Verschoor, Jan Adrianus2020-09-172020-09-172020-09-302020*S2020http://hdl.handle.net/2263/76180Dissertation (MSc (Biochemistry))--University of Pretoria, 2020.Despite the immense amount of research, development, and implementation for new TB diagnostics, diagnosing TB in resource-poor countries remains a challenge, especially in children and people living with HIV. A delayed or missed diagnosis of active infection is one of the major causes of transmission and mortality. Major progress has been made in laboratory-based diagnostics, but point-of-care (POC) diagnostics that are critical in high burdened and resource-limited areas remain elusive. Blood based antibody biomarker tests are superior to sputum-based tests, but are compromised by HIV co-infection, which reduces the ability of the patient to produce antibodies or to contain the pathogen in the lungs. TB patients produce antibodies directed against M. tuberculosis mycolic acids (MA), a pathogen-derived lipid. Anti-MA antibodies arise without T cell help and are thereby not affected by HIV co-infection. They can therefore be used as biomarkers for detection of TB in patient sera. The Mycolic acids Antibody Real-Time Inhibition (MARTI) test has the potential to accurately detect patient low affinity anti-MA antibodies as biomarker for active TB. A point of care version of MARTI is envisioned to be a lateral flow immunoassay named Mycolic Acid Lateral-flow Immuno-Assay (MALIA). MALIA makes use of recombinant monoclonal antibodies (gallibodies) that are specific to MA to compete with patient biomarker antibodies for binding to the MAs on a test line to indicate TB. The aim of this study is to demonstrate proof of principle that MALIA is feasible, both in terms of function and affordability. For affordability, the up scaled counter current distribution (CCD) purification of the most expensive element, MA, was attempted with success. However, quantification of the yield of MA post purification resulted in a 37% loss after acetone precipitation, even though MA has never been shown to be soluble in acetone. The probing of the acetone waste with gallibodies in ELISA confirmed the presence of antigenic MA. An immuno-blot test on silica TLC plates was developed, showing that the MAs lose antigenicity when separated by means of TLC. Whether this was due to separating mycolate from its organic counter-ion was investigated, but gave inconclusive results, but indicated that MA remains in acetone solution for hours after heating to 90 C and cooling to room temperature The solubility limit of MA in acetone was determined to be 0.25 mg/ml before falling out of solution after four hours of cooling. The discovery of acetone soluble MA overcomes the challenges of solvent compatibility in automated MA printing for MALIA, while simultaneously enhancing the antigenic conformation of MA on silica. Silica on TLC plate format proved to be a workable and affordable solid phase substrate for lateral flow immunoassay. Proof of principle of a workable and affordable MALIA for POC TB screening was thereby provided.en© 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDTuberculosisMycolic acidsPoint of careDissertation