The detection of antibodies to Mycobacterium tuberculosis by microplate enzyme-linked immunosorbent assay (ELISA).
ABSTRACT The detection of antibodies to Mycobacterium tuberculosis by enzyme-linked immunosorbent assay has proved to be a potentially useful technique for the serodiagnosis of tuberculosis. The technique is capable of full automation. The use of a purified antigen should further improve the sensitivity of the method.
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ABSTRACT: In order to identify immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active tuberculosis (TB), we designed an M. tuberculosis fusion 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 in Escherichia coli BL21 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.Clinical and vaccine Immunology: CVI 02/2012; 19(4):536-44. · 2.60 Impact Factor
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ABSTRACT: Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations. A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant. The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.PLoS ONE 01/2012; 7(7):e40213. · 3.73 Impact Factor
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ABSTRACT: Bovine tuberculosis represents one of the very important infectious diseases in Egypt and the world. It has zoonotic importance and causes severe economic losses. Accurate and rapid diagnosis considered as the milestone for control of the disease. In this study ELISA technique was used for confirmation of positive reactors cows that tested with single intradermal tuberculin test, to detect false positive reactors. Bovine PPD and ST.CF antigens have been used as two different coating antigens for ELISA technique. 3747 cattle from dairy farms in five different governorates were subjected to the single intradermal cervical tuberculin test whereas 78 (2.24%) proved positive reactors to tuberculin. These positive reactors tested with ELISA. 64 (82.05%) animals were positive by ELISA coated with ST-CF, while by using bovine PPD as coating antigen 58 (74.35%) animals were positive. The previous results indicated that ELISA test showed higher sensitivity and specificity using ST-CF as coating antigen than in case of bovine PPD coating antigen.Beni-Suef Veterinary Medical Journal. 02/2013; 22(1687-7926):126-129.