Foetal bovine serum (fbs)

NDP-MSH was purchased from Bachem (Bubendorf, Switzerland). Ultrapure LPS (E. coli, 0111:B4) and Pam₃CSK₄ were purchased from Invivogen (San Diego, CA, USA). Fluorescent microspheres (Molecular Probes, F8823) were kindly provided by Dr. Candolfi (INBIOMED, UBA-CONICET). Foetal bovine serum (FBS) was obtained from Natocor (Córdoba, Argentina). DMEM/F-12, DMEM, L-Glutamine and antibiotics were purchased from Invitrogen Life technologies (CA, USA). All other media and supplements were obtained from Sigma-Aldrich Corporation, unless specified otherwise.

Flat-bottom 96 well micro titre half-area plates were coated in triplicate with 25 μL of 1 μg/mL of sFhKT or the recombinant antigens (rFhKT1.1, rFhKT1.3, rFhKT4) in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4°C. After three washes with 100 μL of PBS-0.05% Tween 20 (PBST), 50 μL/well of blocking buffer,10% foetal bovine serum (FBS) (Natocor, Córdoba, Argentina) diluted in PBS was added and incubated for 1 h at 37°C. After washing three times with PBST, 25 uL of serum samples from sheep in six dilutions:2 × 10⁻³; 1 × 10⁻³ ; 5 × 10⁻⁴ ; 2.5 × 10⁻⁴ ; 1.25 × 10⁻⁴ ; 6.25 × 10⁻⁵ in PBST, 1% of FBS of PBS were added. After washing three times, 25 μL/well of HRP-conjugated donkey anti-sheep IgG (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:50.000 in 1% of FBS of PBS, was added and the plates were incubated for 1 h at 37°C. Following five washes with PBST, 50 μL/well of 3,3′,5,5′, tetramethylbenzidine (TMB; Sigma Aldrich, St. Louis, MO, USA) was added and the plates were incubated at room temperature for 4 min. The reaction was stopped by the addition of 25 μL/well of 1 M sulphuric acid. Optical density was measured at 450 nm (OD450) [15 (link)].