ELISA Quantification of Serum SP-D

The levels of SP‐D were determined in serum as a measure of intravascular leakage (Gaunsbaek et al., 2013 (link)). Diluted serum specimens were subjected to an enzyme linked immunosorbent assay (ELISA; R&D Systems) for measurement of SP‐D levels. Briefly, microwells were coated with 4 μg/ml capture antibody overnight at 4°C. After washing the wells, the nonspecific sites were blocked with 1% BSA solution in Dulbecco's phosphate‐buffered saline (DPBS) for 1 h at room temperature. The wells were washed and incubated with the mouse SP‐D standard (62.5–4000 pg/ml) and serum specimens (1:10 and 1:50) diluted in 1% BSA solution in DPBS, overnight at 4°C. The next day, the wells were washed and incubated with 125 ng/ml detection antibody. The immune complex was then incubated with 1:40 diluted streptavidin‐horse radish peroxidase for 2 h at room temperature, and developed by adding tetramethylbenzidine substrate solution (Sigma). The reaction was stopped by adding 2 N H₂SO₄. Finally, the optical density was read spectrophotometrically at 450 and 540 nm, and subtracted per the manufacturer's instructions to correct for optical imperfections in the plate.