The Rv1419 PCR fragment representing the entire ORF was generated with specific primers engineered to introduce NdeI e XhoI restriction enzymes sites into the resulting PCR product, using Mtb H37Rv DNA as template: NdeI, sense (5′-GGAATTCCATATGGGTGAATTACGGTTGG-3′) and XhoI, antisense (5′-CCGCTCGAGTCATTACGGCACGCTATCCC-3′). PCR was performed (4 min at 94°C, PD0325901 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C for 36 cycles) and sequence was confirmed by DNA sequencing. E. coli BL21(DE3) was grown at 37°C to an A600(nm) of 0.6, and the expression was performed in the presence

of 1 mM isopropylthiogalactoside. Following 4 h induction, cells were harvested by centrifugation and resuspended in 10 mM Na2HPO4, 10 mM NaH2PO4, 0.5 M NaCl, and 10 mM of imidazole (lysis buffer). Cells were lysed by sonication three times at 30% of amplitude and centrifuged at 5400×g, 4°C for 20 min. rec-sMTL-13 was recovered as inclusion bodies and resuspended in lysis buffer containing 8 M urea. rec-sMTL-13 was purified by nickel affinity chromatography (GE Healthcare, Brazil) under denaturing conditions, dialyzed, and resuspended in PBS. Subcellular fractions from Mtb H37Rv were used. Whole cell lysate, CFP, membrane, and cell wall fractions were obtained by strain

growth to a late-log phase (day 14) in GAS medium as described elsewhere 14, 48, 49. Balb/c mice were i.p. immunized with rec-sMTL-13 (4×20 μg) plus AluGel followed by one (20 μg) i.v. injection with the lectin at weekly intervals. click here Splenocytes were fused with Ag8XP3653 myeloma cells (kindly provided by Prof. Carlos Zanetti/UFSC) in a 5:1 ratio using PEG 50% as fusogen. Cells were then cultured in RPMI 1640 medium (Invitrogen, Brazil) supplemented with 20% FBS (Hyclone, USA) and hybridomas were selected

using 0.1 mM hypoxanthine, 4×10−4 M aminopterine and 0.016 mM thymidine. Hybridoma supernatants were screened by ELISA, in which purified rec-sMTL-13 was used as the capture antigen (see Detection of Ab against Interleukin-3 receptor sMTL-13 by ELISA ). Out of the initial 900 clones screened, 12 positive clones were selected based on production of higher titers of Ab against the lectin. Of these, one clone was subcloned by limited dilution and Ig class and subclass were found to be IgG1κ as determined by the SBA Clonotyping System/HRP (Southern Biotech, USA). The UFPR Animal Experimental Ethics Committee has approved the study protocol (23075.031314/2008-41). Polystyrene microplates (Biosystems, Brazil) were coated overnight with sMTL-13 (5 μg/mL) diluted in 0.06 M carbonate buffer (pH9.6). Microplates were blocked, washed, and incubated with supernatants from hybridome cultures for 40 min at 37°C. Plates were then incubated with HRP-goat anti-mouse IgG (SC Biotechnology, USA; 1:1,200) for 40 min at 37°C. Color development was performed by adding ABTS® Peroxidase substrate (KPL, USA).