Limulus amebocyte lysate assay

H1N1 influenza virus, A/PR/34/8 (PR8), was passaged in Madin-Darby canine kidney (MDCK, ATCC, #CCL-34™, Manassas, VA) cells. Virus was grown in MDCK cells in DMEM/F12 with ITS+ (BD Biosciences, Franklin Lakes, NJ) exposed to trypsin, harvested at 72 hours postinfection, and titered by plaque assay in MDCK cells. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The lower limit of detection of this assay is 0.1 EU/ml or approximately 20 pg/ml LPS. For determination of viral titers in infected mice, whole mouse lungs were collected and homogenized in 1 ml of ice-cold PBS. Solid debris was pelleted by centrifugation, and viral titer was determined using a standard plaque assay on MDCK cells.

Three dosR regulon-encoded (Rv1737c, Rv2029c, Rv2628), two Resuscitation Promoting (Rv0867c and Rv2389c) antigens from Mycobacterium tuberculosis (Mtb), recognized as immunogenic in a previous study [29 (link)], and the RD1 fusion Protein ESAT6-CFP10 (E6-C10) were used in the present study. The recombinant proteins were produced and QC-ed by CLMCF and THMO. The production, quality control, preparation and storage of the antigens was previously described [24 (link), 39 (link)]. Briefly, genes were amplified by PCR and cloned by Gateway Technology (Invitrogen, San Diego, CA) in a bacterial expression vector containing an N-terminal histidine tag. The proteins were overexpressed in Escherichia coli BL21(DE3) and purified, as described previously [40 (link)]. Purity and size were checked by gel electrophoresis and Western blotting with anti-His antibodies and anti-E. coli antibodies. Residual endotoxin levels were determined by a Limulus amebocyte lysate assay (Cambrex) and were found to be below 50 IU/mg recombinant protein. Recombinant antigens were freeze-dried and shipped at ambient temperature to the Colombian research site, prepared as described [40 (link)] aliquoted and stored at −80 °C until further use. The purified protein extract (RT50) (PPD) from Staten Serum Institute (Copenhagen, Denmark) was also used in this study.

H1N1 influenza virus, A/PR/34/8 (PR8), was passaged in Madin–Darby canine kidney (MDCK) cells. Virus was grown in MDCK cells in DMEM/F12 with ITS+ (BD Biosciences, Franklin Lakes, NJ) and trypsin, harvested at 72 h postinfection, and titered by plaque assay in MDCK cells. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The lower limit of detection of this assay is 0.1 EU/ml or approximately 20 pg/ml LPS. For determination of viral titers in infected mice, whole mouse lungs were collected and homogenized in 1 ml of ice cold PBS. Solid debris was pelleted by centrifugation and viral titer was determined using a standard plaque assay on MDCK cells [22 (link)]. Results were expressed as PFU/ml of extract.

H1N1 influenza virus, A/PR/34/8 (PR8), was passaged in Madin-Darby canine kidney (MDCK) cells. Virus was propagated in MDCK cells in DMEM/F12 with ITS+ (BD Biosciences, Franklin Lakes, NJ) and trypsin, harvested at 72 h post-infection and titered by plaque assay in MDCK cells. The virus was stored in aliquots at −80 °C. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The lower limit of detection of this assay is 0.1 EU/mL or approximately 20 pg/mL LPS.

RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin, L-Glutamine were purchased from Euroclone, Devon, UK. Fycoll was purchased from Cederlane Labs and Percoll from Amersham Bioscience, Pittsburgh, PA, USA. Recombinant human granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-13 (IL-13) were purchased from ProSpec TechnoGene, East Brunswick, NJ, USA. All reagents contained <0.125 endotoxin units/mL, as checked by the Limulus Amebocyte Lysate assay (Cambrex, East Rutherford, NJ, USA). LPS from Escherichia coli strain 055:B5 was obtained from Sigma–Aldrich, Milano, Italy. Wortmannin was purchased from Tocris Biosciences, Bristol, UK.