Preparation and epitope analysis of monoclonal antibodies against African swine fever virus DP96R protein.

BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.

Virtual crossmatching and epitope analysis in kidney transplantation: What the physician involved in kidney transplantation should know?

Solid-phase single antigen bead (SAB) assay for detection of anti-human leukocyte antigen (HLA) antibodies and high-resolution HLA typing have enabled tremendous progress in virtual crossmatch (VXM) technology in recent years. However, misinterpretation of the SAB assay may result in detrimental consequences after kidney transplantation. Meanwhile, epitope analysis could be an effective method to estimate immunizing eplets, which may provide ancillary information for better understanding of the SAB assay. To perform epitope analysis appropriately, it is necessary to understand the basic principles related to histocompatibility testing and the characteristics of the SAB assay. Therefore, knowledge of the properties and limitations of the SAB assay is critical. In this review, we aim to describe the fundamental concepts regarding immunobiological assessment, including HLA, anti-HLA antibodies, and SAB assay, and explain epitope analysis using examples.

Epitope Analysis of Murine and Chimeric Monoclonal Antibodies Recognizing the Cancer Testis Antigen PRAME.

We investigated the epitope specificity of different monoclonal antibodies recognizing the cancer testis antigen PRAME. Antibody 5D3 binds to the fragment of PRAME corresponding to 160-180 amino acid residues. Antibodies 6H8 and F11 bind to the fragment corresponding to 180-200 amino acid residues of PRAME. These antibodies retained the ability to recognize these PRAME fragments after chimerization.

Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody.

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.

Zinc transporter 8 (ZnT8) autoantibody epitope specificity and affinity examined with recombinant ZnT8 variant proteins in specific ZnT8R and ZnT8W autoantibody-positive type 1 diabetes patients.

Variant-specific zinc transporter 8 autoantibodies (ZnT8A) against either arginine (R) or tryptophan (W) at amino acid (aa) position 325 of the zinc transporter 8 (ZnT8) has been identified in type 1 diabetes (T1D) patients. Reciprocal cross-over tests revealed differences in half-maximal binding to indicate variable affinity of patient ZnT8 autoantibodies. Insufficient recombinant ZnT8 variant proteins have precluded detailed analyses of ZnT8 autoantibody affinity. The aims in the present study were to (i) generate recombinant ZnT8R- and ZnT8W-aa275-369 proteins; (ii) test the ZnT8R- and ZnT8W-aa275-369 proteins in reciprocal competitive radiobinding assays (RBA) against ZnT8R- and ZnT8W-aa268-369 labelled with (35) S-methionine; and (iii) determine the specificity and affinity of sera specific for either ZnT8 arginine (R) or ZnT8 tryptophan (W) autoantibodies in newly diagnosed T1D patients. The results demonstrate, first, that it was possible to produce recombinant human MBP-ZnT8-aa275-369 protein purified to homogeneity for RBA reciprocal competition experiments. Secondly, high-titre ZnT8WA sera diluted to half maximal binding showed significant specificity for respective variants of either ZnT8R or ZnT8W. Thirdly, ZnT8WA-positive sera showed high affinity for ZnT8W compared to ZnT8RA for ZnT8R. These data demonstrate that T1D patients may have single amino acid-specific autoantibodies directed against either ZnT8R or ZnT8W and that the autoantibody affinity to the respective variant may be different. Further studies are needed to assess the mechanisms by which variant-specific ZnT8A of variable affinity develop and their possible role in the pathogenic process leading to the clinical onset of T1D.

HLA molecular mismatches and induced donor-specific tolerance in combined living donor kidney and hematopoietic stem cell transplantation.

Introduction: We investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT). Methods: All patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection. Results: Follow-up time of this cohort was 6-13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30-0.61] vs. 0.24 [IQR, 0.18-0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM. Conclusion: Our results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings. Clinical trial registration: https://clinicaltrials.gov/study/NCT00498160, identifier NCT00498160.

Molecular characterization and serodiagnostic evaluation of the Echinococcus ortleppi recombinant glutaredoxin 1 protein for cystic echinococcosis in buffalo (Bubalus bubalis).

Cystic echinococcosis (CE), caused by the metacestode of Echinococcus granulosus sensu lato (s.l.), adversely affects the physiology of the vital organs in which they grow. Condemnation of meat causes substantial economic loss to the livestock industry. Conventionally the infection is detected by necropsy as serological diagnosis of the infection in livestock is ambiguous. Identification of specific diagnostic antigens would be a substitute for the cyst fluid antigens which lack adequate diagnostic sensitivity and specificity. BLAST analysis supported by the negligible pairwise nucleotide distance of the 389 nt COX1, 489 nt NAD1, and 425 nt ITS1 with the related sequences of E. ortleppi ascertained the association of E. ortleppi with CE in buffaloes. Given the extensive distribution of glutaredoxin 1 in every developmental stage of Echinococcus granulosus s.l that makes it an ideal serodiagnostic antigen for CE, we expressed the 14 kDa E. ortleppi glutaredoxin 1 (rEoGrx1) protein in E. coli BL21 (DE3) and tested a total of 225 sera samples, including 126 sera samples from the necropsy-positive buffalo, by the rEoGrx1 IgG-ELISA. The ELISA could detect a total of 82/126 sera samples as positive. The diagnostic sensitivity and specificity of the rEoGrx1 IgG-ELISA were 65.1 % and 51.5 %, respectively. The protein showed serological cross-reaction against Fasciola gigantica, Toxoplasma gondii, and Sarcocystis sp. The in silico bioinformatics analysis of the E. ortleppi, F. gigantica, and T. gondii glutaredoxin sequences revealed fully conserved amino acids at positions 11 and 21, the substitution of conserved amino acids at positions 14 and 6, and semi-conserved substitutions at positions 3 and 4, respectively. The findings partly explain the molecular basis of the serological cross-reactivity of the protein.

Statistical Analysis and Tokenization of Epitopes to Construct Artificial Neoepitope Libraries.

Epitopes are specific regions on an antigen's surface that the immune system recognizes. Epitopes are usually protein regions on foreign immune-stimulating entities such as viruses and bacteria, and in some cases, endogenous proteins may act as antigens. Identifying epitopes is crucial for accelerating the development of vaccines and immunotherapies. However, mapping epitopes in pathogen proteomes is challenging using conventional methods. Screening artificial neoepitope libraries against antibodies can overcome this issue. Here, we applied conventional sequence analysis and methods inspired in natural language processing to reveal specific sequence patterns in the linear epitopes deposited in the Immune Epitope Database (www.iedb.org) that can serve as building blocks for the design of universal epitope libraries. Our results reveal that amino acid frequency in annotated linear epitopes differs from that in the human proteome. Aromatic residues are overrepresented, while the presence of cysteines is practically null in epitopes. Byte pair encoding tokenization shows high frequencies of tryptophan in tokens of 5, 6, and 7 amino acids, corroborating the findings of the conventional sequence analysis. These results can be applied to reduce the diversity of linear epitope libraries by orders of magnitude.

Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

Erratum: Clinical significance of shared T cell epitope analysis in early de novo donor-specific anti-HLA antibody production after kidney transplantation and comparison with shared B cell epitope analysis.

[This corrects the article DOI: 10.3389/fimmu.2021.621138.].

Antibody-Antigen Binding Interface Analysis in the Big Data Era.

Antibodies have become the Swiss Army tool for molecular biology and nanotechnology. Their outstanding ability to specifically recognise molecular antigens allows their use in many different applications from medicine to the industry. Moreover, the improvement of conventional structural biology techniques (e.g., X-ray, NMR) as well as the emergence of new ones (e.g., Cryo-EM), have permitted in the last years a notable increase of resolved antibody-antigen structures. This offers a unique opportunity to perform an exhaustive structural analysis of antibody-antigen interfaces by employing the large amount of data available nowadays. To leverage this factor, different geometric as well as chemical descriptors were evaluated to perform a comprehensive characterization.

Clinical Significance of Shared T Cell Epitope Analysis in Early De Novo Donor-Specific Anti-HLA Antibody Production After Kidney Transplantation and Comparison With Shared B cell Epitope Analysis.

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4+ T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4+ T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4+ memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4+ memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4+ memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4+ memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation.

The Cancer Epitope Database and Analysis Resource: A Blueprint for the Establishment of a New Bioinformatics Resource for Use by the Cancer Immunology Community.

Recent years have witnessed a dramatic rise in interest towards cancer epitopes in general and particularly neoepitopes, antigens that are encoded by somatic mutations that arise as a consequence of tumorigenesis. There is also an interest in the specific T cell and B cell receptors recognizing these epitopes, as they have therapeutic applications. They can also aid in basic studies to infer the specificity of T cells or B cells characterized in bulk and single-cell sequencing data. The resurgence of interest in T cell and B cell epitopes emphasizes the need to catalog all cancer epitope-related data linked to the biological, immunological, and clinical contexts, and most importantly, making this information freely available to the scientific community in a user-friendly format. In parallel, there is also a need to develop resources for epitope prediction and analysis tools that provide researchers access to predictive strategies and provide objective evaluations of their performance. For example, such tools should enable researchers to identify epitopes that can be effectively used for immunotherapy or in defining biomarkers to predict the outcome of checkpoint blockade therapies. We present here a detailed vision, blueprint, and work plan for the development of a new resource, the Cancer Epitope Database and Analysis Resource (CEDAR). CEDAR will provide a freely accessible, comprehensive collection of cancer epitope and receptor data curated from the literature and provide easily accessible epitope and T cell/B cell target prediction and analysis tools. The curated cancer epitope data will provide a transparent benchmark dataset that can be used to assess how well prediction tools perform and to develop new prediction tools relevant to the cancer research community.

EPIphany-A Platform for Analysis and Visualization of Peptide Immunoarray Data.

Antibodies are critical effector molecules of the humoral immune system. Upon infection or vaccination, populations of antibodies are generated which bind to various regions of the invading pathogen or exogenous agent. Defining the reactivity and breadth of this antibody response provides an understanding of the antigenic determinants and enables the rational development and assessment of vaccine candidates. High-resolution analysis of these populations typically requires advanced techniques such as B cell receptor repertoire sequencing, mass spectrometry of isolated immunoglobulins, or phage display libraries that are dependent upon equipment and expertise which are prohibitive for many labs. High-density peptide microarrays representing diverse populations of putative linear epitopes (immunoarrays) are an effective alternative for high-throughput examination of antibody reactivity and diversity. While a promising technology, widespread adoption of immunoarrays has been limited by the need for, and relative absence of, user-friendly tools for consideration and visualization of the emerging data. To address this limitation, we developed EPIphany, a software platform with a simple web-based user interface, aimed at biological users, that provides access to important analysis parameters, data normalization options, and a variety of unique data visualization options. This platform provides researchers the greatest opportunity to extract biologically meaningful information from the immunoarray data, thereby facilitating the discovery and development of novel immuno-therapeutics.

Identification of a novel calcium activated potassium channel from Leishmania donovani and in silico predictions of its antigenic features.

Visceral Leishmaniasis is a major neglected tropical disease with increasing incidences of drug resistance. This has led to the search for a suitable drug target for chemotherapeutic intervention. Potassium channels are a family of membrane proteins which play a vital role in homeostasis and any perturbation in them alters cell survival which makes them an attractive target. To characterize a calcium-activated potassium channel from Leishmania donovani (LdKCa), a putative ion-channel like protein which showed sequence similarity with other Trypanosoma cruzi putative potassium channels was selected. It was cloned and expressed with a histidine tag. MALDI confirmed that it is a potassium channel. Homology model of LdKCa was generated by I-TASSER. It is a transmembrane protein localized in the plasma membrane as predicted by DeepLoc tool. In silico analyses of the protein showed that it is a small conductance calcium activated potassium channel. Point mutation in conserved signature domain 'TXGYGD' affects the protein function as predicted by heat map analysis. The LdKCa model predicted amino acids S207, T208 and M236 as ligand-binding sites. The sequence HSLRGRSARVIQLAWRLRKARKVGPHAPSLKQKVYTLVLSWLLT was the highest scoring B-cell epitope. The highest scoring T-cell epitope was RLYSVIVYL. This study may provide new insights into antigenicity features of leishmanial calcium-activated potassium channels.

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach.

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe++ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.

Analysis of T and B Cell Epitopes to Predict the Risk of de novo Donor-Specific Antibody (DSA) Production After Kidney Transplantation: A Two-Center Retrospective Cohort Study.

Risk prediction of de novo donor specific antibody (DSA) would be very important for long term graft outcome after organ transplantation. The purpose of this study was to elucidate the association of eplet mismatches and predicted indirectly recognizable HLA epitopes (PIRCHE) scores with de novo DSA production. Our retrospective cohort study enrolled 691 living donor kidney transplantations. HLA-A, B, DRB and DQB eplet mismatches and PIRCHE scores (4 digit of HLA-A, B, DR, and DQ) were determined by HLA matchmaker (ver 2.1) and PIRCHE-II Matching Service, respectively. Weak correlation between eplet mismatches and PIRCHE scores was identified, although both measurements were associated with classical HLA mismatches. Class II (DRB+DQB) eplet mismatches were significantly correlated with the incidence of de novo class II (DR/DQ) DSA production [8/235 (3.4%) in eplet mismatch ≤ 13 vs. 92/456 (20.2%) in eplet mismatch ≥ 14, p < 0.001]. PIRCHE scores were also significantly correlated with de novo class II DSA production [26/318 (8.2%) in PIRCHE ≤ 175 vs. 74/373 (19.8%) in PIRCHE ≥ 176, p < 0.001]. Patients with low levels of both class II eplet mismatches and PIRCHE scores developed de novo class II DSA only in 4/179 (2.2%). Analysis of T cell and B cell epitopes can provide a beneficial information on the design of individualized immunosuppression regimens for prevention of de novo DSA production after kidney transplantation.

Autoantibodies Against the Immunodominant Bullous Pemphigoid Epitopes Are Rare in Patients With Dermatitis Herpetiformis and Coeliac Disease.

Dermatitis herpetiformis (DH) is an extraintestinal manifestation of coeliac disease (CD). Patients with DH have an elevated risk of development of another autoimmune blistering skin disease, bullous pemphigoid (BP). In this study we investigated whether patients with DH and CD (mean age for both 49 years) have circulating autoantibodies against BP180, the major BP autoantigen. ELISA tests showed that only a few DH (3/46) and CD (2/43) patients had BP180-NC16A IgG autoantibodies. Immunoblotting found that more than half of the DH samples contained IgG autoantibodies against full-length BP180. Epitope mapping with 13 fusion proteins covering the BP180 polypeptide revealed that in DH and CD patients, IgG autoantibodies did not target the NC16A or other epitopes typical of BP but recognized other intracellular and mid-extracellular regions of BP180. None of the analyzed DH and CD patients with either ELISA or immunoblotting positivity had IgG or IgA reactivity against the cutaneous basement membrane in indirect immunofluorescence analysis or skin symptoms characteristic of BP. Although only a minority of middle-aged DH patients had IgG autoantibodies against the immunodominant epitopes of BP180, our results do not exclude the possibility that intermolecular epitope spreading could explain the switch from DH to BP in elderly patients.

Development of a Chimeric Vaccine Against Pseudomonas aeruginosa Based on the Th17-Stimulating Epitopes of PcrV and AmpC.

Pulmonary infection caused by Pseudomonas aeruginosa (PA) has created an urgent need for an efficient vaccine, but the protection induced by current candidates is limited, partially because of the high variability of the PA genome. Antigens targeting pulmonary Th17 responses are able to provide antibody-independent and broad-spectrum protection; however, little information about Th17-stimulating antigens in PA is available. Herein, we identified two novel PA antigens that effectively induce Th17-dependent protection, namely, PcrV (PA1706) and AmpC (PA4110). Compared to intramuscular immunization, intranasal immunization enhanced the protection of rePcrV due to activation of a Th17 response. The Th17-stimulating epitopes of PcrV and AmpC were identified, and the recombinant protein PVAC was designed and generated by combining these Th17-stimulating epitopes. PVAC was successfully produced in soluble form and elicited broad protective immunity against PA. Our results provide an alternative strategy for the development of Th17-based vaccines against PA and other pathogens.

Epitope Analysis Aids in Transplant Decision Making by Determining the Clinical Relevance of Apparent Pre-Transplant Donor Specific Antibodies (DSA).

Pre-transplantation work-up on a patient with end stage renal disease using Single Antigen Bead (SAB) testing showed significant anti-HLA-B*44:02 (>5,000 MFI) and anti-HLA-B*44:03 (>1,000 MFI) antibodies, with persistence on quarterly testing. No significant Class II anti-HLA antibodies were present. The patient received a potential offer from a living unrelated-donor expressing HLA-B*44:02. Based on the presence of anti-HLA-B*44:02 antibody, the crossmatch (XM) was predicted to be positive. However, the actual fluorescence cytometry crossmatch (FCXM) was negative. FCXM and Complement Dependent Cytotoxicity-XM (CDC-XM) studies with three surrogate donors who expressed HLA-B*44:02 (and no other potential confounding HLA types) were also negative. Additional assays were performed for detecting anti-HLA antibodies. Immucor® LSA® SAB analyses also revealed presence of anti-HLA-B*44:02 and anti-HLA-B*44:03 antibodies. However, One Lambda® Antigen Trays, C1q analysis, and iBeads®, did not detect elevated anti-HLA-B*44:02 and/or anti-HLA-B*44:03 antibodies. An extensive evaluation of all exposed and non-exposed epitopes expressed by the patient and the donor was performed to identify the non-shared epitopes between them. The donor specific antibody (DSA) pattern detected would be expected to conform to non-shared epitopes; however, non-shared "exposed" epitopes were not present in the DSA antibody pattern. Whereas, the apparent DSA antibody pattern consisted of antibodies to "non-exposed" epitopes. Altogether, it was concluded that the anti-HLA-B*44:02 antibody detected by SAB testing was directed against some denatured component(s) (non-exposed) of the HLA antigen attached to the SAB, and would not be clinically significant. The patient received the transplant and the post-transplant course has been uneventful for greater than 5 years. This case emphasizes: (1) A significant number of SAB may have denatured HLA molecules attached to them (2) The DSA and non-DSA anti-HLA antibody patterns should be evaluated for the expected epitope components to determine the clinical relevance. Additional testing should be considered to help with these analyses (3) The FCXM remains the "gold standard" for making the final decision to transplant, since the "stringent" use of a "predicted" positive XM using apparent DSA detected by SAB analysis may exclude XM-compatible donors.