The production of monoclonal antibodies against esat6 of mycobacterium tuberculosis.
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Date
2016
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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.
Description
Master’s degree. University of KwaZulu-Natal, Durban.