CD169 Fc Binding to Liposomes Assay

The liposomes (25 µM phosphate) were coated in ethanol on Immuno MaxiSorp plates (NUNC, Roskilde, Denmark) and incubated overnight. The plates were blocked in 1% bovine serum albumin (BSA; Fraction V, Fatty acid free, Calbiochem, San Diego, CA, USA) in phosphate-buffered saline (PBS) and incubated with CD169 Fc or its mutant form (CD169 Fc R97A) (2 μg/mL) for 1 h at room temperature (kindly provided by Prof. Dr. P.R. Crocker, University of Dundee) [41 (link)]. Peroxidase-labelled goat anti-human IgG was added for 30 min at room temperature. After a final washing step, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma-Aldrich) was added as a substrate, and the optical density (OD) was measured in a microplate absorbance spectrophotometer (Biorad, Hercules, CA, USA) at 450 nm.