Abstract:
Tuberculosis (TB) is a curable infectious disease, but remains a persistent scourge of mankind. It is caused by Mycobacterium tuberculosis (M. tb) that is a master of survival in the human host, where it can lie dormant for decades, build up resistance to antibiotics and await conditions for relapsing into active disease. It demands a global strategy to manage the epidemic, of which the single most important element is fast and affordable accurate diagnosis. To date, the only reliable diagnosis of active disease depends on sputum samples, which consequently leads to misdiagnosis of extrapulmonary TB, paediatric TB and TB in immune compromised populations such as HIV infected individuals. Because of this, the World Health Organization (WHO) and Foundation for Innovative New Diagnostics (FIND) called for a non-sputum-based triage test for the diagnosis of active tuberculosis (TB) with specific target product profiles (TPP) to eradicate TB by 2035. Our TB research group is focused on the development of TB diagnostics based on the detection of surrogate marker anti-mycolic acids (anti-MA) antibodies in patient blood samples at the point of care level. Mycolic acids (MA) are cell wall waxes from the mycobacterial infectious agent. The group has created recombinant monoclonal antibodies against MA, selected from a semi-synthetic chicken phage displayed antibody gene library, which was found to have inadequate avidity for application in sensitive TB diagnosis. In this study, attempts were made to increase avidity of the recombinant antibodies by creation of oligomers of their antigen binding short chain variable (scFv) fragments. Two strategies were applied to this end: first, shortening the synthetic linker between the heavy and light variable domains of the anti-MA scFv’s and second, fusing the scFv genes with the core streptavidin gene that can be expressed to generate spontaneous non-covalent tetramers. Both approaches yielded oligomeric antigen binding proteins with significantly improved avidity for MA binding compared to that of scFv. The best of these were selected to demonstrate their utility in an originally designed lateral flow immunoassay, which opens the way for its further development into that much needed diagnostic device for point of care screening for active TB in disease burdened communities and health centres.
