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- Creators: Department of Chemistry and Biochemistry
- Creators: School of Molecular Sciences
- Resource Type: Text
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
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
Sickle Cell Disease (SCD) is a prevalent genetic disease in Africa, and specifically in Kenya. The lack of available relevant disease education and screening mean that most don't understand the importance of getting testing and many children die before they can get prophylactic care. This project was designed to address the lack of knowledge with supplemental educational materials to be partnered with an engineering capstone project that provides a low cost diagnostic test.
ContributorsShawver, Jamie Christine (Author) / Caplan, Michael (Thesis director) / Snyder, Jan (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Structure is a critical component in drug development. This project supports antibody- facilitated structure determination for the following eleven membrane proteins: the human histamine and dopamine G protein-coupled receptors (HRH4 and DRD2) involved in a wide variety of pathologies such as allergies, inflammation, asthma, pain along with Parkinson's and schizophrenia respectively, the human cystic fibrosis transmembrane conductance regulator (CFTR), the human NaV1.8 voltage-gated sodium ion channel, the human TPC2 two-pore channel, the SARS virus proteins 3a, E and M, the MERS virus protein E and M, and the malarial chloroquine resistance transporter (PfCRT). Serum antibodies against these proteins were generated by genetic immunization, and both in vitro and in vivo expressed membrane proteins were created to characterize the serum antibodies. Plasmid clones were generated for genetic immunization, in vitro protein expression, and in vivo expression (HEK293T transfection). Serum antibodies were generated by genetic immunization of mice by gene gun. Genetic immunization promotes an immune response that allows for the generation of antibodies in the absence of purified protein. In vitro expression was accomplished through the novel technique: in vitro translation with hydrophobic magnetic beads (IVT-HMB). Transfections were performed using the HEK293T cell line to express the protein in vivo. The generated protein was then used in gel electrophoresis and silver stain and/or Western blot analyses to identify and visualize the proteins. These expressed proteins will allow for forthcoming characterization of the generated antibodies. The resulting antibodies will in turn enable structure determination of these important membrane proteins by co-crystallization.
ContributorsDrotar, Beniamin (Author) / Fromme, Petra (Thesis director) / Hansen, Debra T. (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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
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