Evaluating Gal-3 Binding to Neo-Glycoproteins

The affinity of Gal-3 for neo-glycoproteins 9–16 was determined using ELISA as reported previously [15 (link),27 (link),30 (link),49 (link)]. For the immobilization of the respective neo-glycoproteins or non-modified BSA (negative control), we used F16 Maxisorp NUNC-Immuno Modules (Thermo Scientific, Roskilde, Denmark). Per well, an amount of 5 pmol was incubated overnight at a working concentration of 0.1 µM (PBS). Then the wells were blocked with bovine serum albumin (2% w/v) diluted in PBS (1 h, room temperature). Afterwards, recombinant Gal-3 in varying concentration (total volume 50 µL) was added and incubated for 1 h. Detection of bound Gal-3 was achieved using anti-His₆-IgG1 antibody from mouse conjugated with horseradish peroxidase (Roche Diagnostics, Mannheim, Germany) diluted in PBS (1:2000, 50 µL, 1 h, room temperature). TMB One (Kem-En-Tec, Taastrup, Denmark) substrate solution was utilized to initiate reaction of IgG-conjugated peroxidase. The reaction was stopped by adding 3 M hydrochloric acid (50 µL). The binding signal of bound galectin was measured with a spectrophotometer (Spectra Max Plus, Molecular Devices, Sunnyvale, CA, USA) at an optical density of 450 nm. Obtained data were analyzed using SigmaPlot 10 software (Systat Software GmbH, Erkrath, Germany).
In the competitive ELISA design, the F16 Maxisorp NUNC-Immuno Modules (Thermo Scientific, Roskilde, Denmark) were coated overnight with ASF (Sigma Aldrich, Steinheim, Germany; 0.1 µM in PBS, 50 µL, 5 pmol per well) and blocked with BSA (2% w/v) diluted in PBS (1 h, room temperature). Afterwards, a mixture of the respective compound 1–16 in varying concentrations together with Gal-3 (total volume 50 µL; 5 µM final Gal-3 concentration) were added and incubated for 1 h. Detection of bound Gal-3 and data analysis were performed as described above.