Part I. Highly sensitive hybridization assays for prostate-specific antigenmRNA based on time-resolved fluorescence and bioluminescence. Part II. Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase and kinase activity.

摘要

Bioanalytical assays, namely nucleic acid hybridization assays and immunoassays, provide powerful tools for scientific investigation. In order for such techniques to be useful in a routine clinical setting, the procedures should be highly sensitive, simple and efficient to perform, and adaptable to automation. The objective of my doctoral research was to develop clinically applicable bioanalytical assays for the determination of cancer-associated analytes. More specifically, the first pan of this DISSERTATION describes hybridization assays which were developed for the detection and quantification of prostate-specific antigen (PSA) mRNA, characteristic of prostate cancer cells. By using the polymerase chain reaction (PCR) combined with time-resolved fluorometric or bioluminescent detection systems, these assays can detect mRNA representative of one PSA-expressing cell amidst one million PSA-negative cells. These methods can facilitate the early detection of prostate cancer cells in the bloodstream, thereby aiding in the correct staging and treatment of prostate cancer patients. The quantitative PCR method, which was developed by analyzing PSA mRNA in parallel with the mRNA of the housekeeping gene, $eta$-actin, allows for the monitoring of relative changes in PSA mRNA levels. This method may aid in the study of prostate cancer by permitting the correlation of PSA mRNA levels in patients, over time, with types of treatment, aggressiveness of tumours and patient outcomes. The second part of the DISSERTATION describes fluorometric and time-resolved immunofluorometric assays developed for the determination of the oncogenically relevant protein-tyrosine kinase (PTK) and phosphatase (PTP) activities. The first assay developed for the determination of PTP activity utilized Tb$sp{3+}$ and conventional fluorometry. Since Tb$sp{3+}$ forms fluorescent complexes with phosphotyrosine (P-Tyr) but not tyrosine (Tyr), PTP activity (ie. the dephosphorylation of P-Tyr) was determined by measuring the decrease in fluorescence. For the time-resolved immunofluoromeuic PTP and PTK assays, synthetic substrates containing Tyr residues were immobilized onto microtitre wells. The P-Tyr groups (formed upon incubation with PTK, or remaining following the PTP reaction) were detected using an anti-phosphotyrosine antibody and an alkaline phosphatase-conjugated secondary antibody. The methods proposed here are safer, more practical, and offer superior sensitivity to established isotopic methods. Source: Dissertation Abstracts International, Volume: 59-08, Section: B, page: 3899. Adviser: Theodore K. Christopoulos. Thesis (Ph.D.)--University of Windsor (Canada), 1996.