A novel magneto immunoassay was developed to detect intracellular PSA in LNCAP cells. This project was conducted to investigate methods of rapid intracellular protein measurement using a magneto-immuno assay. Cell cultures were established using Jurkat and ECV304 cells and were used as a continuous source of intracellular proteins. As the project progressed LNCAP cells were used as a model for intracellular PSA. To release intracellular protein, experiments were performed to develop a novel method of cell lysis using paramagnetic particles (PMPs). To accomplish this, eight different lysis methods were initially evaluated using a quantitative approach measuring total protein and lactate dehydrogenase LDH activity. A qualitative approach was adapted to study the protein by western blotting and environmental scanning electron microscopy (ESEM). A novel method utilizing a sonicator probe in conjunction with paramagnetic particles to lyse Jurkat cells was investigated; two types of particles were investigated (1 urn and 2.8 urn Dynabeads). This method was compared with other traditional lysing methods such as freeze-thawing, detergent, heat treatment and a sonicator water bath. The results showed that chemical lysis method was the most efficient for releasing protein and yielded the highest LDH activity. But chemical lysis method was not able to release measurable PSA from LNCAP cells. A higher amount of PSA was released when sonicating LNCAP cells with super paramagnetic particles energised with a sonicator probe. Two sizes of paramagnetic particles (2.8 and 1 urn) and particles of the same size with different coating were evaluated. The size of the paramagnetic particles had a significant effect on the sonication process. ESEM studies showed that the cells tend to lose their shape and integrity when sonicated by the sonicator alone. Moreover comparing the micrographs obtained when sonicating the cells with 1 urn particles a membranous structures was observed while a coagualtive structures were observed when sonicating the cells with 2.8 urn particles. The second part of the thesis describes an investigation into a novel magneto immunoassay, using paramagnetic particles as a label for PSA detection, using a prototype devices developed at UWE. The method developed relies on the specificity of antibody-antigen interaction between antigen and antibody on PMPs and the surface of specifically designed reaction vessel containing the PMP to be captured on the surface. The captured PMP was then detected using magnetometer which has a resonant coil situated below the reaction surface. Using this approach a faster and cheaper detection of PSA could be achieved. A disposable reaction chamber, using low sample volume is added advantages in this method. Two detection systems were evaluated, a resonant coil magnetometer (RCM) detection system and a fixed frequency magnetometer (FFM), both developed at UWE. The latter system was used to investigate the effect of testosterone on LNCAP cells and the release of intracellular PSA. The results show that androgens cause a positively enhanced LNCAP cell growth rate, which consecutively leads to an increase in LNCAP intracellular PSA production. Correlation of serum samples using patient's samples from the Bristol Royal Infirmary was performed. The FFM system gave 90 % sensitivity and specificity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:573576 |
| Date | January 2009 |
| Creators | Sharif, Elham Abdullatif Modh |
| Publisher | University of the West of England, Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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