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- Genre: Academic theses
Description
Measles is a contagious, vaccine-preventable disease that continues to be the leading
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2016
Description
Energy Expenditure (EE), a key diagnostic measurement for treatment of obesity, is measured via indirect calorimetry method through breath biomarkers of CO2 production and/or O2 consumption rates (VCO2 and/or VO2, respectively). Current technologies are limited due to prevailing designs requiring wearable facial accessories that present accuracy, precision, and usability concerns with regards to free living measurement. A novel medical device and smart home system, named Smart Pad, has been developed, with the capability of energy expenditure assessment via VCO2 measured from a room’s CO2 concentration. The system has 3 distinct capabilities: contactless EE measurement, air quality optimization via actuation of room ventilation, and efficiency optimization via ventilation actuation of only human-occupied environments. The Smart Pad shows accuracy of 90% for 14-19 minutes of resting measurement and accuracy of 90% for 4.8-7.0 minutes of exercise measurement after calibrating for air exchange rate (λ [hour-1]) using a reference method. Without reference instrument calibration, the Smart Pad system shows average accuracy of nearly 100% with correlations of Y=1.02X, R=0.761 for high resolution measurements and Y=1.06X, R=0.937 for averaged measurements over 50-60 minutes. In addition, the Smart Pad validation for contactless EE measurement has been performed in different environments, including a vehicle, medical office, a private office, and an ambulatory enclosure with rooms, ranging in volume from 3.1 m3 to 18.8m3. It was concluded that contactless EE measurements can be accurately performed in all tested scenarios with both low and high air exchange environments with λ ranging from 1.5 Hours-1 to 10.0 Hours -1. The system represents a new way to assess EE of individuals under free-living conditions in an unobstructive, passive, and accurate manner, and it is comparable or better in single breath gas measurement accuracy (with comparisons sourced from FDA data) than other medical devices (e.g. Vyntus CPXTM, MasterScreen CPXTM, Oxycon ProTM, and MedGemTM) which were 510(k) cleared by the FDA for prescription use in metabolic/cardiopulmonary diagnostics.
ContributorsSprowls, Mark (Author) / Forzani, Erica (Thesis advisor) / Destaillats, Hugo (Committee member) / Kulick, Doina (Committee member) / Nikkhah, Mehdi (Committee member) / Raupp, Gregory (Committee member) / Arizona State University (Publisher)
Created2021
Description
Abnormally low or high blood iron levels are common health conditions worldwide and can seriously affect an individual’s overall well-being. A low-cost point-of-care technology that measures blood iron markers with a goal of both preventing and treating iron-related disorders represents a significant advancement in medical care delivery systems. Methods: A novel assay equipped with an accurate, storable, and robust dry sensor strip, as well as a smartphone mount and (iPhone) app is used to measure total iron in human serum. The sensor strip has a vertical flow design and is based on an optimized chemical reaction. The reaction strips iron ions from blood-transport proteins, reduces Fe(III) to Fe(II), and chelates Fe(II) with ferene, with the change indicated by a blue color on the strip. The smartphone mount is robust and controls the light source of the color reading App, which is calibrated to obtain output iron concentration results. The real serum samples are then used to assess iron concentrations from the new assay and validated through intra-laboratory and inter-laboratory experiments. The intra-laboratory validation uses an optimized iron detection assay with multi-well plate spectrophotometry. The inter-laboratory validation method is performed in a commercial testing facility (LabCorp). Results: The novel assay with the dry sensor strip and smartphone mount, and App is seen to be sensitive to iron detection with a dynamic range of 50 - 300 µg/dL, sensitivity of 0.00049 µg/dL, coefficient of variation (CV) of 10.5%, and an estimated detection limit of ~15 µg/dL These analytical specifications are useful for predicting iron deficiency and overloads. The optimized reference method has a sensitivity of 0.00093 µg/dL and CV of 2.2%. The correlation of serum iron concentrations (N=20) between the optimized reference method and the novel assay renders a slope of 0.95, and a regression coefficient of 0.98, suggesting that the new assay is accurate. Lastly, a spectrophotometric study of the iron detection reaction kinetics is seen to reveal the reaction order for iron and chelating agent. Conclusion: The new assay is able to provide accurate results in intra- and inter- laboratory validations and has promising features of both mobility and low-cost.
ContributorsSerhan, Michael (Author) / Forzani, Erica (Thesis advisor) / Raupp, Gregory (Committee member) / Acharya, Abhinav (Committee member) / Hu, Tony (Committee member) / Smith, Barbara (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work focuses on qualifying the performance of an optoelectrical measurement system designed to analyze ribonucleic acid (RNA) within a micro sample. The system is capable of measuring light intensity converted to voltage versus time and is a fast, inexpensive, and portable method for rapid detection of biologics such as SARS-CoV-2 virus, or Covid-19 disease. The measurement system consists of a microfluidic chip and a point of care fluorescent reader.The intent of this research is to measure consistency and robustness of the fluorescent reader combined with the microfluidic chip. The consistency and the robustness of the fluorescent reader within the duty cycle of the system power and the measurement system were analyzed with Six Sigma methods. Control charts, analysis of variance (ANOVAs), and variance components calculations were implemented to characterize the reader system. Through the process of this analysis, baseline characteristics were measured and documented providing valuable data for the improved instrument design.
The existing microfluidic chip is a prototype that works in combination with the reader based on fluorescent detection. Baseline studies were required to define any issues related to microfluidic autofluorescence. Multiple designs were tested to measure reduction in autofluorescence in the microfluidics. It was found that certain designs performed better than others. One approach for improvement in the microfluidic chip may be achieved by characterizing and source controlling materials, optimizing layers, mask apertures, and mask orientations to determine reliability in the measurable output through the fluorescent reader. Since the reader and the microfluidic are designed to work together, any future studies should explore testing where the two components are considered a coupled system.
ContributorsShabtai, Bat-El (Author) / Blain Christen, Jennifer (Thesis advisor) / Abbas, James (Thesis advisor) / Maass, Eric (Committee member) / Beeman, Scott (Committee member) / Arizona State University (Publisher)
Created2021
Description
Chimeric antigen receptor (CAR) T-cell therapies present transformative potentials for progressive and refractory cancer treatment. However, therapy-associated neuronal toxicities, cytokine release syndromes, relapse rates, and the complex responses of patients and medical management have increased the cost of patient care. Prompt point-of-care (POC) quantification of circulating CAR T-cells and associated cytokines could enhance safety, simplify patients' management, and decrease patient care costs. While effective, existing standard detection methods, such as Enzyme-Linked Immunosorbent Assay (ELISA), quantitative Polymerase Chain Reaction(qPCR), and Flow cytometry, are not conducive to quick POC testing due to their complexity and expense. This research introduces a centrifuge-free Rapid Optical Imaging (ROI)-based platform to quantify CAR T-cells and therapy-related cytokine (Interleukin-6) from a single drop of whole blood. Through machine learning, label-free ROI-based CAR T-cell detection has been improved for accuracy compared with fluorescent staining results, and the morphological characteristics of CAR-T cells have been applied to attribute for differentiation and reduce false positives. This multi-layered microfluidic chip integrates cell and cytokines separation, collection, and detection steps, reducing the need for centrifugation or staining procedures. The microfluidic channel system separates white blood cells from whole blood after red blood cell agglutination and membrane filtration. The non-agglutinated samples are then extracted into a subchannel with a functionalized sensor surface for CAR-T-specific detection. Calibration curves were established using blood samples spiked with varying CAR-T cell concentrations. Another subchannel, featuring dual-layer membrane filtration, has been designed for cytokine detection using gold nanoparticle-labeled detection antibodies. Cytokine concentrations are digitally measured by tracking the number of gold nanoparticles in designated zones. This platform aims to offer a rapid and cost-efficient prognostic tool for timely assessment of key molecular and cellular biomarkers of CAR-T therapy patients, facilitating timely and evidence-based treatment adjustments.
ContributorsYu, Nanxi (Author) / Wang, Shaopeng SW (Thesis advisor) / Forzani, Erica EF (Thesis advisor) / Borges, Chad CB (Committee member) / Liu, Yan YL (Committee member) / Arizona State University (Publisher)
Created2023
Description
Human Papillomavirus (HPV) is the most commonly transmitted STI and isresponsible for an estimated 5% of cancer cases worldwide. HPV infection is implicated
in 70% of cervical cancer incidence and is also responsible for a variety of oropharyngeal
and anogenital cancers. While vaccination has greatly reduced the cervical cancer
burden in developed countries, HPV infection remains high in developing countries due
to high cost and poor access to healthcare. Several studies have highlighted the
presence of anti-HPV antibodies following infection and their potential use as
biomarkers for developing novel screening methods. Progression from initial infection to
cancer is slow, thus presenting an opportunity for effective screening programs.
Biomarker screening is an important area of cancer detection and Lateral Flow Assays
(LFA) are a low cost, easy to use alternative to other screening methods that require
extensive training and laboratory space. Therefore, this project proposes as a hypothesis
that the development of an LFA screening for HPV specific IgG can provide clinically
relevant data for the early detection of cervical dysplasia. This project adapts an LFA in a
multiplexed format for fluorescence-based serologic detection of HPV specific IgG in
patient plasma.
ContributorsJohns, William (Author) / Anderson, Karen (Thesis advisor) / Lake, Douglas (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2023