Anti-gp120 and anti-p24 aptamers: potential for use as flow cytometry reagents

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dc.contributor.authorMalatji, Kanyane Bridgett
dc.date.accessioned2019-09-12T12:45:51Z
dc.date.available2019-09-12T12:45:51Z
dc.date.issued2018
dc.descriptionA Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine by Research. Johannesburg, 2018en_ZA
dc.description.abstractIntroduction: Aptamers are nucleic acids selected by systematic evolution of ligands by exponential enrichment (SELEX). They have potential as alternatives to antibodies in research and diagnosis. Their main advantages over antibodies are that they are nonimmunogenic and relatively inexpensive to produce. The aim of this study was to generate fluorescein isothiocyanate (FITC) conjugated gp120 aptamers as well as synthesize p24 aptamers and to potentially use them for detecting human immunodeficiency virus (HIV-1) infected cells. Methods: The gp120 aptamer was conjugated with FITC by incubation with 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) and imidazole. The conjugation and binding to the glycoprotein was confirmed using flow cytometry and capture assay. Aptamers against p24 were selected using SELEX and their sequences elucidated by next generation sequencing. Results: The conjugation of the gp120 aptamer with FITC increased fluorescence emission 24 -fold from baseline. Similar data were obtained when beads coupled with HIV-1 gp120 or whole viruses were detected with the FITC-conjugated aptamer by flow cytometry and capture assay, respectively. When compared to a commercially available antibody (biotinylated anti-gp120 polyclonal antibody), the FITC-conjugated aptamer showed better emission of fluorescence. During the synthesis of p24 aptamers an enrichment of binding sequences was observed. Furthermore, 90,500 sequences were identified by deep sequencing out of the initial 1014 ss-DNA library. Conclusion: A FITC-conjugated gp120 aptamer that can bind the glycoprotein on coated beads or on a whole viral particle was generated. The aptamer is a potential low cost reagent for use in HIV/AIDS research or diagnosis. Furthermore, the selection of p24 aptamers was performed and sequences that bind the target were identifieden_ZA
dc.description.librarianMT 2019en_ZA
dc.format.extentOnline resource (100 leaves)
dc.identifier.citationMalatji, Kanyane Bridgett (2018) Anti-gp120 and anti-p24 aptamers:potential for use as flow cytometry reagents, University of the Witwatersrand, Johannesburg, <http://hdl.handle.net/10539/28096>
dc.identifier.urihttps://hdl.handle.net/10539/28096
dc.language.isoenen_ZA
dc.subject.meshHIV infections--complications
dc.subject.meshFlow cytometry
dc.subject.meshHIV infections
dc.titleAnti-gp120 and anti-p24 aptamers: potential for use as flow cytometry reagentsen_ZA
dc.typeThesisen_ZA
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