Fetuin Terminal Sialic Acid Analysis

An ELLA was performed to analyze the influence of glycoprotein coupling conditions on terminal sialic acid residues of fetuin. 200 µL of a 5 µg x mL⁻¹ fetuin solution (in PBS) was immobilized on 96-well adsorptive microtiter plates (Nunc Immuno Maxisorp, VWR, Langenfeld, Germany) overnight at 4 °C, blocked with BSA, and washed with PBS-Tween (0.05% v/v), as described above. The adsorbed fetuin was then subjected to 50 µL of buffers used for Sepharose coupling (see Section 4.3) with PBS pH 7.5 as a control for one hour at room temperature. After washing three times, samples were incubated with 50 µL of 2 µg x mL⁻¹ biotinylated Maackia amurensis lectin II (MALII, Roche, Mannheim, Germany) and Sambucus nigra elderberry bark lectin (EBL, Roche, Mannheim, Germany) in lectin buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM CaCl₂ ·2H₂O) for one hour. Afterward, for one hour, lectin binding was detected with streptavidin-peroxidase (1:5000 in PBS). The absorption was measured with TMB as described.