A homemade sandwich ELISA was used to quantify TGFBI levels in serum samples. MaxiSorp 96-well microtiter plates (Nunc, GmbH, Germany) were coated with 100 µL of anti-TGFBI antibody (clone 4G9A10; for details see Supplemental Data), at the concentration of 1 µg/mL in carbonate buffer, and incubated at 4 °C overnight. Coated wells were washed three times with PBS/Tween-20 (0.05%) and blocked with 200 µL of 10% FBS/PBS at 37 °C for 2h. After washing three times as before, 100 µL of serum sample (1:20 in PBS) was added to each well. Serial dilutions of human recombinant TGFBI (Targetome SA), from 0 µg/mL to 5 µg/mL, were prepared in PBS to serve as calibration curve. Plates were incubated at 37 °C for 1h. After washing, 100 µL of anti-TGFBI antibody (1:500 in 10% FBS/PBS; clone 4G6B10, detailed in Supplemental Data) was added to each well at 37 °C for 1h, followed, after washing, by incubation with 100 µL of anti-mouse IgG (1:3000 in 10% FBS/PBS; Dako; cat. no. P0260) at 37 °C for 1h. After washing, wells were incubated with 30% H2O2 and 1 mM 2,2'-Azinobis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) solution, and the optical density was read using a Filter Max F5 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.

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