Abstract
Erythropoietin (EPO) is a glycoprotein, secreted mainly by the kidneys that regulates erythrocyte production. Clinically it is used to treat certain anemias, but elite athletes misuse it, despite prohibition of the practice by the World Anti-Doping Agency (WADA). As endogenous and recombinant forms of EPO only vary in the extent of their glycosylation, current assays used to detect this growth factor fail to completely discriminate between the two forms. Thus, there is a need for the development of assays that specifically measure only the recombinant forms. The humoral immune repertoire of camels is unique, in that they produce heavy-chain-only antibodies (HCAbs) which exhibit a broad antigen-binding repertoire. Exploiting this natural immune capability of camels may produce antibodies with unique specificities and affinities for utilization in novel assays. This study aimed to raise camelid HCAbs and heavy chain variable fragments (VHH fragments) directed against recombinant human EPO for future use in specific immunoassay development.1. Polyclonal HCAbs were affinity (Protein G) purified, conjugated to horse radish peroxidase and used in an immunoassay to detect human urinary EPO in a volunteer injected with Epoietin. 2. RNA was isolated from the peripheral blood lymphocytes of these animals and used to produce VHH fragments, or single domain antibodies, by phage display technology, and selected for antigen binding. The camel-derived polyclonal HCAb against rhEPO distinguishes between endogenous and exogenous EPO, but, this needs to be confirmed in a larger cohort and after further assay development. This suggests that camelid polyclonal HCAbs, and VHH fragments generated from a phage display library, have the potential to form the basis for an assay specific for recombinant EPO.
| Date of Award | 2 Dec 2021 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | David J Morgan (Supervisor) & Wael Kafienah (Supervisor) |
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