Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics

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dc.contributor.advisorBlackburn, Jonathan
dc.contributor.authorAmos-Brown, Bianca
dc.date.accessioned2019-02-08T14:40:11Z
dc.date.available2019-02-08T14:40:11Z
dc.date.issued2018
dc.date.updated2019-02-08T14:38:56Z
dc.description.abstractTuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements.
dc.identifier.apacitationAmos-Brown, B. (2018). <i>Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics</i>. (). University of Cape Town ,Faculty of Health Sciences ,Division of Chemical and Systems Biology. Retrieved from http://hdl.handle.net/11427/29452en_ZA
dc.identifier.chicagocitationAmos-Brown, Bianca. <i>"Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics."</i> ., University of Cape Town ,Faculty of Health Sciences ,Division of Chemical and Systems Biology, 2018. http://hdl.handle.net/11427/29452en_ZA
dc.identifier.citationAmos-Brown, B. 2018. Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Amos-Brown, Bianca AB - Tuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements. DA - 2018 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2018 T1 - Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics TI - Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics UR - http://hdl.handle.net/11427/29452 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/29452
dc.identifier.vancouvercitationAmos-Brown B. Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics. []. University of Cape Town ,Faculty of Health Sciences ,Division of Chemical and Systems Biology, 2018 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/29452en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Chemical and Systems Biology
dc.publisher.facultyFaculty of Health Sciences
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherChemical Biology
dc.titleIsolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhD
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