Substrate for hrp

Ten micrograms of each recombinant protein or 20 μg total proteins from ISE6 tick cells were loaded onto a 12% SDS-polyacrylamide pre-cast gel (Life Science, Hercules, CA, USA) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MI, USA) for 2 h at room temperature (RT), and washed four times with TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Tween 20). Purified rabbit IgG were used at a 1:500 dilution in TBS, and the membrane was incubated overnight at 4°C and washed four times with TBS. The membrane was then incubated with an anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:1,000 in TBS with 3% BSA. The membrane was washed five times with TBS and finally developed with TMB (3,3′, 5,5′- tetramethylbenzidine) stabilized substrate for HRP (Promega, Madrid, Spain) according to the manufacturer recommendations.

ESAT-6 was purchased from Sino Biological Inc. (Beijing, China). Anti-ESAT-6 was obtained from Santa Cruz Biotechnology (USA), biotinylated anti-mouse-IgG was from Invitrogen (USA), streptavidin-HRP was procured from Thermo-Scientific (Japan), bovine serum albumin (BSA) was from Promega (USA), and. 16 kDa and Ag85B proteins were obtained from CalBioreagents (USA) and abcan (USA), respectively. ELISA coating buffer (5x) was from Biolegend (Japan). It composed of 0.1 M sodium carbonate (7.13 g NaHCO₃, 1.59 g Na₂CO₃ in 1.0 L; pH 9.5). Substrate for HRP was purchased from Promega and biotin was obtained from Thermo Fisher Scientific, Malaysia. ELISA plates were purchased from Becton Dickinson (France), and the ELISA reader and Tween-20 were from R & M Chemicals (U.K.).