Designing Fusion Molecules from Antigens of Mycobacterium tuberculosis to Enhance Serodiagnostic Sensitivity in Latent TB Infection and Active TB State

The development of rapid, cost-effective and reliable diagnostic tools is essential to effectively control tuberculosis (TB) especially in TB endemic regions having poor socioeconomic status. Detection of multiple antibodies against antigens of Mycobacterium tuberculosis ( Mtb ) by fusion molecules may improve the sensitivity of TB serodiagnosis. In this study, two fusion molecules, Rv2626c-Rv2031c and Rv2626c-tnRv1827 were constructed using the native Rv2626c, Rv2031c and Rv1827 after predicting B-cell epitopes location through in silico analysis. All the native and the fusion proteins were expressed in Escherichia coli in a soluble form and purified. To evaluate their serodiagnostic potential, ELISA was performed to detect antibodies in plasma samples obtained from 116 active TB (ATB) patients and 90 latent TB (LTB) individuals. Sensitivity of Rv2626c, Rv2031c and Rv1827 were 31.0, 30.2 and 33.6% for ATB group and 36.7, 34.4 and 2.2% for LTB group, respectively. Whereas, Rv2626c-tnRv1827 and Rv2626c-Rv2031c showed improved sensitivities of 53.5% and 50.9% in the ATB group and 37.8 and 63.3% in the LTB group, respectively. The sensitivities of fusion molecules were found close to the expected value of combined sensitivities of the two individual antigens, confirming that the epitopes in the fusion molecule were sufficiently exposed for antigen–antibody interaction. Their secondary structure composition determined by circular dichroism spectroscopy was in agreement with that obtained through molecular modelling analysis. The fusion molecule Rv2626c-Rv2031c appears to be a potential preliminary screening candidate, which can be used in the construction of fusion molecules containing three or more Mtb antigens with higher sensitivity for clinically acceptable serodiagnosis of TB.