2.10. CD6 Competition ELISA

A CD6 competition ELISA was performed according to Willrodt et al. [7]. Briefly, a 96-well plate (Nunc) was coated overnight at 4 °C with hALCAM (5 µg/mL) in PBS. The plate was blocked with 2% milk–PBS and subsequently incubated for 2 h at RT with a fixed amount of murine CD6-Fc (0.25 μg/mL, 727-CD, R&D Systems) and decreasing concentrations of antibody in PBS (2000 nM to 0.002 nM). Bound CD6-Fc was detected using HRP-coupled antihuman IgG antibody (A2554, Sigma-Aldrich). Peroxidase activity was detected by adding 100 µL of TMB substrate (77248, Biolegend), and the colorimetric reaction was stopped by adding 50 µL of 1 M H₂SO₄, followed by absorbance measurements at 450 nm and 570 nm using a plate reader (Tecan).