Resolution of triplet components by electrophoresis
Following electrophoresis of platelet-extracted triplets obtained as described above in 6% polyacrylamide gel tubes in Tris-glycine pH 8.3, one gel was stained with Coomassie Brilliant blue G-250 and the bands of O-glycoproteins, albumin and antibody, identified by electrophoretic mobility of corresponding samples isolated from plasma [1], were cut and electroeluted separately. The two glycoprotein bands were also eluted together where required.
ELISA for ligand binding and inhibition assays     Protein or lectin was coated on Nunc MAXISORB microplate wells (maximum 1 µg in 200 µl PBS) by incubation at 37 °C for 3 h or overnight at 4 °C. Wells were washed with PBS containing 0.05 % Tween-20 (PBST) and blocked by incubation for 30 min at 37 °C with PBS containing 0.5 % Tween-20. After another wash with PBST wells were incubated for 2 h at 4 °C with 200 µl PBST containing HRP-labeled or free primary ligands pre-incubated with or without specific sugars. In cases where wells were incubated with primary ligands, wells were washed and treated with HRP-labeled secondary ligands. After washing again thrice with PBST plate-bound HRP was measured by adding  200 µl OPD (0.5 mg/ml) in 0.1 M citrate-phosphate buffer, pH 5.0 containing 0.03% H2O2 for 15 min, followed by addition of 50 µl 12.5 % H2SO4 to stop the reaction and absorbance measured at 490 nm in BIOTEK ELx800 ELISA reader.