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Title: Studies of the IgE receptor using sequence-specific peptides, antibodies and other probes
Author: Gao, Bin
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1994
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Sequence-specific peptides of the individual subunits of the high affinity IgE receptor (FcԑRI), a tetrameric complex of one alpha, one beta and two identical disulphide-linked gamma subunits involved in allergic diseases, were synthesised by solid phase synthesis and the antibodies against these peptides were raised in rabbits. The antibodies were purified and characterised by different immunological methods including ELISA, CELISA, flow cytometry and Western blots. The antibodies not only reacted with the synthetic peptides but also, successfully, bound to the natural FceRI on RBL-2H3 cells. The sequence-specific antibodies against individual subunits were able to recognise the authentic alpha, beta and gamma subunits by Western blots, and provided an unique tool for investigating the structure and functions of the receptor. The antibodies to both terminals of the beta subunit bound to intact cells and yielded a modified topological model for the beta subunit to that proposed in 1988. Dimers of the beta subunit in the RBL-2H3 were observed for the first time with anti-beta antibodies by Western blotting, and an extra 24 kD protein was detected by an antibody against the gamma cytoplasmic fragment but not by the antibodies induced by the whole synthetic gamma subunit. Antisera, especially those raised against the beta subunit of FcRI, triggered degranulation and calcium influx in RBL-2H3 cells. This indicated that either the beta subunit might contribute to signal pathways directly or the biochemical events were triggered by unknown substances in the antisera. The coupling of avidin to biotinylated IgE on RBL-2H3 stimulated a similar cellular response to that elicited by conventional hapten-IgE-receptor coupling. The degranulation of the cells induced by pervanadate, an inhibitor of tyrosine phosphatase, suggested that dephosphorylation is also involved in signal transduction of the receptor- mediated degranulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Immunological methods