Title of Thesis

Development Of Serodiagnostic Test For Tuberculosis By Using Mycobacterium Tuberculosis Specific Antigens

Author(s)

Irum Nawaz Awan

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 (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.

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2.1 Tuberculosis (tb) Disease
2.2 Immune Responses Against Tb
2.3 Mycobacterium Tuberculosis, The Causative Agent
2.4 Bcg Vaccination
2.5 Regions Of Differentiation (rd)
2.6 Tb Pathogenesis
2.7 Diagnostic Tests
2.8 Afb Smear Microscopy And Culture
2.9 Radiological Examinations
2.10 Tuberculin Skin Test (tst)
2.11 T Spot Tb And Quantiferon-tb Gold Test
2.12 Nucleic Acid Amplification Tests (nats)
2.13 Serodaignostic Potential Of Antigens

3.1 Plasmids
3.2 Transformation In Bl21de3plyss Cells And Restriction
3.3 Expression Studies And Sds-page
3.4 Bulk Expression
3.5 Western Blotting
3.6 Purification Of Expressed Proteins
3.7 Dialysis
3.8 Patient Selection For Plasma Sample Collection
3.9 Multiplex Microbead Immunoassay

4.1 Transformation Into Expression Strain Bl21de3plyss
4.2 Restriction Analysis
4.3 Expression Studies
4.4 Bulk Expression Of Proteins
4.5 Purification Of Expressed Proteins By Affinity Chromatography And Purity Check By Sds-page
4.6 Western Blotting
4.7 Multiplex Microbead Immunoassay

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