Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis

• Published in: Tuberculosis (Edinburgh, Scotland) Vol. 112; pp. 27 - 36
• Main Authors: Dhiman, Abhijeet, Haldar, Sagarika, Mishra, Subodh Kumar, Sharma, Neera, Bansal, Anjali, Ahmad, Yusra, Kumar, Amit, Sharma, Tarun Kumar, Tyagi, Jaya Sivaswami
• Format: Journal Article
• Language: English
• Published: Scotland Elsevier Ltd 01.09.2018; Elsevier Science Ltd
• Subjects: Affinity; Antibodies; Antigens, Bacterial - cerebrospinal fluid; Antigens, Bacterial - genetics; Aptamer; Aptamer linked immobilized sorbent assay; Aptamers; Aptamers, Nucleotide - chemical synthesis; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Bacteria; Bacterial Proteins - cerebrospinal fluid; Bacterial Proteins - genetics; Biological evolution; Biomarkers; Biomarkers - cerebrospinal fluid; Case-Control Studies; Cerebrospinal fluid; Deoxyribonucleic acid; Detection; Diagnosis; Diagnostic systems; DNA; Enzyme-linked immunosorbent assay; Humans; Medical diagnosis; Meningitis; Mycobacterium tuberculosis; Mycobacterium tuberculosis - metabolism; Polyclonal antibodies; Predictive Value of Tests; Reagents; Reproducibility of Results; SELEX Aptamer Technique; Tuberculosis; Tuberculosis, Meningeal - cerebrospinal fluid; Tuberculosis, Meningeal - diagnosis; Tuberculosis, Meningeal - microbiology; Tuberculous meningitis; Aptamer; Mycobacterium tuberculosis; Detection; Aptamer linked immobilized sorbent assay; Tuberculous meningitis
• ISSN: 1472-9792; 1873-281X; 1873-281X
• DOI: 10.1016/j.tube.2018.07.004

Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis and its diagnosis remains a challenge even today due to the lack of an adequate test. HspX antigen of Mycobacterium tuberculosis was previously established as a reliable diagnostic biomarker for TBM in an ELISA test format using anti-HspX polyclonal antibodies. Towards overcoming the limitations of batch-to-batch variation and challenges of scalability in antibody generation, we utilized Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop high affinity DNA aptamers against HspX as an alternative diagnostic reagent. Post-SELEX optimization of the best-performing aptamer candidate, H63, established its derivative H63 SL-2 M6 to be superior to its parent. Aptamer H63 SL-2 M6 displayed a specific and high affinity interaction with HspX (Kd ∼9.0 × 10−8 M). In an Aptamer Linked Immobilized Sorbent Assay (ALISA), H63 SL-2 M6 significantly differentiated between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ∼100% sensitivity and ∼91% specificity. Notably, ALISA exhibited comparable performance with previously reported antibody-based ELISA and qPCR. Altogether, our findings establish the utility of HspX aptamer for the reliable diagnosis of TBM and pave the way for developing an aptamer-based point-of-care test for TBM.