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- Creators: Barrett, The Honors College
- Resource Type: Text
- Status: Published
Description
Background: Measles virus (MV) infections are the main cause of vaccine-preventable death in children younger than 5 years. The World Health Organization (WHO) has estimated there are over 20 million cases of measles every year. Currently, diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. These commercial assays measure reactivity against the immunodominant N antigen and can have a false negative rates of 20-30%. Centralized testing by clinical labs can delay rapid screening in an outbreak setting. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to five individual MV proteins representing 85% of the measles proteome. Methods: MV genes were subcloned into pANT_cGST vector to generate C-terminal GST fusion proteins. Single MV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured using a sandwich ELISA for GST expression using relative light units (RLUs) as readouts. Single MV antigens were used as bait to determine the IgG-dependent reactivity in 12 serum samples obtained from immunized animals with previously determined neutralization titer (NT) and the correlation between NT and ELISA reactivity was determined. Results: Protein expression of five measles genes of interest, M, N, F, H, and L, was measured. L exhibited the strongest protein expression with an average RLU value of 4.34 x 10^9. All proteins were expressed at least 50% greater than control (2.33 x 10^7 RLU). As expected, reactivity against the N was the highest, followed by reactivity against M, F, H and L. The best correlation with NT titer was reactivity against F (R^2 = 0.62). Conclusion: These data indicate that the expression of single MV genes M, N, F, H, and L are suitable antigens for serologic capture analysis of measles immunity.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis director) / Reyes del Valle, Jorge (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may be able to detect high-grade cervical intraepithelial neoplasia (CIN3). Biomarkers have potential as a rapid, point-of-care HPV screening tool for low resource areas in the way that traditional cytology cannot, and HPV DNA testing is not yet able to.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
ContributorsResnik, Jack Isiah (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Purushothaman, Immanuel (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.
ContributorsGoulder, Alison Leigh (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Cheng, Julia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Methicillin-Resistant Staphylococcus aureus (MRSA) infections are a major challenge to healthcare professionals. Treatment of MRSA is expensive, and otherwise avoidable deaths occur every year in the United States due to MRSA infections. Additionally, such infections lengthen patients’ stays in hospitals, keeping them out of work and adversely affecting the economy. Beta lactam antibiotics used to be highly effective against S. aureus infections, but resistance mechanisms have rendered methicillin, oxacillin, and other beta lactam antibiotics ineffective against these infections. A promising avenue for MRSA treatment lies in the use of synthetic antibodies—molecules that bind with specificity to a given compound. Synbody 14 is an example of such a synbody, and has been designed with MRSA treatment in mind. Mouse model studies have even associated Syn14 treatment with reduced weight loss and morbidity in MRSA-infected mice. In this experiment, in vitro activity of Syn 14 and oxacillin was assessed. Early experiments measured Syn 14 and oxacillin’s effectiveness in inhibiting colony growth in growth media, mouse serum, and mouse blood. Syn14 and oxacillin had limited efficacy against USA300 strain MRSA, though interestingly it was noted that Syn14 outperformed oxacillin in mouse serum and whole mouse blood, indicating the benefits of its binding properties. A second experiment measured the impact that a mix of oxacillin and Syn 14 had on colony growth, as well as the effect of adding them simultaneously or one after the other. While use of either bactericidal alone did not show a major inhibitory effect on USA300 MRSA colony growth, their use in combination showed major decreases in colony growth. Moreover, it was found that unlike other combination therapies, Syn14 and oxacillin did not require simultaneous addition to MRSA cells to achieve inhibition of cell growth. They merely required that Syn14 be added first. This result suggests Syn14’s possible utility in therapeutic settings, as the time insensitivity of synergy removes a major hurdle to clinical use—the difficulty in ensuring that two drugs reach an affected area at the same time. Syn14 remains a promising antimicrobial agent, and further study should focus on its precise mechanism of action and suitability in clinical treatment of MRSA infections.
ContributorsMichael, Alexander (Author) / Diehnelt, Chris (Thesis director) / Stafford, Phillip (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
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
Description
The research objective is to maintain the A4 nanobody stability during dialysis. Various dialysis buffers were tested and compared, including PBS with varying amounts of the detergent, Tween: low, high, none. Furthermore, PBS, Tris, and HEPES, were tested and compared. PBS without Tween was the worst for preserving A4 stability. PBS was determined to be a better dialysis buffer than Tris or HEPES. To find the optimum buffer, other buffers will be tested and compared with PBS; methods such as gravity filtration and lyophilization will be considered as alternatives to dialysis.
ContributorsTao, Kevin Huang (Author) / Sierks, Michael (Thesis director) / Williams, Stephanie (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
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
Mycobacterium tuberculosis is the primary bacteria responsible for tuberculosis, one of the most dangerous diseases, and top causes of death worldwide, as identified by the World Health Organization in a 2018 report. Diagnostic tools currently exist for identifying those who carry active or latent versions of the infection including chest radiographs, a Mantoux tuberculin skin test, or an acid-fast bacilli smear of sputum samples. These methods are standard in the medical community of high income countries, but pose challenges for lower-income regions of the world as well as vulnerable populations. The need for a rapid, inexpensive, and non-invasive method of tuberculosis detection is evident by the ten million that contracted and 1.6 million that died from TB in 2017 alone. In our study, we used a previously developed nanoplasmon-enhanced scattering technology combined with dark field microscopy in order to investigate the potential for a blood-based TB detection assay. Twenty-eight capture antibodies were screened using cell culture exosomes and human serum samples to identify candidates for a TB-derived exosome biomarker. Four antibodies demonstrated potential for distinguishing negative controls from positive controls in this study: anti-AG85, anti-AG85B, anti-SodA, anti-Ald. These capture antibodies displayed significant differences (p<0.05) for both cell culture exosomes and human serum samples on more than one occasion. The work is significant in its ability to distinguish potential capture antibody targets, and future experimentation may yield a technology ready for clinical settings to address the gap in current TB detection methods.
ContributorsWalls, Robert (Author) / Hu, Tony (Thesis director) / Fan, Jia (Committee member) / School of Molecular Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Monoclonal antibody therapy focuses on engineering immune cells to target specific peptide sequences indicative of disease. An impediment in the continued advancement of this market is the lack of an efficient, inexpensive means of characterization that can be broadly applied to any antibody while still providing high-density data. Many characterization methods address an antibody's affinity for its cognate sequence but overlook other important aspects of binding behavior such as off-target binding interactions. The purpose of this study is to demonstrate how the binding intensity between an antibody and a library of random-sequence peptides, otherwise known as an immunosignature, can be evaluated to determine antibody specificity and polyreactivity. A total of 24 commercially available monoclonal antibodies were assayed on 125K and 330K peptide microarrays and analyzed using a motif clustering program to predict candidate epitopes within each antigen sequence. The results support the further development of immunosignaturing as an antibody characterization tool that is relevant to both therapeutic and non-therapeutic antibodies.
ContributorsDai, Jennifer T. (Author) / Stafford, Phillip (Thesis director) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05