3 years ago

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

DeGroote MA, Russell T, Hraha T, Dobos K, Ochsner UA, Sterling DG, Kruh-Garcia NA, Rice T, Green LS, Janjic N
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 sub-nanomolar affinity for M. tuberculosis (Mtb) proteins (antigen 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 sub-picomolar range in 40% serum. Binding to native Mtb proteins was confirmed by using Mtb culture filtrate proteins, 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 (p<0.0001) differences in the median Mtb signals and in specific pathogen markers such as antigen 85B (A85B). Samples where many Mtb 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 (p<0.0276), particularly in TB patients with HIV co-infection. In conclusion, direct Mtb antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, sub-picomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of Mtb SOMAmers using other platforms and sample types is warranted.

Publisher URL: https://www.ncbi.nlm.nih.gov/pubmed/28794178

DOI: PubMed:28794178

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