The immobilization of BTLA protein in a microcolumn was performed using the NHS-activated acid-coupled Sepharose 4B (Sigma Aldrich). BTLA protein was dissolved in 100 μl of coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) and was added to 0.06 g NHS-activated Sepharose in 100 μl of coupling buffer. The coupling reaction was performed for 1 hour in 25°C and the mixture was transferred into the microcolumn (MoBiTec, Goettingen, Germany). The microcolumn with immobilized protein was washed alternately with 10 ml of solution blocking buffer (0.1 M amino ethanol, 0.5 M NaCl, pH 8.3) and washing buffer (0.1 M CH3COONa, 0.5 M NaCl, pH 4). This step was repeated 4 times. The microcolumn was stored in PBS (pH 7.4).