Mtb h37rv lysate

Mycobacteria-, Ag85B- and ESAT-6-specific antibodies in serum were measured by indirect enzyme-linked immunosorbent assay using Mtb H37Rv lysate (BEI Resources), recombinant Ag85B (BEI Resources), or recombinant ESAT-6 (BEI Resources) to coat and AP-labeled anti-mouse IgG, IgG1, and IgG2c for detection (SouthernBiotech).

Frozen PBMC were thawed and seeded in 96 well plates at a density of 0.5–1 x 10⁶/well. Cells were activated with 10 μg/ml Mtb H37Rv lysate (BEI Resources, Manassas, VA, USA) and 5 μg/ml PHA (Remel, Thermo Fisher Scientific, Lenexa, KS, USA) for 16 hours. Along with stimulants, 1X monensin (BD Biosciences, USA) and 1X Brefeldin (BD Biosciences, USA) solution and 1 μg/ml BD Fast Immune CD28/49d (BD Biosciences, USA) were also added. Unstimulated cells were used as controls. At the end of 16 hrs, cells were first stained with an antibody cocktail comprising Avid, anti-CD45RA APC-H7, anti-CD127 PerCP Cy5.5, anti-HLA-DR FITC and anti-CD25 BV421 at 4°C for 10 minutes. Cells were then fixed with 1X FACS Lyse (BD Biosciences, USA), permeabilised with 1X FACS Perm (BD Biosciences, USA) and stained with an antibody cocktail of anti-CD3 BV570, anti-CD4 BUV395, anti-IFNγ Alexa Fluor 700 (clone B27, BD PHArmingen, BD Biosciences, USA), anti-IL-2 PE (clone MQ117H12, BD Biosciences, USA), anti-IL17A BV605 (clone BL168, Biolegend, San Diego, CA, USA), anti-IL-22 PECy7 (clone 22URT1, eBiosciences, San Diego, CA, USA) and anti-IL-10 BV786 (clone JES3-9D7, BD Biosciences, USA) at room temperature for 30 minutes. Cells were washed, fixed with 1% paraformaldehyde and acquired on BD FACS Aria Fusion using appropriate compensation controls. Data was analysed using FlowJo.