α galactosidase from green coffee beans

To assess the specificity of anti-α-Gal Abs in turkeys, the Galα1-3Gal-HSA antigen (Dextra Laboratories, Reading, UK) was immobilized on an ELISA plate (50 ng/well), and treated or not with α-galactosidase from green coffee beans (Sigma-Aldrich, St. Louis, MO, USA), following the procedure described elsewhere [36 (link)]. Before the treatment, the enzyme was centrifuged at 10,000× g for 10 min at 4 °C, to remove the ammonium sulfate. The supernatant was discarded and 100 mM potassium phosphate buffer (pH 6.5) was added to the pellet, so the final concentration of the enzyme solution was 50 mU/100 µL. The plate was then incubated at 37 °C for 24 h in a humidified plastic chamber to avoid evaporation. After the incubation, wells were washed five times with 150 µL of PBST and the indirect ELISA was performed as described above. Sera samples from the turkeys treated with E. coli O86:B7 (n = 5), E. coli BL21 (n = 5) and PBS (n = 5) were randomly selected and used in the specificity assay. The statistical differences of sera reactivity against treated and non-treated antigen were evaluated using the Wilcoxon signed rank test in the GraphPad 5 Prism program (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant when p < 0.05.

Glycolipids were digested by Endoglycoceramidase I (EGCase I) using a ratio of 1 µg Glycolipids per 1 milliunit enzyme in 1x EGCase I Reaction buffer (New England Biolabs, MA, USA) in PBS for 37°C for 16 hours. Precipitated enzyme was removed after heat inactivation for 20 min at 65°C. 2 µg/ml pig kidney tissue lysates were digested with α-Galactosidase from green coffee beans (Sigma Aldrich) at 10 U/ml in 100 mM potassium phosphate buffer, pH 6.5 for 3 hours at room temperature (RT). Ammonium sulfate was removed from α-Galactosidase preparation before digest by pelleting the enzyme through a centrifugation step at 15,000xg for 10 min at 4°C. The supernatant was collected and the pellet resuspended in an equal volume of potassium phosphate buffer. S. aureus lysate was digested by adding 5 µl of whole lysate to 5 µl potassium phosphate buffer containing 20 U/ml α-Galactosidase (end concentration 10 U/ml) and further processed as described before. For EGCase I digestion, 10 µl bacterial lysate was digested in 1x EGCase I Reaction buffer diluted with PBS and 1 µl EGCase I as described above (end volume 20 µl).