Rotherham, Lia S.Maserumule, CharlotteDheda, KeertanTheron, JacquesKhati, MakobetsaHoshino, Yoshihiko2012-11-272012-11-272012-10-04Rotherham LS, Maserumule C, Dheda K, Theron J, Khati M (2012) Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples. PLoS ONE 7(10): e46862. DOI:10.1371/journal.pone.00468621932-620310.1371/journal.pone.0046862http://hdl.handle.net/2263/20497We thank Megan Lucas (Colorado State University) for providing us with the CFP-10 and ESAT-6 expression plasmids, as well as the anti-CFP-10 antibody. We thank Lionel Gresh for his help with the ELONA optimisation, Marisa Joubert and Stoyan Stochev for their help with the protein work.Conceived and designed the experiments: LSR CM KD JT MK. Performed the experiments: LSR CM. Analyzed the data: LSR CM KD JT MK. Contributed reagents/materials/analysis tools: KD. Wrote the paper: LSR CM KD JT MK.BACKGROUND : Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens. METHODS : DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis. RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p,0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (KD) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden’s index and 35% and 95%, respectively, using a rule-in cut-point. CONCLUSION : This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible.FIGURE S1. Expression and purification of CFP-10 and ESAT-6. (A) SDS analysis of purified CFP-10. Lane 1, Precision Plus ProteinTM Kaleidoscope Standards (BioRad); and lane 2, eluate from Ni-NTA column indicating the presence of pure monomeric CFP-10. (B) Immunoblot analysis of the purified CFP-10 protein, indicating that the anti-CFP-10 polyclonal antibody reacted specifically with CFP-10 (lane 1), but not with ESAT-6 (negative control, lane 2). (C) SDS-PAGE analysis of purified ESAT-6. Lane 1, Precision Plus ProteinTM Kaleidoscope Standards (BioRad); and lane 2, eluate from Ni-NTA column indicating the presence of pure monomeric ESTA-6. (D) Immunoblot analysis of the purified ESAT-6 protein, indicating that the anti-ESAT-6 monoclonal antibody reacted specifically with ESAT-6 (lane 1), but not with CFP-10 (negative control, lane 2).FIGURE S2. Complex formation by purified recombinant CFP-10 and ESAT-6 proteins. (A) Native polyacrylamide gel of purified recombinant ESAT-6 (lane 1) and CFP-10 (lane 2) proteins, and a mixture of the individual proteins (lane 3). (B) Confirmation of CFP-10.ESAT-6 complex formation by surface plasmon resonance (SPR). ESAT-6 was injected on a CFP-10 surface from t0 to t600, followed by removal of unbound ESAT-6 protein. A control experiment was likewise performed on a CM5 sensor chip devoid of CFP-10.FIGURE S3. Binding affinity of solid phase-synthesised ssDNA aptamers. In an ELONA the selected aptamers showed binding to CFP-10 and CFP-10.ESAT-6, but not to ESAT-6. These results are in agreement with those presented in Fig. 5, indicating that chemical synthesis of the aptamers does not influence their binding ability. Data are presented as means 6 standard deviations of the mean.FIGURE S4. Binding of folded and unfolded aptamer CSIR 2.11 to CFP-10. One batch of CSIR 2.11 was refolded and a second batch was used directly after thawing in the ELONA. No significant differences were observed in the binding capabilities of the folded and the unfolded aptamers, indicating that a refolding step is not necessary for binding of the aptamer to the target protein. Data are presented as means 6 standard deviation of the mean.FIGURE S5. Binding of selected ssDNA aptamers to lysates of oral cavity bacteria. An ELONA was used to test binding of the selected ssDNA aptamers to lysates prepared from M. tuberculosis, M. smegmatis, M. bovis BCG, P. aeruginosa, S. aureus, C. xerosis and S. pyogenes.FIGURE S6. Evaluation of sputum samples using CSIR 2.11 as a detection reagent. The aptamer was tested on three groups of samples (A) Definite TB, (B) Latent TB and TB negative and (C) healthy laboratory volunteers. Using Youden’s index, the cut-point for positive samples was set at an OD450 of 0.2 and is indicated by the dotted line. Using the rule-in disease, the cutpoint for positive samples is an OD450 of 0.8 and is demarcated by the solid line. Data are presented as means 6 standard deviation of the mean.FIGURE S7. Evaluation of sputum samples using a control aptamer CSIR 3.13 as a detection reagent. Controls included were an aptamer-alone control, a sputum-alone control, a positive control (CFP-10 plus CSIR 2.11), as well as sputum samples that were detected by a ssDNA aptamer (CSIR 3.13) that was selected using the same library but against a different target. Data are represented as means 6 standard deviation of the means.FIGURE S8. Comparison of CSIR 2.11 and CSIR 2.21 using sputum samples. The sensitivity and specificity of CSIR 2.11 and a more specific aptamer, CSIR 2.21, were compared using 28 sputum samples. Using Youden’s index, the cut-point for positive samples was set at an OD450 of 0.1 and is indicated by the solid line. Using the rule-in disease, the cut-point for positive samples is an OD450 of 0.62 and is demarcated by the dotted line. Data are presented as means 6 standard deviation of the mean. Samples that gave a positive result when using CSIR 2.21as a detection molecule are denoted as CSIR 2.21 positive, while negative samples are denoted as CSIR 2.21 negative. Samples that gave a positive result when using CSIR 2.11 as a detection molecule are denoted as CSIR 2.11 positive, while negative samples are denoted CSIR 2.11 negative.en© 2012 Rotherham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted useTuberculosis (TB)Article