CD169-Liposome Binding Assay

Liposomes were diluted in ethanol to a final concentration of 25 µM phospholipids and coated on Immuno MaxiSorp plates (NUNC, Roskilde, Denmark). 1% BSA (BSA; Fraction V, Fatty acid free, Calbiochem, San Diego, CA, USA) diluted in PBS was used to block coated plates. Next, samples were incubated with CD169 Fc or its mutant form (CD169 Fc R97A) (2 μg/mL) for 1 hour at room temperature (kindly provided by Prof. Dr. P.R. Crocker, University of Dundee) (57 (link)). Then, peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch, Ely, UK) was added for an additional 30 minutes and plates were washed, TMB (Sigma Aldrich, Darmstadt, Germany) was added as a substrate and the optical density (OD) measured in a microplate absorbance spectrophotometer (Biorad, Hercules, CA, USA) at 450 nm.