Abstract
Coronary artery disease (CAD) leads to serious health issues by narrowing the coronary arteries. Due to the limitations and potential errors of invasive imaging techniques, non-invasive methods and specific biomarkers are crucial for the early diagnosis of vascular narrowing. Antibodies could serve as novel diagnostic markers in this context.
This Master's thesis initially analyzes antibody reactivities against pathogens using microarray technology. The aim of this work is to establish the Phage Immunoprecipitation Sequencing method (PhIP-Seq) to identify specific antibody responses against CAD-specific proteins. PhIP-Seq combines phage display technology with high-throughput DNA sequencing and could enhance the discovery of new biomarkers for CAD diagnosis. The analysis involved plasma and IgG samples from 160 patients from Wiener Neustadt, divided into stenosis, sclerosis, and control groups. Microarray analyses identified Klebsiella pneumoniae as a potential biomarker for CAD.
For the PhIP-Seq method, a phage display library was created following the Novagen protocol. The characterization of the constructed phage library was performed by Sanger sequencing of selected phage plaques and deep sequencing (Illumina). This confirmed that the plaques contained relevant proteins from the library and that all expected inserts were present. However, only 27% of the reads could be successfully mapped, while the remaining 73% could not be mapped, possibly due to mutations or amplification errors.
Immunoprecipitation with the phages and patient plasma was performed. The resulting antibody-phage complexes were barcoded via PCR and prepared for sequencing. Although the PCR was functional, the barcode integration was insufficient, affecting sequencing as there were not enough barcoded fragments for cluster formation. Optimization of PCR conditions and reagents is needed to enable successful sequencing in future experiments.
This Master's thesis initially analyzes antibody reactivities against pathogens using microarray technology. The aim of this work is to establish the Phage Immunoprecipitation Sequencing method (PhIP-Seq) to identify specific antibody responses against CAD-specific proteins. PhIP-Seq combines phage display technology with high-throughput DNA sequencing and could enhance the discovery of new biomarkers for CAD diagnosis. The analysis involved plasma and IgG samples from 160 patients from Wiener Neustadt, divided into stenosis, sclerosis, and control groups. Microarray analyses identified Klebsiella pneumoniae as a potential biomarker for CAD.
For the PhIP-Seq method, a phage display library was created following the Novagen protocol. The characterization of the constructed phage library was performed by Sanger sequencing of selected phage plaques and deep sequencing (Illumina). This confirmed that the plaques contained relevant proteins from the library and that all expected inserts were present. However, only 27% of the reads could be successfully mapped, while the remaining 73% could not be mapped, possibly due to mutations or amplification errors.
Immunoprecipitation with the phages and patient plasma was performed. The resulting antibody-phage complexes were barcoded via PCR and prepared for sequencing. Although the PCR was functional, the barcode integration was insufficient, affecting sequencing as there were not enough barcoded fragments for cluster formation. Optimization of PCR conditions and reagents is needed to enable successful sequencing in future experiments.
| Originalsprache | Englisch |
|---|---|
| Gradverleihende Hochschule |
|
| Betreuer/-in / Berater/-in |
|
| Publikationsstatus | Veröffentlicht - 9 Sept. 2024 |
Research Field
- Molecular Diagnostics
Schlagwörter
- Antikörper Reaktivitäten
- Biomarker
- Koronare Arterienerkrankung
- Microarray-Technologie
- Phage Immunoprecipitation Sequencing