Quantification of Plasma Immune Markers

Plasma concentrations of IgG, IgM and soluble CD52 were quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocols (Human CD52 ELISA kit, Aviva Systems Biology, San Diego, CA, USA; Human IgG ELISA quantitation set, Bethyl Laboratories, Montgomery, TX, USA; Human IgM ELISA quantitation set, Bethyl Laboratories, Montgomery, TX, USA). For quantitation of soluble CD52, plasma samples were diluted 1:10. Duplicates were measured for each sample. The optical density values were obtained on a SpectraMax M3 (Molecular Devices, San Jose, CA, USA) at wavelength of 450 nm. Quantification of α2,3 sialylation was determined as previously described (20 (link)). Briefly, plates were coated with MAA-II (Vector Labs, Burlingame, CA, USA) at 20 µg/ml overnight at 4°C, washed twice with PBS, blocked for 1 h at RT with 1% BSA in PBS and plated with 20 µg/ml of CD52-Fc or control Fc prior to incubation with anti-IgG Fc HRP antibody (Bethyl Laboratories, 1:1000 dilution). After two washes with PBS, TMB was added and color development stopped by addition of 0.5M H₂SO₄.