High-Sensitivity Custom-Made ELISA

Heparin plasma was thawed and measured using a custom-made ELISA against human soluble sortilin. In short, 96-well plate Nunc Maxisorp was coated with anti–human soluble sortilin [custom made against EC domain as described by Petersen et al. (32)] 1 μg per well in 55 mM NaHCO₃ pH 9.8. The wells were subsequently blocked with 5% bovine serum albumin (BSA) (Sigma A7888) in phosphate-buffered saline (PBS) (pH 7.4). Washing was done in PBS-T (pH 7.4). Sample, internal control, and standard were incubated overnight at 4°C. Standard curve was made from 0.625 to 160 ng/mL of purified human soluble sortilin (21). The in-house generated detection antibody (MABN1792, Sigma) was diluted in PBS-T with 1% BSA to 1 μg/mL. One hundred microliters of the solution was added to each well and allowed to incubate at 37°C for 1 h. Finally, plates were incubated with peroxidase-conjugated goat anti–mouse immunoglobulin G (Dako) for 30 min at room temperature and rinsed five times before addition of o-phenylenediamine dihydrochloride (Dako) mixed with hydrogen peroxide. The reaction was stopped using 0.5 M sulfuric acid, and absorbance was subsequently measured at 490 nm.

To investigate if the anticoagulant used (heparin vs. EDTA) affected the measurable sortilin level, a subset of 44 Dan-NICAD individuals was randomly selected. In a paired design, sortilin was measured again in both EDTA and heparin plasma samples using the same approach as described above.