Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications

Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified apt...

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Bibliographic Details
Published in:Journal of clinical microbiology 2017-10, Vol.55 (10), p.3072-3088
Main Authors: Russell, Theresa M, Green, Louis S, Rice, Taylor, Kruh-Garcia, Nicole A, Dobos, Karen, De Groote, Mary A, Hraha, Thomas, Sterling, David G, Janjic, Nebojsa, Ochsner, Urs A
Format: Article
Language:English
Subjects:
Acyltransferases - analysis
Antigens, Bacterial - analysis
Antigens, Bacterial - immunology
Aptamers, Peptide - metabolism
Bacterial Proteins - analysis
Bacterial Proteins - blood
Bacterial Proteins - urine
Humans
Immunologic Tests - methods
Mycobacteriology and Aerobic Actinomycetes
Mycobacterium tuberculosis - immunology
Protein Binding - physiology
Tuberculosis, Pulmonary - diagnosis
Tuberculosis, Pulmonary - microbiology
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Summary:Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native proteins was confirmed by using culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant ( < 0.0001) differences in the median signals and in specific pathogen markers, such as antigen 85B. Samples where many aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals ( < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of SOMAmers using other platforms and sample types is warranted.
ISSN:0095-1137
1098-660X
DOI:10.1128/JCM.00469-17