Total proteins from Mtb subcellular fractions (10 or 50 μg per lane) were subjected to SDS-PAGE (Invitrogen, USA) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in tris-buffered saline containing 0.1% Tween RG7420 cell line 20 (TBS-T) and probed with the monoclonal supernatant (Clone 276.B7/IgG1κ) or anti-19 KD lipoprotein mAb (clone IT-19; kindly provided by Dr. Antônio Rothfuchs, NIH/NIAID-TVTRM Contract) at 1:1000 dilution followed by incubation with HRP-conjugated secondary Ab (1:2000). Detection was

performed by ECL analysis (Pierce, USA). Thirty-four patients with active pulmonary TB in the Division of Respiratory Diseases of the Central Public Health Clinic of Juiz de Fora, Minas Gerais State and 11 active-diseased patients from Hospital Octávio Mangabeira, Bahia, Brazil were selected. Only those patients with detectable AFB in the sputum bacilloscopy or culture-confirmed disease and who had selleck undergone clinical and chest X-ray examinations, as prescribed by the Brazilian Ministry of Health, were included in the study. AIDS, diabetes, hepatitis, hypertension, pregnancy, and alcoholism were exclusion

criteria. All patients included in the study have been confirmed to present negative bacilloscopy following treatment. Thirty-eight healthy BCG-vaccinated, which constituted the endemic control (EC) group formed by medical students and staff from UFJF, five foreign PPD-negative non-BCG-vaccinated subjects (the non-endemic group) and six PPD-negative BCG-vaccinated individuals were included in the control groups without prior history of Mtb infection. All patients and control

subjects have been informed of the study and have given consent for blood sampling. The UFJF Medical Ethics as well as the Oswaldo Cruz Foundation Committees have approved the study protocols (UFJF-1495.186.2008; CPqGM-219 (CAAE) 2221.0.000.225-06). Histological sections from pleural TB patients or control leprosy patients were deparaffinized in xylene, rehydrated in alcohol and water. Quenching of endogenous peroxidase was performed with a 1.5% hydrogen peroxide-methanol solution for 20 min. Sections were incubated Megestrol Acetate with normal goat serum (30 min 37°C) and then exposed to monoclonal anti-sMTL-13 supernatant (Clone 276.B7). Incubations with biotinylated goat anti-mouse Ab with streptavidin−HRP complex (Vectastain Elite ABC reagent, Vector Laboratories, CA, USA) were performed for 30 min at 37°C. Positive reactions was detected with 3,3′-diaminobenzidine (Dako Cytomation, CA, USA), followed by Harris’s hematoxylin counterstaining. Sections were examined microscopically and images were acquired using a Sight DS-5M-L1 digital camera (Nikon, Melville, NY, USA) connected to an Eclipse 50i light microscope (Nikon). Maxsorb plates (Nunc, Denmark) were coated with rec-sMTL-13 in carbonate buffer overnight at 4°C. Plates were washed with PBS/0.05% Tween-20.