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- All Subjects: Human Papillomavirus
- Creators: Anderson, Karen S
- Creators: Ewaisha, Radwa Mohamed Emadeldin Mahmoud
Description
Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.
ContributorsEwaisha, Radwa Mohamed Emadeldin Mahmoud (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas F (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2018
Description
Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings.
This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV.
The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use.
The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost.
The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.
This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV.
The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use.
The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost.
The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.
ContributorsObahiagbon, Uwadiae (Author) / Blain Christen, Jennifer M (Thesis advisor) / Anderson, Karen S (Committee member) / Goryll, Michael (Committee member) / Smith, Barbara S. (Committee member) / Arizona State University (Publisher)
Created2018
Description
Recent studies have shown that human papillomavirus (HPV) plays a role in development of cancers, one of which is head and neck cancer. There is strong and consistent molecular evidence demonstrating that human papillomavirus (HPV) is an etiological cause of these oropharyngeal cancers. Despite the introduction of HPV vaccines, there is still an increase in human papillomavirus associated OPC (HPVOPC) and it is expected that the incidence of head and neck cancer, specifically oropharyngeal cancer (OPC) will increase. The aim of this study is to utilize human papillomavirus (HPV) seropositivity for rapid detection of HPV early specific antigen-antibodies using a lateral flow assay.
Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.
Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.
ContributorsLadipo, Evelyn (Author) / Anderson, Karen S (Thesis advisor) / Hogue, Brenda G (Committee member) / Hou, Ching-Wen (Committee member) / Arizona State University (Publisher)
Created2019