The production of monoclonal antibodies against esat6 of mycobacterium tuberculosis.

Abstract

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), was responsible for 9.6 million cases and 1.5 million deaths globally. Therefore, early TB diagnosis remains a high priority to mitigate the consequences of poor health outcomes and continuous disease transmission. The strategy in detecting the TB antigen instead of antibodies elicited against TB is preferred as immunosuppression in patients co-infected with HIV, makes antibody detection unfavourable. Monoclonal antibodies (MAbs) to TB-specific markers are therefore an attractive option for use in ELISAs and lateral flow tests that effectively meet the criteria for a rapid antigen detection test. Consequently, the objective for the present study was to produce TB-specific MAbs that would aid in the development and optimization of an ELISA for the rapid detection of TB. In order to meet these objectives this study focused on the production and characterization of MAbs that specifically target the early secretory antigenic target 6 (ESAT6) protein from M. tuberculosis. PCR was performed on M. tuberculosis H37Ra DNA, using primers specific to the esat6 gene. PCR products were inserted into pGEM-T vector followed by ligation into the expression vector pGEX6P-1 for transformation into Escherichia coli (E. coli) strain XL-1 Blue. ESAT6 GST protein was expressed and purified by glutathione sepharose affinity chromatography. Thereafter, recombinant ESAT6 GST protein was used to immunise 10 Balb/C mice and stable hybridoma cell lines were generated. The specificity of three monoclonal antibodies were confirmed and identified as anti-ESAT6 DE2-1, anti- ESAT6 KE10-1 and KE10-2. Hybridomas showing cross-reactivity to non-specific antigens, were excluded from the study. Ouchterloney Double-Diffusion was employed and the three MAbs were subtyped as an IgG and two IgM’s respectively. The multi-epitopic nature of ESAT6 is a desirable characteristic in diagnostic assay development. This characteristic was demonstrated by ELISA using anti-ESAT6 DE2-1 as a coating MAb and anti-ESAT6 KE10-1, conjugated to horse radish peroxidase, as the detection antibody. The generation of anti-ESAT6 MAbs in this study, have demonstrated their potential for use in the development of a rapid TB diagnostic test. This is critical in TB management, treatment of the disease, reducing TB transmission and incidence. Future work must therefore be aimed at the development of a diagnostic test, for use at the point-of-care, which would complement the TB diagnostic algorithm.