title: Generation of Mycobacterium tuberculosis-specific recombinant antigens             and evaluation of the clinical value of antibody detection for serological diagnosis             of pulmonary tuberculosis.
abstract: 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.
authors: Center for Translational Medicine and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu 210093, PR China.
authors: Xia
authors: X
authors: Zhang
authors: Zhonglan
authors: Z
authors: Su
authors: Xiangrong
authors: X
authors: Zhang
authors: Chunmei
authors: C
authors: Hu
authors: Jun
authors: J
authors: Yu
authors: Qian
authors: Q
authors: Gao
authors: Hongwei
authors: H
authors: Wang
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000907:Antibodies, Bacterial / Q000032:analysis / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000097:blood; D007075:Immunoglobulin M / Q000032:analysis / Q000097:blood; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012372:ROC Curve; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012698:Serologic Tests; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*; D055815:Young Adult
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; C072681:Mycobacterium tuberculosis antigens