{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/343950"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/343950","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Characterisation of Alloantibody-Human Leukocyte Antigen Interactions to Improve Immunological Risk Assessment in Solid Organ Transplantation","abstract":"Antibody mediated rejection remains a major challenge in solid organ transplantation, where the development of immunoassays for characterisation a recipient’s donor HLA-specific antibodies has revolutionised the field of solid organ transplantation. These immunoassays are essential for patient evaluation, immune monitoring, and antibody-related immunological risk assessment, however uncertainties concerning data interpretation often prevents determination of an antibody’s clinical significance. The initial aim of this research was to expand the knowledge on the properties of HLA-specific antibodies that govern their pathogenic potential. Luminex single antigen beads (SABs), SAB-C1q, flow cytometry (FC) and complement dependent cytotoxicity (CDC) assays were used to determine the reactivity and complement fixing capacities of human monoclonal antibodies, whilst biolayer interferometry (BLI) was used for real-time quantification of alloantibody-HLA kinetics. Outputs from each immunoassay were found to be dependent on antibody concentration, [Ab], where the degree of CDC was proportional to the antibody’s affinity, KD. The strongest affinity for each antibody was measured with the sensitising antigen. Solid-phase assays offered a higher sensitivity of antibody detection, however stronger interactions could not be distinguished from one another. In attempt to further assess the relationship between antibody-HLA interaction affinity and effector function, the ability to establish an in vitro model of alloantibody-mediated endothelial cell activation and tissue injury was explored. Lastly, using the principles of microfluidic diffusional sizing and Bayesian inference, microfluidic antibody affinity profiling (MAAP) was developed to enable in-solution, simultaneous determination of the KD and [Ab] of antibodies directly in patient serum samples. Quantification of purified monoclonal antibodies spiked into both PBS and blank serum provided a proof-of-principle before moving into HLA antibody-incompatible transplant sera. Here, MAAP was able to quantify the affinity and concentration of antibodies in real-life patient sera, whilst also providing examples of how this information may be used as a tool in clinic to improve transplant-related decision-making processes. Overall, this work provides evidence for the importance of antibody abundance and affinity in clinically relevant humoral alloresponses and, through development of MAAP, outlines a path towards in depth profiling of antibody responses in patient sera.","abstract_html":"Antibody mediated rejection remains a major challenge in solid organ transplantation, where the development of immunoassays for characterisation a recipient’s donor HLA-specific antibodies has revolutionised the field of solid organ transplantation. These immunoassays are essential for patient evaluation, immune monitoring, and antibody-related immunological risk assessment, however uncertainties concerning data interpretation often prevents determination of an antibody’s clinical significance. The initial aim of this research was to expand the knowledge on the properties of HLA-specific antibodies that govern their pathogenic potential. Luminex single antigen beads (SABs), SAB-C1q, flow cytometry (FC) and complement dependent cytotoxicity (CDC) assays were used to determine the reactivity and complement fixing capacities of human monoclonal antibodies, whilst biolayer interferometry (BLI) was used for real-time quantification of alloantibody-HLA kinetics. Outputs from each immunoassay were found to be dependent on antibody concentration, [Ab], where the degree of CDC was proportional to the antibody’s affinity, KD. The strongest affinity for each antibody was measured with the sensitising antigen. Solid-phase assays offered a higher sensitivity of antibody detection, however stronger interactions could not be distinguished from one another. In attempt to further assess the relationship between antibody-HLA interaction affinity and effector function, the ability to establish an in vitro model of alloantibody-mediated endothelial cell activation and tissue injury was explored. Lastly, using the principles of microfluidic diffusional sizing and Bayesian inference, microfluidic antibody affinity profiling (MAAP) was developed to enable in-solution, simultaneous determination of the KD and [Ab] of antibodies directly in patient serum samples. Quantification of purified monoclonal antibodies spiked into both PBS and blank serum provided a proof-of-principle before moving into HLA antibody-incompatible transplant sera. Here, MAAP was able to quantify the affinity and concentration of antibodies in real-life patient sera, whilst also providing examples of how this information may be used as a tool in clinic to improve transplant-related decision-making processes. Overall, this work provides evidence for the importance of antibody abundance and affinity in clinically relevant humoral alloresponses and, through development of MAAP, outlines a path towards in depth profiling of antibody responses in patient sera.","abstract_has_math":false,"creators":["Priddey, Ashley"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Kosmoliaptsis, Vasilis"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-08-01","date_published":"2022-08-01","updated_at":"2026-07-22T22:24:30Z","subjects":["Affinity","Alloantibody","Antibody","HLA","Microfluidics"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.91374","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Kosmoliaptsis, Vasilis"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["NHS Blood and Transplant Research Unit"]},{"key":"dc:creator","label":"Author","values":["Priddey, Ashley"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-08-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/343950"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Affinity","Alloantibody","Antibody","HLA","Microfluidics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.91374"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/93d44ee5-7deb-45a9-a8c1-846daf2eec9e/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Antibody mediated rejection remains a major challenge in solid organ transplantation, where the development of immunoassays for characterisation a recipient’s donor HLA-specific antibodies has revolutionised the field of solid organ transplantation. 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Solid-phase assays offered a higher sensitivity of antibody detection, however stronger interactions could not be distinguished from one another. In attempt to further assess the relationship between antibody-HLA interaction affinity and effector function, the ability to establish an in vitro model of alloantibody-mediated endothelial cell activation and tissue injury was explored. Lastly, using the principles of microfluidic diffusional sizing and Bayesian inference, microfluidic antibody affinity profiling (MAAP) was developed to enable in-solution, simultaneous determination of the KD and [Ab] of antibodies directly in patient serum samples. Quantification of purified monoclonal antibodies spiked into both PBS and blank serum provided a proof-of-principle before moving into HLA antibody-incompatible transplant sera. Here, MAAP was able to quantify the affinity and concentration of antibodies in real-life patient sera, whilst also providing examples of how this information may be used as a tool in clinic to improve transplant-related decision-making processes. Overall, this work provides evidence for the importance of antibody abundance and affinity in clinically relevant humoral alloresponses and, through development of MAAP, outlines a path towards in depth profiling of antibody responses in patient sera."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["b6edbeb6e20826dc35c6dca7a6444a33"]},{"key":"dc:title","label":"Title","values":["Characterisation of Alloantibody-Human Leukocyte Antigen Interactions to Improve Immunological Risk Assessment in Solid Organ Transplantation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Kosmoliaptsis, Vasilis"],"dc:contributor.sponsor":["NHS Blood and Transplant Research Unit"],"dc:creator":["Priddey, Ashley"],"dc:date.issued":["2022-08-01"],"dc:description.abstract":["Antibody mediated rejection remains a major challenge in solid organ transplantation, where the development of immunoassays for characterisation a recipient’s donor HLA-specific antibodies has revolutionised the field of solid organ transplantation. 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Solid-phase assays offered a higher sensitivity of antibody detection, however stronger interactions could not be distinguished from one another. In attempt to further assess the relationship between antibody-HLA interaction affinity and effector function, the ability to establish an in vitro model of alloantibody-mediated endothelial cell activation and tissue injury was explored. Lastly, using the principles of microfluidic diffusional sizing and Bayesian inference, microfluidic antibody affinity profiling (MAAP) was developed to enable in-solution, simultaneous determination of the KD and [Ab] of antibodies directly in patient serum samples. Quantification of purified monoclonal antibodies spiked into both PBS and blank serum provided a proof-of-principle before moving into HLA antibody-incompatible transplant sera. Here, MAAP was able to quantify the affinity and concentration of antibodies in real-life patient sera, whilst also providing examples of how this information may be used as a tool in clinic to improve transplant-related decision-making processes. Overall, this work provides evidence for the importance of antibody abundance and affinity in clinically relevant humoral alloresponses and, through development of MAAP, outlines a path towards in depth profiling of antibody responses in patient sera."],"dc:format.checksum.md5":["b6edbeb6e20826dc35c6dca7a6444a33"],"dc:identifier.doi":["10.17863/CAM.91374"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/93d44ee5-7deb-45a9-a8c1-846daf2eec9e/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/343950"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["Affinity","Alloantibody","Antibody","HLA","Microfluidics"],"dc:title":["Characterisation of Alloantibody-Human Leukocyte Antigen Interactions to Improve Immunological Risk Assessment in Solid Organ Transplantation"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:30Z"}