DNA Aptamer Selection against Mycobacterium tuberculosis Antigen 85 Complex Using Microplate-based SELEX

Antigen 85 complex (Ag85c) secreted by Mycobacterium tuberculosis is a promising TB biomarker due to its abundance in the blood sera of active TB patients. This study aimed to identify an aptamer against Ag85c antigen using a microplate-based protein-SELEX process. The study also aimed to determine the enriched sequence via capillary electrophoresis sequencing and perform binding affinity and cross-reactivity study using direct enzyme-linked oligonucleotide assay. The binding kinetics of the successful aptamer was further studied using surface plasmon resonance (SPR). The aptamer sequence was identified by capillary electrophoresis sequencing and denoted as H1. Aptamer H1 has a strong affinity with apparent Kd (based on EC50 obtained via ELONA) equal to 0.00272 nM and specificity to the target Ag85c compared with a non- specific aptamer, H2 with apparent Kd (EC50) of 0.383 nM. SPR showed a strong binding affinity of 20.93 nM (U-value = 12) between aptamer H1 and the target antigen TB Ag85c. The results showed that aptamer H1 has a strong potential as a bio-probe for developing bioassays and biosensors for TB diagnostics.