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Title: Development of high-throughput, humanised rat basophilic leukaemia reporter systems for assessment of allergic senisitisation
Author: Wan, Daniel
ISNI:       0000 0004 6423 3989
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2015
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In vivo methods of allergy diagnosis may be uncomfortable for patients, have a risk of causing anaphylaxis, and results may be affected by drugs used to treat allergies. In vitro methods avoid these issues, however current high-throughput in vitro methods of allergy diagnosis involve measurements of allergen-specific IgE levels, which may not correspond to symptomatic allergy. Allergen-specific IgE may not always be capable of crosslinking its receptor FcERI upon allergen exposure, an important mechanism for mast cell and basophil degranulation, and release of mediators responsible for an allergic reaction. In this project, new high throughput in vitro methods for allergy diagnosis were tested using rat basophilic leukaemia (RBL) cell lines. Using RBL cell lines expressing human FcsRIa, four inducible fluorescent gene reporter systems were generated: NFATp-DsRed-Express2, RnIL4p-DsRed-Express2, RnCCL2p-DsRed-Express2 and RnTNFop-DsRed-Express2. Of these reporter systems, only NFATp-DsRed-Express2, transfected into RBL-703/21 cells, was highly responsive to FcERI crosslinking. From this cell line, a stably-transfected, highly responsive cell clone was isolated and expanded. This clone was also used in proof-of-principle experiments to demonstrate functionality in allergen microarrays. Results of these assays were comparable to the EXiLE system (an inducible luciferase reporter), and the "traditional" Beta-hexosaminidase assay. An attempt to generate another assay system based on bimolecular fluorescence complementation, to assess FcERI crosslinking, was unsuccessful. A stably-transfected RBL-2H3 cell line expressing high levels of an NPY-mRFP fusion protein was also generated, and offers a simpler and faster degranulation assay to the commonly-used Beta-hexosaminidase assay. A future clone stably-transfected with human FcERIa may be used to assess allergic sensitisation using human sera.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available