Quantification of post-translationally modified p53 in single cells
File(s)
Author(s)
Squires, James Alexander
Type
Thesis
Abstract
This work outlines efforts towards the development of immunosorbent assays for quantifying post-translationally modified p53, a key tumour suppressor protein, in single cancer cells. These assays utilised the microfluidic antibody capture (MAC) chip to selectively detect post-translationally modified p53 at single molecule sensitivity. Three assays for post-translationally modified p53 were developed; for acetylated-p53, ubiquitinated-p53 and phosphorylated-p53. The assay for acetylated p53, utilising click chemistry, was unsuccessful, as was an approach using antibodies. The assay for ubiquitinated p53 was successful in cell lysate, though could not be made successful in single cells. However, an assay for phosphorylated-p53 was successful and was multiplexed alongside a previously developed total p53 assay to elucidate the effects of a range of drugs on the phosphorylation and total p53 expression in single cells from immortalised cancer cell lines. This assay was then successfully applied, alongside the total p53 assay, to clinically relevant patient-derived xenograft material, the results of which were published in the peer-reviewed literature due to its novelty. This constituted one of the first examples of the measurement of a potentially clinically relevant biomarker in tumour derived material at single cell sensitivity. Further development of the MAC chip aimed at improving its throughput and range of accessible analytes could allow its use as a diagnostic or prognostic device when linked to clinically relevant outcomes such as remission, progression free survival, overall survival or measures of quality of life. This would be a significant contribution towards the personalisation of cancer treatment.
Version
Open Access
Date Issued
2017-10
Date Awarded
2018-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Klug, David
Tate, Edward
Ces, Oscar
Sponsor
Engineering and Physical Sciences Research Council
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)