Development of a Novel Immunoassay for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease affecting one third of the world's population and is the leading cause of death worldwide from a single infective agent. Mycobacterium tuberculosis (MTB) is the main causative organism in humans, whilst Mycobacterium bovis is the primary cause of TB in animals. M. bovis can also cause TB in humans, zoonotic TB (ZTB), with animals acting as reservoirs of infection. The incidence of ZTB is considered to be low, but is likely to be underestimated as both organisms cause similar symptoms, are >99.95% similar at the genomic level, and can only be speciated using molecular techniques which are rarely undertaken in high TB burden countries. Consequently, human TB infections are routinely considered to be caused by MTB. Speciation is essential as M. bovis is intrinsically resistant to pyrazinamide, a first line anti-TB drug, thus misdiagnosis of ZTB as HTB could result in treatment failure and contribute to development of antibiotic resistance. To date, no rapid immunological based diagnostic assay can differentiate at the species level. Development of immunological assays requires species specific binders. Here we present the development of novel binders to M. tuberculosis H37Rv and M. bovis AF2122/97, both highly pathogenic strains of mycobacteria. The mycobacteria were inactivated using gamma irradiation to preserve the structure of their outer surface. Whole cells and ethanol extracted antigens were used as targets for binder production. In addition, key cell surface and secreted antigens were identified from the literature and either obtained or synthesized, and also used as targets. A panel of binders, including polyclonal and monoclonal antibodies, and phage display derived peptides, were produced and evaluated by ELISA. Selected binders, when combined in precise combinations, were capable of differentiating between M. bovis and M. tuberculosis, resulting in species-specific diagnosis of TB in humans. The ELISA data indicates that both M. bovis and MTB could be detected at approximate concentrations of 1 x 104 CFU/ mL and there was no cross-reactivity with the other Mycobacterium spp. tested. A further aim of this research is to translate the ELISA based assay into a low-cost paper-based format for use in high burden, resource poor countries to enable accurate diagnosis and direct treatment of TB disease in regions of the world where TB is most prevalent. Combining the binders with functionalized nanomaterials such as gold nanoparticles (AuNPs) will produce bioconjugates which can be used as an alternative to conventional biochemical sensing due to their unique optical and chemical properties. The result will be a paper based diagnostic device with the potential to enable more sensitive, species-specific diagnosis of TB disease and point-of-care testing.