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Generation of Mycobacterium tuberculosis-specific recombinant antigens and evaluation of the clinical value of antibody detection for serological diagnosis of pulmonary tuberculosis

Xia Zhang, Zhonglan Su, Xiangrong Zhang, Chunmei Hu, Jun Yu, Qian Gao, Hongwei Wang
International Journal of Molecular Medicine 2013, 31 (3): 751-7
Rapid diagnosis of pulmonary tuberculosis (TB) infection is critical in clinical practice. To establish an effective serological diagnostic technique, we generated the several Mycobacterium tuberculosis (MTB)-specific immunogenic antigens and evaluated the clinical benefits of detection of immunoglobulin G (IgG) and IgM antibodies raised against these target antigens for the diagnosis of patients with active TB. The genes encoding the MTB-specific antigens 6-kDa early secretory antigenic target of MTB (ESAT-6), 10-kDa culture filtrate protein (CFP-10), ESX-1 substrate protein C (ESPC), 14KD/38KD and ESAT-6/14KD/38KD, were amplified from the MTB genome by PCR. Prokaryotic vectors were constructed for the expression of the individual MTB antigens. The target recombinant protein was expressed in Escherichia coli (BL21/DE3) and purified using immobilized metal affinity chromatography (IMAC). An ELISA based immunoassay was set up using these target antigens for the diagnosis of active TB. The detection samples included 98 patients with active TB and 102 healthy control volunteers. The cutoff OD value for IgG and IgM antibodies was selected according to a receiver operating characteristic (ROC) analysis. The sensitivity, specificity and positive likelihood ratio were also determined. We successfully cloned, expressed and purified the ESAT-6, CFP-10, ESPC, 14KD/38KD and ESAT‑6/14KD/38KD recombinant antigens of MTB. The mean levels of IgG antibodies were significantly higher in patients with pulmonary TB compared with control groups. The target MTB-specific antigens can distinguish a TB infection from a non-TB infection, showing significant difference in statistics (P<0.001). The sensitivity of the IgG test ranged from 69.4% (ESAT-6) to 77.6% (ESAT-6/14KD/38KD) in the active TB patients; the specificity of assays varied from 78.4% (CFP-10) to 90.2% (14KD+38KD) in the healthy control groups. The IgM antibody test can not distinguish a TB infection from a non-TB healthy control. In conclusion, clinical use of the ESAT-6, CFP-10, ESPC, 14KD/38KD and ESAT-6/14KD/38KD antigens based on serodiagnostic IgG assay is of significant value for the rapid diagnosis of TB and for the discrimination between active TB patients and healthy controls.


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