Compositions and methods for donor-specific antibody screening and tissue typing

Abstract

The invention features compositions and methods for donor-specific antibody screening and tissue typing.

Description

[0001] COMPOSITIONS AND METHODS FOR DONOR-SPECIFIC ANTIBODY SCREENING AND TISSUE TYPING

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 30, 2025, is named “51820-002W02_Sequence_Listing_4_30_25” and is 17,310 bytes in size.

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims the benefit of U.S. Patent Application Serial No. 63 / 649,758, filed on May 20, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0006] BACKGROUND OF THE INVENTION

[0007] Each mammalian species has a single chromosomal region that encodes the highly polymorphic major histocompatibility antigens, and this region on the human chromosome 6 codes the HLA genes. Similarly, in mice, the murine leukocytic antigen is found on chromosome 17 and expressed on the surface of murine cells as histocompatibility 2 (H2). HLA is highly polymorphic; therefore, it is a major immunologic target of organ rejection when mismatched between the donor and the recipient. The number of HLA antigen mismatches in A, B, and DR loci correlates with allograft survival; the higher the number of mismatches, the greater the risk of allograft rejection.

[0008] The MHC locus encodes a highly polymorphic complex protein that is divided mainly into MHC classes I and II. Functional MHC Class I glycoproteins are composed of a heterodimer between a 45 kDa polymorphic a chain and a 12 kDa constant p2-microglobulin.

[0009] Both T cell-mediated and antibody-mediated effector mechanisms have an essential role in kidney transplant rejection. T cell-mediated rejection is caused by recipient T lymphocytes that respond to donor HLA antigens expressed in the transplanted organ.

[0010] After transplantation, donor-derived antigens are delivered to the recipient’s draining lymph nodes and activate an alloimmune response. A subset of CD4+ T cells called follicular helper T cells (Tfh) are activated and promote differentiation of B cells into antibody-secreting plasma cells. Plasma cells produce donor-targeting antibodies against HLA and non-HLA antigens, which can deposit in allograft kidney and cause injury via complement-dependent and independent mechanisms. Currently, the major obstacle to achieving successful allograft outcomes is the occurrence of antibody-mediated rejection.

[0011] The presence of preformed antibodies against donor antigens, either HLA or non-HLA, can be a potential cause of allograft injury after transplantation, and hence, it is important to perform crossmatching prior to transplantation. These antibodies can develop as a result of exposure to human antigens through activities like blood transfusion, pregnancy, or a previous transplant. For the purposes of crossmatching, donor T lymphocytes, which express class I but not class II HLA, are used as a surrogate target for detection of circulating anti-class I (HLA-A and -B) antibodies in the recipient. A positive cytotoxic crossmatch of recipient serum with donor T lymphocytes indicates the presence of preformed donor- specific anti-HLA class I antibodies and is usually predictive of an acute vasculitic event termed hyperacute rejection. This finding represents the only widely accepted absolute immunologic contraindication for kidney transplantation.

[0012] More recently, solid-phase assays have emerged as alternatives, utilizing purified HLA molecules in place of donor HLA-presenting cells. For instance, Luminex-based cross-matching (LUMXM) has been extensively employed over the last few decades. Advancement in HLA typing, and rapid antibody characterization have expedited the adoption of virtual crossmatching (VXM) in most US centers. In this technology, immunologic compatibility is assessed by comparing the patient's alloantibody profile with the donor's histocompatibility antigens represented on HLA-coated beads. VXM potentially reduces the ischemia time of transplanted organs and, theoretically, can lessen the costs and complications of organ transplantation.

[0013] However, the use of HLA beads presents a significant challenge due to the possibility of HLA denaturation during the purification steps or as a result of chemical cross-linking or adsorption of HLA to the surface of the bead. Furthermore, usually truncated, refolded HLA, with missing transmembrane and cytoplasmic domains, is utilized in the manufacturing of HLA beads, potentially leading to the exposure of otherwise inaccessible cryptic epitopes. The process of immobilization of truncated HLA molecules on beads can alter surface epitopes that are typically accessible to antibodies in the full-length native HLA structure, thus resulting in misleading false-positive and false-negative reactivity. A false positive result may also lead to a serious pitfall, as patients on the waiting list may get declined for potentially compatible organ offers. Additionally, the solid phase single antigen bead assay results utilizing LUMINEX®, are reported as a numerical number called mean fluorescence intensity (MFI).

[0014] Hence, there exists a distinct and unmet need to develop screening tools with considerable sensitivity that can provide more specific identification of anti-HLA antibodies.

[0015] SUMMARY OF THE INVENTION

[0016] In one aspect, the invention features a human leukocyte antigen-nanodisc (HLA-ND) complex including: (a) a human leukocyte antigen (HLA); (b) a lipid composition; and (c) a membrane scaffold protein (MSP).

[0017] In some embodiments, the HLA is a class I HLA. In some embodiments, the HLA class I is HLA- A, HLA-B, or HLA-C.

[0018] In some embodiments, the HLA is a class II HLA. In some embodiments, the class II HLA is HLA- DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO.

[0019] In some embodiments, the lipid composition forms a bilayer.

[0020] In some embodiments, the lipid composition includes a detectable label. In some embodiments, the detectable label is rhodamine, fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), 4',6-diamidino-2-phenylindole (DAPI), coumarin, cyanine, xanthene, naphthalene, oxadiazole, anthracene, pyrene, oxazine, acridine, arylmethine, tetrapyrroles, Alexa Fluor compounds, or boron-dipyrromethene (BODIPY), or derivatives thereof.

[0021] In some embodiments, the MSP includes the amino acid sequence of NW9 (SEQ ID NO: 1 ) or NW6 (SEQ ID NO: 3). In some embodiments, the HLA-ND is between about 5 nm and about 10 nm. In some embodiments, the HLA-ND is about 8 nm.

[0022] In some embodiments, there is at least one HLA per nanodisc. In some embodiments, the complex consists of an HLA selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA- DQ, HLA-DR, HLA-DM, and HLA-DO.

[0023] In some embodiments, the HLA-ND complex of further includes a bead (for example, a microsphere bead).

[0024] In some embodiments, the HLA-ND complex further includes (i) a first reporter fragment linked to an anti-HLA antibody capture agent; and (ii) a second reporter fragment linked to: (a) an HLA capture agent; (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

[0025] In some embodiments, the anti-HLA antibody capture agent includes an anti-IgG antibody or antigen-binding fragment thereof.

[0026] In some embodiments, the anti-IgG binding fragment thereof is a Fab, scFv, nanobody, or mini protein.

[0027] In some embodiments, the anti-HLA antibody capture agent includes the amino acid sequence of PLG binder (SEQ ID NO: 5), S-PG binder (SEQ ID NO: 7), or PG-S binder (SEQ ID NO: 9).

[0028] In some embodiments, the HLA capture agent is an anti-HLA antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the HLA capture agent includes the amino acid sequence of 128-1 binder (SEQ ID NO: 11 ) or 128-2 binder (SEQ ID NO: 13).

[0030] In some embodiments, the reporter is a luminescence-based reporter, a fluorescence-based reporter, or an absorbance-based reporter.

[0031] In some embodiments, the luminescence-based reporter is luciferase.

[0032] In some embodiments, the HLA-ND complex further includes an anti-HLA antibody.

[0033] In another aspect, the invention features a HLA-ND complex library, including one or more of the HLA-ND complexes disclosed herein.

[0034] In some embodiments, the HLA-ND complex library includes between about 106-1012HLA-ND complexes.

[0035] In another aspect, the invention features a method of producing an HLA-ND complex, including: (i) providing an HLA; and (ii) incubating the HLA with an MSP and a lipid composition.

[0036] In some embodiments, the MSP includes the amino acid sequence of NW9 or NW6.

[0037] In some embodiments, the HLA-ND is between about 5 nm and about 10 nm. In some embodiments, the HLA-ND is about 8 nm.

[0038] In some embodiments, the method includes isolating the HLA from the plasma membrane fraction of a cell including the HLA. In some embodiments, the cell is a monoallelic B cell or a native cell. In some embodiments, the method includes the steps of: (i) incubating a mixture including the HLA, MSP, lipid composition, and detergent for between about 30 minutes to about 120 minutes; and (ii) removing the detergent. In another aspect, the invention features a method of detecting an anti-HLA antibody, including: (a) providing a sample from a subject; (b) contacting the sample with an HLA-ND complex disclosed herein; and (c) determining the presence of a signal from the reporter, thereby detecting the anti-HLA antibody.

[0039] In another aspect, the invention features a method of predicting whether an organ transplant recipient is at risk of hyperacute rejection, the method including: (a) providing a sample from the organ transplant recipient; (b) contacting the sample with an HLA-ND complex including an HLA that corresponds to an HLA of the donor of the transplanted organ; and (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient is at risk of hyperacute rejection.

[0040] In another aspect, the invention features a method of monitoring the development of donorspecific antibodies (DSAs) in a subject who received an organ transplant, the method including: (a) providing a sample from the organ transplant recipient; (b) contacting the sample with an HLA-ND complex including an HLA that corresponds to an HLA of the donor of the transplanted organ; and (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient has developed DSAs.

[0041] In some embodiments, the sample is a serum sample or a blood sample.

[0042] In another aspect, the invention features a kit including: (i) an HLA-ND complex disclosed herein; (ii) a first reporter fragment linked to an anti-HLA antibody capture agent; and (iii) a second reporter fragment linked to: (a) an HLA capture agent; (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

[0043] Advantageously, the compositions and methods described herein allow for the incorporation of human leukocyte antigen (HLA) (e.g., a full-length mature HLA or a fragment thereof) into nanodiscs to enhance stability and preserve its structure and function. When HLA is inserted into a nanodisc, it will be surrounded by a lipid bilayer, providing an environment that approximates its native state. Noticeably, the incorporation of HLA into nanodisc (HLA-NDs) prevents the oligomerization or aggregation of HLA, with a membrane scaffold protein (MSP) belt acting as a bumper case to prevent aggregation. Moreover, HLA is extracted from cell membranes, circumventing any structural alteration associated with conventional purification and refolding from bacterial insoluble inclusion bodies. Unlike refolded HLA, membranal HLA adopts a native, peptide bound conformation. When a peptide dissociates from the HLA molecule on the surface of a living cell, the molecule undergoes a conformational change, followed by rapid internalization. Consequently, empty HLA molecules that lack a peptide are swiftly removed from the cell surface. This mechanism serves to prevent the acquisition of peptides from the surrounding extracellular fluid.

[0044] Additionally, the MSP can be utilized for HLA-ND immobilization over beads and integration with single antigen-bead (SAB) technologies. The nanodisc-containing membrane proteins may be immobilized over solid beads. The immobilization is mediated through tagged MSP rather than the enclosed membrane protein. Advantageously, membrane proteins are more active and stable in a lipid bilayer than in detergents. A further advantage provided herein is a novel assay to monitor potential development of donorspecific antibodies (DSA) that is simple, rapid, and cost-effective. Being a homogenous assay, accurate read-outs are yielded without the need to process the sample. There is no need for multiple steps of incubation and wash cycles, and the results interpretation is simple and straightforward. These features make the assay suitable and convenient for point-of-care detection. There is no need for sophisticated equipment to measure the read-outs. The generated signal can be measured by conventional platereaders or even standard smart phone cameras, opening opportunities for self-testing at home.

[0045] The compositions and methods provided herein overcome the disadvantages of conventional bead-based assays. For example, the compositions and methods provided herein no longer requires surface immobilization of HLA over beads (as in LUMINEX® assays), washing steps or processing patients’ samples. This is useful as immobilization of HLA over beads modifies its tertiary structure, revealing cryptic epitopes that are normally considered inaccessible to antibodies. This potentially results in aberrant false positive. This is critical as some patients are denied transplantation based on anti-HLA antibodies falsely identified by the SAB assay.

[0046] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0047] Definitions

[0048] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0049] The term “capture agent” as used herein refers to any molecule with the ability to bind to a target molecule (e.g., a target anti-HLA antibody or a target HLA). Suitable capture agents include, but are not limited to antibodies or antigen-binding fragments thereof (e.g., a single-chain fragment variable (scFv), Fab, Fab', F(ab')2, nanobody, or mini protein). Mini proteins are described in Watson et al., Nature 620, 1089-1100 (2023) and Cao et al, Nature 605, 551 -560 (2022).

[0050] As used herein, the term “contacting” refers to placement in direct physical association, including both in solid and liquid form.

[0051] As used herein, “detectable label” refers to one or more markers, signals, or moieties which are attached, incorporated or associated to a molecule which emit an optical signal that is readily detected by methods known in the art including fluorescence, luminescence, absorbance and the like. Detectable labels include fluorophores, radioisotopes, chromophores, enzymes, dyes, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like.

[0052] As used herein, the terms “detecting” and “detection” include qualitative or quantitative measurements or both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels.

[0053] As used herein, the term “fragment” in the context of a reporter refers to a portion of the linear sequence of the reporter. In some embodiments, the reporter fragment alone does not have functional reporter activity. In some embodiments, one reporter fragment in physical proximity of another reporter fragment together have functional reporter activity. As used herein, the term “HLA-ND complex” refers to a composition comprising (a) a human leukocyte antigen (HLA); (b) a lipid composition; and (c) a membrane scaffold protein (MSP).

[0054] As used herein, the term “incubation” refers to a method wherein a duration is provided for a reaction to happen. An incubation step may be included with a method described herein to provide for a duration wherein, e.g., an HLA, a MSP, and a lipid composition may associate with each other to form an HLA-ND.

[0055] As used herein, the term “lipid composition” refers to a composition including one or more lipids. The lipid composition may include a mixture of different unique lipids (e.g., one, two, three, or more unique lipids).

[0056] As used herein, the terms “membrane scaffold protein” (MSP) and “belt protein” are used interchangeably and refer to a protein that self-assembles with lipids into membrane bilayers. MSPs are amphipathic, with one part of its structure more or less hydrophilic and facing the aqueous solvent and the other part more or less hydrophobic and facing the center of the hydrophobic bilayer that is to be stabilized.

[0057] As used herein, the term “nanodisc” refers to a discoidal lipid bilayer which is “belted" or "ringed” by a membrane scaffold protein.

[0058] As used herein, the term “percent (%) identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0059] 100 x (fraction of A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0060] As used herein, the term “reporter” refers to a molecule that can generate a detectable signal. A detectable signal may be any optical signal that is readily detected by methods known in the art including luminescence, fluorescence, absorbance and the like. The reporter may be split into a first fragment and a second fragment, that, when in proximity, form a functional reporter. As used herein, the term “functional reporter” refers to a reporter that fulfills the normal function of a reference reporter (e.g., an intact reporter that is not split).

[0061] As used herein, the term “sample” means any biological or other fluids. An exemplary sample is a serum sample or a blood sample obtained from a subject (for example, a human patient).

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0065] Figure 1 shows a schematic representation of the general isolation, nanodisc assembly and purification of native MHC I, II and non-MHC nanodisc complexes from murine Balb / c mouse splenocytes.

[0066] Figure 2 shows a characterization of native MHC II nanodisc library isolated from Balb / c mouse splenocytes. (A) Size exclusion chromatography. (B) Western blot. (C) Silver-stain SDS-PAGE. (D) Negative-stain EM. (E) The list of mass spec- identified peptides and proteins. The results confirm the successful incorporation of MHC II into nanodisc. MHC (excised from gel) was digested into peptides using trypsin. The peptides are then separated by liquid chromatography which is coupled to the mass spectrometer. (F) Representative MS / MS spectra showing the quality of mass spectrometry data of 2 identified peptides. The b- and y- ions derived from the 2 peptides ion are listed in the right.

[0067] Figure 3 shows a production and binding characterization of human HLA-nanodiscs. (A) A schematic showing the production of human HLA-nanodisc complexes utilizing monoallelic cell lines. Monoallelic HLA A6801 and HLA A201 cells were utilized to produce HLA-I nanodiscs. (B) Flow cytometry analysis shows the binding between HLA A6801 nanodiscs, and FITC-labeled HLA-ABC IgG antibodies immobilized over beads. Empty nanodiscs (control) did not show any binding. (B-right) A histogram showing the binding between the beads decorated with FITC-labeled HLA-ABC antibodies and HLA-nanodisc made from A6801 cell line (Red). Empty nanodiscs (Blue) were used as a negative control.

[0068] (C) Flow cytometry analysis shows the binding between HLA A201 nanodiscs, and PerCP-e flour 710- labeled HLA-A201 IgG antibody immobilized over beads. HLA-A6801 nanodiscs and empty nanodiscs did not show significant binding to anti HLA-A201 antibodies, (c-right) A histogram showing the binding between the beads decorated with PerCP-e flour 710-labeled HLA-A201 antibodies and HLA-nanodiscs made from A201 cell line (Red). HLA-nanodiscs made from A6801 cell line (orange) and empty nanodiscs (Blue) were used as controls. Both empty nanodiscs and HLA-nanodiscs contain PE-Rhodamine lipids.

[0069] (D) Flow cytometry plots of the Rhodamine HLA-A201 nanodisc incubated with anti-HLA-A201 positive human serum, demonstrating selective binding of HLA-201 ND to the anti-HLA A201 antibody in the serum. Empty nanodisc incubated with anti-HLA-A201 positive serum demonstrated no binding, (d-right) A histogram showing the binding between the HLA-A201 nanodisc and anti-HLA antibody in the serum (red). Empty nanodiscs (green) were used as controls.

[0070] Figure 4 shows there were no detectable denatured HLA among the NDs library. (A) Binding analysis between native / denatured ND library (made from cells expressing HLA A201 ) with HC-10 antibodies by microscale thermophoresis. Upon denaturation, NDs bind to HC-10 antibody (Blue line). (B) flow cytometry analysis shows the binding of acid-denatured rhodamine labelled NDs library to HC-10 Ab decorated beads. For native NDs library, binding was comparable to the background signal associated with non-specific binding to the beads in absence of HC-10 antibodies. (C) Quantification of b, showing average % of rhodamine-positive beads ± SEM.

[0071] Figure 5 shows a luminescence biosensor for the detection of anti-HLA antibodies. (A) The principle of our detection technology. In the presence of anti-HLA antibodies, antibodies will bind to HLA embedded in nanodisc and form HLA-antibody-nanodisc complex. The complex will bring the two luciferase fragment proteins into proximity and assemble an active enzyme. The active enzyme will convert a substrate to light that can be detected using a plate reader or portable device. In the absence of anti-HLA antibodies, the HLA embedded in nanodisc will bind to only one split fragment, which is not enough to assemble an active enzyme, thus, no light signal will be detected. (B) Coomassie blue-stained SDS-PAGE showing the purity of s-PLG. Right: Protein structure of s-PLG as predicted by AlphaFold, c Coomassie blue-stained SDS-PAGE showing the purity of L-128. Right: L-128 structure as predicted by AlphaFold. (D-F) Binding analysis of antibody / HLA, antibody / s-PLG, and HLA / L-128 by microscale thermophoresis. (G) Emission spectra of the bright blue bioluminescence. (H) Bioluminescence noise evaluation of luciferase probes. (J) Evaluation of detection limit with varying concentrations of HLA antibody.

[0072] Figure 6 is a schematic showing a full-length HLA nanodisc bead-based assay. (A) HLA will be assembled into nanodisc upon isolation from cell membrane. HLA-nanodiscs will be immobilized on oligonucleotide labelled XMAP® beads. (B) Compatibility of HLA nanodiscs with current multiplexing measurements.

[0073] Figure 7 is a schematic showing different strategies for a homogenous, beads-free assay. (A) Luciferase complementation is mediated via two separate binders that binds HLA and anti-HLA antibodies. (B) Luciferase subunit is genetically fused into the MSP of the nanodisc. (C) The small subunit of luciferase will be utilized as a peptide tethered on the nanodisc. Palmitoylated luciferase subunit is used to present luciferase subunit for complementation.

[0074] Figure 8 is a schematic showing a complementation-independent Amplified Luminescent Proximity Homogenous Assay (ALPHA) based assay. An anti-HLA antibody binder is linked to donor moiety, with the ability to generate reactive singlet oxygens upon excitation. NW9 is linked to acceptor dye that emits intense light only in response to singlet oxygen produced by excited donor in proximity.

[0075] DETAILED DESCRIPTION OF THE INVENTION

[0076] The disclosure provides HLA-nanodisc complexes (HLA-ND), resulting from the incorporation of HLA (e.g., natural, full-length HLA) into lipid bilayer nanodiscs in a native-like membrane environment, effectively maintaining the structure and preventing denaturation. HLA-ND complexes may be immobilized on beads to perform donor-specific antibody (DSA) screening using serum obtained from transplantation recipients. Further provided herein are compositions and methods for a bead-free homogenous assay for the detection of anti-HLA antibodies using HLA-ND complexes.

[0077] I. Human leukocyte antigen-nanodisc complex

[0078] The disclosure provides human leukocyte antigen-nanodisc complexes (HLA-ND) including: (a) a human leukocyte antigen (HLA); (b) a lipid composition; and (c) a membrane scaffold protein (MSP). In some embodiments, the HLA-ND complex further includes: (i) a first reporter fragment linked to an anti- HLA antibody capture agent; and (ii) a second reporter fragment linked to (a) an HLA capture agent; (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) a lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter. In some embodiments, the HLA-ND complex further includes an anti-HLA antibody.

[0079] When HLA is inserted into a nanodisc, it will be surrounded by a lipid bilayer, providing an environment that approximates its native state. Therefore, nanodiscs preserve the structure and function of HLA. Noticeably, the incorporation of HLA into nanodiscs prevents the oligomerization or aggregation of HLA, with the MSP belt acting as a bumper case to prevent aggregation. Moreover, HLA may be extracted from cell membranes, circumventing any structural alteration associated with conventional purification and refolding from bacterial insoluble inclusion bodies. Unlike refolded HLA, membranal HLA adopts a native, peptide bound conformation. When a peptide dissociates from the HLA molecule on the surface of a living cell, the molecule undergoes a conformational change, followed by rapid internalization. Consequently, empty HLA molecules are swiftly removed from the cell surface. This mechanism serves to prevent the acquisition of peptides from the surrounding extracellular fluid.

[0080] In some embodiments, the HLA-ND complex is composed of a nanometer-sized phospholipid bilayer patch encircled by typically two copies of a MSP and typically one HLA.

[0081] The small size of the nanodisc encourages the incorporation of only a single copy of HLA per nanodisc; since the small area of the nanodisc is not enough to accommodate other membrane proteins alongside HLA in the same nanodisc (co-incorporation). In some embodiments, the nanodisc is between about 1 nm to about 50 nm (e.g., about 1 nm to about 5 nm, about 5 nm to about 10 nm, about 10 nm to about 15 nm, about 15 nm to about 20 nm, about 20 nm to about 25 nm, about 25 nm to about 30 nm, about 30 nm to about 35 nm, about 35 nm to about 40 nm, about 40 nm to about 45 nm, or about 45 nm to about 50 nm). In some embodiments, the nanodisc is between about 5 nm to about 10 nm. In some embodiments, the nanodisc is about 8 nm.

[0082] In some embodiments, provided herein is a HLA-ND complex library, comprising one or more of the HLA-ND complexes disclosed herein. In some embodiments, the HLA-ND complex library comprises between about 103-1015HLA-ND complexes. In some embodiments, the HLA-ND complex library comprises between about 106-1012HLA-ND complexes. In some embodiments, the HLA-ND complex library comprises about 109HLA-ND complexes. 1. HLA

[0083] The HLA-ND complexes described herein include a human leukocyte antigen (HLA). HLAs expressed on nucleated cells are the main targets of recipient’s anti-HLA antibodies upon solid organ transplantation. The HLA may be a full mature HLA protein.

[0084] In some embodiments the HLA-ND complex includes one HLA per nanodisc.

[0085] The HLA may be any HLA known in the art. In some embodiments, the HLA may be a class I HLA (e.g., HLA-A, HLA-B, or HLA-C). Exemplary class I HLAs include HLA A6801 , HLA A201 , and HLA B702. In some embodiments, the HLA may be a class II HLA (e.g., HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO).

[0086] 2. Lipid composition

[0087] The HLA-ND complexes described herein include a lipid composition. Any lipid may be used. In some embodiments, the lipid composition may include one or more lipids that form a lipid bilayer. In some embodiments, the lipid composition may include one or more non-bilayer forming lipids or hydrophobic molecules as labels.

[0088] In some embodiments, the lipid composition includes a phospholipid. For example, the phospholipid may be a phosphatidylcholine. Exemplary phosphatidylcholines include 1 -palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1 ,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-dioleoyl-sngycero-3-phosphocholine (DOPC), and N-oleoyl-D-erythro-sphingosylphosphorylcholine (18:1 SM).

[0089] In some embodiments, the phospholipid is a phosphatidylglycerol. Exemplary phosphatidylglycerols include 1 -palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1 -glycerol)) (POPG),1 ,2- dimyristolyl-sn-glycero-3-phosphoglycerol (DMPG), and 1 ,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG).

[0090] In some embodiments, the phospholipid is a glycerophospholipid, such as a glycerophospholipid derived from lipids found in thermophile bacteria e.g., Archaeobacteria). These phospholipids can enhance HLA-ND complex stability and shelf-life.

[0091] In some embodiments, the phospholipid is a phosphatidylethanolamine (PE).

[0092] In some embodiments, the lipid composition includes a detectable label. The detectable label is a marker, signal, or moiety attached, incorporated or associated to a lipid which emits an optical signal that is readily detected by methods known in the art including fluorescence, luminescence, absorbance and the like. Detectable labels include fluorophores, radioisotopes, chromophores, enzymes, dyes, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. In some embodiments, the detectable label is rhodamine, fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), 4',6-diamidino-2-phenylindole (DAPI), coumarin, cyanine, xanthene, naphthalene, oxadiazole, anthracene, pyrene, oxazine, acridine, arylmethine, tetrapyrroles, Alexa Fluor compounds, or boron-dipyrromethene (BODIPY), or derivatives thereof. In some embodiments, the detectable label is rhodamine. In some embodiments, phosphatidylethanolamine (PE) is linked to a detectable label (e.g., rhodamine). In some embodiments, the lipid composition may include one or more unique lipids. For example, the lipid composition may include a phosphatidylcholine (e.g., POPC), a phosphatidylglycerol (e.g., POPG), and a phosphatidylethanolamine (e.g., PE including a detectable label (e.g., rhodamine)). In some embodiments, the lipid composition may include about 60% POPC, about 40% POPG, and about 10% Rhodamine PE.

[0093] 3. Membrane scaffold protein

[0094] The HLA-ND complexes described herein include a membrane scaffold protein (MSP). An MSP is a protein that self-assembles with lipids into membrane bilayers. The MSP is amphipathic, with one part of its structure more or less hydrophilic and facing the aqueous solvent and the other part more or less hydrophobic and facing the center of the hydrophobic bilayer that is to be stabilized. The MSP may be useful for prevent oligomerization or aggregation of the HLA in the nanodisc.

[0095] In some embodiments, the HLA-ND complex includes at least two MSP copies (e.g., two, three, four, or five MSP copies). In some embodiments, the HLA-ND complex includes two MSP copies.

[0096] In some embodiments, the MSP may include 1 -3 cysteine residues to facilitate labeling or immobilization. In some embodiments, the MSP may include affinity tags (e.g., Avi tag, His tag, strep tag, or flag tag) to enable immobilization or purification. In some embodiments, the MSP does not include an affinity tag. In some embodiments, the MSP may be human-based or mouse-based.

[0097] Any suitable MSP may be used. In some embodiments, the MSP may include the amino acid sequence of NW9 (SEQ ID NO: 1 ) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of NW9. The sequence of NW9 is provided below: GSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEP LGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALE DLRQGLLPVLESFKVSFLSALEEYTKKLNTQLPGTGAAALEHHHHHH (SEQ ID NO: 1 )

[0098] In some embodiments, the NW9 may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the NW9 comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. The sequence of SEQ ID NO: 2 is provided below: MGSSHHHHHHENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQ KKWQEEMELYRQKVEPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAK ATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQLPGTGAAALEHHHHHH (SEQ ID NO: 2)

[0099] In some embodiments, the MSP may include the amino acid sequence of NW6 (SEQ ID NO: 3) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of NW6. The sequence of NW6 is provided below: GSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYSDELRQRLAARLEALKENGGA RLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQLPGTGAAALEHHHHH H (SEQ ID NO: 3)

[0100] In some embodiments, the NW6 may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the NW6 comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4. The sequence of SEQ ID NO: 4 is provided below: MGSSHHHHHHENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYSDELR QRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT QLPGTGAAALEHHHHHH (SEQ ID NO: 4)

[0101] 4. Reporter

[0102] In some embodiments, the HLA-ND complex further includes a first reporter fragment linked to an anti-HLA antibody capture agent and a second reporter fragment linked to (a) an HLA capture agent, (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) a lipid head group or lipid tail.

[0103] The reporter may be any molecule that can generate a detectable signal. A detectable signal may be any optical signal that is readily detected by methods known in the art including luminescence, fluorescence, absorbance and the like.

[0104] The reporter may be split into a first fragment and a second fragment that, when in proximity, form a functional reporter. The first fragment and second fragment only form a functional reporter in the presence of an HLA antibody.

[0105] Any split enzyme may be used as the reporter. In some embodiments, the reporter is a split luciferase. For example, the first complementary unit of luciferase may be linked to an anti-HLA antibody capture agent, and the second complementary unit of luciferase (e.g., a 12 amino acid-long fragment of luciferase) may be linked to (a) an HLA capture agent, (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) a lipid head group or lipid tail. The luciferase fragment may be linked to the lipid using any method known in the art, such as terminal palmitoylation.

[0106] In some embodiments, the reporter is sHRP or a split fluorescent protein.

[0107] In some embodiments, the reporter is an Amplified Luminescent Proximity Homogenous Assay (ALPHA) reporter. This technology is based on channeling of single oxygen species, generated from a photoexcitable moiety “donor”, to a “acceptor” located in close proximity; in order to induce a chemiluminescent signal. The donor consists of a photosensitizer that excites ambient oxygen into single state when irradiated at 680nm. Once excited, the donor generates more than 60,000 oxygen singlet molecules each second, with a short half-life time that allows for a diffusion distance of 200 nm maximum. This diffusion limit permits excitation of acceptors present only in proximity, i.e. on the same HLA-ND complexes. Thus, background signal is rendered very minimal. In some embodiments, the anti-HLA antibody capture agent may be linked to donor moiety, with the ability to generate reactive singlet oxygens upon excitation, and the MSP may be linked to acceptor dye that emits intense light only in response to singlet oxygen produced by excited donor in proximity. a. Anti-HLA antibody capture agent

[0108] The anti-HLA antibody capture agent may be any molecule that binds to an anti-HLA antibody. In some embodiments, the anti-HLA antibody capture agent includes an anti-IgG antibody or antigenbinding fragment thereof.

[0109] For example, an anti-HLA antibody capture agent may be adopted from immunoglobulin binding proteins. Several proteins with a high affinity for mammalian immunoglobulins (Igs) have been isolated from Gram-positive bacteria, including protein A from Staphylococcus aureus, protein G from group C and G streprococci, protein L from Finegoldia magna and the M-protein from group A streptococci. A unique Ig binder in Mycoplasma, protein M, has a structure that is different from Gram-positive Ig binding proteins. These proteins can bind to different Igs without the requirement of antigen-binding sites. This non-immune binding mechanism is thought to protect bacteria from the action of the complement system. In some embodiments, different fusion proteins between different Ig binders may be developed to generate multi-valent binders. This may maximize the binding potential since different Ig-binding proteins bind to different parts of the antibody.

[0110] In some embodiments, the anti-HLA antibody capture agent may include 1 -3 cysteine residues to facilitate labeling or immobilization. In some embodiments, the anti-HLA antibody capture agent may include affinity tags (e.g., Avi tag, His tag, strep tag, or flag tag) to enable immobilization or purification. In some embodiments, the anti-HLA antibody capture agent does not include an affinity tag.

[0111] In some embodiments, the anti-HLA antibody capture agent may include the amino acid sequence of PLG binder (SEQ ID NO: 5) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of PLG binder. PLG binder is a chimeric protein composed of the Ig-light-chain-binding domain from Peptostreptococcus magnus protein L and IgG-Fc binding repeats from streptococcal protein G. The sequence of PLG-binder is provided below: GSVTGYRLFEEILVSQGSSGGGGSGGGGSSGVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKK DNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALK KDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLL AKENGKYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFAEATAEAYRYADL LAKENGKYTADLEDGGYTINIRFAGKKVDETYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGE WTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGE WTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGV WTYDDATKTFTVTEMV (SEQ ID NO: 5)

[0112] In some embodiments, the PLG-binder may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the PLG-binder comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 6. The sequence of SEQ ID NO: 6 is provided below: MGSSHHHHHHENLYFQGSVTGYRLFEEILVSQGSSGGGGSGGGGSSGVTIKANLIFANGSTQTAEFKGT FEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKG TFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFK GTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEF KGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFAGKKVDETYKLILNGKTLKGETTTEAVDAAT AEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATA EKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAE KAFKQYANDNGVDGVWTYDDATKTFTVTEMV (SEQ ID NO: 6)

[0113] In some embodiments, the anti-HLA antibody capture agent may include the amino acid sequence of S-PG binder (SEQ ID NO: 7) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of S-PG binder. The sequence of S-PG binder is provided below: GMKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGK TLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKT LKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTESQGSSGGGGSGGGGSSGVTGYR LFEEIL (SEQ ID NO: 7)

[0114] In some embodiments, the S-PG binder may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the S-PG binder comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 8. The sequence of SEQ ID NO: 8 is given below: MGSSHHHHHHENLYFQGMKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVID ASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVI DASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKT FTVTESQGSSGGGGSGGGGSSGVTGYRLFEEIL (SEQ ID NO: 8)

[0115] In some embodiments, the anti-HLA antibody capture agent may include the amino acid sequence of PG-S binder (SEQ ID NO: 9) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of PG-S binder. The sequence of PG-S binder is provided below: GSVTGYRLFEEILVSQGSSGGGGSGGGGSSGMKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD ATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDA TKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDAT KTFTVTE (SEQ ID NO: 9)

[0116] In some embodiments, the PG-S binder may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the PG-S binder comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 10. The sequence of SEQ ID NO: 10 is given below: MGSSHHHHHHENLYFQGSVTGYRLFEEILVSQGSSGGGGSGGGGSSGMKGETTTEAVDAATAEKVFK QYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFK QYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFK QYANDNGVDGVWTYDDATKTFTVTE (SEQ ID NO: 10)

[0117] In some embodiments, any mini protein or mini binder against anti-HLA antibody can be designed and used as the anti-HLA antibody capture agent. b. HLA capture agent

[0118] The HLA capture agent may be any molecule that binds to an HLA. In some embodiments, the HLA capture agent includes an anti-HLA antibody or antigen-binding fragment thereof (e.g., a singlechain fragment variable (scFv), Fab, Fab', F(ab')2, nanobody, or mini protein).

[0119] To generate an HLA capture agent, monoclonal pan HLA antibodies may be adopted, such as W6 / 32 and EMR-8-5. An exemplary HLA capture agent is 128 binder (e.g., 128-1 binder or 128-2 binder). 128 binder is a single-chain variable fragment (scFv) derived from W6 / 32 antibody (pan HLA class I- reactive monoclonal antibody).

[0120] In some embodiments, the HLA capture agent may include 1 -3 cysteine residues to facilitate labeling or immobilization. In some embodiments, the HLA capture agent may include affinity tags (e.g., Avi tag, His tag, strep tag, or flag tag) to enable immobilization or purification. In some embodiments, the HLA capture agent does not include an affinity tag.

[0121] In some embodiments, the HLA capture agent may include the amino acid sequence of 128-1 binder (SEQ ID NO: 11 ) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of 128-1 binder. The sequence of 128-1 binder is provided below: GSQVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWSGGSTDYNAAFISR LSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFTTSTSAWFAYWGQGTLVTVSAGGGGSGGGGSGGGG SGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFT GSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPWTFGGGTKLEIRQGSSGGGGSGGGGSSGVFTLEDF VGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFK VVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSML FRVTINS (SEQ ID NO: 11 )

[0122] In some embodiments, the 128-1 binder may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the 128-1 binder comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 12. The sequence of SEQ ID NO: 12 is given below: MGWSCIILFLVATATGVHSGSSHHHHHHENLYFQGSQVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYG VHWVRQPPGKGLEWLGVIWSGGSTDYNAAFISRLSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFTTST SAWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVS NDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPWTF GGGTKLEIRQGSSGGGGSGGGGSSGVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQ RIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYE GIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 12) In some embodiments, the HLA capture agent may include the amino acid sequence of 128-2 binder (SEQ ID NO: 13) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of 128-2 binder. The sequence of 128-2 binder is provided below: GSQVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWSGGSTDYNAAFISR LSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFTTSTSAWFAYWGQGTLVTVSAGGGGSGGGGSGGGG SGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFT GSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPWTFGGGTKLEIRQGSSGGGGSGGGGSSGVFTLEDF VGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFK VVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSML FRVTINS (SEQ ID NO: 13)

[0123] In some embodiments, the 128-2 binder may comprise an affinity tag (e.g., Avi tag, His tag, strep tag, or flag tag). For example, the 128-2 binder comprising an affinity tag may include the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 14. The sequence of SEQ ID NO: 14 is given below: MGSSHHHHHHENLYFQGSQVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLG VIWSGGSTDYNAAFISRLSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFTTSTSAWFAYWGQGTLVTVS AGGGGSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKL LIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPWTFGGGTKLEIRQGSSGGG GSGGGGSSGVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIP YEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLW NGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 14)

[0124] In some embodiments, any mini protein or mini binder against HLA can be designed and used as the HLA capture agent. In some embodiments, the HLA capture agent may be a mini binder that recognizes the constant p2-microglobulin or MSP itself.

[0125] II. Kit

[0126] Further provided herein are kits including (a) an HLA-ND complex; (b) a first reporter fragment linked to an anti-HLA antibody capture agent; and (c) a second reporter fragment linked to (i) an HLA capture agent, (ii) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (iii) a lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter. The HLA-ND complex, reporter, anti-HLA antibody capture agent, and HLA capture agent may be as described herein.

[0127] III. Methods of production

[0128] The disclosure further provides methods of producing an HLA-ND complex described herein, including: (a) providing an HLA; and (b) incubating the HLA with a MSP and a lipid composition described herein. In some embodiments, the methods describe the isolation and incorporation of full-length, unmodified HLAs into phospholipid bilayer nanodiscs to form stable HLA-ND complexes. Inclusion of HLA into nanodiscs does not alter HLA recognition or binding by anti-HLA antibodies. Importantly, membrane proteins are more active and stable in a lipid bilayer than in detergents. HLA-ND complexes bind only to corresponding antibodies. Additionally, the constituents of the nanodisc do not induce non-specific binding to serum antibodies.

[0129] In order to solve the problem of altered HLA conformation associated with immobilization of truncated HLA on beads, unaltered, full-length HLA proteins may be isolated from the cell surfaces and incorporated into phospholipid bilayer nanodiscs, resulting in stable HLA-nanodisc complexes in a nativelike membrane environment. Small membrane scaffold proteins may be used to generate nanometerscale nanodiscs (e.g., between about 1 nm to about 50 nm (e.g., about 8 nm)), which encourages the incorporation of only a single copy of HLA per nanodisc; since the small area of the nanodisc is not enough to accommodate other membrane proteins alongside HLA in the same nanodisc (coincorporation).

[0130] The methods described herein circumvent HLA denaturation associated with conventionally purified HLA, without inducing denaturation of HLA. The purification and enclosure of HLA within the nanodiscs does not induce alteration in HLA folding.

[0131] 1. HLA preparation

[0132] HLAs may be isolated using any method known in the art. In some embodiments, HLAs may be isolated from monoallelic B cell lines, such as human monoallelic B cell lines. These cell lines may be generated by transducing B cell lines with retroviral vectors (e.g., EBV-transformed B721 .221 cells), each of which codes for a single HLA allele. In some embodiments, the HLA may be a class I HLA (e.g., HLA- A, HLA-B, or HLA-C). In some embodiments, the class I HLA may be HLA A6801 , HLA A201 , and HLA B702. In some embodiments, the HLA may be a class II HLA (e.g., HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO).

[0133] In some embodiments, HLAs may be isolated from HLA-A*68:01 , HLA-A*02:01 , or HLA-B*07:02 transformed B cells. In some embodiments, the HLA may be isolated from native cells, such as human native cells.

[0134] The solubilized membrane protein fraction (e.g., a solubilized plasma membrane protein fraction) may be collected according to standard methods known in the art. For example, cell pellets may be harvested, gently homogenized, and centrifuged to collect the membrane fraction. Mild solubilization of membrane proteins can be induced with a detergent-containing buffer (e.g., cholate). The use of the plasma membrane fraction ensures only HLA displayed on the cell surface are incorporated and avoids any misfolded proteins. The native lipids from the cell membrane may be retained in the final nanodisc, which can preserve the proper structure and conformation of the HLA.

[0135] The HLA preparation methods described herein allow for the preservation of native post translational modifications, such as glycosylation.

[0136] In some embodiments, an affinity tag can be amended to the HLA to facilitate the purification without the use of low pH. 2. Lipid composition preparation

[0137] In some embodiments, the methods of producing an HLA-ND complex include preparing a lipid composition. The lipid composition may include one or more lipids. In some embodiments, the lipid composition includes one or more lipids that forms a bilayer. In some embodiments, the lipid composition may include one or more non-bilayer forming lipids or hydrophobic molecules as labels.

[0138] In some embodiments, the lipid composition includes a phospholipid. For example, the phospholipid may be a phosphatidylcholine. Exemplary phosphatidylcholines include 1 -palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1 ,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-dioleoyl-sngycero-3-phosphocholine (DOPC), and N-oleoyl-D-erythro-sphingosylphosphorylcholine (18:1 SM).

[0139] In some embodiments, the phospholipid is a phosphatidylglycerol. Exemplary phosphatidylglycerols include 1 -palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1 -glycerol)) (POPG),1 ,2- dimyristolyl-sn-glycero-3-phosphoglycerol (DMPG), and 1 ,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG).

[0140] In some embodiments, the phospholipid is a phosphatidylethanolamine (PE).

[0141] In some embodiments, the lipid composition includes a detectable label. Exemplary detectable labels include rhodamine. In some embodiments, the phosphatidylethanolamine (PE) includes a detectable label (e.g., rhodamine).

[0142] In some embodiments, the lipid composition may include one or more unique lipids. For example, the lipid composition may include a phosphatidylcholine (e.g., POPC), a phosphatidylglycerol (e.g., POPG), and a phosphatidylethanolamine (e.g., PE including a detectable label (e.g., rhodamine)). In some embodiments, the lipid composition may include about 60% POPC, about 40% POPG, and about 10% Rhodamine PE.

[0143] To form the lipid composition, lipids may be dissolved in chloroform, mixed, and dried under nitrogen. The dried lipids may be stored in a desiccator under vacuum overnight.

[0144] 3. MSP preparation

[0145] MSPs may be expressed and purified according to methods known in the art. For example, the MSP may be expressed in bacterial cells (e.g., E. coli cells) and purified using affinity chromatography (e.g., Ni-NTA affinity chromatography).

[0146] 4. Nanodisc assembly

[0147] Nanodiscs are synthesized according to standard methods know in the art. In one example, for nanodisc assembly, a stock solution of the lipid composition may be prepared by dissolving the dried lipid mixture in a buffer. The buffer may contain between about 1 mM and about 1 M detergent (e.g., about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 50 mM, about 50 mM to about 100 mM, about 100 mM to about 500 mM, or about 500 mM to about 1 M). In some embodiments, the buffer may contain 100 mM detergent. The detergent may be any suitable detergent known in the art (e.g., Na-cholate). The lipid mixture may be dissolved using sonication in a water bath for between about 10 minutes to about 60 minutes (e.g., about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, or about 50 minutes to about 60 minutes). In some embodiments, the lipid mixture may be dissolved using sonication in a water bath for about 30 minutes.

[0148] The stock solution of the lipid composition may contain between about 1 mM to about 1 M lipid (e.g., about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 50 mM, about 50 mM to about 100 mM, about 100 mM to about 500 mM, or about 500 mM to about 1 M). In some embodiments, the stock solution of the lipid composition may contain about 33 mM lipid.

[0149] The lipid solution may then be mixed with the MSP solution to form a final molar ratio of between about 10:1 to about 1000:1 lipid :MSP (e.g., about 10:1 to about 50:1 , about 50:1 to about 100:1 , about 100:1 to about 500:1 , or about 500:1 to about 1000:1 lipid :MSP) . In some embodiments, the molar ratio of lipid :MSP may be about 100:1 .

[0150] To favor the incorporation of only one copy of HLA per nanodisc, small NW9 or NW6 nanodiscs (~8 nm in diameter) may be used. The small area of the nanodisc allows the incorporation of a single HLA molecule per nanodisc and hampers the co-incorporation of other membrane proteins alongside HLA in the same nanodisc.

[0151] 5. Incorporation of HLA into nanodiscs

[0152] For incorporation of HLA into nanodiscs, forming HLA-ND complexes, solubilized membrane fractions containing HLA proteins may be incubated with the nanodisc (including the lipid composition and an MSP) and a detergent.

[0153] The MSP: lipid composition: detergent ratio may be about 1 :100:155. In some embodiments, the MSP may be present at a concentration of between about 0.1 mM and about 10 mM (e.g., between about 0.1 mM and about 0.5 mM, between about 0.5 mM and about 1 mM, between about 1 mM and about 5 mM, or between about 5 mM and about 10 mM).

[0154] In some embodiments, the lipid composition may be present at a concentration of between about 10 mM and about 1000 mM (e.g., between about 10 mM and about 50 mM, between about 50 mM and about 100 mM, between about 100 mM and about 500 mM, or between about 500 mM and about 1000 mM).

[0155] In some embodiments, the detergent may be present at a concentration of between about 10 mM and about 1000 mM (e.g., between about 10 mM and about 50 mM, between about 50 mM and about 100 mM, between about 100 mM and about 500 mM, or between about 500 mM and about 1000 mM).

[0156] In some embodiments, the MSP may be present at a concentration of about 0.2 mM, the lipid composition may be present at a concentration of about 12 mM, and the detergent may be present at a concentration of about 30 mM.

[0157] The mixture may be incubated on ice for between about 30 minutes to about 120 minutes (e.g., about 30 minutes to about 45 minutes, about 45 minutes to about 60 minutes, about 60 minutes to about 75 minutes, about 75 minutes to about 90 minutes, about 90 minutes to about 105 minutes, or about 105 minutes to about 120 minutes). In some embodiments, the mixture may be incubated on ice for about 60 minutes.

[0158] Following incubation, the detergent may then be removed using any method known in the art. For example, the detergent may be removed by adsorption over nonpolar polystyrene adsorbent beads. The mixture may then be filtered and purified (e.g., using a gel filtration column or by immunoaffinity capture).

[0159] The purity and successful incorporation of HLA into nanodiscs may be assessed by silver-stain, SDS-PAGE and Western blotting will be employed. HLA-ND complexes may be further analyzed by electron microscopy imaging and be subjected to mass spectrometric analysis. Stability of HLA in nanodiscs of different sizes and lipid ratios may be assessed by size exclusion chromatography and thermal shift assay.

[0160] IV. Methods of detection

[0161] The disclosure further provides methods of detecting anti-HLA antibodies, including (i) contacting a sample with an HLA-ND complex described herein and (ii) determining the presence of a signal from the reporter, thereby detecting the anti-HLA antibody. These methods are useful in the detection of anti- HLA antibodies and to perform virtual crossmatching before and after solid organ transplantation. In some embodiments, the methods are useful in determining maternal-fetal HLA compatibility.

[0162] Solid organ transplantation remains as the only treatment for most patients with end-stage organ failure. However, the outcomes of those patients remain poor due to the risk of acute and chronic rejection. HLAs expressed on nucleated cells are the main targets of recipient’s anti-HLA antibodies upon solid organ transplantation. Anti-HLA antibodies can develop either pre- or post-transplantation. HLA- specific antibodies that develop pre-transplantation (due to exposure to human antigens through blood transfusion, pregnancy, or previous transplant) can lead to hyperacute rejection and immediate graft loss, if directed against the donor. These anti-HLA antibodies, specific against donor antigens, are described as Donor Specific Antibodies (DSA). DSA formed post-transplantation can also cause chronic antibody- mediated rejection (ABMR) which is now recognized as the leading cause of allograft failure in solid organ transplantation, contributing to 64% of graft failure. Hence, DSA is an established biomarker predicting allograft rejection.

[0163] The incidence of ABMR is likely to rise due to the growing use of organs from deceased human donors, a factor linked to a higher risk of ABMR development. To prevent hyperacute rejection, patientdonor compatibility can be directly tested using the complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometric crossmatch (F-XM). Both approaches are cell-based where donor cells are cultured with serum from recipients to determine the presence of DSA. These assays are laborious and time-consuming, leading to delays in performing the transplant, increasing the cold ischemia time, and potentially worsening the outcomes of the allograft.

[0164] Technological advances have made it possible to isolate and purify HLA antigens and to immobilize them to solid matrices (beads), so that HLA antibodies specificities can be determined by standard flow cytometry or a more developed LUMINEX® system. These multiplexed single antigen bead (SAB) assays, which have a unique HLA molecule coupled to a particular bead, have good sensitivity, and allows for characterization of anti-HLA antibody specificity. Since testing can be performed prior to the identification of a donor, this has quickly led to the adoption of “virtual” crossmatching (VXM) for rapid determination of whether a candidate possesses an HLA antibody that is directed against a particular donor. VXM compares the molecular HLA typing of the donor with the detailed antibody profile of the recipient to allow for a pivotal decision over HLA compatibility. Because of its improved ability to identify DSAs, V-XM reduced the ischemia time of transplanted organs and potentially reduced complications and costs.

[0165] However, the current method of purifying and immobilizing HLA antigens on beads can lead to the modification of the tertiary structure of the molecules and exposure of cryptic epitopes thus, resulting in aberrant false positive and false negative reactivity. With the increasing reliance on virtual crossmatching (comparing HLA antibodies identified using the single antigen bead assay to the donor HLA typing to identify DSA) in lieu of a physical crossmatch, the accuracy of HLA antibody identification is even more critical.

[0166] In some embodiments, the disclosure provides a method of predicting whether an organ transplant recipient is at risk of hyperacute rejection, the method including: (a) providing a sample from the organ transplant recipient; (b) contacting the sample with an HLA-ND complex including an HLA that corresponds to an HLA of the donor of the transplanted organ; and (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient is at risk of hyperacute rejection.

[0167] In some embodiments, the disclosure provides a method of monitoring the development of DSAs in a subject who received an organ transplant, the method including: (a) providing a sample from the organ transplant recipient; (b) contacting the sample with an HLA-ND complex including an HLA that corresponds to an HLA of the donor of the transplanted organ; and (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient has developed DSAs.

[0168] The sample may comprise any biological sample or other fluids. In some embodiments, the sample is a serum sample or a blood sample obtained from a subject (for example, a human patient). In some embodiments, the sample is a tissue sample or a biopsy sample obtained from a subject (for example, a human patient).

[0169] 1. Bead-based assay

[0170] In some embodiments, the methods involve HLA-ND complex immobilization over beads. To keep HLA completely unaltered, HLA-ND complex immobilization does not involve any manipulation of HLA proteins. Instead, the immobilization will be mediated through tagged MSPs rather than the enclosed HLA membrane protein.

[0171] The methods are compatible with xMAP® technology utilized in the LUMINEX® SAB assay. MAGPLEX-TAG™ microsphere beads may be used to immobilize HLA-ND complexes. These beads are color-coded, oligonucleotide-coupled and dyed into spectrally distinct regions. This allows for identification of individual beads in xMAP instrument.

[0172] In order to immobilize HLA-ND complexes over MAGPLEX-TAG™ beads, MSPs may be modified with complementary oligonucleotide. For example, MSPs may be engineered to carry cysteine amino acids that will be used for conjugation with 5’-amino-modified oligonucleotides, using the bifunctional Sulfo-SMCC cross-linker. The oligo-conjugated nanodiscs may then be purified by size exclusion chromatography or by using spin-column concentrator with appropriate molecular weight cut offs. The results may be analyzed (i) statistically, by establishing a fluorescence level (cutoff) that is significantly above background; and using it to assess a positive / negative result of our assay; (ii) practically, by correlating the observed fluorescence values with a known crossmatch result, to calculate the degree and extent of any false-positives / false-negatives; and (Hi) clinically, based on statistical and practical data, to identify a threshold level that predicts immediate / early graft loss.

[0173] 2. Bead-free assay

[0174] In some embodiments, the methods are bead-free. Since developing a cost-effective point of care assay to monitor DSA is an important clinical unmet need, the methods described herein provide a novel, beads-free, homogenous assay for the rapid detection of anti-HLA antibodies utilizing HLA-ND complexes. The nanodiscs are tolerant to a wide range of pH levels, salts and resistant to proteolysis, enabling the use of nanodisc with biological samples. Being a homogenous assay, accurate read-outs are yielded without the need to process the sample. There is no need for multiple steps of incubation and wash cycles, and the results interpretation is simple and straightforward. The generated signal can be measured by conventional plate-readers or even standard smart phone cameras, opening opportunities for self-testing at home.

[0175] In some embodiments, the method for detecting an anti-HLA antibody includes: (a) providing a sample from a subject; (b) contacting the sample with an HLA-ND complex described herein; and (c) determining the presence of a signal from the reporter, thereby detecting the anti-HLA antibody.

[0176] The bead-free assay involves the complementation of a split reporter (e.g., a split luciferase) on the HLA-anti-HLA antibody complex. A functional reporter (e.g., luciferase) may be split into two fragments. One fragment may be fused to the anti-HLA antibody. Another fragment may be fused to an HLA capture agent, an MSP at the N-terminus, C-terminus, or at a cysteine residue, or a lipid head group or lipid tail. Only in the presence of the HLA antibody complex, an active reporter will be formed, consequently generate a detectable signal.

[0177] In some embodiments, prior to determining the presence of a signal from the reporter, the HLA- ND complex may be incubated with the sample for, e.g., between about 1 minute to about 60 minutes (e.g., about 1 minute to about 10 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, or about 50 minutes to about 60 minutes). In some embodiments, the HLA-ND complex may be incubated with the sample for about 10 minutes. In some embodiments, the incubation is performed at about room temperature.

[0178] In some embodiments, prior to determining the presence of a signal from the reporter, a substrate for the reporter is added. For example, when the reporter is luciferase, luciferase substrate may be added prior to measuring luminescence. EXAMPLES

[0179] Table of Contents

[0180] Example 1. Materials and methods. isolation of murine Baib / c spienocytes

[0181] Balb / c mouse spleen tissue was used as an alloantigen source; spienocytes were extracted and mashed, then filtered through 70-micron filters to eliminate debris before being reconstituted in cold PBS. Spleen cells were washed with 10 ml of cold PBS, and RBCs were lysed for 60-120 seconds in 2 ml of LCK lysate solution per spleen, then rinsed with 10 ml of cold PBS. Finally, the cell pellets were kept at - 80 °C for ongoing studies.

[0182] Major histocompatibility complex membrane protein solubilization and purification

[0183] Cell pellets were thawed in a cold room for 30 minutes, then resuspended in 5 ml of cold buffer A (20 mM Tris-HCI, PH 8.0, 150 NACL, 0.5 mM PMSF, and 1 mM protease inhibitor). Cell homogenizing was done using a manual homogenizer for 15 min, then centrifuged at 15 000 RPM and 4 °C for 15 min. Cell suspension was prepared by dissolving the cell pellet in 5 ml of cold buffer A (20 mM Tris-HCI, PH 8.0, 150 NACL, 100 mM Na cholate). The suspension was centrifuged at 15 000 RPM for 10 min at 4 °C; the supernatant solution containing the solubilized membrane MHC proteins was collected, and purified proteins were centrifuged using a 30 MWCO cut-off concentrator tube at 7000 RPM and 4 °C for 20 min. Concentrated purified proteins were collected and used for MHC nanodisc complex preparation.

[0184] Membrane scaffold protein (MSP) expression and purification.

[0185] MSP1 D1 plasmids were purchased from Addgene and expressed in E. coli BL21 (DE3) cells with minor modifications. When the OD600 approached 0.9, expression was stimulated by 1 mM isopropyl-D- 1 -thiogalactopyranoside (IPTG) in LB medium at 37°C. At 4 hours after induction, cells were collected and resuspended in 20 mM Tris-HCI [pH 8.0], 150 mM NaCI (pH 7.4), and 1 mM PMSF, and lysed by sonication in the presence of 1% Triton X-100 for purification. The lysate was cleared by centrifugation at 15 000 x g for 30 minutes at 4 °C and then batch-bound for 2 hours at 4 °C with Ni2+-NTA resin. Following that, four column volumes of each of the following were used to wash the resin: (1 ) 20 mM Tris- HCI [pH 8.0], 150 mM NaCI, 1% Triton X-100; (2) 20 mM Tris-HCI [pH 8.0], 150 mM NaCI, 50 mM sodium cholate; (3) 20 mM Tris-HCI [pH 8.0], 150 mM NaCI; and (4) 20 mM Tris-HCI [pH 8.0], 150 mM 20 mM imidazole (5) Tris-HCI [pH 8.0], 150 mM NaCI, and 500 mM imidazole were used to elute MSP1 D1 , fractions were collected and were checked via SDS-PAGE, concentrated and stored at -20 °C until needed. The concentrations of MSP were measured spectrophotometrically at 280 nm using a nanodrop instrument.

[0186] Preparation of lipid solutions for nanodiscs

[0187] The nanodisc lipid composition for major histocompatibility complex incorporation was optimized at 60% POPC and 40% POPG and 10% of Rhodamine PE in case of generation of Rhodamine fluorescent nanodisc complexes. POPC and POPG were dissolved in chloroform and mixed in the desired ratio and dried under nitrogen. The dried lipids were stored in a desiccator under vacuum overnight. For nanodisc assembly, a stock solution (32.8 mM) was prepared by dissolving the dried lipid mixture in buffer A (20 mM Tris-HCI [pH 7.4], 150 mM NaCI, 0.5 mM EDTA, 0.01% NaN3) with 100 mM Na-cholate by sonication in a water bath for 30 min until the solution turned clear. The lipid solution was mixed with MSP1 E3D1 in 20 mM Na-cholate and Tris buffer in a final molar ratio of 100:1 (phospholipid: MSP).

[0188] Major histocompatibility complex incorporation into nanodiscs: assembly and purification

[0189] Before use, all buffers and stock solutions were thoroughly degassed. The B cell lysate was added to the purified MSP1 D1 and POPC-POPG / sodium cholate solubilized mixture, and the final MSP1 D1 : lipid: detergent ratio was adjusted to 1 :100:155 (0.2 mM, 12 mM, 31 .5 mM). After the mixture was incubated on ice for 1 hour, the detergent was removed by adding 80% (w / v) Bio-Beads SM-2 (BioRad) and incubating on ice for 1 hour, followed by gently rotating at 4°C for 4 hours. The disc preparation was filtered through 0.45-mm nitrocellulose filter tubes, rotated at 4 °C for two additional hours, and then purified on a gel filtration column (Superdex 200 10 / 300 GL) pre-equilibrated with 20 mM Tris-HCI [pH 8.0] and 150 mM NaCI. Purity was assessed by SDS-PAGE, and peak fractions were collected and concentrated.

[0190] Ni-NTA metal affinity chromatography

[0191] The nanodisc-lysate mixture was filtered through a 0.22 pm syringe filter to remove the Bio-Rad hydrophobic beads. Ni-NTA beads (1 mL) were equilibrated with buffer B (20 mM Tris pH 7.4, 150 mM NaCI, and 20 mM imidazole) and incubated for 1 hour at 4 °C with the nanodisc lysate mixture. The resin was then washed with 2 column volumes of buffer A and 2 column volumes of buffer B and before eluting protein with 2 column volumes of elution buffer (20 mM Tris pH 7.4, 150 mM NaCI, 500 mM imidazole). The eluted protein was concentrated by ultrafiltration against a 10 kDa cutoff membrane. A portion of concentrated Nickel-NTA-purified nanodisc sample was saved for assay of purity.

[0192] Antibody-agarose bead conjugation

[0193] An appropriate ratio of antibody to bead was made. 50 ul (0.3 mg) of anti-MHC class I antibodies (Bio X Cell) and 40 ul (0.45 mg) of anti-MHC class II (Bio X Cell) antibodies was added to 350 ul of Gamma-Bind Sepharose beads supplied by GE Healthcare to allow crosslinking through Protein G resin using dimethylpimelimidate (DMP) as a crosslinker. At 4 °C, the columns were rocked overnight, then washed three times with 0.2 M sodium borate (PH 9.0), and finally spun at 300-500 g to pellet the beads. The supernatant was carefully removed, and a tiny aliquot was collected for SDS-PAGE analysis.

[0194] Antibody Quenching

[0195] The produced 0.2M ethanolamine in 0.2 Na Borate, pH 8.0 quenching reagent was used to rinse the beads once and remove any remaining DMP. Reconstituted beads were incubated for one to two hours at room temperature in 1 ml of 0.2 M ethanolamine (pH 8.0). A sample was obtained to test the effectiveness. In order to eliminate uncoupled IgG antibodies after each wash, the column was washed with 0.58 percent v / v acetic acid + 150 mM NaCI for 3-5 column volumes, followed by 3-5 column volumes of cold PBS, and then spun at 300-500 g to pellet beads. The column was preserved in PBS at 2°C to 8°C after the supernatant was gently removed. 0.01 percent NaN3 was then added. To ensure efficiency, an SDS-PAGE gel was performed.

[0196] MHC-ND separation using MHC / / / / antibody column

[0197] The column was equilibrated with 2-3 column volumes of PBS before collecting 100-ul aliquot samples of MHC I and II. Antibody bound to gamma beads washed with 0.1 mM glycine to remove nonspecific bonds before re-equilibration with a 5-column volume of 20 mM Tris-base 100 NACL buffer. At least 400 ul of the MHC-ND library were added and incubated for 1 hour at room temperature or overnight at 4 °C. Then the column was washed with a 5-column volume of 20 mM Tris Base 100 NACL buffer and elution with 100 mM glycine was done, followed by PH adjustment with 1 M Tris PH 8 and a final buffer PH 3. After elution, the medium was washed three times using cleaning buffer (1 M acetic acid, pH 2.5), followed by re-equilibration with 2 to 3 column volumes of binding buffer, and kept in 20% ethanol at 2°C to 8°C.

[0198] Western Blot for murine MHC I and II antigen complex detection

[0199] Equal quantities of proteins as well as a molecular weight marker were loaded into the wells of the SDS-PAGE gel (5-15%). The gel was run for 15 minutes at 250 V. Before producing the stack, the PVDF membrane was activated with methanol for 1 minute and then washed with transfer buffer. The transfer time and voltage were optimized; a 1 -hour transfer at 24 V was used. Membrane blocking was performed for 1 hour at room temperature or overnight at 4 °C using a non-fat dry milk solution buffer. In blocking buffer, the membrane was incubated with suitable dilutions of anti-mouse MHC Class II (l-A / l-E) monoclonal antibody (M5 / 114.15.2) Catalog # 14-5321 -82 (1 :3000) and Mouse HLA Class 1 ABC Monoclonal Antibody Catalog # MBS3014478 (1 :2000). Overnight incubation at 4°C is recommended. The membrane was washed three times in TBST buffer for five minutes each time. At room temperature, the membrane was incubated for 1 hour with proper dilutions of goat anti-rat IgG (H+L) secondary antibody (HRP Catalog # 31470) or goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (HRP Catalog # G-21040 (1 :5000) in blocking buffer for 1 h at room temperature, and the membrane was washed three times in TBST buffer for five minutes each time. For signal development, excess reagent was removed and the membrane was covered in transparent plastic wrap, then an image was acquired using darkroom development techniques for chemiluminescence.

[0200] Cell lines

[0201] The HEK293T and W632 hybridoma cell lines were purchased from ATCC. Monoallelic B cell lines were given as a gift from the Derin Keskin laboratory at the Dana-Farber Cancer Institute, Harvard Medical School, and were allowed to grow in our laboratory at 37 °C under CO2 for 48-72 h before being gently harvested and stored at 80 °C.

[0202] Cell culture, transfection, and cell lysate formation

[0203] B lymphoblastoid B cell transfected by EBV-transformed B721 .221 cells with retroviral vectors, each of which codes for a single MHC class I allele, such as HLA A6801 , B0702, and HLA A201 , cells were harvested from four 150 mm culture dishes and stored at -80°C.

[0204] Low pH-based denaturation of HLA-nanodiscs

[0205] Nanodisc-HLA was exposed to low pH. First, 20 pl of nanodisc-HLA (13 pM in 1xPBS) was mixed with 180 pl of acetic acid (5%, pH=2.5) and kept at room temperature for 40 minutes. Then, 200 pl of the mixture was further mixed with 1800 pl of 1xPBS, resulting in 130 nM of nanodisc-HLA with a pH of 7.4 for microscale thermophoresis (MST) measurement and flow cytometry.

[0206] Binding affinity analysis of antibody / HLA, antibody / sPLG, and HLA / 128 using microscale thermophoresis (MST)

[0207] The binding affinity of antibody / HLA, antibody / sPLG, and HLA / 128 was measured using MST. The measurements were performed on a Monolith NT.115 system (NanoTemper Technologies). The fluorescence signal of the antibody or 128 was measured using the Monolith His-Tag labeling 2ndgeneration kit RED-tris-NTA (NanoTemper Technologies). The samples were prepared in PBS buffer containing 0.05% Tween 20. The HLA or antibody was titrated in two-fold dilution steps. For measurements, the samples were filled into premium-coated capillaries. Measurements were performed at 2% light-emitting diode (LED) and 20% MST power, 30 s laser on, and 5 s laser off. Fluorescence was excited at 605-645 nm, and emission was detected at 680-685 nm. MST measurement was repeated three times, and the results were analyzed using MO Affinity Analysis software (NanoTemper Technologies).

[0208] Bioluminescence measurements of antibody

[0209] For the analysis of anti-HLA antibodies, the assay was performed in a black 96-well plate, and the bioluminescence was recorded by the plate reader. 10 pL of sPLG, 10 pL of 128, and 10 pL of nanodisc-HLA (1 .6 pM) were mixed, followed by the addition of 10 pL of anti-HLA antibodies at different concentrations, and the subsequent incubation of the mixture for 10 min at room temperature. Then, 80 |al_ of luciferase substrate (40 pM, Nano-Gio® In Vivo Substrate, Promega) was added, and the bioluminescence was quantified 5 min later using (Discovery, Promega) with 0.5 s integration time.

[0210] Imaging of gels and blots

[0211] Silver-stained polyacrylamide gels and immuno-stained western blots were imaged using a BioRad Chemi-Doc™Touch system. Imaging of immunostained blots was performed using the instrument’s standard chemiluminescent application with autoexposure; silver-stained gels were imaged using white light transillumination with autoexposure.

[0212] Flowcytometry analysis

[0213] The following antibodies were used for decorating UltraComp compensation beads (Invitrogen) at room temperature for 30 minutes: Anti-IgG FITC, W632 clone (Anti-Hu HLA-ABC Invitrogen); Anti-IgG A2 PerCP-eFlour 710, BB7.2 clone (Anti-Hu HLA-A2 Invitrogen). Goat FITC- labeled Anti-human Fc- IgG (GOXHU FC affinity XADS, Invitrogen), Flow cytometry analysis was performed on a Cytek Aurora spectral analyzer, and data were analyzed with FlowJo, version 10 (FlowJo LLC).

[0214] AlphaFold structure prediction and visualization

[0215] AlphaFold was employed to predict the sPG and 128 structures. The computation was performed using ChimeraX which ran it on Google Colab servers and the protein structures were visualized using PyMOL.

[0216] Example 2. Preparation and characterization of native MHC library.

[0217] First, murine Balb / c mouse splenocytes were used as a source of the major histocompatibility complex MHC I and II antigens (MHC system is known as HLA in humans). Subsequently, a protocol for the isolation and incorporation of native MHCs into nanodiscs was developed. Nanodiscs are a synthetic self-assembled membrane model that is composed of a nanometer-sized phospholipid bilayer patch encircled by two copies of an a-helical amphipathic belt protein, termed a membrane scaffold protein. When a membrane protein is inserted into a nanodisc, it becomes surrounded by a lipid bilayer, creating an environment that closely resembles its native state. Therefore, nanodiscs effectively maintain the structure and function of membrane proteins.

[0218] Figure 1 shows the workflow for preparing MHC I and II as well as non-MHC nanodisc membrane proteins library. To favor the incorporation of only one copy of MHC molecules per nanodisc, small NW9 nanodiscs (~8 nm in diameter) were used. The small area of the nanodisc allows the incorporation of a single MHC molecule per nanodisc and hampers the co-incorporation of other membrane proteins alongside MHC in the same nanodisc. Initially, Balb / c splenocytes were processed to obtain full length MHC proteins, which were subsequently incubated with NW9 belt protein to generate stable MHC nanodisc complexes from the entire cell membrane, as outlined in the isolation protocol. Next, anti-mouse MHC I (H-2Kd / H-2Dd), and MHC II (l-A / l-E) antibodies were covalently linked to the protein-A modified agarose beads to prepare MHC I and II separation columns, as illustrated in Figure 1 . Subsequently MHC-nanodiscs were eluted from the columns using acidic buffer and the eluted fractions were neutralized immediately by addition of Tris buffer.

[0219] To evaluate the purity and successful incorporation of native MHCs into nanodiscs, various analytical techniques were employed, including Western blot, silver-stain SDS-PAGE, electron microscopy imaging and mass spectrometry analyses. Figure 2 presents the characterization data for MHC ll-ND. Western blot shows a band around 60 KDa corresponding to the monomeric MHC II (ap) and another band around 30 KDa corresponding to either a or subunits of MHC II (Figure 2b). Silver-stain shows a band around 20 KDa that corresponds to NW9, another around 60 KDa corresponds to monomeric MHC II (ap) and a faint band around 30 KDa corresponds to either a or p subunit of MHC II (Figure 2c). No other bands were visible in the silver-stain SDS-PAGE indicating that MHC II is the main protein incorporated. Negative-stain EM images confirmed the assembly of the nanodiscs (Figure 2d). Mass spectrometry analysis was conducted to identify amino acid composition and sequence of the peptides, confirming the successful isolation and incorporation of native MHC II into nanodiscs (Figure 2e). The search utilizing the murine proteome library verified the presence of MHC proteins, with H2-Ab1 , H2-Ea and H2-Eb1 alleles being the most abundant MHC class II isolates as shown in Figures 2e and 2f. The results here are described for mouse MHC proteins but are readily applicable to human HLA proteins.

[0220] Example 3. Isolation of full-length HLA I from monoallelic B cell lines and incorporation into nanodiscs.

[0221] Next, the production of full-length human HLA nanodisc complexes utilizing monoallelic B cell lines was pursued. These B cell lines were generated by transducing EBV-transformed B721 .221 cells with retroviral vectors, each of which codes for a single MHC class I allele, such as HLA A6801 , HLA A201 and HLA B702. The single antigens expressed on a mammalian cell line permit a simplified procedures for the isolation and production of monoallelic HLA-nanodisc preparations, which would be used to detect or isolate antibodies one at a time from sensitized serum containing multiple antibodies and more precisely determine antibody specificity.

[0222] Briefly, HLA A6801 and HLA A201 cells were harvested, gently homogenized, centrifuged, and the membrane fraction was collected and incubated with buffer containing sodium cholate to facilitate the solubilization of membrane proteins. The solubilized membrane fractions were then incubated with the scaffold protein, NW9, a mixture of POPC / POPG lipids and 10% rhodamine lipids. Next, Bio-beads were added to remove the detergent and initiate the assembly of Rhodamine-labeled HLA A6801 and HLA A201 nanodiscs (Figure 3a). The binding between the HLA A6801 Rhodamine-labeled nanodisc and the anti-HLA ABC class I antibody was verified using a high-resolution flow cytometry, as shown in Figure 3b. Rhodamine HLA-I nanodisc complexes bind to the beads decorated with the FITC IgG anti-HLA ABC antibody. In contrast, Rhodamine labeled empty nanodiscs (control) did not show any binding. Next, the specificity of HLA-nanodisc in detecting antibodies against specific alleles that belongs to the same gene was tested. To achieve this, a PerCP-eflour 710 labelled anti-HLA-A2 antibody was inclubated with Rhodamine-labeled HLA-A6801 NDs, HLA-A201 NDs, and Rhodamine empty nanodiscs (control). Only the HLA-A201 NDs complex showed binding to PerCP-eflour 710 IgG anti-HLA-A2 antibody, as presented in Figure 3c.

[0223] Example 4. Donor specific antibody screening using HLA nanodisc complexes.

[0224] Initially, HLA-A201 nanodisc complex were utilized in order to ensure that HLA retains its binding capacity and specificity towards serum anti-HLA antibodies. Positive DSA serum from individuals with anti-HLA-A201 antibodies, and serum with negative DSA, as determined by a high-resolution LUMINEX® assay, were obtained and used in our analysis. Rhodamine-labeled HLA-A201 nanodiscs were incubated with the serum for one hour. Next, FITC-labeled anti-human-Fc IgG antibodies immobilized on beads were used to capture the HLA-nanodisc-antibody complexes. As anticipated, binding and immobilization of HLA-ND complexes over the beads occurred with HLA-A201 positive serum, while no significant binding was observed between HLA-A201 nanodiscs and negative DSA serum or between empty nanodiscs and anti-HLA-A201 positive serum, as illustrated in Figure 3d. This finding suggests that the assembly of HLA in nanodiscs does not hinder HLA / antibody binding, and importantly, the nanodisc constituents do not induce nonspecific binding to serum antibodies in the samples tested.

[0225] Example 5. Incorporation of HLA Into nanodisc preserves its native conformation.

[0226] To assess potential denaturation of the HLA molecules during the NDs library preparation, experiments were conducted to examine the interactions with mAb HC-10, known to react only with denatured HLA. The HC-10 antibody is known to specifically bind to a well-defined epitope sequence that is normally inaccessible due to the interaction between the p2-microglobulin and intact HLA molecules. Denaturation of HLA results in the loss of interaction with p2-microglobulin, allowing for HC-10 binding. Initially, HC-10 antibody was immobilized on polystyrene beads and subsequently labeled the entire NDs library with rhodamine for flow cytometry identification (Figure 4a). Co-incubation of the prepared NDs library with HC-10 beads demonstrated minimal binding, with a very low percentage (0.4%) of rhodaminepositive beads. Subsequently, denaturation was induced by subjecting the NDs library to low pH (5% acetic acid, pH 2.5) for 40 minutes at room temperature, followed by neutralization by (0.5M Tris-HCI, pH 9.0) buffer. As anticipated, a substantial proportion of HC-10-coated beads exhibited fluorescence, indicating the presence and binding of denatured HLA-ND complexes to HC-10 mAb (Figure 4a, b). Additionally, the microscale thermophoresis (MST) assay was utilized to assess the binding between HC- 10 and HLA-ND complexes in solution in the absence of any beads. The MST results show that HLA-ND complexes exhibited negligible binding affinity to the HC-10 mAb and that this binding substantially increased after denaturation, as shown in Figure 4c. These results collectively demonstrate that the purification and incorporation of HLA within NDs does not induce alteration in HLA folding.

[0227] Example 6. Developing a homogenous and rapid assay fo the detection of anti-HLA antibodies.

[0228] As monitoring DSA post transplantation is currently highly recommended, cost remains an obstacle toward wide implementation. Developing an inexpensive point of care assay to monitor DSA is an important clinical unmet need. Hence, encouraged by the successful MHC nanodisc incorporation and the binding results, a simple, rapid, sensitive, and homogenous assay for the detection of anti-HLA antibodies was developed herein. The assay uses a split-luciferase bioluminescent biosensor, and it is based on protein complementation. A functional luciferase protein is split into two fragments, which are then fused to two different binders that can bind to the anti-HLA antibody and HLA class I antigens, separately, forming S-PLG and L-128 as two split luciferase probes, as shown in Figure 5a. PLG binder is a chimeric protein composed of the Ig-light-chain-binding domain from Peptostreptococcus magnus protein L and IgG-Fc binding repeats from streptococcal protein G 17. 128 binder is single-chain variable fragment (scFv) derived from W6 / 32 antibody (pan HLA class l-reactive monoclonal antibody). The HLA antibody-HLA-nanodisc complex brings the split luciferase fragments into close proximity to form a functional luciferase that generates a bright blue bioluminescent signal upon exposure to its substrate. NanoLuc is employed as the luciferase enzyme to react with the luciferin substrate and generate a bioluminescence signal with high stability for HLA antibody detection. The assay is straightforward to perform and involves only mixing the patient's sample with the two luciferase probes and nanodisc-HLA, followed by the addition of the luciferin substrate for bioluminescence readout.

[0229] Protein S-PLG was expressed in E. coli, and L-128 was expressed in mammalian cells. SDS- PAGE analysis demonstrates the high purity and the correct sizes for the two proteins (Figure 5b). Alpha Fold was employed to predict the protein structures 18, 19. Initially, commercially available human HLA- A02:01 and anti-HLA A2 antibody were used to confirm that L-128 and S-PLG fusion proteins retain their binding activities after modifying them with luciferase fragments. The microscale thermophoresis (MST) results show that the affinity equilibrium constants of anti-HLA Ab / HLA, anti-HLA Ab / S-PLG, and HLA / L- 128 are 2.3 nM, 80 nM, and 3.3 nM, respectively (Figure 5d,e,f). When the two complementary luciferase probes (L-128 and S-PLG) and samples containing HLA antibodies are mixed with the luciferin substrate, they generate bright blue bioluminescence with an emission wavelength of 455 nm (Figure 5g), indicating the functionality of the complementary luciferase protein. Using the luciferase probes, a detection limit of 2.5 nM was achieved (Figure 5i).

[0230] Example 7. Development of a bead-based assay with unaltered full-length HLA in nanodlsos

[0231] To circumvent all conformational changes that are associated with the conventional purification of recombinant HLA, HLA protein can be directly extracted from the surface of human-expressing cells. To stabilize HLA in a native-like environment, HLA can be assembled into lipid nanodiscs. Unlike conventional immobilization technique, which includes modifications of HLA, the nanodisc can be used to immobilize HLA-ND over detection beads. This approach overcomes the critical drawbacks of the currently available technologies, while still enabling for multiplexing detection of antibodies against different HLA types (Figure 6).

[0232] Membrane HLA proteins can be extracted into lipid nanodiscs, followed by immobilization on detection beads. The generated HLA-NDs-beads can be used to validate the assay against a library of patients’ serum, in comparison to the currently available beads-based assay.

[0233] Cell expression systems (mammalian and bacterial), immobilized-affinity chromatography purification, size exclusion chromatography, MST, SDS PAGE, immunoblotting, EM, and mass spectroscopic analysis methods may be used in connection with the assay. Example 8. Development of a bead-tree, cost-effective assay to monitor DSA development Monitoring of potential development of donor-specific antibodies is critical to avoid posttransplantation graft failure. Described herein is a cost-effective, rapid, and simple assay for point-of-care monitoring of anti-HLA antibodies.

[0234] Multivalent binders to anti-HLA antibodies are designed and validated. Then, the binder is used for a luciferase complementation assay (Figure 7). The full potential of nanodiscs components are used to develop the assay. The assay depends on the proximity of PLG protein to the nanodisc, which occurs only when an anti-HLA antibody exists. First, PLG can be engineered to be fused with an enzymatically inert subunit of luciferase. Taking advantage of the short length of the second complementary unit of luciferase, which is only 12 amino acids-long, a modified NW9 belt protein with this unit encoded at its C or N terminal end can be engineered (Figure 7A). In parallel, the 12 amino acids peptide can be synthesized and tethered onto the lipid of the nanodiscs. There are a number of well-standardized techniques to tether peptides on lipids, including terminal palmitoylation, (Figure 7B).

[0235] Cell expression systems (mammalian, insect, or bacterial), immobilized-metal affinity chromatography (IMAC) purification, size exclusion chromatography, MST, BLI and luminescence detection methods may be used in connection with the assay.

[0236] Example 9. Complementation-independent ALPHA assay

[0237] In some embodiments, a complementation-independent Amplified Luminescent Proximity Homogenous Assay (ALPHA) assay may be used. This technology is based on channeling of single oxygen species, generated from a photoexcitable moiety “donor”, to a “acceptor” located in close proximity; in order to induce a chemiluminescent signal. The donor consists of a photosensitizer that excites ambient oxygen into single state when irradiated at 680nm. Once excited, the donor generates more than 60,000 oxygen singlet molecules each second, with a short half-life time that allows for a diffusion distance of 200 nm maximum. This diffusion limit permits excitation of acceptors present only in proximity, i.e. on the same HLA-NDs. Thus, background signal is rendered very minimal (Figure 8).

[0238] OTHER EMBODIMENTS

[0239] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0240] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0241] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0242] E1 . A human leukocyte antigen-nanodisc (HLA-ND) complex comprising:

[0243] (a) a human leukocyte antigen (HLA);

[0244] (b) a lipid composition; and (c) a membrane scaffold protein (MSP).

[0245] E2. The HLA-ND complex of E1 , wherein the HLA is a class I HLA.

[0246] E3. The HLA-ND complex of E2, wherein the HLA class I is HLA-A, HLA-B, or HLA-C.

[0247] E4. The HLA-ND complex of E1 , wherein the HLA is a class II HLA.

[0248] E5. The HLA-ND complex of E4, wherein the class II HLA is HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or

[0249] HLA- DO.

[0250] E6. The HLA-ND complex of any one of E1 -E5, wherein the lipid composition forms a bilayer.

[0251] E7. The HLA-ND complex of any one of E1 -E6, wherein the lipid composition comprises a detectable label.

[0252] E8. The HLA-ND complex of E7, wherein the detectable label is rhodamine, fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), 4',6-diamidino-2- phenylindole (DAPI), coumarin, cyanine, xanthene, naphthalene, oxadiazole, anthracene, pyrene, oxazine, acridine, arylmethine, tetrapyrroles, Alexa Fluor compounds, or boron-dipyrromethene (BODIPY), or derivatives thereof.

[0253] E9. The HLA-ND complex of any one of E1 -E8, wherein the MSP comprises the amino acid sequence of NW9 (SEQ ID NO: 1 ) or NW6 (SEQ ID NO: 3).

[0254] E10. The HLA-ND complex of any one of E1 -E9, wherein the HLA-ND is between about 5 nm and about 10 nm.

[0255] E11 . The HLA-ND complex of E10, wherein the HLA-ND is about 8 nm.

[0256] E12. The HLA-ND complex of any one of E1 -E11 , wherein there is at least one HLA per nanodisc.

[0257] E13. The HLA-ND complex of E12, wherein the complex consists of an HLA selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

[0258] E14. The HLA-ND complex of any one of E1 -E13, further comprising a bead (for example, a microsphere bead).

[0259] E15. The HLA-ND complex of any one of E1 -E13, further comprising:

[0260] (i) a first reporter fragment linked to an anti-HLA antibody capture agent; and

[0261] (ii) a second reporter fragment linked to:

[0262] (a) an HLA capture agent;

[0263] (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or

[0264] (c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

[0265] E16. The HLA-ND complex of E15, wherein the anti-HLA antibody capture agent comprises an anti- IgG antibody or antigen-binding fragment thereof.

[0266] E17. The HLA-ND complex of E16, wherein the anti- IgG binding fragment thereof is a Fab, scFv, nanobody, or mini protein.

[0267] E18. The HLA-ND complex of any one of E15-E17, wherein the anti-HLA antibody capture agent comprises the amino acid sequence of PLG binder (SEQ ID NO: 5), S-PG binder (SEQ ID NO: 7), or PG-S binder (SEQ ID NO: 9).

[0268] E19. The HLA-ND complex of any one of E15-E18, wherein the HLA capture agent is an anti-HLA antibody or antigen-binding fragment thereof. E20. The HLA-ND complex of E19, wherein the HLA capture agent comprises the amino acid sequence of 128-1 binder (SEQ ID NO: 11 ) or 128-2 binder (SEQ ID NO: 13).

[0269] E21 . The HLA-ND complex of any one of E15-E20, wherein the reporter is a luminescence-based reporter, a fluorescence-based reporter, or an absorbance-based reporter.

[0270] E22. The HLA-ND complex of E21 , wherein the luminescence-based reporter is luciferase.

[0271] E23. The HLA-ND complex of any one of E1 -E22, further comprising an anti-HLA antibody.

[0272] E24. A HLA-ND complex library, comprising one or more of the HLA-ND complexes of any one of E1 - E23.

[0273] E25. The HLA-ND complex library of E24, comprising between about 106-1012HLA-ND complexes.

[0274] E26. A method of producing an HLA-ND complex, comprising:

[0275] (i) providing an HLA; and

[0276] (ii) incubating the HLA with an MSP and a lipid composition.

[0277] E27. The method of E26, wherein the MSP comprises the amino acid sequence of NW9 or NW6.

[0278] E28. The method of E26 or E27, wherein the HLA-ND is between about 5 nm and about 10 nm.

[0279] E29. The method of E28, wherein the HLA-ND is about 8 nm.

[0280] E30. The method of any one of E26-E29, wherein the method comprises isolating the HLA from the plasma membrane fraction of a cell comprising the HLA.

[0281] E31 . The method of E30, wherein the cell is a monoallelic B cell or a native cell.

[0282] E32. The method of any one of E26-E31 , wherein the method comprises the steps of:

[0283] (i) incubating a mixture comprising the HLA, MSP, lipid composition, and detergent for between about 30 minutes to about 120 minutes; and

[0284] (ii) removing the detergent.

[0285] E33. A method of detecting an anti-HLA antibody, comprising:

[0286] (a) providing a sample from a subject;

[0287] (b) contacting the sample with the HLA-ND complex of any one of E1 -E23; and

[0288] (c) determining the presence of a signal from the reporter, thereby detecting the anti-HLA antibody.

[0289] E34. A method of predicting whether an organ transplant recipient is at risk of hyperacute rejection, the method comprising:

[0290] (a) providing a sample from the organ transplant recipient;

[0291] (b) contacting the sample with an HLA-ND complex comprising an HLA that corresponds to an HLA of the donor of the transplanted organ; and

[0292] (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient is at risk of hyperacute rejection.

[0293] E35. A method of monitoring the development of donor-specific antibodies (DSAs) in a subject who received an organ transplant, the method comprising:

[0294] (a) providing a sample from the organ transplant recipient;

[0295] (b) contacting the sample with an HLA-ND complex comprising an HLA that corresponds to an HLA of the donor of the transplanted organ; and (c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient has developed DSAs.

[0296] E36. The method of any one of E33-E35, wherein the sample is a serum sample or a blood sample.

[0297] E37. A kit comprising: (i) an HLA-ND complex of any one of E1 -E23;

[0298] (ii) a first reporter fragment linked to an anti-HLA antibody capture agent; and

[0299] (iii) a second reporter fragment linked to:

[0300] (a) an HLA capture agent;

[0301] (b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or (c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

[0302] Other embodiments are within the following claims.

Claims

CLAIMS1 . A human leukocyte antigen-nanodisc (HLA-ND) complex comprising:(a) a human leukocyte antigen (HLA);(b) a lipid composition; and(c) a membrane scaffold protein (MSP).

2. The HLA-ND complex of claim 1 , wherein the HLA is a class I HLA.

3. The HLA-ND complex of claim 2, wherein the HLA class I is HLA-A, HLA-B, or HLA-C.

4. The HLA-ND complex of claim 1 , wherein the HLA is a class II HLA.

5. The HLA-ND complex of claim 4, wherein the class II HLA is HLA-DP, HLA-DQ, HLA-DR, HLA-DM, orHLA- DO.

6. The HLA-ND complex of claim 1 , wherein the lipid composition forms a bilayer.

7. The HLA-ND complex of claim 1 , wherein the lipid composition comprises a detectable label.

8. The HLA-ND complex of claim 7, wherein the detectable label is rhodamine, fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), 4',6-diamidino-2-phenylindole (DAPI), coumarin, cyanine, xanthene, naphthalene, oxadiazole, anthracene, pyrene, oxazine, acridine, arylmethine, tetrapyrroles, Alexa Fluor compounds, or boron-dipyrromethene (BODIPY), or derivatives thereof.

9. The HLA-ND complex of claim 1 , wherein the MSP comprises the amino acid sequence of NW9 (SEQ ID NO: 1 ) or NW6 (SEQ ID NO: 3).

10. The HLA-ND complex of claim 1 , wherein the HLA-ND is between about 5 nm and about 10 nm.11 . The HLA-ND complex of claim 10, wherein the HLA-ND is about 8 nm.

12. The HLA-ND complex of claim 1 , wherein there is at least one HLA per nanodisc.

13. The HLA-ND complex of claim 12, wherein the complex consists of an HLA selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

14. The HLA-ND complex of claim 1 , further comprising a bead (for example, a microsphere bead).

15. The HLA-ND complex of claim 1 , further comprising:(i) a first reporter fragment linked to an anti-HLA antibody capture agent; and(ii) a second reporter fragment linked to:(a) an HLA capture agent;(b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or(c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

16. The HLA-ND complex of claim 15, wherein the anti-HLA antibody capture agent comprises an anti- IgG antibody or antigen-binding fragment thereof.

17. The HLA-ND complex of claim 16, wherein the anti- IgG binding fragment thereof is a Fab, scFv, nanobody, or mini protein.

18. The HLA-ND complex of claim 15, wherein the anti-HLA antibody capture agent comprises the amino acid sequence of PLG binder (SEQ ID NO: 5), S-PG binder (SEQ ID NO: 7), or PG-S binder (SEQ ID NO: 9).

19. The HLA-ND complex of claim 15, wherein the HLA capture agent is an anti-HLA antibody or antigenbinding fragment thereof.

20. The HLA-ND complex of claim 19, wherein the HLA capture agent comprises the amino acid sequence of 128-1 binder (SEQ ID NO: 1 1 ) or 128-2 binder (SEQ ID NO: 13).21 . The HLA-ND complex of claim 15, wherein the reporter is a luminescence-based reporter, a fluorescence-based reporter, or an absorbance-based reporter.

22. The HLA-ND complex of claim 21 , wherein the luminescence-based reporter is luciferase.

23. The HLA-ND complex of claim 1 , further comprising an anti-HLA antibody.

24. A HLA-ND complex library, comprising one or more of the HLA-ND complexes of claim 1 .

25. The HLA-ND complex library of claim 24, comprising between about 106-1012HLA-ND complexes.

26. A method of producing an HLA-ND complex, comprising:(i) providing an HLA; and(ii) incubating the HLA with an MSP and a lipid composition.

27. The method of claim 26, wherein the MSP comprises the amino acid sequence of NW9 or NW6.

28. The method of claim 26, wherein the HLA-ND is between about 5 nm and about 10 nm.

29. The method of claim 28, wherein the HLA-ND is about 8 nm.

30. The method of claim 26, wherein the method comprises isolating the HLA from the plasma membrane fraction of a cell comprising the HLA.31 . The method of claim 30, wherein the cell is a monoallelic B cell or a native cell.

32. The method of claim 26, wherein the method comprises the steps of:(i) incubating a mixture comprising the HLA, MSP, lipid composition, and detergent for between about 30 minutes to about 120 minutes; and(ii) removing the detergent.

33. A method of detecting an anti-HLA antibody, comprising:(a) providing a sample from a subject;(b) contacting the sample with the HLA-ND complex of claim 1 ; and(c) determining the presence of a signal from the reporter, thereby detecting the anti-HLA antibody.

34. A method of predicting whether an organ transplant recipient is at risk of hyperacute rejection, the method comprising:(a) providing a sample from the organ transplant recipient;(b) contacting the sample with an HLA-ND complex comprising an HLA that corresponds to an HLA of the donor of the transplanted organ; and(c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient is at risk of hyperacute rejection.

35. A method of monitoring the development of donor-specific antibodies (DSAs) in a subject who received an organ transplant, the method comprising:(a) providing a sample from the organ transplant recipient;(b) contacting the sample with an HLA-ND complex comprising an HLA that corresponds to an HLA of the donor of the transplanted organ; and(c) determining the presence of a signal from the reporter, wherein the presence of a signal indicates that the transplant recipient has developed DSAs.

36. The method of claim 33, wherein the sample is a serum sample or a blood sample.

37. A kit comprising:(i) an HLA-ND complex of claim 1 ;(ii) a first reporter fragment linked to an anti-HLA antibody capture agent; and(iii) a second reporter fragment linked to:(a) an HLA capture agent;(b) the MSP at the N-terminus, C-terminus, or at a cysteine residue; or(c) the lipid head group or lipid tail; and wherein the first and second reporter fragments together form a functional reporter.

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