H fabp

The serum levels of KIM-1 (R&D, USA) and H-FABP (Abcam, UK) and NAG activities (Abcam, UK) were measured by commercially available ELISA and activity assay kits following the manufacturer’s recommendations. Briefly, for the KIM-1 and H-FABP ELISAs, 50 μl of serum sample was added to plates precoated with a monoclonal antibody and incubated for 2 h at room temperature. After washing three times with wash buffer, 200 μl human serum KIM-1/H-FABP conjugate antibody was added to each well and incubated for another 2 h. After washing 3 times with buffer, the plates were incubated with 200 µl substrate solution for 30 min, and 50 µl stopping buffer was added to terminate the reaction. Serum KIM-1/H-FABP concentrations were assessed by detecting absorbance at 450 nm using an ELISA instrument (Thermo FC, US). For the serum NAG activity assay, 70 μl of serum sample was added to the plates, after which 55 μl of NAG substrate was added and incubated for 30 minutes; the reaction was terminated with 25 µl of stopping buffer, and the plate was incubated for 10 minutes in the dark. The serum NAG activity was calculated based on absorbance detection at OD 400 nm.