Quantifying Soluble Galectin-1 by ELISA

Soluble Gal1 was determined using an in-house Enzyme-Linked Immunosorbent Assay (ELISA) as described (34 (link)). In brief, high-binding 96-well microplates (Costar; Corning) were coated with capture Ab (2 μg/mL purified rabbit anti-human Gal1 polyclonal IgG) in 0.1 M sodium carbonate, pH 9.5. After incubation for 18 h at 4 °C, wells were rinsed three times with wash buffer (0.05% Tween-20 in PBS) and incubated for 1 h at room temperature with blocking solution (2% Bovine Serun Albumin (BSA)  in Phosphate buffer saline (PBS)). Samples and standards (100 μL) were diluted in 1% BSA–Tween-20 and incubated for 18 h at 4 °C. Plates were then washed and incubated with 100 ng/mL biotinylated detection Ab (purified rabbit anti-human Gal1 polyclonal IgG) for 1 h. Plates were rinsed three times before adding 0.33 μg/mL HRP-labeled streptavidin (Sigma-Aldrich) for 30 min. After washing, 100 μL 3,3’,5,5’-Tetramethylbenzidine (TMB) solution (0.1 mg/mL tetramethylbenzidine and 0.06% H₂O₂ in citrate–phosphate buffer, pH 5.0) was added to plates. The reaction was stopped by adding 2N H₂SO₄. Optical densities were determined at 450 nm in a Multiskan MS Microplate Reader (Thermo Fisher Scientific). A standard curve ranging from 2.5 to 160 ng/mL human rGal1 was run in parallel.