dc.contributor.author |
Rotherham, Lia S.
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|
dc.contributor.author |
Maserumule, Charlotte
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|
dc.contributor.author |
Dheda, Keertan
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dc.contributor.author |
Theron, Jacques
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dc.contributor.author |
Khati, Makobetsa
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dc.contributor.editor |
Hoshino, Yoshihiko |
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dc.date.accessioned |
2012-11-27T06:30:36Z |
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dc.date.available |
2012-11-27T06:30:36Z |
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dc.date.issued |
2012-10-04 |
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dc.description |
We 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. |
en_US |
dc.description |
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. |
en_US |
dc.description.abstract |
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. |
en_US |
dc.description.sponsorship |
This work was supported by the Department of Science & Technology (http://www.dst.gov.za/) and the Technology Innovation Agency (http://www.
tia.org.za/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. |
en_US |
dc.description.uri |
www.plosone.org |
en_US |
dc.format.extent |
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).
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.format.extent |
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.
(TIF) |
en_US |
dc.identifier.citation |
Rotherham 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.0046862 |
en_US |
dc.identifier.issn |
1932-6203 |
|
dc.identifier.other |
10.1371/journal.pone.0046862 |
|
dc.identifier.uri |
http://hdl.handle.net/2263/20497 |
|
dc.language.iso |
en |
en_US |
dc.publisher |
Public Library of Science |
en_US |
dc.rights |
© 2012 Rotherham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use |
en_US |
dc.subject |
TB |
en_US |
dc.title |
Selection and application of ssDNA aptamers to detect active TB from sputum samples |
en_US |
dc.type |
Article |
en_US |