Abstract
BackgroundThe 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1∶1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown.MethodsIn the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera.ResultsOne linear B-cell epitope (KWDAT) consistent with the 162nd–166th sequence of CE and the 57th–61st sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals.ConclusionThere was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.
Highlights
Tuberculosis (TB) remains one of the major global health problems, in most developing countries
In the present report we describe the identification of functional epitopes of CFP10-ESAT-6 (CE) protein by phage display
After alignment of the 9 sequences of these peptides against the amino acid sequence of CE protein, one consensus amino acid sequence WDAT contained by clones E5,E6,E7,E9 and E18 was obtained, which was totally consistent with the 163rd–166th sequence of CE (Figure 1) and the 58th–61st sequence of ESAT-6 protein
Summary
Tuberculosis (TB) remains one of the major global health problems, in most developing countries. Estimates are that one-third of the world’s population is currently infected with Mycobacterium tuberculosis (MTB). In developing countries, the diagnosis of TB still largely depends on the isolation of the slow-growing MTB [2]. Serological tests that are currently commercially available for TB diagnosis continue to produce inconsistent and imprecise estimates of sensitivity and specificity, and a recent meta-analysis of these tests developed very poor quality of evidence to support diagnosis [4]. WHO warned against the utility of current serological tests in the immunodiagnosis of TB and strongly recommended that they must not be used for the diagnosis of pulmonary and extra-pulmonary TB [5,6]. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown
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