Methods of assessing urine samples for antibodies of interest

Abstract

Provided are methods of assessing a urine sample for antibodies of interest. In some instances, the methods comprise contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, and any combination thereof. Such methods further comprise assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more types of HLA antigens. According to some embodiments, the urine sample was obtained from a kidney transplant recipient, and the methods optionally further comprise monitoring the kidney transplant recipient (e.g., for rejection of the kidney transplant) based on the assessing. Antigen arrays that find use in practicing the methods of the present disclosure are also provided.

Description

[0001] METHODS OF ASSESSING URINE SAMPLES FOR ANTIBODIES OF INTEREST

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 676,781 , filed July 29, 2024, which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] Kidney transplantation is the optimal treatment option for individuals with end-stage-renal- disease (ESRD). Even though there has been significant improvement in how the transplanted kidneys are maintained and monitored, long-term survival of transplanted kidneys remains a major challenge. After transplantation, transplanted kidneys endure injuries, of both immune and non-immune assaults. To date, most studies in kidney transplantation have focused on the analysis of immune cell infiltration gene and protein expression in the kidney and blood. However, the complex mechanism of organ injury and failure in kidney transplantation is still yet to be fully understood. Better methods for early detection of transplant injury are highly desired in order to move towards the goal of increasing long-term survival of transplanted kidneys to decrease the burden of organ shortage for kidney transplantation.

[0006] SUMMARY

[0007] Provided are methods of assessing a urine sample for antibodies of interest. In some instances, the methods comprise contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA- DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof. Such methods further comprise assessing the urine sample for anti- HLA antibodies by assessing for binding of antibodies to the one or more types of HLA antigens. According to some embodiments, the urine sample was obtained from a kidney transplant recipient, and the methods optionally further comprise monitoring the kidney transplant recipient (e.g., for rejection of the kidney transplant) based on the assessing. Antigen arrays that find use in practicing the methods of the present disclosure are also provided.

[0008] BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1A-1 C: (A) Study schematic to identify urine Ab markers for graft injury in kidney transplantation. (B) Summary of study samples, which included blood samples collected from healthy control (no injury), samples from transplant recipients with no allograft injury, and samples with 3 different allograft injuries, namely, acute rejection, chronic injury, and BK virus nephropathy. (C) IgG antibodies were identified against 1752 reactive antigens in the urine. Antigens were mapped for their antigenicity with kidney compartments reported previously, which resulted in 28 glomerulus-specific antigens, 32 inner cortex-specific antigens, 65 outer cortex- specific antigens, seven medulla-specific antigens, 43 papillary tips, and 1 14 renal pelvis-specific antigens.

[0010] FIG. 2A-2B: (A) A heatmap showing unsupervised clustering of 20 significantly increased antibodies in transplantation with no injury compared to healthy control. (B) A volcano plot displaying the most significantly increased antibodies in kidney transplant with no injury samples.

[0011] FIG. 3A-3B: (A) A volcano plot displaying IgG antibodies that were increased in transplant injuries, including acute rejection, chronic allograft injury and BK virus nephropathy of kidney transplant recipients when compared to healthy control. (B) A volcano plot displaying IgG antibodies that were increased in transplant injuries, including acute rejection, chronic allograft injury and BK virus nephropathy of kidney transplant recipients when compared to transplant recipients with no graft injury.

[0012] FIG. 4A-4B: (A) A volcano plot displaying IgG antibodies that were increased in acute rejection compared to healthy control. (B) A volcano plot displaying IgG antibodies that were increased acute rejection compared to transplant recipients with no graft injury.

[0013] FIG. 5A-5C: (A) A heat map that lists increased antibodies in BKVN urine compared to healthy urine. (B) A volcano plot displaying IgG antibodies that were increased in BK virus infection in kidney transplantation compared to healthy control. (C) A volcano plot displaying IgG antibodies that were increased in BK virus infection in kidney transplantation when compared to transplant recipients with no graft injury.

[0014] DETAILED DESCRIPTION

[0015] Before the methods and antigen arrays of the present disclosure are described in greater detail, it is to be understood that the methods and antigen arrays are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and antigen arrays will be limited only by the appended claims.

[0016] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and antigen arrays. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and antigen arrays, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and antigen arrays.

[0017] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0018] 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 methods and antigen arrays belong. Although any methods and antigen arrays similar or equivalent to those described herein can also be used in the practice or testing of the methods and antigen arrays, representative illustrative methods and antigen arrays are now described.

[0019] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and antigen arrays are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0020] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0021] It is appreciated that certain features of the methods and antigen arrays, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and antigen arrays, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and antigen arrays and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0022] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0023] METHODS OF ASSESSING URINE SAMPLES FOR ANTIBODIES OF INTEREST

[0024] Aspects of the present disclosure include methods of assessing urine samples for antibodies of interest. In a first aspect, provided are methods of assessing a urine sample for anti-human leukocyte antigen (anti-HLA) antibodies, the methods comprising contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof. Such methods further comprise assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more types of HLA antigens. In a second aspect, provided are methods of assessing a urine sample for antibodies, the methods comprising contacting an antigen array with a urine sample, wherein the antigen array comprises one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM1 12A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof. Such methods further comprise assessing the urine sample for the antibodies by assessing for binding of antibodies to the one or more antigens.

[0025] The methods of the present disclosure find a variety of uses and are based at least in part on the unexpected finding that antibodies associated with antibody-mediated kidney transplant rejection and kidney graft injury are detectable in urine. There has been substantial effort to understand the complex mechanism of graft rejection and acceptance. Recently developed “omic” methods are powerful for interrogating complex biological processes to study disease causes and mechanisms. Most studies in kidney transplantation have focused on the analysis of gene and protein expression in the kidney and blood. The Human Leukocyte Antigen (HLA) system is important in organ and tissue transplantation in that HLA antibodies play a crucial role in organ transplant rejection, particularly in the context of antibody-mediated rejection (AMR). Demonstrated for the first time herein is quantitative identification of IgG antibodies against HLA and non-HLA (nHLA) antigens in the urine collected from kidney transplant recipients with graft injuries, including acute rejection. Several of the HLA and nHLA antigens specifically mounted an antibody response at the time of graft injury and acute rejection. Thus, in some instances, the methods find use in noninvasive monitoring of a kidney transplant recipient (e.g., for rejection of the kidney transplant and / or injury to the kidney graft) which constitute an improvement over existing invasive approaches. Anti-HLA Antibody Assessment

[0026] As summarized above, aspects of the present disclosure include methods of assessing a urine sample for anti-human leukocyte antigen (anti-HLA) antibodies. The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important part of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized to present antigenic peptides to the T-cell receptor (TCR) on T cells.

[0027] Class I MHC molecules are present as transmembrane glycoproteins on the surface of all nucleated cells. Intact class I molecules consist of an alpha heavy chain bound to a beta-2 microglobulin molecule. The heavy chain consists of 2 peptide-binding domains, an immunoglobulin (Ig)-like domain, and a transmembrane region with a cytoplasmic tail. The heavy chain of the class I molecule is encoded by genes at HLA-A, HLA-B, and HLA-C loci. T cells that express CD8 molecules react with class I MHC molecules. These T cells often have a cytotoxic function, requiring them to be capable of recognizing any infected cell. Because every nucleated cell expresses class I MHC molecules, all infected cells can act as antigen-presenting cells for CD8 T cells (CD8 binds to the nonpolymorphic part of the class I heavy chain). Some class I MHC genes encode nonclassical (ie, not CD8-binding) MHC molecules, such as HLA-G (which may play a role in protecting the fetus from the maternal immune response) and HLA-E (which presents peptides to certain receptors on natural killer [NK] cells).

[0028] Class II MHC molecules are usually present only on professional antigen-presenting cells (B cells, macrophages, dendritic cells, Langerhans cells), thymic epithelium, and activated (but not resting) T cells. Most nucleated cells can be induced to express class II MHC molecules by interferon (IFN)-gamma. Class II MHC molecules consist of 2 polypeptide (alpha [a] and beta [p]) chains; each chain has a peptide-binding domain, an Ig-like domain, and a transmembrane region with a cytoplasmic tail. Both polypeptide chains are encoded by genes in the HLA-DP, HLA-DQ, or HLA-DR region of chromosome 6. T cells reactive to class II molecules express CD4 and are often helper cells.

[0029] The MHC class III region of the genome encodes several molecules important in inflammation; they include complement components C2, C4, and factor B; tumor necrosis factor (TNF)-alpha; lymphotoxin; and 3 heat shock proteins, which help protect cells from stress and maintain protein homeostasis.

[0030] Individual serologically defined antigens encoded by the class I and class II gene loci in the HLA system are given standard designations (e.g., HLA-A1 , -B5, -C1 , -DR1 ). Alleles defined by DNA sequencing are named to identify the gene, followed by an asterisk, numbers representing the allele group (often corresponding to the serologic antigen encoded by that allele), a colon, and numbers representing the specific allele (e.g., A*02:01 , DRB1 *01 :03, DQA1 *01 :02). Sometimes additional numbers are added after a colon to identify allelic variants that encode identical proteins, and after another colon, other numbers are added to denote polymorphisms in introns or in 5' or 3' untranslated regions (e.g., A*02:101 :01 :02, DRB1 *03:01 :01 :02).

[0031] The MHC class I and class II molecules are the most immunogenic antigens that are recognized during rejection of an allogeneic transplant. The strongest determinant is HLA-DR, followed by HLA-B and HLA-A. These three loci are therefore the most important in the context of transplant compatibility and rejection.

[0032] The methods of assessing a urine sample for anti-HLA antibodies comprise contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA- DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof. In some instances, the HLA antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens. According to some embodiments, the HLA antigen array comprises 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 1 1 or fewer, or 10 or fewer types of HLA antigens.

[0033] The HLA antigen array may further comprise one or more non-HLA antigens, wherein the assessing comprises assessing the urine sample for antibodies specific for the one or more non- HLA antigens by assessing for binding of antibodies to the one or more non-HLA antigens. Nonlimiting examples of non-HLA antigens which may be present on the HLA antigen array include AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof. Further no-limiting examples of non-HLA antigens which may be present on the HLA antigen array include CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof. Further no-limiting examples of non-HLA antigens which may be present on the HLA antigen array include BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof. In some instances, the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens.

[0034] According to some embodiments, the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0035] Non-HLA Antibody Assessment

[0036] Aspects of the present disclosure include methods of assessing a urine sample for antibodies of interest, e.g., non-HLA antibodies. In some embodiments, such methods comprise contacting an antigen array with a urine sample, wherein the antigen array comprises one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, F0XP4, GADD45G, G0RASP1 , HCLS1 , IVD, 0TUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKN0X2, PPIA, RAB3D, R0R1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof. The methods further comprise assessing the urine sample for the antibodies by assessing for binding of antibodies to the one or more antigens. In some instances, the antigen array comprises 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more of the antigens. In some embodiments, the antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer of the antigens. In certain embodiments, the antigen array comprises 35 or fewer, 30 or fewer, 25 or fewer, or 20 or fewer of the antigens.

[0037] Array Formats

[0038] The HLA antigen array may be provided in any suitable format. In some embodiments, the array comprises the antigens on a single substrate or solid support. Non-limiting examples of substrates and solid supports include a micro-well plate, a slide (e.g., an epoxysilane-coated or nitrocellulose-coated glass slide), a stick, a chip, and the like. In other embodiments, the array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen. The term “solid support” means an insoluble material having a surface to which reagents or materials can be attached, e.g., so that they can be readily separated from a solution. An antigen array may comprise a collection of solid supports having an average greatest dimension of 1000 micrometers (pm) or less. In some embodiments, the collection of solid supports has an average greatest dimension of 750 pm or less, 500 pm or less, 250 pm or less, 100 pm or less, 1 pm or less, 0.75 pm or less, 0.50 pm or less, 0.25 pm or less, or 0.1 pm or less. In certain embodiments, the particulate solid supports have an average greatest dimension of from about 0.50 pm to about 500 pm, e.g., from about 0.75 pm to about 250 pm, e.g., about 1 pm.

[0039] A variety of materials can be used as solid supports. Support materials include any material that can act as a support for indirect or direct attachment of the antigens. Suitable materials include, but are not limited to, organic or inorganic polymers, natural and synthetic polymers, including, but not limited to, agarose, cellulose, nitrocellulose, cellulose acetate, other cellulose derivatives, dextran, dextran-derivatives and dextran co-polymers, other polysaccharides, glass, silica gels, gelatin, polyvinyl pyrrolidone, rayon, nylon, polyethylene, polypropylene, polybutylene, polycarbonate, polyesters, polyamides, vinyl polymers, polyvinylalcohols, polystyrene and polystyrene copolymers, polystyrene cross-linked with divinylbenzene or the like, acrylic resins, acrylates and acrylic acids, acrylamides, polyacrylamides, polyacrylamide blends, co-polymers of vinyl and acrylamide, methacrylates, methacrylate derivatives and co-polymers, other polymers and co-polymers with various functional groups, latex, butyl rubber and other synthetic rubbers, silicon, glass, paper, natural sponges, insoluble protein, surfactants, metals, metalloids, magnetic materials, and any combinations thereof.

[0040] The solid supports may be any suitable shape, including but not limited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or non-geometric shape.

[0041] In certain embodiments, the solid supports are beads. As used herein, the term “bead” refers to a small mass that is generally spherical or spheroid in shape. According to some embodiments, a bead as used herein has an average diameter of from about 0.50 pm to about 500 pm, e.g., from about 0.75 pm to about 250 pm, e.g., about 1 pm.

[0042] Additionally, and for purposes herein, the solid supports may be magnetically responsive, e.g., by virtue of comprising one or more paramagnetic and / or superparamagnetic substances, such as for example, magnetite. Such paramagnetic and / or superparamagnetic substances may be embedded within the matrix of the solid supports, and / or may be disposed on an external and / or internal surface of the bead.

[0043] Any suitable strategies for stably associating antigens to the surface of a solid support may be employed. For example, the surface may be functionalized (or “activated” / “derivatized”) with reactive groups to which the antigen may bind to become bound to the surface of the solid support. The surface may be functionalized with any useful / convenient reactive group, including but not limited to thiol groups (-SH), amine groups (-NH2), carboxyl groups (-COO), and / or the like.

[0044] Bioconjugation strategies that find use in binding antigens to surfaces are described in Hermanson, “Bioconjugate Techniques,” Academic Press, 3rd edition, 2013, Haugland, 1995, Methods Mol. Biol. 45:205-21 ; Brinkley, 1992, Bioconjugate Chemistry 3:2, and elsewhere.

[0045] Reactions for the modification of silanol groups are known and include, but are not limited to, modification of a SiO2surface to present amines (e.g., by reaction with aminopropyl trimethoxysilane (APTMS)) or ethoxides (e.g., by reaction with 3-glycidyloxypropyl- trimethoxysilane (GPTMS)). Reagents also exist to incorporate sulfhydryls, carboxyl and other useful reactive groups for conjugation.

[0046] The antigens may already include a functional group useful for reacting with a reactive group present on the surface of a solid support, or such a functional group may be provided to the antigens or solid support. Functional groups that may be employed include, but are not limited to, active esters, isocyanates, imidoesters, hydrazides, amino groups, aldehydes, ketones, photoreactive groups, maleimide groups, alpha-halo-acetyl groups, epoxides, azirdines, and the like. Reagents such as iodoacetamides, maleimides, benzylic halides and bromomethylketones react by S-alkylation of thiols to generate stable thioether products. For example, at pH 6.5-7.5, maleimide groups react with sulfhydryl groups to form stable thioether bonds. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide by the nucleophile. Because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa(~8.3, depending on protein structural context). Thiols also react with certain amine-reactive reagents, including isothiocyanates and succinimidyl esters. The TS-Link series of reagents are available for reversible thiol modification.

[0047] With respect to amine reactive groups, primary amines exist at the N-terminus of polypeptide chains and in the side-chain of lysine (Lys, K) amino acid residues. Among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic, making them ready targets for conjugation with several reactive groups. For example, NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS). Also by way of example, imidoester crosslinkers react with primary amines to form amidine bonds. Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases. As such, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8-10.

[0048] Numerous other synthetic chemical groups will form chemical bonds with primary amines, including but not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, carbodiimides, anhydrides, and fluorophenyl esters. Such groups conjugate to amines by either acylation or alkylation.

[0049] In general, a solid support may first be reacted with a liquid phase component comprising the one or more antigens of interest under suitable binding conditions such that the antigens are sufficiently immobilized to the support. Optionally, immobilization of the antigens to the support can be enhanced by first coupling the antigens to a protein with better binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to the Agl, are well known to those of ordinary skill in the art. See, e.g., Brinkley, M.A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30:117-124; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0050] Approaches for fabricating protein arrays are known in the art and include those described in Sutandy et al. (2013) Curr Protoc Protein Sci. 72(1 ):271 1-27116; Berrade et al. (201 1 ) Pharm Res. 28(7):1480-1499; and Joos (2004) “Protein Arrays: Methods and Protocols” Expert Review of Proteomics 1 (3):261 -262; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0051] Assessment for the Presence of Antibodies in Urine

[0052] A variety of suitable approaches for assessing for the presence of antibodies of interest in biological samples are known and may be implemented when practicing the methods of the present disclosure.

[0053] In some embodiments, assessing for the presence or absence of the antibodies is carried out using a sandwich assay. For example, the antigens may be attached to a solid surface (e.g., as an array) for capturing the antibodies (if present in the urine sample), and one or more detection reagents are added that bind (e.g., specifically bind) to the captured antibodies. In certain embodiments, a detection reagent is a detection antibody (e.g., anti-human IgG antibody when the urine sample was obtained from a human subject) that binds to an epitope of the captured antibodies that is different from the binding site to which the antigen binds. As a result, the antibody (if present in the urine sample) is “sandwiched” between the antigen and the detection reagent. The detection reagents may include detectable labels such that assessing for the presence or absence of binding of antibodies to antigens on the array involves detecting the labels of the detection reagents. According to certain embodiments, a secondary detection reagent is employed. Suitable secondary reagents include labeled secondary antibodies (e.g., fluorescently labeled antibodies, magnetic labeled antibodies, etc.), secondary antibodies linked to an enzyme that catalyzes the conversion of a substrate to a detectable product, and the like. Examples of labels which permit direct measurement of immunocomplexes include radiolabels, such as3H or125l, fluorophores, dyes, beads, chemiluminescents, colloidal particles, and the like. Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a colored or fluorescent product. The label may be detected directly (e.g., a radioisotope) or by binding to one or more additional molecules (e.g., an epitope which binds to a labeled antibody). Non-limiting examples of fluorescent compounds which may be employed include fluorochromes such as Fluorescein (FITC) or Alexa 488, Cy 3, or Cy 5 and green fluorescent protein, as well as phycoerythrin (PE) PE-Cy5, PerCP, ECD, and the like. In some embodiments, a secondary antibody (e.g., secondary anti-human IgG antibody when the urine sample was obtained from a human subject) is labeled with a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like.

[0054] According to some embodiments, the assessing is conducted by an enzyme-linked immunosorbent assay (ELISA). ELISA assays for detecting urinary antibodies have been developed and include those described in Nagaoka et al. (2021 ) Vaccines 9:778 9(7):778. doi: 10.3390 / vaccines9070778; Itoh et al. (2001 ) Am. J. Trap. Med. Hyg. 65:362-365; and Alemohammad et al. (1993) J. Clin. Microbiol. 31 :2174-2177; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0055] Additional details and design considerations for sandwich and other assays that find use in practicing the methods of the present disclosure are described, e.g., in Cox et al. (2014) Immunoassay Methods, Eli Lilly & Company and the National Center for Advancing T ranslational Sciences.

[0056] In certain embodiments, the assessing is by flow cytometry. Bead-based multiplexed flow cytometry assays and reagents for detecting antibodies in biological samples are known and include the LABScreen methodology and reagents available from Thermo Scientific / One Lambda. In one non-limiting example, a panel of color-coded microbeads coated with different antigens can be combined in one suspension in a single test. The biological sample to be interrogated is incubated with the beads and any human antibodies in the sample that bind to the antigens are labeled with R-Phycoerythrin (PE) conjugated antihuman IgG. Beads comprising labeled antibodies are then detected using a flow cytometer according to the manufacturer’s instructions. With the benefit of the present disclosure, one of ordinary skill in the art may adapt such approaches for assessing urine samples.

[0057] Kidney Transplant Applications

[0058] According to any of the methods of the present disclosure, the urine sample may have been obtained from a kidney transplant recipient. The methods of the present disclosure are based at least in part on the unexpected finding that antibodies associated with antibody- mediated kidney transplant rejection and kidney graft injury are detectable in urine. Demonstrated for the first time herein is quantitative identification of IgG antibodies against HLA and non-HLA (nHLA) antigens in the urine collected from kidney transplant recipients with graft injuries, including acute rejection. Several of the HLA and nHLA antigens specifically mounted an antibody response at the time of graft injury and acute rejection.

[0059] Accordingly, in some instances, when the urine sample was obtained from a kidney transplant recipient and the antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof, the methods further comprise monitoring for rejection of the kidney transplant based on the assessing. The monitoring may indicate rejection of the kidney transplant. When the monitoring indicates rejection of the kidney transplant, in some instances, the methods further comprise treating the kidney transplant recipient for rejection of the kidney transplant. In certain embodiments, treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient. According to some embodiments, treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (I VIG), rituximab, bortezomib, or any combination thereof to the kidney transplant recipient. In some instances, treating the kidney transplant recipient comprises splenectomy.

[0060] In some instances, the monitoring indicates the absence of rejection of the kidney transplant. When the monitoring indicates the absence of rejection of the kidney transplant, in certain embodiments, the methods further comprise modifying a treatment of the kidney transplant recipient based on the absence of rejection of the kidney transplant. The modifying may comprise ceasing or reducing administration of an immunosuppressive agent to the kidney transplant recipient. According to some embodiments, the modifying comprises ceasing or reducing administration of one or more of IVIG, rituximab, and / or bortezomib to the kidney transplant recipient. Details regarding strategies and agents for treating kidney transplant rejection of different classes / grades (e.g., according to the Banff classification system) are described in Alasfar et al. (2023) J Clin Med. 12(15):4927 doi: 10.3390 / jcm12154927, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0061] In certain embodiments, when the urine sample was obtained from a kidney transplant recipient and the antigen array comprises one or more non-HLA antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof, the methods further comprise monitoring the kidney transplant for graft injury. In some instances, such monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN). When the monitoring indicates the presence of AR, CAN, and / or BKVN, the methods may further comprise treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

[0062] ANTIGEN ARRAYS

[0063] Aspects of the present disclosure further include antigen arrays.

[0064] In certain embodiments, provided are HLA antigen arrays comprising two or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof, wherein the HLA antigen array comprises 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer types of HLA antigens. According to some embodiments, the HLA antigen array comprises two or three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0065] The HLA antigen array may further comprise one or more non-HLA antigens. Non-limiting examples of non-HLA antigens which may be present on the HLA antigen array include one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof. Further non-limiting examples of non-HLA antigens which may be present on the HLA antigen array include one or more antigens chosen from CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof. Further non-limiting examples of non-HLA antigens which may be present on the HLA antigen array include one or more antigens chosen from BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof. In certain embodiments, the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens. According to some embodiments, the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0066] In certain embodiments, provided are antigen arrays comprising two or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof, wherein the array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens. Such an antigen array may comprise 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more of the antigens. The antigen array may comprise one or more HLA antigens. For example, the antigen array may comprise one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof. Such an array may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0067] The antigen arrays may be provided in any convenient format, including any of the array formats described in the section above relating to methods of assessing urine samples for antibodies of interest. For example, all antigens of the antigen array may be present on a single substrate, e.g., glass slide, micro-well plate, etc. In other embodiments, the antigen array comprises a plurality of solid supports (e.g., beads), wherein each solid support comprises a single type of antigen.

[0068] KITS

[0069] Aspects of the present disclosure further include kits. In some embodiments, a kit includes one or more arrays (e.g., two or more arrays) of the present disclosure.

[0070] The kits may include instructions for using the one or more arrays. For example, the kits may comprise instructions for using the one or more arrays to monitor a kidney transplant recipient, e.g., for kidney transplant rejection and / or graft injury.

[0071] The kits may further comprise one or more reagents that find use in the contacting and / or assessing steps of the methods of the present disclosure. For example, a kit may further comprise a detection reagent for detecting urinary antibodies bound to one or more antigens on the array. In one non-limiting example, a kit of the present disclosure further comprises an anti-human IgG antibody (which may be detectably labeled) for use in detecting urinary antibodies (present in a urine sample obtained from a human kidney transplant recipient) bound to antigens on the array.

[0072] The instructions included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0073] Non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

[0074] 1 . A method of assessing a urine sample for anti-human leukocyte antigen (anti-HLA) antibodies, the method comprising: contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA- DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof; and assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more types of HLA antigens.

[0075] 2. The method of clause 1 , wherein the HLA antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0076] 3. The method of clause 1 or 2, wherein the HLA antigen array comprises 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer types of HLA antigens.

[0077] 4. The method of any one of clauses 1 -3, wherein the HLA antigen array further comprises one or more non-HLA antigens, and wherein the assessing comprises assessing the urine sample for antibodies specific for the one or more non-HLA antigens by assessing for binding of antibodies to the one or more non-HLA antigens.

[0078] 5. The method of clause 4, wherein the non-HLA antigens comprise one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, F0XP4, GADD45G, G0RASP1 , HCLS1 , IVD, 0TUB2, SDCCAG3, and any combination thereof.

[0079] 6. The method of clause 4 or 5, wherein the non-HLA antigens comprise one or more antigens chosen from CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof.

[0080] 7. The method of any one of clauses 4-6, wherein the non-HLA antigens comprise one or more antigens chosen from BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof.

[0081] 8. The method of any one of clauses 4-7, wherein the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens.

[0082] 9. The method of any one of clauses 1 -8, wherein the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0083] 10. The method of any one of clauses 1-9, wherein all antigens of the HLA antigen array are present on a single substrate.

[0084] 11 . The method of any one of clauses 1 -9, wherein the HLA antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

[0085] 12. The method of clause 11 , wherein the solid supports are beads.

[0086] 13. The method of any one of clauses 1-12, wherein the urine sample was obtained from a kidney transplant recipient.

[0087] 14. The method of clause 13, further comprising monitoring the kidney transplant based on the assessing.

[0088] 15. The method of clause 14, wherein monitoring the kidney transplant comprises monitoring for rejection of the kidney transplant.

[0089] 16. The method of clause 15, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant.

[0090] 17. The method of clause 16, wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

[0091] 18. The method of clause 16 or 17, wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (IVIG) , rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

[0092] 19. The method of any one of clauses 16-18, wherein treating the kidney transplant recipient comprises splenectomy. 20. The method of clause 15, wherein the monitoring indicates the absence of rejection of the kidney transplant, and wherein the method further comprises modifying a treatment of the kidney transplant recipient based on the absence of rejection of the kidney transplant.

[0093] 21 . The method of clause 20, wherein the modifying comprises ceasing or reducing administration of an immunosuppressive agent to the kidney transplant recipient.

[0094] 22. The method of clause 20 or 21 , wherein the modifying comprises ceasing or reducing administration of one or more of I VIG, rituximab, and / or bortezomib to the kidney transplant recipient.

[0095] 23. The method of clause 13, wherein the HLA antigen array comprises one or more of the non-HLA antigens of clause 5, clause 6, or both, and wherein the method further comprises monitoring the kidney transplant for graft injury.

[0096] 24. The method of clause 23, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

[0097] 25. A method of assessing a urine sample for antibodies, the method comprising: contacting an antigen array with a urine sample, wherein the antigen array comprises one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof; and assessing the urine sample for the antibodies by assessing for binding of antibodies to the one or more antigens.

[0098] 26. The method of clause 25, wherein the antigen array comprises 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more of the antigens.

[0099] 27. The method of clause 25 or 26, wherein the antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0100] 28. The method of any one of clauses 25-27, wherein the urine sample was obtained from a kidney transplant recipient.

[0101] 29. The method of clause 28, further comprising monitoring the kidney transplant based on the assessing.

[0102] 30. The method of clause 29, wherein monitoring the kidney transplant comprises monitoring the kidney transplant for graft injury. 31 . The method of clause 30, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

[0103] 32. The method of any one of clauses 25-31 , wherein the antigen array further comprises one or more HLA antigens, and wherein the method further comprises assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more HLA antigens.

[0104] 33. The method of clause 32, wherein the antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof.

[0105] 34. The method of clause 33, wherein the antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0106] 35. The method of any one of clauses 32-34, wherein the urine sample was obtained from a kidney transplant recipient.

[0107] 36. The method of clause 35, wherein the method further comprises monitoring for rejection of the kidney transplant based on the assessing for binding of antibodies to the one or more HLA antigens.

[0108] 37. The method of clause 36, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant.

[0109] 38. The method of clause 37, wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

[0110] 39. The method of clause 37 or 38, wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (I VIG) , rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

[0111] 40. A method of monitoring a kidney transplant recipient for rejection of the kidney graft and / or injury to the kidney graft, the method comprising performing the method of any one of clauses 13-24 or 28-39.

[0112] 41 . The method of clause 40, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant. 42. The method of clause 41 , wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

[0113] 43. The method of clause 41 or 42, wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (IVIG) , rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

[0114] 44. The method of any one of clauses 40-43, wherein monitoring the kidney transplant comprises monitoring the kidney transplant for graft injury.

[0115] 45. The method of clause 44, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

[0116] 46. An HLA antigen array comprising two or more types of HLA antigens chosen from HLA- B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA- DRB3, and any combination thereof, wherein the HLA antigen array comprises 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer types of HLA antigens.

[0117] 47. The HLA antigen array of clause 46, wherein the HLA antigen array comprises two or three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0118] 48. The HLA antigen array of clause 46 or 47, wherein the HLA antigen array further comprises one or more non-HLA antigens.

[0119] 49. The HLA antigen array of clause 48, wherein the non-HLA antigens comprise one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof.

[0120] 50. The HLA antigen array of clause 48 or 49, wherein the non-HLA antigens comprise one or more antigens chosen from CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof.

[0121] 51 . The HLA antigen array of any one of clauses 48-50, wherein the non-HLA antigens comprise one or more antigens chosen from BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof.

[0122] 52. The HLA antigen array of any one of clauses 48-51 , wherein the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens. 53. The HLA antigen array of any one of clauses 46-52, wherein the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0123] 54. The HLA antigen array of any one of clauses 46-53, wherein all antigens of the HLA antigen array are present on a single substrate.

[0124] 55. The HLA antigen array of any one of clauses 46-53, wherein the HLA antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

[0125] 56. The HLA antigen array of clause 55, wherein the solid supports are beads.

[0126] 57. An antigen array comprising two or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof, wherein the array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

[0127] 58. The antigen array of clause 57, wherein the antigen array comprises 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more of the antigens.

[0128] 59. The antigen array of clause 57 or 58, wherein the antigen array further comprises one or more HLA antigens.

[0129] 60. The antigen array of clause 59, wherein the antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA- DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof.

[0130] 61 . The antigen array of clause 60, wherein the antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

[0131] 62. The antigen array of any one of clauses 57-61 , wherein all antigens of the antigen array are present on a single substrate.

[0132] 63. The antigen array of any one of clauses 57-61 , wherein the antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

[0133] 64. The antigen array of clause 63, wherein the solid supports are beads.

[0134] The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL

[0135] Example 1 - A large number of HLA and nHLA Abs are detected in transplant urine.

[0136] Urine antibodies were identified using a microarray-based platform printed with 9,480 human antigens targeting HLA and nHLA antigens. All values in the dataset were Iog2 transformed. Antigens with values lower than Iog2(500) [-8.97] were dropped from the dataset in injury cases and preserved in stable graft and healthy controls. After data cleaning measures were applied, 7,728 antigens were removed, and 1 ,752 values persisted.

[0137] The repertoire of antibodies in the urine is highly representative of kidney antigens. Datasets from previously published reports were used to map antigens represented by the urine Abs and antigens were compared against reactive antigens of kidney vs. urine proteins identified using a high throughput mass spectrometry-based proteomics. Of the 1752 reactive antigens with IgG Abs in the urine, 28 glomerulus-specific antigens, 32 inner cortex-specific antigens, 65 outer cortex-specific antigens, seven medulla-specific antigens, 43 papillary tips antigens, and 114 renal pelvis-specific antigens were mapped for their antigenicity with kidney compartments reported previously

[0015] (Fig. 1 C).

[0138] Example 2 - Antibodies against HLA epitopes could be identified in urine

[0139] Antibodies were quantified against HLA antigens that included major histocompatibility complex, class I, B (HLA-B), HLA class I histocompatibility antigen, Cw-7 alpha chain, major histocompatibility complex, class II, DM alpha (HLA-DMA), major histocompatibility complex, class II, DO alpha (HLA-DOA), major histocompatibility complex, class II, DO beta (HLA-DOB), major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1 ), HLA class II histocompatibility antigen, DQ(5) alpha chain, HLA class II histocompatibility antigen, DQ(3) alpha chain, major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1), major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1 ), HLA class II histocompatibility antigen, DR alpha chain, major histocompatibility complex, class II, DR alpha (HLA-DRA), major histocompatibility complex, class II, DR beta 1 (HLA-DRB1 ), major histocompatibility complex, class II, DR beta 1 (HLA-DRB1 ), major histocompatibility complex, class II, DR beta 1 (HLA- DRB1 ), major histocompatibility complex, class II, DR beta 3 (HLA-DRB3). A complete list of HLA and nHLA antigens profiled is available in Li et al. (2009) PNAS 106(1 1 ):4148-4153, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0140] Example 3 - Antibodies specific to kidney transplantation without a specific injury were identified IgG antibodies were identified in circulation that are specific to kidney transplant recipients without any graft injury (AR, BKVN, or CAN). An unsupervised clustering differentiated the majority (4 / 5) of healthy controls from transplant patients (Fig. 2A). The antibodies that contributed to this separation were against the human antigens AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM1 12A, F0XP4, GADD45G, G0RASP1 , HCLS1 , IVD. The antibodies were also against unnamed proteins resulting from C14orf104, and C3orf54. These proteins were enriched in the activation of caspases through apoptosis (FDR= 1 ,45e-05). As shown in the volcano plot, the most significant antigens against which the antibodies were increased included SDCCAG3, GADD45G, CCDC69, DDI1 , and OTUB2 (Fig. 2B). These proteins are enriched in p53 signaling pathway (FDR=0.0021 ).

[0141] Example 4 - Antibodies specific to kidney transplantation with injury were identified as compared to healthy control and transplant recipients without injury

[0142] Data analysis was performed to list the most significant antibodies increased in transplant injury, including AR, CAN, and BKV, and these antibodies were compared against antibodies identified in healthy control and transplant recipients with stable grafts (STA) who have good functioning kidneys with no injury. Compared to healthy controls, a number of antibodies against human antigens were identified, including MAPK7, RAB3D, ZCCH8, PKNOX2, and RUNX1 (Fig. 3A). The same transplant injury cases with AR, CAN, and BKV, when compared against stable grafts (STA), resulted in a different set of antibodies increased that included the antibodies against CARD9, TERF1 , SYK, CLIP4, and FILIP1 (Fig. 3B).

[0143] Example 5 - Antibodies specific to kidney transplantation with acute rejection were identified compared to healthy control and transplant recipients without injury

[0144] The antibody data was analyzed to identify the most significantly increased antibodies in the cases of AR, CAN, and BKV infection. Antibodies against RAB3D, SAPS2, DOH , TLE4, RUNX1 T1 , ZCCHC8, RUNX1 T1 were significantly increased in AR compared to the healthy control (Fig. 4A). CARD9 alone significantly increased, as specific to AR compared to STA (Fig. 4B).

[0145] Example 6 - Antibodies specific to kidney transplantation with BK virus nephropathy were identified compared to healthy control and transplant recipients without injury

[0146] The antibody data was analyzed to identify the most significantly increased antibodies in the case of BKVN. Antibodies against SAPS2, LARP2, PKNOX2, BTN3A3, SNURF, CDKN1 B were among the top elevated Abs in the BKVN urine compared to the urine from the healthy control (Fig. 5A). FCGR3A, RPL36, and FKG44 were the top 3 Abs increased in BKVN urine compared to STA urine. (Fig. 5B).

[0147] Example 7 - Antibodies in the urine map to urine proteins associated with kidney injury

[0148] Leveraging available published data on kidney transplant injury enriched protein database

[0026] , urine Abs were mapped to see if any urine Abs were mapping with kidney transplant enriched urine proteins. Mapping resulted in an overlap of 16 proteins- SERPINF1 , PPIA, LMNA, MYL6, PKM2, IGHM, FCGR3A, CD300LG, TGFBR2, MDH1 , HSP90B1 , CHGB, CADM1 , CHMP2B, R0R1 , and CTTN. Of these 16 proteins, TGFBR2 and FCGR3A are associated with transforming growth factor complex and are known to be involved in tissue remodeling when kidney transplants are damaged

[0027] ,

[0149] Materials and Methods

[0150] Patient and study samples. Urine samples were collected from 45 kidney transplant (KT) patients from Lucile Packard Children’s Hospital, Stanford University, following IRB- approved protocols. Urine from 5 age-matching healthy individuals were also included in the study. Each urine sample from the transplant cohort was matched with a biopsy collected at the time of urine collection. Second morning void mid-stream urine samples were collected and centrifuged for 20 min at 2000 x g at room temperature. All the urine samples used in this study were collected prior to biopsy was done. The supernatant was adjusted to a pH of 7.0. The samples were frozen until time for use.

[0151] Among KT samples, the sample distribution was as follows: acute rejection (AR; n=16), chronic allograft nephropathy (CAN; n=10), BKV nephropathy (BKV; n=8) and normal graft function without significant pathology (STA; n=11 ) (Fig. 1 B). All the study samples were collected from pediatric and young adult recipients transplanted between 2000 and 201 1 at Lucile Packard Children’s Hospital of Stanford University. The ethics committee of Stanford University Medical School and UCSF Medical Center approved the study. All patients / guardians provided informed consent to participate in the research, in full adherence to the Declaration of Helsinki. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism. A summary of demographic information of kidney transplant patients and a summary of study samples and analysis scheme is provided in Table 1 .

[0152] All 45 kidney biopsies were blindly analyzed by a Stanford University pathologist and were graded by the Banff classification [18-20] for acute rejection. Intragraft C4d stains were performed [21 , 22] to assess for acute humoral rejection (AHR) [23, 24], “Graft injury” in this study was defined as a greater than 20% increase in serum creatinine from its previous steady-state baseline value and an associated biopsy that was pathological. Acute rejection (AR) was defined at minimum, as per Banff Schema, a tubulitis score >1 accompanied with an interstitial inflammation score >1 . Chronic allograft nephropathy (CAN) was defined at minimum as a tubular atrophy score >1 accompanied by an interstitial fibrosis score >1 . BKV nephropathy (BKVN) was defined as positivity of polyomavirus PCR in peripheral blood, together with a positive SV40 stain in the concomitant renal allograft biopsy. Normal (STA) allografts were defined by an absence of significant injury pathology as defined by Banff schema.

[0153] Data generation. ProtoArray® Human Protein Microarray v4.0 and v4.1 (Invitrogen, Carlsbad, CA) was used for this study. The slides contained a total of approximately 8200 antigens printed on each slide. The complete list of antigens used to measure Ab levels is provided in Li et al. (2009) PNAS 106(1 1 ):4148-4153, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The proteins included in the arrays belong to different cellular location and with a wide variety of functions. For the detection of immune response, the manufacturer’s protocol was used with slight changes

[0001] . Briefly, the microarray slides were blocked with 5.0 mL Blocking Buffer (100 mM Sod. Phosphate pH 7.4, 200 mM NaCI, 0.08% Triton X-100, 25% Glycerol, 20 mM Reduced Glutathione, 1.0 mM DTT, 1% Hammarstein Grade Caesin) with gentle agitation in 4-well trays for 1 hr at 4 °C. After the blocking step, the Blocking Buffer was removed by aspiration and a 5.0 mL diluted serum (1 :150) in PBST Buffer (1 X PBS, 1% Hammerstein Grade casein, 0.1% Tween 20) was added onto the plate to incubate for 90 min with gentle agitation at 4 °C. In the next step, the serum sample was removed and the plates were washed 5 times with 5.0 mL fresh PBST Buffer with 5 minute incubations per wash with gentle agitation. A 5.0 mL portion of secondary antibody (anti-human antibody-Alexa Fluor® 647 conjugate) diluted in PBST Buffer was added and the plates were incubated for 90 minutes with gentle agitation at 4 °C. The secondary antibody solution was removed by aspiration. The plates were then washed for 5 times with 5.0 mL fresh PBST Buffer, 5 minute incubations per wash with gentle agitation. At the end, the slides were dried by centrifugation and scanned using Axon GenePix 4000B Scanner (Molecular Devices. Sunnyvale, CA). The data was acquired by using microarray analysis software GenePix pro 6.0 (Molecular devices, Sunnyvale, CA). The slides were scanned at 635nm with a PMT gain of 600, a laser power of 100% and a focus point of 0 pm. The “.gal” files were obtained from a Protoarray central portal on the Invitrogen website (www.invitrogen.com / ProtoArray) by submitting the barcode of each protein microarray.

[0154] Data analysis. Signal intensities per spot for each individual array were obtained using GenePx pro 6.0. Prospector Analyzer® 5.2(lnvitrogen Inc) was used to subtract the background, to normalize and to analyze GenePix Results (GPR) files for each array. A standard cut-off Z- Score of 3.0 was used. Individual antigen reactivity was ranked based on Z-score above or below the mean signal for each array. Arrays from patients of a distinct clinical phenotype were analyzed as a group. Group analyses (> 5 per group as suggested by the manufacturer) were made by comparing 2 sets of individual antibody level for every antigen present on the array using M- statistics of the Prospector Analyzer® with Robust-Linear-Normalization (RLM) normalization method

[0025] . The initial dataset has 9480 unique readouts. All values in the dataset were Iog2 transformed. Readouts with values lower than Iog2(500) [-8.97] were dropped from the dataset in injury cases and preserved in stable graft and healthy controls. After data cleaning measures were applied 7728 readouts were removed and 1752 values persisted. The cleaned dataset was then Min-Max Scaled between a range of zero to one.

[0155] Two samples that clustered beyond their category or predominated absolute expression for readouts were dropped from analyses. The ANOVA p-values were corrected for multiple hypotheses using Benjamini-Hochberg correction. Hypergeometric enrichment, univariate and multivariate analysis, and logistic regression modeling were performed using customized algorithms. Ingenuity® software version 7.5 (Ingenuity Systems, Inc. Redwood City, CA) was used to map target antigens to canonical pathways within the Ingenuity Knowledge base.

[0156] Mapping of urine Abs representing kidney compartment-specific antigens and kidney allograft-specific urine proteins. Publicly available data on the reactivity of kidneyspecific antigens was used to map urine Abs identified in the study described herein. Previously reported 3835 kidney antigens

[0015] were mapped with 1752 urine Abs reported in this report. To assess the representation of urine Abs from urine proteins enriched in kidney transplant injury urine proteomics, the previously published proteomics dataset

[0026] was used.

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Claims

WHAT IS CLAIMED IS:1 . A method of assessing a urine sample for anti-human leukocyte antigen (anti-HLA) antibodies, the method comprising: contacting an HLA antigen array with a urine sample, wherein the HLA antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA- DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof; and assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more types of HLA antigens.

2. The method of claim 1 , wherein the HLA antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

3. The method of claim 1 , wherein the HLA antigen array comprises 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer types of HLA antigens.

4. The method of claim 1 , wherein the HLA antigen array further comprises one or more non-HLA antigens, and wherein the assessing comprises assessing the urine sample for antibodies specific for the one or more non-HLA antigens by assessing for binding of antibodies to the one or more non-HLA antigens.

5. The method of claim 4, wherein the non-HLA antigens comprise one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof.

6. The method of claim 4, wherein the non-HLA antigens comprise one or more antigens chosen from CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof.

7. The method of claim 4, wherein the non-HLA antigens comprise one or more antigens chosen from BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof.

8. The method of claim 4, wherein the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens.

9. The method of claim 1 , wherein the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

10. The method of claim 1 , wherein all antigens of the HLA antigen array are present on a single substrate.11 . The method of claim 1 , wherein the HLA antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

12. The method of claim 11 , wherein the solid supports are beads.

13. The method of claim 1 , wherein the urine sample was obtained from a kidney transplant recipient.

14. The method of claim 13, further comprising monitoring the kidney transplant based on the assessing.

15. The method of claim 14, wherein monitoring the kidney transplant comprises monitoring for rejection of the kidney transplant.

16. The method of claim 15, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant.

17. The method of claim 16, wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

18. The method of claim 16, wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (IVIG), rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

19. The method of claim 16, wherein treating the kidney transplant recipient comprises splenectomy.

20. The method of claim 15, wherein the monitoring indicates the absence of rejection of the kidney transplant, and wherein the method further comprises modifying a treatment of the kidney transplant recipient based on the absence of rejection of the kidney transplant.21 . The method of claim 20, wherein the modifying comprises ceasing or reducing administration of an immunosuppressive agent to the kidney transplant recipient.

22. The method of claim 20, wherein the modifying comprises ceasing or reducing administration of one or more of IVIG, rituximab, and / or bortezomib to the kidney transplant recipient.

23. The method of claim 13, wherein the HLA antigen array comprises one or more of the non-HLA antigens of claim 5, claim 6, or both, and wherein the method further comprises monitoring the kidney transplant for graft injury.

24. The method of claim 23, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

25. A method of assessing a urine sample for antibodies, the method comprising: contacting an antigen array with a urine sample, wherein the antigen array comprises one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof; and assessing the urine sample for the antibodies by assessing for binding of antibodies to the one or more antigens.

26. The method of claim 25, wherein the antigen array comprises 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more of the antigens.

27. The method of claim 25, wherein the antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

28. The method of claim 25, wherein the urine sample was obtained from a kidney transplant recipient.

29. The method of claim 28, further comprising monitoring the kidney transplant based on the assessing.

30. The method of claim 29, wherein monitoring the kidney transplant comprises monitoring the kidney transplant for graft injury.31 . The method of claim 30, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

32. The method of claim 25, wherein the antigen array further comprises one or more HLA antigens, and wherein the method further comprises assessing the urine sample for anti-HLA antibodies by assessing for binding of antibodies to the one or more HLA antigens.

33. The method of claim 32, wherein the antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA- DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof.

34. The method of claim 33, wherein the antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

35. The method of claim 32, wherein the urine sample was obtained from a kidney transplant recipient.

36. The method of claim 35, wherein the method further comprises monitoring for rejection of the kidney transplant based on the assessing for binding of antibodies to the one or more HLA antigens.

37. The method of claim 36, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant.

38. The method of claim 37, wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

39. The method of claim 37, wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (IVIG), rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

40. A method of monitoring a kidney transplant recipient for rejection of the kidney graft and / or injury to the kidney graft, the method comprising performing the method of any one of claims 13-24 or 28-39.41 . The method of claim 40, wherein the monitoring indicates rejection of the kidney transplant, and wherein the method further comprises treating the kidney transplant recipient for rejection of the kidney transplant.

42. The method of claim 41 , wherein treating the kidney transplant recipient comprises administering an effective amount of an immunosuppressive agent or an increased dosage thereof to the kidney transplant recipient.

43. The method of claim 41 , wherein treating the kidney transplant recipient comprises administering an effective amount of IV immunoglobulin (IVIG), rituximab, bortezomib, or any combination thereof to the kidney transplant recipient.

44. The method of claim 40, wherein monitoring the kidney transplant comprises monitoring the kidney transplant for graft injury.

45. The method of claim 44, wherein the monitoring indicates the presence of acute rejection (AR), chronic allograft nephropathy (CAN), and / or BK virus associated nephropathy (BKVN), and wherein the method further comprises treating the kidney transplant recipient for the AR, CAN, and / or BKVN.

46. An HLA antigen array comprising two or more types of HLA antigens chosen from HLA- B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof, wherein the HLA antigen array comprises 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer types of HLA antigens.

47. The HLA antigen array of claim 46, wherein the HLA antigen array comprises two or three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

48. The HLA antigen array of claim 46, wherein the HLA antigen array further comprises one or more non-HLA antigens.

49. The HLA antigen array of claim 48, wherein the non-HLA antigens comprise one or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, and any combination thereof.

50. The HLA antigen array of claim 48, wherein the non-HLA antigens comprise one or more antigens chosen from CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof.51 . The HLA antigen array of claim 48, wherein the non-HLA antigens comprise one or more antigens chosen from BTN3A3, CDKN1 B, LARP2, PKNOX2, SAPS2, SNURF, and any combination thereof.

52. The HLA antigen array of claim 48, wherein the HLA antigen array comprises 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, or 40 or fewer non-HLA antigens.

53. The HLA antigen array of any one of claims 46-52, wherein the HLA antigen array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

54. The HLA antigen array of any one of claims 46-52, wherein all antigens of the HLA antigen array are present on a single substrate.

55. The HLA antigen array of any one of claims 46-52, wherein the HLA antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

56. The HLA antigen array of claim 55, wherein the solid supports are beads.

57. An antigen array comprising two or more antigens chosen from AKTIP, ASPA, BIRC4, CASC4, CCDC69, CDK10, CHCHD2, CHKA, CUTED1 , CRYBA4, DDI1 , EPM2AIP1 , FAM112A, FOXP4, GADD45G, GORASP1 , HCLS1 , IVD, OTUB2, SDCCAG3, CADM1 , CARD9, CD300LG, CHGB, CHMP2B, CLIP4, CTTN, FCGR3A, FILIP1 , HSP90B1 , IGHM, LMNA, MAPK7, MDH1 , MYL6, PKM2, PKNOX2, PPIA, RAB3D, ROR1 , RUNX1 , SERPINF1 , SYK, TERF1 , TGFBR2, ZCCH8, and any combination thereof, wherein the array comprises 75 or fewer, 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer antigens.

58. The antigen array of claim 57, wherein the antigen array comprises 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more of the antigens.

59. The antigen array of claim 57, wherein the antigen array further comprises one or more HLA antigens.

60. The antigen array of claim 59, wherein the antigen array comprises one or more types of HLA antigens chosen from HLA-B, HLA-DOA, HLA-DOB, HLA-DPA1 , HLA-DQA1 , HLA- DQB1 , HLA-DRA, HLA-DRB1 , HLA-DRB3, and any combination thereof.61 . The antigen array of claim 60, wherein the antigen array comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight, or each of the types of HLA antigens.

62. The antigen array of any one of claims 57-61 , wherein all antigens of the antigen array are present on a single substrate.

63. The antigen array of any one of claims 57-61 , wherein the antigen array comprises a plurality of solid supports, wherein each solid support comprises a single type of antigen.

64. The antigen array of claim 63, wherein the solid supports are beads.

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