Des h37 ra

Mouse splenocytes were isolated as described above. 5 × 10⁵ splenocytes were plated in 96-well U-bottom plates (Greiner) in R10 medium and incubated in the presence or absence of F(ab’)₂ fragments with 0.001, 0.01, and 0.1 μg/ml dried, inactivated M. tuberculosis (Des. H37 Ra, Difco Laboratories) suspended in R10 medium for 12 h under SCCC.

Dried, inactivated M. tuberculosis (Des. H37 Ra, Difco Laboratories, Detroit, USA) was suspended and diluted to a final concentration of 50 mg/ml in 50 mM carbonate buffer (pH 9.6). Per well, 50 μl diluted M. tuberculosis were coated overnight at 4 °C to flat-bottom 96-well plates (MaxiSorp®, Nunc). After washing extensively with PBS containing 0.1 % Tween20 (wash buffer), wells were blocked with PBS containing 1 % bovine serum albumin and 0.05 % Tween20 (blocking buffer) for 2 h at RT. Thereafter, all liquid was removed, and IVIG, F(ab’)₂, or Rituximab (Mabtera, Roche) were diluted to 1, 0.1, 0.01, and 0.001 μg/ml with blocking buffer and added to the wells. After incubation for 1 h at RT, plates were washed with wash buffer and incubated for 1 h with biotinylated mouse-anti-human IgG (BD, clone G18-145) diluted in blocking buffer to a final concentration of 0.5 μg/ml. Wells were washed again and incubated with horseradish peroxidase (HRP) coupled streptavidin (Mabtech). 1-StepTM Ultra TMB-ELISA Substrate Solution (ThermoFischer Scientific) was used to detect HRP activity. The reaction was stopped by addition of 2 M NaCl, and colorimetric signals were evaluated by measuring the optical density at 450 nm.