavy chain and light chain (SEQ ID NOs: 1, 2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1, are or are not from the same antigen binding fragment clone. In some aspects, a HCDR1, HCDR2, HCDR3, and LCDR1 each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR3, and LCDR1 of CP 16 or CP 17 heavy chain and light chain (SEQ ID NOs: 1, 2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1 are or are not from the same antigen binding fragment clone.

[0012] In some aspects, a heavy chain variable region comprises an amino acid sequence of, of at least, or of at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to a heavy chain variable region of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises an amino acid sequence of, of at least, or of at most 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to the light chain variable region of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7). In some aspects, a heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises the amino acid sequence of a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0013] In some aspects, an antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and the light chain comprises an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) sequence identity to CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0014] In some aspects, an antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP 16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and the light chain comprises the amino acid sequence of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7). In some aspects, an antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP 16 heavy chain (SEQ ID NO: 2), and the light chain comprises the amino acid sequence of CP16 light chain (SEQ ID NO: 1). In some aspects, an antibody comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP17 heavy chain (SEQ ID NO: 8), and the light chain comprises the amino acid sequence of CP 17 light chain (SEQ ID NO: 7). In some aspects, an antibody or antigen binding fragment is human, chimeric, or humanized. In some aspects, an antibody or antigen binding fragment is not human, chimeric, or humanized.

[0015] In some aspects, an antibody or antigen binding fragment binds to a calprotectin complex with a KD of about 10'4M / L to about 10'14M / L, or about 10'6M / L to about 10'12M / L. In some aspects, an antibody or antigen binding fragment binds to a calprotectin complex with an EC50 less than about 1 nM to about 1000 nM, or less than about 1 nM to about 100 nM. Insome aspects, an antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with a KD of about 10'4M / L to about 10'14M / L, or about 10'6M / L to about 10'12M / L. In some aspects, an antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex with a KD of about 10’4M / L to about 10'14M / L, or about 10'6M / L to about 10'12M / L. In some aspects, an antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with an EC50 of less than about 1 nM to about 1000 nM, or less than about 1 nM to about 100 nM. In some aspects, an antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex with an EC50 of less than about 1 nM to about 1000 nM, or less than about 1 nM to about 100 nM. In some aspects, an antibody or antigen binding fragment is a neutralizing antibody or antigen binding fragment. In some aspects, an antibody or antigen binding fragment is not a neutralizing antibody or antigen binding fragment. In some aspects, an antibody is or is not a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody. In some aspects, an antigen binding fragment is or is not a single chain variable fragment (scFv), F(ab’)2, Fab’, fragment antigen binding protein (Fab), Fv, or rlgG. In some aspects, an antigen binding fragment comprises, consists essentially of, or consists of a Fab.

[0016] In some aspects, also provided herein is a polypeptide comprising an antibody or antigen binding fragment disclosed herein. In some aspects, a polypeptide comprises at least two antigen binding fragments. In some aspects, a polypeptide is or is not multivalent. In some aspects, a polypeptide is or is not bispecific. In some aspects, a polypeptide comprises an antibody or antigen binding fragment that is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system. In some aspects, a reporter system comprises or does not comprise a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system. In some aspects, a reporter system comprises or does not comprises a split luciferase reporter system. In some aspects, a split luciferase reporter system comprises or does not comprises a NANOBIT® (PROMEGA™) luciferase reporter system, and the fragments of the split luciferase comprise or do not comprise SmBiT and LgBiT from NANOLUCIFERASE® (NANOLUC®, PROMEGA™). In some aspects, an antibody or antigen binding fragment and a fragment of a split luciferase can be linked with a peptide linker that is less than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids (or any range derivable therein). In some aspects, an antibody or antigen binding fragment anda fragment of a split luciferase can be linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS- DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, an antibody or antigen binding fragment and a fragment of a split luciferase can be linked at the C-terminus of the antibody or antigen binding fragment.

[0017] In some aspects, a polypeptide comprises a CP 16 heavy chain linked to SmBiT or LgBiT. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker that is, is at least, or is at most 2, 5, 10, 12, 15, 20, or 25 amino acids in length. In some aspects, a CP 16 heavy chain can be linked to SmBiT or LgBiT with a linker peptide comprising, consisting of, or consisting essentially of GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a CP16 heavy chain can be linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a CP 16 heavy chain can be linked to LgBiT with linker peptide GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a polypeptide comprises a CP16 light chain linked to SmBiT or LgBiT. In some aspects, a CP 16 light chain can be linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0018] In some aspects, a polypeptide comprises a CP 17 heavy chain linked to SmBiT or LgBiT. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker that is, is at least, or is at most 2, 5, 10, 12, 15, 20, or 25 amino acids in length. In some aspects, a CP 17 heavy chain can be linked to SmBiT or LgBiT with linker peptide GS- DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a CP17 heavy chain can be linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a polypeptide comprises a CP 17 light chain linked to SmBiT or LgBiT. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker that is, is at least, or is at most 2, 5, 10, 12, 15, 20, or 25 amino acids in length. In some aspects, a CP17 light chain can belinked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK- GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0019] Also provided herein, in some aspects, are compositions comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies, antigen binding fragments, and / or polypeptides disclosed herein. Also provided herein, in some aspects, are nucleic acids encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies, antigen binding fragments, and / or polypeptides disclosed herein. In some aspects, a nucleic acid is or is not comprised in a vector. In some aspects, a nucleic acid and / or vector are or are not comprised in a host cell. In some aspects, a host cell is or is not a prokaryotic cell or a eukaryotic cell. In some aspects, a host cell is or is not a bacterial cell, fungal cell, mammalian cell, or insect cell. In some aspects, a host cell is or is not A. coli. Also provided herein, in some aspects, are methods of making a cell comprising introducing a nucleic acid(s) and / or vectors described herein into a cell. Also provided herein, in some aspects, are methods of culturing a cell under conditions that allow for expression of a polypeptide from a nucleic acid. In some aspects, methods provided herein further comprise isolating an expressed polypeptide from a host cell.

[0020] Also provided herein, in some aspects, are composition comprising, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system. In some aspects, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system. In some aspects, a composition comprises more than one polypeptide, and the polypeptides bind to the same epitope, or to different epitopes. In some aspects, the polypeptides bind in a calcium dependentmanner. In some aspects, the polypeptides bind in a calcium independent manner. In some aspects, the polypeptides bind in a calcium dependent and / or a calcium independent manner.

[0021] In some aspects, a composition comprises a reporter system comprising or not comprising a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system. In some aspects, a reporter system comprises a split luciferase reporter system. In some aspects, a split luciferase reporter system comprises or does not comprise a NANOBIT® (PROMEGA™) luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NANOLUCIFERASE® (NANOLUC®, PROMEGA™).

[0022] In some aspects, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of a split luciferase that are linked with a peptide linker that is less than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of a split luciferase can be linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides can be linked at the C-terminus to at least one fragment of the split luciferase. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP I 7 Fabs.

[0023] In some aspects, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16-LgBiT). In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, at least one CP 16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS- DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP 16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP16 heavy chain is linked to LgBiT with linker peptide GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP16 light chain is linked to SmBiT or LgBiT.

[0024] In some aspects, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP I 7 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT(CP17-LgBiT). In some aspects, at least one CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS- DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a composition comprises at least one CP 17 heavy chain linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a composition comprises at least one CP 17 heavy chain linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a composition comprises at least one CP 17 light chain linked to SmBiT or LgBiT.

[0025] In some aspects, a composition comprises a concentration of CP16-SmBiT and / or CP17-SmBiT that is , is at least, or is at most about 1 to 1000 mM, 1 to 500 mM, or 2 to 100 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 15 to 60 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP16-SmBiT that is, is at least, or is at most about 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, or 55 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP17-SmBiT that is, is at least, or is at most about 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, or 30 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 1 to 1000 mM, 1 to 500 mM, or 2 to 100 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 5 to 25 nM (or any value derivable therein). In some aspects, a composition comprises a concentration of CP16-LgBiT is, is at least, or is at most about 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, or 20 nM (or any range or value derivable therein). In some aspects, a composition comprises a concentration of CP17-LgBiT is, is at least, or is at most about 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, or 20 nM (or any range or value derivable therein).

[0026] In some aspects, a composition comprises at least one of CP16-LgBiT and / or CP 17- LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT. In some aspects, a composition comprises: a) CP16-LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17- LgBiT and CP16-SmBiT; or d) CP17-LgBiT and CP17-SmBiT. In some aspects, a composition comprises a ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT that is not 1 : 1. In some aspects, a composition comprises a lower concentration of CP16-LgBiTand / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT. In some aspects, a composition comprises a ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or2:l (or any range or value derivable therein). In some aspects, a composition comprises a ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 3:4, 3:5, 3:7, 3:8, 1:3, 3:10, 3:11, 1:4, 3:13, 3:14, 1:5, 3:16, 3:17, 1:6, 3:19, 3:20, or 1:7 (or any range or value derivable therein). In some aspects, a composition comprises about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT. In some aspects, a composition comprises about 15 nM CP16-LgBiT and about 50 nM CP16-SmBiT. In some aspects, a composition comprises about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT. In some aspects, a composition comprises about 15 nM CP16-LgBiT and about 25 nM CP16-SmBiT.

[0027] In some aspects, a composition further comprises Ca2+. In some aspects, a composition does not comprise Ca2+. In some aspects, a composition comprises at least, at most, or about 2 mM Ca2+. In some aspects, a composition comprises or does not comprises furimazine. In some aspects, a composition comprises or does not comprises TBST, BSA, and / or CaCh. In some aspects, a composition comprises TBST at about pH 6-8, about 0.01-1% BSA, and / or about 0.5-5 mM CaCh (or any value derivable therein). In some aspects, a composition comprises TBST at about pH 7.4, about 0.2% BSA, and / or about 2 mM CaCh.

[0028] Also provided herein, in some aspects, are methods for evaluating a sample from a subject, comprising the step of contacting a biological sample from the subject (including extracts of the sample), with at least one antibody, antigen binding fragment, polypeptide, and / or composition disclosed herein. In some aspects, a method further comprises the step of incubating the antibody, antigen binding fragment, polypeptide, and / or composition with the biological sample, under conditions that allow for the binding of the antibody, antigen binding fragment, polypeptide, and / or polypeptide of the composition to bind to antigens in the biological sample. In some aspects, a method further comprises the step of detecting the binding of an antigen to the antibody, antigen binding fragment, polypeptide, and / or polypeptide of the composition. In some aspects, detecting comprises measuring of a signal produced by a reporter system.

[0029] Also provided herein, in some aspects, are methods of measuring the concentration of calprotectin complex in a biological sample, the methods comprising the steps of contactingthe biological sample with an antibody, antigen binding fragment, polypeptide and / or composition disclosed herein, and measuring the level of interaction between the antibody, antigen binding fragment, Fab, and / or polypeptide with the calprotectin complex. In some aspects, a method comprises use of at least one antibody, antigen binding fragment, and / or polypeptide that is operatively linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system, or the composition comprises an antibody, antigen binding fragment, polypeptide, or Fab that is operatively linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system can be utilized to produce a detectable level of a chemiluminescence signal when the reporter system is reconstituted. In some aspects, measuring comprises or does not comprise measuring a level of luminescence. In some aspects, an antibody, antigen binding fragment, polypeptide, and / or Fab of a composition is or is not in solution or is or is not linked to a solid support. In some aspects, a solution comprises or does not comprise furimazine, TBST, BSA, and / or CaCh. In some aspects, a solid support comprises or does not comprises a lateral flow assay strip, or a paper. In some aspects, a biological sample, or an extract thereof, comprises or does not comprises serum, plasma, whole blood, and / or stool.

[0030] Also provided herein, in some aspects, are methods for detecting calprotectin in a sample, the methods comprising the steps of, i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more polypeptides that comprise an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP 17 light chain (SEQ ID NOs: 1 and 7); wherein the CP 16 and / or CP 17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating. In some aspects, a sample comprises, or comprises an extract of, serum, plasma, whole blood, and / or stool. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more Fabs that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8) and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); and wherein the CP16 and / or CP17heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system. In some aspects, methods comprise use of more than one polypeptide, and the polypeptides bind to the same epitope, or to different epitopes. In some aspects, the polypeptides bind in a calcium dependent and / or independent manner. In some aspects, the polypeptides bind in a calcium dependent manner. In some aspects, the polypeptides bind in a calcium independent manner.

[0031] In some aspects of methods disclosed herein, a reporter system comprises or does not comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system. In some aspects, a reporter system comprises or does not comprises a split luciferase reporter system. In some aspects, a split luciferase reporter system comprises or does not comprises a NANOBIT® (PROMEGA™) luciferase reporter system, and fragments of the split luciferase comprise, consist essentially of, or consist of SmBiT and LgBiT from NANOLUCIFERASE® (NANOLUC®, PROMEGA™). In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of a split luciferase are linked with a peptide linker that is, is at most, or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs and at least one fragment of the split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked at the C-terminus of the heavy chain or light chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs.

[0032] In some aspects, in methods comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16- LgBiT). In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, at least one CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP 16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP16 light chain is linked to SmBiT or LgBiT. Insome aspects, at least one CP 16 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP16 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP 16 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0033] In some aspects, in methods comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17- LgBiT). In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, at least one CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP17 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP 17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP17 light chain is linked to SmBiT or LgBiT. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, at least one CP 17 light chain is linked to SmBiT or LgBiT with linker peptide GS- DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a least one CP17 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, at least one CP 17 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0034] In some aspects, methods provided herein comprise incubating a sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 1- 1000 nM, 2-500 nM, or 2-100 nM (or any range or value derivable therein). In some aspects, methods comprise incubating a sample with a concentration of CP16-SmBiT and / or CP17- SmBiT that is, is at least, or is at most about 15-60 nM (or any value derivable therein). In some aspects, methods comprise incubating a sample with a concentration of CP16-SmBiT that is, is at least, or is at most about 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, or 55 nM (or any range or value derivable therein). In some aspects, methods provided herein comprise incubating a sample with a concentration of CP17-SmBiTthat is, is at least, or is at most about 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, or 30 nM (or any range or value derivable therein).

[0035] In some aspects, methods provided herein comprise incubating a sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 1-1000 nM, 1-500 nM, 1-100 nM, or 2-50 nM (or any range or value derivable therein). In some aspects, methods provided herein comprise incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 5-25 nM (or any value derivable therein). In some aspects, methods provided herein comprise incubating the sample with a concentration of CP16-LgBiT that is, is at least, or is at most about 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, or 20 nM (or any range or value derivable therein). In some aspects, methods provided herein comprise incubating the sample with a concentration of CP17-LgBiT that is, is at least, or is at most about 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, or 20 nM (or any range or value derivable therein). In some aspects, methods provided herein comprise incubating a sample (or extract thereof) with at least one of CP16-LgBiT and / or CP17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

[0036] In some aspects, methods provided herein comprise use of a combination of a) CP16-LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16- SmBiT; and / or d) CP17-LgBiT and CP17-SmBiT. In some aspects, methods provided herein comprise a ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT that is or is not 1:1. In some aspects, methods provided herein comprise incubating a sample with a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT. In some aspects, ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CPI 7-SmBiT is, is at least, or is at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or2:l (or any range or value derivable therein). In some aspects, ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, oris at most about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 3:4, 3:5, 3:7, 3:8, 1:3, 3:10, 3:11, 1:4, 3:13, 3:14, 1:5, 3:16, 3:17, 1:6, 3 : 19, 3 :20, or 1 :7 (or any range or value derivable therein). In some aspects, methods comprise incubating a sample with about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT (or any value derivable therein). In some aspects, methods comprise incubating a sample with about 15 nM CP16-LgBiT and about 50 nM CP16-SmBiT. In some aspects, methods comprise incubating a sample with about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT(or any value derivable therein). In some aspects, methods comprise incubating a sample with about 15 nM CP16-LgBiT and about 25 nM CP16-SmBiT.

[0037] In some aspects, methods provided herein further comprise contacting and incubating a sample with Ca2+. In some aspects, the incubating with Ca2+is at concentration of, of at least, or of at most 2 mM. In some aspects, methods provided herein further comprise or do not comprise contacting and incubating a sample with furimazine. In some aspects, methods further comprise or do not comprise contacting and / or incubating a sample with TBST, BSA, and / or CaCh. In some aspects, the incubating comprises TBST at about pH 6-8, BSA at about 0.01-1%, and / or CaCh at about 0.5-5 mM (or any value derivable therein). In some aspects, incubating with TBST is at about pH 7.4, BSA is at about 0.2%, and / or CaCh is at about 2 mM. In some aspects, the incubating is for, for at least, or for at most 30 seconds to 5 hours, 1 minute to 1 hour, 2 to 30 minutes, or about 15 minutes to 30 minutes (or any range or value derivable therein).

[0038] In some aspects, methods provided herein comprise quantitatively measuring a signal. In some aspects, methods provided herein comprise quantitatively measuring luminescence. In some aspects, measuring of a signal is quantitative, and the signal correlates linearly with increasing calprotectin concentrations. In some aspects, methods comprise detecting a luminescence signal in a liquid solution. In some aspects, methods comprise detecting a luminescence signal on a solid support. In some aspects, a luminescence measuring is mediated through imaging. In some aspects, imaging is by a consumer grade camera. In some aspects, methods provided herein are performed in a point-of-care and / or at-home setting.

[0039] In some aspects, levels of calprotectin measured using methods provided herein are indicative of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diseases or disorders. In some aspects, levels of calprotectin in a sample are quantified and compared to a negative or positive control. In some aspects, levels of calprotectin in a sample are noted as abnormal when the detected signal is, or is greater than about 1.25-1000-fold (or any range or value derivable therein), or 2-200- fold (or any range or value derivable therein) higher than the negative control.

[0040] Also provided herein, in some aspects, are methods of treating a disease or disorder in a subject, the method comprising performing a method of measuring calprotectin provided herein and tailoring a treatment regimen based upon levels of calprotectin detected. In some aspects, a disease or disorder is or is not characterized by inflammation. In some aspects, a disease or disorder comprises or does not comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, cancer, rheumatoid arthritis, tuberculosis (TB), and / or COVID-19 infection. In some aspects, a disease or disorder comprises IBD, andmeasurements of calprotectin levels in serum, plasma, whole blood, and / or stool, or extracts thereof, indicate disease severity and / or disease progression / remission. In some aspects, a disease or disorder comprises TB, and measurements of calprotectin levels in serum, plasma, whole blood, and / or stool, or extracts thereof, indicate disease severity and / or disease progre ssi on / remi ssi on .

[0041] Also provided herein, in some aspects, are engineered stabilized calprotectin complexes comprising S100A9 linked to S100A8 to form a heterodimeric fusion protein. In some aspects, S100A9 and S100A8 are linked by a peptide linker, e.g., a GGS peptide linker. In some aspects, a calprotectin complex is further linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tags. In some aspects, a calprotectin complex is linked to a SNAP -tag protein for selective biotinylation.

[0042] Also provided herein, in some aspects, are polypeptides comprising an antibody or antigen binding fragment linked with a fragment of a split luciferase. In some aspects, a linker comprises, consists essentially of, or consists of a peptide linker. In some aspects, a split luciferase comprises a NANOBIT® (PROMEGA™) system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NANOLUC® (PROMEGA™) luciferase. In some aspects, a polypeptide comprises an antibody or antigen binding fragment and a fragment of a split luciferase that are linked with a peptide linker that is, is at least, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 5 amino acids, or 2 amino acids. In some aspects, a polypeptide comprises an antibody or antigen binding fragment and the fragment of a split luciferase that are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS- DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13). In some aspects, a polypeptide comprises an antibody or antigen binding fragment that binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes. In some aspects, an antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex. In some aspects, an antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex. In some aspects, a polypeptide comprises an antibody or antigen binding fragment comprising an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the heavy chain and / or light chain of CP 16 and / or CP 17 (SEQ ID NOs: 1, 2, 7, and 8). In some aspects, a polypeptide comprisesan antibody or antigen binding fragment comprises the heavy chain and / or light chain of CP 16 and / or CP17 (SEQ ID NOs: 1, 2, 7, and 8). In some aspects, an antigen binding fragment comprises a Fab.

[0043] Also provided herein, in some aspects, are methods for identifying a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP 17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the disease state based upon the level of calprotectin detected. In some aspects, a disease state comprises or does not comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, and / or cancer.

[0044] Also provided herein, in some aspects, are methods for measuring the efficacy of a therapeutic intervention (e.g., treatment regimen) for treating a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morecomponents of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the drug as effective or not effective for treatment of the disease state based upon the level of calprotectin detected. In some aspect, a disease state comprises or does not comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, and / or cancer.

[0045] Also provided herein, in some aspects, are methods of classifying the severity of a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence of, of at least, or of at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to the CP 16 and / or CP 17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the severity of a disease state based upon the level of calprotectin detected. In some aspects, a disease state comprises or does not comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, and / or cancer.

[0046] In some aspects, also provided herein are kits comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibody, antigen binding fragment, polypeptide, composition, and / or engineered stabilized calprotectin complex disclosed herein. In some aspects, kits further comprise additional reagents packaged individually and / or collectively. In some aspects, kits comprise furimazine, TBST, BSA, and / or CaCh.

[0047] Methods, systems, compositions, and kits are provided that involve engineered polypeptides for screening, identifying, diagnosing, and / or quantifying calprotectin, and subsequently treating a condition (e.g., a disease) accordingly.

[0048] In particular aspects, methods, systems, compositions, and kits comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more reporter systems (e.g., reporter systems comprising enzymes that provide a detectable signal / label such as a luminescent reporter and / or a fluorescent reporters, detectable tags, etc.) that can be split, and which only produce signal upon reconstitution (e.g., through close proximity) of the split reporter.

[0049] In particular aspects, reporter systems do not readily produce a detectable signal when split, and only become readily detectable when reconstituted, e.g., through proximity / co- localization.

[0050] One, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components can be attached to each portion or domain such that an interaction between components can be qualified and / or quantitated based on detectable activity (e.g., luminescence, fluorescence, enzymatic activity, etc.).

[0051] In specific aspects, the methods, systems, compositions and kits comprise an interaction domain (e.g., a Fab or portion thereof), such as CP16 and / or CP17. In certain aspects, portions of CP16 and / or CP17 are linked, directly or indirectly, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0052] In some aspects, the different targets are capable of physically and / or chemically interacting with one another. The term “interaction component” refers to a chemical moiety or entity that is capable of physically and / or chemically interacting with another interaction component. In specific aspects, the interaction components are polypeptides (and / or peptides) that specifically bind one another or a polypeptide and a split reporter system that can interact when the polypeptides are maintained in close proximity with each other.

[0053] A measurement of calprotectin can be quantified by a change in signal (e.g., luminescence) of about, at least about or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150% and / or by about, at least about, or at most about 2x, 3x, 4x, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, HOx, 120x, 130x, 140x, 150x, 160x, 170x, 180x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x, 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 410x, 420x, 430x, 440x, 450x, 460x, 470x, 480x, 490x, 500x, 600x, 700x, 800x, 900x, lOOOx (or any range derivable therein) relative to a control. The change may be an increase or a decrease in signal dependent upon the control (e.g., relative to a positive or negative control).

[0054] As used herein the specification, “a” or “an” may mean one or more or 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more.

[0055] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0056] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0057] As used herein “another” may mean at least a second or more.

[0058] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0059] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention. As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that aspects or aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0060] “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0061] It is specifically contemplated that any limitation discussed with respect to one aspect or aspect of the inventions may apply to any other aspect or aspect of the inventions. Furthermore, any composition of the inventions may be used in any method of the inventions, and any method of the inventions may be used to produce or to utilize any composition of the inventions. Any aspect or aspect discussed with respect to one aspect of the disclosure appliesto other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0062] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0063] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0064] Aspect 1 is an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 having at least 80% sequence identity to the LCDR1 from a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0065] Aspect 2 is the antibody or antigen binding fragment of aspect 1, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acid sequence of an HCDR1, HCDR2, and HCDR3 of CP16 or CP I 7 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence of the LCDR1 from the light chain variable region of CP16 or CP I 7 light chain (SEQ ID NOs: 1 and 7).

[0066] Aspect 3 is the antibody or antigen binding fragment of aspect 1 or 2, wherein the HCDR1, HCDR2, HCDR3, and LCDR1 each comprise an amino acid sequence that has at least 80% sequence identity to an HCDR1, HCDR2, HCDR3, and LCDR1 of CP I 6 or CP I 7 heavy chain and light chain (SEQ ID NOs: 1, 2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1 are from the same antigen binding fragment clone.

[0067] Aspect 4 is the antibody or antigen binding fragment of aspect 1 or 2, wherein the HCDR1, HCDR2, HCDR3, and LCDR1 each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR3, and LCDR1 of CP I 6 or CP I 7 heavy chain and light chain (SEQ ID NOs: 1,2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1 are from the same antigen binding fragment clone.

[0068] Aspect 5 is the antibody or antigen binding fragment of any one of aspects 1-4, wherein the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0069] Aspect 6 is the antibody or antigen binding fragment of aspect 5, wherein the heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises the amino acid sequence of a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0070] Aspect 7 is the antibody or antigen binding fragment of any one of aspects 1-6, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence with at least 70% sequence identity to CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and the light chain comprises an amino acid sequence with at least 70% sequence identity to CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0071] Aspect 8 is the antibody or antigen binding fragment of aspect 7, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and the light chain comprises the amino acid sequence of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0072] Aspect 9 is the antibody or antigen binding fragment of aspect 8, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP16 heavy chain (SEQ ID NO: 2), and the light chain comprises the amino acid sequence of CP16 light chain (SEQ ID NO: 1).

[0073] Aspect 10 is the antibody or antigen binding fragment of aspect 8, wherein the antibody comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP17 heavy chain (SEQ ID NO: 8), and the light chain comprises the amino acid sequence of CP17 light chain (SEQ ID NO: 7).

[0074] Aspect 11 is the antibody or antigen binding fragment of any one of aspects 1-10, wherein the antibody or antigen binding fragment is human, chimeric, or humanized.

[0075] Aspect 12 is the antibody or antigen-binding fragment of any one of aspects 1-11, wherein the antibody or antigen binding fragment binds to a calprotectin complex with a KD of about 10-6 M / L to about 10-12 M / L.

[0076] Aspect 13 is the antibody or antigen-binding fragment of any one of aspects 1-12, wherein the antibody or antigen binding fragment binds to a calprotectin complex with an EC50 of less than about 1 nM to about 100 nM.

[0077] Aspect 14 is the antibody or antigen-binding fragment of any one of aspects 1-13, wherein the antibody or antigen binding fragment binds to a calprotectin (SI 00 A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with a KD of about 10-6 M / L to about 10-12 M / L.

[0078] Aspect 15 is the antibody or antigen-binding fragment of any one of aspects 1-13, wherein the antibody or antigen binding fragment binds to a calprotectin (SI 00 A8 / 9)2 tetramer complex with a KD of about 10-6 M / L to about 10-12 M / L.

[0079] Aspect 16 is the antibody or antigen-binding fragment of any one of aspects 1-14, wherein the antibody or antigen binding fragment binds to a calprotectin (SI 00 A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with an EC50 of less than about 1 nM to about 100 nM.

[0080] Aspect 17 is the antibody or antigen-binding fragment of any one of aspects 1-14, wherein the antibody or antigen binding fragment binds to a calprotectin (SI 00 A8 / 9)2 tetramer complex with an EC50 of less than about 1 nM to about 100 nM.

[0081] Aspect 18 is the antibody or antigen binding fragment of any one of aspects 1-17, wherein the antibody or antigen binding fragment is a neutralizing antibody or antigen binding fragment.

[0082] Aspect 19 is the antibody or antigen binding fragment of any one of aspects 1-17, wherein the antibody or antigen binding fragment is not a neutralizing antibody or antigen binding fragment.

[0083] Aspect 20 is the antibody or antigen binding fragment of any one of aspects 1-19, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

[0084] Aspect 21 is the antigen binding fragment of any one of aspects 1-19, wherein the antigen binding fragment is a single chain variable fragment (scFv), F(ab’)2, Fab’, fragment antigen binding protein (Fab), Fv, or rlgG.

[0085] Aspect 22 is the antigen binding fragment of aspect 21 , wherein the antigen binding fragment is a Fab.

[0086] Aspect 23 is a polypeptide comprising the antibody or antigen binding fragment of any one of aspects 1-22.

[0087] Aspect 24 is the polypeptide of aspect 23, wherein the polypeptide comprises at least two antigen binding fragments.

[0088] Aspect 25 is the polypeptide of aspect 23 or 24, wherein the polypeptide is multivalent.

[0089] Aspect 26 is the polypeptide of any one of aspects 23-25, wherein the polypeptide is bispecific.

[0090] Aspect 27 is the polypeptide of any one of aspects 23-26, wherein the antibody or antigen binding fragment are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0091] Aspect 28 is the polypeptide of aspect 27, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0092] Aspect 29 is the polypeptide of aspect 27 or 28, wherein the reporter system comprises a split luciferase reporter system.

[0093] Aspect 30 is the polypeptide of aspect 29, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0094] Aspect 31 is the polypeptide of aspect 29 or 30, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is, is less than, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 10 amino acids, 5 amino acids, or 2 amino acids.

[0095] Aspect 32 is the polypeptide of any one of aspects 29-31, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0096] Aspect 33 is the polypeptide of any one of aspects 29-32, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked at the C-terminus of the antibody or antigen binding fragment.

[0097] Aspect 34 is the polypeptide of any one of aspects 29-33, wherein the polypeptide comprises a CP 16 heavy chain linked to SmBiT or LgBiT.

[0098] Aspect 35 is the polypeptide of aspect 34, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0099] Aspect 36 is the polypeptide of aspect 34, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0100] Aspect 37 is the polypeptide of any one of aspects 34-36, wherein the CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0101] Aspect 38 is the polypeptide of any one of aspects 34-37, wherein the CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0102] Aspect 39 is the polypeptide of any one of aspects 34-37, wherein the CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0103] Aspect 40 is the polypeptide of any one of aspects 29-33, wherein the polypeptide comprises a CP 16 light chain linked to SmBiT or LgBiT.

[0104] Aspect 41 is the polypeptide of aspect 40, wherein the CP 16 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0105] Aspect 42 is the polypeptide of any one of aspects 29-33, wherein the polypeptide comprises a CP 17 heavy chain linked to SmBiT or LgBiT.

[0106] Aspect 43 is the polypeptide of aspect 42, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0107] Aspect 44 is the polypeptide of aspect 42, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0108] Aspect 45 is the polypeptide of any one of aspects 42-45, wherein the CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0109] Aspect 46 is the polypeptide of any one of aspects 42-45, wherein the CP17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0110] Aspect 47 is the polypeptide of any one of aspects 29-33, wherein the polypeptide comprises a CP 17 light chain linked to SmBiT or LgBiT.

[0111] Aspect 48 is the polypeptide of aspect 47, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0112] Aspect 49 is the polypeptide of aspect 47 or 48, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0113] Aspect 50 is the polypeptide of any one of aspects 47-49, wherein the CP 17 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0114] Aspect 51 is a composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies, antigen binding fragments, and / or polypeptides according to any one of aspects 1-50.

[0115] Aspect 52 is a nucleic acid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibody, antigen binding fragment, and / or polypeptide of any one of aspects 1-50.

[0116] Aspect 53 is a vector comprising the nucleic acid of aspect 52.

[0117] Aspect 54 is a host cell comprising the nucleic acid or vector of aspects 52 or 53.

[0118] Aspect 55 is the host cell of aspect 54, wherein the host cell is a prokaryote or a eukaryote.

[0119] Aspect 56 is the host cell of aspect 54 or 55, wherein the host cell is a bacterial cell, fungal cell, mammalian cell, or insect cell.

[0120] Aspect 57 is the host cell of any one of aspect 54-56, wherein the host cell is E. coli.

[0121] Aspect 58 is a method of a making a cell comprising transferring the nucleic acid(s) of aspect 52 or the vector of aspect 53 into a cell.

[0122] Aspect 59 is the method of aspect 58, wherein the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.

[0123] Aspect 60 is the method of aspect 59, wherein the method further comprising isolating the expressed polypeptide.

[0124] Aspect 61 is a composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / orCP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0125] Aspect 62 is the composition of aspect 61 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0126] Aspect 63 is the composition of aspect 61 or 62, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope, or to different epitopes.

[0127] Aspect 64 is the composition of any one of aspects 61-63, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium dependent manner.

[0128] Aspect 65 is the composition of any one of aspects 61-63, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium independent manner.

[0129] Aspect 66 is the composition of any one of aspects 61-65, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0130] Aspect 67 is the composition of any one of aspects 61-66, wherein the reporter system comprises a split luciferase reporter system.

[0131] Aspect 68 is the composition of aspect 67, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0132] Aspect 69 is the composition of aspect 67 or 68, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is, is less than, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 5 amino acids, or 2 amino acids.

[0133] Aspect 70 is the composition of any one of aspects 67-69, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ IDNO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0134] Aspect 71 is the composition of any one of aspects 67-70, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides is linked at the C-terminus to at least one fragment of the split luciferase.

[0135] Aspect 72 is the composition of any one of aspects 67-71, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs.

[0136] Aspect 73 is the composition of any one of aspects 67-72, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16-LgBiT).

[0137] Aspect 74 is the polypeptide of aspect 73, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0138] Aspect 75 is the composition of aspect 73 or 74, wherein at least one CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0139] Aspect 76 is the composition of any one of aspects 73-75, wherein at least one CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0140] Aspect 77 is the composition of any one of aspects 73-76, wherein at least one CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0141] Aspect 78 is the composition of any one of aspects 73-77, wherein at least one CP16 light chain is linked to SmBiT or LgBiT.

[0142] Aspect 79 is the composition of any one of aspects 67-78, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17-LgBiT).

[0143] Aspect 80 is the composition of aspect 79, wherein at least one CP 17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK- GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0144] Aspect 81 is the composition of aspect 78 or 79, wherein at least one CP 17 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0145] Aspect 82 is the composition of aspect 78 or 79, wherein at least one CP 17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0146] Aspect 83 is the composition of any one of aspects 67-82, wherein at least one CP 17 light chain is linked to SmBiT or LgBiT.

[0147] Aspect 84 is the composition of any one of aspects 67-83, wherein the concentration of CP16-SmBiT and / or CP17-SmBiT is, is greater than, or at most about 2-100 nM.

[0148] Aspect 85 is the composition of any one of aspects 67-84, wherein the concentration of CP16-SmBiT and / or CP17-SmBiT is, is greater than, or is at most about 15-60 nM.

[0149] Aspect 86 is the composition of any one of aspects 67-85, wherein the concentration of CP16-SmBiT is, is at least, or is at most about 50 nM.

[0150] Aspect 87 is the composition of any one of aspects 67-86, wherein the concentration of CP17-SmBiT is, is at least, or is at most about 25 nM.

[0151] Aspect 88 is the composition of any one of aspects 67-87, wherein the concentration of CP16-LgBiT and / or CP17-LgBiT is, is at least, or is at most about 2-100 nM.

[0152] Aspect 89 is the composition of any one of aspects 67-88, wherein the concentration of CP16-LgBiT and / or CP17-LgBiT is, is at least, or is at most about 5-25 nM.

[0153] Aspect 90 is the composition of any one of aspects 67-89, wherein the concentration of CP16-LgBiT is, is at least, or is at most about 15 nM.

[0154] Aspect 91 is the composition of any one of aspects 67-90, wherein the concentration of CP17-LgBiT is, is at least, or is at most about 15 nM.

[0155] Aspect 92 is the composition of any one of aspects 61-91 comprising at least one of CP16-LgBiT and / or CP17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

[0156] Aspect 93 is the composition of any one of aspects 61-92 comprising a) CP16- LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16- SmBiT; or d) CP17-LgBiT and CP17-SmBiT.

[0157] Aspect 94 is the composition of aspect 92 or 93, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

[0158] Aspect 95 is the composition of aspect 93 or 94, comprising a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT.

[0159] Aspect 96 is the composition of any one of aspects 93-96, wherein the ratio of CP16- LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 : 1.1, 1 : 1.2, 1 :1.3, 1 :1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3: 10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

[0160] Aspect 97 is the composition of any one of aspects 92-96, comprising about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT.

[0161] Aspect 98 is the composition of aspect 97, comprising about 15 nM CP16-LgBiT and about 50 nM CP16-SmBiT.

[0162] Aspect 99 is the composition of any one of aspects 92-96, comprising about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT.

[0163] Aspect 100 is the composition of aspect 99, comprising about 15 nM CP16-LgBiT and about 25 nM CP16-SmBiT.

[0164] Aspect 101 is the composition of any one of aspects 61-100, further comprising Ca2+.

[0165] Aspect 102 is the composition of aspect 101, comprising at least 2 mM Ca2+.

[0166] Aspect 103 is the composition of any one of aspects 61-102, further comprising furimazine.

[0167] Aspect 104 is the composition of any one of aspects 61-103, further comprising TBST, BSA, and CaC12.

[0168] Aspect 105 is the composition of aspect 104, comprising TBST pH 7.4, 0.2% BSA, and 2 mM CaC12.

[0169] Aspect 106 is a method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subject, or extract thereof, with at least one antibody, antigen binding fragment, polypeptide, or composition of any one of aspects 1-105.

[0170] Aspect 107 is the method of aspect 106, wherein the method further comprises incubating the antibody, antigen binding fragment, polypeptide, or composition with the biological sample, or extract thereof, under conditions that allow for the binding of the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition to bind to antigens in the biological sample or extract thereof.

[0171] Aspect 108 is the method of any one of aspects 106-107, wherein the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition.

[0172] Aspect 109 is the method of aspect 108, wherein the detecting comprises measuring of a signal produced by a reporter system.

[0173] Aspect 110 is a method of measuring the concentration of calprotectin complex in a biological sample, the method comprising contacting the biological sample with the antibody, antigen binding fragment, or polypeptide of any one of aspects 1-51, or the composition of anyone of aspects 61-105, and measuring the level of interaction between the antibody, antigen binding fragment, Fab, or polypeptide with the calprotectin complex.

[0174] Aspect 111 is the method of aspect 110, wherein the at least one antibody, antigen binding fragment, or polypeptide is operatively linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system, or the composition comprises an antibody, antigen binding fragment, polypeptide, or Fab that is operatively linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0175] Aspect 112 is the method of any one of aspects 110-111, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system can produce a detectable level of a chemiluminescence signal when the reporter system is reconstituted.

[0176] Aspect 113 is the method of any one of aspects 110-112, wherein the measuring comprises measuring a level of luminescence.

[0177] Aspect 114 is the method of any one of aspects 106-113, wherein the antibody, antigen binding fragment, polypeptide, or Fab of the composition is in solution or is linked to a solid support.

[0178] Aspect 115 is the method of aspect 114, wherein the solution comprises furimazine, TBST, BSA, and / or CaC12.

[0179] Aspect 116 is the method of aspect 114, wherein the solid support comprises a lateral flow assay strip, or a paper.

[0180] Aspect 117 is the method of any one of aspects 106-116, wherein the biological sample, or an extract thereof, comprises serum, plasma, whole blood, or stool.

[0181] Aspect 118 is a method for detecting calprotectin in a sample, the method comprising:

[0182] i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system;

[0183] ii) incubating the sample and polypeptides for a period of time; and

[0184] iii) measuring levels of a signal produced by the reporter system during and / or after the incubating.

[0185] Aspect 119 is the method of aspect 118, wherein the sample comprises, or comprises an extract of, serum, plasma, whole blood, or stool.

[0186] Aspect 120 is the method of any one of aspects 118-119, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8) and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); and wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0187] Aspect 121 is the method of any one of aspects 118-120, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope.

[0188] Aspect 122 is the method of any one of aspects 118-120, comprising more than one polypeptide, and wherein the polypeptides bind to different epitopes.

[0189] Aspect 123 is the method of any one of aspects 118-122, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium-dependent manner.

[0190] Aspect 124 is the method of any one of aspects 118-122, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium-independent manner.

[0191] Aspect 125 is the method of aspect 119 or 120, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0192] Aspect 126 is the method of any one of aspects 119-125, wherein the reporter system comprises a split luciferase reporter system.

[0193] Aspect 127 is the method of aspect 126, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0194] Aspect 128 is the method of aspect 126 or 127, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is, is at least, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 5 amino acids, or 2 amino acids.

[0195] Aspect 129 is the method of any one of aspects 126-128, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs and at least one fragment of the split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0196] Aspect 130 is the method of any one of aspects 126-129, wherein the 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked at the C-terminus of the heavy chain or light chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs.

[0197] Aspect 131 is the method of any one of aspects 126-130, comprising 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more CP 16 Fabs, wherein at least one CP 16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16-LgBiT).

[0198] Aspect 132 is the polypeptide of aspect 131, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0199] Aspect 133 is the method of aspect 131 or 132, wherein at least one CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0200] Aspect 134 is the method of any one of aspects 131-133, wherein at least one CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0201] Aspect 135 is the method of any one of aspects 131-134, wherein at least one CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0202] Aspect 136 is the method of any one of aspects 131-135, wherein at least one CP16 light chain is linked to SmBiT or LgBiT.

[0203] Aspect 137 is the method of aspect 136, wherein at least one CP16 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK- GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0204] Aspect 138 is the method of any one of aspects 136-137, wherein at least one CP16 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0205] Aspect 139 is the method of any one of aspects 136-138, wherein at least one CP16 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0206] Aspect 140 is the method of any one of aspects 126-139, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP 17 Fabs, wherein at least one CP 17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17-LgBiT).

[0207] Aspect 141 is the method of aspect 140, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0208] Aspect 142 is the method of aspect 140 or 141, wherein at least one CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0209] Aspect 143 is the method of any one of aspects 136-142, wherein at least one CP17 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0210] Aspect 144 is the method of any one of aspects 136-143, wherein at least one CP17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0211] Aspect 145 is the method of any one of aspects 126-144, wherein at least one CP17 light chain is linked to SmBiT or LgBiT.

[0212] Aspect 146 is the polypeptide of aspect 145, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0213] Aspect 147 is the method of aspect 145 or 146, wherein at least one CP17 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0214] Aspect 148 is the method of any one of aspects 145-147, wherein at least one CP17 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0215] Aspect 149 is the method of any one of aspects 145-148, wherein at least one CP17 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0216] Aspect 150 is the method of any one of aspects 126-149, comprising incubating the sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 2-100 nM.

[0217] Aspect 151 is the method of any one of aspects 126-146, comprising incubating the sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 15-60 nM.

[0218] Aspect 152 is the method of any one of aspects 126-150, comprising incubating the sample with a concentration of CP16-SmBiT that is about 50 nM.

[0219] Aspect 153 is the method of any one of aspects 126-152, comprising incubating the sample with a concentration of CP17-SmBiT that is about 25 nM.

[0220] Aspect 154 is the method of any one of aspects 126-153, comprising incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 2-100 nM.

[0221] Aspect 155 is the method of any one of aspects 126-154, comprising incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 5-25 nM.

[0222] Aspect 156 is the method of any one of aspects 126-155, comprising incubating the sample with a concentration of CP16-LgBiT that is about 15 nM.

[0223] Aspect 157 is the method of any one of aspects 126-156, comprising incubating the sample with a concentration of CP17-LgBiT that is about 15 nM.

[0224] Aspect 158 is the method of any one of aspects 119-157, comprising at least one of CP16-LgBiT and / or CP17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

[0225] Aspect 159 is the method of any one of aspects 119-158, comprising a) CP16- LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16- SmBiT; or d) CP17-LgBiT and CP17-SmBiT.

[0226] Aspect 160 is the method of aspect 158 or 159, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

[0227] Aspect 161 is the method of aspect 159 or 160, comprising incubating the sample with a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT.

[0228] Aspect 162 is the method of any one of aspects 159-161, wherein the ratio of CP 16- LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 : 1.1, 1 : 1.2, 1 :1.3, 1 :1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3: 10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

[0229] Aspect 163 is the method of any one of aspects 158-162, comprising incubating the sample with, with at least, or with at most about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT.

[0230] Aspect 164 is the method of aspect 163, comprising incubating the sample with about 15 nM CP16-LgBiT and about 50 nM CP16-SmBiT.

[0231] Aspect 165 is the method of any one of aspects 158-162, comprising incubating the sample with, with at least, or with at most about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT.

[0232] Aspect 166 is the method of aspect 165, comprising incubating the sample with about 15 nM CP16-LgBiT and about 25 nM CP17-SmBiT.

[0233] Aspect 167 is the method of any one of aspects 119-166, further comprising contacting and incubating the sample with Ca2+.

[0234] Aspect 168 is the method of aspect 167, wherein the incubating with Ca2+ is at a concentration of at least 2 mM.

[0235] Aspect 169 is the method of any one of aspects 119-168, further comprising contacting and incubating the sample with furimazine.

[0236] Aspect 170 is the method of any one of aspects 119-169, further comprising contacting and incubating the sample with TBST, BSA, and / or CaC12.

[0237] Aspect 171 is the method of aspect 170, wherein the incubating with TBST is at about pH 7.4, BSA is at about 0.2%, and CaC12 is at about 2 mM.

[0238] Aspect 172 is the method of any one of aspects 118-171, wherein the incubating is for, for at least, or for at most about 2 to 30 minutes.

[0239] Aspect 173 is the method of any one of aspects 118-172, wherein the incubating is for about 15 to 30 minutes.

[0240] Aspect 174 is the method of any one of aspects 118-173, wherein the measuring comprises quantitatively measuring luminescence.

[0241] Aspect 175 is the method of any one of aspects 118-174, wherein the measuring of signal is quantitative, and the signal correlates linearly with increasing calprotectin concentrations.

[0242] Aspect 176 is the method of any one of aspects 118-175, wherein the method comprises detecting the luminescence signal in a liquid solution.

[0243] Aspect 177 is the method of any one of aspects 118-176, wherein the method comprises detecting the luminescence signal on a solid support.

[0244] Aspect 178 is the method of aspects 174-177, wherein the luminescence measuring is mediated through imaging.

[0245] Aspect 179 is the method of aspect 178, wherein the imaging is by a consumer grade camera.

[0246] Aspect 180 is the method of any one of aspects 118-179, wherein the method is performed in a point-of-care and / or at-home setting.

[0247] Aspect 181 is the method of any one of aspects 118-180, wherein the levels of calprotectin measured are indicative of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diseases or disorders.

[0248] Aspect 182 is the method of any one of aspects 118-181, wherein the levels of calprotectin in a sample are quantified and compared to a negative or positive control.

[0249] Aspect 183 is the method of aspect 182, wherein the levels of calprotectin in a sample are noted as abnormal when the detected signal is or is at least about 2-200 fold higher than the negative control.

[0250] Aspect 184 is a method of treating a disease or disorder in a subject, comprising performing the method of any one of aspects 118-183, and tailoring treatment based upon levels of calprotectin detected.

[0251] Aspect 185 is the method of aspect 184, wherein the disease or disorder is characterized by inflammation.

[0252] Aspect 186 is the method of aspect 184 or 185, wherein the disease or disorder comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, cancer, rheumatoid arthritis, tuberculosis (TB), and / or COVID-19 infection.

[0253] Aspect 187 is the method of aspect 186, wherein the disease or disorder comprises IBD, and measurements of calprotectin levels in serum, plasma, whole blood, and / or stool, or extracts thereof, indicate disease severity and / or disease progression / remission.

[0254] Aspect 188 is an engineered stabilized calprotectin complex comprising S100A9 linked to S100A8 to form a heterodimeric fusion protein.

[0255] Aspect 189 is the engineered stabilized calprotectin complex of aspect 188, wherein the S100A9 and S100A8 are linked by a GGS peptide linker.

[0256] Aspect 190 is the engineered stabilized calprotectin complex of aspect 188 or 189, wherein the complex is further linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tags.

[0257] Aspect 191 is the engineered stabilized calprotectin complex of aspect 190, wherein the complex is linked to a SNAP-TAG protein for selective biotinylation.

[0258] Aspect 192 is a polypeptide comprising an antibody or antigen binding fragment linked with a fragment of a split luciferase.

[0259] Aspect 193 is the polypeptide of aspect 192, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0260] Aspect 194 is the polypeptide of aspect 192 or 193, wherein the split luciferase comprises a NANOBIT system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NANOLUC luciferase.

[0261] Aspect 195 is the polypeptide of any one of aspects 192-194, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is, is at least, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 5 amino acids, or 2 amino acids.

[0262] Aspect 196 is the polypeptide of any one of aspects 192-195, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ IDNO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0263] Aspect 197 is the polypeptide of any one of aspects 192-196, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes.

[0264] Aspect 198 is the polypeptide of any one of aspects 192-197, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium-dependent manner.

[0265] Aspect 199 is the polypeptide of any one of aspects 192-197, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium-independent manner.

[0266] Aspect 200 is the polypeptide of any one of aspects 192-199, wherein the antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex.

[0267] Aspect 201 is the polypeptide of any one of aspects 192-200, wherein the antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex.

[0268] Aspect 202 is the polypeptide of any one of aspects 192-201, wherein the antibody or antigen binding fragment comprises an amino acid sequence at least 80% identical to the heavy chain and / or light chain of CP16 and / or CP17 (SEQ ID NOs: 1, 2, 7, and 8).

[0269] Aspect 203 is the polypeptide of any one of aspects 192-202, wherein the antibody or antigen binding fragment comprises the heavy chain and / or light chain of CP 16 and / or CP 17 (SEQ ID NOs: 1, 2, 7, and 8).

[0270] Aspect 204 is the polypeptide of any one of aspects 192-203, wherein the antigen binding fragment comprises a Fab.

[0271] Aspect 205 is a method for identifying a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the disease state based upon the level of calprotectin detected.

[0272] Aspect 206 is the method of aspect 205, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0273] Aspect 207 is a method for measuring the efficacy of a regimen for treating a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the treatment regimen as effective or not effective for treatment of the disease state based upon the level of calprotectin detected.

[0274] Aspect 208 is the method of aspect 207, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0275] Aspect 209 is a method for classifying the severity of a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and classifying the severity of a disease state based upon the level of calprotectin detected.

[0276] Aspect 210 is the method of aspect 209, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0277] Aspect 211 is a kit comprising the antibody, antigen binding fragment, polypeptide, composition, means for performing the method, and / or engineered stabilized calprotectin complex according to any one of the preceding aspects.

[0278] Aspect 212 is the use of the antibody, composition, method, engineered stabilized calprotectin complex, polypeptide, and / or kit of any one of the preceding aspects for medicinal and / or biomedical research purposes.

[0279] Aspect 213 is an Fab comprising the heavy chain and light chain of CP 16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

[0280] Aspect 214 is an Fab comprising the heavy chain and light chain of CP 16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0281] Aspect 215 is an Fab comprising the heavy chain and light chain of CP17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

[0282] Aspect 216 is an Fab comprising the heavy chain and light chain of CP 17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0283] Aspect 217 is a composition comprising a first Fab and a second Fab, wherein the first Fab comprises the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT by a linker peptide comprising SEQ ID NO: 13; and the second Fab comprises the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0284] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0285] Aspect 1A is an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 having at least 80% sequence identity to the LCDR1 from a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0286] Aspect 2A is the antibody or antigen binding fragment of aspect 1A, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acidsequence of a HCDR1, HCDR2, and HCDR3 of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence of the LCDR1 from the light chain variable region of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0287] Aspect 3 A is the antibody or antigen binding fragment of aspect 1 A or 2A, wherein the HCDR1, HCDR2, HCDR3, and LCDR1 each comprise an amino acid sequence that has at least 80% sequence identity to an HCDR1, HCDR2, HCDR3, and LCDR1 of CP 16 or CP 17 heavy chain and light chain (SEQ ID NOs: 1, 2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1 are from the same antigen binding fragment clone.

[0288] Aspect 4A is the antibody or antigen binding fragment of aspect 1 A or 2A, wherein the HCDR1, HCDR2, HCDR3, and LCDR1 each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR3, and LCDR1 of CP16 or CP17 heavy chain and light chain (SEQ ID NOs: 1, 2, 7, and 8), and wherein the HCDR1, HCDR2, HCDR3, and LCDR1 are from the same antigen binding fragment clone.

[0289] Aspect 5A is the antibody or antigen binding fragment of any one of aspects 1A- 4A, wherein the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0290] Aspect 6A is the antibody or antigen binding fragment of aspect 5A, wherein the heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of CP16 or CP17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises the amino acid sequence of a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

[0291] Aspect 7A is the antibody or antigen binding fragment of any one of aspects 1A- 6A, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence with at least 70% sequence identity to CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and the light chain comprises an amino acid sequence with at least 70% sequence identity to CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0292] Aspect 8A is the antibody or antigen binding fragment of aspect 7A, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP16 or CP17 heavy chain (SEQ IDNOs: 2 and 8), and the light chain comprises the amino acid sequence of CP16 or CP17 light chain (SEQ ID NOs: 1 and 7).

[0293] Aspect 9A is the antibody or antigen binding fragment of aspect 8A, wherein the antibody or antigen binding fragment comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP16 heavy chain (SEQ ID NO: 2), and the light chain comprises the amino acid sequence of CP16 light chain (SEQ ID NO: 1).

[0294] Aspect 10A is the antibody or antigen binding fragment of aspect 8 A, wherein the antibody comprises a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of CP17 heavy chain (SEQ ID NO: 8), and the light chain comprises the amino acid sequence of CP17 light chain (SEQ ID NO: 7).

[0295] Aspect 11 A is the antibody or antigen binding fragment of any one of aspects 1 A- 10A, wherein the antibody or antigen binding fragment is human, chimeric, or humanized.

[0296] Aspect 12A is the antibody or antigen-binding fragment of any one of aspects 1 A- 11 A, wherein the antibody or antigen binding fragment binds to a calprotectin complex with a KD of about 10A-6 M / L to about 10A- 12 M / L.

[0297] Aspect 13A is the antibody or antigen-binding fragment of any one of aspects 1A- 12 A, wherein the antibody or antigen binding fragment binds to a calprotectin complex with an EC50 of less than about 1 nM to about 100 nM.

[0298] Aspect 14A is the antibody or antigen-binding fragment of any one of aspects 1 A- 13 A, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with a KD of about 10A-6 M / L to about 10A-12 M / L.

[0299] Aspect 15A is the antibody or antigen-binding fragment of any one of aspects 1A- 13 A, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex with a KD of about 10A-6 M / L to about 10A- 12 M / L.

[0300] Aspect 16A is the antibody or antigen-binding fragment of any one of aspects 1A- 14 A, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with an EC50 of less than about 1 nM to about 100 nM.

[0301] Aspect 17A is the antibody or antigen-binding fragment of any one of aspects 1A- 14 A, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex with an EC50 of less than about 1 nM to about 100 nM.

[0302] Aspect 18A is the antibody or antigen binding fragment of any one of aspects 1A- 17A, wherein the antibody or antigen binding fragment is a neutralizing antibody or antigen binding fragment.

[0303] Aspect 19A is the antibody or antigen binding fragment of any one of aspects 1A- 17A, wherein the antibody or antigen binding fragment is not a neutralizing antibody or antigen binding fragment.

[0304] Aspect 20A is the antibody or antigen binding fragment of any one of aspects 1 A- 19A, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

[0305] Aspect 21 A is the antigen binding fragment of any one of aspects 1 A-19A, wherein the antigen binding fragment is a single chain variable fragment (scFv), F(ab’)2, Fab’, fragment antigen binding protein (Fab), Fv, or rlgG.

[0306] Aspect 22 A is the antigen binding fragment of aspect 21 A, wherein the antigen binding fragment is a Fab.

[0307] Aspect 23A is a polypeptide comprising the antibody or antigen binding fragment of any one of aspects 1A-22A.

[0308] Aspect 24A is the polypeptide of aspect 23 A, wherein the polypeptide comprises at least two antigen binding fragments.

[0309] Aspect 25A is the polypeptide of aspect 23A or 24A, wherein the polypeptide is multivalent.

[0310] Aspect 26A is the polypeptide of any one of aspects 23A-25A, wherein the polypeptide is bispecific.

[0311] Aspect 27A is the polypeptide of any one of aspects 23 A-26A, wherein the antibody or antigen binding fragment are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0312] Aspect 28A is the polypeptide of aspect 27A, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0313] Aspect 29A is the polypeptide of aspect 27A or 28A, wherein the reporter system comprises a split luciferase reporter system.

[0314] Aspect 30A is the polypeptide of aspect 29A, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0315] Aspect 31A is the polypeptide of aspect 29A or 30A, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is, is less than, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 10 amino acids, 5 amino acids, or 2 amino acids.

[0316] Aspect 32A is the polypeptide of any one of aspects 29A-31 A, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0317] Aspect 33 A is the polypeptide of any one of aspects 29A-32A, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked at the C-terminus of the antibody or antigen binding fragment.

[0318] Aspect 34A is the polypeptide of any one of aspects 29A-33A, wherein the polypeptide comprises a CP 16 heavy chain linked to SmBiT or LgBiT.

[0319] Aspect 35 A is the polypeptide of aspect 34A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0320] Aspect 36A is the polypeptide of aspect 34A, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0321] Aspect 37A is the polypeptide of any one of aspects 34A-36A, wherein the CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0322] Aspect 38A is the polypeptide of any one of aspects 34A-37A, wherein the CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0323] Aspect 39A is the polypeptide of any one of aspects 34A-37A, wherein the CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0324] Aspect 40A is the polypeptide of any one of aspects 29A-33A, wherein the polypeptide comprises a CP 16 light chain linked to SmBiT or LgBiT.

[0325] Aspect 41 A is the polypeptide of aspect 40A, wherein the CP16 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ IDNO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0326] Aspect 42A is the polypeptide of any one of aspects 29A-33A, wherein the polypeptide comprises a CP 17 heavy chain linked to SmBiT or LgBiT.

[0327] Aspect 43 A is the polypeptide of aspect 42A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0328] Aspect 44A is the polypeptide of aspect 42A, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0329] Aspect 45 A is the polypeptide of any one of aspects 42A-45A, wherein the CP 17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0330] Aspect 46A is the polypeptide of any one of aspects 42A-45A, wherein the CP 17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0331] Aspect 47A is the polypeptide of any one of aspects 29A-33A, wherein the polypeptide comprises a CP 17 light chain linked to SmBiT or LgBiT.

[0332] Aspect 48A is the polypeptide of aspect 47A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0333] Aspect 49A is the polypeptide of aspect 47A or 48A, wherein the linker comprises, consists essentially of, or consists of a peptide linker that is 2, 5, 10, 12, 15, 20, or 25 amino acids in length.

[0334] Aspect 50A is the polypeptide of any one of aspects 47A-49A, wherein the CP 17 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0335] Aspect 51 A is a composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies, antigen binding fragments, and / or polypeptides according to any one of claims 1- 50.

[0336] Aspect 52A is a nucleic acid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibody, antigen binding fragment, and / or polypeptide of any one of claims 1-50.

[0337] Aspect 53 A is a vector comprising the nucleic acid of claim 52.

[0338] Aspect 54A is a host cell comprising the nucleic acid or vector of claim 52 or 53.

[0339] Aspect 55A is the host cell of claim 54, wherein the host cell is a prokaryote or a eukaryote.

[0340] Aspect 56A is the host cell of claim 54 or 55, wherein the host cell is a bacterial cell, fungal cell, mammalian cell, or insect cell.

[0341] Aspect 57A is the host cell of any one of aspects 54A-56A, wherein the host cell is E. coli.

[0342] Aspect 58A is a method of making a cell comprising transferring the nucleic acid(s) of claim 52 or the vector of claim 53 into a cell.

[0343] Aspect 59 A is the method of aspect 58 A, wherein the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.

[0344] Aspect 60 A is the method of aspect 59 A, wherein the method further comprises isolating the expressed polypeptide.

[0345] Aspect 61A is a composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0346] Aspect 62A is the composition of aspect 61 A comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0347] Aspect 63A is the composition of aspect 61A or 62A, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope, or to different epitopes.

[0348] Aspect 64A is the composition of any one of aspects 61A-63A, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium dependent manner.

[0349] Aspect 65 A is the composition of any one of aspects 61A-63A, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium independent manner.

[0350] Aspect 66A is the composition of any one of aspects 61 A-65A, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0351] Aspect 67A is the composition of any one of aspects 61 A-66A, wherein the reporter system comprises a split luciferase reporter system.

[0352] Aspect 68A is the composition of aspect 67A, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0353] Aspect 69A is the composition of aspect 67A or 68A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is, is less than, or is at most 25 amino acids, 20 amino acids, 15 amino acids, 12 amino acids, 5 amino acids, or 2 amino acids.

[0354] Aspect 70A is the composition of any one of aspects 67A-69A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS- DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK- GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13).

[0355] Aspect 71 A is the composition of any one of aspects 67A-70A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides are linked at the C-terminus to at least one fragment of the split luciferase.

[0356] Aspect 72A is the composition of any one of aspects 67A-71 A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs.

[0357] Aspect 73A is the composition of any one of aspects 67A-72A, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16-LgBiT).

[0358] Aspect 74A is the polypeptide of aspect 73 A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0359] Aspect 75A is the composition of aspect 73A or 74A, wherein at least one CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ IDNO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0360] Aspect 76A is the composition of any one of aspects 73 A-75A, wherein at least one CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0361] Aspect 77A is the composition of any one of aspects 73 A-76A, wherein at least one CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0362] Aspect 78A is the composition of any one of aspects 73 A-77A, wherein at least one CP 16 light chain is linked to SmBiT or LgBiT.

[0363] Aspect 79A is the composition of any one of aspects 67A-78A, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17-LgBiT).

[0364] Aspect 80A is the composition of aspect 79A, wherein at least one CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0365] Aspect 81A is the composition of aspect 78A or 79A, wherein at least one CP17 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0366] Aspect 82A is the composition of aspect 78 A or 79A, wherein at least one CP 17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0367] Aspect 83 A is the composition of any one of aspects 67A-82A, wherein at least one CP 17 light chain is linked to SmBiT or LgBiT.

[0368] Aspect 84A is the composition of any one of aspects 67A-83A, wherein the concentration of CP16-SmBiT and / or CP17-SmBiT is, is greater than, or at most about 2-100 nM.

[0369] Aspect 85A is the composition of any one of aspects 67A-84A, wherein the concentration of CP16-SmBiT and / or CP17-SmBiT is, is greater than, or is at most about 15- 60 nM.

[0370] Aspect 86A is the composition of any one of aspects 67A-85A, wherein the concentration of CP16-SmBiT is, is at least, or is at most about 50 nM.

[0371] Aspect 87A is the composition of any one of aspects 67A-86A, wherein the concentration of CP17-SmBiT is, is at least, or is at most about 25 nM.

[0372] Aspect 88A is the composition of any one of aspects 67A-87A, wherein the concentration of CP16-LgBiT and / or CP17-LgBiT is, is at least, or is at most about 2-100 nM.

[0373] Aspect 89A is the composition of any one of aspects 67A-88A, wherein the concentration of CP16-LgBiT and / or CP17-LgBiT is, is at least, or is at most about 5-25 nM.

[0374] Aspect 90A is the composition of any one of aspects 67A-89A, wherein the concentration of CP16-LgBiT is, is at least, or is at most about 15 nM.

[0375] Aspect 91A is the composition of any one of aspects 67A-90A, wherein the concentration of CP17-LgBiT is, is at least, or is at most about 15 nM.

[0376] Aspect 92A is the composition of any one of aspects 61A-91A comprising at least one of CP16-LgBiT and / or CP17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

[0377] Aspect 93A is the composition of any one of aspects 61 A-92A comprising a) CP16- LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16- SmBiT; or d) CP17-LgBiT and CP17-SmBiT.

[0378] Aspect 94A is the composition of aspect 92A or 93 A, wherein the ratio of CP16- LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

[0379] Aspect 95A is the composition of aspect 93A or 94A, comprising a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17- SmBiT.

[0380] Aspect 96A is the composition of any one of aspects 93 A-96A, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3: 10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

[0381] Aspect 97A is the composition of any one of aspects 92A-96A, comprising about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT.

[0382] Aspect 98A is the composition of aspect 97 A, comprising about 15 nM CP16- LgBiT and about 50 nM CP16-SmBiT.

[0383] Aspect 99A is the composition of any one of aspects 92A-96A, comprising about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT.

[0384] Aspect 100 A is the composition of aspect 99 A, comprising about 15 nM CP 16- LgBiT and about 25 nM CP16-SmBiT.

[0385] Aspect 101A is the composition of any one of aspects 61A-100A, further comprising Ca2+.

[0386] Aspect 102A is the composition of aspect 101 A, comprising at least 2 mM Ca2+.

[0387] Aspect 103A is the composition of any one of aspects 61A-102A, further comprising furimazine.

[0388] Aspect 104A is the composition of any one of aspects 61A-103A, further comprising TBST, BSA, and CaC12.

[0389] Aspect 105A is the composition of aspect 104A, comprising TBST pH 7.4, 0.2% BSA, and 2 mM CaC12.

[0390] Aspect 106A is an engineered stabilized calprotectin complex comprising S100A9 linked to S100A8 to form a heterodimeric fusion protein.

[0391] Aspect 107A is the engineered stabilized calprotectin complex of aspect 106A, wherein the S100A9 and S100A8 are linked by a GGS peptide linker.

[0392] Aspect 108 A is the engineered stabilized calprotectin complex of aspect 106A or 107A, wherein the complex is further linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tags.

[0393] Aspect 109A is the engineered stabilized calprotectin complex of aspect 108 A, wherein the complex is linked to a SNAP-TAG protein for selective biotinylation.

[0394] Aspect 110A is a polypeptide comprising an antibody or antigen binding fragment that binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes linked with a fragment of a split luciferase wherein the split luciferase comprises a NANOBIT system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NANOLUC luciferase.

[0395] Aspect 111A is the polypeptide of aspect 110A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0396] Aspect 112A is the polypeptide of any one of aspects 110A-111A, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is, is at least, or is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0397] Aspect 113A is the polypeptide of any one of aspects 110A-112A, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0398] Aspect 114A is the polypeptide of any one of aspects 110A-113A, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium dependent manner.

[0399] Aspect 115A is the polypeptide of any one of aspects 110A-114A, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium independent manner.

[0400] Aspect 116A is the polypeptide of any one of aspects 110A-115A, wherein the antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex.

[0401] Aspect 117A is the polypeptide of any one of aspects 110A-116A, wherein the antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex.

[0402] Aspect 118A is the polypeptide of any one of aspects 110A-117A, wherein the antibody or antigen binding fragment comprises an amino acid sequence at least 80% identical to the heavy chain and / or light chain of CP16 and / or CP17 (SEQ ID NOs: 1, 2, 7, and 8).

[0403] Aspect 119A is the polypeptide of any one of aspects 110A-118A, wherein the antibody or antigen binding fragment comprises the heavy chain and / or light chain of CP 16 and / or CP 17 (SEQ ID NOs: 1, 2, 7, and 8).

[0404] Aspect 120A is the polypeptide of any one of aspects 110A-119A, wherein the antigen binding fragment comprises a fragment antibody (Fab).

[0405] Aspect 121A is a fragment antibody (Fab) comprising the heavy chain and light chain of CP 16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

[0406] Aspect 122A is a fragment antibody (Fab)comprising the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0407] Aspect 123A is a fragment antibody (Fab)comprising the heavy chain and light chain of CP 17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

[0408] Aspect 124A is a fragment antibody (Fab)comprising the heavy chain and light chain of CP17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0409] Aspect 125A is a composition comprising a first fragment antibody (Fab) and a second Fab, wherein the first Fab comprises the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT by a linker peptide comprising SEQ ID NO: 13; and the second Fab comprises the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter LgBiT by a linker peptide comprising SEQ ID NO: 13.

[0410] Aspect 126A is a kit comprising the antibody, antigen binding fragment, polypeptide, composition, fragment antibody (Fab), and / or engineered stabilized calprotectin complex according to any one of the preceding aspects.

[0411] Aspect 127A is the kit of aspect 126A, wherein the kit further comprises one or more negative or positive control samples.

[0412] Aspect 128A is the kit of aspect 126A or 127A, wherein the kit comprises an ELISA for detecting calprotectin.

[0413] Aspect 129A is the kit of any one of aspects 126A-128A, wherein the calprotectin antibody or binding fragment is operatively linked to a solid support.

[0414] Aspect 130A is the kit of any one of aspects 126A-129A, wherein the kit comprises at least two anti-calprotectin antibodies, at least two anti-calprotectin antibody binding fragments, or one anti-calprotectin antibody and one anti-calprotectin antibody binding fragment.

[0415] Aspect 131 A is the kit of any one of aspects 126A-130A, wherein the one or more anti-calprotectin antibodies or calprotectin antibody binding fragments is linked to a detectable label.

[0416] Aspect 132A is the kit of any one of aspects 126A-131A, further comprising buffers, solvents, and / or containers.

[0417] Aspect 133A is a method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subject, or extract thereof, with at least one antibody, antigen binding fragment, polypeptide, fragment antibody (Fab), or composition of any one of aspects 1 A- 132 A.

[0418] Aspect 134A is the method of aspect 133A, wherein the method further comprises incubating the antibody, antigen binding fragment, polypeptide, or composition with the biological sample, or extract thereof, under conditions that allow for the binding of the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition to bind to antigens in the biological sample or extract thereof.

[0419] Aspect 135A is the method of any one of aspects 133A-134A, wherein the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition.

[0420] Aspect 136A is the method of aspect 135A, wherein the detecting comprises measuring of a signal produced by a reporter system.

[0421] Aspect 137A is a method of measuring the concentration of calprotectin complex in a biological sample, the method comprising contacting the biological sample with theantibody, antigen binding fragment, polypeptide, composition, or kit component of any one of aspects 1 A-132A, and measuring the level of interaction between the antibody, antigen binding fragment, Fab, or polypeptide with the calprotectin complex.

[0422] Aspect 138A is the method of aspect 137A, wherein the at least one antibody, antigen binding fragment, or polypeptide is operatively linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system, or the composition comprises an antibody, antigen binding fragment, polypeptide, or Fab that is operatively linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0423] Aspect 139A is the method of any one of aspects 137A-138A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system can be produce a detectable level of a chemiluminescence signal when the reporter system is reconstituted.

[0424] Aspect 140 A is the method of any one of aspects 137A-139A, wherein the measuring comprises measuring a level of luminescence.

[0425] Aspect 141 A is the method of any one of aspects 133 A-140A, wherein the antibody, antigen binding fragment, polypeptide, or Fab of the composition is in solution or is linked to a solid support.

[0426] Aspect 142A is the method of aspect 141A, wherein the solution comprises furimazine, TBST, BSA, and / or CaC12.

[0427] Aspect 143A is the method of aspect 141A, wherein the solid support comprises a lateral flow assay strip, or a paper.

[0428] Aspect 144A is the method of any one of aspects 133A-143A, wherein the biological sample, or an extract thereof, comprises serum, plasma, whole blood, or stool.

[0429] Aspect 145A is a method for treating a subject with an inflammatory disorder, the method comprising administering a treatment for the inflammatory disorder to a subject that has had the level of calprotectin evaluated in a biological sample from the subject using any of the antibodies, antigen binding fragments, polypeptides, compositions, fragment antibodies (Fab), kit component, and / or engineered stabilized calprotectin complexes of aspects 1 A-132A.

[0430] Aspect 146A is the method of aspect 145A, wherein the biological sample comprises a serum, plasma, blood, or fecal sample.

[0431] Aspect 147A is the method of any one of aspects 145A-147A, wherein the level of calprotectin in the biological sample from the subject has been quantitated.

[0432] Aspect 148A is the method of aspect 147A, wherein the level of calprotectin is normalized.

[0433] Aspect 149A is the method of any one of aspects 145A-148A, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is greater than the level of a calprotectin in a control sample.

[0434] Aspect 150A is the method of any one of aspects 145A-148A, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is less than the level of a calprotectin in a control sample.

[0435] Aspect 151A is the method of any one of aspects 145A-148A, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is not significantly different than the level of calprotectin in a control sample.

[0436] Aspect 152A is the method of any one of aspects 145A-151A, wherein the control comprises the level of calprotectin that is representative of the level of calprotectin in a subject with an inflammatory disorder.

[0437] Aspect 153 A is the method of any one of aspects 145A-151A, wherein the control comprises the level of calprotectin that is representative of the level of calprotectin in a subject without an inflammatory disorder.

[0438] Aspect 154A is the method of any one of aspects 145A-153A, wherein the inflammatory disorder comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID- 19 infection, tuberculosis (TB), and / or cancer.

[0439] Aspect 155 A is the method of aspect 154A, wherein the treatment comprises an anti-inflammatory medication, an immunomodulator, an immunosuppressant, a small molecule, a biologic, an antibiotic, an antidiarrheal, a pain reliever, a vitamin, a dietary support, and / or a surgery.

[0440] Aspect 156A is the method of aspect 154A or 155 A, wherein the treatment comprises isoniazid, rifampin, rifabutin, rifapentine, pyrazinamide, and / or ethambutol.

[0441] Aspect 157A is a method for detecting calprotectin in a sample, the method comprising:

[0442] i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating.

[0443] Aspect 158A is the method of aspect 157A, wherein the sample comprises, or comprises an extract of, serum, plasma, whole blood, or stool.

[0444] Aspect 159A is the method of aspect 157A or 158A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8) and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); and wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

[0445] Aspect 160A is the method of any one of aspects 157A-159A, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope.

[0446] Aspect 161A is the method of any one of aspects 157A-159A, comprising more than one polypeptide, and wherein the polypeptides bind to different epitopes.

[0447] Aspect 162A is the method of any one of aspects 157A-161A, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium dependent manner.

[0448] Aspect 163A is the method of any one of aspects 157A-161A, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium independent manner.

[0449] Aspect 164A is the method of aspect 158A or 159A, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

[0450] Aspect 165A is the method of any one of aspects 158A-164A, wherein the reporter system comprises a split luciferase reporter system.

[0451] Aspect 166A is the method of aspect 165 A, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

[0452] Aspect 167A is the method of aspect 165A or 166A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0453] Aspect 168A is the method of any one of aspects 165A-167A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs and at least one fragment of the split luciferase are linked with a peptide linker comprising, consisting of, or consisting essentially of GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ IDNO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0454] Aspect 169A is the method of any one of aspects 165A-168A, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked at the C-terminus of the heavy chain or light chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs.

[0455] Aspect 170A is the method of any one of aspects 165A-169A, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16-LgBiT).

[0456] Aspect 171A is the polypeptide of aspect 170A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0457] Aspect 172A is the method of aspect 170A or 171A, wherein at least one CP16 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0458] Aspect 173 A is the method of any one of aspects 170A-172A, wherein at least one CP16 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0459] Aspect 174A is the method of any one of aspects 170A-173A, wherein at least one CP16 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0460] Aspect 175A is the method of any one of aspects 170A-174A, wherein at least one CP 16 light chain is linked to SmBiT or LgBiT.

[0461] Aspect 176A is the method of aspect 175A, wherein at least one CP16 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK- GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0462] Aspect 177A is the method of any one of aspects 175A-176A, wherein at least one CP16 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0463] Aspect 178A is the method of any one of aspects 175A-177A, wherein at least one CP16 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0464] Aspect 179A is the method of any one of aspects 165A-178A, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17-LgBiT).

[0465] Aspect 180A is the method of aspect 179A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0466] Aspect 181A is the method of aspect 179A or 180A, wherein at least one CP17 heavy chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0467] Aspect 182A is the method of any one of aspects 175A-181A, wherein at least one CP17 heavy chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0468] Aspect 183A is the method of any one of aspects 175A-182A, wherein at least one CP17 heavy chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0469] Aspect 184A is the method of any one of aspects 165A-183A, wherein at least one CP 17 light chain is linked to SmBiT or LgBiT.

[0470] Aspect 185 A is the polypeptide of aspect 184A, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

[0471] Aspect 186A is the method of aspect 184A or 185A, wherein at least one CP17 light chain is linked to SmBiT or LgBiT with linker peptide GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0472] Aspect 187A is the method of any one of aspects 184A-186A, wherein at least one CP17 light chain is linked to SmBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0473] Aspect 188 A is the method of any one of aspects 184A-187A, wherein at least one CP17 light chain is linked to LgBiT with linker peptide GS-DYKDDDDK-GS (SEQ ID NO: 13).

[0474] Aspect 189A is the method of any one of aspects 165A-188A, comprising incubating the sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 2-100 nM.

[0475] Aspect 190A is the method of any one of aspects 165A-185A, comprising incubating the sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 15-60 nM.

[0476] Aspect 191A is the method of any one of aspects 165A-189A, comprising incubating the sample with a concentration of CP16-SmBiT that is about 50 nM.

[0477] Aspect 192A is the method of any one of aspects 165A-191A, comprising incubating the sample with a concentration of CP17-SmBiT that is about 25 nM.

[0478] Aspect 193 A is the method of any one of aspects 165A-192A, comprising incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 2-100 nM.

[0479] Aspect 194A is the method of any one of aspects 165A-193A, comprising incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 5-25 nM.

[0480] Aspect 195 A is the method of any one of aspects 165A-194A, comprising incubating the sample with a concentration of CP16-LgBiT that is about 15 nM.

[0481] Aspect 196A is the method of any one of aspects 165A-195A, comprising incubating the sample with a concentration of CP17-LgBiT that is about 15 nM.

[0482] Aspect 197A is the method of any one of aspects 158A-196A, comprising at least one of CP16-LgBiT and / or CP17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

[0483] Aspect 198A is the method of any one of aspects 158A-197A, comprising a) CP16- LgBiT and CP16-SmBiT; b) CP16-LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16- SmBiT; or d) CP17-LgBiT and CP17-SmBiT.

[0484] Aspect 199A is the method of aspect 197A or 198 A, wherein the ratio of CP16- LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

[0485] Aspect 200A is the method of aspect 198 A or 199A, comprising incubating the sample with a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16- SmBiT and / or CP17-SmBiT.

[0486] Aspect 201A is the method of any one of aspects 198A-200A, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3: 10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

[0487] Aspect 202A is the method of any one of aspects 197A-201A, comprising incubating the sample with, with at least, or with at most about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT.

[0488] Aspect 203 A is the method of aspect 202A, comprising incubating the sample with about 15 nM CP16-LgBiT and about 50 nM CP16-SmBiT.

[0489] Aspect 204A is the method of any one of aspects 197A-201A, comprising incubating the sample with, with at least, or with at most about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT.

[0490] Aspect 205A is the method of aspect 204A, comprising incubating the sample with about 15 nM CP16-LgBiT and about 25 nM CP16-SmBiT.

[0491] Aspect 206A is the method of any one of aspects 158A-205A, further comprising contacting and incubating the sample with Ca2+.

[0492] Aspect 207A is the method of aspect 206A, wherein the incubating with Ca2+ is at a concentration of at least 2 mM.

[0493] Aspect 208A is the method of any one of aspects 158A-207A, further comprising contacting and incubating the sample with furimazine.

[0494] Aspect 209A is the method of any one of aspects 158A-208A, further comprising contacting and incubating the sample with TBST, BSA, and / or CaC12.

[0495] Aspect 210A is the method of aspect 209A, wherein the incubating with TBST is at about pH 7.4, BSA is at about 0.2%, and CaC12 is at about 2 mM.

[0496] Aspect 211 A is the method of any one of aspects 157A-210A, wherein the incubating is for, for at least, or for at most about 2 to 30 minutes.

[0497] Aspect 212A is the method of any one of aspects 157A-211A, wherein the incubating is for about 15 to 30 minutes.

[0498] Aspect 213 A is the method of any one of aspects 157A-212A, wherein the measuring comprises quantitatively measuring luminescence.

[0499] Aspect 214A is the method of any one of aspects 157A-213A, wherein the measuring of signal is quantitative, and the signal correlates linearly with increasing calprotectin concentrations.

[0500] Aspect 215A is the method of any one of aspects 157A-214A, wherein the method comprises detecting the luminescence signal in a liquid solution.

[0501] Aspect 216A is the method of any one of aspects 157A-214A, wherein the method comprises detecting the luminescence signal on a solid support.

[0502] Aspect 217A is the method of aspects 213A-216A, wherein the luminescence measuring is mediated through imaging.

[0503] Aspect 218A is the method of aspect 217A, wherein the imaging is by a consumergrade camera.

[0504] Aspect 219A is the method of any one of aspects 157A-218A, wherein the method is performed in a point-of-care and / or at-home setting.

[0505] Aspect 220A is the method of any one of aspects 157A-219A, wherein the levels of calprotectin measured are indicative of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diseases or disorders.

[0506] Aspect 221 A is the method of any one of aspects 157A-220A, wherein the levels of calprotectin in a sample are quantified and compared to a negative or positive control.

[0507] Aspect 222A is the method of aspect 221A, wherein the levels of calprotectin in a sample are noted as abnormal when the detected signal is or is at least about 2-200 fold higher than the negative control.

[0508] Aspect 223 A is a method of treating a disease or disorder in a subject, comprising performing the method of any one of aspects 157A-222A, and tailoring treatment based upon levels of calprotectin detected.

[0509] Aspect 224 A is the method of aspect 223 A, wherein the disease or disorder is characterized by inflammation.

[0510] Aspect 225 A is the method of aspect 223 A or 224A, wherein the disease or disorder comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, cancer, rheumatoid arthritis, tuberculosis (TB), and / or COVID-19 infection.

[0511] Aspect 226A is the method of aspect 225 A, wherein the disease or disorder comprises IBD, and measurements of calprotectin levels in serum, plasma, whole blood, and / or stool, or extracts thereof, indicate disease severity and / or disease progression / remission.

[0512] Aspect 227A is a method for identifying a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and iv) classifying the disease state based upon the level of calprotectin detected.

[0513] Aspect 228A is the method of aspect 227A, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0514] Aspect 229A is a method for measuring the efficacy of a regimen for treating a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and iv) classifying the treatment regimen as effective or not effective for the treatment of the disease state based upon the level of calprotectin detected.

[0515] Aspect 230 A is the method of aspect 229 A, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0516] Aspect 231 A is a method for classifying the severity of a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and iv) classifying the severity of a disease state based upon the level of calprotectin detected.

[0517] Aspect 232A is the method of aspect 231 A, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

[0518] Aspect 233 A is the use of the antibody, composition, method, engineered stabilized calprotectin complex, polypeptide, and / or kit of any one of the preceding aspects for medicinal and / or biomedical research purposes.

[0519] Other objects, features and advantages of the present inventions will become apparent from the following detailed description. It should be understood, however, that thedetailed description and the specific examples, while indicating specific aspects of the inventions provided herein, are given by way of illustration only, since various changes and modifications within the spirit and scope of the inventions will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0520] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The inventions provided herein may be better understood by reference to the drawings with the detailed description of specific aspects presented herein.

[0521] FIGs. 1A-1D, Fabs that bound recombinant human calprotectin (rCAL) were developed. FIG. 1A shows a scheme of phage display approach used to discover Calprotectin binders. The native recombinant calprotectin heterodimer (“rCAL”, shown in orange, left), was engineered into a stabilized heterodimer (stabilized calprotectin, “sCAL”, shown in blue, right), where S100A9 was fused with the N-terminal of SI 00 A8 via linker 2 (4xGGS linker; SEQ ID NO: 22) to ensure proper complex formation. sCAL also contained a SNAP -tag system (“SNAP”, shown in gray) fused to the N-terminus of human S100A9 via linker 1 (LVPRGSPAGGSP; SEQ ID NO: 21) for immobilization via biotinylation. Phage display was then performed for 5 rounds using this synthetic protein system. PDB S100A8 / 9: lxk4, SNAP: 3kzy. FIG. IB displays the amino acid sequences of the CDRs of 2 unique Fabs (CP16 and CP 17) identified from phage display (SEQ ID NOs: 1, 2, 7, and 8 respectively). FIG. 1C shows the EC50 of CP 16 and CP 17 from multipoint ELISA with sCAL. Data represent mean ± std. dev. (n = 3 replicates). FIG. ID shows results of an exemplary Immunoabsorbance luminescence (immunoLUC) assay measuring the binding of sCAL and rCAL (1 ng / pl), which was coated directly on the plate surface, with NANOLUC® (PROMEGA™) fused Fabs. The same concentration of BSA and SNAP protein were also coated in different wells and examined for their binding towards Fab-NLucs. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments). P-values represent unpaired t-test with Welch correction, ns = not significant. In the bar graph of FIG. ID, the bars, from left to right, represent BSA, SNAP, sCAL, and rCAL, for each group (CP16-NLuc and CP17-NLuc).

[0522] FIGs. 2A-2E, Fab-Enabled Split Luciferase Calprotectin Assay (FESCA) allowed accurate and rapid quantification of rCAL. FIG. 2A is a schematic representation of an exemplary FESCA. Two Fabs bound to the same calprotectin complex, bringing together the split parts of a luciferase enzyme (e.g., LgBiT and SmBiT (VTGYRLFEEIL) fragments ofNANOLUC® (PROMEGA™) luciferase), resulting in the reconstitution of active luciferase and luminescent signal. FIG. 2B shows a general workflow of an exemplary FESCA to quantify the rCAL concentration in a sample. FIG. 2C provides an evaluation of different Fab sensor pairs for FESCA. For each reaction, 3.325 nM Fab-SmBiT and 3.325 nM Fab-LgBiT were incubated with 1 ng / pL rCAL, BSA, or SNAP protein using the protocol as outlined in FIG. 2B. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments for BSA and rCAL; n = 2 replicates for SNAP). For each group, the bars, from left to right, represent BSA, rCAL, and SNAP experimental conditions. FIG. 2D shows a dose curve of an exemplary FESCA with varying calprotectin concentrations using CP16-SmBiT / CP16-LgBiT (50 nM / 15 nM) showing both relative luminescence normalized to blank (left y-axis) and raw luminescence (right y-axis) from the plate reader. FIG. 2E shows a dose curve of an exemplary FESCA with varying calprotectin concentrations using with CP17-SmBiT / CP16-LgBiT (25 nM / 15 nM) showing both relative luminescence normalized to blank (left y-axis) and raw luminescence (right y-axis) from the plate reader. For FIG. 2D and FIG. 2E, data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments).

[0523] FIGs. 3A-3G, Solution-based FESCA was suitable for measuring Calprotectin in blood and blood-derived products. FIG. 3A shows a schematic of workflow of FESCA with human serum. Following spiking in different amounts of rCAL into serum, a lOx dilution with buffer (TBST with 0.2% BSA and 2 mM CaC12) was performed before FESCA. FIG. 3B displays rCAL-dependent luminescence of FESCA in serum. FIG. 3C shows a schematic of workflow of FESCA with human plasma. Following spiking in different amounts of rCAL into plasma, a lOx dilution with buffer (TBST with 0.2% BSA and 2 mM CaC12) was performed before FESCA assay. FIG. 3D displays rCAL-dependent luminescence of FESCA in plasma. FIG. 3E shows a schematic of workflow of FESCA with human whole blood. Following spiking in different amounts of rCAL into the whole blood, a lOx dilution with buffer (TBST with 0.2% BSA and 2 mM CaC12) was performed before FESCA. FIG. 3F displays rCAL- dependent luminescence of FESCA in whole blood. In FIGs. 3B, 3D, and 3F, the luminescence signal was shown as relative luminescence normalized to blank control (left y-axis) and raw luminescence reading from plate reader (right y-axis). FIG. 3G shows a combination plot from FIGs. 3B, 3D, and 3F. For FIGs. 3B and 3D, data represent mean ± std. dev. (n = 6 replicates in 2 independent experiments). For FIG. 3F, data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments). For FIG. 3G, triangles represent whole blood data points, circles represent serum data points, and squares represent plasma data points.

[0524] FIGs. 4A-4D, Solution-based FESCA and paper-based FESCA measured calprotectin in stool extract. FIG. 4A shows a schematic of workflow for FESCA with stool extract. Stool extract was used for FESCA without dilution. FIG. 4B displays rCAL-dependent luminescence of FESCA in stool extract compared to buffer, showing raw luminescence, revealing signal suppression by stool extract. Numbers in grey indicate the relative luminescence in stool extract compared to blank stool extract. FIG. 4C shows a scheme of paper-based FESCA. CP16-SmBiT was adsorbed on cellulose filter paper, a sample was applied to the paper, the paper was rinsed, incubated with CP16-LgBiT and furimazine, and then luminescence imaged. FIG. 4D is an exemplary image by a chemiluminescent imager (e.g., LI-COR™ imager) of paper-based FESCA as described in FIG. 4C, comparing samples added in buffer or stool extract, each with or without rCAL spiked in. The numbers under chemiluminescence image indicate the relative luminescence compared to blank buffer. The number in parentheses indicated the fold change compared to blank stool extract.

[0525] FIGs. 5A-5C, Imaging-based FESCA detected Calprotectin in varied biological samples. FIG. 5A shows a scheme of imaging-based in-solution FESCA by cell phone or plate reader. Samples are arrayed in a 384-well plate in a 1 : 1 ratio with FESCA mixed and imaged in a darkroom (top) or read by a plate reader (bottom). FIG. 5B displays a dose curve of calprotectin in buffer arrayed on a plate in alternating wells and processed as shown in FIG. 5A. The luminescence signal is shown as relative luminescence normalized to blank control (lefty-axis) and raw luminescence reading from a smartphone camera (e.g., IPHONE®, APPLE™) (right y-axis). FIG. 5C displays a dose curve of calprotectin in lOx diluted serum or plasma arrayed on a plate in alternating wells and processed as shown in FIG. 5A. Plots show raw luminescence reading from a smartphone camera (e.g., IPHONE®, APPLE™), and the numbers indicate the relative luminescence compared to corresponding blank control. Images taken by a smartphone camera (e.g., IPHONE® 13 Pro, APPLE™) with default night mode and quantified by Imaged. Data represent mean ± std. dev. (n=4 individual replicates from 2 independent experiments).

[0526] FIGs. 6A-6F, Calprotectin binders were characterized. FIG. 6A shows analysis of the biotinylation of sCAL for the selection, which was visualized by SDS-PAGE gel electrophoresis and Coomassie blue staining. FIG. 6B shows analysis of the formation of S100A8 / 9 dimer in rCAL and sCAL by ELISA. 10 ng / pL rCAL, sCAL, BSA, or SNAP protein was coated on the 96-well black immunoplates, and then examined by monoclonal antibody to S100A9 + S100A8 [27E10], Anti -mouse HRP was then used for colorimetric ELISA with TMB substrate. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments).FIG. 6C show confirmation of rCAL and sCAL with commercial calprotectin ELISA. 10, 50, 200 ng / ml rCAL (left columns), sCAL (second columns), SNAP (third columns), or BSA (right columns) were examined by commercial calprotectin ELISA (e.g., BUHLMANN fCAL® ELISA) assay according to manufacturer’s instruction. Data represent mean ± std. dev. (n = 2 replicates in 1 independent experiment) with all replicates plotted. FIG. 6D shows phage titer after round 4 and round 5 of phage display selections for sCAL. FIG. 6E shows single-point phage ELISA of CP 16 and CP 17, which were selected from the phage display libraries. Data represent mean ± std. dev. (n = 2 replicates in 1 independent experiment) with all replicates plotted. For each groups, the bars from left to right represent SNAP, BSA, and sCAL. FIG. 6F shows analysis of the purification of Fabs (CP16 and CP17) and split-luciferase-fused (e.g., NANOLUC®-fused) Fabs (CP16-NLuc and CP17-NLuc), which was visualized by SDS- PAGE gel electrophoresis and Coomassie blue staining.

[0527] FIGs. 7A-7B, Surface Plasmon Resonance (SPR) analysis was performed on the binding of CP17 with sCAL. FIG. 7A shows binding curves of various amounts of CP 17 to sCAL immobilized on an SPR chip. FIG. 7B shows the binding parameters of CP 17 to sCAL fitted from the binding curves in FIG. 7A.

[0528] FIGs. 8A-8B, Dose-response curves were created for the binding of CP16 and CP17 Fabs with rCAL from immunoabsorbent luminescence (immunoLUC) assay results. FIG. 8A shows results of ImmunoLUC assay measuring the dose-dependent binding of a gradient of rCAL (0.1-10 ng / pL), which were coated directly on the plate surface, with CP16-NLuc. The same concentration gradient of SNAP protein was also coated in different wells and examined for their binding towards CP16-NLuc. Signals are shown as relative luminescence normalized to blank. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments). FIG. 8B shows results of ImmunoLUC assay measuring the dosedependent binding of a gradient of rCAL (0.1-10 ng / pL), which were coated directly on the plate surface, with CP17-NLuc. The same concentration gradient of SNAP protein was also coated in different wells and examined for their binding towards CP17-NLuc. Signals are shown as relative luminescence normalized to blank. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments) with all replicates plotted.

[0529] FIGs. 9A-9C, Competitive immunoabsorbent luminescence (competitive immunoLUC) assays were performed using CP16, CP17, CP16-NLuc, and CP17-NLuc on rCAL. FIG. 9A shows a schematic representation of competitive immunoLUC assay. The well surfaces were precoated with 1 ng / pL rCAL, blocked by varying concentrations of unmodified Fabs, incubated with Fab-NLuc, and then luminescence read after washing. FIG.9B displays results of competitive immunoLUC assay with CP16-NLuc. 0 pg / pL, 0.003 pg / pL, 0.01 pg / pL, and 0.03 pg / pL (0, 62.5, 208, 625 nM, respectively) CP16 or CP17 were used to compete with 10 nM CP16-NLuc on the well surface coated with 1 ng / pL rCAL. Data represent mean ± std. dev. (n = 2 replicates in 1 representative experiment). FIG. 9C displays results of competitive immunoLUC with CP17-NLuc. 0 pg / pL, 0.003 pg / pL, 0.01 pg / pL, and 0.03 pg / pL (0, 62.5, 208, 625 nM, respectively) CP I 6 or CP I 7 were used to compete with 10 nM CP 17- NLuc on the well surface coated with 1 ng / pL rCAL. Data represent mean ± std. dev. (n = 2 replicates in 1 representative experiment) with all replicates plotted.

[0530] FIGs. 10A-10E, CP16 and CP17 split Nanoluc fusions were created and analyzed. FIG. 10A shows analysis of the purification of CP16-LgBiT, CP16-SmBiT, CP17- LgBiT, and CP17-SmBiT, which was visualized by SDA-PAGE gel electrophoresis and Coomassie blue staining. FIG. 10B shows yield and number of assays per protein preparation calculated based on the yields of each protein obtained from IL preparations. The numbers of assays were calculated according to the optimized in-solution exemplary FESCA protocol shown in Fig 2B. FIG. 10C shows exemplary design of Fab heavy chain fusion with LgBiT or SmBiT. Linker A and Linker B (shown in blue) connect heavy chain-GS-FLAG tag (FLAG labelled in green; SEQ ID NO: 20) and LgBiT or SmBiT, respectively. FIG. 10D shows sequences of linkers with different length (SEQ ID NOs: 14-16, which were fused with GS- Flag Tag SEQ ID NO: 20 to create SEQ ID NOs: 17-19). FIG. 10E is a heat map of the signal- to-noise ratio (luminescence at 1 ng / pL rCAL divided by luminescence in blank buffer) of CP16-LgBiT / CP17-SmBiT with different linker lengths. For each reaction, 3.325 nM CP17- SmBiT and 3.325 nM CP16-LgBiT were incubated with 1 ng / pL rCAL using the protocol as outlined in Fig. 2B. Data represent mean (n = 4 replicates in 2 independent experiments). LU: luminescence with arbitrary unit.

[0531] FIGs. 11A-11F, Concentrations optimization of FESCA with CP16- SmBiT / CP16-LgBiT and CP17-SmBiT / CP16-LgBiT was performed. FIGs. 11A-11C Concentration optimization of CP16-SmBiT / CP16-LgBiT with 0.3 ng / pL, 1 ng / pL, or 3 ng / pL rCAL. FIG. 11A displays luminescence signal of FESCA on rCAL using 3.325 nM CP16- LgBiT and different concentrations of CP16-SmBiT. FIG. 11B displays luminescence signal of FESCA on rCAL using 50 nM CP16-SmBiT and different concentrations of CP16-LgBiT. FIG. 11C displays the plots (relative luminescence multiplied by the luminescence difference with blank control) with 50 nM CP16-SmBiT and different concentrations of CP16-LgBiT under 0.3 ng / pL, 1 ng / pL, and 3 ng / pL rCAL. The optimal CP16-LgBiT concentration was indicated by line and number (shown in red). FIGS. 11D-11F Concentration optimization ofCP17-SmBiT / CP16-LgBiT with 0.3 ng / pL, 1 ng / pL, or 3 ng / pL rCAL. FIG. 11D displays luminescence signal of FESCA on rCAL using 3.325 nM CP16-LgBiT and different concentrations of CP17-SmBiT. FIG. HE displays luminescence signal of FESCA on rCAL using 25 nM CP17-SmBiT and different concentrations of CP16-LgBiT. FIG. 1 IF displays the plots of (relative luminescence multiplied by the luminescence difference with blank control) 25 nM CP17-SmBiT and different concentrations of CP16-LgBiT under 0.3 ng / pL, 1 ng / pL, and 3 ng / pL rCAL. The optimal CP16-LgBiT concentration was indicated by line and number (shown in red). In FIGs. 11 A, 1 IB, 1 ID, and 1 IE, the optimal concentration was indicated by red arrow, and data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments). AU: arbitrary unit. In FIGs. 11C and 1 IF, only the means are shown.

[0532] FIGs. 12A-12D, The effect of Ca2+on FESCA was determined. FIG. 12A shows dose response curves of FESCA with CP16-SmBiT / CP16-LgBiT, in which the reactions were performed in buffer with 2 mM Ca2+or without any complemented Ca2+. The luminescence signal was shown as relative luminescence normalized to blank control. FIG. 12B shows dose response curve of FESCA with CP17-SmBiT / CP16-LgBiT, in which the reactions were performed in the buffer with 2 mM Ca2+or without any complemented Ca2+. The luminescence signal was shown as relative luminescence normalized to blank control. FIG. 12C is a schematic representative of Ca2+rescue with FESCA. A concentration gradient of rCAL was challenged by three different treatments: (1) incubation with 2 mM Ca2+(left), (2) incubation with 2 mM Ca2+followed by incubation with 2 mM EDTA (middle); or (3) incubation with 2 mM Ca2+then incubated with 2 mM EDTA followed by incubation with another 2 mM Ca2+(right). FIG. 12D shows dose response curves of FESCA with CP16-SmBiT / CP16-LgBiT on the rCALs treated with 2 mM Ca2+, with 2 mM Ca2+and 2 mM EDTA, and with 2 mM Ca2+, 2 mM EDTA and 2 mM Ca2+in tandem. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments) with all replicates plotted.

[0533] FIG. 13, Native calprotectin levels in plasma (1.59 ng / pl), serum (4.67 ng / pl), whole blood (plasma, 1.324 ng / pl), and stool samples (1.08 ng / pl in 2 mL filtered extract; 216 pg / g stool) used in this disclosure were quantified by a commercial ELISA kit (e.g., BUHLMANN fCAL® ELISA).

[0534] FIGs. 14A-14B, Additional characterizations of FESCA in blood were performed. FIG. 14A shows rCAL-dependent luminescence of FESCA in whole blood. The luminescence signal was shown as raw luminescence counts as measured by plate reader. rCAL-dependent luminescence of FESCA in the plasma obtained from the same whole blood sample was also shown as a comparison. FIG. 14B shows luminescence of FESCA on Creactive protein (CRP) and rCAL. Signals were shown as relative luminescence normalized to blank. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments) with all replicates plotted. P-values represent unpaired t-test with Welch correction, ns = not significant. ****: p< o.OOOl.

[0535] FIGs. 15A-15C, The effect of stool matrix on FESCA was analyzed. FIG. 15A shows a schematic representation of the workflow used to evaluate the effect of stool matrix to luciferase (e.g., NANOLUCIFERASE®, PROMEGA™) system and FESCA sensors. CP 16- NLuc, CP16-SmBiT, CP16-LgBiT or the mixture of CP16-SmBiT and CP16-LgBiT was incubated with stool extract, furimazine was then added, and luminescence read after 15-min. FIG. 15B shows luminescence of a concentration gradient of CP16-full-length NLuc in buffer and stool extract. Data represent mean ± std. dev. (n = 4 replicates in 2 independent experiments) with all replicates plotted. P-values represent unpaired t-test with Welch correction, ns = not significant. FIG. 15C shows luminescence of FESCA sensors in buffer and stool extract. Data represent mean ± std. dev. (n = 2 replicates in 1 independent experiment) with all replicates plotted.

[0536] FIGs. 16A-16C, An exemplary lateral flow strip-based FESCA was characterized. FIG. 16A shows a schematic representation of workflow of lateral flow, strip based FESCA. The sample with rCAL was mixed with 50 nM biotinylated CP16-SmBiT and 15 nM CP16-LgBiT, furimazine was added, and then the strip analyzed using a streptavi din- modified lateral flow assay. FIG. 16B shows analysis of the biotinylation of CP16-SmBiT on SDS-PAGE, which was visualized by Coomassie blue staining. FIG. 16C shows an exemplary image of luminescence from strip based FESCA with concentration gradient of rCAL, with the positions of sample pad and streptavidin band labeled.

[0537] FIG. 17, Provides results of additional replicates of paper based FESCA with stool extract as shown in Fig 4E. The numbers under chemiluminescence image indicated the relative luminescence compared to blank buffer. The number in parentheses indicated the relative luminescence compared to blank stool extract.

[0538] FIGs. 18A-18B, Imaging-based FESCA in serum was performed using a chemiluminescent (e.g., LICOR®) gel imager. FIG. 18A shows an Image by chemiluminescent imager (e.g., LLCOR® gel imager) (with 2-minutes exposure) of FESCA assay in the 10-diluted serum (same batch and processing as FIGs. 3A-3B), which were spiked with different concentration of rCAL (0-50 ng / pL) before dilution. FIG. 18B shows quantification of the luminescence signal in FIG. 18A by ImageJ.

[0539] FIGs. 19A-19B, Are raw photos of imaging-based FESCA taken with a consumer cell phone camera showing buffer, serum, and plasma samples, as described in FIG. 5. FIG. 19A shows raw images of FESCA in buffer shown in FIG. 5B. Images were captured by a consumer camera (e.g., smartphone, e.g., IPHONE® 13 Pro, APPLE™) in darkness (top) and in brightfield (bottom). Four individual replicates were obtained from two independent experiments. FIG. 19B shows raw images of FESCA in 10-fold diluted serum and plasma shown in FIG. 5C. Images were captured by a consumer camera (e.g., smartphone, e.g., IPHONE® 13 Pro, APPLE™) in darkness (top) and in brightfield (bottom). Four individual replicates were obtained from two independent experiments. Samples were arrayed in alternating wells.

[0540] FIGs. 20A-20D, Provide results of imaging-based FESCA taken with a smartphone (e.g., IPHONE®, APPLE™) of whole blood samples. FIG. 20A shows a schematic representation of workflow to test human whole blood with imaging based FESCA with a smartphone (e.g., an IPHONE®, APPLE™). Following spiking in different amounts of rCAL into the whole blood, a lOx dilution with buffer (TBST with 0.2% BSA and 2 mM CaCh) was performed before FESCA and imaging. FIG. 20B shows a dose curve of calprotectin in 1 Ox-diluted whole blood arrayed on a plate in alternating wells and processed as shown in FIG. 20A. Images were taken by a smartphone (e.g., IPHONE® 13 Pro, APPLE™) with default night mode. FIG. 20C provides quantification of FIG. 20B by Imaged. Plots show raw luminescence reading from a smartphone (e.g., IPHONE®, APPLE™). FIG. 20D shows raw images of FESCA in 10-fold diluted whole blood shown in FIG. 20B. Images were captured by a smartphone (e.g., IPHONE® 13 Pro, APPLE™) in darkness (top) and in brightfield (bottom). 2 Individual replicates were obtained from 1 independent experiment.

[0541] FIG. 21, Correlation of native calprotectin level from ELISA with in-solution FESCA. Shown are luminescence readings and calprotectin levels from 4 samples from Tuberculosis patients and 4 samples from healthy controls. Data represent mean (n = 2 replicates in 1 independent experiment).

[0542] FIGs. 22A-22B, show ImmunoLUC assays measuring the dose-dependent binding of a gradient of rCAL (0.1-10 ng / pL). rCAL were coated directly on the plate surface, in the buffer with or without 2 mM CaCh. FIG. 22A shows results with CP16-NLuc. FIG. 22B shows results with CP17-NLuc. Signals are shown as raw luminescence intensity. AU: arbitrary unit. Data represent mean ± std. dev. (n = 5 replicates in 2 independent experiments) with all replicates plotted.

[0543] FIG. 23, shows real-time monitoring luminescence signal from FESCA when challenged with a dose gradient of rCAL (0-10 ng / pL). After 30-minutes incubation of FESCA sensors (CP16-SmBiT / CP16-LgBiT) and rCAL, the luminescence was monitored immediately after furimazine was added. Signals are shown as raw luminescence intensity. AU: arbitrary unit. Data represent mean ± std. dev. (n = 3 replicates in 1 independent experiment) with all replicates plotted.

[0544] FIG. 24, shows imaged-based FESCA by laboratory gel imager. Dose curve of calprotectin in buffer arrayed on a plate in alternating wells and processed as shown in Fig 5A.DETAILED DESCRIPTION

[0545] Calprotectin (aka S100A8 / 9 complex or MRP8 / 18 complex) is a metal ion-binding protein complex that is an abundant molecular component of the innate immune system (see e.g., PMID: 29925260). Calprotectin can be released from neutrophils and epithelial cells at sites of infection or inflammation, where it can inhibit pathogen growth by sequestrating environmental transition metal ions, such as Ni2+, Zn2+, and Mn2+(see e.g., PMID: 18276893, 29925260). Metal ion sequestration can be achieved by the Ca2+-induced tetramerization of two calprotectin S100A8 / 9 dimers to form the metal-chelating (S100A8 / 9)2 heterotetramer (see e.g., PMID: 10536818, and 23082970). While many configurations of calprotectin have been identified (see e.g., PMID: 2071612, and 30380834), only the (S100A8 / 9)2 heterotetramer is resistant to protease digestion, allowing it to maintain its functionality upon pathogenic onslaught (see e.g., PMID: 16690079, 29925260, and 26925211). Calprotectin’ s biostability and correlation with host immunity make it a useful marker of infection or inflammation.

[0546] Clinically, calprotectin levels, especially fecal calprotectin levels, have been established as a biomarker used for distinguishing active inflammatory bowel disease (IBD) from irritable bowel syndrome (IBS), a disease with some similar symptoms as IBD (see e.g., PMID: 30840605, 17924558, and 32054445). Generally, fecal calprotectin levels of less than 50 pg / g indicate that active IBD is unlikely, while levels greater than 200 pg / g are predictive of IBD (see e.g., PMID: 28420947). Moreover, fecal calprotectin levels correlate with disease severity and are associated with disease progression, providing both patients and clinicians valuable insights into the dynamics of the disease, and can stratify patients in remission (<150- 200 pg / g), to moderate or severe ulcerative colitis (>150-200 pg / g) (see e.g., PMID: 30840605). Fecal calprotectin has a high negative predictive value for recurrence after surgery in Crohn’s disease, (see e.g., PMID: 25620670). Recent studies have also revealed that serum / plasma / perspiration calprotectin levels, similar to fecal calprotectin, can also reflect thedisease activity of IBD (see e.g., PMID: 34690210, 31528300, 23845231, and 27596694), providing a potential approach to monitor IBD progression without cumbersome / inconvenient stool collection. Outside of IBD, elevated serum calprotectin has been associated with other systemic inflammation diseases and cancers (see e.g., PMID: 9292145), such as rheumatoid arthritis (RA) (see e.g., PMID: 26242859, and 27363504), and poor COVID-19 outcomes (see e.g., PMID: 37787461). Therefore, tracking calprotectin levels across various biological samples -ranging from stool samples to blood samples- would significantly aid in diagnosing disease, monitoring disease progression, assessing the effectiveness of treatment interventions, and / or instructing appropriate disease intervention regimens.

[0547] Currently, clinical testing for calprotectin levels, especially fecal calprotectin levels, relies on enzyme-linked immunosorbent assays (ELISAs), which require trained personnel and expensive equipment, and are therefore limited to laboratory settings. In addition, data collection of fecal calprotectin levels is usually impeded by low patient compliance to the generally inconvenient fecal immunochemical testing requirements (see e.g., PMID 30684199), and specifically among patients with IBD (see e.g., PMID 36776647), with the necessity of sample transportation to the clinic often acting as a primary deterrent (see e.g., PMID: 36776647, and 28570431), making monitoring the disease progression and guiding management challenging for both the physicians and the patients. Therefore, improved clinical tests, particularly at-home testing, for calprotectin levels would be of high diagnostic value and clinical utility.

[0548] Recently, a lateral flow assay (LFA) for calprotectin was developed using a novel calprotectin-binding peptide (see e.g., PMID: 37198182), opening new opportunities for novel calprotectin assay development. However, colorimetric LFAs generate semi quantitative results, and are therefore less suitable for quantifying the range of calprotectin levels associated with disease progression and / or remission. Therefore, a convenient, quantitative, and inexpensive method for quantifying calprotectin levels at the point-of-care or even at-home situations would be desirable.

[0549] In some aspects, provided herein are exemplary compositions, methods, and kits comprising and / or associated with a Fab-Enabled Split Luciferase Calprotectin Assay (FESCA), a novel assay for the convenient and quantitative assessment of calprotectin levels. In certain aspects, compositions and methods provided herein are not limited to reporter systems comprising the exemplary split luciferases. In some aspects, FESCA comprises commercial luciferase technologies (e.g., NANOBIT® technology, PROMEGA™) (see e.g., PMID: 26569370), in which two halves of a split luciferase, LgBiT and SmBiT, are broughttogether to reconstitute an active luciferase by fused binding modules. To develop the exemplary FESCA, the inventors utilized phage display to generate two fragment antigen binding proteins (Fabs), CP16 and CP17, which bind to two different regions of the calprotectin complex. The CP16 and / or CP17 Fabs were then linked with an exemplary split luciferase reporter system (e.g., NANOLUC®, PROMEGA™ LgBiT and SmBiT luciferase fragments), which can be expressed in good yields from E. coli. After optimizing the protein designs and experimental conditions, the inventors developed FESCA protocols suitable for use with various samples, which involves mixing a biological sample with the assay components and then measuring luminescence. As shown herein, FESCA detected physiologically relevant calprotectin levels with an up to 100-fold dynamic range in the context of buffer, serum, plasma, whole blood, and / or stool extract, both in-solution and in paper-based assay formats. In addition, the output luminescence signal was strong enough to not only be measured by a plate reader and / or a laboratory charged-coupled device (CCD) camera, but the luminescence was suitable for measurement with a consumer-grade cell phone camera. As provided herein, FESCA is an easy to use, low-cost, and quantitative method to measure calprotectin levels in various physiologically relevant patient samples.I. METHODS AND COMPOSITIONSA. Antibodies

[0550] Aspects of the disclosure relate to antibodies, antigen binding fragments thereof, or polypeptides capable of specifically binding to calprotectin complexes. In some aspects, calprotectin complexes may include S100A8 monomers, S100A9 monomers, S100A8 homodimers, S100A9 homodimers, S100A8 / 9 heterodimers, and / or (S100A8 / 9)2 heterotetramers. In some aspects, calprotectin complexes may exclude S100A8 monomers, S100A9 monomers, S100A8 homodimers, S100A9 homodimers, S100A8 / 9 heterodimers, and / or (S100A8 / 9)2 heterotetramers. In some aspects, calprotectin complexes comprise, consist of, or consist essentially of S100A8 / 9 heterodimers and / or (S100A8 / 9)2 heterotetramers.

[0551] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.

[0552] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more epitopes that are capable of interacting with different antibodies.

[0553] The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0554] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockb erg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y, incorporated herein by reference in its entirety. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81 :3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82: 178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986), each incorporated herein by reference in its entirety. Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0555] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0556] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be solely from a single source or may be “chimeric,” that is, different portions of the antibody may be from two different antibodies. For example, the variable or CDR regions may be from a rat or murine source, while the constant region can be from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, andmuteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013, incorporated herein by reference in its entirety).

[0557] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (K) and lambda (X). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0558] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the aminoterminus of the polypeptide, and the CH domains are at the carboxy-terminus, with the CH3 being closest to the — COOH end. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (5), gamma (y), alpha (a), or epsilon (a) chains, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgMl and IgM2. IgA subtypes include IgAl and IgA2.1. Types of Antibodies

[0559] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab')2, Fab', Fab, Fv, and the like), including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.

[0560] The term “monomer” means an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies. The term “dimer” means an antibody containing two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein. The term “multimer” means an antibody containing more than two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein.

[0561] The term “bivalent antibody” means an antibody that comprises two antigenbinding sites. The two binding sites may have the same antigen specificities, or they may be bi-specific, meaning the two antigen-binding sites have different antigen specificities.

[0562] Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more distinct epitopes. In some aspects, bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen. In some aspects, bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies”. Single domain antibodies can be sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. W02010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which are specifically incorporated herein by reference in their entirety.

[0563] Bispecific antibodies can be constructed as: a whole IgG, Fab'2, Fab'PEG, a diabody, or alternatively as scFv. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti -idiotypic reaction. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-1553 (1992), each of which are specifically incorporated by reference in their entirety.

[0564] In certain aspects, the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind a different antigen. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Accordingly, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.

[0565] In some aspects, multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art. One such example is diabodies that are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites. The linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2: 1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001), each of which is incorporated herein by reference in its entirety).

[0566] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be advantageous because they can be readily constructed and expressed in E. coli. Diabodies (and other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94 / 13804, incorporated herein by reference in its entirety) from libraries. If one arm of the diabody is kept constant, for instance, with a specificity directed against a protein, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39: 167-173, 2017), each of which is hereby incorporated by reference in their entirety.

[0567] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities. See, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0568] The part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.” The paratope consists of the amino acid residues that make contact with the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, Framework Regions (FR). The hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “Complementarity Determining Region (CDR).” The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have high primary sequencesimilarity / consensus. The consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions. The hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur. CDRs in the VL domain are identified as LI, L2, and L3, with LI occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs may also be given the names CDR-L1 (or LCDR1), CDR-L2 (or LCDR2), and CDR-L3 (or LCDR3). The L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain arranged linearly in the primary structure, and separated from each other by Framework Regions. The amino terminal (N- terminal) end of the VL chain is named FR1. The region identified as FR2 occurs between LI and L2 hypervariable loops. FR3 occurs between L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which includes three CDRs identified as CDR-H1 (or HCDR1), CDR-H2 (or HCDR2), and CDR-H3 (or HCDR3). The majority of amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of the framework regions (approximately 85%). The three dimensional, or tertiary, structure of an antibody molecule is such that the framework regions are more internal to the molecule and provide the majority of the structure, with the CDRs on the external surface of the molecule.

[0569] Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed for identification of the CDRs of antibodies: Kabat (as described in T. T. Wu and E. A. Kabat, “An Analysis Of The Sequences Of The Variable Regions Of Bence Jones Proteins And Myeloma Light Chains And Their Implications For Antibody Complementarity,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989, incorporated herein by reference in its entirety); and IMGT (as described in M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003, incorporated herein by reference in its entirety). These methods each include unique numbering systems for-the identification of the amino acid residues that constitute the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen occur in the CDRs, although in some cases, residues within the framework regions contribute to antigen binding.

[0570] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include:

[0571] 1) Computational predictions of the tertiary structure of the antibody / epitope binding interactions based on the chemical nature of the amino acid sequence of the antibody variable region and composition of the epitope.

[0572] 2) Hydrogen-deuterium exchange and mass spectroscopy

[0573] 3) Polypeptide fragmentation and peptide mapping approaches in which one generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides for the epitope.

[0574] 4) Antibody Phage Display Library analysis in which the antibody Fab fragment encoding genes of the mammal are expressed by bacteriophage in such a way as to be incorporated into the coat of the phage. This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific VH and VL domains as appropriate.

[0575] In certain aspects, affinity matured antibodies are enhanced with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CDRs thereof that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s). Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio / Technology 10:779 (1992), incorporated herein by reference in its entirety, describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and / or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009), incorporated herein by reference in its entirety, in conjugation with computation methods as demonstrated in Tiller et al., Front. Immunol. 8:986 (2017), incorporated herein by reference in its entirety.

[0576] Chimeric immunoglobulins are the products of fused genes from different species; “humanized” chimeras generally have the framework region (FR) from human immunoglobulins and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CDRs are from a non-human source.

[0577] In certain aspects, portions of the heavy and / or light chain are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 (1984), incorporated herein by reference in its entirety. For methods relating to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1985), each of which are specifically incorporated herein by reference in their entirety. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are all hereby incorporated by reference for all purposes.

[0578] In some aspects, minimizing the antibody polypeptide sequence from the non- human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues from non-antigen recognizing regions of the non-human antibody are modified to be homologous to corresponding residues in a human antibody or isotype. One example is the “CDR-grafted” antibody, in which an antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CDRs from a particular species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies from another species or belonging to another antibody class or subclass. For use in humans, the V region composed of CDR1, CDR2, and partial CDR3 for both the light and heavy chain variance region from a non-human immunoglobulin, are grafted with a human antibody framework region, replacing the naturally occurring antigen receptors of the human antibody with the non-human CDRs. In some instances, corresponding non-human residues replace framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1 : 105 (1998); Harris, Biochem. Soc. Transactions 23;1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239: 1534-36 (1988), each incorporated herein by reference in its entirety.

[0579] Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0580] Polyclonal antibody preparations typically include different antibodies against different determinants (epitopes). In order to produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generally with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the sera of the host. The polyclonal antibody can be affinity purified against the antigen rendering it monospecific.

[0581] Monoclonal antibodies or “mAb” refer to an antibody obtained from a population of homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.2. Functional Antibody Fragments and Antigen-Binding Fragments

[0582] Certain aspects provided herein relate to antibody fragments, such as antibody fragments that bind to calprotectin. The term functional antibody fragment includes antigenbinding fragments of an antibody that retain the ability to specifically bind to an antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and / or light chain (VL); and in some aspects, include constant region heavy chain 1 (CHI) and light chain (CL). In some aspects, they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains. Aspects of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CHI domains; (ii) the Fd fragment type constituted with the VH and CHI domains; (iii) the Fv fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015), each of which are incorporated by reference.

[0583] Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 complementarity determining regions (CDRs) from a light chain variable region. Fusions of CDR-containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein.

[0584] The term Fab fragment (also “Fab” or “FAB”) means a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL and CHI domains. The term Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes the VL, VH, CL and CHI domains and all or part of the hinge region. The term F(ab')2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab' fragments linked by a disulfide bridge at the hinge region. An F(ab')2 fragment includes, for example, all or part of the two VH and VL domains, and can further include all or part of the two CL and CHI domains.

[0585] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs. An Fd fragment can further include CHI region sequences.

[0586] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CHI domains. The VL and VH include, for example, the CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference. The term (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain comprises self-associating a- helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”

[0587] A single domain antibody is an antigen-binding fragment containing only a VH or the VL domain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0588] Fragment Antigen Binding Region (Fab) - Fab polypeptides of the disclosure include the Fab antigen binding fragment of an antibody. Unless specifically stated otherwise,the term “Fab” relates to a polypeptide excluding the Fc portion of the antibody. The Fab may be conjugated to a polypeptide comprising other components, such as reporter systems, fragments of a reporter system, detectable moieties, further antigen binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and / or accessory proteins. In some aspects, a Fab is not conjugated to a polypeptide comprising other components, such as reporter systems, fragments of a reporter system, detectable moieties, further antigen binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and / or accessory proteins. Fab polypeptides can be generated using conventional techniques known in the art and are well-described in the literature.

[0589] Fragment Crystallizable Region (Fc) - An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are included.3. Polypeptides with Antibody CDRs and Scaffolding Domains that Display the CDRs

[0590] Antigen-binding peptide scaffolds, such as complementarity-determining regions (CDRs), can be used to generate protein-binding molecules in accordance with the aspects provided herein. Generally, a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and / or be easy to produce, express, and purify. See e.g., Skerra, J Mol Recognit, 13: 167-87 (2000), incorporated herein by reference in its entirety.

[0591] The protein scaffolds can be sourced from, but not limited to: fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”. Such proteins are described in US Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. W02006 / 056464, each of which are specifically incorporated herein by reference in theirentirety. In some aspects, scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used.4. Antibody Binding

[0592] The term “selective binding agent” refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab', F(ab')2, single chain antibodies, peptides, peptide fragments and proteins.

[0593] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Immunologically reactive” means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample. The term “immune complex” refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.

[0594] In some aspects, samples from a subject may be immunologically reactive, and compositions provided herein (e.g., comprising CP16 and / or CP17) may form immune complexes with a molecule in the sample (e.g., calprotectin) a. Affinity / Avidity

[0595] The term “affinity” refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (Ka or ka sometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody to its antigen relative to the binding strength of the antibody to an unrelated amino acid sequence. Affinity can be expressed as, for example, 20-fold greater binding ability of the antibody to its antigen then to an unrelated amino acid sequence. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and / or selective binding agent.

[0596] There are several experimental methods that can be used by one skilled in the art to evaluate the binding affinity of any given antibody or selective binding agent for its antigen. This is generally done by measuring the equilibrium dissociation constant (KD or Kd), using the equation KD = koff / kon = [A][B] / [AB], The term koff is the rate of dissociation between the antibody and antigen per unit time, and is related to the concentration of antibody and antigen present in solution in the unbound form at equilibrium. The term kon is the rate ofantibody and antigen association per unit time, and is related to the concentration of the bound antigen-antibody complex at equilibrium. The units used for measuring the KD are mol / L (molarity, or M), or concentration. The Ka (affinity constant) of an antibody is the opposite of the KD, and is determined by the equation Ka = 1 / KD. Examples of some experimental methods that can be used to determine the KD value are: enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE).

[0597] Antibodies deemed useful in certain aspects may have an affinity constant (Ka) of about, at least about, or at most about 106, 107, 108, 109, or 1010M or any range or value derivable therein. Similarly, in some aspects, antibodies may have a dissociation constant of about, at least about, or at most about 10'6, 10'7, 10'8, 10'9, 10'10M, or any range or value derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies. An antibody of the disclosure can be said to “specifically bind” its target antigen when the dissociation constant (KD) is < 108M. An antibody of the disclosure can be said to specifically bind an antigen with “high affinity” when the KD is <5* 109M, and with “very high affinity” when the KD is <5* 1010M. b. Epitope Specificity

[0598] The epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity. In the case of protein or polypeptide antigens, the epitope is the specific residues (or specified amino acids or protein segment) that the antibody binds with high affinity. An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity. For purposes of this specification and the accompanying claims, the terms “epitope” and “antigenic determinant” are used interchangeably to refer to the site on an antigen to which B and / or T cells can respond or recognize. Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide, or quaternary interactions in a complex. An epitope typically includes at least 3, and typically 5-10 amino acids in a unique spatial conformation.

[0599] Epitope specificity of an antibody can be determined in a variety of ways. One approach, for example, involves testing a collection of overlapping peptides of 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are immobilized in separate wells of amicrotiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments. An antibody or antigen-binding fragment can be screened for binding to each of the various peptides, for example to confirm which peptide the antibody or antigenbinding fragment is binding to. The epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding. c. Modification of Antibody Antigen-Binding Domains

[0600] It is understood that the antibodies and / or antigen-binding fragments of the present disclosure may be modified, such that they are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of interest in the present disclosure (e.g., calprotectin complexes). Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.

[0601] As discussed herein, minor variations in the amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain about, at least, or at most 70%, 71%, 72%, 73%, 74%, or 75%, or more sequence identity. In some aspects, variations in the amnio acid sequence maintain about, at least, or at most 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or any range or value derivable therein, sequence identity. In particular, in certain aspects conservative amino acid replacements are expressly contemplated.

[0602] Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families based on the chemical nature of the side chain; e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). For example, it is reasonable to expect that an isolatedreplacement of a leucine moiety with an isoleucine or valine moiety, or a similar replacement of an amino acid with a structurally related amino acid in the same family, will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art.

[0603] Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Standard methods to identify protein sequences that fold into a known three-dimensional structure are available to those skilled in the art; Dill and McCallum., Science 338: 1042-1046 (2012), incorporated herein by reference in its entirety. Several algorithms for predicting protein structures and the gene sequences that encode these have been developed, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / guide / proteins / ) and at the Bioinformatics Resource Portal (on the World Wide Web at expasy.org / proteomics). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure.

[0604] Framework modifications can be made to antibodies to decrease immunogenicity, for example, by “backmutating” 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more framework residues to a corresponding germline sequence.

[0605] It is also contemplated that the antigen-binding domain may be multi-specific or multivalent by multimerizing the antigen-binding domain with VH and VL region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific).5. Chemical Modification of Antibodies

[0606] In some aspects, also contemplated are glycosylation variants of antibodies and / or antigen binding fragments thereof, wherein the number and / or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Glycosylation of the polypeptides can be altered, for example, by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861, each incorporated herein by reference in its entirety). In certain aspects, antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more new N-linked glycosylation sites are created. Antibodies typically have an N-linked glycosylation site in the Fc region.

[0607] Additional antibody variants include cysteine variants, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0608] In some aspects, the polypeptides can be pegylated to stabilize the polypeptide and / or increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more PEG groups become attached to the polypeptide. Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0154316 and EP 0401384, each incorporated herein by reference in its entirety. In some aspects, an antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modifiedwith, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylene polyols, and polyvinyl alcohols. As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins. a. Conjugation

[0609] Derivatives of the antibodies and antigen binding fragments that are described herein are also provided. The derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment. The derivatized antibody can comprise or exclude, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead) or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fractions thereof that may be utilized in a reporter system (e.g., split luciferase, split fluorescent proteins, etc.), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), and / or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). In certain aspects, compositions, methods, and / or kits provided herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies or antigen binding fragments linked, directly or indirectly, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fractions (fragments) of a reporter system, such as but not limited to, split luciferase reporter systems.

[0610] In some aspects, an antibody or an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins. In some aspects, polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e.g., EP 0322094 and EP 0486525.

[0611] In some aspects, the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide. Additional suitable conjugated molecules can include ribonuclease (RNase), DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences. In some aspects, functional nucleic acid moleculesmay act as effectors, inhibitors, modulators, and / or stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.

[0612] In some aspects of this disclosure, it will be useful to detectably or therapeutically label a Fab polypeptide or protein G Fab-binding domain. Methods for conjugating polypeptides to these agents are known in the art. For the purpose of illustration only, polypeptides can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled polypeptides can be used for diagnostic techniques, either in vivo, or in an isolated test sample or in methods described herein.

[0613] As used herein, the term "label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. In some aspects, luminescence or fluorescence assays comprise split enzymatic components, which alone are not active, but when brought together in close proximity (e.g., localized at the same calprotectin complex) and provided with a substrate, are active.

[0614] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and describedfor example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6. sup. th ed.), incorporated herein by reference in its entirety. Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0615] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine (TRITC), eosin, erythrosine (Red No. 3), coumarin, methylcoumarins, pyrene, Malachite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6. sup. th ed.), incorporated herein by reference in its entirety.

[0616] In another aspect, a fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0617] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens / polypeptides, e.g., rhodamine / anti-rhodamine, biotin / avidin and biotin / streptavidin.

[0618] The coupling of polypeptides to low molecular weight haptens can increase the sensitivity of the antibody in an assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See, Harlow and Lane (1988) supra.

[0619] In some aspects, disclosed herein are polypeptides (e.g., antibodies, antigen binding domains, etc.) that are linked to at least one agent to form an antibody conjugate or payload. In order to increase the efficacy of antibody molecules as diagnostic (e.g., for use in an assay) or therapeutic agents, it can be desirable to link (directly or indirectly) or covalently bind or complex at least one desired molecule or moiety, such as a reporter system or fragment thereof. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like. By contrast, a reporter molecule is defined asany moiety that may be detected using an assay. Non-limiting examples of reporter molecules that can be conjugated or excluded from conjugation to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands, and / or fragments thereof. In certain aspects, reporter molecules may be split into two or more fragments, and linked (directly or indirectly) to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides (e.g., antibodies, antigen binding domains, etc.). In specific aspects, reporter systems comprise fragmented luciferase reporter molecules and variants thereof. b. Conjugate Types

[0620] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more portions thereof. “Detectable labels” are compounds and / or elements that can be detected due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to be detected, and / or further quantified if desired. Detectable labels may themselves constitute a reporter system, or may be split into two or more fragments and employed as part of a reporter system, alone, or in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional components (e.g., substrates, ions, buffers, etc.). Examples of detectable labels include, but are not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. Particular examples of labels are, but not limited to, luciferases (e.g., including but not limited to, NANOLUC® Luciferase, Renilla Luciferase, and Firefly Luciferase), horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and a- or P-galactosidase. Antibody conjugates include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme to generate a colored or otherwise detectable (e.g., luminescent) product upon contact with a substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The uses of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference. Molecules containing azido groups may also be used to formcovalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).

[0621] In some aspects, contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In this way, the agent of interest can be targeted directly to cells bearing cell surface antigen. The antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein. In some aspects, an antibody may be conjugated to various therapeutic substances in order to target a cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens.

[0622] In antibody drug conjugates (ADC), an antibody (Ab) is conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more drug moi eties (D) through a linker (L). The ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Antibody drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate. In yet another aspect, the antibody may be conjugated to a “receptor” (such as streptavidin) for utilization in tumor or cancer cell pre-targeting wherein the antibodyreceptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0623] Examples of an antibody-drug conjugates known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605- 614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26: 151-172 (1997); U.S. Pat. No. 4,975,278, each incorporated herein by reference in its entirety). In contrast, systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the target tumor cells (Baldwin et al., Lancet 1 :603-5 (1986); Thorpe, (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: AReview,” In: Monoclonal Antibodies ‘84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506, each incorporated herein by reference in its entirety). Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21 : 183-87 (1986), incorporated herein by reference in its entirety).

[0624] In certain aspects, ADC include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Antibody-containing fusion proteins may comprise or exclude peptides added to facilitate purification or identification of the antibody (e.g., poly-His). An antibody polypeptide also can be linked to the FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio / Technology 6: 1204 (1988), and U.S. Pat. No. 5,011,912, each incorporated herein by reference in its entirety. Oligomers that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibody polypeptides may be employed as antagonists. Oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibody polypeptides are contemplated for use. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. In certain aspects, oligomers comprise multiple antibody polypeptides joined via covalent or non-covalent interactions between peptide moi eties fused to the antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides from antibodies are among the peptides that can promote oligomerization of antibody polypeptides attached thereto, as described in more detail below.c. Conjugation Methodology

[0625] Several methods are known in the art for the attachment or conjugation of an antibody or antigen binding fragment to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bos(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). In some aspects, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5, 196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., 1987).

[0626] In some aspects, conjugation occurs during translation of an RNA molecule into a polypeptide, where the conjugate is a polypeptide and is linked to the antibody or antigen binding fragment through a peptide bond, either directly or through a linker peptide.B. Antibody Production

[0627] Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutics are well known in the art as disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745 (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference), each incorporated herein by reference in its entirety. These antibodies may be polyclonal or monoclonal antibody preparations,monospecific antisera, human antibodies, hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question. In certain aspects, polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various aspects can also be synthesized in solution or on a solid support in accordance with conventional techniques. See, for example, Stewart and Young, (1984); Tam et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.

[0628] Other methods of making antibodies are through molecular display technologies. These include technologies such as phage display and yeast surface display, have been the major driving force of the development (Winter, 1994; Boder, 2000; Viti, 2000; Bradbury, 2011, each incorporated herein by reference in its entirety). These enable display of singlechain Fv (scFv) and Fab fragments on the surface of microorganisms and links the phenotype of a variant to its cognate DNA and thus amino acid sequence (McCafferty, 1990; Kang, 1991; Barbas, 1991, each incorporated herein by reference in its entirety). Using these techniques, it is possible to produce a library of an antibody fragment that contains billions of variants and identify variants with a desired binding function from the library in a HTP manner. Using variants of these techniques, it is possible to evolve a pool of antibody fragments to increase the specificity of the antibody fragments to a particular antigen, through multiple iterations of selection.

[0629] In the standard antibody architecture, six surface loops, three each from the heavychain and light-chain variable domains constitute the complementarity determining regions (CDRs). Thus, the antibody and its fragments can be considered as "molecular scaffolds" for presenting CDRs. The sequence diversity of CDRs is the primary source of antibody affinity and specificity. In recombinant antibody libraries, the diversity of CDRs can be either derived from natural immune systems or generated by random mutagenesis, and Fv libraries of both types have been successfully used (Barbas, 1992; Knappik, 2000, each incorporated herein by reference in its entirety).

[0630] Yeast surface display is another important technical development in antibody engineering. Although the library size achievable with this technique is much smaller than that with phage display, yeast surface display coupled with fluorescence activated cell sorter (FACS) allows for HTP, quantitative characterization of antibody affinity, specificity and expression efficiency (Boder, 2000). The major strengths of phage display include the large size of a library and rapid completion of the selection and propagation cycle (< 2 days). Yeastdisplay provides a highly quantitative readout of binding and expression levels using FACS and the ability to precisely define the selection stringency based on equilibrium and kinetic binding parameters as well as expression levels. The integration of these two display methods can provide for an optimal selection of clones that not only are high affinity binders but are stable and express well.

[0631] The end products of recombinant antibody screening are the genes encoding Fv and Fab fragments with desired binding characteristics. Bacterial expression systems for Fv and Fab with good yields have been established [Borrebaeck, 1992], Thus, the phage display clones can be quickly reformatted into an expression cassette for the production of soluble Fv and Fab fragments. In some aspects, phage display clones can be included in expression cassettes for the production of soluble Fv and Fab fragments in various cell lines, including prokaryote or eukaryote cell lines. In some aspects, the expression cassette may be codon optimized for production of the soluble Fv and Fab fragments in the cell line selected.

[0632] In general, at least 15- 20 residues of CDR residues contribute to antibody binding to its target. Full combinatorial randomization of this many sites together far exceeds the limits of phage and yeast display library diversity. Since the number of sites cannot be significantly reduced, new library strategies have been developed that reduce the 20 natural amino acid genetic code to a smaller subset, while maintaining high functional diversity (Fellouse, 2007). These libraries using a greatly reduced genetic code can produce high affinity Fab and scFv molecules. Although several reduced genetic code libraries have been constructed and tested, the concept has not been optimized for different target classes and especially for the smaller scaffold camelids VHH and FN3 domains (D2). For screening of these novel libraries, a combination of phage display and yeast surface display can be utilized for optimal efficiency. In molecular display approaches, generally only the variable regions of the antigen binding loops (CDRs) of the Fab domain are diversified. The main architecture of the domain is held constant. This main architecture can be based on a fully human antibody scaffold. This circumvents the need to humanize the scaffold, as is required by methods involving animal immunizations.

[0633] Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or overall functional properties relative to being a treatment for infection. Thus, monoclonal antibodies may havealterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid.C. Antibodies, Antigen Binding Fragments, and Polypeptides

[0634] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. The term polypeptide also includes an antibody fragment described herein as well as antibody domains, such as HCDR1 (Heavy chain complementarity determining region 1), HCDR2 (Heavy chain complementarity determining region 2), HCDR3 (Heavy chain complementarity determining region 3), LCDR1 (Light chain complementarity determining region 1), LCDR2 (Light chain complementarity determining region 2), LCDR3 (Light chain complementarity determining region 3), HFRW1 (Heavy chain framework region 1), HFRW2 (Heavy chain framework region 2), HFRW3 (Heavy chain framework region 3), HFRW4 (Heavy chain framework region 4), LFRW1 (Light chain framework region 1), LFRW2 (Light chain framework region 21), LFRW3 (Light chain framework region 3), LFRW4 (Light chain framework region 4), VH (Heavy chain variable region), VL (Light chain variable region), CH (Heavy chain constant region), or CL (Light chain constant region).

[0635] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term“recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0636] In specific aspects, provided herein are antigen binding fragment polypeptides that bind with high specificity to calprotectin. In some aspects, the antigen binding fragment polypeptides may comprise a heavy chain and / or a light chain. In some aspects, the antigen binding fragment polypeptide comprises a CP 16 heavy chain amino acid sequence (SEQ ID NO: 2), or a sequence of, of at least, or of at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range or value derivable therebetween) identity thereto. In some aspects, the antigen binding fragment polypeptide comprises a CP16 light chain amino acid sequence (SEQ ID NO: 1), or a sequence of, of at least, or of at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range or value derivable therebetween) identity thereto. In some aspects, the antigen binding fragment polypeptide comprises a CP17 heavy chain amino acid sequence (SEQ ID NO: 8), or a sequence of, of at least, or of at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range or value derivable therebetween) identity thereto. In some aspects, the antigen binding fragment polypeptide comprises a CP 17 light chain amino acid sequence (SEQ ID NO: 7), or a sequence of, of at least, or of at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range or value derivable therebetween) identity thereto. In some aspects, the antigen binding fragment polypeptide comprises a CP 16 heavy chain and light chain amino acid sequence. In some aspects, the antigen binding fragment polypeptide comprises a CP 17 heavy chain and light chain amino acid sequence. In some aspects, compositions, methods, and / or kits provided herein comprise a CP 16 and / or CP 17 heavy chain and / or light chain amino acid sequence, or a variant or conjugate thereof. In certain aspects, a polypeptide comprising a CP 16 heavy chain amino acid sequence is conjugated (linked) either directly or indirectly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties. In certain aspects, a polypeptide comprising a CP 16 light chain amino acid sequence is conjugated (linked) either directly or indirectly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties. In certain aspects, a polypeptide comprising a CP 17 heavy chain amino acid sequence is conjugated (linked) either directly or indirectly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties. In certain aspects, a polypeptide comprising a CP17 light chain amino acid sequence is conjugated (linked) either directly or indirectly to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties.In certain aspects, a detectable moiety comprises, consists of, or consists essentially of a luciferase enzyme or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fragments of a luciferase enzyme.

[0637] In certain aspects the size of an antibody, antigen binding fragment, protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0638] The antibody, antigen binding fragment, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be, be at least, or be at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range or value therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1-22.

[0639] In some aspects, the antibody, antigen binding fragment, protein, or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57. 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82. 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571,572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685,686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704,705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742,743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761,762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837,838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875,876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913,914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951,952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970,971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000, (or any derivable range therein) of SEQ ID NOs: 1-22.

[0640] In some aspects, the antibody, antigen binding fragment, or polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613,, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670,, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708,, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727,, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746,, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784,785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803,804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841,842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879,880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917,918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955,956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974,975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993,994, 995, 996, 997, 998, 999, or 1000, (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 1-22.

[0641] In some aspects, the antibody, antigen binding fragment, protein, or polypeptide may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 4243, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54^ 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 6768, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 9293, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550,551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645,646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721,722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759,760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778,779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797,798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816,817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873,874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911,912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949,950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968,969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987,988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 1-22 that are, are at least, or are at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of SEQ ID NOs: 1-22.

[0642] In some aspects there is a nucleic acid molecule, antibody, antigen binding fragment, protein, or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,I l l, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756,757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775,776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851,852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889,890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965,966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984,985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of any of SEQ ID NOs: 1-22 and comprising, comprising at least, or comprising at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543,, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657,, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714,, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733,, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752,, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771,, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809,, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847,, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885,886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904,905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923,924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942,943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980,981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids or nucleotides of any of SEQ ID NOs: 1-22.

[0643] In some aspects, the amino acid at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, or 400 of the heavy chain, light chain, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFRW1, HFRW2, HFRW3, HFRW4, LFRW1, LFRW2, LFRW3, or LFRW4 of any of SEQ ID NOs: 1-22 is substituted with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0644] In some aspects, a polypeptide (e.g., antibody, antibody fragment, Fab, etc.) of the disclosure comprises a CDR that is, is at least, or is at most 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (or any range or value derivable therein) in sequence to one of SEQ ID NOs: 3-6 or 9-12. In some aspects, a polypeptide comprises 1, 2, and / or 3 CDRs from one of SEQ ID NOs: 3-6 or 9-12. The CDR may be one that has been determined by Kabat, IMGT, or Chothia. In further aspects, a polypeptide may have CDRs that have 1, 2, and / or 3 amino acid changes (e.g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to these 1, 2, or 3 CDRs. In some aspects, a polypeptide comprises additionally or alternatively, an amino acid sequence that is, is at least, or is at most 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.

[0645] From amino to carboxy terminus the CDRs are CDR1, CDR2, and CDR3. In some aspects, a polypeptide may have CDRs that have 1, 2, and / or 3 amino acid changes (e.g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to CDR1, CDR2, or CDR3. In some aspects, the CDRs of SEQ ID NOs: 3-6 or 9-12 may further comprise 1, 2, 3, 4, 5, or 6 additional amino acids at the amino or carboxy terminus of the CDR. The additional amino acids may be from the heavy and / or light chain framework regions of SEQ ID NOs: 1, 2, 7, or 8, that are shown as immediately adjacent to the CDRs. Accordingly, aspects relate to polypeptides comprising an HCDR1 (i.e., CDR-H1), HCDR2 (i.e., CDR-H2), HCDR3( i.e., CDR-H3), LCDR1 (i.e., CDR-L1), LCDR2 (i.e., CDR-L2), and / or LCDR3 (i.e., CDR-L3) with at least or at most or exactly 1, 2, 3, 4, 5, 6 or 7 amino acids at the amino end of the CDR or at the carboxy end of the CDR, wherein the additional amino acids are the 1, 2, 3, 4, 5, 6, or 7 amino acids of SEQ ID NOs: 1, 2, 7, or 8 that are shown as immediately adjacent to the CDRs. Other aspects relate to antibodies comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CDRs, wherein the CDR is a fragment of SEQ ID NOs: 1-12 and wherein the fragment lacks 1, 2, 3, 4, or 5 amino acids from the amino or carboxy end of the CDR. In some aspects, the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the carboxy end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the amino end of the CDR. In some aspects, the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the amino end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the carboxy end of the CDR. In further aspects, an antibody may be alternatively or additionally humanized in regions outside the CDR(s) and / or variable region(s). In some aspects, a polypeptide comprises additionally or alternatively, an amino acid sequence that is,is at least, or is at most 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any range or value derivable therebetween) identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.

[0646] In other aspects, a polypeptide or protein comprises 1, 2, 3, 4, 5, or 6 CDRs from either or both of the light and heavy variable regions of CP16 and / or CP17, or SEQ ID NOs: 1, 2, 7, or 8, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and / or 3 amino acid changes with respect to these CDRs. In some aspects, parts or all of the antibody sequence outside the variable region have been humanized. In some aspects, parts or all of the antibody sequence outside the variable region are not humanized. A protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and / or 1 or 2 polypeptides similar to a light chain polypeptide.

[0647] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes (e.g., genes encoding the calprotectin complex) have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art

[0648] In certain aspects, it is contemplate...

Claims

CLAIMSWhat is claimed is:

1. An antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 having at least 80% sequence identity to the LCDR1 from a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

2. The antibody or antigen binding fragment of claim 1, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acid sequence of an of a HCDR1, HCDR2, and HCDR3 of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence of the LCDR1 from the light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

3. The antibody or antigen binding fragment of claim 1, wherein the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

4. The antibody or antigen binding fragment of claim 3, wherein the heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of CP 16 or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and / or the light chain variable region comprises the amino acid sequence of a light chain variable region of CP 16 or CP 17 light chain (SEQ ID NOs: 1 and 7).

5. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is human, chimeric, or humanized.

6. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen binding fragment binds to a calprotectin complex with a KD of about 10'6M / L to about 10'12M / L.

7. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen binding fragment binds to a calprotectin complex with an EC50 of less than about 1 nM to about 100 nM.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with a KD of about 10'6M / L to about 10'12M / L.

9. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen binding fragment binds to a calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex with an EC50 of less than about 1 nM to about 100 nM.

10. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is a neutralizing antibody or antigen binding fragment.

11. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is not a neutralizing antibody or antigen binding fragment.

12. The antibody or antigen binding fragment of claim 1, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

13. The antigen binding fragment of claim 1, wherein the antigen binding fragment is a single chain variable fragment (scFv), F(ab’)2, Fab’, fragment antigen binding protein (Fab), Fv, or rlgG.

14. The antigen binding fragment of claim 13, wherein the antigen binding fragment is a Fab.

15. A polypeptide comprising the antibody or antigen binding fragment of claim 1.

16. The polypeptide of claim 15, wherein the polypeptide comprises at least two antigen binding fragments.

17. The polypeptide of claim 15, wherein the polypeptide is multivalent.

18. The polypeptide of claim 15, wherein the polypeptide is bispecific.

19. The polypeptide of claim 15, wherein the antibody or antigen binding fragment are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

20. The polypeptide of claim 19, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

21. The polypeptide of claim 19, wherein the reporter system comprises a split luciferase reporter system.

22. The polypeptide of claim 21, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

23. The polypeptide of claim 21 , wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

24. The polypeptide of claim 21, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

25. The polypeptide of claim 21, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked at the C-terminus of the antibody or antigen binding fragment.

26. The polypeptide of claim 21, wherein the polypeptide comprises a CP16 heavy chain linked to SmBiT or LgBiT.

27. A composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies, antigen binding fragments, and / or polypeptides according to any one of claims 1-26.

28. A nucleic acid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibody, antigen binding fragment, and / or polypeptide of any one of claims 1-26.

29. A vector comprising the nucleic acid of claim 28.

30. A host cell comprising the nucleic acid or vector of claim 28.

31. The host cell of claim 30, wherein the host cell is a prokaryote or a eukaryote.

32. The host cell of claim 30, wherein the host cell is a bacterial cell, fungal cell, mammalian cell, or insect cell.

33. The host cell of claim 30, wherein the host cell is E. coli.

34. A method of a making a cell comprising transferring the nucleic acid(s) of claim 28 or the vector of claim 29 into a cell.

35. The method of claim 34, wherein the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.

36. The method of claim 35, wherein the method further comprising isolating the expressed polypeptide.

37. A composition comprising, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

38. The composition of claim 37 comprising, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise the CP16 and / or CP17 heavy chain (SEQ ID NOs: 2 and 8), and the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 heavy chains are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system.

39. The composition of claim 37, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope, or to different epitopes.

40. The composition of claim 37, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium dependent manner.

41. The composition of claim 37, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium independent manner.

42. The composition of claim 37, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

43. The composition of claim 37, wherein the reporter system comprises a split luciferase reporter system.

44. The composition of claim 43, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

45. The composition of claim 43, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

46. The composition of claim 43, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker comprising GS-DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS- DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

47. The composition of claim 43, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides is linked at the C-terminus to at least one fragment of the split luciferase.

48. The composition of claim 43, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs.

49. The composition of claim 43, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16- LgBiT).

50. The composition of claim 49, wherein at least one CP 16 light chain is linked to SmBiT or LgBiT.

51. The composition of claim 43, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17- LgBiT).

52. The composition of claim 43, wherein at least one CP17 light chain is linked to SmBiT or LgBiT.

53. The composition of claim 43, wherein the concentration of CP16-SmBiT and / or CP17- SmBiT is about 2-100 nM.

54. The composition of claim 43, wherein the concentration of CP16-LgBiT and / or CP17- LgBiT is, is at least, or is at most about 2-100 nM.

55. The composition of claim 37, comprising at least one of CP16-LgBiT and / or CP17- LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

56. The composition of claim 37 comprising a) CP16-LgBiT and CP16-SmBiT; b) CP16- LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16-SmBiT; or d) CP17-LgBiT and CP17- SmBiT.

57. The composition of claim 55, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

58. The composition of claim 56, comprising a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT.

59. The composition of claim 56, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 :1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3:10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

60. The composition of claim 55, comprising about 1-25 nM CP16-LgBiT and about 1.925- 50 nM CP16-SmBiT.

61. The composition of claim 55, comprising about 1-25 nM CP16-LgBiT and about 0.9625-37.5 nM CP17-SmBiT.

62. The composition of claim 37, further comprising Ca2+.

63. The composition of claim 62, comprising at least 2 mM Ca2+.

64. The composition of claim 37, further comprising furimazine.

65. The composition of claim 37, further comprising TBST, BSA, and CaCh.

66. An engineered stabilized calprotectin complex comprising S100A9 linked to S100A8 to form a heterodimeric fusion protein.

67. The engineered stabilized calprotectin complex of claim 66, wherein the S100A9 and S100A8 are linked by a GGS peptide linker.

68. The engineered stabilized calprotectin complex of claim 66, wherein the complex is further linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tags.

69. The engineered stabilized calprotectin complex of claim 68, wherein the complex is linked to a SNAP-TAG protein for selective biotinylation.

70. A polypeptide comprising an antibody or antigen binding fragment that binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes linked with a fragment of a split luciferase wherein the split luciferase comprises a NANOBIT system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NANOLUC luciferase.

71. The polypeptide of claim 70, wherein the linker comprises, consists essentially of, or consists of a peptide linker.

72. The polypeptide of claim 70, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker that is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

73. The polypeptide of claim 70, wherein the antibody or antigen binding fragment and the fragment of a split luciferase are linked with a peptide linker comprising GS-DYKDDDDK- GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS-DYKDDDDK-GS (SEQ ID NO: 13).

74. The polypeptide of claim 70, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium dependent manner.

75. The polypeptide of claim 70, wherein the antibody or antigen binding fragment binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calprotectin complex epitopes in a calcium independent manner.

76. The polypeptide of claim 70, wherein the antibody or antigen binding fragment binds the calprotectin (S100A8 / 9)2 tetramer complex and / or S100A8 / 9 heterodimer complex.

77. The polypeptide of claim 70, wherein the antibody or antigen binding fragment comprises an amino acid sequence at least 80% identical to the heavy chain and / or light chain of CP 16 and / or CP 17 (SEQ ID NOs: 1, 2, 7, and 8).

78. The polypeptide of claim 70, wherein the antigen binding fragment comprises a fragment antibody (Fab).

79. A fragment antibody (Fab) comprising the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

80. A fragment antibody (Fab)comprising the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

81. A fragment antibody (Fab)comprising the heavy chain and light chain of CP17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter by a linker peptide comprising SEQ ID NO: 13.

82. A fragment antibody (Fab)comprising the heavy chain and light chain of CP17 (SEQ ID NOs: 7 and 8) linked to a split luciferase reporter SmBiT or LgBiT by a linker peptide comprising SEQ ID NO: 13.

83. A composition comprising a first fragment antibody (Fab) and a second Fab, wherein the first Fab comprises the heavy chain and light chain of CP 16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter SmBiT by a linker peptide comprising SEQ ID NO: 13; and thesecond Fab comprises the heavy chain and light chain of CP16 (SEQ ID NOs: 1 and 2) linked to a split luciferase reporter LgBiT by a linker peptide comprising SEQ ID NO: 13.

84. A kit comprising the antibody, antigen binding fragment, polypeptide, composition, fragment antibody (Fab), and / or engineered stabilized calprotectin complex according to any one of the preceding claims.

85. The kit of claim 84, wherein the kit further comprises one or more negative or positive control samples.

86. The kit of claim 84, wherein the kit comprises an ELISA for detecting calprotectin.

87. The kit of claim 84, wherein the calprotectin antibody or binding fragment is operatively linked to a solid support.

88. The kit of claim 84, wherein the kit comprises at least two anti-calprotectin antibodies, at least two anti-calprotectin antibody binding fragments, or one anti-calprotectin antibody and one anti-calprotectin antibody binding fragment.

89. The kit of claim 84, wherein the one or more anti-calprotectin antibodies or calprotectin antibody binding fragments is linked to a detectable label.

90. The kit of claim 84, further comprising buffers, solvents, and / or containers.

91. A method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subject, or extract thereof, with at least one antibody or antigen binding fragment of claim 1.

92. The method of claim 91 , wherein the method further comprises incubating the antibody, antigen binding fragment, polypeptide, or composition with the biological sample, or extract thereof, under conditions that allow for the binding of the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition to bind to antigens in the biological sample or extract thereof.

93. The method of claim 91, wherein the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment, polypeptide, or polypeptide of the composition.

94. The method of claim 93, wherein the detecting comprises measuring of a signal produced by a reporter system.

95. A method of measuring the concentration of calprotectin complex in a biological sample, the method comprising contacting the biological sample with the antibody, antigen binding fragment, polypeptide, composition, or kit component of any one of claims 1-90, and measuring the level of interaction between the antibody, antigen binding fragment, Fab, or polypeptide with the calprotectin complex.

96. The method of claim 95, wherein the measuring comprises measuring a level of luminescence.

97. The method of claim 91 or 95, wherein the antibody, antigen binding fragment, polypeptide, or Fab of the composition is in solution or is linked to a solid support.

98. The method of claim 97, wherein the solution comprises furimazine, TBST, BSA, and / or CaCh.

99. The method of claim 97, wherein the solid support comprises a lateral flow assay strip, or a paper.

100. The method of claim 91 or 95, wherein the biological sample, or an extract thereof, comprises serum, plasma, whole blood, or stool.

101. A method for treating a subject with an inflammatory disorder, the method comprising administering a treatment for the inflammatory disorder to a subject that has had the level of calprotectin evaluated in a biological sample from the subject using at least one antibody or antigen binding fragment of claim 1.

102. The method of claim 101, wherein the biological sample comprises a serum, plasma, blood, or fecal sample.

103. The method of claim 101, wherein the level of calprotectin in the biological sample from the subject has been quantitated.

104. The method of claim 103, wherein the level of calprotectin is normalized.

105. The method of claim 101, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is greater than the level of a calprotectin in a control sample.

106. The method of claim 101, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is less than the level of a calprotectin in a control sample.

107. The method of claim 101, wherein the subject has or has been determined to have a level of calprotectin in the biological sample that is not significantly different than the level of calprotectin in a control sample.

108. The method of claim 101, wherein the control comprises the level of calprotectin that is representative of the level of calprotectin in a subject with an inflammatory disorder.

109. The method of claim 101, wherein the control comprises the level of calprotectin that is representative of the level of calprotectin in a subject without an inflammatory disorder.

110. The method of claim 101, wherein the inflammatory disorder comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

111. The method of claim 110, wherein the treatment comprises an anti-inflammatory medication, an immunomodulator, an immunosuppressant, a small molecule, a biologic, an antibiotic, an anti diarrheal, a pain reliever, a vitamin, a dietary support, and / or a surgery.

112. The method of claim 110, wherein the treatment comprises isoniazid, rifampin, rifabutin, rifapentine, pyrazinamide, and / or ethambutol.

113. A method for detecting calprotectin in a sample, the method comprising, i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; andiii) measuring levels of a signal produced by the reporter system during and / or after the incubating.

114. The method of claim 113, wherein sample comprises, or comprises an extract of, serum, plasma, whole blood, or stool.

115. The method of claim 113, comprising more than one polypeptide, and wherein the polypeptides bind to the same epitope.

116. The method of claim 113, comprising more than one polypeptide, and wherein the polypeptides bind to different epitopes.

117. The method of claim 113, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium dependent manner.

118. The method of claim 113, comprising more than one polypeptide, and wherein the polypeptides bind to an epitope in a calcium independent manner.

119. The method of claim 114, wherein the reporter system comprises a gene expression reporter system, a fluorescence reporter system, a luminescence reporter system, a chemiluminescence reporter system, and / or a substrate detection reporter system.

120. The method of claim 114, wherein the reporter system comprises a split luciferase reporter system.

121. The method of claim 120, wherein the split luciferase reporter system comprises a NANOBIT luciferase reporter system, and the fragments of the split luciferase comprise SmBiT and LgBiT from NanoLuciferase (NANOLUC).

122. The method of claim 120, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked with a peptide linker that is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

123. The method of claim 120, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs and at least one fragment of the split luciferase are linked with a peptide linker comprising GS- DYKDDDDK-GGSGGGGSGGSSSGG (SEQ ID NO: 19), GS-DYKDDDDK-GGGGSGGGGS (SEQ ID NO: 18), GS-DYKDDDDK-GGGGS (SEQ ID NO: 17), or GS- DYKDDDDK-GS (SEQ ID NO: 13).

124. The method of claim 120, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides and at least one fragment of the split luciferase are linked at the C-terminus of the heavy chain or light chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fabs.

125. The method of claim 120, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP16 Fabs, wherein at least one CP16 heavy chain is linked to SmBiT (CP16-SmBiT) or LgBiT (CP16- LgBiT).

126. The method of claim 125, wherein at least one CP16 light chain is linked to SmBiT or LgBiT.

127. The method of claim 120, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CP17 Fabs, wherein at least one CP17 heavy chain is linked to SmBiT (CP17-SmBiT) or LgBiT (CP17- LgBiT).

128. The method of claim 120, wherein at least one CP17 light chain is linked to SmBiT or LgBiT.

129. The method of claim 120, comprising incubating the sample with a concentration of CP16-SmBiT and / or CP17-SmBiT that is, is at least, or is at most about 2-100 nM.

130. The method of claim 120, comprising incubating the sample with a concentration of CP16-LgBiT and / or CP17-LgBiT that is, is at least, or is at most about 2-100 nM.

131. The method of claim 113, comprising at least one of CPI 6-LgBiT and / or CP 17-LgBiT, and at least one CP16-SmBiT and / or CP17-SmBiT.

132. The method of claim 113, comprising a) CP16-LgBiT and CP16-SmBiT; b) CP16- LgBiT and CP17-SmBiT; c) CP17-LgBiT and CP16-SmBiT; or d) CP17-LgBiT and CP17- SmBiT.

133. The method of claim 131, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is not 1 : 1.

134. The method of claim 131, comprising incubating the sample with a lower concentration of CP16-LgBiT and / or CP17-LgBiT relative to CP16-SmBiT and / or CP17-SmBiT.

135. The method of claim 131, wherein the ratio of CP16-LgBiT and / or CP17-LgBiT to CP16-SmBiT and / or CP17-SmBiT is, is at least, or is at most about 1 : 1, 1 :1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9, 1 :2, 3:4, 3:5, 3:7, 3:8, 1 :3, 3:10, 3: 11, 1 :4, 3: 13, 3: 14, 1 :5, 3: 16, 3: 17, 1 :6, 3: 19, 3:20, or 1 :7.

136. The method of claim 131, comprising incubating the sample with, with at least, or with at most about 1-25 nM CP16-LgBiT and about 1.925-50 nM CP16-SmBiT.

137. The method of claim 113, further comprising contacting and incubating the sample with Ca2+.

138. The method of claim 137, wherein the incubating with Ca2+is at concentration of at least 2 mM.

139. The method of claim 113, further comprising contacting and incubating the sample with furimazine.

140. The method of claim 113, further comprising contacting and incubating the sample with TBST, BSA, and / or CaCl2.

141. The method of claim 113, wherein the incubating is for, for at least, or for at most about 2 to 30 minutes.

142. The method of claim 113, wherein the measuring comprises quantitatively measuring luminescence.

143. The method of claim 113, wherein the method comprises detecting the luminescence signal in a liquid solution.

144. The method of claim 113, wherein the method comprises detecting the luminescence signal on a solid support.

145. The method of claim 142, wherein the luminescence measuring is mediated through imaging.

146. The method of claim 145, wherein the imaging is by a consumer grade camera.

147. The method of claim 113, wherein the method is performed in a point-of-care and / or at-home setting.

148. The method of claim 113, wherein the levels of calprotectin in a sample are quantified and compared to a negative or positive control.

149. The method of claim 148, wherein the levels of calprotectin in a sample are noted as abnormal when the detected signal is or is at least about 2-200 fold higher than the negative control.

150. A method of treating a disease or disorder in a subject, comprising performing the method of claim 113, and tailoring treatment based upon levels of calprotectin detected.

151. The method of claim 150, wherein the disease or disorder is characterized by inflammation.

152. The method of claim 150, wherein the disease or disorder comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, cancer, rheumatoid arthritis, tuberculosis (TB), and / or COVID-19 infection.

153. The method of claim 152, wherein the disease or disorder comprises IBD, and measurements of calprotectin levels in serum, plasma, whole blood, and / or stool, or extracts thereof, indicate disease severity and / or disease progression / remission.

154. A method for identifying a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and iv) classifying the disease state based upon the level of calprotectin detected.

155. The method of claim 154, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

156. A method for measuring the efficacy of a regimen for treating a disease state in a subj ect comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; and iv) classifying the treatment regimen as effective or not effective for treatment of the disease state based upon the level of calprotectin detected.

157. The method of claim 156, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

158. A method for classifying the severity of a disease state in a subject comprising, detecting levels of calprotectin in a sample from the subject by: i) contacting the sample with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides that comprise an amino acid sequence at least 70% identical to the CP 16 and / or CP 17 heavy chain (SEQ ID NOs: 2 and 8), and an amino acid sequence at least 70% identical to the CP16 and / or CP17 light chain (SEQ ID NOs: 1 and 7); wherein the CP16 and / or CP17 amino acid sequences are linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components of a reporter system; ii) incubating the sample and polypeptides for a period of time; and iii) measuring levels of a signal produced by the reporter system during and / or after the incubating; andiv) classifying the severity of a disease state based upon the level of calprotectin detected.

159. The method of claim 158, wherein the disease state comprises inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis, Crohn’s disease, inflammatory diseases, rheumatoid arthritis, COVID-19 infection, tuberculosis (TB), and / or cancer.

160. Use of the antibody, composition, method, engineered stabilized calprotectin complex, polypeptide, and / or kit of any one of the preceding claims for medicinal and / or biomedical research purposes.

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