Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients with Active Tuberculosis

Kumar, Gyanendra; Dagur, Pradeep K.; Singh, Prashant Kumar; Shankar, Hari; Yadav, V. S.; Katoch, Vishwa Mohan; Bajaj, Bharat; Gupta, Rahul; Sengupta, Utpal; Joshi, Beenu

doi:10.1007/s00005-009-0055-4

Cited by 36 publications

(17 citation statements)

References 40 publications

(39 reference statements)

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“…There are several new diagnostic techniques, such as nucleic acid amplification and immune reactions based on the cell-mediated immune response (10). Detection of antibodies using serodiagnostic tests is a rapid, easy-to-perform, user-friendly method which does not require a living cell.…”

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confidence: 99%

Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

Chen

2012

Clin. Vaccine Immunol.

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ABSTRACTIn order to identify immunodominant antigens ofMycobacterium tuberculosisthat may be used in the serodiagnosis of active tuberculosis (TB), we designed anM. tuberculosisfusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed inEscherichia coliBL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.

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confidence: 99%

Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

Chen

2012

Clin. Vaccine Immunol.

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“…Similarly, all strains shown to export CFP-10 at high levels were virulent, whereas strains with less CFP-10 in supernatants were avirulent. ESAT-6 and CFP-10 have been reported to be strong targets for humoral and cell-mediated immune response (11). RV3615, also known as ESX-1 substrate protein C (ESPC), is a highly immunodominant RD1-dependent secreted antigen specific for MTB infection (12).…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

et al. 2013

International Journal of Molecular Medicine

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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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“…A study done in China [10] reported a sensitivity and specificity of 66.7% and 81.8%, respectively for ESAT-6-CFP-10 antigen. Another study from India reported a sensitivity and specificity of 64.9% and 88.9% for rESAT-6 and 66% and 85.2% for rCFP-10 antigen, respectively [18]. In these studies, they used healthy controls to determine specificity which might have resulted in a higher specificity [7].…”

Section: Discussionmentioning

confidence: 99%

Serodiagnostic Performance of Resat-6-CFP-10 in the Diagnosis of Pulmonary Tuberculosis in Ethiopia

Belay¹,

Bjune²,

Amen³

2011

Mycobact Diseases

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Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients with Active Tuberculosis

Cited by 36 publications

References 40 publications

Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

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

Serodiagnostic Performance of Resat-6-CFP-10 in the Diagnosis of Pulmonary Tuberculosis in Ethiopia

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