Lectin-Based ELISA

In order to detect specific glycan structures exposed on IgG, a Lectin-based ELISA assay was performed on the serum samples (Sjöwall et al., 2015; Stümer et al., 2017). Specific glycosylation patterns were evaluated by biotin-labelled lectins: aleuria aurantia lectin (AAL), lens culinaris agglutinin (LCA), O-glycosidically linked galactose/N-acetylgalactosamine (GalNAc) ligand jacalin (JAC) and sambucus nigra lectin (SNA) (Vector Laboratories, United States).

Protein A from Staphylococcus aureus (Sigma-Aldrich) was diluted in coating buffer (0.1 M Na₂Co₃/NaHCO₃, pH 9,6) to 20 μg/ml and applied onto 96-well MaxiSorpTM microtitre plates (Nunc, Roskilde, Denmark; F96) at 4° overnight (50 µL/well). Alternatively, Fab2-fragment of goat anti-human Fab-specific IgG (Jackson Laboratories Immunoresearch, West Grove, PA, United States) was used at 2 μg/ml.

A blocking buffer (60 µL/well) was then applied onto the plates at room temperature (RT) for 1 h. As blocking buffer, we used 3% deglycosylated gelatin with 0.1% CaCl2 and 0.1% MgCl2. Gelatin was achieved by treatment with periodic acid for 24 h and subsequent dialysis against TBS-Ca-Mg until a pH -value of 7,4 was reached. Sera were diluted 1:500 in washing solution (TBS-Ca-Mg 0.05% Tween-20) and incubated at RT for 3 h (50 µL/well). After 3 washes with washing solution (150 µL/well), plates were incubated at RT for 1 h with biotin-labelled lectins diluted in TBS Ca-Mg (50 mcl/well), at various concentrations (AAL 100 ng/ml, LCA 100 ng/ml, GalNac -Jacalin 50 ng/ml, SNA 50 ng/ml). After washing, HRP-conjugated streptavidin in TBS Ca-Mg was incubated for ½ h at RT (50 µL/well), then washed again and Substrate Solution (TMB) was added (75 µL/well). After 10–15 min stopped with H₂SO₄ (37.5 mcl/well). Optical density values were obtained with the ELISA-reader employing a 450 nm filter. Each serum was tested in duplicate; the final value was obtained by the mean of the two results minus blank. Three samples were used as inter-assay controls. Each test was performed in blind, results were expressed as Optical Density (OD).