2.2. PCSK9 ELISA

Blood samples were collected from 12 to 24 h after the admission of the patients to the intensive care unit. EDTA was used as the anticoagulant, and the plasma was subsequently prepared. The human PCSK9 DuoSet ELISA (R&D Systems; Wiesbaden, Nordenstadt, Germany) was used as suggested by the provider (the dilution of plasma was 1:100). The capture antibody was diluted in phosphate-buffered saline (PBS) and was then used to coat a 96-well microplate. The sealed plate was incubated overnight at room temperature. The capture antibody was removed the following morning and the microplate was washed three times with PBS. Blocking was achieved by incubation with reagent dilution solution for 1 h at room temperature. After 3 times washing, 100 µL of the diluted plasma was added to the wells. A seven-point standard curve ranging from 125 pg/mL to 8000 pg/mL, respectively, was prepared and processed in parallel with the samples. Each standard sample and each blood sample was analyzed in duplicate and for calculations the mean values were used. The covered microplate was incubated for 2 h at room temperature. After washing for three times the detection antibody was added, and the microplate was incubated for 2 h at room temperature and washed again. Then, the streptavidin-horse radish peroxidase solution was added for 20 min. After washing, the substrate solution was added and then incubated for 20 min in the dark. After adding the stop solution the optical density was measured at 450 nm. The wave length correction was set to 540 nm. The values of the blanks, which were wells treated identically as all other wells but without the addition of plasma, were subsequently subtracted.