Development And Identification Of Monoclonal Antibodies Against ESAT-6Protein Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis(M.tb)6kDa early secreted antigenic target, ESAT-6, is a small secreted protein found from the early secreted culture filter of M.tb and crucial for virulence and immunity. Gene Rv3875encoding ESAT-6, which lacks a signal sequence, locates within the RD1of M.tb genome and encoding95amino acid (aa). RD1can be found only in pathogenic Mycobacterium bovis (M.bovis) and M.tb, but absent in attenuated BCG.ESAT-6protein, had both T-cell and B-cell epitopes, can induce potent cellular and humoral immune response. The most advantage immune response of ESAT-6protein in the phase of M.tb infection is cellular immune response. In the first phase of infection, ESAT-6was consistently found to be a very important target for the T cell response, and this antigen could induced high levels release of interferon (IFN-y). ESAT-6has been proposed as a desirable antigen candidate for vaccine and diagnosis of M.tb infection. Monoclonal antibodies (MAbs) with high specifivity and sensitivity is necessary in the research and application of ESAT-6protein. However, ESAT-6protein has a lower molecular weight and relatively fewer B-cell epitopes, it is hard to develop MAbs against ESAT-6following the standard procedure of creating murine B lymphocyte hybridomas.In this study, the recombinant proteins His-CFP10-ESAT-6(in brief called His-CE) and GST-ESAT-6were expressed and purified as the immunogen antigen and detecting, respectively, to develop the monoclonal antibodies (MAbs) against ESAT-6antigen of M.tb. MAbs against ESAT-6were identified by indirect ELISA after three rounds of subcloning with limiting dilution method, finally, three hyridoma cell lines secreting MAbs against ESAT-6were obtained and named4E1,4D7and4G8, respectively. Isotype analysis revealed4E1,4D7and4G8were IgGl. The indirect ELISA results showed that the titers of three MAbs were about107in ascetic fluids and104in culture supernatant. In MAbs indirect ELISA assay, three MAbs only react with recombinant ESAT-6proteins and natural ESAT-6antigen from M.tb H37Rv, not react with any other recombinant proteins or BCG without ESAT-6.Western blotting analysis revealed that all three MAbs against ESAT-64E1,4D7and4G8could react with recombinant protein rHis-CE and rGST-ESAT-6specifically and present specific band. In addition, three MAbs could react with natural ESAT-6antigen from M.tb H37Rv.The indirect ELISA and competitive ELISA were conducted to define the epitopes recognized by three MAbs. The result revealed that all of them could bind the1-20aa peptides of ESAT-6protein.The recombinant plasmid pCMV-HA-ESAT-6was transfected into HEK293T cells to detect the expression of ESAT-6protein in the cells by the indirect immunofluorescence assay with MAbs against ESAT-6. The expression of ESAT-6protein was detected and significant specific fluorescence on the cell membrane.GST pull down test was operated to capture ESAT-6protein from M.tb H37Rv and lysate of pET-30a-ESAT-6/BL21(DE3) through rGST-CFP10. Western blotting analysis revealed that MAb against ESAT-6could react with ESAT-6from M.tb H37Rv and lysate of pET-30a-ESAT-6/BL21(DE3) captured by rGST-CFP10.In short, three hybridoma cell lines secreted M.tb ESAT-6MAbs were acquired, provided vaulable tool for the studying about the mechanisms of infection and the diagnostic methods of tuberculosis. In addition, three MAbs could react with recombinant ESAT-6proteins and ESAT-6proteins expressed in HEK293T cell, as well as natural ESAT-6antigen from M.tb H37Rv specifically.