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- All Subjects: Biochemistry
- Member of: Theses and Dissertations
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- Status: Published
Current methods for IgG antibody detection include enzyme immunoassays (EIA) such as the commercially available Diamedix Immunosimplicity® Measles IgG test kit and the Diamedix Immunosimplicity® Mumps IgG test kit. EIAs generally provide high sensitivity and strong specificity, however, there is a need for rapid screening of measles and mumps specific immunity in outbreak and resource-limited areas which could be solved by use a point-of-care (POC) platform.
This study aims to optimize a point-of-care device for the multiplexed detection of MeV, MuV, and RuV IgG antibodies in sera and to compare the sensitivity to commercial enzyme immunoassays. The IgG antibody levels to MeV and MuV were measured using EIA test kits for a total of 44 healthy serum samples. Of the samples, 6% were seronegative for MeV-specific IgG antibodies and 75% were seronegative for MuV-specific antibodies, showing low correlation of IgG antibody levels between both viruses.
To improve the sensitivity of the POC device, multiple conjugated fluorescent secondary antibodies were tested with different surface chemistries. Signal detection was measured using the pre-developed four-site slide reader. Preliminary data show that Nile Red microspheres provide robust signal detection and should be the secondary antibody of choice when sera are tested for IgG antibodies using the POC platform in future work.
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.
DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.
The purpose of this experiment is to deliver DNA origami barrels loaded with Cas13d-gRNA binary complexes to HPV-16 and HPV-18 cervical cancer lines to make the cancer mortal. The production of Cas 13d has proven successful with a concentration of ~ 1mg/mL, but the activity assay performed has not shown conclusive evidence of Cas13d and guide RNA binary complex formation or activity. Successful annealing of the DNA origami barrel has been quantified by an agarose gel, but further quantification by TEM is in progress. Overall, steady progress is being made towards the goal of targeting HPV16 E6/E7 pre-mRNA with CRISPR/Cas13d.