Dynamic evolution and immunoreactivity of aptamers binding to polyclonal antibodies against MPT64 antigen of...

Antibody responses can be useful markers of tuberculosis infection. However, the established immunoassay diagnostic method is limited by antigenic variability. Replacing the recombinant proteins with aptamers may overcome these antigenic challenges. In this study, we systematically monitored the selection process of aptamers against anti-MPT64 antibodies of Mycobacterium tuberculosis to obtain more aptamers for developing a multisite system to increase the sensitivity of TB serological diagnosis. Twelve high-affinity aptamers with distinctive secondary structures were obtained by analyzing the dynamic evolution of aptamers against anti-MPT64 antibodies in the process of system evolution of ligands by exponential enrichment (SELEX). Pocket and stem-loops were found to be the basis of these aptamers binding to antibodies. Point mutations of highly conserved nucleotides in the pocket and stem-loop structures resulted in decreased affinity of aptamers to targets. To test the potential of these aptamers for future use in a serological diagnostic tool, three high-affinity aptamers with different epitope specificities were applied as capture aptamer in an enzyme-linked immunosorbent assay (ELISA) with sera of TB patients. The results showed that three aptamers all effectively bound anti-MPT64 antibodies from TB patients and had high specificity and sensitivity. These aptamers with high immunoreactivity in human sera may represent an efficient and promising analogue of MPT64 and have potential to substitute MPT64 as a nucleic acid antigen in the serological diagnosis of TB. Moreover, these aptamers with different epitope specificities may facilitate the development of a sandwich assay platform or a multisite system to effectively capture more targets in sera.