Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis |
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Authors: | Conrad E. Chan Bryan Z. Zhao Amaury Cazenave-Gassiot Shyue-Wei Pang Anne K. Bendt Markus R. Wenk Paul A. MacAry Brendon J. Hanson |
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Affiliation: | 2. Immunology Program and Centre for Life Sciences, National University of Singapore, Singapore,117456 Singapore;;4. Life Sciences Institute, Centre for Life Sciences, National University of Singapore, Singapore,117456 Singapore;;11. Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore, 117510 Singapore |
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Abstract: | Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 105 CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers. |
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Keywords: | phage display mycolic acid biomarker antibody-based assay |
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