Multiplex ABO antibody assays and methods

Abstract

The present application provides a multiplex assay for identifying a subject's ABO histo-blood subtype antibody profile, the multiplex assay comprising incubating a biological sample from the subject with a bead composition containing Luminex® beads individually linked to type I, type II, type III, and type IV ABO-A and ABO-B subtype glycan antigens. Each of the antigen-linked beads contains bovine serum albumin (BSA)-linked ABO subtype antigens bound to the corresponding bead, and the amount of BSA-linked ABO subtype antigen is 1.0 x 10 6 Approximately 5 μg per bead. Compositions and kits for performing multiplex assays are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 425,637, entitled "Multiplex ABO Antibody Assay and Method," filed November 15, 2022, which is incorporated herein by reference in its entirety.

[0002] Technical Field This application relates to the field of ABO blood group antibody detection. More specifically, this application relates to assays and methods for detecting and quantifying anti-ABO antigen subtype antibodies, for example, in the fields of organ and cell transplantation and blood transfusion. This method and assay allows for the simultaneous detection and accurate evaluation of antibodies against multiple ABO antigen subtype structures using a glycan assay. This technology can also be used to detect glycan structures associated with xenotransplantation. [Background technology]

[0003] More than a century has passed since Karl Landsteiner first described the ABO blood grouping (Landsteiner 2001). Although further knowledge of the A, B, and H antigen structures (H antigen defining blood group O) has been gained since then, detection of ABO antibodies in clinical practice and related research still relies on a variation of the hemagglutination method described in 1901. Hemagglutination is known to suffer from a lack of sensitivity and poor reproducibility (Datta et al. 2021; Denomme and Anani 2020; Kang, Lim, and Baik 2014). Distinguishing between IgG and IgM isotypes by agglutination is also tedious, making it impossible to define ABH glycan-subtype specificity. However, ABO hemagglutination titers are routinely used to inform the clinical management of transfusions and ABO-incompatible (ABOI) transplants.

[0004] Knowledge of ABH glycobiology is evolving, including the sequencing of genes encoding glycosyltransferases that decorate human cells and tissues with these carbohydrate structures (Lane 2016; de Mattos 2016; Oriol et al. 1992; Pendu et al. 1989). Six glycan subtypes (I-VI) are known to exist for each A, B, and H antigen, as shown in Table 1.

[0005] The six subtypes of each of the three major ABH antigens are shown in Table 1.

[0006] [Table 1]

[0007] A, B, and H subtype glycans are not equally represented in various cells and tissues. The most biologically relevant antigens are reported to be A-II, III, IV, and B-II (Bentall et al. 2021; Clausen and Hakomori 1989; Jeyakanthan et al. 2016; de Mattos 2016). In ABO-A individuals, assuming the more common A1 subgroup, subtype A-II is the only A antigen found on vascular endothelium, while erythrocytes and (some) epithelial cells are additionally decorated with A-III and A-IV glycans. Tissues from ABO-B individuals appear to possess only B-II glycans, although not all tissues have been well studied; this function is hindered by the limited availability of B subtype-specific monoclonal antibodies (Clausen et al. 1985; Jeyakanthan et al. 2015; Ravn and Dabelsteen 2000).

[0008] ABO A2 is a common subgroup of the ABO-A blood group. Approximately 20% of all ABO-A individuals have ABO-A2, rather than the more common A1. ABO-A2 individuals have only ABO-A-II structures on all their cells and tissues (Svensson et al. 2009). For this reason, ABO-A2 kidney donors are used for transplants to ABO-O and ABO-B recipients, who have disproportionately longer waiting times compared with ABO-A and ABO-AB recipients. This increasing clinical practice for the use of A2 donors requires new technologies that can specifically detect antibodies against the A-II glycan structure and exclude antibodies against the clinically irrelevant A-III and A-IV antigen structures.

[0009] Some glycobiology studies have used A- and B-type trisaccharides as surrogates for A- and B-subtype glycans, but these surrogate glycans are not biologically relevant, so the significance of anti-A-trisaccharide-specific and anti-B-trisaccharide-specific antibodies is unclear (Pochechueva et al. 2011; Stussi et al. 2005).

[0010] Naturally occurring antibodies against non-self ABH glycans present a major immunological barrier in transplantation and transfusion. Because hemagglutination assays do not distinguish between glycan subtype specificities of ABO antibodies, fully characterizing antibody profiles remains challenging, leading to inaccurate risk assessment. Subtype specificity is important for assessing ABO antibodies in ABOi transplantation due to tissue-specific glycan presentation on endothelial and epithelial cells as well as red blood cells (Bentall et al. 2021; Jeyakanthan et al. 2016). To improve the precision and accuracy of ABO antibody detection, an ABH-glycan microarray was previously developed (WO 2013 / 029181). However, this method was not implemented in clinical laboratories due, in part, to a lack of readily available equipment, a lack of expertise in this method in a clinical laboratory environment, and a lack of proper optimization (Bentall et al. 2021; Daga et al. 2021; Jeyakanthan et al. 2015, 2016). Furthermore, achieving standardization within and between laboratories using this previous method has been a challenge (Jeyakanthan et al. 2016; Muthana and Gildersleeve 2016).

[0011] Luminex® methods and technology are now widely used in clinical and laboratory antibody detection assays. Individual polystyrene beads (also known interchangeably as microspheres or microbeads) are conjugated to target antigens, and up to 500 beads can be distinguished from one another by their different color intensities. After incubation of patient serum (or plasma) with the antigen-conjugated beads, phycoerythrin (PE)-labeled secondary antibodies are used to detect serum antibodies bound to each individual bead, and the Luminex® instrument reports the PE fluorescence intensity of each bead. Instrument settings are automatically determined during calibration, making the method highly reproducible within and between clinical laboratories. Indeed, this rapid and more sophisticated antibody detection technology has become the clinical standard in histotyping laboratories worldwide for the detection of antibodies against human leukocyte antigens (HLA) to support solid organ and hematopoietic stem cell transplantation (HSCT) (El-Awar, Lee, and Terasaki 2005; Sullivan, Gebel, and Bray 2017; Tait et al. 2013).

[0012] The existence of Luminex® equipment and expertise in HLA histocompatibility laboratories makes this highly standardized method a natural fit for ABO histocompatibility antibody profiling. However, difficulties with proper loading and reproducible stable binding of ABH-glycans, for example, mean that the use of this method for ABO histocompatibility antibody detection has not been successfully implemented.

[0013] The preceding information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. It is not necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2013 / 029181

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[0015]

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[0016] The present application aims to provide a multiplex ABO antibody assay and method useful for accurate and comprehensive detection and characterization of ABO antibodies. One objective of the present assay system and method is to provide a specific and sensitive means for detecting ABO-related compatibility between donors and recipients, immune risk assessment, and pre- and post-transplant management in clinical settings of transplantation and similar aspects of blood transfusion. The present method and system provides improved accuracy and ABO glycan specificity in pre-transplant assessment of donor and recipient compatibility and in planning intentional ABO-incompatible transplants with maximum safety. The present method and system are further useful for monitoring antibodies in post-transplant patients to rapidly identify early stages of rejection, thus enabling prompt clinical intervention to prevent or minimize damage from antibody-mediated injury. The present method and assay system can also facilitate improved matching between recipient and donor blood products in the event of transfusion. Identification of unexpected transfusion reactions and atypical ABO blood group antibodies will reduce the number of cases in which the wrong blood product is provided to an individual. The method can also be used to screen whole blood donors, which can be used in trauma injury situations and requires the use of ABO-O donors who have been shown to have low levels of anti-A and anti-B antibodies.

[0017] According to one aspect of the present application, there is provided a method for identifying an ABO histo-blood subtype antibody profile of a subject, comprising: (a) incubating a biological sample obtained from a subject with a bead composition, the bead composition comprising: (i) a plurality of subsets of Luminex® beads, each subset of the plurality individually binding to a type I, type II, type III, type IV ABO-A or ABO-B subtype glycan antigen; (ii) a first control subset of Luminex® beads coupled to a negative control antigen (e.g., BSA) to serve as a negative control; (iii) a second control subset of Luminex® beads that are not bound to antigen and serve as an additional negative control; and (iv) a third control subset of Luminex® beads coupled to a positive control antigen (e.g., galactose-α-1,3-galactose) to serve as a positive control incubating the mixture; (b) washing the incubated mixture from step (a); and (c) incubating the washed mixture from step (b) with secondary anti-IgG and anti-IgM antibodies, wherein the secondary antibodies are labeled with fluorescent labels; (d) washing the incubated mixture from step (c) to remove unbound secondary antibody; (e) measuring fluorescence associated with a subset of Luminex® beads; (f) generating an ABO antigen subtype antibody profile of the subject from the fluorescence data collected in step (e); Including, A subset of beads bound to ABO-A and ABO-B subtype glycans each contained bovine serum albumin (BSA)-linked ABO subtype antigens bound to the corresponding beads, with the amount of BSA-linked ABO subtype antigens being 1.0 × 10 6 Approximately 5 μg per bead.

[0018] According to one embodiment there is provided a kit for identifying an ABO histo-blood subtype antibody profile of a subject, comprising: (a) a bead composition comprising: (i) a plurality of subsets of Luminex® beads, each subset of the plurality individually binding to a type I, type II, type III, or type IV ABO-A or ABO-B subtype glycan antigen; (ii) a first control subset of Luminex® beads coupled to a negative control antigen (e.g., BSA) to serve as a negative control; (iii) a second control subset of Luminex® beads that are not bound to antigen and serve as an additional negative control; and (iv) a third control subset of Luminex® beads coupled to a positive control antigen (e.g., galactose-α-1,3-galactose) to serve as a positive control A bead composition comprising: (b) Instructions for use of a kit for carrying out the above method. A kit is provided comprising:

[0019] In some embodiments, the bead composition for use in the above methods or as a component of the kit defined above further comprises two subsets of Luminex® beads individually coupled to BSA-linked type V ABO-A and ABO-B subtype glycan antigens, and / or the bead composition further comprises two subsets of Luminex® beads individually coupled to BSA-linked type VI ABO-A and ABO-B subtype glycan antigens, and / or the bead composition further comprises subsets of Luminex® beads individually coupled to BSA-linked type I, type II, type III, type IV, type V and / or type VI ABO-H glycan antigens.

[0020] In some embodiments, the bead composition for use in the above method or as a component of the kit defined above further comprises one or more subsets of Luminex® beads individually coupled to BSA-linked antigens present in the donor animal for xenotransplantation, where the antigens present in the donor animal are optionally N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or both.

[0021] For a better understanding of the present application and other aspects and further features described herein, reference is made to the following description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0022] [Figure 1] An overview of the ABO antibody detection assay of the present invention and example results from one ABO-A individual are shown. Individual Luminex® beads were conjugated to each ABO-A and ABO-B glycan subtype antigen. Negative control beads included beads conjugated to BSA and beads that did not bind any target. Positive control beads were conjugated to α-Gal glycan. Serum or plasma was added to duplicate wells for the addition of either IgG or IgM secondary antibodies. This example ABO antibody profile was from an individual with an ABO-A1 / O genotype who had a hemagglutination titer of 1 / 32 against ABO-B red blood cells and a negative hemagglutination titer against ABO-A1 red blood cells. [Figure 2] The titration results to select the required glycan-antigen conjugate concentration (μg of BSA-linked antigen per ml of bead suspension containing 1.0×10 6 beads / ml) are shown graphically. [Figure 3] FIG. 10 is an example of graphical confirmation that A and B glycan subtype binding was highly consistent across beads. [Figure 4]A graphical comparison of this multiplex assay on glass slides with a previous ABH-glycan microarray assay was shown, demonstrating that the Luminex® assay was able to detect antibodies with specificity for ABO-A subtype glycans with greater sensitivity than the microarray assay. This increased sensitivity was most clearly observed at lower antibody concentrations. [Figure 5] Illustrated are assay results of 24 individual runs of a positive control serum (these results are presented as mean + / - 95% confidence interval) showing the excellent reproducibility of the multiplex assay described herein. [Figure 6] Figure 6 graphically compares IgG and IgM antibodies by blood type, demonstrating the presence of a wide range of IgG and IgM anti-ABO antibodies in healthy adult individuals across ABO-O, -A, and -B blood types. ABO-O individuals (n = 68) produced higher levels of IgG anti-A than ABO-B individuals (n = 17), and higher levels of IgG anti-B compared to ABO-A individuals (n = 48). In contrast, IgM isotype anti-A and anti-B antibodies did not vary based on ABO blood type. This figure also highlights the differences in anti-A-II, III, and IV IgG antibody levels observed in ABO-O individuals. [Figure 7] A graph shows assay results using sera from ABO-O individuals showing no significant differences in levels of ABO antibodies between females (n=47) and males (n=21) (anti-A shown as the average of A-II, III, and IV subtype specificity, anti-B only B-II subtype specificity). [Figure 8]A comparison of antibodies with specificity for A and B trisaccharides with antibodies specific for cell- and tissue-associated tetrasaccharide glycan subtypes A-II, -III, and -IV and B-II is shown. This comparison shows that ABO-A individuals commonly have high levels of IgG and IgM anti-A-trisaccharide antibodies (i.e., biologically irrelevant antibodies with reactivity against "self" A), along with relatively low antibody levels to A-II, III, and IV subtype glycans. In contrast, ABO-B individuals are less likely to have either anti-B-trisaccharide or anti-B-II antibodies. [Figure 9] The graph shows the results of assays of healthy sera (n=132) that showed no difference between IgG and IgM anti-α-Gal antibody levels, nor did they differ by gender (n=83 women, n=49 men). [Figure 10] The graph shows the results of sera tested in parallel by hemagglutination and analysis using multiplex assays (n=119), demonstrating a wide range of IgG and IgM antibody levels within each hemagglutination titer. [Figure 11] A comparison of IgG and IgM antibody levels against hemagglutination titers in ABO-O individuals only (n=54) is shown graphically and demonstrates the same variability as shown in FIG. [Figure 12] 10A-10C graphically illustrate that using a multiplex assay according to one embodiment of the present application, pan-reactive anti-H antibodies in Bombay phenotype subjects can be readily visualized, further facilitating the definition of antibody isotype (IgG and IgM) patterns in these subjects. [Figure 13] The results of the assay of kidney transplant candidates evaluated for ABO-A incompatible transplants (n=88) are shown graphically, demonstrating the wide distribution of anti-H antibodies among these candidates. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0025] The term "comprising" as used herein is understood to mean that the list below is not exhaustive and may or may not include any other additional suitable items, e.g., one or more further features, components and / or ingredients, as appropriate.

[0026] Throughout this specification, references to "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "additional embodiments," or "further embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] As used herein, the term "ABO antigen subtype" refers to the glycan subtypes of A, B, and H(O) antigens. ABO histo-blood group antigens are glycan structures that are always found as part of a larger glycan structure, and adjacent residues allow the carbohydrate antigen to be classified into subtypes. Each of these ABO antigens exists as one of six subtypes, as shown in Table 1 above. Additional subtypes may be identified in the future. The present assays and methods can be adapted to utilize information regarding the presence, absence, or amount of such as yet unidentified subtypes.

[0028] As used herein, the term "anti-ABO antigen subtype antibody profile" refers to antibodies present in a sample or subject that have specificity for an ABO histoblood group antigen subtype. A basic profile includes information regarding the presence or absence of at least one anti-ABO antigen subtype antibody; a full profile can further include information regarding the absolute or relative amounts of antibodies present in a sample or subject. A profile can further include information regarding the isotypes of anti-ABO antigen subtype antibodies present. In one example, an anti-ABO antigen subtype antibody profile includes information regarding the presence or absence, or absolute or relative amounts, and / or isotypes, of anti-ABO antigen subtype antibodies specific for multiple ABO histoblood group antigen subtypes, such as multiple of the 18 major ABO histoblood group antigen subtypes. In another example, an anti-ABO antigen subtype antibody profile includes information regarding the presence or absence, or absolute or relative amounts, of anti-ABO antigen subtype antibodies specific for the 18 major ABO histoblood group antigen subtypes.

[0029] As used herein, the term "ABO subgroup" is used to refer to subgroups within the broad ABO blood grouping. These subgroups can be characterized by qualitative and quantitative differences in ABO antigen profiles.

[0030] As used herein, the term "biological sample" refers to a blood- or tissue-derived sample containing ABO subtype antigens or anti-ABO antigen subtype antibodies, including, but not limited to, organs, tissues, and red blood cells [ABO subtype antigens], as well as serum, plasma, and blood (i.e., whole blood) and other blood products (e.g., IVIG) and monoclonal antibodies [anti-ABO antigen subtype antibodies]. The biological sample may be a patient sample or a healthy individual considered a control.

[0031] Abbreviations used herein include the following: ABOi ABO incompatibility AHG anti-human globulin BSA Bovine serum albumin α-Gal galactose-α-1,3-galactose DTT Dithiothreitol HLA human leukocyte antigen HSCT hematopoietic stem cell transplantation MFI Mean Fluorescence Intensity PE Phycoerythrin IVIG (intravenous IgG immunoglobulin)

[0032] The present inventors have developed a multiplex assay for characterizing A, B, and H glycan subtype-specific antibodies and defining antibody isotypes in patient or control samples, such as human serum samples or monoclonal antibodies. In some embodiments, the multiplex assay is a bead-based assay in which multiple subsets of beads are used, each subset of beads having a unique associated detection label (e.g., a unique embedded fluorescence). In some aspects, the multiplex assay is a Luminex®-based assay, as exemplified herein as an example of such a bead-based assay.

[0033] This multiplex assay overcomes many of the limitations associated with current clinical hemagglutination assays typically used to assess A and B antibody levels, due to the inability of hemagglutination assays to determine glycan subtype-specific antibodies in patient samples. The method and system are optimized to facilitate reproducible and reliable results from patient samples (or monoclonal antibody preparations) and different testing sites (e.g., clinical or laboratory). This multiplex assay is useful, for example, for ABH histocompatibility assessment for organ and cell transplantation and transfusion medicine, as well as for research in the field of glycoimmunology.

[0034] The methods and multiplex assays described herein provide a means for enabling physicians and other clinicians to accurately assess ABO compatibility or incompatibility and, consequently, safely establish boundaries for transplantation or transfusion. Use of the methods and systems of the present invention can reduce adverse reactions resulting from the unintended or accidental use of incompatible blood products and organs / tissues / cells, and help enable the safe use of intentional incompatibilities, thus saving lives.

[0035] The present method and multiplex assay system address a major limitation of hemagglutination: A, B, and H glycan subtypes are differentially expressed on red blood cells and various tissues. This has important implications for both transplant and transfusion compatibility. In one embodiment, the present method involves the use of a device having multiple ABO histo-blood group subtype antigens bound to a carrier, such as a glycan microarray or macroarray. The device is then used to qualitatively or quantitatively measure antibodies to ABO antigen subtypes. This method is useful, for example, because it can enable the degree of transplant and transfusion compatibility to be assessed more accurately than currently available methods.

[0036] According to one aspect, there is provided an immunological method for determining the anti-ABO antigen subtype antibody profile of a subject, the method comprising determining the presence or absence of antibodies to ABH antigen subtypes in a biological sample obtained from the subject. Typically, the biological sample is serum or plasma (but may also be from a blood product or monoclonal antibody preparation).

[0037] The method includes obtaining a biological sample from a subject with A, B, and / or H glycan subtype antibodies, incubating the sample with a plurality of multiplexed A, B, and H antigen subtypes, and detecting binding complexes formed by binding of the plurality of multiplexed A, B, and H antigen subtypes with the subtype antibodies present in the sample. The bound antibodies are then detected in a manner that allows the identity of each anti-A, B, or H subtype-specific antibody to be determined. The level of each subtype antibody is also determined by the level of fluorescent intensity output, for example, using a Luminex® instrument.

[0038] A multiplex assay uses a composition comprising a combination of subsets of beads, each subset coupled to a different A, B, or H subtype antigen, thereby allowing for the simultaneous detection and measurement of A, B, or H subtype-specific antibodies in a sample. In certain embodiments, the assay utilizes Luminex® beads available from Luminex.

[0039] Figure 1 provides a broad overview of the present application's ABO antibody detection multiplex assay and examples of results obtained from an ABO-A individual. Briefly, in this example, the assay uses individual A and B subtype glycans bound to beads or individual subsets of beads, and although not shown in this example, H glycans may also be included. The method involves incubating the glycan-bound beads with a biological sample (such as a plasma or serum sample from a patient), washing the mixture to remove unbound antibodies, and then detecting the presence of antibodies in the sample bound to the glycans on the beads. The bound antibodies are detected using a secondary detection antibody bearing a fluorescent label (e.g., phycoerythrin). The mean fluorescence intensity (MFI) of each subset of beads is detected as a measure of the ABO antibodies present in the biological sample, for example, using a Luminex® instrument.

[0040] In some embodiments, the secondary antibody may be an anti-human antibody (i.e., one that recognizes and binds to a human antibody) or may be an antibody with specificity for any human antibody of a specific isotype, which may be, but is not limited to, an anti-human IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgD and / or IgA antibody, or any combination of these antibodies.

[0041] In certain embodiments, the method uses both an IgG-specific secondary antibody and an IgM-specific secondary antibody for detection. Optionally, the method further uses an IgA-specific secondary antibody for detection.

[0042] We have found that IgG and IgM ABO-A and -B antibodies vary significantly within each ABO-A and -B titer as detected by hemagglutination. The relative amounts of these antibodies within the detected antibody profile provide additional information useful for characterizing compatibility or incompatibility between donor and recipient (for transplantation or transfusion).

[0043] In determining a subject's anti-A, B, or H subtype-specific antibody profile, it is advantageous to test for the presence or absence of antibodies to most or all of the ABO antigen subtypes. According to one embodiment, a composition comprising a combination of bead subsets includes a subset of beads individually coupled to type I, type II, type III, or type IV ABO-A or ABO-B subtype glycan antigens, respectively. Antibodies to type V subtype antigens have a degree of cross-reactivity with type III and type IV subtype antigens, and therefore do not necessarily include beads coupled to type V subtype antigens. Similarly, antibodies to type VI subtype antigens have a degree of cross-reactivity with type II subtype antigens, and therefore do not necessarily include beads coupled to type VI subtype antigens. However, in some embodiments, a composition comprising a combination of bead subsets includes a subset of beads individually coupled to type I, type II, type III, type IV, type V, and type VI ABO-A or ABO-B subtype glycan antigens, respectively. The inclusion of subtypes I-VI may enable the definition of clinically relevant antibody profiles.

[0044] In some embodiments, the antigen-bound bead composition further comprises one or more bead subsets, each bound to an ABO-H type I, type II, type III, type IV, type V, or type VI subtype glycan antigen. Use of such compositions in the multiplex assays described herein can be useful for characterizing a subject's A, B, and H antibody profile. This can be particularly beneficial, for example, for identifying subjects with the Bombay phenotype, who lack a normal fucosyltransferase 1 (FUT1) gene. Detecting Bombay phenotype individuals is important to avoid complications during transplantation or transfusion. However, standard tests for the ABO blood group system (e.g., hemagglutination) erroneously identify these individuals as having type O blood. Anti-H immunoglobulins can activate the complement cascade, resulting in lysis of red blood cells while they are still circulating, causing an acute hemolytic reaction. This can be avoided by using the compositions and assays described herein to classify subjects and identify the Bombay phenotype.

[0045] In alternative embodiments, the multiplex assays described herein can be adapted for use in risk assessment of recipients for xenotransplantation. In this embodiment, the composition of antigen-coupled beads comprises subsets of beads individually coupled to ABO-H and / or xenoglycan antigens expressed on the donor animal / organ. In some embodiments, the composition comprises a subset of beads coupled to non-human sialic acids. In some non-limiting embodiments, the composition of antigen-coupled beads comprises a subset of beads coupled to N-glycolylneuraminic acid (Neu5Gc) and a subset of beads coupled to N-acetylneuraminic acid (Neu5Ac). Neu5Gc and Neu5Ac are sialic acid molecules synthesized by the gene cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH).

[0046] Each antigen-coupled bead composition further comprises a control subset of beads coupled to a positive control antigen and another control subset of beads coupled to a negative control antigen.

[0047] In certain embodiments, the positive control antigen is the galactose-α-1,3-galactose (α-Gal) epitope (α-Gal-(1→3)-β-Gal-(1→(3)4)-GlcNAc-R), which is found on glycolipids and glycoproteins of non-primate mammals and New World monkeys. All humans are known to produce antibodies specific to α-Gal. By applying the optimized binding technology of the present invention to this antigen on a subset of beads, it is possible to confirm that the assay is functioning properly by noting the presence of this positive control signal, which indicates the formation of a binding complex on a designated bead address within the panel. Furthermore, incorporation of this antigen can also be useful to provide a surrogate for normal levels of antibody in a sample.

[0048] Negative control beads are essential for this assay to ensure that nonspecific reactivity to the platform itself does not affect the assay output. In this assay, these beads are designed based on known potential targets of assay nonspecificity, as was done in the histocompatibility assay. In one example, the negative control antigen is bovine serum albumin (BSA). BSA is also used to prepare glycan-protein conjugates for binding glycan antigens to beads. Therefore, the use of BSA as a negative control is useful to confirm that the antibodies in the sample are binding to the glycan antigen and not the protein used in the conjugate. Secondary negative control beads are not bound to any antigen and are used to assess nonspecific reactivity to the bead surface. These controls are essential for clinical testing and are required to meet the clinical laboratory accreditation standards defined by accreditation organizations.

[0049] Binding of glycan antigens to beads As mentioned above, the A, B, and H antigens are glycans. The synthesis of glycans using the prerequisite alkene aglycone has been previously reported (Jeyakanthan et al. 2015, 2016; Meloncelli and Lowary 2009, 2010; Meloncelli, West, and Lowary 2011). The glycan antigens are then attached to bead glycan-protein conjugates. To prepare the glycan-protein conjugates, glycan subtype antigens were synthesized according to the previously described synthesis method, and BSA was linked to the amines in the glycan antigen structure.

[0050] According to some embodiments, a bead composition is provided comprising multiple subsets of Luminex® beads, each of the multiple subsets individually binding to ABO-A or ABO-B subtype glycan antigens of type I, type II, type III, type IV, type V, and / or type VI.

[0051] In a particular example, the composition comprises the following BSA-linked type I, type II, type III, and type IV ABO-A or ABO-B subtype glycan antigens bound to respective subsets of Luminex® beads. A type Iα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, A type II α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, A type IIIα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, A type IVα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, B type Iα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, B type IIα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, B type IIIα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, and B type IVα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA.

[0052] In other embodiments, the bead composition comprises a subset of Luminex® beads bound to BSA-linked type V ABO-A and ABO-B subtype glycan antigens. In some examples, the BSA-linked type V ABO-A and ABO-B subtype glycan antigens are A type Vα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA and B type Vα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA.

[0053] In other embodiments, the bead composition comprises a subset of Luminex® beads bound to BSA-linked type VI ABO-A and ABO-B subtype glycan antigens (with or without beads bound to BSA-linked type VI ABO-A and ABO-B subtype glycan antigens). In some examples, the BSA-linked type VI ABO-A and ABO-B subtype glycan antigens are: A type VIα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA, and B type VIα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA.

[0054] In other embodiments, the bead composition comprises a subset of Luminex® beads individually coupled to BSA-linked Type I, Type II, Type III, Type IV, Type V, and Type VI ABO-H subtype glycan antigens. In some examples, the BSA-linked Type I, Type II, Type III, Type IV, Type V, and Type VI ABO-H subtype glycan antigens are as follows:

[0055] Additional bead(s) are coupled to galactose-α-1,3-galactose (α-Gal) and BSA as positive and negative control beads, respectively. Blood group A- and B-trisaccharides can also be coupled to individual beads. For example, type VI ABO-A and ABO-B subtype glycan antigens are: H type Iα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, H type IIα-L-Fucp-(1→2)-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, H type IIIα-L-Fucp-(1→2)-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, H type IVα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, H type Vα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-Galp-BSA, and H type VIα-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA.

[0056] We have found that to provide reproducible results using Luminex® beads, the loading ratio of glycan antigens on the beads must be adjusted significantly higher than the standard loading ratio. In particular, using titration studies, we have found that a loading ratio of 1.0 × 10 6 We found that approximately 5 μg of BSA-linked ABO subtype antigen was required per bead, approximately five times the amount recommended by the manufacturer. Higher loading values ​​are typically avoided to minimize nonspecific binding during the assay. However, without wishing to be bound by theory, this specific loading concentration may be required due to the unique structure and properties of glycan-protein conjugates. Because the protein-to-bead ratio differs from strict protein binding protocols, a specific antigen-to-bead ratio is essential. Insufficient levels of antigen on each bead will result in overall reduced assay sensitivity. Accurate transplant risk assessment requires high sensitivity of the antibody detection platform as well as consistent levels of antigen from bead to bead. The consistency of antigen binding is shown in Figure 3.

[0057] Use of multiplex assays in ABO matching for transplantation Improved matching in transplants is achieved using the currently described methods and multiplex assays by measuring potential transplant recipients' antibodies against tissue-specific ABO histo-blood group glycan subtype antigens. This antibody profile information, combined with knowledge of antigen subtype expression in the donor's organ or tissue, is used to determine the degree of compatibility for such transplants. This multiplex assay not only measures antibodies against all 18 ABO antigen subtypes, as well as assay control targets, but also allows for the identification of different antibody isotypes in a quantitative and reproducible manner.

[0058] Different cells and tissues express different A and B antigen subtypes with varying distribution. While A, B, and H antigen subtypes are known to be expressed on various tissue surfaces, clinicians have only recently recognized that this subtype expression can vary between different tissues in the same individual. Simply considering a donor's ABO blood type and assessing anti-A and anti-B antibody levels using (red blood cell) hemagglutination, as currently practiced when assessing donor organ or tissue compatibility, completely ignores the known variance in A and B glycan subtypes expressed in different donor organs and tissues. The lack of glycan subtype-specific antibody information does not allow for accurate transplant immune risk assessment and may unnecessarily exclude individuals from access to ABO-incompatible transplants, thus contributing to disparities in transplant access.

[0059] In one embodiment of the present application, potential recipients can be evaluated for suitability for ABO-incompatible organ or tissue transplantation using this multiplex assay. The relevant anti-ABO glycan subtype-specific antibody profile will be determined. Patients can be considered for ABOI transplantation in relation to the relevant tissue / organ-specific antigen subtypes. In situations where low antibody levels against the relevant ABO antigen subtypes are present on the donor organ, the transplant may be considered a low immunological risk. This ability to determine subtype-specific antibodies is particularly important when the detection of antibodies in a hemagglutination assay that are actually unrelated to the organ / tissue being transplanted can negate eligibility for an intended ABO-incompatible transplant.

[0060] After transplantation, the patient could be further monitored for antibodies against donor A or B glycan subtypes present on the donor organ using the multiplex assay of the present application. Any development of antibodies or an increase in antibody levels could be detected, and appropriate clinical measures could be enacted. Confirmation of the absence or low level of antibodies against tissue target antigens would similarly help to avoid unnecessary therapeutic interventions that may be caused by the detection of (irrelevant) antibodies in hemagglutination assays.

[0061] In another embodiment, potential recipients can be evaluated for compatibility with ABO-compatible transplants using this multiplex assay. In this case, patient serum antibodies are assessed to identify unexpected serological reactions potentially due to less common patient ABO subgroups. This evaluation would be accomplished using this multiplex assay to identify and characterize any antibodies present against all or a subset of the 18 ABO antigen subtypes. It is only important that low levels of antibodies against ABO antigen subtypes are present on the donor organ. In ABO-compatible transplants, this analysis is not as critical as in intentional ABO-incompatible transplants. However, in rare cases, unexpected antibody-mediated reactions occur and are thought to contribute to chronic graft damage. This method allows many of these reactions to be identified in advance, minimizing the risk. Ideally, serum from the organ donor should also be evaluated for antibody levels against ABO antigen subtypes. This approach would help identify unexpected ABO incompatibilities before transplantation, acting as a fail-safe.

[0062] In another embodiment, potential recipients can be evaluated for compatibility with xenotransplantation of organs from non-human donors. In this case, the patient's serum antibodies are evaluated to identify potential reactivity to the donor organ due to the presence of antibodies against one or more xenoantigens found in the donor organ and / or expressed by the donor animal. This evaluation will be accomplished using the present multiplex assay to identify and characterize any antibodies present against one or more xenoantigens. Again, absent or low levels of antibodies against one or more xenoantigens are important.

[0063] Use of multiplex assays for ABO matching in blood transfusion Improved matching in transfusions can be achieved by measuring potential recipients' serum antibodies against ABO histoblood group glycan subtypes present in donor products prior to transfusion using the currently provided multiplex assay. This information can then be used to aid in the accurate identification and pathogenesis of unexpected transfusion reactions and to assist in identifying the most compatible blood product. One potential application is the detection of A and B antibodies in the context of ABO-incompatible platelet transfusions, a common clinical practice. In other cases, discrepancies are caused by rare ABO subgroups. Therefore, the ability to quantify anti-A and anti-B antibodies at the subtype and isotype level would be valuable to clinical laboratory technicians and physicians, facilitating the selection of compatible blood products. As new A, B, and H glycan subtypes are identified, this multiplex assay may be easily adaptable to account for newly discovered blood group antigen variants. Specifically, the bead composition can be adapted and expanded to incorporate additional antigen-binding beads whose antigens represent newly discovered variants.

[0064] In order to gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only, and therefore should not be construed as limiting the scope of the invention in any way. [Example]

[0065] Example 1: ABO subtype antigen binding to Luminex® beads This example summarizes the specific procedure for ABO subtype antigen binding to Luminex® beads and the optimization of the procedural conditions. The basic procedure is adapted from the August 2016 Rev E guidelines of the Luminex User Manual for the xMAP® Antibody Coupling Kit.

[0066] Methods and Results To couple the BSA-linked ABO subtype antigens, the coupling process was carried out using the xMAP® 40-50016 Antibody Coupling Kit and Luminex® MagPlex® beads (e.g., MagPlex®-C beads, region 012).

[0067] The ABO subtype antigens were A and B tetrasaccharide antigens (A-VI) synthesized using methods previously developed in the Lowary laboratory at the University of Alberta (Meloncelli PJ et al. 2010, Meloncelli PJ et al. 2011, and Meloncelli PJ et al. 2009) and linked to BSA. A detailed overview of the glycans used can be found in Jeyakanthan et al. 2016. All antigens were reconstituted to 2 mg / ml in 14 Ω water and stored at -80°C until thawed for use.

[0068] [Table 2]

[0069] Joining Procedure Luminex MagPlex® beads were conjugated to individual ABO-A and ABO-B subtype glycans prepared as previously described (Jeyakanthan et al. 2015, 2016; Meloncelli and Lowary 2009, 2010; Meloncelli et al. 2011).

[0070] Briefly, A and B glycan subtype I-VI tetrasaccharide antigens were synthesized, and bovine serum albumin (BSA) was linked to the amines in these carbohydrate structures. The conjugation procedure was performed according to the protein conjugation protocol recommended by the Luminex bead manufacturer (Angeloni et al. 2016). The antigen-to-bead ratio was optimized for this glycan-protein antigen target using titration studies. The xMAP Antibody Conjugation (AbC) Kit® was used for further standardization of the conjugation procedure, as detailed below.

[0071] ABO-A and ABO-B subtype glycan binding to beads The required amount of Luminex® beads was resuspended and washed using the activation buffer from the binding kit. The beads were incubated with Sulfo-NHS (N-hydroxysulfosuccinimide) reagent and freshly prepared EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) reagent in the dark at room temperature for 20 minutes. The activated beads were washed with the activation buffer to remove excess sulfo-NHS and EDC. 1.0 × 10 BSA-linked ABO subtype antigens were added to the washed beads in the activation buffer. 6 5 μg was added per bead and incubated for approximately 2 hours in the dark at room temperature with mixing on a tube rotator. The ABO subtype antigen-bound beads were washed using Wash buffer from the Coupling Kit and then counted.

[0072] The optimal antigen binding concentration was determined by titration using total amounts of antigen of 1 μg, 5 μg, and 50 μg and relevant monoclonal antibody concentrations up to 16 μg / ml. As shown in Figure 2, the optimal concentration of BSA-conjugated glycan for bead binding was approximately 5 μg / ml (1.0 × 10 6 It was determined that the total volume of the spheroid contained 10 ...

[0073] Additional beads were coupled to galactose-α-1,3-galactose (α-Gal) and BSA (Dextra, UK) as positive and negative control beads, respectively, using the same procedure as above. Blood group A- and B-trisaccharides were also coupled to individual beads (Dextra, UK).

[0074] Confirmation of bead-antigen binding Antigen binding to the beads was confirmed using a panel of monoclonal antibodies with either pan-reactivity to ABO-A and ABO-B glycan structures or specificity for individual ABO-A and -B glycan subtype structures. The amount of antigen bound to the beads was consistently comparable between beads, as shown in Figure 3. Further details, including the specific monoclonal antibodies used, are provided in Appendix A, the conjugation and binding confirmation standard operating protocol (SOP).

[0075] Microarray antibody analysis The monoclonal antibodies used in the bead-binding confirmation protocol were also run in parallel in an ABH-glycan microarray assay as previously described (Jeyakanthan et al. 2015, 2016). The antigens bound to the glass array slides were identical to those bound to Luminex beads. The Luminex assay detected low concentrations of antibody with greater sensitivity compared to the glycan array (Figure 4).

[0076] Application of the assay using healthy control serum Antigen-conjugated beads were used to characterize anti-A and anti-B antibody (and related antibody) profiles in serum or plasma samples from healthy adult individuals (n=143; ABO-O: n=68; ABO-A: n=48; ABO-B: n=17; ABO-AB: n=10) as described in the Luminex ABO Antibody Detection SOP (Appendix B). Briefly, 50 μL of diluted serum or plasma was incubated with pooled single-antigen beads and then incubated at room temperature with gentle agitation. The beads were washed three times before the addition of either an anti-human IgG or IgM secondary antibody. The secondary antibody concentration was also optimized for this method. The plates were again incubated at room temperature with gentle agitation and washed three more times. The beads were resuspended in 80 μL of buffer and acquired on a Luminex® 200 instrument or a FlexMap 3D® Luminex instrument. When acquired on FlexMap 3D®, mean fluorescence intensity (MFI) results were divided by 1.67 to achieve MFI comparability between instruments. Each run included a positive and negative control serum.

[0077] The results of the assay were highly reproducible as shown in Figure 5, and substantiated the results of the positive control sample tested across 24 individual runs.

[0078] A comparison of IgG and IgM antibodies by blood type is shown in Figure 6. Here, and throughout the results, antibody analysis focused on antibodies specific for the A-II, -III, and -IV and B-II glycan subtypes because these subtypes have been reported to be the most biologically relevant glycan targets (Bentall et al. 2021; Clausen and Hakomori 1989; Jeyakanthan et al. 2016). The levels of antibodies with specificity for these A and B subtypes were highly variable between individuals. Furthermore, sera from ABO-O individuals contained significantly higher levels of IgG isotype antibodies against A-II, -III, -IV, and B-II glycans than sera from ABO-B and ABO-A individuals, respectively, whereas there were no differences in the levels of IgM isotype antibodies between sera from individuals with different ABO blood types. No significant differences associated with gender were observed (Figure 7).

[0079] Antibodies against the A and B trisaccharide targets were compared with antibodies with specificity for cell- and tissue-associated tetrasaccharide glycan subtypes A-II, -III, and -IV and B-II. While more than half of ABO-A individuals had detectable IgG and IgM antibodies to the A-trisaccharide, less than 10% of ABO-A blood group healthy controls showed reactivity to A-tetrasaccharide-targeted beads (Figure 8). This same finding was not observed for B-trisaccharide glycans versus B-II subtype glycans. This observation confirmed that the trisaccharide target is not an acceptable surrogate for tetrasaccharide glycan subtype antigens.

[0080] Similar levels of α-Gal antibodies of the IgG versus IgM isotype were detected, and this observation remained true when α-Gal antibodies were analyzed by gender, as shown in Figure 9 .

[0081] Hemagglutination ABO antibody titer test Traditional ABO titer testing was performed on individuals who were also tested by the Luminex ABO antibody assay. Sufficient samples for agglutination testing were available for 119 of 143 healthy controls (ABO-O n=54, ABO-A n=43, ABO-B n=16, ABO-AB n=6). Serially diluted serum / plasma (50 μL) was incubated with 25 μL of 1% ABO-A1 and ABO-B reagent red blood cells (Refercell®, Immucor) in a 96-well tray at room temperature. The plate was mixed and incubated for 1 hour. Agglutination was read with an ELISPOT™ reader (CTL). The agglutination titer was reported as the last dilution showing visual agglutination. These agglutination scores were compared to the levels of tetrasaccharide-specific antibodies detected by the Luminex assay.

[0082] Within each anti-A and anti-B ABO hemagglutinin titer, there was a high degree of variability in the IgG and IgM antibodies detected by the Luminex assay. As shown in the IgM-only data in Figure 10, although there was an overall increase in IgM antibody levels at each titer, there was overlap in the levels of IgG and IgM antibodies at each titer. In two cases, anti-A and anti-B tetrasaccharide antibodies were detected when negative antibody titers were reported, and these results were confirmed repeatedly. The figure shows this same comparison only for ABO-O individuals. Thus, not only is it impossible to determine the relevant subtype specificity in the hemagglutinin assay, but each titer demonstrates high variability in antibody levels as well as overlap in antibody levels across many titer results.

[0083] Application of the assay to subjects with the Bombay phenotype Using H antigen-conjugated beads, this Luminex®-based assay was used to characterize the profile of anti-H antibodies (IgG and IgM) in serum or plasma samples from subjects with the Bombay phenotype. The results are shown in Figure 12.

[0084] Application of the assay to kidney transplant candidates Using H antigen-conjugated beads, this Luminex®-based assay was used to characterize the anti-H antibody profile in kidney transplant candidates in serum or plasma samples from kidney transplant candidates being evaluated for ABO-A incompatible transplants. These results demonstrate the value of this assay in evaluating xenotransplant recipients, as xenotransplant panels using this technology can include glycan targets for A, B, and H, as well as other antigens associated with xenotransplantation, such as Neu5Gc and Neu5Ac, and positive and negative control beads bearing α-Gal. Additional glycans associated with xenotransplantation can be easily incorporated into this assay by conjugating the glycan antigens to a separate subset (or subsets) of beads for inclusion in the assay composition.

[0085] Antibody analysis statistical methods All data were tested for normality using the Shapiro-Wilk, Anderson-Darling, and Kolmogorov-Smirnov tests. If results were not normally distributed, nonparametric analyses, such as the Mann-Whitney and Wilcoxon tests, were used for paired and unpaired data, respectively. The only normally distributed data was the positive control comparison shown in Figure 5. GraphPad Prism 9.3.1 was used for analysis and data graphing.

[0086] Consideration This example describes the use of an embodiment of the multiplex assay of the present application to measure serum antibodies with specificity for ABO-A and -B glycans and demonstrate its reproducibility and utility in the study of samples from healthy adults. The need for better methods for detecting ABO antibodies has been widely reported (Denomme and Anani 2020). In the field of histocompatibility, similar tools have been used for HLA antibody detection for nearly 20 years. However, until the development of this multiplex assay, attempts to develop a multiplex ABO evaluation / detection assay were unsuccessful.

[0087] Clinical laboratories currently lack reproducible assays for the detection and accurate characterization of glycan subtype-specific IgG and IgM antibodies to support ABOI transplantation and transfusion. It is widely acknowledged that hemagglutination assays are poorly standardized, making interlaboratory comparison of titer data difficult (Bentall et al. 2016; Daga et al. 2021; Denomme and Anani 2020; JP, J, and LJ 2008; Kang et al. 2014). As demonstrated herein, this Luminex®-based assay is highly reproducible and enables accurate characterization of the isotype and subtype specificity of anti-A and anti-B antibodies, thus overcoming these barriers in clinical ABO antibody evaluation.

[0088] The ease of isotype differentiation of ABO antibodies also opens up the possibility of understanding the risk of IgG versus IgM ABO antibodies in clinical ABO-incompatible (ABOI) organ and cell transplantation. Some studies have used dithiothreitol (DTT) and the addition of antihuman globulin (AHG) to distinguish IgG from IgM ABO antibodies, but this practice is inconsistent. DTT is known to dissociate IgM pentamers but is not specific for IgM antibodies alone. AHG titration methods are also inconsistent, with some laboratories including a DTT treatment and others not performing this treatment prior to ABO antibody titration testing (Kahlyar et al. 2022).

[0089] It is widely recognized that there is a discrepancy in organ transplant waiting times based on a patient's ABO blood type. Patients with blood types O and B wait longer for transplants from ABO-compatible donors compared with patients with blood types A and AB, creating inequities in access to solid organ transplants. There is also a growing need for transplant registries to consider ABOI transplants due to the increasing number of patients with elevated levels of HLA antibodies and insufficient access to HLA-matched organs (Hussey, Parameshwar, and Banner 2007). The success of ABOI transplants suggests that unfavorable blood types ABO-O and ABO-B, as well as HLA-sensitive patients, would benefit from access to ABOI donors (Fan et al. 2004; de Weerd and Betjes 2018; West et al. 2001). However, well-recognized limitations of hemagglutination titer testing present a barrier to consistent and reproducible ABO antibody assessment within and across programs, and therefore accurate risk assessment, and use in national transplant registries.

[0090] Increased knowledge of subtype-specific antibodies in the context of ABOI transplantation will facilitate advances in transplant clinical practice and other areas of research. Because antibodies directed against endothelial versus epithelial cells can be readily characterized, improved histopathological examination of ABOI grafts can be performed. The pathology of transplant rejection is constantly evolving, and this additional knowledge can be applied to retrospective and prospective studies (Bruneval et al. 2017; Haas et al. 2017; Kobashigawa et al. 2018). Immune memory has been identified as a critical gap in transplant risk assessment but can be difficult to study (Tambur et al. 2018). This Luminex®-based assay can be easily incorporated into an immune memory assay for simultaneous assessment of HLA and ABO B cell memory (Karahan et al. 2018).

[0091] While the field of ABOI transplantation would clearly benefit from the use of this assay, there are also transfusion medicine applications. There is an increasing demand for whole blood transfusions from ABO-O donors, but naturally occurring anti-A and anti-B antibodies can cause transfusion reactions (Yazer and Spinella 2018). Currently, female whole blood donors are excluded due to reports that women have more ABO antibodies than men, but the results of this study did not demonstrate any gender-based differences in the study population. The ABO blood type barrier frequently crosses platelet transfusions and IVIG, yet transfusion medicine laboratories lack a consistent approach for measuring ABO antibodies in these blood products.

[0092] The field of ABO glycobiology is hindered by the lack of readily available monoclonal antibodies with specificity for individual glycan subtypes. This Luminex®-based assay allows for unambiguous characterization of A and B glycan monoclonal antibodies, as shown in the binding confirmation data from this assay development. This bead (microsphere) panel has been expanded to include H disaccharide and tetrasaccharide targets. H panel beads have been manufactured (data not shown) and can be included with A and B beads for simultaneous antibody determination. The specificities of monoclonal antibodies commonly used in clinical transfusion medicine are also not well defined, and this Luminex®-based assay can be used for this purpose.

[0093] There may be in vivo glycan modifications to the A and B tetrasaccharides that are not shown in the bead composition used in this example, however, if additional related glycans are determined to be biologically relevant, this composition can be readily expanded to include such targets using the same techniques applied here.

[0094] In this example, the secretory status of the subjects in this study was not evaluated. Subtype I glycans have been reported to be secreted in individuals with the corresponding fucosyltransferase 2 (FUT2) genotype. AI and BI subtype-specific antibody patterns suggesting a FUT2 non-secretor genotype were observed in some individuals (data not shown). Secretory factor status is not known to be related to transplantation or routine transfusion practices, but this can be further studied using this Luminex®-based assay.

[0095] Additionally, the H panel described above can be used to assess A, B, and H antibody profiles in individuals with the Bombay phenotype who lack a normal fucosyltransferase 1 (FUT1) gene. This is shown in Figure 12, which demonstrates the ability of the present Luminex®-based assay to identify pan-reactive H reactivity in subjects with the Bombay phenotype.

[0096] Transplantation has recently observed major advances in the field of xenotransplantation (Cowan and Tector 2017; Montgomery et al. 2022; Porrett et al. 2022). This Luminex®-based assay can be incorporated into recipient risk assessment algorithms because ABO-O pigs may have different ABO-H and associated glycans decorating cells and tissues compared to humans (Milland and Sandrin 2006). While the xenografts used in these recent transplants were derived from α-Gal knockout animals, there are other potential carbohydrate targets of interest, and this reproducible Luminex®-based assay can be expanded to include other xenoglycans of interest, such as, but not limited to, Neu5Gc. Figure 13 illustrates the use of this assay in xenotransplantation to assess the presence of anti-H antibodies in kidney transplant candidates.

[0097] conclusion In summary, this example demonstrates that the present Luminex®-based assay is a highly reproducible ABO antibody detection assay that can be easily incorporated into clinical histocompatibility laboratories. The data presented herein demonstrate the high variability of ABO antibody profiles and the inadequacy of red blood cell titration assays for reporting the complexity of these antibodies. Detection of organ / tissue-irrelevant antibodies by red blood cell agglutination also unnecessarily excludes individuals from undergoing ABO-incompatible transplants. The present Luminex®-based assay can characterize A- and B-glycan subtype-specific antibodies in human serum and define antibody isotypes, overcoming many of the limitations of red blood cell agglutination assays. This glycan-specific antibody detection assay can be used for allograft-specific determination of ABO antibodies and immune risk assessment in the context of ABOI transplantation and transfusion, potentially significantly advancing these fields. This method also readily lends itself to basic glycobiology research in multicenter studies.

[0098] References Angeloni,Stephan,Robert Cordes,Sherry Dunbar,Carlos Garcia,Grant Gibson,Charles Martin,and Valerie Stone.2016.XMAP Cookbook:A Collection of Methods and Protocols for Developing Multiplex Assays with XMAP Technology.3rd ed.Austin,TX:Luminex Corporation. Bentall,Andrew,Mylvaganam Jeyakanthan,Manjit Braitch,Christopher W.Cairo,Todd L.Lowary,Stephanie Maier,Anne Halpin,Bruce Motyka,Lu Zou,Lori J.West,and Simon Ball.2021.“Characterization of ABH-Subtype Donor-Specific Antibodies in ABO-A-Incompatible Kidney Transplantation.”American Journal of Transplantation 21(11):3649-62. Clausen,H.and S.Hakomori.1989.“ABH and Related Histo-Blood Group Antigens;Immunochemical Differences in Carrier Isotypes and Their Distribution.”Vox Sanguinis 56(1):1-20. Clausen,H.,S.B.Levery,E.Nudelman,S.Tsuchiya,and S.Hakomori.1985.“Repetitive A Epitope(Type 3 Chain A)Defined by Blood Group A1-Specific Monoclonal Antibody TH-1:Chemical Basis of Qualitative A1 and A2 Distinction.”Proceedings of the National Academy of Sciences of the United States of America 82(4):1199-1203. Daga,Sunil,Shimon Hussain,Nithya Krishnan,David Lowe,Manjit Braitch,Prashanth Patel,Andrew Bentall,Simon Ball,Daniel A.Mitchell,Robert Higgins,Ian Skidmore,Daniel Zehnder,and David Briggs.2021. “Immunoglobulin Isotype Compositions of ABO Specific Antibodies Are Dependent on the Individual Patient Blood Group and Blood Group Specificity:Results from a Healthy Donor Cohort.”Journal of Immunological Methods 494:113053. Datta,S.S.,S.Basu,M.Reddy,K.Gupta,and S.Sinha.2021.“Comparative Evaluation of the Conventional Tube Test and Column Agglutination Technology for ABO Antibody Titration in Healthy Individuals:A Report from India.”Immunohematology 37(1):25-32. Denomme,Gregory A.and Waseem Q.Anani.2020.“ABO Titers:Harmonization and Identifying Clinically Relevant ABO Antibodies.”Transfusion 60(3):441-43. El-Awar,N.,J.Lee,and P.I.Terasaki.2005.“HLA Antibody Identification with Single Antigen Beads Compared to Conventional Methods.”Human Immunology 66(9):989-97. Jeyakanthan,M.,P.J.Meloncelli,L.Zou,T.L.Lowary,I.Larsen,S.Maier,K.Tao,J.Rusch,R.Chinnock,N.Shaw,M.Burch,K.Beddows,L.Addonizio,W.Zuckerman,E.Pahl,J.Rutledge,K.R.Kanter,C.W.Cairo,J.M.Buriak,D.Ross,I.Rebeyka,and L.J.West.2016.“ABH-Glycan Microarray Characterizes ABO Subtype Antibodies:Fine Specificity of Immune Tolerance After ABO-Incompatible Transplantation.”American Journal of Transplantation 16(5):1548-58. Jeyakanthan,M.,K.Tao,L.Zou,P.J.Meloncelli,T.L.Lowary,K.Suzuki,D.Boland,I.Larsen,M.Burch,N.Shaw,K.Beddows,L.Addonizio,W.Zuckerman,B.Afzali,D.H.Kim,M.Mengel,A.M.J.Shapiro,and L.J.West.2015.“Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups:Implications for Organ Transplantation.”American Journal of Transplantation(10):2602. Kang,Seon Joo,Young Ae Lim,and Sae Yun Baik.2014.“Comparison of ABO Antibody Titers on the Basis of the Antibody Detection Method Used.”Annals Of Laboratory Medicine 34(4):300-306. Landsteiner,K.2001.“[Agglutination phenomena of normal human blood].”Wiener klinische Wochenschrift 113(20-21):768-69. Lane,William.2016.“Next Generation Sequencing for Blood Group Antigen Profiling.”Blood 128(22):SCI-24 LP-SCI-24. de Mattos,Luiz Carlos.2016.“Structural Diversity and Biological Importance of ABO,H,Lewis and Secretor Histo-Blood Group Carbohydrates.”Revista Brasileira de Hematologia e Hemoterapia 38(4):331-40. Meloncelli,Peter J.and Todd L.Lowary.2009.“Synthesis of ABO Histo-Blood Group Type V and VI Antigens*.”Australian Journal of Chemistry 62(6):558-74. Meloncelli,Peter J.and Todd L.Lowary.2010.“Synthesis of ABO Histo-Blood Group Type I and II Antigens.”Carbohydrate Research 345(16):2305-22. Meloncelli,Peter J.,Lori J.West,and Todd L.Lowary.2011.“Synthesis and NMR Studies on the ABO Histo-Blood Group Antigens:Synthesis of Type III and IV Structures and NMR Characterization of Type I-VI Antigens.”Carbohydrate Research 346(12):1406-26. Muthana,Saddam M.and Jeffrey C.Gildersleeve.2016.“Factors Affecting Anti-Glycan IgG and IgM Repertoires in Human Serum.”Scientific Reports 6(1):19509. Oriol,R.,R.Mollicone,P.Coullin,A.M.Dalix,and J.J.Candelier.1992.“Genetic Regulation of the Expression of ABH and Lewis Antigens in Tissues.”APMIS.Supplementum 27:28-38. Pendu,J.Le,A.M.Dalix,R.Mollicone,K.Crainic,and R.Oriol.1989.“EXPRESSION OF ABH,LEWIS AND RELATED TISSUE ANTIGENS IN THE HUMAN THYMUS.”International Journal of Immunogenetics 16(1):19-32. Pochechueva,Tatiana,Alexander Chinarev,Marianne Spengler,Elena Korchagina,Viola Heinzelmann-Schwarz,Nicolai Bovin,and Robert Rieben.2011.“Multiplex Suspension Array for Human Anti-Carbohydrate Antibody Profiling.”Analyst 136(3):560-69. Ravn,V.and E.Dabelsteen.2000.“Tissue Distribution of Histo-Blood Group Antigens.”APMIS:Acta Pathologica,Microbiologica,et Immunologica Scandinavica 108(1):1-28. Stussi,G.,K.Huggel,H.U.Lutz,U.Schanz,R.Rieben,and J.D.Seebach.2005.“Isotype-Specific Detection of ABO Blood Group Antibodies Using a Novel Flow Cytometric Method.”British Journal of Haematology 130(6):954-63. Sullivan,Harold C.,Howard M.Gebel,and Robert A.Bray.2017.“Understanding Solid-Phase HLA Antibody Assays and the Value of MFI.”Human Immunology. Svensson,L.,L.Rydberg,L.C.De Mattos,and S.M.Henry.2009.“Blood Group A1 and A2 Revisited:An Immunochemical Analysis.”Vox Sanguinis 96(1):56-61. Tait, Brian D., Caner Susal, Howard M. Gebel, Peter W. Nickerson, Andrea A. Zachary, Frans HJClaas, Elaine F. Reed, Robert A. Bray, Patricia Campbell, Jeremy R. Chapman, P. Toby Coates, Robert B. Colvin, Emanuele Cozzi, Ilias INDoxiadis, Susan V Fuggle, John Gill, Denis Glotz, Nils Lachmann, Thalachallour Mohanakumar, Nicole Suciu-Foca, Suchitra Sumitran-Holgersson, Kazunari Tanabe, Craig J. Taylor, Dolly B. Tyan, Angela Webster, Adriana Zeevi, and Gerhard Opelz.2013. “Consensus Guidelines on the Testing and Clinical Management Issues Associated with HLA and Non-HLA Antibodies in Transplantation.”Transplantation 95(1):19-47. *****

[0099] Appendix A Standard Operating Procedure: Luminex® Bead Binding Verification

[0100] Purpose: This SOP outlines a procedure for assessing the success / efficiency of conjugation of A or B glycan subtypes and controls (BSA and αGal) to Luminex beads. 1 This step is post-binding and must be performed before the beads are used. It should be performed in parallel with the latest lot / batch of beads. This binding confirmation should use separate QC runs of human negative and positive controls, as well as other recently tested sera as further QC for the new beads.

[0101] This protocol can also be used for quality control of stored beads or for release / lot QC on new Novoclone™ and secondary antibody shipments. Note that specific concentrations of the Novoclone™ reagent are not provided by the vendor, and vendor QC for this reagent is performed by hemagglutination. Thus, there may be some lot-to-lot variability with this Luminex® platform, making lot QC on Luminex® beads very important for the Novoclone™ reagent.

[0102] material: [Table 3]

[0103] Specimen: This SOP provides a binding confirmation summary for the ABO-BSA-antigen-conjugated Luminex® beads that will be used later to test the specimen, so no specimen is used at this stage of the Luminex® bead-based ABH antigen assay. Only the monoclonal antibody is required.

[0104] Safety: Please refer to specific SDS for detailed information.

[0105] Quality Control (QC): Previously characterized antibodies are used for this purpose. New lot numbers or new antibodies must be tested in parallel with previously validated conjugated bead lots / batches (or in parallel with new reagents). PBS-PBN wells are included to ensure there is no non-specific fluorescence for any particular bead.

[0106] procedure: [Table 4] JPEG2025541547000006.jpg208153JPEG2025541547000007.jpg235153JPEG2025541547000008.jpg104154

[0107] Procedural Precautions Aseptic conditions are not required for these procedures.

[0108] Calculating the number of beads needed The Excel worksheet calculates the number of beads according to the following calculation: 2500 beads per well (including 2 wells to allow for pipetting losses of reagents) Bead Calculation C1V1=2500 beads where: C1 = concentration of beads in the original stock solution of coupled beads (beads / µL); V1 = volume of beads added to each well from the original stock solution (µL); Exemplary Calculations C1V1=2500 beads where C1 = concentration of beads / mL (e.g., 3.75 x 10 5 ); V1 = x (determined per well, in μL); (3.75×10 5 beads / mL)(x) = 2500 beads x = 6.7 μL beads per well, then diluted to 50 μL

[0109] Preparation of 1 / 100 secondary antibody Prepared in a 5 mL Falcon tube (Number of wells + 1) x 100 μL = total volume required Exemplary calculation: (14-1) x 100 μL = 1500 μL 1 / 1000 1 / 1000=x / 1500μL x=1.5 μL 1.5μL - 1500μL PBS-TBN

[0110] Appendix B Luminex Human Sample ABO Antibody Detection the purpose This SOP outlines the procedure for measuring IgG and IgM ABO subtype-specific antibodies using Luminex single antigen beads. This assay is based on the method described by Jeyakantham et al. 1 The microarray method was adapted for Luminex from that published by .

[0111] The assay uses a Luminex bead-based panel in which ABO-A and ABO-B subtype I-VI glycans are conjugated to individual Luminex beads, and the panel also includes additional beads for bovine serum albumin (BSA), galactose-α-1,3-galactose (α-Gal) glycans, and A- and B-trisaccharides.

[0112] Phycoerythrin (PE) secondary antibodies specific for human IgG and IgM are used to detect ABO antibodies bound to individual beads, which can be acquired on a Luminex 200™ or FlexMap 3D® Luminex instrument.

[0113] material [Table 5]

[0114] Specimens: This method has been validated for use with serum, heparin, and EDTA plasma. Samples do not require EDTA treatment.

[0115] Safety: The beads' individual SDS and wash buffers are stored at West Labs. There are no safety concerns with these reagents.

[0116] Quality Control: The assay includes a negative control serum and a positive control serum. AB-1: ABO-AB individual plasma unit obtained from the University of Alberta Transfusion Medicine Laboratory VP-5: A pool of two ABO-O plasma units obtained from the University of Alberta Transfusion Medicine Laboratory

[0117] Control beads include: BSA-conjugated beads (negative control / background beads) Antigen-free beads (negative control / background beads) α-Gal (as a positive control bead)

[0118] Measures: Universal precautions should be observed when handling human specimens.

[0119] procedure [Table 6] JPEG2025541547000011.jpg233153JPEG2025541547000012.jpg184153

[0120] Procedural Notes: Aseptic conditions are not required for this SOP. The optimal dilutions of serum / plasma samples and PE-labeled anti-human IgG and IgM were determined by checkerboard experiments. *****

[0121] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0122] The invention thus described, it will be apparent that it may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be apparent to those skilled in the art are intended to be included within the scope of the following claims.

Claims

1. 1. A method for identifying an ABO histo-blood subtype antibody profile of a subject, comprising: (a) incubating a biological sample obtained from said subject with a bead composition, said bead composition comprising: (i) a plurality of subsets of Luminex® beads, each subset of said plurality individually binding to a type I, type II, type III, type IV ABO-A or ABO-B subtype glycan antigen; (ii) a first control subset of Luminex® beads coupled to a negative control antigen and serving as a negative control; (iii) a second control subset of Luminex® beads that are not bound to antigen and serve as an additional negative control; and (iv) A third control subset of Luminex® beads coupled to a positive control antigen and serving as a positive control. incubating; (b) washing the incubated mixture from step (a); (c) incubating the washed mixture from step (b) with secondary anti-IgG and anti-IgM antibodies, where the secondary antibodies are labeled with fluorescent labels; (d) washing the incubated mixture from step (c) to remove unbound secondary antibody; (e) measuring the fluorescence associated with said subset of Luminex® beads; (f) generating an ABO antigen subtype antibody profile of the subject from the fluorescence data collected in step (e); Including, The subset of beads bound to the ABO-A and ABO-B subtype glycans each contain bovine serum albumin (BSA)-linked ABO subtype antigens bound to corresponding beads, and the amount of the BSA-linked ABO subtype antigens is 1.0 x 10 ６ Approximately 5 μg per bead.

2. BSA-linked type I, type II, type III, type IV ABO-A or ABO-B subtype glycan antigens are: A type I α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, A type II α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, A type III α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, A type IVα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, B type Iα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, B type II α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, B type III α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, and B type IVα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA The method of claim 1, wherein

3. The bead composition further comprises two subsets of Luminex® beads coupled to BSA-linked type V ABO-A and ABO-B subtype glycan antigens, the amount of the BSA-linked type V ABO subtype antigens being 1.0 x 10 ６ 3. The method of claim 1 or 2, wherein the amount is about 5 μg per bead.

4. the BSA-linked type V ABO-A and ABO-B subtype glycan antigens are A type Vα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA and B type Vα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA The method of claim 3, wherein

5. The bead composition further comprises two subsets of Luminex® beads coupled to BSA-linked type VI ABO-A and ABO-B subtype glycan antigens, the amount of the BSA-linked type V ABO subtype antigens being 1.0 x 10 ６ The method of any one of claims 1 to 4, wherein the amount is about 5 μg per bead.

6. the BSA-linked type VI ABO-A and ABO-B subtype glycan antigens are A type VIα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA, and B type VIα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA The method of claim 5, wherein

7. The bead composition further comprises a subset of Luminex® beads individually coupled to BSA-linked Type I, Type II, Type III, Type IV, Type V, and Type VI ABO-H subtype glycan antigens, wherein the amount of the BSA-linked ABO-H subtype antigens is 1.0 x 10 ６ The method of any one of claims 1 to 6, wherein the amount is about 5 μg per bead.

8. the BSA-linked type I, type II, type III, type IV, type V, and type VI ABO-H subtype glycan antigens are H type Iα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, H type II α-L-Fucp-(1→2)-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, H type III α-L-Fucp-(1→2)-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, H type IVα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, H type Vα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-Galp-BSA, and H type VIα-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA The method of claim 7, wherein

9. 9. The method of any one of claims 1 to 8, wherein the bead composition further comprises one or more subsets of Luminex® beads individually coupled to BSA-linked antigens present in a donor animal for xenotransplantation, wherein the antigens present in the donor animal are optionally N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or both.

10. The method according to any one of claims 1 to 9, wherein the positive control antigen is galactose-α-1,3-galactose (α-Gal).

11. The method of any one of claims 1 to 10, wherein the negative control is BSA.

12. 1. A kit for identifying an ABO histo-blood subtype antibody profile of a subject, comprising: (a) a bead composition comprising: (i) a plurality of subsets of Luminex® beads, each subset of said plurality individually binding to a type I, type II, type III, type IV ABO-A or ABO-B subtype glycan antigen; (ii) a first control subset of Luminex® beads coupled to a negative control antigen and serving as a negative control; (iii) a second control subset of Luminex® beads that are not bound to antigen and serve as an additional negative control; and (iv) A third control subset of Luminex® beads that are coupled to a positive control antigen and act as positives. a bead composition comprising: (b) Instructions for use of a kit for carrying out the method of claim 1. Includes a kit.

13. the BSA-linked type I, type II, type III, type IV ABO-A or ABO-B subtype glycan antigen is A type I α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, A type II α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, A type III α-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, A type IVα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, B type Iα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, B type II α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, B type III α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, and B type IVα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-GalpNAc-BSA The kit of claim 12, wherein

14. The bead composition further comprises two subsets of Luminex® beads coupled to BSA-linked type V ABO-A and ABO-B subtype glycan antigens, the amount of the BSA-linked type V ABO subtype antigens being 1.0 x 10 ６ 14. The kit of claim 12 or 13, wherein the amount is about 5 μg per bead.

15. the BSA-linked type V ABO-A and ABO-B subtype glycan antigens are A type Vα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA and B type Vα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-β-D-Galp-BSA The kit of claim 14, wherein

16. The bead composition further comprises two subsets of Luminex® beads coupled to BSA-linked type VI ABO-A and ABO-B subtype glycan antigens, the amount of the BSA-linked type V ABO subtype antigens being 1.0 x 10 ６ The kit of any one of claims 12 to 15, wherein the amount is about 5 μg per bead.

17. the BSA-linked type VI ABO-A and ABO-B subtype glycan antigens are A type VIα-D-GalpNAc-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA, and B type VIα-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA 17. The kit of claim 16, wherein:

18. The bead composition further comprises a subset of Luminex® beads individually coupled to BSA-linked Type I, Type II, Type III, Type IV, Type V, and Type VI ABO-H subtype glycan antigens, wherein the amount of the BSA-linked ABO-H subtype antigens is 1.0 x 10 ６ The kit of any one of claims 12 to 17, wherein the amount is about 5 μg per bead.

19. the BSA-linked type I, type II, type III, type IV, type V, and type VI ABO-H subtype glycan antigens are H type Iα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GlcpNAc-BSA, H type II α-L-Fucp-(1→2)-β-D-Galp-(1→4)-β-D-GlcpNAc-BSA, H type III α-L-Fucp-(1→2)-β-D-Galp-(1→3)-α-D-GalpNAc-BSA, H type IVα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GalpNAc-BSA, H type Vα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-Galp-BSA, and H type VIα-L-Fucp-(1→2)]-β-D-Galp-(1→4)-β-D-Glcp-BSA 19. The kit of claim 18, wherein

20. 20. The kit of any one of claims 12 to 19, wherein the bead composition further comprises one or more subsets of Luminex® beads individually coupled to BSA-linked antigens present in a donor animal for xenotransplantation, the antigens present in the donor animal optionally being N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), or both.

21. The kit according to any one of claims 11 to 18, wherein the positive control antigen is galactose-α-1,3-galactose (α-Gal).

22. The kit according to any one of claims 11 to 19, wherein the negative control antigen is BSA.

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