Abstract:
The effective removal and control of tuberculosis (TB) disease can be
achieved with early and accurate diagnosis. It accounts for a majority of deaths and
loss of health status thus damaging the economy. The present diagnostics for TB
are not very effective as their sensitivity and specificity are low. Therefore the tests
with more diagnostic value need to be developed. Thus a study was planned to
develop an indigenous technology by exploiting biotechnology tools, and a new
emerging technique i.e. multiplex microbead immunoassay (MMIA). Six potential
recombinant antigenic genes: ag85a, ag85b, ag85c, cfp-10, esat-6 and hspx of
Mycobacterium tuberculosis (M. tuberculosis) were selected for this purpose and
respective genes were transformed into expression strain Bl21DE3pLysS for
overexpression of proteins. Expression of each antigen was optimized for various
conditions like concentration of isopropyl beta-D-1-thiogalactopyranoside (IPTG),
time and temperature. Expression of protein was then confirmed by Western
blotting. After confirmation, proteins were overexpressed in bulk cultures and
purified by using immobilized metal affinity chromatography (IMAC) by using
Histidine-tag (His-tag). The purified proteins were quantified and used to coat on
microbeads at different concentrations and were used for analysis of collected
blood samples. The blood samples of TB patients and healthy controls were
collected from Federal TB Centre, Rawalpindi, from the students of Pir Mehr Ali
Shah - Arid Agriculture University Rawalpindi (PMAS-AAUR), Rawalpindi,
Punjab, Pakistan and healthy controls from USA. The collected human blood
samples were divided as tuberculin skin test negative (TST -ve) healthy controls,
from Pakistan and USA (group 1), TST positive (group 2), reactivated TB patients
xx(group 3) and time points of active TB patients who were diagnosed and were
under treatment (group 4). The coated microbeads were then used to analyze the
presence of antibodies against M. tuberculosis in the collected blood samples. The
results of group 1, Pakistan and USA group (TST negative) showed in general the
absence of antibodies against any of the six antigens used in the MMIA. In the
group 2 (TST positive), low median fluorescence intensity (MFI) values were
detected against all antigens. Further in group 3 (reactivated TB patients) highest
MFI values were observed against all antigens whereas in group 4 (active TB
patient time points) MFI values were higher than group 2 but lower than group 3.
This shows MMIA is very specific in detection of TB. Therefore, based on this it
may be concluded that these antigens can be used to develop MMIA. However, use
of more antigens and standardization is required.