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Generation and application of antibodies for detection of prostate cancer biomarkers

Zapatero-Rodríguez, Julia (2016) Generation and application of antibodies for detection of prostate cancer biomarkers. PhD thesis, Dublin City University.

Prostate cancer is one of the main health problems in men–over 3,300 new cases are diagnosed each year in Ireland alone. Serum Prostate Specific Antigen (PSA) is the main biomarker used to aid in the early detection of prostate cancer. However, its use has resulted in a high rate of overdiagnosis and overtreatment. Finding alternative, more sensitive and specific biomarkers/biomarker panels is now a priority. HER2 is a promising candidate biomarker for this panel, as it is related with cancer progression and survival. This research describes the construction of an avian-derived HER2-specific scFv library, from which highly specific antibodies were isolated and characterised. After extensive optimization of assay conditions, a competitive ELISA for HER2 detection was developed, but the sensitivity required to detect HER2 within the clinical-relevant range could not be achieved. To circumvent this, fluorescence-based HER2 detection was investigated, but did not resulted in an improved sensitivity. Thus, an electrochemical impedance spectroscopy (EIS)-based immunosensor was designed for highly sensitive detection of HER2. Nanomaterial-based electrochemical detection exhibited great sensitivity for HER2 from serum samples, with a LOD of 0.01 ng/mL. ‘In-house’-generated anti-HER2 and anti-fPSA scFv antibodies were also incorporated in a centrifugal microfluidic ‘lab-on-a-disc’ platform for the simultaneous detection of a panel of prostate cancer biomarkers. ELISA-based detection of HER2 and fPSA was demonstrated on a compact, automated and robust CD-like device that employs purely rotational fluidic flow control for the entire assay protocol, from plasma separation to signal production. This work provides an insight about the potential of microfluidic technologies for multi-marker assay-based detection. Finally, the application of a magneto-immunoassay for the detection of fPSA was explored in collaboration with The Gwent Group Ltd. Anti-fPSA scAb antibodies were immobilised on magnetic beads for the capture and easy separation of this antigen from the sample. A ‘proof-of-concept’ amperometric magneto-immunosensor for the detection of fPSA within the clinical-relevant ranges (0.5–10 ng/mL) was developed.
Item Type:Thesis (PhD)
Date of Award:December 2016
Supervisor(s):O'Kennedy, Richard
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
Funders:Marie Curie EU
ID Code:21588
Deposited On:07 Apr 2017 12:28 by Richard O'Kennedy . Last Modified 19 Jul 2018 15:09

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