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Optimizing Dialysis Conditions for A4 Stability

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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.

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.

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Date Created
2015-05

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Targeting Traumatic Brain Injury: Using Phage Display to Create a Specific Antibody for Neurocan Chondroitin Sulfate Proteoglycan Recognition

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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 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.

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Date Created
2015-05

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TB or Not TB: Methodologies and Clinical Validation of Tuberculosis Detection Using Nanoplasmon-Enhanced Scattering and Dark Field Microscopy

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

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.

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Date Created
2019-05

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Characterization of Designed Ankyrin Repeat Proteins for Neurodegenerative Disease Diagnostics

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The following paper discusses the potential for Designed Ankyrin Repeat Proteins (DARPin) use as a diagnostic tool for neurodegenerative diseases in particular Alzheimer's disease (AD) and Parkinson's disease (PD). The two structures investigated for AD and PD were ADC7 and

The following paper discusses the potential for Designed Ankyrin Repeat Proteins (DARPin) use as a diagnostic tool for neurodegenerative diseases in particular Alzheimer's disease (AD) and Parkinson's disease (PD). The two structures investigated for AD and PD were ADC7 and PDC1. Plasmid transformation was performed in order to grow the DARPin in E. coli for simple expression. Following growth and purification the proteins were validated using SDS-PAGE, Western Blot, BCA and indirect sandwich ELISA using transgenic mouse brain tissue. Targeted functionality of the DARPin structure was utilized during characterization methods to ensure the efficacy of the protein as a diagnostic for the respective disease targets. Both the ADC7 and PDC1 demonstrated improved binding with transgenic mice compared to wild type with a maximum 1.8 and 1.7 relative ratio, respectively. Additionally, both of the proteins demonstrated exclusive binding to their disease target and did not provide false positive results.

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2016-12