Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients

Abstract

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn’t distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, p < 0.0001) and 0.821 (95% CI 0.752–0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB’s POCT by antibody-based diagnosis.

Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals’ blood cells in combination with CpG-DNA

“…For this purpose, we purified secreted M. tuberculosis proteins from M. tuberculosis culture filtrates according to previous established methods 31 . The purified proteins were identified as MPT 32 (Rv1860, or Apa), MPT 45 (Rv0129c, Antigen 85C, or fbpC), MPT 46 (Rv3914, TrxC), MPT53 (Rv2878c, or mpt53), MPT 59 (Rv1886c, Antigen 85B, or fbpB), and MPT 63 (Rv1926c, mpt63) by mass spectrometry 32 .…”

“…PstS1 was observed the immunodominant 38 kDa protein 46 and a recent report suggested anti-PstS1 antibodies contribute to host protection in humans 47 . We recently reported antibody levels against native M. tuberculosis proteins including Apa and PstS1, but the corresponding E. coli recombinant proteins were unable to detect pulmonary tuberculosis patients 32 . Exact reasons for the strong immunogenicity of post-translationally modified proteins of M. tuberculosis can be complex, but this is fundamentally due to accuracy of immune responses.…”

“…The antigens were purified utilizing FPLC with DEAE-Sepholose column as descrived previously 31 . The obtained antigens were confirmed by SDS-PAGE and then analyzed by mass spectrometry (Triplet OF 5600 + , SCIEX, Tokyo, Japan) after trypsin digestion and then verified by Mascot version 2.6 using the M. tuberculosis (Strain ATCC 25618/H37Rv) database 32 .…”