Advancing Tuberculosis Antigen Detection by Selecting Sandwich-Compatible Aptamers Against the Mycobacterium tuberculosis ESAT-6/CFP-10 Heterodimer Using Library-Immobilized Strategies

Tuberculosis (TB) represents the world’s leading infectious disease killer, surpassing COVID-19. Despite being preventable and curable, a substantial proportion of cases remain undiagnosed or unreported, sustaining transmission. The detection of antigens such as the Mycobacterium tuberculosis ESAT-6/CFP-10 heterodimer, which are secreted at different stages of infection, is a promising screening strategy. Traditional antigen tests relying on antibodies suffer from significant problems of specificity and sensitivity. Aptamers, selected in an in vitro technique termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), are short, single-stranded oligonucleotides that fold into unique 3D structures, allowing them to bind to specific targets, including proteins, cells, or small molecules, with high affinity and specificity. However, conventional SELEX methods often yield suboptimal aptamers partly due to target immobilization that can promote nonspecific interactions and distort protein conformation. Here, we employed two library-immobilized strategies to generate aptamers against the M. tuberculosis ESAT-6/CFP-10 heterodimer. We first performed capture-SELEX to select aptamers against the unconstrained, conformationally native heterodimer target in solution. These selected aptamers were then exploited in a second SELEX, where aptamer-target complexes were “baited” by a target-specific antibody to isolate sandwich-compatible aptamers for downstream assay development. The selected aptamers exhibited high nanomolar affinity regardless of library and selection strategy employed, with bait-SELEX-derived candidates emerging after fewer selection rounds. These aptamers were successfully applied in antibody/aptamer hybrid sandwich assays, supporting their potential for TB antigen detection in nonsputum samples.