System and method for detection of 14-3-3 ETA protein in capillary blood

Abstract

Aspects of the disclosure provide for devices and methods of use thereof for detecting the presence of 14-3-3η protein in capillary blood samples from subjects suspected of having or at risk of developing arthritic conditions. In an example, a method includes obtaining a capillary blood sample from such a subject, and applying the capillary blood sample to a lateral flow device under conditions sufficient for formation of an immunocomplex comprising an immobilized anti-14-3-3η antibody coupled to a 14-3-3η protein that is further coupled to a labeled anti-14-3-3η antibody, wherein formation of the immunocomplex, and visualization thereof, is indicative of the arthritic condition.

Description

SYSTEM AND METHOD FOR DETECTION OF 14-3-3 ETA PROTEIN IN CAPILLARYBLOOD CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 570,489,filed March 27, 2024, which is incorporated herein by reference in its entirety. FIELD OF DISCLOSURE

[0002] The present disclosure relates generally to systems and methods of detecting 14-3-3ηprotein for use in determining and / or monitoring arthritic conditions in subjects suspected of having, or at risk of developing, arthritic conditions. Specifically, the invention relates to a lateral flow assay capable of detecting 14-3-3η protein in capillary blood samples, for use as a point-of- care diagnostic tool. BACKGROUND OF THE DISCLOSURE

[0003] 14-3-3 proteins are a family of conserved intracellular regulatory molecules that areubiquitously expressed in eukaryotes, and may be considered evolved members of the Tetratrico Peptide Repeat superfamily. They have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. Indeed, more than 100 signaling proteins have been reported as 14-3-3 ligands. They generally have 9 or 10 alpha helices, and usually form homo- and / or hetero-dimer interactions along their amino-termini helices. The 14-3-3 proteins contain a number of known domains, including regions for divalent cation interaction, phosphorylation & acetylation, and proteolytic cleavage, among others. There are seven distinct genetically encoded isoforms in this family that are known to be expressed in mammals, designated as 14-3-3α / β (alpha / beta), 14-3-3δ / ξ (delta / zeta), 14-3-3ε (epsilon), 14-3- 3γ (gamma), 14-3-3η (eta), 14-3-3τ / θ (tau / theta), and 14-3-3σ (sigma / stratifin).

[0004] Arthritis, or arthralgia, generally refers to inflammatory disorders of the joints of thebody, and is usually accompanied by pain, swelling and stiffness. Arthritis may result from any of several causes including infection, trauma, degenerative disorders, metabolic disorders or disturbances or other unknown etiologies. Osteoarthritis (OA) is a common form of non- inflammatory arthritis that may occur following trauma to a joint, following an infection of a Page 1 of 66 1104081999\1\AMERICASjoint or simply as a result of aging. Osteoarthritis is also known as degenerative joint disease. Rheumatoid arthritis (RA) is traditionally considered a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It is a disabling and painful inflammatory condition which can lead to substantial loss of mobility due to pain and joint destruction.

[0005] An association between 14-3-3η protein and rheumatoid arthritis (RA) was reportedfor the first time by Kilani et al. (J Rheumatol.2007;34(8):1650‐1657). Briefly, it was found that serum 14-3-3η protein was present at significantly higher levels in the synovial fluid and serum of patients with arthritis compared to healthy individuals, and a positive association was found between 14-3-3η and matrix metalloproteinases (MMPs). MMPs have been confirmed to be responsible for the structure erosion of RA, as well as being a potential indicator of progressive joint damage and disease activity (Galil SM et al., Int J Rheum Dis.2016;19(4):377‐384; Ma JD et al., Arthritis Res Ther.2015;17:289; Green MJ et al., Rheumatology.2003;42(1):83‐88). These findings suggest that 14-3-3η may be relevant to the course of cartilage and bone destruction, via regulation of the expression of MMPs. As a biomarker of arthritic conditions, serum 14-3-3η protein levels are also associated to some extent with inflammatory responses and joint damage (Maksymowych WP et al., Arthritis Res Ther.2014;16(2):R99).

[0006] The causative agents for RA, AS and OA are currently not well defined. However,the physiological events associated with progression of the disease, from prolonged periods of swelling and inflammation caused by excessive synovial fluid accumulation in the joints, through degradation and deterioration of the cartilage and underlying bone tissues by degradative enzyme activities, and the proliferation of fibroblast-like synoviocyte (FLS) cells resulting in permanent structural damage, are known. If detected early enough, the potential long-term deleterious effects of disease can be reversed, or at least minimized, with appropriate physical and medical therapies. The importance of proper diagnosis, particularly in the early stages of disease, is often underestimated by primary care physicians, but may prevent serious, lifelong arthritic complications for patients. Specifically, in early arthritis there is a window of opportunity, during which the disease can be treated before irreversible joint erosions evolve. This window is usually open about three to six months after onset of disease.

[0007] Point-of-care (POC) testing refers to near-patient detection techniques. Compared toconventional laboratory testing, POC testing is advantageous in that results can be obtained Page 2 of 66 1104081999\1\AMERICASwithin minutes, as compared to conventional methods that can take hours or days in centralized laboratories. Accordingly, POC testing can enable more rapid clinical decision making in the process of diagnosis, treatment choice and monitoring, and prognosis. Unfortunately, however, no POC test options for the detection of 14-3-3η protein have been described to date. Existing tests require the use of serum samples, or synovial fluid, and the level of 14-3-3η protein in serum is at least five times lower than that of synovial fluid.

[0008] Moreover, and significantly, POC testing typically requires the use of capillary blood,and there are well known inconsistencies in protein levels between capillary versus venous blood samples (Astrand R et al., Clinical Chemistry and Laboratory Medicine.2012; 50(6): 1055- 1061). Indeed, measurement of biomarkers in biofluids using POC testing methodology faces numerous challenges including but not limited to detection sensitivity, difficulties in sample preparation, potential need for multiplexed devices for diagnostic confirmation, and the like, and even commercialized assays can require optimization prior to employment for routine use (Khelifa L et al., Lab Chip.2022; 22: 2451-2475). Accordingly, it is at best uncertain whether 14-3-3η can be reproducibly detected in capillary blood, let alone whether a 14-3-3η POC test that relies on capillary blood samples can be accurately and reliably used as a diagnostic, prognostic, and / or theranostic tool for arthritic conditions. SUMMARY OF DISCLOSURE

[0009] The present disclosure provides methods, devices and kits for assaying the level of 14-3-3η protein in a subject.

[0010] In a first aspect, a method of assaying the level of 14-3-3η protein in a subject suspectedof having or at risk of developing an arthritic condition is provided, comprising: a) contacting a capillary blood sample from a subject with a capture agent associated with a substrate, wherein the capture agent can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, and wherein the substrate is a portion of a lateral flow immunochromatographic cassette; and b) detecting the binding of the capture agent to 14-3-3η protein with a detection agent that binds to said 14-3-3η protein within the lateral flow immunochromatographic cassette to produce a visible test line and determining whether the level of 14-3-3η protein is at or above a threshold level based on the resulting test line intensity, where the capture agent comprises one or more anti-14-3-3η Page 3 of 66 1104081999\1\AMERICASantibodies, the detection agent comprises one or more labeled anti-14-3-3η antibodies, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent.

[0011] In another aspect, a method is provided for treating an arthritic disease in a subject,comprising where at least one capillary blood sample taken from the subject is tested for the presence of 14-3-3η in a lateral flow assay, and wherein an arthritis therapy is initiated in the event that the level of 14-3-3η protein in the at least one capillary blood sample is at or above a threshold level within the lateral flow immunochromatographic cassette.

[0012] In another aspect, a method is provided for treating a patient with arthritis, comprisinga) testing for the presence of 14-3-3η protein in a capillary blood sample of a patient within a lateral flow immunochromatographic cassette; b) identifying the patient as having 14-3-3η protein in the capillary blood sample; and c) initiating treatment of the patient having a capillary blood sample that is positive for 14-3-3η protein.

[0013] In another aspect, a method is provided for detecting a reduction in, or absence of,therapeutic efficacy of an arthritis therapy in a subject that has been administered the arthritis therapy, comprising: a) detecting in a first capillary blood sample obtained from the subject at a first time point the presence or absence of at least a threshold level of 14-3-3η protein within a lateral flow immunochromatographic cassette; and b) detecting in a second capillary blood sample obtained from the subject at a second time point the presence or absence of at least a threshold level of 14-3-3η protein within the lateral flow immunochromatographic cassette, where an increase in the level of 14-3-3η protein and / or an increase in test line intensity in the second capillary blood sample indicates a reduction in, or absence of, therapeutic efficacy of the arthritis therapy.

[0014] In another aspect, a method is provided for detecting a reduction in therapeutic efficacyof an arthritis therapy in a subject that has been administered the arthritis therapy, comprising performing a lateral flow assay to determine whether two or more capillary blood samples taken at different time points from the subject contain a level of 14-3-3η protein that is at least a threshold level; wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette; and wherein an increase in or the presence of the threshold level Page 4 of 66 1104081999\1\AMERICASof 14-3-3η protein in the two or more samples indicates a reduction in, or absence of, therapeutic efficacy.

[0015] In another aspect, a device is provided for assaying the level of 14-3-3η protein in acapillary blood sample from a subject, the device comprising: a capture agent associated with a substrate, wherein the capture agent comprises one or more anti-14-3-3η antibodies that can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, and wherein the substrate is a portion of a lateral flow immunochromatographic cassette comprising, in order of flow, a buffer zone, a sample receiving zone comprising a sample pad, a conjugate pad comprising a detection agent comprising one or more labeled antibodies, and a test zone comprising said substrate, configured such that: the buffer zone is located upstream of the sample receiving zone and the test zone is located downstream of the sample receiving zone, upon flow of the sample through the lateral flow immunochromatographic cassette, labeled 14-3-3η protein from the sample will bind with the capture agent in the test zone to form a detectable complex producing the test line, as part of a running buffer sweep; wherein the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent; preferably wherein the capture agent is an antibody produced by hybridoma clone AUG4-ETA-8F10 or an antigen binding fragment thereof, and the detection agent is an antibody produced by hybridoma clone AUG3-CKNS-7F8 or an antigen binding fragment thereof.

[0016] In another aspect, a method is provided for detecting 14-3-3η protein in a sample,comprising contacting the sample receiving pad of a device according to the present disclosure with a capillary blood sample from a subject, followed by contacting the buffer zone of the device with buffer, such that 14-3-3η protein present in the sample will bind with labeled antibodies in the conjugate pad, and the labeled 14-3-3η protein will bind with the capture agent in the test zone to form a detectable complex producing a visible test line, as part of a running buffer sweep; and determining the presence and / or amount of 14-3-3η protein in the sample based on the resulting test line intensity. In embodiments, a positive test line intensity is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η.

[0017] In another aspect, a kit for assaying the level of 14-3-3η protein in a capillary bloodsample obtained from a subject, comprises: a device of the present disclosure, and a running buffer. Page 5 of 66 1104081999\1\AMERICASINCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a graph showing correlation between concentration of 14-3-3η in capillaryblood versus venous blood sample.

[0020] FIG. 2 depicts a simplified lateral flow assay format used in Example 3 for proof-of-concept.

[0021] FIG. 3 depicts a proof of concept lateral flow design for feasibility testing.

[0022] FIG. 4 depicts the results of blood leeching experiments conducted with varioustypes of materials for use with lateral flow devices of the present disclosure.

[0023] FIG. 5 depicts images of lateral flow devices of the present disclosure andcorresponding line signal intensity data illustrating the detection of 14-3-3η protein from whole blood sample.

[0024] FIG. 6 depicts images of lateral flow devices of the present disclosure with test linesignal intensity for different concentrations of 14-3-3η.

[0025] FIG. 7 depicts an exemplary lateral flow device of the present disclosure.

[0026] FIG. 8 depicts line signal intensity data for varying test line concentration.

[0027] FIG. 9 depicts line signal intensity data for varying 14-3-3η concentration.

[0028] FIG. 10 depicts a reference guide for evaluating test line intensity.

[0029] FIG. 11 depicts the results of test line intensity evaluation for serum samples using areference guide to group 14-3-3η concentration.

[0030] FIG. 12 depicts the correlation between 14-3-3η concentration and group based on areference guide for 14-3-3η spiked whole blood. Page 6 of 66 1104081999\1\AMERICASDETAILED DESCRIPTION I. Definitions

[0031] For purposes of interpreting this specification, the following definitions will apply,and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0032] “About” as used herein when referring to a measurable value such as an amount, atemporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0033] As used herein, “w / v” refers to the weight of the component in a given volume ofsolution.

[0034] “Ranges”: throughout this disclosure, various aspects of the disclosure can bepresented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0035] “Arthritic condition,” “arthritis,” and “arthralgia” are used interchangeably, andgenerally refer to, except where indicated, an inflammatory disorder of the joints of the body. Pain, swelling, stiffness, and difficulty of movement are frequently associated with arthritic conditions. Arthritis consists of more than 100 different conditions. These can be anything from relatively mild forms to crippling systemic forms. An arthritic condition may result from any of several causes, including infection, trauma, degenerative disorders, metabolic disorders or Page 7 of 66 1104081999\1\AMERICASdisturbances, or other unknown etiologies. An arthritic condition may be more specifically described according to the subtype, for example, rheumatoid arthritis, and other conditions where there may be joint involvement such as: mixed connective tissue disease (MCTD), crystal induced arthritis, reactive arthritis, spondyloarthropathy (e.g. ankylosing spondylitis), osteoarthritis, sarcoidosis, palindromic rheumatism, post traumatic arthritis, malignancy related arthritis, septic arthritis, lyme arthritisbacterial, infectious arthritis, etc. Arthritis may further accompany other identified disorders, including gout, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, etc. Well-defined arthritic condition refers to knowledge regarding the type of arthritis and its stage, e.g., onset, remission, relapse etc.

[0036] “Immunological binding” and “formation of an immune complex” and “formation ofan immunocomplex” are used interchangeably and as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties can be quantified using methods well known in the art. For example, see Davies et al. (1990) Annual Rev. Biochem.59:439-473. An antibody, or antigen-binding fragment thereof, is said to “specifically bind,” “immunologically bind,” and / or is “immunologically reactive” if it reacts at a detectable level (within, for example, a lateral flow assay) with ligand, and does not react detectably with other ligands, including other isoforms, under similar conditions.

[0037] “Antibody” refers to a composition comprising a protein that binds specifically to acorresponding antigen and has a common, general structure of immunoglobulins. The term antibody specifically covers polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species. Typically, an antibody will comprise at least two heavy chains and two light chains interconnected by disulfide bonds, which when combined form a binding domain that interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3, and may be of the mu, delta, gamma, alpha or epsilon isotype. Similarly, the light chain is comprised of a light chain variable region (VL) and a Page 8 of 66 1104081999\1\AMERICASlight chain constant region (CL). The light chain constant region is comprised of one domain, CL, which may be of the kappa or lambda isotype. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The heavy chain constant region mediates binding of the immunoglobulin to host tissue or host factors, particularly through cellular receptors such as the Fc receptors (e.g., FcγRI, FcγRII, FcγRIII, etc.). As used herein, antibody also includes an antigen binding portion of an immunoglobulin that retains the ability to bind antigen. These include, as examples, F(ab), a monovalent fragment of VL CL and VH CH antibody domains; and F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term antibody also refers to recombinant single chain Fv fragments (scFv) and bispecific molecules such as, e.g., diabodies, triabodies, and tetrabodies (see, e.g., U.S. Pat. No.5,844,094).

[0038] “Antigen” is to be construed broadly and refers to any molecule, composition, orparticle that can bind specifically to an antibody. An antigen has one or more epitopes that interact with the antibody, although it does not necessarily induce production of that antibody.

[0039] “Contacting” as used herein includes in solution and solid phase, for examplecontacting a capillary blood sample with capture agent, or a detection agent, for example contacting a sample with an antibody or antigen-binding fragment thereof. Contacting also refers to, for example, contacting a capillary blood sample to a sample pad of a lateral flow device.

[0040] “Effective amount,” or “therapeutically effective amount,” as used herein refers to anamount of therapeutic agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease, for example an arthritic condition. When administered to a subject, a dosage will generally be used that will achieve target tissue / cell concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and / or underlying causes of an arthritic condition. Page 9 of 66 1104081999\1\AMERICAS

[0041] “Epitope” refers to a determinant capable of specific binding to an antibody. Epitopesare chemical features generally present on surfaces of molecules and accessible to interaction with an antibody. Typical chemical features are amino acids and optionally sugar moieties, having three-dimensional structural characteristics as well as chemical properties including charge, hydrophilicity, and lipophilicity. Conformational epitopes are distinguished from non- conformational epitopes by loss of reactivity with an antibody following a change in the spatial elements of the molecule without any change in the underlying chemical structure.

[0042] “Humanized antibody” refers to an immunoglobulin molecule containing a minimalsequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. A humanized antibody will also encompass immunoglobulins comprising at least a portion of an immunoglobulin constant region (Fc), generally that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Reichmann et al, Nature 332:323-329 (1988)).

[0043] “Isotype” refers to an antibody class defined by its heavy chain constant region.Heavy chains are generally classified as gamma, mu, alpha, delta, epsilon and designated as IgG, IgM, IgA, IgD, and IgE. Variations within each isotype are categorized into subtypes, for example subtypes of IgG are divided into IgG1, IgG2, IgG3, and IgG4, while IgA is divided into IgA1 and IgA2. The IgY isotype is specific to birds.

[0044] “Monoclonal antibody” or “monoclonal antibody composition” refers to a preparationof antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Page 10 of 66 1104081999\1\AMERICAS

[0045] The term “human monoclonal antibody” includes antibodies displaying a singlebinding specificity which have variable and / or constant regions (if present) derived from human immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

[0046] “Single chain Fv” or “scFv” refers to an antibody comprising the VH and VL regionsof an antibody, wherein these domains are present in a single polypeptide chain. Generally, an scFv further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0047] "Autoantibodies" are endogenous antibodies that specifically bind self antigens, i.e., anormal tissue component. An autoantibody is produced in response to a naturally occurring antigen of the same body (e.g., 14-3-3η protein) that produces the autoantibody.

[0048] “Subject” and “patient” are used interchangeably and refer to, except where indicated,mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species.

[0049] “Recombinant antibody” refers to all antibodies produced by recombinant techniques.These include antibodies obtained from an animal that is transgenic for the immunoglobulin locus, antibodies expressed from a recombinant expression vector, and antibodies created, prepared, and expressed by splicing of any immunoglobulin gene sequence to any other nucleic acid sequence.

[0050] “14-3-3” and “14-3-3 protein” are used interchangeably and refer to at least onemember of the 14-3-3 family of conserved intracellular regulatory molecules that are ubiquitously expressed in eukaryotes. There are seven distinct genetically encoded isoforms of the 14-3-3 proteins that are known to be expressed in mammals, with each isoform comprising between 242- 255 amino acids. The seven 14-3-3 protein isoforms are designated as 14-3-3α / β (alpha / beta), 14- 3-3δ / ξ (delta / zeta), 14-3-3ε (epsilon), 14-3-3γ (gamma), 14-3-3η (eta), 14-3-3τ / θ (tau / theta), and 14-3-3σ (sigma / stratifin).14-3-3 proteins have a high degree of sequence similarity, and are known to undergo post-translational processing, e.g., phosphorylation, etc. See, e.g., Megidish et al. (1998) J. Biol. Chem.273: 21834-45. Consequently, anti-14-3-3η antibodies may bind to and / or Page 11 of 66 1104081999\1\AMERICASrecognize a 14-3-3η protein that has been modified, e.g., by natural (e.g., post-translational) or chemical processes.

[0051] "Diagnostic" means identifying the presence or nature of a pathologic condition in asubject based on the presence or absence and / or amount of 14-3-3η protein(s), and / or based on the presence or absence and / or amount of autoantibodies to 14-3-3η protein(s). A diagnostic method makes a correlation between the presence or absence and / or amount of 14-3-3η protein(s), and / or presence or absence, and / or amount of autoantibodies directed against 14-3-3η protein(s), and the occurrence of a specific disease, e.g., rheumatoid arthritis, or a group of diseases (e.g., arthritic conditions).

[0052] "Prognostic" means determining the potential progression or outcome of a pathologiccondition and / or disease process in a subject, with or without treatment, based on the presence / absence and / or amount of 14-3-3η protein(s), and / or based on the presence or absence and / or amount of autoantibodies to 14-3-3η protein(s). A prognostic method makes a correlation between the presence or absence and / or amount of 14-3-3η protein(s), and / or based on the presence or absence and / or amount of autoantibodies to 14-3-3η protein(s), and the progression and / or likely outcome of an arthritic condition.

[0053] "Theranostic" means determining the potential and / or likely reaction to a proposedtreatment protocol for a pathologic condition and / or disease process in a subject, based on the presence / absence and / or amount of 14-3-3η protein(s), and / or based on the presence or absence and / or amount of autoantibodies to 14-3-3η protein(s), and tailoring an appropriate treatment for the subject based on the results. A theranostic method makes a correlation between the presence / absence and / or amount of 14-3-3η protein(s), and / or based on the presence or absence and / or amount of autoantibodies to 14-3-3η protein(s), and the likely efficacy of different treatment options on the arthritic condition in the subject.

[0054] “Screening” as used herein refers to the application of a procedure or test to people whoas yet have no symptoms of a particular disease, for the purpose of determining their likelihood of having the disease. Those who have a positive result from the screening test will typically need further evaluation with subsequent diagnostic tests or procedures.

[0055] “Initiating an arthritis therapy” may comprise obtaining a venous blood, serum orsynovial fluid sample from the patient and performing a laboratory-based ELISA for one or more Page 12 of 66 1104081999\1\AMERICASarthritis markers, including 14-3-3η, to confirm the arthritis diagnosis prior to administering the desired arthritis therapy. II. Lateral Flow Device

[0056] In embodiments, 14-3-3η levels in a capillary blood sample may be assessed via alateral flow device of the present disclosure. Various lateral flow assay methods may be utilized to test for the presence, absence, and / or quantity of, 14-3-3η protein, generally in its natural configuration. In one embodiment, a “sandwich” assay method may rely on one or more anti-14- 3-3η antibodies immobilized on a solid support, which forms part of a complex that also includes a labeled anti-14-3-3η antibody or other labeled biomolecule capable of specifically binding to 14- 3-3η protein. In such an example assay methodology, the one or more immobilized anti-14-3-3η antibodies may specifically bind to an epitope that is different from the labeled anti-14-3-3η antibody or other labeled biomolecule. This may enable an ability to detect 14-3-3η protein in capillary blood by measuring a presence, and in some examples an amount, of the resultant immunocomplex.

[0057] Lateral flow devices, also referred to herein as lateral flow immunochromatographiccassettes or test strips, or simply test strips, of the present disclosure comprise a solid support on which a sample receiving area or sample pad, and a test zone, or capture pad, are supported. The solid support material is one which is capable of supporting the sample pad and capture pad and for providing for the capillary flow of sample out from the sample pad to the capture pad when the lateral flow test strip is exposed to an appropriate solvent or buffer which acts as a carrier liquid for the sample. General classes of materials that may be used as support include organic or inorganic polymers, and natural and synthetic polymers. More specific examples of suitable solid supports include, without limitation, glass fiber, cellulose, nylon, crosslinked dextran, various chromatographic papers and nitrocellulose. One particularly useful material is nitrocellulose.

[0058] It is to be understood that an anti-14-3-3η antibody or other biomolecule capable ofspecifically recognizing 14-3-3η can be immobilized at the test line, and a control biomolecule can be immobilized at the control line. The choice of control biomolecule can be readily selected by the skilled person. In one example, the control biomolecule can be biotin-coupled bovine serum albumin (BSA). In another example the control biomolecule can be Goat anti-Mouse IgG. The conjugate pad can include one or more antibodies or other biomolecules that specifically recognize 14-3-3η protein, linked to a detectable label. The conjugate pad additionally includes one or more Page 13 of 66 1104081999\1\AMERICASantibodies or other biomolecules that specifically recognize the control biomolecule, linked to a detectable label. In an example where the control biomolecule is biotin-coupled BSA, streptavidin linked to a detectable label may be included in the conjugate pad for use in detecting the control biomolecule.

[0059] Sample applied to the sample pad may flow via capillary action through the conjugatepad, to the capture pad, before being absorbed by the absorption pad.14-3-3η protein in the sample can be recognized by, e.g., anti-14-3-3η antibody or other biomolecule coupled to a detectable label, at the conjugate pad. The one or more antibodies or other biomolecules (e.g., streptavidin linked to a detectable label) capable of recognizing the control biomolecule, may migrate through the conjugate pad. 14-3-3η protein bound by the labeled anti-14-3-3η antibody or other labeled biomolecule may migrate to the capture pad, where an immunocomplex is formed at the test line between the immobilized anti-14-3-3η antibody or other biomolecule, the 14-3-3η protein, and the anti-14-3-3η antibody or other biomolecule coupled to a detectable label. At the control line, the one or more antibodies or other biomolecules coupled to a detectable label which specifically recognize the control biomolecule are bound thereto. In this way, 14-3-3η protein in a capillary blood sample may be detected using a lateral flow assay of the present disclosure. Addition of running buffer upstream of the sample receiving zone ensures that the capillary blood sample and detectable label flow to the test zone.

[0060] In embodiments, the conjugate pad may be treated with a solution that may include butis not limited to one or more of sodium borate, dextran, BSA, Tween-20, sodium azide, and the like. In examples, such a solution may be in a pH range of 7.5-8.5, for example pH 8.0. The drying of the labeled anti-14-3-3η antibody or other biomolecule and the labeled antibody or other biomolecule capable of specifically binding to the control biomolecule onto the conjugate pad may be conducted at temperatures ranging from about 35-60℃, for example about 35℃, 40℃, 45℃, 50℃, 55℃, or about 60℃.

[0061] The capillary blood sample applied to the sample pad may in some examples be dilutedin appropriate buffer (e.g., running buffer comprising one or more of HEPES, CaCl2, NaCl, NaN3, Tween-20 and 0.01% polyvinyl alcohol) and applied to sample pad. The dilution may be 1:1 sample-to-buffer, or the sample may be diluted by less (e.g., 1:0.5, 1:0.25, 1:0.1, 1:0.05, 1:0.01, etc.), or in some examples more (e.g., 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:2, 1:3, etc.). In some Page 14 of 66 1104081999\1\AMERICASembodiments, the capillary blood sample may not be diluted at all before being applied to sample pad.

[0062] Detectable labels useful in the lateral flow devices of the present disclosure includecompounds which are conjugated directly or indirectly to another biomolecule, e.g., an anti-14-3- 3η antibody as herein disclosed, to facilitate detection of an immunocomplex to which the labeled biomolecule is bound. Specific non-limiting examples of labels include fluorescent tags / particles, enzymatic linkages, radioactive isotopes (e.g., 14C, 32P, 125I, 3H isotopes and the like), microspheres, nanoparticles, and the like.

[0063] In embodiments, nanoparticles may comprise one or more of metal nanoparticles (e.g.,gold nanoparticles), magnetic nanoparticles, silica nanoparticles, and latex nanoparticles. In embodiments, the fluorescent particles can include one or more of Alexa fluoro 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, ruby (tris (2,2-bipyridyl) ruthenium), FITC (fluorescein isothiocyanate) rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl- rhodamine), Texas Red, DAPI (4,6-diamidino -2-phenylindole, coumarin, and the like, or may be time-resolved fluorescence (TRF).

[0064] In embodiments, the detection agent is detectably labeled with a label selected from thegroup consisting of a gold nanoparticle, a carbon nanoparticle, a colored latex bead, an enzyme, a fluorescent label, a luminescent label, and a radioactive label. In embodiments, the gold nanoparticle comprises a gold nanoshell, e.g. BioReady Gold Nanoshells – Carboxyl, PN GSXR150-5M (nanoComposix, San Diego, CA).

[0065] As discussed, sample pads of the lateral flow devices as herein disclosed initiallyreceive the capillary blood sample, and may optionally serve to remove particulates from the sample. Sample pads of the present disclosure may be comprised of various materials including but not limited to glass fiber, woven fibers, non-woven fibers, cellulosic fibers, and the like. A cellulose sample pad may be beneficial if a large bed volume (e.g., about 250 µl / cm2) is a factor in a particular embodiment. Sample pads may be treated with one or more release agents, such as buffers, salts, proteins, detergents, and surfactants. Such release agents may be useful, for example, to promote resolubilization of conjugate-pad constituents, and to block non-specific binding sites in other components of a lateral flow device, such as a nitrocellulose membrane. Representative release agents include but are not limited to trehalose or glucose (1%-5%), PVP or PVA (0.5%- Page 15 of 66 1104081999\1\AMERICAS2%), Tween 20 or Triton X-100 (0.1%-1%), casein (1%-2%), SDS (0.02%-5%), and PEG (0.02%- 5%).

[0066] The conjugate pads of the present disclosure serve to, among other things, hold adetection agent as discussed. Suitable materials for the conjugate pad include glass fiber, polyester, paper, surface modified polypropylene, and the like. It is within the scope of this disclosure that, in embodiments, a detection agent may be applied externally, for example from a dropper bottle, in which case a lateral flow device may need not contain a conjugate pad (see, e.g., U.S. Pat. No. 4,740,468).

[0067] Detection reagent(s), such as labeled anti-14-3-3η antibodies (or other biomolecules)and labeled biomolecules capable of recognizing a control biomolecule, may be released into solution upon application of / contact with the capillary blood sample. The conjugate pad may be treated with one or more substances to influence release of the detector reagent into solution. For example and without limitation, the conjugate pad may be treated with PVA or PVP (about 0.5% to about 2%) and / or Triton X-100 (about 0.5%). Other release agents may include but are not limited to hydroxypropylmethyl cellulose, SDS, Brij 35, and β-lactose. A mixture of two or more release agents may be used in any given application. In a particular disclosed embodiment, a detection reagent present in the conjugate pad is a gold-conjugated antibody, or a plurality of different gold-conjugated antibodies. In some examples, different antibodies serving as different detectors may be labeled with different detector labels.

[0068] In embodiments, sample pad and / or conjugate pad materials may be selected based onan ability to reduce blood leeching in lateral flow devices of the present disclosure. More specifically, sample pad and / or conjugate pad materials may be selected for an ability to rapidly separate plasma from whole blood samples for use in lateral flow applications as herein disclosed, thereby retaining blood cells while allowing serum to flow rapidly. Examples include but are not limited to CytoSep® 1660, CytoSep® 1662, CytoSep® 1663, CytoSep® 1667 (HV), and CytoSep® 1668 (HV+) (Ahlstrom-Munksjö, Helsinki, Finland). In a preferred embodiment, the sample pad and / or conjugate pad may comprise CytoSep® 1668 (HV+).

[0069] In embodiments, the invention provides a device for assaying the level of 14-3-3ηprotein in a capillary blood sample from a subject, the device comprising: a capture agent associated with a substrate, wherein the capture agent comprises one or more anti-14-3-3η antibodies and can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, Page 16 of 66 1104081999\1\AMERICASand wherein the substrate is a portion of a lateral flow immunochromatographic cassette comprising, in order of flow, a buffer zone, a sample receiving zone comprising a sample pad, a conjugate pad comprising a detection agent comprising one or more labeled antibodies, and a test zone comprising said substrate, configured such that: the buffer zone is located upstream of the sample receiving zone and the test zone is located downstream of the sample receiving zone, upon flow of the sample through the lateral flow immunochromatographic cassette, labeled 14-3-3η protein from the sample will bind with the capture agent in the test zone to form a detectable complex producing the test line, as part of a running buffer sweep; wherein the capture agent is an anti-14-3-3η antibody, the detection agent is a labeled anti-14-3-3η antibody, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent.

[0070] In exemplary embodiments, the capture agent is an antibody produced by hybridomaclone AUG4-ETA-8F10 or an antigen binding fragment thereof, and the detection agent is an antibody produced by hybridoma clone AUG3-CKNS-7F8 or an antigen binding fragment thereof.

[0071] In exemplary embodiments, the detection agent is detectably labeled with a goldnanoshell (GNS). In exemplary embodiments, 3-7F8 mAb conjugate (detection carrying the GNS) concentration at A825ranged 20-60 at peak wavelength of the absorbance spectrum. The antibody GNS loading quantity for the conjugation is 18 ug of antibody per mL of GNS, with GNS being also at A825 = 20. In other words, the starting concentration of the GNS is A825 = 20 and the final concentration of conjugate is A825= 20.4-8F10 mAb (capture at the test line) concentration range is 1-5 mg / mL.

[0072] In exemplary embodiments, the sample receiving zone of the lateral flowimmunochromatographic cassette comprises a 26mm Millipore GFDX sample pad, a 10mmMillipore GFDX conjugate pad, and a 12 mm Ahlstrom 1668 stack pad. In another exemplary embodiment, the sample receiving zone of the lateral flow immunochromatographic cassette comprises a 26mm Ahlstrom 1668 Sample Pad, a 10mm Millipore GFDX Conjugate Pad, and a 12 mm Ahlstrom 1668 Stack Pad sample pad. In embodiments, the lateral flow immunochromatographic cassette is a MDI Device-22-port cassette.

[0073] In another aspect, the invention provides a kit for assaying the level of 14-3-3η protein ina capillary blood sample obtained from a subject, comprising: a device according to the present disclosure, and a running buffer. Page 17 of 66 1104081999\1\AMERICASAnti-14-3-3η antibodies and antigen-binding fragments thereof

[0074] In embodiments of the lateral flow devices of the present disclosure, one or both of animmobilized biomolecule capable of specifically binding to 14-3-3η protein in its natural configuration (i.e., a capture agent) and / or a labeled biomolecule also capable of specifically binding to 14-3-3η protein in its natural configuration (i.e. a detection agent), may be antibodies, or antigen-binding fragments thereof.

[0075] Anti-14-3-3η antibodies of the present disclosure refer to antibodies capable of (i)binding specifically to human 14-3-3η protein in its native configuration, as evidenced by, for example, immunoprecipitation, and (ii) binding selectively to human 14-3-3η protein over other human 14-3-3 protein isoforms.

[0076] By specifically binding to a human 14-3-3η protein in its “natural configuration” ismeant an ability to bind to 14-3-3 η protein as encountered in vivo. This may be evidenced, for example, by the ability of antibody to immunoprecipitate 14-3-3η protein from a biological sample, preferably a capillary blood sample.

[0077] By “selective for said human 14-3-η protein over other human 14-3-3 protein isoforms”is meant an ability to bind specifically to human 14-3-3η protein and to bind preferentially to 14- 3-3η as compared to other human 14-3-3 protein isoforms under the same conditions. Selectivity may be evidenced, for example, using an ELISA assay, which may be done using, for example, supernatant from hybridoma clones, and may extend similarly to lateral flow devices of the present disclosure. A control (e.g., pre-immune serum) is preferably used. A “selective” antibody is capable of recognizing 14-3-3η and generating a higher signal against 14-3-3η as compared to other 14-3-3 isoforms, preferably at least a 1.5 fold, more preferably at least a 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, or higher signal as compared to other isoforms. In a preferred embodiment, a selective antibody has an ability to selectively immunoprecipitate 14-3-3η as compared to other 14-3-3 isoforms. In a preferred embodiment, an anti-14-3-3η antibody exhibits selectivity for said human 14-3-3η protein over human 14-3-3 alpha, beta, delta, epsilon, gamma, tau, and zeta proteins.

[0078] In embodiments, an anti-14-3-3η antibody of the invention is capable of binding to anepitope comprising a peptide selected from the group consisting of 14-3-3η loop peptides, 14-3- Page 18 of 66 1104081999\1\AMERICAS3η helix peptides, and 14-3-3η non-helix peptides, with η loop peptides being especially preferred (see Table 1). Table 1.14-3-3η epitopesPage 19 of 66 1104081999\1\AMERICASSEQ ID NO: 21 210-218 non-helix TLNEDSYKDE ID N 22 77 4 h li KKLEKVKAK

[0079] Monospecific anti-14-3-3η antibodies useful in the present invention include thosegenerated and prepared according to the disclosure of WO 2009 / 067811, the contents of which is incorporated herein in its entirety. Particularly preferred antibodies are those which bind to and recognize 14-3-3η but do not bind with or cross-react with any of the other 14-3-3 isoforms. Such antibodies include those produced by hybridoma clones AUG3-CKNS-2D5, AUG3-CKNS-7F8, and AUG4-ETA-8F10, as disclosed in WO 2009 / 067811.

[0080] For use in lateral flow devices of the present invention, it is generally preferred that acapture antibody immobilized at a test line specifically binds to a different 14-3-3η epitope than a labeled antibody included as a component of the conjugate pad that also specifically binds 14-3- 3η protein. Accordingly, in an embodiment the capture antibody may comprise an antibody produced by one of hybridoma clones AUG-CKNS-2D5, AUG3-CKNS-7F8, or AUG3-CKNS- 7H8, and the labeled antibody may comprise an antibody produced by hybridoma clone AUG4- Page 20 of 66 1104081999\1\AMERICASETA-8F10. In an alternative embodiment, the capture antibody may comprise an antibody produced by hybridoma clone AUG4-ETA-8F10, and the labeled antibody may comprise an antibody produced by one of hybridoma clones AUG-CKNS-2D5, AUG3-CKNS-7F8, or AUG3- CKNS-7H8.

[0081] Exemplary antibody sequences are provided in Table 2 below (CDR sequences areunderlined in HCVR, LCVR, heavy chain, and light chain sequences; leader sequences are shown in bold): Table 1: 14-3-3η Antibody Sequences V W I EPage 21 of 66 1104081999\1\AMERICASDTAVYYCARATATLYWG DTAVYYCSPVFGYWGQG QGTLVTVSA TTLTVSS K K T D G V W I E G K K T D GPage 22 of 66 1104081999\1\AMERICASMFFLMAVVTGVNSEVQL MFFLMAVVTGVNSEVQL QQSGAELVKPGASVKLSC QQSGAELVKPGASVKLSC Q I E G I T Y T D G

[0082] In embodiments, the capture antibody is the 4-8F10 antibody produced by thehybridoma AUG4-ETA-8F10, or an antigen-binding fragment thereof.

[0083] In embodiments, the capture antibody comprises a HCDR1 having the sequence of SEQID NO: 42 (GFNIKDTY), a HCDR2 having the sequence of SEQ ID NO: 43 (IDPANGNT), and a HCDR3 having the sequence of SEQ ID NO: 44 (SPVFGY). In preferred embodiments, the capture antibody comprises a LCDR1 having the sequence of SEQ ID NO: 45 (QSVLYSSNQKNY), a LCDR2 having the sequence of SEQ ID NO: 46 (WAS), and a LCDR3 having the sequence of SEQ ID NO: 47 (HQYLSSWT). In embodiments, the capture antibody comprises a HCDR1 having the sequence of SEQ ID NO: 42 (GFNIKDTY), a HCDR2 having the sequence of SEQ ID NO: 43 (IDPANGNT), a HCDR3 having the sequence of SEQ ID NO: 44 (SPVFGY), a LCDR1 having the sequence of SEQ ID NO: 45 (QSVLYSSNQKNY), a LCDR2 Page 23 of 66 1104081999\1\AMERICAShaving the sequence of SEQ ID NO: 46 (WAS), and a LCDR3 having the sequence of SEQ ID NO: 47 (HQYLSSWT). In embodiments, the capture antibody comprises a heavy chain variable region (HCVR) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48 (EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRID PANGNTKYDPKFQGKATITADTSSDTAYLQLSSLTSEDTAVYYCSPVFGYWGQGTTLTV SS). In embodiments, the capture antibody comprises a light chain variable region (LCVR) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 49 (NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKL EIK). In embodiments, the capture antibody comprises a heavy chain variable region / light chain variable region (HCVR / LCVR) sequence pair comprising a HCVR sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48 and a LCVR sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 49. In embodiments, the capture antibody comprises a HCVR / LCVR sequence pair comprising SEQ ID NOs: 48 / 49. In embodiments, the capture antibody comprises a heavy chain (HC) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52 (EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRID PANGNTKYDPKFQGKATITADTSSDTAYLQLSSLTSEDTAVYYCSPVFGYWGQGTTLTV SSAKTT PP). In embodiments, the capture antibody comprises a light chain (LC) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 53 (NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSWTFGGGTKL EIKRAD AAPTVSIFPPS). In embodiments, the capture antibody comprises a HC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52 and a LC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 53. In embodiments, the capture antibody comprises a HC / LC sequence pair comprises the sequences of SEQ ID NOs: 52 / 53. In embodiments, the capture antibody comprises the six CDRs of a HCVR / LCVR sequence pair comprising SEQ ID NOs: 48 / 49. In embodiments, Page 24 of 66 1104081999\1\AMERICASthe capture antibody comprises a HC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56 and a LC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57.

[0084] In embodiments, the detection agent comprises a detection antibody, e.g. a labeledantibody or antigen-binding fragment thereof. In embodiments, the detection antibody is the 3-7F8 antibody produced by the hybridoma AUG3-CKNS-7F8.

[0085] In embodiments, the detection antibody comprises a heavy chain complementaritydetermining region 1 (HCDR1) having the sequence of SEQ ID NO: 34 (GFNIKDTY), a HCDR2 having the sequence of SEQ ID NO: 35 (IDPANGNT), and a HCDR3 having the sequence of SEQ ID NO: 36 (ARATATLY). In embodiments, the detection antibody comprises a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 37 (QSIVHSNGNTY), a LCDR2 having the sequence of SEQ ID NO: 38 (KVS), and a LCDR3 having the sequence of SEQ ID NO: 39 (QGSHVPYT). In embodiments, the detection antibody comprises a HCDR1 having the sequence of SEQ ID NO: 34 (GFNIKDTY), a HCDR2 having the sequence of SEQ ID NO: 35 (IDPANGNT), a HCDR3 having the sequence of SEQ ID NO: 36 (ARATATLY), a LCDR1 having the sequence of SEQ ID NO: 37 (QSIVHSNGNTY), a LCDR2 having the sequence of SEQ ID NO: 38 (KVS), and a LCDR3 having the sequence of SEQ ID NO: 39 (QGSHVPYT). In embodiments, the detection antibody comprises a heavy chain variable region (HCVR) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 (EVQLQQSGAELVKPGASVKLSCTDSGFNIKDTYMHWVKQRPEQGLEWIGKID PANGNTKYDPKFQGKATITSDTSSNTAYLQLSSLTSEDTAVYYCARATATLYWGQGTL VTVSA). In embodiments, the detection antibody comprises a light chain variable region (LCVR) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 41 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGT KLEIK). In embodiments, the detection antibody comprises a heavy chain variable region / light chain variable region (HCVR / LCVR) sequence pair comprising a HCVR sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 and a LCVR sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence Page 25 of 66 1104081999\1\AMERICASidentity to SEQ ID NO: 41. In embodiments, the detection antibody comprises a heavy chain variable region / light chain variable region (HCVR / LCVR) sequence pair comprising SEQ ID NOs: 40 / 41. In embodiments, the detection antibody comprises a heavy chain (HC) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50 (EVQLQQSGAELVKPGASVKLSCTDSGFNIKDTYMHWVKQRPEQGLEWIGKID PANGNTKYDPKFQGKATITSDTSSNTAYLQLSSLTSEDTAVYYCARATATLYWGQGTL VTVSAAT TTAP). In embodiments, the detection antibody comprises a light chain (LC) comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 51 (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGT KLEIK RADAAPTVSIFPPS). In embodiments, the detection antibody comprises a HC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50 and a LC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 51. In embodiments, the detection antibody comprises a HC / LC sequence pair comprises the sequences of SEQ ID NOs: 50 / 51. In embodiments, the detection antibody comprises the six CDRs of a HCVR / LCVR sequence pair comprising SEQ ID NOs: 40 / 41. In embodiments, the detection antibody comprises a HC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 54 and a LC having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 55.

[0086] In embodiments, the capture agent can be present at a test line concentration of about1.0 mg / mL to about 5.0 mg / mL. In embodiments, the capture agent can be present at a test line concentration of about 1.0 mg / mL to about 5.0 mg / mL, about 3.0 mg / mL to about 5.0 mg / mL, about 4.0 mg / mL to about 5.0 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, about 2.0 mg / mL, about 2.5 mg / mL, about 3.0 mg / mL, about 3.5 mg / mL, about 4.0 mg / mL, about 4.1 mg / mL, about 4.2 mg / mL, about 4.3 mg / mL, about 4.4 mg / mL, about 4.5 mg / mL, about 4.6 mg / mL, about 4.7 mg / mL, about 4.8 mg / mL, about 4.9 mg / mL, about 5.0 mg / mL, or any range or value therebetween. In embodiments, the capture agent can be present at a test line concentration of about 4.8 mg / mL. Page 26 of 66 1104081999\1\AMERICAS

[0087] In embodiments, the detection agent can be present at an OD from about 20 to about60 (A825). In some embodiments, the detection agent can be present at an OD from about 20 to about 60, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 20 to about 25, about 25 to about 60, about 25 to about 50, about 25 to about 40, about 25 to about 30, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 60, about 40 to about 50, about 50 to about 60, about 20, about 25, about 30, about 40, about 50, about 60, or any range or value therebetween (A825). In embodiments, the detection agent can be present at an OD of about 20 (A825).

[0088] In embodiments, the capture agent is dispensed at about 0.08 µL / mm. In someembodiments, the detection agent is dispensed at about 2.5 µL / mm. Autoantibodies

[0089] It is known that autoantibodies can be generated against epitopes of 14-3-3η protein.The presence of autoantibodies directed against 14-3-3η protein detectable in capillary blood may be useful in the screening, diagnosis, prognosis, and / or treatment monitoring of an arthritic condition. Accordingly, in embodiments a lateral flow device of the present disclosure may rely on the detection of autoantibodies against 14-3-3η protein to provide an indication of whether a subject suspected of having, or at risk for developing, an arthritic condition, is positive for 14-3- 3η protein.

[0090] In embodiments, a capture agent for a lateral flow assay may comprise an anti-14-3-3ηantibody or antigen-binding fragment thereof, immobilized at a test line of a lateral flow device. In such an embodiment, a detection agent may comprise a labeled antibody, antigen-binding fragment thereof, or some other biomolecule capable of binding to autoantibodies directed against the 14-3-3η protein. In a capillary blood sample, 14-3-3η protein may be bound to anti-14-3-3η autoantibodies. When applied to a sample pad of a lateral flow device according to the subject invention, a complex of the 14-3-3η protein bound to autoantibody may migrate through the conjugate pad, where the detection agent (e.g., a labeled anti-human antibody or antigen-binding fragment thereof) may recognize and bind to the autoantibody that is in turn bound to 14-3-3η protein. Then, at the test line, the immobilized anti-14-3-3η antibody or antigen-binding fragment thereof may specifically bind the 14-3-3η protein that is coupled to anti-14-3-3 autoantibody that is in turn coupled to the detection agent. In this way, a lateral flow assay of the present disclosure may report a level of 14-3-3η protein bound by autoantibody. Page 27 of 66 1104081999\1\AMERICAS

[0091] The above embodiments detect autoantibody bound to 14-3-3η protein in a capillaryblood sample. In other embodiments, lateral flow assays of the present disclosure are capable of detecting the presence of autoantibody against 14-3-3η protein, where said autoantibody is not bound to the 14-3-3η protein in the capillary sample, or wherein conditions of the lateral flow device are such that the 14-3-3η protein in the capillary sample does not remain bound to the autoantibody while in contact with the sample pad and / or conjugate pad, but wherein conditions are permissive for autoantibody binding to the 14-3-3η protein at the capture pad. In one such embodiment, a capture agent for a lateral flow assay may comprise a 14-3-3η protein, or fragment thereof, immobilized at a test line. In such an embodiment, autoantibody directed against 14-3-3η protein may bind to the immobilized 14-3-3η protein, or fragment thereof, and may be visualized by a detection agent (e.g., labeled anti-human antibody or antigen-binding fragment thereof, such as IgG, IgA and / or IgM) capable of recognizing the autoantibody.

[0092] In embodiments, a capture agent for a lateral flow assay may comprise a 14-3-3η proteinor fragment thereof that comprises an epitope shared between a plurality of 14-3-3 protein isoforms, or may comprise an epitope unique to one or a subset of 14-3-3 isoforms. “Shared” as used herein refers to a fragment or epitope in common between two or more 14-3-3 protein isoforms. In preferred embodiments, the 14-3-3 protein or fragment thereof comprises a 14-3-3 eta and / or gamma epitope. In one embodiment, the 14-3-3 protein of fragment thereof comprises a 14-3-3 eta epitope shared by at least one other 14-3-3 isoform, e.g.14-3-3 gamma. In another embodiment, the 14-3-3 eta epitope is unique to 14-3-3 eta. In some embodiments, the 14-3-3η protein or fragment thereof comprises an epitope selected from the sequences in Table 3 below: List of Peptides Peptide Name Sequence AA locationPage 28 of 66 1104081999\1\AMERICASPeptide Name Sequence AA location VISSIEQKTMADGNEKKLEKVKAYR-AHX-

[0093] Methods of making antibodies to 14-3-3η protein involve protocols available to thoseskilled in the art, and have been described in detail in WO 2009067811, WO 2010102412, and WO 2013 / 056377, the contents of each of which is herein expressly incorporated by reference in their entirety for all purposes.

[0094] Briefly, monoclonal antibodies may be produced by generation of hybridomas inaccordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as an enzyme-linked immunosorbent assay (ELISA), to identify one or more hybridomas that produce an antibody that specifically binds with particular portion(s) of 14- 3-3η protein as disclosed herein. As a representative example, a 14-3-3η protein or fragment thereof may be used to immunize non-human hosts, e.g., donkey, goat, sheep, guinea pig, hamster, rabbit, rat and mouse, to obtain polyclonal and monoclonal antibodies that react with the 14-3-3 protein or a fragment thereof.

[0095] The peptide immunogens additionally may contain a cysteine residue at the carboxylterminus, and may be conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are well known in the art. A full-length polypeptide of the present invention may be used as the immunogen, or, alternatively, antigenic peptide fragments of the polypeptides may be used. An antigenic peptide of a polypeptide of the present invention comprises at least 7 continuous amino acid residues and encompasses an epitope Page 29 of 66 1104081999\1\AMERICASsuch that an antibody raised against the peptide forms a specific immune complex with the polypeptide. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0096] Monoclonal antibodies may be generated by other methods known to those skilled inthe art of recombinant DNA technology. As an alternative to preparing monoclonal antibody- secreting hybridomas, a monoclonal antibody to a polypeptide of the present invention may be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a particular polypeptide related to the present invention to thereby isolate immunoglobulin library members that bind to such polypeptides.

[0097] Techniques and commercially available kits for generating and screening phage displaylibraries are well known to those skilled in the art. Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in the literature. For example, the "combinatorial antibody display" method is well known and was developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies. After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and / or framework 1 (FR1) sequences, as well as primers to a conserved 3' constant region, can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies; a similar strategy has also been used to amplify human heavy and light chain variable regions from human antibodies.

[0098] Polyclonal sera and antibodies may be produced by immunizing a suitable subject witha polypeptide of the present invention. The antibody titer in the immunized subject may be monitored over time by standard techniques, such as with ELISA using immobilized protein. If desired, the antibody molecules directed against a polypeptide of the present invention may be isolated from the subject or culture media and further purified by well-known techniques, such as protein A chromatography, to obtain an IgG fraction. Page 30 of 66 1104081999\1\AMERICAS

[0099] Fragments of antibodies to the polypeptides of the present invention may be producedby cleavage of the antibodies in accordance with methods well known in the art. For example, immunologically active Fab and F(ab')2 fragments may be generated by treating the antibodies with an enzyme such as pepsin.

[0100] Additionally, chimeric, humanized, and single-chain antibodies to the polypeptides ofthe present invention, comprising both human and nonhuman portions, may be produced using standard recombinant DNA techniques and / or a recombinant combinatorial immunoglobulin library. Humanized antibodies may also be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. For example, human monoclonal antibodies (mAbs) may be generated using transgenic mice carrying the human immunoglobulin genes rather than murine immunoglobulin genes. Splenocytes from these transgenic mice immunized with the antigen of interest may then be used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein.

[0101] Chimeric antibodies, including chimeric immunoglobulin chains, may be produced byrecombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted.

[0102] An antibody or an immunoglobulin chain may be humanized by methods known in theart. Humanized antibodies, including humanized immunoglobulin chains, may be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison (1985) Science 229: 1202-07; Oi et al. (1986) BioTechniques 4:214; Queen et al., U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid sequences are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target. The recombinant DNA encoding the humanized antibody, or fragment thereof, then can be cloned into an appropriate expression vector. Page 31 of 66 1104081999\1\AMERICAS

[0103] Humanized or CDR-grafted antibody molecules or immunoglobulins may be producedby CDR grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-25; Verhoeyan et al. (1988) Science 239: 1534; Beidler et al. (1988) J. Immunol.141 :4053-60; Winter, U.S. Pat. No.5,225,539, the contents of all of which are hereby incorporated by reference. Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638 A; Winter, U.S. Pat. No. 5,225,539, the contents of which are hereby incorporated by reference). All of the CDRs of a particular human antibody may be replaced with at least a portion of a nonhuman CDR, or only some of the CDRs may be replaced with nonhuman CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

[0104] Human antibodies may additionally be produced using transgenic nonhuman animalsthat are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. See, e.g., PCT publication WO 94 / 02602. The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. One embodiment of such a nonhuman animal is a mouse, and is termed the XENOMOUSE™ as disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules. In another embodiment, human antibodies of this disclosure can be raised using a mouse referred to as “KM Mouse™” that carries human immunoglobulin sequences on transgenes and transchomosomes, as described in detail in PCT Publication WO 02 / 43478. Another example includes the use of VELOCIMMUNE® Technology (see, e.g., U.S. Pat. No.6,596,541, Regeneron Page 32 of 66 1104081999\1\AMERICASPharmaceuticals, VELOCIMMUNE®. Briefly, the VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antigen-binding protein, e.g., antibody, comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody. Additional examples of transgenic animals that can be used to produce anti-14-3-3 antibodies of the present disclosure include OmniRatTMand OmniMouseTM(see, e.g., Osborn M., et al. (2013) Journal of Immunology 190: 1481-90; Ma B., et al. (2013) Journal of Immunological Methods 400-401: 78-86; Geurts A., et al. (2009) Science 325: 433, U.S. Pat. No.8,907,157; European Pat. No.2152880B1; European Pat. No.2336329B1).

[0105] Monoclonal, chimeric and humanized antibodies that have been modified by, e.g.,deleting, adding, or substituting other portions of the antibody, e.g., the constant region, are also within the scope of the invention. As nonlimiting examples, an antibody can be modified by deleting the constant region, by replacing the constant region with another constant region, e.g., a constant region meant to increase half-life, stability, or affinity of the antibody, or a constant region from another species or antibody class, and by modifying one or more amino acids in the constant region to alter, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement fixation, etc.

[0106] Methods for altering an antibody constant region are known in the art. Antibodies withaltered function, e.g., altered affinity for an effector ligand, such as FcR on a cell, or the CI component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar types of alterations to the murine (or other species) immunoglobulin may be applied to reduce or eliminate these functions, and are known in the art.

[0107] For example, it is possible to alter the affinity of an Fc region of an antibody (e.g., anIgG, such as a human IgG) for an FcR (e.g., Fc gamma Rl), or for Clq binding by replacing the specified residue(s) with a residue(s) having an appropriate functionality on its side chain, or by Page 33 of 66 1104081999\1\AMERICASintroducing a charged functional group, such as glutamate or aspartate, or an aromatic nonpolar residue such as phenylalanine, tyrosine, tryptophan or alanine (see, e.g., U.S. Pat. No.5,624,821).

[0108] Methods for covalent or chemical attachment of antibodies may also be utilized. Cross-linking agents are well known in the art, for example, homo-or heterobifunctional linkers as are well known (see the 2006 Pierce Chemical Company Crosslinking Reagents Technical Handbook, Hermanson, GT, Bioconjugate Techniques, Academic Press, San Diego, CA (1996), Aslam M and Dent AH, Bioconjugation protein coupling techniques for the biomedical sciences, Houndsmills, England Macmillan Publishers (1999), Pierce Applications Handbook & Catalog, Perbio Science, Ermbodegem, Belgium (2003-2004), Haughland, RP, Handbook of Fluorescent Probes and Research Chemicals Eugene, 9th Ed , Molecular Probes, OR (2003), and U.S. Patent No. 5,747,641). Those of skill in the art will appreciate the suitability of various functional groups on the amino acid(s) of an antibody for modification, including cross-linking Suitable examples of chemical crosslinkers used for antibody crosslinking include, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate], SATA [N-succnimidyl S- acethylthio-acetate], hemi-succinate esters of N-hydroxysuccinimide, sulfo-N-hydroxy- succnimide, hydroxybenzotriazole, and p-nitrophenol, dicyclohexylcarbodiimide (DCC), 1-(3- dιmethylamιnopropyl)-3-ethylcarbodιιmιde (ECD), and 1-(3-dιmethylamιnopropyl)-3- ethylcarbodnmide methiodide (EDCI) (see, e.g., U.S. Patent No. 4,526,714, the disclosure of which is fully incorporated by reference herein). Other linking reagents include glutathione, 3- (dιethoxyphosphoryloxy)-1,2,3-benzotrιazιn-4(3H)-one (DEPBT), onium salt-based coupling reagents, polyoxyethylene-based heterobifunctional cross-linking reagents, and other reagents (Haitao, et al , Organ Lett 191 -94 (1999), Albercio et al., J Organic Chemistry 639678-9683 (1998), Arpicco et al., Bioconjugate Chem 8327-337 (1997), Frisch et al., Bioconjugate Chem 7 180-186 (1996), Deguchi et al., Bioconjugate Chem 1032-37 (1998), Beyer et al , J Med Chem 41 2701-2708 (1998), Drouillat et al., J Pharm Sci 8725-30 (1998), Trimble et al., Bioconjugate Chem 8 416-423 (1997)). Techniques for conjugating therapeutic compounds to antibodies are also described in Arnon et al., "Monoclonal Antibodies for lmmunotargeting of Drugs in Cancers Therapy," in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al., ed, pp.243-256, Alan R Liss, lnc (1985), Thorpe, et al. "The Preparation and Cytotoxic Properties of Antibody Toxin Conjugates," Immunol Rev 62119-58 (1982), and Pietersz, GA, "The linkage of cytotoxic drugs to monoclonal antibodies for the treatment of cancer," Bioconjugate Chemistry 1 (2) 89-95 (1990), all references incorporated herein by reference. Page 34 of 66 1104081999\1\AMERICAS

[0109] The anti-14-3-3η antibodies disclosed herein include antibodies conjugated toinorganic or organic compounds, including, by way of example and not limitation, labels as herein disclosed, as well as other proteins, nucleic acids, carbohydrates, steroids, and lipids (see, e.g., Green, et al., Cancer Treatment Reviews, 26269-286 (2000)). In embodiments, the compound may be bioactive. Bioactive refers to a compound having a physiological effect on a cell as compared to a cell not exposed to the compound. A physiological effect is a change in a biological process, including, by way of example and not limitation, DNA replication and repair, recombination, transcription, translation, secretion, membrane turnover, cell adhesion, signal transduction, cell death, and the like. A bioactive compound includes pharmaceutical compounds, in embodiments. IV. Screening, Diagnostic, Prognostic, and Theranostic Methods, and Treatment Monitoring

[0110] In one aspect, the invention provides a method for assaying the level of 14-3-3η proteinin a subject, comprising: a) contacting a capillary blood sample from a subject with a capture agent associated with a substrate, wherein the capture agent can bind labeled 14-3-3η protein inthe capillary blood sample to form a complex, and wherein the substrate is a portion of a lateralflow immunochromatographic cassette; and b) detecting the binding of the capture agent to 14-3- 3η protein with a detection agent that binds to said protein within the lateral flow immunochromatographic cassette to produce a visible test line and determining whether the level of 14-3-3η protein is at or above a threshold level based on the resulting test line intensity, wherein the capture agent comprises one or more anti-14-3-3η antibodies, the detection agent comprises one or more labeled anti-14-3-3η antibodies, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent.

[0111] In embodiments, the lateral flow immunochromatographic cassette comprises, in orderof flow, a buffer zone, a sample receiving zone comprising a sample pad, a conjugate pad comprising the detection agent, and a test zone; wherein the buffer zone is located upstream of the sample receiving zone and the test zone is downstream of the sample receiving zone, and the method comprises adding at least about 25 µl of sample to the sample receiving zone followed by adding at least about 75, 80, 85, 90, 95, 100, 105 or 110 µl of running buffer to the buffer zone, and upon flow of the sample through the lateral flow immunochromatographic cassette, labeled 14-3-3η from the sample will bind with the capture agent in the test zone to form a detectable complex producing the test line, as part of a running buffer sweep. Page 35 of 66 1104081999\1\AMERICAS

[0112] Without being bound by theory, the running buffer enables the sample to move throughthe conjugate pad and to the test zone which can allow the detection agent to bind 14-3-3η protein in the sample and the labeled 14-3-3η protein to bind to the capture agent in the test zone which then produces a signal for detection of the detectable complex at the test line. The flow of the running buffer also enables the reaction at the control line to take place. Without the running buffer addition, the sample will not effectively flow through the lateral flow immunochromatographic cassette..

[0113] In embodiments, a volume of the capillary blood sample added to the lateral flowdevice is about 50µL or less; preferably wherein the volume of the capillary blood sample is between about 25-50 µL.

[0114] In embodiments, an arthritis therapy is initiated if the level of 14-3-3η protein is at orabove the threshold level, preferably wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette. In embodiments, an arthritis therapy is initiated if the level of 14-3-3η protein is above the threshold level, preferably wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette.

[0115] In embodiments, steps a) and b) are performed on at least two capillary blood samplestaken at different time points from the subject, and initiating an arthritis therapy when the patient has at least two consecutive capillary blood samples displaying a level of 14-3-3η protein at or above the threshold level, or displaying an increasing level of 14-3-3η protein and / or an increasing test line intensity in the subsequent sample.

[0116] In embodiments, the subject is a patient undergoing an arthritis therapy, the methodcomprising performing steps a) and b) on at least two capillary blood samples taken at different time points from the patient, and modifying the arthritis therapy when the patient has at least two consecutive capillary blood samples displaying an increasing level of 14-3-3η protein and / or an increasing test line intensity in the subsequent sample. In embodiments, the subject is a patient Page 36 of 66 1104081999\1\AMERICASundergoing an arthritis therapy, the method comprising performing steps a) and b) on at least two capillary blood samples taken at different time points from the patient, and modifying the arthritis therapy when the patient has at least two consecutive capillary blood samples displaying at least the same level of 14-3-3η protein and / or the same test line intensity in the subsequent sample.

[0117] In another aspect, a method of treating an arthritic disease in a subject compriseswherein at least one capillary blood sample taken from the subject is tested for the presence of 14- 3-3η in a lateral flow assay, and wherein an arthritis therapy is initiated in the event that the level of 14-3-3η protein in the at least one capillary blood sample is at or above a threshold level within the lateral flow immunochromatographic cassette.

[0118] In embodiments, wherein at least two capillary blood samples taken at different timepoints from the subject are tested for the presence of 14-3-3η in the lateral flow assay, an arthritis therapy is initiated in the event that the level of 14-3-3η protein in at least two capillary blood samples from the subject are at or above the threshold level within the lateral flow immunochromatographic cassette, or at least two capillary blood samples display an increasing level of 14-3-3η protein and / or an increasing test line intensity within the lateral flow immunochromatographic cassette. In embodiments, the at least two samples are consecutive samples.

[0119] In embodiments, the different time points are at least seven days apart, at least 2, 3, 4,5, 6, 7, 8, 9, or 10 weeks apart, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart. In embodiments, the at least two samples are consecutive samples that are at least 3, 4, 5, 6, 7 or 8 weeks apart.

[0120] In embodiments, testing for the presence of 14-3-3η in a lateral flow assay comprises:a) contacting a capillary blood sample from a subject with a capture agent associated with a substrate, wherein the capture agent can bind labeled 14-3-3η protein in the capillary blood sampleto form a complex, and wherein the substrate is a portion of a lateral flow immunochromatographiccassette; and b) detecting the binding of the capture agent to 14-3-3η protein with a detection agent that binds to said complex within the lateral flow immunochromatographic cassette to produce a visible test line and determining whether the level of 14-3-3η protein is at or above a threshold level based on the resulting test line intensity, wherein the capture agent is an anti-14-3-3η antibody, the detection agent is a labeled anti-14-3-3η antibody, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent. Page 37 of 66 1104081999\1\AMERICAS

[0121] In another aspect, a method of treating a patient with arthritis comprises: a) testing forthe presence of 14-3-3η protein in a capillary blood sample of a patient within a lateral flow immunochromatographic cassette; b) identifying the patient as having 14-3-3η protein in the capillary blood sample; and c) initiating treatment of the patient having a capillary blood sample that is positive for 14-3-3η protein.

[0122] In another aspect, a method of detecting a reduction in, or absence of, therapeuticefficacy of an arthritis therapy in a subject that has been administered the arthritis therapy, the method comprising: a) detecting in a first capillary blood sample obtained from the subject at a first time point the presence or absence of at least a threshold level of 14-3-3η protein within a lateral flow immunochromatographic cassette; and b) detecting in a second capillary blood sample obtained from the subject at a second time point the presence or absence of at least a threshold level of 14-3-3η protein within the lateral flow immunochromatographic cassette, wherein an increase in the level of 14-3-3η protein and / or an increase in test line intensity in the second capillary blood sample, or the presence of at least the threshold level in both the first and second capillary blood samples, indicates a reduction in, or absence of, therapeutic efficacy of the arthritis therapy.

[0123] In embodiments, the threshold level comprises a test line intensity that correlates withat least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η protein in a reference sample within the lateral flow.

[0124] In embodiments, the first capillary blood sample is taken before the subject isadministered the arthritis therapy, and the presence of the threshold level of 14-3-3η protein in both the first and second samples indicates a reduction in, or absence of, therapeutic efficacy.

[0125] In embodiments, the first capillary blood sample and the second capillary blood sampleare separated by at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, or at least two, three, four, five, six months or twelve months.

[0126] In embodiments, the first capillary blood sample and the second capillary blood sampleare separated by at least 5 minutes, at least 15 minutes, at least an hour, at least one, two, three, four, five, six, or seven days, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Page 38 of 66 1104081999\1\AMERICAS

[0127] In embodiments, the method can further comprise selecting an arthritis therapy for thesubject if there is an increase in the level of 14-3-3η protein and / or an increase in test line intensity in the second capillary blood sample. In embodiments, the method can further comprise selecting an arthritis therapy for the subject if there is the level of 14-3-3η protein is at least the threshold level in both the first capillary blood sample and the second capillary blood sample. In certain embodiments, selecting an arthritis therapy comprises evaluating a current therapy of the subject, determining a new therapy for the subject, modifying a current therapy of the subject, or stopping a current therapy of the subject. In certain embodiments, the current therapy comprises administering a DMARD and / or an anti-arthritic biologic to the subject.

[0128] In embodiments, the method further comprises changing the arthritis therapy from aDMARD to an anti-arthritic biologic if the second capillary blood sample has at least the threshold level of 14-3-3η.

[0129] In another aspect, a method of detecting a reduction in therapeutic efficacy of anarthritis therapy in a subject that has been administered the arthritis therapy comprises performing a lateral flow assay to determine whether two or more capillary blood samples taken at different time points from the subject contain a level of 14-3-3η protein that is at least a threshold level; wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette; and wherein an increase in or the presence of the threshold level of 14-3-3η protein in the two or more samples indicates a reduction in, or absence of, therapeutic efficacy.

[0130] In embodiments, the method further comprises selecting an arthritis therapy for thesubject if a threshold level of 14-3-3η protein is determined to be present in at least two capillary blood samples from the subject.

[0131] In embodiments, consecutive time points can be separated by about 2 weeks, 4 weeks,6 weeks, 8 weeks, 12 weeks, 16 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year or more. In certain embodiments, two samples from different time points can be separated by about 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year or more. Page 39 of 66 1104081999\1\AMERICAS

[0132] In one aspect, the invention provides a method for diagnosing an arthritic conditionbased on the presence, absence and / or amount of 14-3-3η protein. In general, the method includes obtaining a capillary blood sample from a subject suspected of having or at risk for developing an arthritic condition, and applying the capillary blood sample to a lateral flow device as herein described, under conditions sufficient for formation of an immunocomplex comprising a capture agent coupled to 14-3-3η protein that is further coupled to a detection agent. In embodiments, the capture agent is an anti-14-3-3η antibody, or antigen-binding fragment thereof, that specifically recognizes 14-3-3η protein. In embodiments, the detection agent is a labeled anti-14-3-3η antibody, or antigen-binding fragment thereof, that specifically recognizes 14-3-3η protein. Formation of the immunocomplex and, optionally an amount of the formed immunocomplex, is indicative of the arthritic condition. In embodiments, the method relies on detection of 14-3-3η protein in the capillary blood sample.

[0133] In one aspect, the invention provides a method for diagnosing an arthritic conditionbased on an indirect measurement of a presence, absence and / or amount of 14-3-3η protein. More specifically, the method involves detecting the presence, absence and / or amount of autoantibody bound to 14-3-3η protein in a capillary blood sample. The autoantibody may be directed to 14-3- 3η protein. In general, the method includes obtaining a capillary blood sample from a subject suspected of having or at risk for developing an arthritic condition, and applying the capillary blood sample to a lateral flow device of the present disclosure under conditions sufficient for formation of an immunocomplex comprising a capture agent, the autoantibody bound to 14-3-3η protein, and a labeled detection agent. In embodiments, the capture agent is an anti-14-3-3η antibody, or antigen-binding fragment thereof, that specifically recognizes 14-3-3η protein. In embodiments, the detection agent is a labeled anti-human antibody (e.g., anti-human IgG, IgA and / or IgM), or antigen-binding fragment(s) thereof, that is capable of recognizing the autoantibody. Formation of the immunocomplex and optionally an amount of the formed immunocomplex, is indicative of the arthritic condition.

[0134] In another aspect, provided is another method for diagnosing an arthritic conditionbased on an indirect measure of a presence or absence and / or amount of 14-3-3η protein. The method involves the use of a detection agent to detect 14-3-3η protein bound to autoantibody in a capillary blood sample. The autoantibody may be directed to 14-3-3η protein. In general, the method includes obtaining a capillary blood sample form a subject suspected of having or at risk Page 40 of 66 1104081999\1\AMERICASfor developing an arthritic condition, and applying the capillary blood sample to a lateral flow device of the present disclosure under conditions sufficient for formation of an immunocomplex comprising a capture agent capable of recognizing the autoantibody, autoantibody bound to 14-3- 3η protein, and the detection agent that specifically recognizes 14-3-3η protein. In embodiments, the detection agent is a labeled anti-14-3-3η antibody, or antigen-binding fragment thereof, that specifically recognizes 14-3-3η protein. In embodiments, the capture agent is an anti-human antibody (e.g., anti-human IgG, IgA and / or IgM), or antigen-binding fragment(s) thereof, that is capable of recognizing the autoantibody. In embodiments, the capture agent is 14-3-3η protein or an antibody-binding fragment thereof.

[0135] In another aspect, provided is another method for diagnosing an arthritic conditionbased on an indirect measure of a presence or absence and / or amount of 14-3-3η protein. The method involves detection of the presence of autoantibody directed against 14-3-3η protein. In general, the method includes obtaining a capillary blood sample form a subject suspected of having or at risk for developing an arthritic condition, and applying the capillary blood sample to a lateral flow device of the present disclosure under conditions sufficient for formation of an immunocomplex comprising a capture agent, the autoantibody, and a labeled detection agent. In embodiments, the capture agent is 14-3-3η protein, or a fragment thereof. In any of the above- mentioned embodiments, the labeled detection agent may be an anti-human antibody (e.g., IgG, IgA, and / or IgM) capable of recognizing autoantibody in the capillary sample.

[0136] In the methods described above, concentration of the detection agent at the test line isindicative of the presence of an arthritic condition. Typically, concentration of the detection agent at the test line creates a line that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of capture agent immobilized on the membrane is selected to generate a visually discernable pattern when the capillary blood sample contains a level of 14- 3-3η protein and / or autoantibody directed there against sufficient to generate the positive signal in the lateral flow assays described herein. In embodiments, an extent to which a capillary blood sample is positive for the presence of 14-3-3η and / autoantibody directed there against, is quantifiable.

[0137] In embodiments, the methodologies as described above are useful in the screeningand / or diagnosis of spondyloarthropathy (e.g. ankylosing spondylitis), Behçet's disease, childhood arthritis, Diffuse Idiopathic Skeletal Hyperostosis (DISH), Felty's Syndrome, fibromyalgia, gout, Page 41 of 66 1104081999\1\AMERICASgranulomatosis with polyangiitis (GPA), infectious arthritis, inflammatory arthritis, Mixed Connective Tissue Disease (MCTD), myositis (dermatomyositis, polymyositis), osteoarthritis, Paget's Disease, polymyalgia rheumatica with Giant Cell Arteritis, pseudogout, psoriatic arthritis, Raynaud's Phenomenon, reactive arthritis, rheumatoid arthritis, scleroderma, Sjögren syndrome, and / or systemic lupus erythematosus. In embodiments, the methodologies as described above are useful in the screening and / or diagnosis of rheumatoid arthritis (RA). In embodiments, the methodologies as described above are useful in the screening and / or diagnosis of ankylosing spondylitis (AS). The general methodologies mentioned above can additionally or alternatively be used as prognostic indicators. For example, reliance on a lateral flow assay as herein disclosed can enable a determination about the progression of and / or likely outcome of an arthritic condition.

[0138] In embodiments, any of the above methodologies may also rely on alternativebiological samples such as serum, plasma, and / or synovial fluid. In embodiments, diagnosis and / or prognosis may additionally include reliance on one or more additional markers assayed from a biological sample from a subject. For example, one or more other markers of arthritis, or prognostic indicators, etc., may be assayed in addition to reliance on the presence / absence and / or amount of 14-3-3η protein and / or presence / absence and / or amount of autoantibodies directed there against, to improve clinical diagnostic and / or prognostic sensitivity. The other markers may be proteins or nucleic acids. In embodiments, one or more other markers can be included as part of a lateral flow device of the present disclosure, so that the assaying of multiple markers can be done simultaneously on a single lateral flow device. In other examples, other assays well known to those skilled in the art can be used in conjunction with the LFT provided herein. The other markers can include but are not limited to one or more of matrix metalloproteinases (MMP) proteins or nucleic acids or other factors which are commonly used as indicators for arthritis, e.g., anti-CCP, anti- rheumatoid factor (RF), C-reactive protein (CRP), serum amyloid A (SAA), IL-6, SIOO, osteopontin, RF, MMP- 1, MMP-3, hyaluronic acid, sCD14, angiogenesis markers and products of bone, cartilage or synovium metabolism (e.g. CTX-1 and CTX - II), etc. Methods for isolating and assaying nucleic acids based on reference sequences are well known in the art, as are methods for detecting proteins of interest within a patient sample. In embodiments, the biological sample can be serum, plasma, capillary blood, or synovial fluid.

[0139] The general methodologies mentioned above can additionally or alternatively be usedas theranostic indicators. For example, reliance on a lateral flow assay to determine Page 42 of 66 1104081999\1\AMERICASpresence / absence and / or amount of 14-3-3η protein and / or presence / absence and / or amount of autoantibodies directed there against (with or without information pertaining to other markers) as herein disclosed can be used to determine or infer a likely efficacy of various treatment options on an arthritic condition in a subject.

[0140] Treatment methodologies for managing arthritic conditions are known, and, inembodiments can include but are not limited to administration of a therapeutically effective amount of one or more therapeutic agents. The one or more therapeutic agents may be selected from aspirin, nonsteroidal anti-inflammatory agents, corticosteroids, cyclooxygenase 2 specific inhibitors (CSIs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs), penicillamine, Rituximab (monoclonal anti-CD20 antibody), cyclosporine, Infliximab (monoclonal anti tumor necrosis factor antibody), Leflunomide (pyrimidine synthesis inhibitor), Anakinra (recombinant human interleukin-1 receptor antagonist), Etanercept (protein therapy based on a binding fragment of the tumor necrosis factor alpha receptor), Adalimumab (monoclonal antitumor necrosis antibody), and others as described in WO2013056377, the contents of which is incorporated by reference herein in its entirety. In embodiments, the arthritis therapy can comprise administering a disease-modifying anti-rheumatic drug (DMARD) to the subject. In embodiments, the DMARD is selected from the group consisting of apremilast, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, tofacitinib, and combinations thereof. In embodiments, the arthritis therapy can comprise administering an anti-arthritic biologic to the subject. In embodiments, the anti-arthritic biologic is selected from the group consisting of a tumor necrosis factor-α inhibitor (TNF-α inhibitor), a B-cell inhibitor, an interleukin inhibitor, a selective co-stimulation modulator, and combinations thereof. In embodiments, the TNF-α inhibitor is selected from the group consisting of adalimumab, certolizumab, etanercept, golimumab, and infliximab. In embodiments, the B-cell inhibitor is selected from the group consisting of belimumab and rituximab. In embodiments, the interleukin inhibitor is selected from the group consisting of anakinra, canakinumab, guselkumab, ixekizumab, Risankizumab, sarilumab, secukinumab, tocilizumab, and ustekinumab. In embodiments, the selective co-stimulation modulator is abatacept.

[0141] In embodiments, selecting an arthritis therapy comprises evaluating a current therapyof the subject, determining a new therapy for the subject, modifying a current therapy of the Page 43 of 66 1104081999\1\AMERICASsubject, or stopping a current therapy of the subject. In embodiments, modifying a current therapy comprises increasing a dose of arthritis therapy. In embodiments, determining a new therapy comprises administering another DMARD or anti-arthritic biologic to the subject. In embodiments, determining a new therapy comprises changing the arthritis therapy from a DMARD to an anti- arthritic biologic.

[0142] In embodiments, a treatment methodology can additionally or alternatively includeadministration of a therapeutically effective amount of one or more of the anti-14-3-3η antibodies as herein disclosed. For example, anti-14-3-3η antibodies, or antigen-binding fragments thereof, as herein disclosed can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an anti-14-3-3η antibody and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or emulsifying agents, preservatives, or buffers, can be included which may enhance the shelf life or effectiveness of the anti-14-3-3η antibodies. Additional details as to compositions, administration routes, and dosages of anti-14-3-3η antibodies are described in detail in PCT publication WO 2009 / 067811, the contents of which is hereby incorporated by reference in its entirety.

[0143] In some embodiments, a treatment methodology may additionally or alternativelyinclude a recommendation to the subject of one or more anti-arthritic lifestyle modifications. Such modifications may include changes to lifestyle, habits and practices intended to alleviate the symptoms of rheumatoid arthritis. Examples include changes to physical activity (e.g., increase in exercise), changes that lead to a reduction in body mass index (BMI), reduction or cessation of smoking, increase in vitamin and / or mineral intake (e.g., increased vitamin D intake), reduction in stress levels, increased quality of sleep, and the like.

[0144] Accordingly, in embodiments, a lateral flow assay to determine presence, absenceand / or amount of 14-3-3η protein and / or presence, absence and / or amount of autoantibodies Page 44 of 66 1104081999\1\AMERICASdirected there against (with or without information pertaining to other markers) as herein disclosed can be used to determine a likely efficacy of any one or more of the treatment options mentioned above on an arthritic condition in a subject. For example, in embodiments the continued presence, absence and / or amount of 14-3-3η protein and / or continued presence, absence and / or amount of autoantibodies directed there against (with or without information pertaining to other markers) can be used as a biomarker to infer whether a particular treatment regimen is working for a particular patient, or not. In embodiments where it is determined that a particular treatment regimen is not working, or is not working sufficiently as desired, the treatment regimen may be altered and the lateral flow assay as herein described can again be used to assess effectiveness of the new treatment regimen.

[0145] In an aspect, the invention provides methods for monitoring treatment of an arthriticcondition. In embodiments, the arthritic condition is selected from the group comprising or consisting of spondyloarthropathy (e.g. ankylosing spondylitis), Behçet's disease, childhood arthritis, Diffuse Idiopathic Skeletal Hyperostosis (DISH), Felty's Syndrome, fibromyalgia, gout, granulomatosis with polyangiitis (GPA), infectious arthritis, inflammatory arthritis, Mixed Connective Tissue Disease (MCTD), myositis (dermatomyositis, polymyositis), osteoarthritis, Paget's Disease, polymyalgia rheumatica with Giant Cell Arteritis, pseudogout, psoriatic arthritis, Raynaud's Phenomenon, reactive arthritis, rheumatoid arthritis, scleroderma, Sjögren syndrome, and / or systemic lupus erythematosus. In embodiments, the arthritic condition is rheumatoid arthritis. In embodiments, the arthritic condition is ankylosing spondylitis. In embodiments, the methods involve, via a lateral assay as herein disclosed, determining the presence and / or amount of 14-3-3η protein and / or presence and / or amount of autoantibodies directed there against (with or without information pertaining to other markers) as herein disclosed, and in turn monitoring effectiveness of a particular treatment regimen. In embodiments, the monitoring may include, via a lateral assay as herein disclosed, determining the presence, absence and / or amount of 14-3-3η protein and / or presence, absence and / or amount of autoantibodies directed there against (with or without information pertaining to other markers). In this way, the lateral flow assay(s) of the present disclosure can be used to monitor treatment efficacy for arthritic conditions, preferably rheumatoid arthritis.

[0146] Also provided herein are kits for evaluating an arthritic condition. In a preferredexample, the arthritic condition is rheumatoid arthritis. Such kits typically comprise two or more Page 45 of 66 1104081999\1\AMERICAScomponents necessary for performing a diagnostic, prognostic, and / or theranostic assay. Components may include compounds, reagents, containers, instructions, and / or equipment. For example and without limitation, a kit can include one or more lateral flow devices as herein disclosed, and one or more capillary blood collection tube(s). In embodiments, a capillary blood collection tube is a Microtainer™ (BD Biosciences, India), a Microvette® (Sarstedt, Germany), or other capillary blood collection device. In embodiments, a kit can include one or more buffers, dilution reagents, and the like. In embodiments, a kit contains instructions for use.

[0147] Accordingly, provided herein are kits comprising at least one lateral flow device fordetecting, in a capillary blood sample, the presence / absence and / or amount of 14-3-3η protein and / or presence / absence and / or amount of autoantibodies directed there against. In embodiments, a kit optionally includes reagents / compositions for detecting the presence of other markers, in a patient sample, which may be a capillary blood sample or other sample (e.g., synovial fluid, plasma, serum, or tissue (e.g., synovial joint, damaged joint tissue, etc.). The reagents / composition for detecting the presence of other markers can include reagents / compositions useful in immunological-based techniques, including but not limited to ELISA, western blot analysis, immunohistochemistry, immunofluorescence, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, precipitation reactions, agglutination assays, complement fixation assays, protein A assays, Immunoelectrophoresis assays, fluorescence activated cell sorting (FACS) analysis, radioimmunoassay, and the like.

[0148] In any of the methods of the present disclosure, the subject can be a human subject. Inany of the methods of the present disclosure, the arthritis is selected from the group comprising or consisting of spondyloarthropathy (e.g. ankylosing spondylitis), Behçet's disease, childhood arthritis, Diffuse Idiopathic Skeletal Hyperostosis (DISH), Felty's Syndrome, fibromyalgia, gout, granulomatosis with polyangiitis (GPA), infectious arthritis, inflammatory arthritis, Mixed Connective Tissue Disease (MCTD), myositis (dermatomyositis, polymyositis), osteoarthritis, Paget's Disease, polymyalgia rheumatica with Giant Cell Arteritis, pseudogout, psoriatic arthritis, Raynaud's Phenomenon, reactive arthritis, rheumatoid arthritis, scleroderma, Sjögren syndrome, and / or systemic lupus erythematosus. In embodiments, the arthritis is rheumatoid arthritis. In embodiments, the arthritis is a spondyloarthropathy, such as ankylosing spondylitis. EXAMPLES Page 46 of 66 1104081999\1\AMERICAS

[0149] The following examples are put forth so as to provide those of ordinary skill in the artwith a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0130] Example 1. Evaluation of 14-3-3η protein venous and capillary levels

[0131] This Example demonstrates that venous and capillary blood samples are substantiallyequivalent in terms of 14-3-3η protein levels.

[0132] In this Example, samples were obtained with a Microvette® (Sarstedt, Germany). 22matched samples were evaluated, fifteen of which were negative and 7 of which were positive. As illustrated in the graph depicted at FIG.1, levels of 14-3-3η protein levels were surprisingly found to be substantially equivalent in terms of amount in capillary and venous samples.

[0133] Example 2. HemaSpot testing

[0134] Serum samples were run on HemaSpot using a single punch or three (3) punch. One-step versus two-step extraction and different buffer conditions were also tested.

[0135] HemaSpot was found to be an unreliable method due to variability with sample flowon the device, high day-to-day variability in testing results and low extraction efficiency compared to pre-punch spotting and extraction with serum. Low positives flipped to negative and extraction efficiency for 3 punches was 20% and 33% for medium and high positives.

[0136] Example 3. Proof of concept and feasibility testing

[0137] Initial testing using ELISA kit reagents and Western blot materials in a simplifiedlateral flow assay showed significant challenges including: very slow and inconsistent sample flow, high non-specific binding (NSB), i.e. false positives, high running background, channeling of sample flow around spotted antibodies and poor sample retention on sample pads.

[0138] The simplified lateral flow assay, which is illustrated in FIG. 2, was performed by thefollowing procedure. No conjugate pad or control line was incorporated into the test strips. The sample—an antigen standard sample—was diluted in sample buffer containing an HRP- Page 47 of 66 1104081999\1\AMERICASconjugate. Diluted sample was then applied to the sample pad. The sample was allowed to flow by capillary action along the membrane. HRP substrate was then applied to the strips for detection. Finally, the results were analyzed, with a positive result being the appearance of a band at the Test Line and a negative result being the lack of a band at the Test Line.

[0139] Example 4. Conjugate type and blood volume

[0140] This Example demonstrates that the type of conjugate and sample volume impact thequality of the LFT assay.

[0141] Screening, selecting, and optimizing the LFT components (cassette, buffers, samplepad, conjugate pad, nitrocellulose (NC) and respective blocking treatments, Ab’s concentrations, and applications, etc.) for the first construct that reproducibly produced sensitivity of 0.36 ng / mL in serum samples was performed. The success of this design, unlike the previous attempts, is attributed to using GNS instead of colloidal gold for the conjugated antibody.

[0142] Initial experiments used colloidal gold particles for conjugates. However, results weredifficult to reproduce. Gold nanoshell (GNS) conjugates (nanoComposix) were used as a detection reagent in all further experiments instead of colloidal gold given that GNS provided improved sensitivity and reproducibility.

[0143] Sensitivity was achieved by increasing the blood volume to 50 µL, which is relativelyhigh for these types of assays, but was considered necessary to achieve the clinically relevant sensitivity and enable differentiating patient disease risk levels. These risk levels are shown in Table 3 below: Table 3. 14-3-3η Correlation Table Between Risk, Serum Concentration and 14-3-3η Status

[0144] This initial construct was able to reproducibly determine the analyte at theconcentration of ~0.4 ng / mL in serum, thus enabling the differentiation of high and very high radiographic risk category from moderate and low in a qualitative assay format. Page 48 of 66 1104081999\1\AMERICAS

[0145] However, issues remained that were related to non-specific background (NSB) andblood leeching from the sample pad into the test window.

[0146] In all of the following Examples, gold nanoshell (GNS) conjugates (nanoComposix)were used as a detection reagent instead of colloidal gold.

[0147] Example 5. Sweep of affinity components to reduce false positives / non-specificbackground

[0148] This example demonstrates that non-specific background, i.e. false positives, can bereduced through modification of the test line concentration and conjugate dispense rate.

[0149] An Arista / BioSigma 3.9mm cassette format was used with test line antibodyconcentrations at 1.4 mg / mL, 1.7 mg / mL or 2.0 mg / mL and conjugate dispense rates of 1.5 µL / mm, 2.0 µL / mm or 2.5 µL / mm. The negative control was running buffer while the positive control was 2 ng / mL 14-3-3 η spiked running buffer. Results were captured using a line signal LFT reader after 20 minutes. As shown in Table 4 below, the currently high test line antibody concentration (clone 4-8F10, 2 mg / mL) boosts test line signal but may contribute to the false positive signal (signal:noise ratio) while the higher conjugate (3-7F8) dispense rate at 2.5 µL / mm did not contribute significantly to non-specific binding and may be beneficial for the assay. Table 4. Signal-to-Noise Ratio for Varying Test Line Concentration and Conjugate Pad Dispense Rate

[0150] Example 6. Evaluation of materials to reduce blood leeching

[0151] This Example demonstrates that particular materials are advantageous over othercommonly used materials in lateral flow assays, to reduce blood leeching, reduce false positives, i.e. reduce non-specific background, and improve consistency of results. Page 49 of 66 1104081999\1\AMERICAS

[0152] A variety of conjugate pad / sample pad materials were compared to determine theirrelative impact on blood leeching in lateral flow devices of the present disclosure. The performance of these materials for preventing or reducing blood leeching upon addition of 50 µL donor blood chased with 70 µL running buffer was assessed. The cassette used was from Chembio (Hauppauge, NY, USA). Briefly, materials tested included glass fiber diagnostic pad (GFDX), 8980 (Ahlstrom-Munksjö), 1667 (Ahlstrom-Munksjö), 1668 HV+ (Ahlstrom- Munksjö), 1663 (Ahlstrom-Munksjö), Whatman MF1 bound glass fiber, and Whatman GF / DVA bound glass fiber. As illustrated at FIG.3, Ahlstrom Cytosep® HV plus 1668 emerged as a candidate for significant reduction of blood leeching. This material uses both physical and chemical separation of red blood cells to achieve the advantageous results.

[0153] The Arista / BioSigma 3.9mm traditional format was also tested using combinations ofa GFDX – 1668 conjugate pad stack or a 1668 – GFDX conjugate pad stack and different ratios of blood to running buffer. The results shown in FIG.4 show significant blood leeching.

[0154] The ChemBio format (5.1 mm) which can be adequate for higher sample volumeswas investigated.

[0155] Example 7. Experimental cassette trials

[0156] This Example demonstrates that lateral flow devices as herein described are capableof detecting 14-3-3η protein in a capillary blood sample. For this Example, strip dimensions were 5.0mm x 80mm. Blood volume tested was 50 µL, and buffer chase volume was 100-150 µL. When the MDI device (MDI Membrane Technologies, Camp Hill, PA) was combined with Ahlstrom grade 1668, it was determined that 50 µL of blood can be loaded to the cassette with minimal blood leeching and low signal-to-noise / false positive signal (FIG.5). FIG.5shows detected 14-3-3η protein, demonstrating feasibility of the disclosed lateral flow devices and methods of use thereof.

[0157] Initial results with MDI Device-2 were promising. When combined with AhlstromGrade 1668 blood separation pad, 50uL of blood can be loaded to the cassette with minimal blood leeching and low-NSB / FP signal. As shown in FIG.5, a combination of Ahlstrom Cytosep 1668 blood filter pad and novel 2-port cassette (MDI-2) improved blood leaching. FIG.5 shows results with 2.5 µL / mm dispensed conjugate and 1.4 mg / mL test lines. Page 50 of 66 1104081999\1\AMERICAS

[0158] A ShenFeng Plastics K116 cassette was also tested with varying concentrations ofrecombinant 14-3-3η diluted to 1, 0.5 or 0.25 ng / mL in blood. Briefly, the test line was 2.0 mg / mL 14-3-3η clone 4-8F10 and the control line was 1 mg / mL goat anti-mouse IgG. The conjugate pad was 10 mm GFDX, 2.5 µL dispensed conjugate pad. The assay format was a K116 3.9mm strip, 10 mm GFDX conjugate pad, 10 mm 1668, 25mm, 1660 SP triple stack.

[0159] A 30 µL sample of blood was added to the blood port and 90 µL running bufferadded to the sample port to chase. Test and control lines were analyzed on the line signal reader at 20 minutes. As shown in FIG.6, a combination of materials can minimize blood leaching, low sample volume capacity may limit sensitivity using this cassette.

[0160] Example 8. Further evaluation of test line concentration and conjugate OD

[0161] This Example demonstrates that lateral flow devices as herein described are capableof detecting 14-3-3η with improved signal to noise ratios based on test line concentration and conjugated OD.

[0162] Two strip constructions were used. One was a 26 mm 1668 sample pad, 10 mmGFDX conjugate pad, 12 mm 1668 stack (ID) while the other was a 25 mm GFDX sample pad, 10 mm GFDX conjugate pad, 12 mm 1668 stack in a MDI Device-22-port cassette (IE). Test lines were at 2.0, 1.5. or 1.0 mg / mL of 3-7F8 Mab, while conjugate conditions were OD 20, OD 40 and )D 60. Conjugate antibody was 3-7F8 and test line antibody was 4-8F10.

[0163] 0 or 0.8 ng / mL 14-3-3η-spiked single-donor whole blood was used. 50 µL of theblood sample was added to the second cassette port.120 µL of running buffer was then added to the first cassette port. Cassettes were analyzed on a line signal reader at 20 minutes.

[0164] Construct sensitivity was further improved by changing the construct elements and byincreasing both the conjugate Ab loading (3-7F8) and test line Ab (4-8F10) concentration (1E prototype). More specifically, the two prototypes were assembled in the same cassette (MDI) but were different in the strip construct. The ID design was: 26mm Ahlstrom 1668 Sample Pad, 10mm Millipore GFDX Conjugate Pad, 12 mm Ahlstrom 1668 Stack Pad sample pad (blood filter). The IE Design was: 26mm Millipore GFDX Sample Pad, 10mm Millipore GFDX Conjugate Pad, 12 mm Ahlstrom 1668 Stack Pad sample pad.

[0165] As shown in Table 5 below, in general, increasing the conjugate OD increased non-specific binding but did not significantly improve sensitivity. Higher test line concentration Page 51 of 66 1104081999\1\AMERICASgenerally improved test line sensitivity without increasing non-specific binding. The IE construction provided better flow and better test line signal to noise ratios, with IE showing better signal sensitivity and modulation with analyte concentration increase. The IE design used in the following Examples included the configuration shown in FIG.7. Line signal intensity data for ID and IE designs with test line concentrations of 2, 3, 4 or 4.8 mg / mL (FIG.8) and 0.4 ng / mL 14-3-3η or a test line of 4.8 mg / mL and varying 14-3-3η concentration (FIG.9) further supported these conclusions. The IE configuration used in further examples had a test line concentration of 4.8 mg / mL and a detection agent at OD20 (A825). The IE configuration used in further examples had a test line dispensed at 0.08 µL / mm and a conjugate dispensed at 2.5 µL / mm. Table 5. Signal-to-Noise Ratio for Varying Test Line Concentration and Conjugate OD 4-8F10 Conc. 14-3-3η Antigen Conc. in (mg / mL) 3-7F8 t DBlood S / N

[0166] Example 9. Evaluation of LFT versus JOINTstat ELISA

[0167] This Example demonstrates concordance between the results from a lateral flow testas described herein and the JOINTstat® ELISA in distinguishing positive and negative samples and suitability of the LFT as a semi-quantifiable / quantifiable method. Page 52 of 66 1104081999\1\AMERICAS

[0168] The 14-3-3ƞ concentration of serum samples determined by JOINTstat® will be usedas reference values to assess performance of LFT.

[0169] The protocol to be used for the study was optimized for 50 µL of whole blood as thesample volume. This study will be performed using 25 µL of serum. Serum provides a less complex matrix for testing and eliminates additional variables that could occur due to spiking antigen in whole blood.

[0170] 53 serum samples with 14-3-3η concentration determined by JOINTstat® were testedon a LFT (IE from Example 8). Among these samples, 19 were selected as negative samples (14- 3-3η < 0.19 ng / ml) and 34 positive samples had concentrations spanning from 0.27 to 28 ng / mL.

[0171] 25 µL of sample was added to the second sample port of the cassette. Samples werechased by adding 95 µL of running buffer to the first sample port. This was repeated every 30 seconds for each sample tested (n=2 for each strip condition). Strips were evaluated by visual inspection at 20 minutes. A reference key card as shown in FIG.10 was used to evaluate test line intensity. The key card scores ranged in intensity from no test line presence (Group 0) to increasing intensity, with the highest intensity indicated by Group 3.

[0172] Acceptable positive tests were those with the visual appearance of both test andcontrol lines, while acceptable negative tests were those with appearance of the control line only. If no control line appeared, the test was considered invalid. In order to be considered positive, the positive sample had to test positive for all replicates. In order to be considered negative, the negative sample had to test negative for all replicates.

[0173] Of the 53 samples tested, all positive samples (34 / 34) tested positive on LFT. For the19 negative samples, 85% (n =16) tested as negative on LFT with 15% (n=3) testing as false positives. This indicates a strong concordance between the two methods.

[0174] Visual scoring of the test lines obtained from the 544 samples showed a stepwisegradation of 14-3-3η levels, which is significant as shown in FIG.11 and Table 6 below which shows a Krusakl-Wallis test for the four intensity groups shown in FIG.11. Table 6. Kurskal-Wallis Test Results Kruskal-Wallis Test ResultPage 53 of 66 1104081999\1\AMERICASExact or approximate P value? ApproximateP v l mm r ****

[0175] These results demonstrate LFT positivity shows concordance with serum JOINTstat®14-3-3η positivity. Because the data indicate a stepwise gradation of 14-3-3η levels which is significant, the LFT can be used for disease prognosis and monitoring by using a reference key card to score intensity of the test line.

[0176] Example 10. Evaluation of spiked whole blood

[0177] This Example demonstrates that a LFT as described herein shows concordance withELISA results in distinguishing positive and negative samples and the suitability of the LFT as a semi-quantifiable / quantifiable method.

[0178] Recombinant 14-3-3η or a 14-3-3η positive serum sample, as tested with ELISA,were spiked into whole blood at concentrations of 0.4, 0.5, 0.6, 0.7, 0.8, 1.6 and 3.2 ng / mL. Each spiked sample was tested on three LFTs (n=3, triplicate) as described in Example 8 (IE).

[0179] Briefly, 50 µL of sample (either positive or negative) was added to the second sampleport of the cassette. Samples were chased by adding 90 µL of running buffer to the first sample port. This was repeated every 30 seconds for each sample tested (n=2 for each strip condition). Strips were evaluated visually at 20 minutes according to the acceptance criteria and reference card described in Example 9. The study was extended to assess 14-3-3η levels in 22 matched finger prick and venous serum samples.

[0180] Whole blood samples spiked at concentrations > 0.5 ng / mL for both recombinantprotein and positive serum tested positive on LFT. Only whole blood samples spiked at concentrations of 0.4 ng / mL tested negative on LFT. This highlights a strong concordance between the two methods (LFT and ELISA).

[0181] The intensity of the test line for the spiked whole blood samples on LFT was visuallyscored using the reference card described in Example 9. The LFT test line for spiked samples Page 54 of 66 1104081999\1\AMERICASshowed linear gradation to the corresponding expected concentration of 14-3-3η spiked in whole blood as shown in FIG.12.

[0182] The correlation between LFT test line intensity and spiked concentrations wasdetermined by Spearman correlation as the data for expected spiked concentrations was not normally distributed. LFT test line intensity and spiked 14-3-3η concentrations showed a strong & significant correlation (p=0.001) as shown in Table 7 below. Table 7. Test Line Intensity Versus Expected 14-3-3η Level Correlation Expected 14-3-3η Levels

[0183] In whole blood testing, LFT correctly identified all samples spiked at concentrations≥0.5 ng / mL. The Spearman correlation showed a strong and significant association (r=0.96 to 0.98, p=0.001) between LFT test line intensity and spiked 14-3-3η levels. Finally, the equivalence testing for finger prick and venous serum samples affirmed the LFT’s reliability, with a strong correlation (p<0.0001), underscoring its potential for near-patient testing.

[0184] A strong and significant correlation between LFT test line intensity and expectedspiked concentrations suggests Semi-Quantitative utility of the LFT method.

[0185] The Examples herein validate LFT as a reliable and semi-quantitative method fordetecting 14-3-3η in finger prick blood, demonstrating strong concordance with traditional ELISA results. ELISA results have been correlated with risk level. The findings support the feasibility of LFT in clinical laboratory medicine for early detection, prognosis, and monitoring of autoimmune diseases, paving the way for enhanced patient accessibility and self-initiated testing.

[0186] The embodiments and examples described above are intended to be merelyillustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims. Page 55 of 66 1104081999\1\AMERICAS

Claims

CLAIMS What is claimed is:

1. A method of assaying the level of 14-3-3η protein in a subject suspected of having or atrisk of developing an arthritic condition, the method comprising: a) contacting a capillary blood sample from a subject with a capture agent associatedwith a substrate, wherein the capture agent can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, and wherein the substrate is a portion of alateral flow immunochromatographic cassette; and b) detecting the binding of the capture agent to 14-3-3η protein with a detection agentthat binds to said within the lateral flow immunochromatographic cassette to produce a visible test line and determining whether the level of 14-3-3η protein is at or above a threshold level based on the resulting test line intensity; wherein the capture agent comprises one or more anti-14-3-3η antibodies, the detection agent comprises one or more labeled anti-14-3-3η antibodies, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent.

2. The method according to claim 1, wherein the lateral flow immunochromatographiccassette comprises, in order of flow, a buffer zone, a sample receiving zone comprising a sample pad, a conjugate pad comprising said detection agent, and a test zone comprising said substrate; wherein the buffer zone is located upstream of the sample receiving zone and the test zone is located downstream of the sample receiving zone, and the method comprises adding at least about 25 µl of sample to the sample receiving zone followed by adding at least about 75, 80, 85, 90, 95, 100, 105 or 110 µl of running buffer to the buffer zone, and upon flow of the sample through the lateral flow immunochromatographic cassette, labeled 14-3-3η protein from the sample will bind with the capture agent in the test zone to form a detectable complex producing the test line as part of a running buffer sweep. Page 56 of 66 1104081999\1\AMERICAS3. The method of claim 1 or 2, wherein the detection agent is detectably labeled with a labelselected from the group consisting of a gold nanoparticle, a carbon nanoparticle, a colored latex bead, an enzyme, a fluorescent label, a luminescent label, and a radioactive label.

4. The method of claim 3, wherein the gold nanoparticle is a gold nanoshell.

5. The method of any one of claims 1-4, wherein the capture agent is present at about 1.0 toabout 5.0 mg / mL, preferably about 4.8 mg / mL.

6. The method of any one of claims 1-5, wherein the detection agent is present about OD 20to about OD 60, preferably OD 20 (A825).

7. The method of any one of claims 1-6, wherein a volume of the capillary blood sampleadded to the lateral flow device is about 50 µL or less; preferably wherein the volume of the capillary blood sample is between about 25-50 µL.

8. The method of any preceding claim, wherein an arthritis therapy is initiated if the level of14-3-3η protein is above the threshold level, preferably wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette.

9. The method of any one of claims 1-7, comprising performing steps a) and b) on at leasttwo capillary blood samples taken at different time points from the subject, and initiating an arthritis therapy when the patient has at least two consecutive capillary blood samples displaying a level of 14-3-3η protein at or above the threshold level, or displaying an increasing level of 14- 3-3η protein and / or an increasing test line intensity in the subsequent sample.

10. The method of any one of claims 1-7, wherein the subject is a patient undergoing anarthritis therapy, the method comprising performing steps a) and b) on at least two capillary blood samples taken at different time points from the subject, and modifying the arthritis therapy Page 57 of 66 1104081999\1\AMERICASwhen the subject has at least two consecutive capillary blood samples displaying an increasing level of 14-3-3η protein and / or an increasing test line intensity in the subsequent sample.

11. The method of any one of claims 1-10, wherein the capture agent comprises a captureantibody having a heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 42, a HCDR2 having the sequence of SEQ ID NO: 43, a HCDR3 having the sequence of SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 45, a LCDR2 having the sequence of SEQ ID NO: 46, and a LCDR3 having the sequence of SEQ ID NO:

47.

12. The method of claim 11, wherein the capture antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 48 / 49 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 52 / 53.

13. The method of any one of claims 1-12, wherein the detection agent comprises a detectionantibody comprising a heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 34, a HCDR2 having the sequence of SEQ ID NO: 35, a HCDR3 having the sequence of SEQ ID NO: 36, a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 37, a LCDR2 having the sequence of SEQ ID NO: 38, and a LCDR3 having the sequence of SEQ ID NO:

39.

14. The method of claim 13, wherein the detection antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 40 / 41 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 50 / 51.

15. A method of treating an arthritic disease in a subject, wherein at least one capillary bloodsample taken from the subject is tested for the presence of 14-3-3η in a lateral flow assay, and wherein an arthritis therapy is initiated in the event that the level of 14-3-3η protein in the at least one capillary blood sample is at or above a threshold level within the lateral flow immunochromatographic cassette. Page 58 of 66 1104081999\1\AMERICAS16. The method of claim 15, wherein at least two capillary blood samples taken at differenttime points from the subject are tested for the presence of 14-3-3η in the lateral flow assay, and wherein an arthritis therapy is initiated in the event that the level of 14-3-3η protein in at least two capillary blood samples from the subject are at or above the threshold level within the lateral flow immunochromatographic cassette, or at least two capillary blood samples display an increasing level of 14-3-3η protein and / or an increasing test line intensity within the lateral flow immunochromatographic cassette.

17. The method of claim 16, wherein the different time points are at least seven days apart, atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks apart, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months apart.

18. The method of any one of claims 15-17, wherein testing for the presence of 14-3-3η in alateral flow assay comprises: a) contacting a capillary blood sample from a subject with a capture agent associatedwith a substrate, wherein the capture agent can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, and wherein the substrate is a portion of alateral flow immunochromatographic cassette; and b) detecting the binding of the capture agent to 14-3-3η protein with a detection agentthat binds to said protein within the lateral flow immunochromatographic cassette to produce a visible test line and determining whether the level of 14-3-3η protein is above or below a threshold level based on the resulting test line intensity; wherein the capture agent comprises one or more anti-14-3-3η antibodies, the detection agent comprises one or more labeled anti-14-3-3η antibodies, and the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent.

19. The method of claim 18, wherein the capture agent comprises a capture antibody havinga heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 42, a HCDR2 having the sequence of SEQ ID NO: 43, a HCDR3 having the sequence of SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) having the Page 59 of 66 1104081999\1\AMERICASsequence of SEQ ID NO: 45, a LCDR2 having the sequence of SEQ ID NO: 46, and a LCDR3 having the sequence of SEQ ID NO:

47.

20. The method of claim 19, wherein the capture antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 48 / 49 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 52 / 53.

21. The method of any one of claims 18-20, wherein the detection agent comprises adetection antibody comprising a heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 34, a HCDR2 having the sequence of SEQ ID NO: 35, a HCDR3 having the sequence of SEQ ID NO: 36, a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 37, a LCDR2 having the sequence of SEQ ID NO: 38, and a LCDR3 having the sequence of SEQ ID NO:

39.

22. The method of claim 21, wherein the detection antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 40 / 41 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 50 / 51.

23. A method of treating a patient with arthritis, the method comprising: a) testing for thepresence of 14-3-3η protein in a capillary blood sample of a patient within a lateral flow immunochromatographic cassette; b) identifying the patient as having 14-3-3η protein in the capillary blood sample; and c) initiating treatment of the patient having a capillary blood sample that is positive for 14-3-3η protein.

24. A method of detecting a reduction in, or absence of, therapeutic efficacy of an arthritistherapy in a subject that has been administered the arthritis therapy, the method comprising: a) detecting in a first capillary blood sample obtained from the subject at a first time point the presence or absence of at least a threshold level of 14-3-3η protein within a lateral flow immunochromatographic cassette; and b) detecting in a second capillary blood sample obtained from the subject at a second time point the presence or absence of at least a threshold level of 14-3-3η protein within the lateral flow immunochromatographic cassette; Page 60 of 66 1104081999\1\AMERICASwherein an increase in the level of 14-3-3η protein and / or an increase in test line intensity in the second capillary blood sample, or the presence of at least the threshold level in both the first and second capillary blood samples, indicates a reduction in, or absence of, therapeutic efficacy of the arthritis therapy.

25. The method of claim 24, wherein the threshold level comprises a test line intensity thatcorrelates with at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η protein in a reference sample within the lateral flow immunochromatographic cassette.

26. The method of claim 24 or 25, wherein the first capillary blood sample is taken before thesubject is administered the arthritis therapy, and wherein the presence of the threshold level of 14-3-3η protein in both the first and second samples indicates a reduction in, or absence of, therapeutic efficacy.

27. The method of any one of claims 24-26, wherein the first capillary blood sample and thesecond capillary blood sample are separated by at least 5 minutes, at least 15 minutes, at least an hour, at least one, two, three, four, five, six, or seven days, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

28. The method of any one of claims 8-27, wherein the arthritis therapy comprisesadministering a disease-modifying anti-rheumatic drug (DMARD) to the subject.

29. The method of claim 28, wherein the DMARD is selected from the group consisting ofapremilast, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, tofacitinib, and combinations thereof.

30. The method of any one of claims 7-27, wherein the arthritis therapy comprisesadministering an anti-arthritic biologic to the subject. Page 61 of 66 1104081999\1\AMERICAS31. The method of claim 30, wherein the anti-arthritic biologic is selected from the groupconsisting of a tumor necrosis factor-α inhibitor (TNF-α inhibitor), a B-cell inhibitor, an interleukin inhibitor, a selective co-stimulation modulator, and combinations thereof.

32. The method of claim 31, wherein the TNF-α inhibitor is selected from the groupconsisting of adalimumab, certolizumab, etanercept, golimumab, and infliximab.

33. The method of claim 31, wherein the B-cell inhibitor is selected from the groupconsisting of belimumab and rituximab.

34. The method of claim 31, wherein the interleukin inhibitor is selected from the groupconsisting of anakinra, canakinumab, guselkumab, ixekizumab, Risankizumab, sarilumab, secukinumab, tocilizumab, and ustekinumab.

35. The method of claim 31, wherein the selective co-stimulation modulator is abatacept.

36. The method of any one of claims 24-35, further comprising selecting an arthritis therapyfor the subject if there is an increase in the level of 14-3-3η protein and / or an increase in test line intensity in the second capillary blood sample.

37. The method of claim 36, wherein selecting an arthritis therapy comprises evaluating acurrent therapy of the subject, determining a new therapy for the subject, modifying a current therapy of the subject, or stopping a current therapy of the subject.

38. The method of claim 37 wherein the current therapy comprises administering a DMARDand / or an anti-arthritic biologic to the subject.

39. The method of claim 37 or 38, wherein modifying a current therapy comprises increasinga dose of the arthritis therapy.

40. The method of claim 37 or 38, wherein determining a new therapy comprisesadministering another DMARD or anti-arthritic biologic to the subject. Page 62 of 66 1104081999\1\AMERICAS41. The method of claim 37 or 38, wherein determining a new therapy comprises changingthe arthritis therapy from a DMARD to an anti-arthritic biologic.

42. The method of any one of claims 1-26, further comprising changing the arthritis therapyfrom a DMARD to an anti-arthritic biologic if the second capillary blood sample has at least the threshold level of 14-3-3η.

43. The method of any of the preceding claims, wherein the subject is a human subject.

44. The method of any of the preceding claims, wherein the arthritis is rheumatoid arthritis.

45. A method of detecting a reduction in therapeutic efficacy of an arthritis therapy in asubject that has been administered the arthritis therapy, the method comprising performing a lateral flow assay to determine whether two or more capillary blood samples taken at different time points from the subject contain a level of 14-3-3η protein that is at least a threshold level; wherein the threshold level is equal to the level of 14-3-3η protein present in a reference sample comprising at least about 0.20 ng / mL, at least about 0.30 ng / mL, at least about 0.40 ng / mL, at least about 0.50 ng / mL, at least about 0.60 ng / mL, at least about 0.70 ng / mL, or at least about 0.80 ng / mL 14-3-3η and the resulting test line intensity within said lateral flow immunochromatographic cassette; and wherein an increase in or the presence of the threshold level of 14-3-3η protein in the two or more samples indicates a reduction in, or absence of, therapeutic efficacy.

46. The method of claim 45, further comprising selecting an arthritis therapy for the subjectif a threshold level of 14-3-3η protein is determined to be present in at least two capillary blood samples from the subject.

47. The method of claim 46, wherein selecting an arthritis therapy comprises evaluating acurrent therapy of the subject, determining a new therapy for the subject, modifying a current therapy of the subject, or stopping a current therapy of the subject. Page 63 of 66 1104081999\1\AMERICAS48. A device for assaying the level of 14-3-3η protein in a capillary blood sample from asubject, the device comprising: a capture agent associated with a substrate, wherein the capture agent comprises one or more anti-14-3-3η antibodies that can bind labeled 14-3-3η protein in the capillary blood sample to form a complex, and wherein the substrate is a portion of a lateral flow immunochromatographic cassette comprising, in order of flow, a buffer zone, a sample receiving zone comprising a sample pad, a conjugate pad comprising a detection agent comprising one or more labeled antibodies, and a test zone comprising said substrate, configured such that: the buffer zone is located upstream of the sample receiving zone and the test zone is located downstream of the sample receiving zone, upon flow of the sample through the lateral flow immunochromatographic cassette, labeled 14-3-3η protein from the sample will bind with the capture agent in the test zone to form a detectable complex producing the test line as part of a running buffer sweep; wherein the capture agent binds to a different epitope of the 14-3-3η protein than the detection agent; preferably wherein the capture agent is an antibody produced by hybridoma clone AUG4-ETA-8F10 or an antigen binding fragment thereof, and the detection agent is an antibody produced by hybridoma clone AUG3-CKNS-7F8 or an antigen binding fragment thereof.

49. The device according to claim 48, wherein the detection agent is detectably labeled witha gold nanoshell.

50. The device according to claim 48 or 49, wherein sample receiving zone of the lateral flowimmunochromatographic cassette comprises a 26mm Ahlstrom 1668 Sample Pad, a 10mmMillipore GFDX Conjugate Pad, and a 12 mm Ahlstrom 1668 Stack Pad sample pad, optionally wherein the lateral flow immunochromatographic cassette is a MDI Device-22-port cassette.

51. The device according to claim 48 or 49, wherein sample receiving zone of the lateral flowimmunochromatographic cassette comprises a 26mm Millipore GFDX Sample Pad, a 10mmMillipore GFDX Conjugate Pad, and a 12 mm Ahlstrom 1668 Stack Pad sample pad, optionally wherein the lateral flow immunochromatographic cassette is a MDI Device-22-port cassette. Page 64 of 66 1104081999\1\AMERICAS52. The device of any one of claims 48-51, wherein the capture agent comprises a captureantibody having a heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 42, a HCDR2 having the sequence of SEQ ID NO: 43, a HCDR3 having the sequence of SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 45, a LCDR2 having the sequence of SEQ ID NO: 46, and a LCDR3 having the sequence of SEQ ID NO:

47.

53. The device of claim 52, wherein the capture antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 48 / 49 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 52 / 53.

54. The device of any one of claims 48-53, wherein the detection agent comprises a detectionantibody comprising a heavy chain complementarity determining region 1 (HCDR1) having the sequence of SEQ ID NO: 34, a HCDR2 having the sequence of SEQ ID NO: 35, a HCDR3 having the sequence of SEQ ID NO: 36, a light chain complementarity determining region 1 (LCDR1) having the sequence of SEQ ID NO: 37, a LCDR2 having the sequence of SEQ ID NO: 38, and a LCDR3 having the sequence of SEQ ID NO:

39.

55. The device of claim 54, wherein the detection antibody comprises a heavy chain variableregion / light chain variable region sequence pair comprising SEQ ID NOs: 40 / 41 or a heavy chain / light chain sequence pair comprising SEQ ID NOs: 50 / 51.

56. The device of any one of claims 48-55, wherein the capture agent is present at about 1.0to about 5.0 mg / mL, preferably about 4.8 mg / mL.

57. The device of any one of claims 48-56, wherein the detection agent is present about OD20 to about OD 60, preferably OD 20 (A825).

58. A kit for assaying the level of 14-3-3η protein in a capillary blood sample obtained froma subject, comprising: a device according to any one of claims 48-57, and a running buffer. Page 65 of 66 1104081999\1\AMERICAS

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