controlling the disease. During the recent years, there has been emphasis on the development of new and rapid tests for the diagnosis of TB such as detection of M. tuberculosis components in clinical specimen using cell-mediated immune (CMI) responses.6 Tuberculin skin test (TST) with purified protein derivative (PPD) as one of the CMI-based tests in detection of TB infection has low specificity.7 There- fore, a new diagnostic test based on specific antigens would be of great value in screening and detection of TB. The availability of the completely sequenced ge- nome of M. tuberculosis has provided tools for the identification of bacterial antigens that are useful in the development of new reagents to diagnose and control TB.7 Genome of M. tuberculosis consists of 16 regions of differences (RD). Among these regions, the RD1 locus which plays a key role in the virulence of M. tuberculosis, is present in clinically pathogenic strains of M. tuberculosis and M. bovis, but is deleted in M. bovis BCG (bacillus Calmette-Guerin) vaccine strains.4 This region contains nine protein coding genes (RV3871-3879c), which probably encodes pro- tective and/or virulent antigens.7,8 Among the major antigens of RD1 locus, early se- cretory antigenic target-6 (ESAT-6) and culture fil- trate protein-10 (CFP-10) are encoded by the genes esxA and esxB respectively and form a 1:1 heterodi- meric complex in vitro. ESAT-6 is the best- characterized protein within the RD1 region. It has been recognized as an important stimulator of T-cells both in vitro and in vivo.9 Also, it has been proposed as a tool for diagnosis of M. tuberculosis infection and frequently used in enzyme-linked immunospot assay (ELISPOT).10,11 This assay is a specific method for identifying M. tuberculosis infection from the re- sponse to BCG vaccination. It has based on produc- tion of interferon gamma (IFN-γ by activated T cells after exposure to M. tuberculosis antigens. The CMI-based tests such as ELISPOT permit ear- ly detection of TB infection in a setting of low ende- micity and provide an inexpensive tool for diagnosis of TB in developing countries. Given the significance of ESAT-6 and CFP-10 secretion in mycobacterial virulence and the importance of these antigens as di- agnostic tools for the detection of TB, we expressed and purified these two antigens as well as their fused form. In order to evaluate their validity for detection of TB patients, commercial ESAT-6 was used and its diagnostic potential compared with the produced re- combinant proteins. Recombinant antigens in diagnosis of TB Materials and Methods The study population (n=60) was divided into two groups: The first group (n=30) comprised of patients who were diagnosed with pulmonary TB (as con- firmed by examination of sputum for acid-fast bacilli and/or culturing), with no history of treatment for the disease. Twenty percent of these patients were Af- ghan immigrants and PPD skin test for all TB patients was performed. Twenty five percent of the patients had negative PPD skin test. All blood samples were obtained from the newly diagnosed sputum smear positive patients reported to the Masih Daneshvari Hospital in Tehran before administration of chemo- therapy. The healthy BCG-vaccinated individuals (n=30) were group number 2. Healthy donors were individuals without prolonged direct contact with TB patients, with no vaccination during 10 years ago or any other clinical TB symptom. HIV positive indi- viduals were excluded from the study. All the patients and normal subjects gave their written informed con- sent prior to participation in the study. Mouse monoclonal ESAT-6 antibody and Glutathi- one S Transferase (GST) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and mouse monoclonal CFP-10 antibody was purchased from Abcam; Glutathione agarose was purchased from Sigma and isopropyl-1-thio-β-D-galactopyranoside (IPTG) was obtained from Fermentas (USA). Com- mercial ESAT-6 was obtained from Staten Serum In- stitute (Copenhagen, Denmark). Purified DNA from different strains of Mycobac- teria including Mycobacterium tuberculosis C (M. tuberculosis C), Mycobacterium tuberculosis DT, Mycobacterium tuberculosis PN, Mycobacterium bo- vis AN5 and Mycobacterium IR 150 were provided from Razi Serum and Vaccine Research Institute (Tehran, Iran). Using bioinformatic analysis, we compared the genome of these strains with M. tuber- culosis H37RV. Native M. tuberculosis C strain, con- sequently was selected for our analysis due to its ge- nomic resemblance to H37RV. Standard procedures for PCR, transformation, cloning and analysis of DNA were used.5-12 All con- structs made by PCR were sequenced to verify their integrity. Esat-6, cfp-10 and esat-6/cfp-10 fusion genes were amplified from genomic DNA of M. tu- berculosis C using the forward and reverse primers shown in Table 1. The PCR products were cloned into pGEX4T1 containing N-terminal GST tag. Re- combinant plasmids were transformed to E. coli WWW.ircmj.com Vol 13 August 2011 557