Progranulin

ELISA plates (Nunc) were coated with 50 μl/well of human proteins in TBS-T (0.1% Tween) overnight at 4° C. The concentrations were: 3 μg/ml for L1CAM (Sino Biological), 1.5 μg/ml for DEL-1 (R&D), 2 μg/ml for angiopoietin-1 (R&D), 0.5 μg/ml for angiopoietin-2 (R&D), 1 μg/ml for vascular endothelial growth factor-A (Peprotech), 3 μg/ml for progranulin (R&D), 0.4 μg/ml for platelet derived growth factor-BB (eBioscience), and 1 μg/ml for hepatocyte growth factor (Sino Biological). Plates were washed and blocked for 1.5 hours at room temperature with 100 μl/well of protein-free blocking buffer (PFB) (0.1% Tween, Pierce, Cat# 37570). Patient sera diluted 1:2000 in PFB-T were added at 50 μl/well in triplicate for 1 hour at 4° C. After washing, 50 μl/well of an HRP-conjugated anti-human IgG (Fab)2 diluted 1:2000 (Southern Biotech, Cat# 6005-05)) was added for 1 hour at room temperature. The plates were washed and 50 μl/well of biotinylated Tyramide (10 μl/ml in amplification diluent concentrate diluted 1:1 with ddH2O, ELAST, PerkinElmer) was added for 30 minutes at room temperature. After washing, wells were incubated with streptavidin-HRP (50 μl/well, concentration of 2 μl/ml) in 1% BSA-PBS-T (0.1% Tween) for 30 min at room temperature. The plate was developed with pNPP substrate (Sigma-Aldrich) and the absorbance measured at 450 nm.