Human progranulin duoset elisa

To determine the human PGRN levels in the serum, frontal cortex, and spinal cord lysates, homemade rabbit anti-human granulin E antibodies were used to coat a 96-well microplate with 100 μL per well and incubate overnight at 4°C. The next day, after washing with 250 μl washing buffer (0.05% Tween® 20 in PBS, pH 7.4) for 3 min, the wells were blocked with blocking buffer (1% BSA in PBS, pH 7.4) for 1 hour, followed by incubation with the proper amount of samples or recombinant PGRN protein (Human Progranulin DuoSet ELISA, R&D Systems) at concentrations of 62.5, 125, 250, 500, 1000, 2000 and 4000 pg/mL for 2 hours. The wells were then washed three times before incubation with the detection Antibody (Human Progranulin DuoSet ELISA, R&D Systems) for 2 hours. Then the plate was incubated with Streptavidin-HRP solution for 20 minutes at room temperature. After 3 washes, 50 μL of TMB substrate solution (Thermo Fisher Scientific) was added into the wells and the plate was incubated for 5–15 min until blue color appeared. The reaction was stopped with 2N H₂SO₄ solution (Thermo Fisher Scientific). The optical density was determined by a microplate reader using the readings at 450 nm and subtracting the readings at 540 nm.