Also, it has been proposed as a tool for diagnosis of infection and frequently used in enzyme-linked immunospot assay (ELISPOT) [8,9]

Also, it has been proposed as a tool for diagnosis of infection and frequently used in enzyme-linked immunospot assay (ELISPOT) [8,9]. Based on these findings, we proposed that ESAT-6 could be acted as a target for developing novel mAbs in TB immunological detection. of 100% in all cases. Our data indicated that the mAb generated in this study can potentially serve as a tool in the laboratory diagnosis of TB. (which is an intracellular pathogen. Fast and accurate diagnosis of TB is an important element in controlling this disease. However, the diagnosis of latent infection remains difficult due to the lack of a simple, reliable test for infection. Currently, infection can be diagnosed with the help of the tuberculin skin test (TST) using purified protein derivative (PPD), but this conventional method has known limitations in accuracy and reliability. Furthermore, interpretation of serial TST results is complicated by nonspecific variation and because of its intradermal application, by potential boosting from precedent tests [2]. Conversion of the PPD reactivity from negative to positive challenges the clinician [3]. Genome of H37Rv consists of 16 regions of differences (RD) ranging from 2 to 12.7 kb, which are deleted in most BCG variants and members of TBC [4]. Nine of the RDs (containing 61 ORFs) are absent from all the BCG strains as well as all virulent strains [4]. One key deletion, RD1, was Acrivastine missing from all the BCG strains but present in all other complex members including [4]. The RD regions are largely responsible for the heterogeneity in their epidemiological and clinical behavior. The RD1 locus plays a key role in the virulence of [5]. Among the major antigens of RD1 locus, comparative genomic studies have revealed two genes, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) exclusively present in several pathogenic mycobacterial species, including and non-tuberculosis species Acrivastine [6]. ESAT-6 is the best characterized protein within the RD1 region. It has been recognized that ESAT-6 can induce a strong T-cell-mediated immune response both in vitro and in vivo [7]. Also, it has been proposed as a tool for diagnosis of infection and frequently used in enzyme-linked immunospot assay (ELISPOT) [8,9]. Based on these findings, we proposed that ESAT-6 could be acted as a target for developing novel mAbs in TB immunological detection. Specific mAbs against may prove to be ideal reagents for diagnosing TB infection. In the present study, we generated and characterized a new mAb that specifically anti ESAT-6 protein. This development has great utility for immunoblotting, IP and ELISA. Moreover, the isotype of this mAb was an IgG2b with a kappa chain. Clinical validation of this mAb showed that it has highly detection and sensitivity rates of ESAT-6 in TB cases. Thus, this mAb will provide a powerful tool for the laboratory diagnosis of TB infection. Materials and methods Bio-synthesis of the target peptide The target peptide consisted of 13 amino acids (aa), ranging from 24 to 36 residues of ESAT-6 protein (aa sequence: SIHSLLDEGKQSL), was synthesized by a conventional solid-state reaction technique (Applied Biosystems, Inc., CA, USA), based on the widely used Fmoc strategy (optimized for antigenicity and specificity), using the Wang resin as the solid support [10]. After synthesis, the peptides were cleaved from the resin and purified in High Performance Liquid Chromatography (HPLC), and the mass spectrometry (MS) was carried out to evaluate the quality of the synthesized peptides (purity 95%). Immunization of mice BALB/c mice were obtained from Shanghai Experimental Animal Centre of the Chinese Academy of Sciences (Shanghai, China). Animal experiments were performed in accordance with the guidelines of the Chinese Council on Animal Care and approved by Hangzhou First Peoples Hospital Committees on Animal Experimentation. The female mice, six weeks old, receiving the regular injections of 0.2 ml ESAT-6 antigen were immunized TM4SF18 one time with a two-week interval, repeated four times. Six weeks after enhanced immunization, the mice were killed and the lymphoblasts were obtained from Acrivastine the spleen for the next fusion stage. Generation of anti-ESAT-6 mAb Mouse myeloma Sp2/0 cells were purchased from American Type Culture Collection (Manassas, VA, USA), and was grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS). One day before fusion, the peritoneal macrophages from the normal BALB/c mice were used as feeder layer cells. Spleen cells from immunized animals were fused with Sp2/0 myeloma cells according to the protocol that had been previously described [11]. More than 100 independent hybridomas were obtained from two fusions. Positive hybridomas clones were selected by coating-antigen ELISA. To obtain the stable mAb-expression hybridomas, a limiting.