Detecting antibodies

Abstract

An example composition includes one or more binding agents and one or more solid-phase substrates bound to the one or more binding agents. The one or more binding agents are or include: (i) a peptide including at least part of a full-length protein, where the peptide is for binding to an antibody, or (ii) a molecule that mimics an ability of the peptide to bind to the antibody.

Description

[0001] DETECTING ANTIBODIES

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of, and priority to, the earlier filing date of U.S.

[0004] Provisional Patent Application No. 63 / 664,345, which was filed June 26, 2024, and which is titled “Detecting Antibodies”. U.S. Provisional Patent Application No. 63 / 664,345 is incorporated herein by reference in its entirety.

[0005] TECHNICAL FIELD

[0006] This specification describes example implementations of techniques for detecting antibodies.

[0007] BACKGROUND

[0008] Certain immunoassays use complex proteins. Examples of complex proteins include AT1R and HNA-3.

[0009] AT1R (angiotensin II receptor type 1) antibodies are protein reagents that bind to ATI R antigens. The presence of AT1R antibodies in a patient sample may indicate that the patient has rejected, or is susceptible to rejecting, a solid organ transplant.

[0010] HNA-3 (human neutrophil antigen-3a) is an antigenic determinant on the Choline gTransporter-like Protein 2 (CTL2). The presence of HNA-3 antibodies in a patient sample may indicate that the patient has experienced, or is susceptible to, an immune-mediated transfusion reaction.

[0011] SUMMARY

[0012] An example composition includes one or more binding agents and one or more solidphase substrates bound to the one or more binding agents. The one or more binding agents are or include: (i) a peptide including an amino acid sequence at least 95 % identical to at least part of a full-length protein, where the peptide is for binding to an antibody, or (ii) a molecule that mimics an ability of the peptide to bind to the antibody. The example composition may include one or more of the following features, either alone or in combination. The one or more binding agents may further include a (i) a second peptide comprising an amino acid sequence at least 95% identical to at least part of the full-length protein or (ii) a second molecule that mimics an ability of the peptide to bind to the antibody. The one or more binding agents may further include the full-length protein. In some implementations, the one or more binding agents may include two or more peptides, or about two to about ten peptides, or corresponding mimics.

[0013] The full-length protein may be or include a multi-membrane spanning protein. The full- length protein may be or include AT1R or HNA3. The peptide may be or include a chain having a shorter length than an entirety of the full-length protein. For example, in this context, a peptide that is or includes a chain having a shorter length than an entirety of the full-length protein may be a relatively small peptide of about 10-30 amino acids, a larger peptide of about 31 to 100 amino acids, or a relatively large peptide of about 101 to 300 amino acids. The one or more solid phase substrates may be or include a polystyrene bead or plate.

[0014] In an example composition, the full-length protein includes AT1R and the peptide includes an amino acid sequence at least 95 % identical to: (i) a portion of AT1R selected from a second loop (ECL2) of AT1R, (ii) SEQ ID NO. 1, (iii) SEQ ID NO. 2, or (iv) fragments thereof.

[0015] In an example composition, the full-length protein includes HNA-3 and the peptide includes an amino acid sequence at least 95 % identical to: (i) a portion of HNA3, (ii) a portion of CTLA 2 selected from the loop, (iii) SEQ ID NO. 3, or (v) fragments thereof.

[0016] An example method is for determining a presence of one or more antibodies in a biological sample obtained from a subject. The method includes contacting the biological sample with any of the foregoing compositions and detecting binding of the one or more of binding agents to the one or more antibodies.

[0017] An example kit includes one or more solid-phase substrates and one or more binding agents. The one or more binding agents may be or include (i) a peptide including at least part of a full-length protein, where the peptide is for binding to an antibody, or (ii) a molecule that mimics an ability of the peptide to bind to the antibody.

[0018] An example method for performing a diagnosis on a subject that has undergone a solid organ transplant includes contacting a biological sample obtained from the subject with any of the foregoing compositions, and measuring levels of one of more antibodies in the sample. Increased levels of the one or more antibodies, compared to reference levels, indicate that the subject has developed, or is at risk of developing, a transplant rejection response in response to the solid organ transplant.

[0019] An example method for diagnosing an immune-mediated transfusion reaction response in a subject that has undergone a transfusion includes contacting a biological sample obtained from the subject with any of the foregoing compositions, and measuring levels of the one of more antibodies in the sample. Increased levels of the one or more antibodies, compared to reference levels, indicate that the subject has developed, or is at risk of developing, an immune-mediated transfusion reaction in response to the transfusion.

[0020] An example method for processing or preparing a human biological sample includes obtaining a biological sample from a human subject at risk for solid organ transplant rejection or an immune-mediated transfusion reaction, contacting the biological sample with any of the foregoing compositions, and quantifying a level of an antibody that binds to the one or more binding agents.

[0021] An example method includes causing a protein that is based on a complex protein and that is bound to a substrate to contact a patient sample containing an antibody and detecting the antibody in the patient sample based on whether the antibody binds to the protein. Detection may be performed using fluorescent, chemiluminescent, or colorimetric detection. The protein may be a construct of the complex protein that is different from the complex protein. The method may include causing a full-length version of the complex protein bound to a substrate to contact the patient sample containing an antibody. Detecting the antibody in the patient sample may also be based on whether the antibody binds to the full-length version of the complex protein. The antibody may be for AT1R or HNA-3.

[0022] An example method includes causing a molecule that mimics an epitope of a complex protein to contact a patient sample containing an antibody and detecting the antibody in the patient sample based on whether the antibody binds to the epitope. The method may include causing a full-length version of the complex protein bound to a substrate to contact the patient sample containing the antibody. Detecting the antibody in the patient sample may also be based on whether the antibody binds to the full-length version of the complex protein. The molecule may be or include a small molecule such as aptamers and amino acid derivatives. Detection may be performed using fluorescent, chemiluminescent, or colorimetric detection. Any two or more of the features described in this specification, including in this summary section, may be combined to form implementations not specifically described in this specification.

[0023] The details of one or more implementations are set forth in the accompanying drawings and the following description. Other features and advantages will be apparent from the description and drawings, and from the claims.

[0024] DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 shows an example AT1R sequence and corresponding domains.

[0026] Fig. 2 shows an example CTL2 sequence and corresponding domains.

[0027] Fig. 3a shows an example AT1R construct in which amino acids 166 through 200 have been removed.

[0028] Fig. 3b shows an example AT1R construct in which amino acids 236 through 305 have been removed.

[0029] Fig. 3c shows an example AT1R construct in which amino acids 61 through 130 and 236 through 305 have been removed.

[0030] Fig. 4 is a flowchart showing operations included in an example process for diagnosing a patient based on results of the example assays described herein.

[0031] Fig. 5 is a flowchart showing operations included in an example process for processing or preparing a biological sample from a patient known to be at risk of organ transplant rejection based on results of the example assays described herein.

[0032] Figs. 6a depicts a full peptide construct for the AT1R protein, and Figs. 6b, 6c, and 6d depict examples of shortened peptide constructs for the AT1R protein.

[0033] Fig. 7 is a graph showing the median fluorescence intensity (MFI) for detecting an AT1R antibody using an example assay.

[0034] Fig. 8 is a graph showing the MFI for detecting an AT1R antibody using an example assay that is different from the assay of Fig. 7.

[0035] Like reference numerals in different figures indicate like elements. DETAILED DESCRIPTION

[0036] Described herein are examples of assays in which structurally complex (or simply, “complex”) proteins or portions thereof are attached to solid-phase (or simply “solid”) substrates.

[0037] An example of a complex protein is a multi-membrane spanning protein. In some implementations, complex proteins include proteins that include one, two, or more membranespanning regions. In some implementations, complex proteins may include two or more hydrophobic transmembrane segments that traverse the lipid bilayer of a cellular or organelle membrane. Multi-membrane spanning proteins may include, but are not limited to, G protein- coupled receptors (GPCRs), ion channels, transporters, and membrane-associated enzymes. Complex proteins may contain extracellular, transmembrane, and cytoplasmic regions, and may exhibit conformational changes in response to ligand binding, voltage, or other stimuli. In some implementations, multi-membrane spanning complex proteins may be expressed recombinantly, purified, reconstituted into lipid bilayers, or presented in cell-derived vesicles or membrane fragments for use in assays, therapeutic screening, or diagnostic applications. Examples of complex proteins include, but are not limited to, AT1R and HNA-3.

[0038] Examples of solid substrates may include, but are not limited to, beads, where examples of beads include magnetic or non-magnetic microspheres. Examples of solid substrates may include, but are not limited to, plates, membrane surfaces, slides, the surface a linear flow assay, nano-, micro-, or macro- titer plates, or microfluidic channels. Examples of materials that may be constituents of the solid substrate may include, but are not limited to, polymeric materials such as polystyrene, polypropylene, polyethylene, polycarbonate, polymethyl methacrylate, and polyvinyl chloride, as well as glass, silica, or silicon-based materials. In implementations, the solid substrate may be a polystyrene bead or plate. In implementations, the solid substrate may include surface functionalization, including surface functionalization by chemical groups such as carboxyl, amine, aldehyde, epoxy, or hydroxyl moieties, which may facilitate covalent or non- covalent attachment of the protein or peptide. The solid substrate may be modified with affinity tags, capture agents (e.g., streptavidin for biotinylated molecules), or linker chemistries (e.g., maleimide-thiol, NHS-ester-amine) to enhance immobilization efficiency, orientation, or stability. The choice of solid substrate and binding chemistry may be selected based on assay format, detection method, or desired binding characteristics. Attachment of an entirety of a complex protein to a substrate can, in some cases, be difficult to implement. For example, attachment of a complex protein to a substrate may disrupt the native structure of the complex protein leading to non-binding of some antibodies or, alternatively, to non-specific binding to cryptic epitopes that are not exposed on the complex protein in its native structure. As background, antibodies include specialized proteins produced by an immune system to identify and to neutralize foreign objects such as bacteria and viruses. The binding of antibodies to complex proteins is an interaction facilitated by the unique structure of the antibody's antigen-binding site. This site is tailored to recognize specific epitopes, which are distinct regions on the surface of the complex protein. When an antibody encounters its corresponding antigen on the complex protein, the antibody may bind to the antigen through interactions such as hydrogen bonds, electrostatic forces, and van der Waals forces.

[0039] Small proteins such as peptides and molecules, examples of which are described herein, may be used to address complications associated with binding an entirety of a complex protein to a substrate. A peptide is a sequence of amino acids linked together by peptide bonds. In some implementations, a peptide that is or includes a chain of amino acids having a length that is shorter than the entire chain of amino acids for a full-length complex protein may be a relatively small peptide of about 10 to 30 amino acids, a larger peptide of about 31 to 100 amino acids, or a relatively large peptide of about 101 to 300 amino acids.

[0040] The example techniques described herein include attaching, to substrates in assays, small proteins, such as peptides, or molecules that mimic full-length complex proteins. The small proteins or molecules may perform the same functions as the original full-length complex protein and, consequently, may be used for detection of antibodies to the full-length complex protein.

[0041] A larger protein, for example, a peptide closer to the full-length of the complex protein is more likely to retain the three dimensional (3D) structure of the original complex protein. However, in some cases, attaching a full-length or nearly full-length protein to the substrate may present problems with maintaining the 3D structure. Relatively small peptides may maintain the antibody binding capabilities of the complex protein while balancing the ability to maintain a secondary structure of the peptide when attaching to different surfaces.

[0042] Antibody-mediated rejection (AMR) is characterized by the immune system's production of antibodies that target and attack a transplanted organ. This form of rejection occurs when the recipient's immune system recognizes the donor organ as foreign and mounts an immune response against it, primarily through the action of antibodies. The antibodies bind to antigens on the surface of the transplanted tissue, leading to inflammation and damage. The binding activates the complement system, a series of proteins that enhance the immune response, resulting in further injury to the organ's blood vessels and tissues. AMR in solid organ transplantation is most often associated with antibodies directed against human leukocyte antigens (HLA) present on a donor organ that differ from the recipient of the donor organ. However, AMR can also occur in the absence of HLA antibodies.

[0043] Other targets, such as AT1R, have been correlated with rejection of the donor organ. AT1R is the main receptor for angiotensin II in the glomerulus and controls arterial blood pressure and salt balance. Detecting the presence of AT1R antibodies may indicate that a solid organ transplant has been rejected in a patient or that a patient that is a candidate for a transplant is susceptible to solid organ transplant rejection. The patient may be a human patient. A solid organ may include any solid organ, such as the kidney, heart, liver, lung, or pancreas. In some implementations, the solid organ may be the kidney. In some implementations, the solid organ may be the heart.

[0044] Some studies have indicated that AT1R may be correlated with rejection and kidney graft loss following a kidney transplant. See A. J. Gareua, “Pre-transplant AT1R antibodies correlate with early allograft rejection, 46 Transplant Immunology, 46:29-35 (February 2018). However, some studies have failed to show a relationship between AT1R and graft rejection. See M. Alijishi, et al., “Rejection and graft outcomes in kidney transplant recipients with and without angiotensin II receptor type 1 antibodies,” Transplant Immunology 76, 101756 (February 2023); B.M. Sorohan, “Angiotensin II type 1 Receptor.

[0045] The structure of AT1R includes an extracellular N-terminus, three intracellular loops (ICL1-3), three extracellular loops (ECL1-3), an amphipathic helix VIII and an intracellular C- terminus. See H. Zhang, et al., “Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography,” Cell. 161 (4): 833— 844 (Apr 23, 2015). The full-length AT1R antibody has a length of 359 amino acids. Anti-ATIR antibodies are thought to bind to the second extracellular loop of AT1R. See D. Dragun, “Angiotensin II Type 1 -Receptor Activating Antibodies in Renal-Allograft Rejection,” The New England Journal of Medicine, 362:6 (Feb. 10, 2005). Assays have been created to detect anti-ATIR antibodies using a peptide, which includes the second extracellular loop. However, peptides do not necessarily adopt the tertiary structure of an intact antigen. Other assays use the full-length antigen. However, these assays may detect the antibody by binding to regions outside the second extracellular loop that may not be clinically important. Peptides of different lengths and corresponding to different portions of the full-length protein provide additional information to determine antibody specificity based on the binding to the different peptides. For example, an assay may include at least two peptides or up to ten peptides corresponding to different regions of a particular full-length protein, or mimics thereof, which may be in addition to the full-length protein itself.

[0046] While it has been established that antibodies to AT1R are clinically important, the data on which this assumption is based has been almost exclusively gathered from one commercial assay on the market. The assumption is that all of the antibodies react with either the AFHYESQ or ENTNIT epitopes on the second extracellular loop of AT1R. See L.S. Meyer, et al., “Angiotensin II Type 1 Receptor Autoantibodies in Primary Aldosteronism,” Hormone and Metabolic Research, 52(6):379-385 (May 13, 2020). However, this assumption may be inaccurate. This inaccurate assumption may explain why AT1R in some patients does not appear to be detrimental.

[0047] Furthermore, current AT1R antibody assays are prone to false positive reactivity. Some implementations of the assays described herein may reduce false reactivity and thus increase the clinical importance of AT1R antibodies. Without being bound by theory, it is believed that the small proteins or small molecules that mimic full-length complex proteins described herein can characterize the reactivity of the AT1R antibodies and provide needed insight into why some AT1R antibodies associate with rejection while others do not.

[0048] Similar to organ transplantation, blood transfusions may also lead to complications if the patient recipient has antibodies against antigens present in the donor blood. One such complication is Transplant Related Acute Lung Injury (TRALI), which is a mild to severe immune-mediated reaction. This condition is believed to be triggered by the transfusion of blood products containing antibodies that react with antigens on the recipient's lung tissue, leading to an inflammatory response. This immune-mediated reaction results in increased permeability of the pulmonary capillaries, causing fluid accumulation in the alveoli and impaired gas exchange.

[0049] TRALI has been attributed to antibodies against either HLA or HNA-3, which is an antigenic determinant on Choline Transporter-like Protein 2 (CTL2). HNA-3a / b is determined by a polymorphism at position 154 in the first extracellular loop where the HNA-3a antigen contains an arginine and the HNA-3b antigen contains a glutamine. Detecting the presence of HLA or HNA-3 antibodies may indicate that a patient that has received a blood transfusion has experienced an immune-mediated transfusion reaction or that a patient that is a candidate for a blood transfusion is susceptible to an immune-mediated transfusion reaction.

[0050] Both AT1R, the sequence for which is shown in Fig. 1, and CTL2, the sequence for which is shown in Fig. 2, include complex protein structures having multiple extracellular loops and transmembrane segments. Because of the complexity of these structures, it may be difficult to attach these structures to a solid support, such as those described below, while maintaining the appropriate tertiary structure for binding to antibodies with similar affinity / avidity to what is observed in vivo. In addition to maintaining the appropriate binding, it may be desirable to characterizes the antibody binding domain on the particular proteins.

[0051] The techniques described herein may provide improved characterization of antibody binding domains for complex proteins, which may lead to an improved understanding of why some antibodies are detrimental in the context of solid organ rejection while some antibodies are not detrimental. For AT1R, the literature suggests that the antibody against the second external loop is clinically important but. to the inventors’ knowledge, this has not been demonstrated using the current assays on market. The techniques described herein may allow one to assess the clinical importance of antibodies against portions of the protein outside the second extracellular loop and to assess whether antibodies against multiple epitopes are more relevant than ones against a single epitope.

[0052] An example assay includes a full-length AT1R protein binding to a solid substrate, examples of which are provided above. This first example assay also includes, as the small protein, peptides comprised of part of the AT1R protein (an “AT1R construct” that is not the whole AT1R protein) binding to the solid substrate. In some implementations, the peptides comprised of part of the AT1R protein include an amino acid sequence that is at least 95 % identical to at least part of the full-length AT1R protein. In some implementations, the percentage identity is 75%, 80%, 85%, 90%, 95%, 97.5%, or 99%.

[0053] In this example assay, the peptides that are bound have the 3D structure of the AT1R protein lacking the second extracellular loop of the full-length AT1R. In some implementations, binding full-length AT1R or an AT1R construct to a solid phase substrate can be implemented through adsorption, covalent binding, or through affinity interaction. Adsorption of proteins to a microtiter plate is a standard method for creating ELISA (enzyme-linked immunosorbent assay) assays. Covalent attachment can be performed using standard EDC ( 1 -Ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride) coupling leading to an amide bond between a carboxyl group on the surface and an amine group on the protein or an amine group on the surface and a carboxyl group on the protein. Affinity binding can occur, e.g., by using an antibody against AT1R. Alternatively, the full-length AT1R or the AT1R construct can be biotinylated and attached to a surface containing avidin or streptavidin. For small molecules, linkers such as PEG (polyethylene glycols) may be used so that the small molecules are distantly removed from the solid phase surface for greater accessibility to antibodies. Other linkers that may be used include polyethylene glycols (PEGs), including short ethylene glycol oligomers (e g., diethylene glycol or triethylene glycol), linear or branched alkyl chains, triazole-containing linkers formed via azide-alkyne cycloaddition, adipic acid dihydrazide (ADH), 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), or other small molecules.

[0054] A patient sample may be applied to the full-length AT1R protein bound to the substrate and to the AT1R protein lacking the second extracellular loop bound to the substrate. The presence or absence of an AT1R antibody in either case may be detected using any of the techniques described herein. An antibody in the patient sample binding to the full-length AT1R, but not to the AT1R lacking the second extracellular loop, indicates that the antibody is specific for the second extracellular loop. Thus, the first example assay may be performed to determine which extracellular loop is the epitope for the antibody.

[0055] Examples of AT1R constructs, which are not the whole AT1R protein, that may be used in the assay in place of the AT1R protein lacking the second extracellular loop include, but are not limited to, the following.

[0056] EXAMPLE 1

[0057] In an example AT1R construct, the second and third cytoplasmic domains of the full- length AT1R protein are joined resulting in the removal of the fourth and fifth transmembrane segments along with the second extracellular loop. The joining of the cytoplasmic domains may occur anywhere within the sequences of the respective domains. An example of this type of AT1R construct is shown in Fig 3a. A schematic diagram conceptually depicting this AT1R construct is shows as Fig. 6b described below. A construct according to example 1 (i.e., with the second external loop removed) was cloned onto the 293T cell line (ATlR-no21oop). Additionally, the full length AT1R was cloned into the 293T cell line (AT1R-FL). The targets were prepared by lysing the cells and binding the targets to Luminex® beads. The AT1R WAS constructed such that the proteins contain a biotin at the C-terminus. The beads were coated with streptavidin, which binds strongly to biotin. The beads were incubated with sera, washed, and captured antibody was detected using phycoerythrin-labeled goat anti-human IgG (immunoglobulin G). Data was collected on a Luminex® 200 instrument which provides the median fluorescence intensity (MFI) shown in Fig. 7. The MFI 701 for ATlR-no21oop was 6760.5 and the MFI 702 for AT1R-FL was 4053, which demonstrated that ATlR-no21oop exhibited a higher MFI than the full length AT1R protein.

[0058] EXAMPLE 2

[0059] In another example AT1R construct, the third and fourth cytoplasmic domains of the full- length AT1R protein are joined resulting in the removal of the sixth and seventh transmembrane segments along with the third extracellular loop. The joining of the cytoplasmic domains may occur anywhere within the sequences of the respective domains. An example of this type of AT1R construct is shown in Fig 3b. A schematic diagram conceptually depicting this AT1R construct is shows as Fig. 6c described below.

[0060] EXAMPLE 3

[0061] In another example AT1R construct, the first and second cytoplasmic domains of the full- length AT1R protein are joined and the third and fourth cytoplasmic domains of the full-length AT1R protein are joined resulting in the removal of the second, third, sixth, and seventh transmembrane segments along with the first and third extracellular loops. The joining of the cytoplasmic domains may occur anywhere within the sequences of the respective domains. An example of this type of AT1R construct is shown in Fig 3c. A schematic diagram conceptually depicting this AT1R construct is shows as Fig. 6d described below.

[0062] As shown in Table 1, the example 1 construct is absent the second extracellular loop (ECL2) and has no ECL2 reactivity, but retains the first extracellular loop (ECL1) and N terminus reactivity. The example 2 construct is absent the third extracellular loop (ECL3) and has no ECL3 reactivity, but retains ECL1, N terminus, and ENCL2 reactivity. The example 3 construct is absent ECL1 and ECL3, but retains ECL2 reactivity.

[0063] Table 1

[0064] Referring to Figs. 3a to 3c, if the AT1R antibody is directed against the second extracellular loop, the antibody will not bind to the structure shown in Fig. 3a. If the AT1R antibody does not bind to the structure shown in Fig. 3b, the antibody could bind to the first or second extracellular loop or the terminus. If the AT1R antibody binds to the structure shown in Fig 3c, the antibody is either directed against the second extracellular loop or the terminus.

[0065] The assay is not limited to use with the sequences / structures of Figs. 3a to 3c as other amino acids that are not related to the extracellular domains may be substituted or deleted from the full-length AT1R protein amnio acid sequence, or non-related sequences may be inserted into the full-length AT1R protein amnio acid sequence as long as the binding of antibodies is not affected.

[0066] EXAMPLE 4

[0067] Another example assay binds, to a solid substrate, one or more peptides covering the AFHYESQ and ENTNIT epitopes that are present in the second extracellular loop of the full- length AT1R protein and may contain peptides covering other extracellular regions of the full- length AT1R protein. Furthermore, this example assay may contain additional peptides covering known mutations in the AT1R protein. An example peptide covering amino acids 166 - 200 as shown below is one example of a peptide the covers both epitopes.

[0068] ATlR aal66-200: HRNVFFIENTNITVCAFHYESQNSTLPIGLGLTKN (SEQ ID NO. 1) In some implementations, a shortened peptide may comprise SEQ ID NO. 1, fragments of SEQ ID NO. 1, or an amino acid sequence having a percentage identify to SEQ ID NO. 1, where the percentage identity is 75%, 80%, 85%, 90%, 95%, 97.5%, or 99% identity with SEQ ID NO. 1.

[0069] A peptide corresponding to amino acids 166 - 200 of the second extracellular loop (SEQ ID NO. 1) was synthesized with a biotin at the N-terminus and attached to Luminex® beads that were coated with streptavidin.

[0070] Five microliters (5pL) of beads were incubated with 10 pL (microliters) to 50 pL sera, washed, and a captured antibody was detected using phycoerythrin-labeled goat anti-human IgG. Data was collected on a Luminex® 200 instrument which provides MFI data shown in FIG. 8 and Table 2. The two sera, 510 and 512 are part of the ASHI (American Society for Histocompatibility and Immunogenics) proficiency test samples where reporting on AT1R using the commercial AT1R Elisa is optional. Serum 510 was reported as positive and serum 512 was reported as negative. Sample 1799 is an in-house sample found to be positive for AT1R (also used in Example 1. above). This data demonstrates that the SEQ ID NO. 1 peptide was selective for AT1R. That is, the assay distinguished between the positive and negative samples. Specifically, the assay data showed higher MFI values corresponding to the known positive status for serum 510 and sample 1799 and lower MFI values corresponding to the known negative status for serum 512. In this example, a threshold of about 750 MFI provides a clear differentiation between the positive and negative samples. Additional tests may be performed using known positive or negative samples to confirm or modify such threshold.

[0071]

[0072] Table 2

[0073] EXAMPLE 5 Another example assay binds, to a solid substrate, one or more peptides covering the

[0074] AFHYESQ and ENTNIT epitopes that are present in the second extracellular loop of the full- length AT1R protein and that may contain peptides covering other extracellular regions of the full-length AT1R protein. In some implementations, this example assay may contain additional peptides covering known mutations in the AT1R protein. An example peptide covering amino acids 166 to 192 as shown below is one example of a peptide that covers both epitopes.

[0075] AT1R aal66-192: HRNVFFIENTNITVCAFHYESQNSTLP (SEQ ID NO. 2)

[0076] In some implementations, a shortened peptide may comprise SEQ ID NO. 2, fragments of SEQ ID NO. 2, or an amino acid sequence having a percentage identify to SEQ ID NO. 2, where the percentage identity is 75%, 80%, 85%, 90%, 95%, 97.5%, or 99% identity with SEQ ID NO. 2.

[0077] In some implementations, one or more of the peptides in the preceding paragraph can be further modified with surrounding sequences to aid in attachment to a solid substrate surface, such as the surface of a carboxylated polystyrene bead or others described herein. For example, one or more lysine residues may be included at one terminus of the peptide sequence to target attachment to a bead. Alternatively, the peptide sequence can be made using a biotin attached for binding to a bead coated in streptavidin. Other attachment methods may also be used.

[0078] The protein construct or peptides, which are not full-length complex proteins, are bound to a solid surface, such as a polystyrene plate, polystyrene bead, or others described herein. The resulting immobilized target is incubated with a patient sample such that any antibody in the sample, such as an AT1R antibody, that has affinity to the bound protein construct or peptides will be captured and can detected using the techniques described herein.

[0079] EXAMPLE 6

[0080] Another example assay uses small molecules that mimic the peptide epitope in the AT1R protein. These are referred to as peptidomimetics. Examples of small molecules that may be used include, but are not limited to, aptamers and amino acid derivatives. Aptamers are examples of single-stranded DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) oligonucleotides that fold into structures that can mimic the epitope in the AT1R protein. Amino acid derivatives such as beta-amino acids may also be used as peptidomimetics. In the assay, the protein, peptides, or peptidomimetics are attached to a solid surface, such as a polystyrene plate, polystyrene bead, or others described herein. The resulting immobilized protein, peptides, or peptidomimetics is incubated with a patient sample such that any antibody in the sample that has affinity to the bound target, such as an AT1R antibody, will be captured and detected.

[0081] The foregoing example assays may be applied to a protein other than AT1R, such as CTL2. CTL2, may be of particular interest in blood transfusion because this protein contains an important polymorphism at position 154 in the first extracellular loop where the HNA-3A antigen contains an arginine and the HNA-3b antigen contains a glutamine. See B.R. Curtis, et al., “HNA-3a-specific antibodies recognize choline transporter-like protein-2 peptides containing arginine, but not glutamine at Position 154,” Transfusion 51 :2168-74 (Oct. 2011). The example assays described herein may be used to screen blood units for the presence or absence of antibodies against HNA-3a or HNA-3b.

[0082] Tn a first example assay, any one of the foregoing AT1R assays may be applied to a protein where CTL2 amino acids 257 - 659 (SEQ ID NO. 3) are removed resulting in a protein that only has the one extracellular domain of interest.

[0083] In a second example assay, any one of the foregoing AT1R assays may be applied to constructs for CTL2 that are similar to what was described for AT1R in which one or more extracellular domains are removed.

[0084] In a third example assay, any one of the foregoing AT1R assays may be applied to peptides covering position 154 of CTL2 including five or more amino acids where the peptides may be modified to aid in attachment to a solid surface.

[0085] In a fourth example assay, any one of the foregoing ATI R assays may be applied to any peptidomimetic that is designed specifically to mimic the behavior, with respect to the ability to bind an antibody, of the intact CLT2 protein containing either the HNA-3a or HNA-3b polymorphism. Examples of peptidomimetics include, but are not limited to, beta-substituted amino acid analogs and single-stranded DNA or RNA oligonucleotides.

[0086] In each of the above four example assays applied to CTL2 (which may also be applied to a protein other than AT1R and CTL2), the protein, peptides, or peptidomimetics are attached to a solid surface, such as a polystyrene plate, a polystyrene bead, or others described herein. The resulting immobilized protein, peptides, or peptidomimetics is incubated with a patient sample such that any antibody in the sample that has affinity to the bound protein, peptides, or peptidomimetics, such as an HNA-3 antibody, will be captured and detected.

[0087] In some implementations, a captured antibody can be detected using a labeled antibody that is specific for human IgG or IgM (immunoglobulin MG). The label for detection can be fluorescent, such as phycoerythrin or fluorescein. In some implementations, chemiluminescent or colorimetric detection methods can be used for detection. The example assays described herein may be implemented as a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a non-competitive immunoassay, a homogenous immunoassay, a heterogenous immunoassay, and / or a reporter-assay, e.g., a luciferase assay. In some implementations, the targets (e.g., protein, peptides, or peptidomimetics) may be bound to particles, such as MicroPlex®, MagPlex® or SeroMAP™ carboxylated microspheres provided by DiaSorin S.p.A, and phycoerythrin may be used for fluorescent detection. In some implementations, the targets may be adsorbed to a polystyrene plate and alkaline phosphatase may be used to produce a change in color that may be read using a standard ELISA plate reader.

[0088] The example assays described herein can be implemented as: (1) assays using a radioactive label, e.g.: (a) radioimmunoassay with competitive binding (RIA) and (b) immunoradiometric assay (IRMA); (2) immunoassays using an enzyme label: (a) enzyme immunoassays (EIA) and (b) enzyme-linked immunosorbent assay (ELISA); or (3) immunoassays using a combination of radioisotope and enzyme labels (ultrasensitive enzyme radio immunoassay) (USERIA).

[0089] In some implementations, the example assays described herein may be applied to any protein that has multiple membrane-spanning regions.

[0090] The assays may be performed on various platforms, including multiplex immunoassay platforms, such as those offered under the Luminex® tradename. Flow Cytometry, ELISA manual or automated systems, or any readers capable of measuring the various signals generated from the assays described herein and in the preceding paragraph may also be used.

[0091] The example assays and variants thereof described herein may be used for performing a diagnosis on a subject, such as human patient, that has undergone a solid organ transplant or transfusion. For example, the solid organ transplant may be a kidney, heart, liver, lung, or pancreas transplant. Referring to Fig. 4, an example method may include obtaining (401) a biological sample such as blood or tissue from the patient. In some implementations, the biological sample may be whole blood, plasma, serum, synovial fluid, amniotic fluid, sputum, pleural fluid, peritoneal fluid, central spinal fluid, urine, saliva, tears or other bodily fluid. The biological sample may be obtained using conventional techniques. The biological sample may be subjected to one or more of the assays described herein. For example, the biological sample may be contacted (e.g., mixed) (402) with a composition that includes a solid-phase substrate such as those described herein and a binding agent bound to, for example coated on, the substrate. The binding agent may be or include one or more binding agents such as (i) a peptide including at least part of a full-length complex protein, where the peptide is for binding to an antibody, or (ii) a molecule that mimics an ability of the peptide to bind to the antibody. The peptide may be or include the entire full-length protein and / or a construct, such as those described herein, that is less than the full-length complex protein or that otherwise differs in structure from the full-length complex protein. The method may include detecting (403) a level of one or more antibodies in the patient sample based, for example, on a measure level of fluorescence, chemiluminescence, or absorbance (colorimetric detection) of the substrate. The detected level(s) is / are compared (404) to one or more reference levels for the antibody(ies). If the measured level(s) exceed the reference level(s), then the assay results may indicate that the subject has developed, or is at risk of developing, a transplant rejection in response to the solid organ transplant (e.g., for AT1R antibodies) or an immune-mediated transfusion reaction in response to a transfusion (e.g., for HNA-3 antibodies). The reference level may be selected based on antibody levels in an individual or population that has been shown not be at risk of developing an organ transplant rejection or an immune-mediated transfusion reaction. The results of the assay may be displayed (405) on a display screen.

[0092] The example assays and variants thereof described herein may be used for processing or preparing a biological sample from a patient known to be at risk of organ transplant rejection. Referring to Fig. 5, an example method may include obtaining (501) the biological sample such as blood or tissue from the patient. The biological sample may be obtained using conventional techniques. The biological sample may be subjected to one or more of the assays described herein. For example, the biological sample may be contacted (e.g., mixed) (502) with a composition that includes a solid-phase substrate such as those described above and a binding agent bound to, for example coated on, the substrate. The method may include detecting (503) a level or quantity of one or more antibodies in the patient sample based, for example, on a measure level of fluorescence, chemiluminescence, or absorbance (colorimetric detection) of the substrate. Results showing the level or quantity of the antibodies may be displayed (504) on a display screen.

[0093] A number of alternative constructs for a shortened peptide or peptide mimic may be prepared for a particular full-length peptide. As described above, in Figs. 6a to 6d, three alternative constructs (Figs. 6b to 6d) for the full-length peptide AT1R are depicted. The topology of AT1R includes an extracellular N-terminus, three intracellular loops (ICL1-3), three extracellular loops (ECL 1-3), an amphipathic helix VIII and an intracellular C-terminus. See H. Zhang; Y. Shibata, “Thermostabilization of the Neurotensin Receptor NTS 1,” J. Mol. Biol. 390(2): 262-77 (2009). Referring to Fig. 6a, these features are illustrated in full-length AT1R protein 601, depicted with respect to a lipid layer 610, including a first intracellular loop 602, a second intracellular loop 603, a third intracellular loop 604, a first extracellular loop 605, a second extracellular loop 606, a third extracellular loop 607, an extracellular N-terminus 608 and an intracellular C-terminus 609. Fig. 6b depicts a first shortened peptide 611 in which extracellular loop 606 is eliminated and second intracellular loop 603 and third intracellular loop 604 are connected without an intervening transmembrane crossing, as indicated by line 612. Fig. 6c depicts a second shortened peptide 613 in which the extracellular loop 607 is eliminated and the third intracellular loop 604 is connected to the intracellular C-terminus 609, as indicated by line 614. Fig. 6d depicts a third shortened peptide 615 in which the first extracellular loop 605 and the third extracellular loop 607 are eliminated and the first intracellular loop 602 is connected to the second intracellular loop 603 as indicated by line 618 and the third intracellular loop 604 is connected to the intracellular C-terminus 609, as indicated by line 617.

[0094] Components of the example assays described herein may be included in a kit, which may contain reagent in liquid or dry for detecting the binding of one or more of binding agents, such as proteins, protein constructs, or molecules, to the antibodies. The kit may include the binding agents and substrates. The kit can also include a calibration or standard such as one or more of a target antibody.

[0095] All or some of the assays described in this specification may be performed automatically and may be configured or controlled by executing, on one or more processing devices, instructions that are stored on one or more non-transitory machine-readable storage media. Examples of non-transitory machine-readable storage media include read-only memory, an optical disk drive, memory disk drive, and random-access memory. All or some of the assays described in this specification may be controlled using a computing system comprised of one or more processing devices and memory storing instructions that are executable by the one or more processing devices to perform various control operations. All or some of the assays described in this specification may be performed manually.

[0096] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," and any variations thereof, are intended to cover a nonexclusive inclusion, such that subject matter described or claimed herein that includes, has, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such subject matter described or claimed herein.

[0097] All publications cited herein are incorporated herein by reference in their entirety.

[0098] All examples described herein are non-limiting.

[0099] In the description and claims provided herein, the adjectives “first”, “second”, “third”, and the like do not designate priority or order unless context suggests otherwise. Instead, these adjectives may be used solely to differentiate the nouns that they modify unless context suggests otherwise.

[0100] Elements of different implementations described may be combined to form other implementations not specifically set forth previously. Elements may be left out of the systems described previously without adversely affecting their operation or the operation of the system in general. Furthermore, various separate elements may be combined into one or more individual elements to perform the functions described in this specification.

[0101] Other implementations not specifically described in this specification are also within the scope of the following claims.

Claims

What is claimed is:

1. A composition comprising: one or more binding agents; and one or more solid-phase substrates bound to the one or more binding agents; wherein the one or more binding agents comprise: a peptide comprising an amino acid sequence at least 95 % identical to at least part of a full-length protein, the peptide for binding to an antibody; or a molecule that mimics an ability of the peptide to bind to the antibody.

2. The composition of claim 1, wherein the one or more binding agents further comprise:(i) a second peptide comprising an amino acid sequence at least 95% identical to at least past of the full-length protein or(ii) a second molecule that mimics an ability of the peptide to bind to the antibody.

3. The composition of claim 1, wherein the one or more binding agents further comprise the full-length protein.

4. The composition of claim 1, wherein the full-length protein comprises a multimembrane spanning protein.

5. The composition of claim 1, wherein the full-length protein comprises AT1R.

6. The composition of claim 1, wherein the full-length protein comprises HNA3.

7. The composition of claim 1, wherein the peptide comprises a chain of shorter length than an entirety of the full-length protein.

8. The composition of claim 1, wherein the one or more solid-phase substrates comprises a polystyrene bead or plate.

9. The composition of claim 1, wherein the full-length protein comprises AT1R and the peptide comprises an amino acid sequence at least 95 % identical to: (i) a portion of AT1R selected from a second loop of AT1R, (ii) SEQ ID NO. 1, (iii) SEQ ID NO. 2, (iv) SEQ ID NO. 3, or (v) fragments of (i), (ii), (iii), or (iv).

10. A method for determining a presence of one or more antibodies in a biological sample obtained from a subject, the method comprising: contacting the biological sample with the composition of any of claims 1 to 7; and detecting binding of the one or more of binding agents to the one or more antibodies.

11. A kit comprising: one or more solid-phase substrates; and one or more binding agents comprising: a peptide comprising at least part of a full-length protein, the peptide for binding to an antibody; or a molecule that mimics an ability of the peptide to bind to the antibody.

12. A method for performing a diagnosis on a subject that has undergone a solid organ transplant, the method comprising: contacting a biological sample obtained from the subject with the composition of any of claims 1 to 9; and measuring levels of one of more antibodies in the sample; wherein increased levels of the one or more antibodies, compared to reference levels, indicate that the subject has developed, or is at risk of developing, a transplant rejection response in response to the solid organ transplant.

13. A method for diagnosing an immune-mediated transfusion reaction response in a subject that has undergone a transfusion, the method comprising: contacting a biological sample obtained from the subject with the composition of any of claims 1 to 9; and measuring levels of the one of more antibodies in the sample;wherein increased levels of the one or more antibodies, compared to reference levels, indicate that the subject has developed, or is at risk of developing, an immune-mediated transfusion reaction in response to the transfusion.

14. A method for processing or preparing a human biological sample, the method comprising: obtaining a biological sample from a human subject at risk for solid organ transplant rejection or an immune-mediated transfusion reaction; contacting the biological sample with the composition of any of claims 1 to 9; and quantifying a level of an antibody that binds to the one or more binding agents.

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