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- Creators: Harrington Bioengineering Program
- Creators: Chen, Qiang
Description
Flavivirus infections are emerging as significant threats to human health around the globe. Among them West Nile(WNV) and Dengue Virus (DV) are the most prevalent in causing human disease with WNV outbreaks occurring in all areas around the world and DV epidemics in more than 100 countries. WNV is a neurotropic virus capable of causing meningitis and encephalitis in humans. Currently, there are no therapeutic treatments or vaccines available. The expanding epidemic of WNV demands studies that develop efficacious therapeutics and vaccines and produce them rapidly and inexpensively. In response, our lab developed a plant-derived monoclonal antibody (mAb) (pHu-E16) against DIII (WNV antigen) that is able to neutralize and prevent mice from lethal infection. However, this drug has a short window of efficacy due to pHu-E16's inability to cross the Blood Brain Barrier (BBB) and enter the brain. Here, we constructed a bifunctional diabody, which couples the neutralizing activity of E16 and BBB penetrating activity of 8D3 mAb. We also produced a plant-derived E16 scFv-CH1-3 variant with equivalent specific binding as the full pHu-E16 mAb, but only requiring one gene construct for production. Furthermore, a WNV vaccine based on plant-derived DIII was developed showing proper folding and potentially protective immune response in mice. DV causes severe hemorrhaging diseases especially in people exposed to secondary DV infection from a heterotypic strain. It is hypothesized that sub-neutralizing cross-reactive antibodies from the first exposure aid the second infection in a process called antibody-dependent enhancement (ADE). ADE depends on the ability of mAb to bind Fc receptors (FcγRs), and has become a major roadblock for developing mAb-based therapeutics against DV. We aim to produce an anti-Dengue mAb (E60) in different glycoengineered plant lines that exhibit reduced/differential binding to FcγRs, therefore, reducing or eliminating ADE. We have successfully cloned the molecular constructs of E60, and expressed it in two plant lines with different glycosylation patterns. We demonstrated that both plant-derived E60 mAb glycoforms retained specific recognition and neutralization activity against DV. Overall, our study demonstrates great strives to develop efficacious therapeutics and potent vaccine candidates against Flaviviruses in plant expression systems.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Huffman, Holly A (Committee member) / Steele, Kelly P (Committee member) / Arizona State University (Publisher)
Created2014
Description
Dental caries also known as tooth decay is a bacterial infection that causes demineralization and destruction of enamel dentin and cementum in the tooth. This bacterium, Streprococcus mutans, feeds on the carbohydrates in the mouth and produces lactic acids that result in dental caries. This thesis discusses the use of plants to produce antibodies, Guy 13 and anti-GTFB to treat this dental disease. We believe these plant-derived antibodies will be effective to treat dental caries and economical to produce.
ContributorsSayegh, Luvenia Crystal (Author) / Chen, Qiang (Thesis director) / Garg, Vikas (Committee member) / Barrett, The Honors College (Contributor) / School of Letters and Sciences (Contributor)
Created2014-12
DescriptionMy main goal for my thesis is in conjunction with the research I started in the summer of 2010 regarding the creation of a TBI continuous-time sensor. Such goals include: characterizing the proteins in sensing targets while immobilized, while free in solution, and while in free solution in the blood.
ContributorsHaselwood, Brittney (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Cook, Curtiss (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2011-12
Description
This project aims to address the current protocol regarding the diagnosis and treatment of traumatic brain injury (TBI) in medical industries around the world. Although there are various methods used to qualitatively determine if TBI has occurred to a patient, this study attempts to aid in the creation of a system for quantitative measurement of TBI and its relative magnitude. Through a method of artificial evolution/selection called phage display, an antibody that binds highly specifically to a post-TBI upregulated brain chondroitin sulfate proteoglycan called neurocan has been identified. As TG1 Escheria Coli bacteria were infected with KM13 helper phage and M13 filamentous phage in conjunction, monovalent display of antibody fragments (ScFv) was performed. The ScFv bind directly to the neurocan and from screening, phage that produced ScFv's with higher affinity and specificity to neurocan were separated and purified. Future research aims to improve the ScFv characteristics through increased screening toward neurocan. The identification of a highly specific antibody could lead to improved targeting of neurocan post-TBI in-vivo, aiding researchers in quantitatively defining TBI by visualizing its magnitude.
ContributorsSeelig, Timothy Scott (Author) / Stabenfeldt, Sarah (Thesis director) / Ankeny, Casey (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was purified, an ELISA was conducted to validate that the antibody was able to bind to the flavivirus fusion loop.
ContributorsPardhe, Mary (Author) / Mason, Hugh (Thesis director) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / School of Life Sciences (Contributor) / Department of Information Systems (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
In this paper, β-estradiol was characterized utilizing electrochemical impedance spectroscopy (EIS) techniques for the purpose of developing a multi-marker fertility sensor. β-estradiol was immobilized onto the surface of gold disk electrodes to find the optimal binding frequency of estradiol and its respective antibody, anti-17β-estradiol, which was determined to be 37.46Hz. At this frequency a logarithmic relationship between concentration and impedance (Z/ohm) was established creating a concentration calibration curve with a slope of 211 ohm/ln(pg mL-1), an R-squared value of 0.986 and a lower limit of detection of 742 fg mL-1. The specificity and cross-reactivity of the antibody with other hormones was tested through interferent and non-target experiments. Signal-to-noise ratio analysis verified that anti-17β-estradiol exhibited minimal chemical reactions with other hormones (SNR< 3) in non-target experiments. Additionally, there were minimal changes in the amount of signal collected during interferent testing, with albumin and follicle stimulating hormone having SNR values greater than 3. These results, along with the unique frequency response of the antibody-target binding reaction, allow for the possibility of using anti-17β-estradiol and β-estradiol for detecting multiple fertility biomarkers on a single sensor.
ContributorsSmith, Victoria Ann (Author) / LaBelle, Jeffrey (Thesis director) / Spano, Mark (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Diabetes mellitus is a disease characterized by many chronic and acute conditions. With the prevalence and cost quickly increasing, we seek to improve on the current standard of care and create a rapid, label free sensor for glycated albumin (GA) index using electrochemical impedance spectroscopy (EIS). The antibody, anti-HA, was fixed to gold electrodes and a sine wave of sweeping frequencies was induced with a range of HA, GA, and GA with HA concentrations. Each frequency in the impedance sweep was analyzed for highest response and R-squared value. The frequency with both factors optimized is specific for both the antibody-antigen binding interactions with HA and GA and was determined to be 1476 Hz and 1.18 Hz respectively in purified solutions. The correlation slope between the impedance response and concentration for albumin (0 \u2014 5400 mg/dL of albumin) was determined to be 72.28 ohm/ln(mg/dL) with an R-square value of 0.89 with a 2.27 lower limit of detection. The correlation slope between the impedance response and concentration for glycated albumin (0 \u2014 108 mg/dL) was determined to be -876.96 ohm/ln(mg/dL) with an R-squared value of 0.70 with a 0.92 mg/dL lower limit of detection (LLD). The above data confirms that EIS offers a new method of GA detection by providing unique correlation with albumin as well as glycated albumin. The unique frequency response of GA and HA allows for modulation of alternating current signals so that several other markers important in the management of diabetes could be measured with a single sensor. Future work will be necessary to establish multimarker sensing on one electrode.
ContributorsEusebio, Francis Ang (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Currently, the management of diabetes mellitus (DM) involves the monitoring of only blood glucose using self-monitoring blood glucose devices (SMBGs) followed by taking interventional steps, if needed. To increase the amount of information that diabetics can have to base DM care decisions off of, the development of an insulin biosensor is explored. Such a biosensor incorporates electrochemical impedance spectroscopy (EIS) to ensure an extremely sensitive platform. Additionally, anti-insulin antibody was immobilized onto the surface of a gold disk working electrode to ensure a highly specific sensing platform as well. EIS measurements were completed with a 5mV sine wave that was swept through the frequency spectrum of 100 kHz to 1 Hz on concentrations of insulin ranging from 0 pM to 100 μM. The frequency at which the interaction between insulin and its antibody was optimized was determined by finding out at which frequency the R2 and slope of the impedance-concentration plot were best. This frequency, otherwise known as the optimal binding frequency, was determined to be 459 Hz. Three separate electrodes were developed and the impedance data for each concentration measured at 459 Hz was averaged and plotted against the LOG (pM insulin) to construct the calibration curve. The response was calculated to be 263.64 ohms/LOG(pM insulin) with an R2 value of 0.89. Additionally, the average RSD was determined to be 19.24% and the LLD was calculated to be 8.47 pM, which is well below the physiological normal range. These results highlight the potential success of developing commercial point-of-care insulin biosensors or multi-marker devices operating with integrated insulin detection.
ContributorsDecke, Zachary William (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Cook, Curtiss (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
Description
Development of a rapid and label-free Electrochemical Impedance Spectroscopy (EIS) biosensor for Cardiovascular Disease (CVD) detection based on Inerluekin-18 (IL-18) sensitivity was proposed to fill the technology gap between rapid and portable CVD point-of-care diagnosis. IL-18 was chosen for this CVD biosensor due to its ability to detect plaque vulnerability of the heart. Custom (hand) made sensors, which utilized a three electrode configuration with a gold disk working electrode, were created to run EIS using both IL-18 and anti-IL-18 molecules in both purified and blood solutions. The EIS results for IL-18 indicated the optimal detection frequency to be 371Hz. Blood interaction on the working electrode increased the dynamic range of impedance values for the biosensor. Future work includes Developing and testing prototypes of the biosensor along with determining if a Nafion based coating on the working electrode will reduce the dynamic range of impedance values caused by blood interference.
ContributorsJha, Amit (Author) / LaBelle, Jeffrey (Thesis director) / Mossman, Kenneth (Committee member) / Frakes, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / Department of Management (Contributor)
Created2013-05
Description
The purpose of this research was to determine and evaluate glutamate oxidase's ability to detect levels of glutamate as part of a working sensor capable of quantifying and detecting stress within the body in the case of adverse neurological events such as traumatic brain injury. Using electrochemical impedance spectroscopy (EIS), a linear dynamic range of glutamate was detected with a slope of 36.604 z/ohm/[pg/mL], a lower detection limit at 12.417 pg/mL, correlation of 0.97, and an optimal binding frequency of 117.20 Hz. After running through a frequency sweep the binding frequency was determined based on the highest consistent reproducibility and slope. The sensor was found to be specific against literature researched non-targets glucose, albumin, and epinephrine and working in dilutions of whole blood up to a concentration of 25%. With the implementation of Nafion, the sensor had a 250% improvement in signal and 155% improvement in correlation in 90% whole blood, illustrating the promise of a working blood sensor. Future work includes longitudinal studies and utilizing mesoporous carbon as the immobilization platform and incorporating this as part of a continuous, multiplexed blood sensor with glucose oxidase.
ContributorsLam, Alexandria Nicole (Author) / LaBelle, Jeffrey (Thesis director) / Ankeny, Casey (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05