*4.3. Lectin Activity Assay*

The lectin activity assay was performed as described earlier (10). Briefly, 150 of porcine stomach mucin (PSM) with concentration of 0.1 mg/mL (0.1 M carbonate buffer, pH 9.5, containing 0.15 M NaCl) was added to each well of a polystyrene 96-well ELISA microtiter plate Maxisorp (Thermo Fisher Scientific, Waltham, MA, USA), incubated at 4 ◦C overnight, washed three times with the buffer containing 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100 (TBS-T) and three times with water. Bovine serum albumin (1 mg/mL) in TBS-T was added as described above. Samples containing recombinant CGL (0.2 mg/mL) were two-fold serially diluted in TBS-T and added in 150 mL aliquots to each well. The plate was incubated at room temperature for 1 h and then washed three times as described above. TBS-T was used as a negative control. Standard assay for alkaline phosphatase activity was carried out as described earlier [10]. One unit of AP activity was defined as the quantity of the enzyme required to release 1.0 μmol of p-nitrophenol from pNPP in 1 min. The specific activity was calculated as units per 1 mg of protein. All lectin activity assays were performed in three independent parallels for three to five times. Data were analyzed using the Student's t-test of the SigmaPlot 2000 version 6.0 program (SPSS Inc.). Differences from controls were considered significant at *p* ≤ 0.05.
