Expression and Purification of Mycobacterium tuberculosis Antigens for Use in Immunoassays for Serodetection of M. tuberculosis Infection in TB Patients

Irum Nawaz Awan, Syed Kumail Ali Rizvi, Muhammad Arif Nadeem Saqib, Mirza Imran Shahzad, Aamer Ali Khattak, Sabira Tahseen, Imran Khan, Resmi Ravindran, Paul A Luciw, Azra Khanum

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Tuberculosis (TB) is a fatal and contagious disease. The annual death toll occurring from TB is approximately 2 million according to World Health Organization (WHO). The removal of disease from global face needs immediate treatment for which early diagnosis is pre-requisite. Existing tests for the diagnosis of TB are not efficient and robust. In the present study Mycobacterium tuberculosis specific six antigens namely cfp-10, esat-6 and hspx, along with three antigens which are components of immunodominant mycolyl transferases ag85a, ag85b, ag85c were expressed and purified to evaluate their potential use in immunoassays like Western blotting and multiplex microbead immunoassay. Protein expression of all six antigenic genes was optimized for time and different concentrations of inducer isopropyl β-D-1-thiogalactopyranoside. Protein products were confirmed by Western blotting and purified through immobilized metal affinity chromatography (IMAC) technique using columns having affinity for His-tag. Each fluorescently labeled set of microbeads were coated with one of the M. tuberculosis specific antigenic proteins and later on human plasma samples of reactivated TB patients along with healthy BCG as well as tuberculin skin test negative controls were tested for presence of antibodies against these antigenic proteins individually in a multiplex format. The results were generated in median fluorescence intensity form which detected antibodies against M. tuberculosis specific antigenic proteins only in reactivated TB patients. This system detected antibodies against four antigenic proteins in 100% of reactivated TB patients. Thus, M. tuberculosis antigens described in this study seem to have purified at the level to be used in the development of immunoassays for the detection of M. tuberculosis infection in TB patients of different categories like active and latent TB.

Original languageEnglish (US)
Pages (from-to)217-226
Number of pages10
JournalPakistan Journal of Zoology
Issue number1
StatePublished - 2012


  • Antibodies
  • Antigens
  • Immunoassay
  • M. tuberculosis
  • Median fluorescence intensity
  • MMIA
  • Multiplex microbead
  • Tuberculosis

ASJC Scopus subject areas

  • Animal Science and Zoology


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