suPAR Measurement by ELISA

For suPAR analysis, enzyme-linked immunosorbent assay (suPARnostic® AUTO Flex ELISA, ViroGates, Birkerod, Denmark) was performed according to manufacturer’s instructions [11 (link)]. Briefly, serum samples diluted 1:10 and peroxidase-conjugated anti-suPAR was mixed and then incubated in an anti-suPAR pre-coated 96-well plate. After 1 h incubation, tetramethylbenzidine substrate was added and the enzymatic reaction was stopped after 20 min by adding 2 N sulfuric acid and read at 450 nm (plate reader Sunrise, Tecan, Männedorf, Switzerland; software Magellan version 7.1, Tecan). All samples were run in duplicates.
A large Danish study of 5 538 adult individuals showed a mean serum concentration of suPAR of 3.51 ng/mL for men and 3.90 ng/mL for women [16 (link)]. The highest value among the controls herein (> 3.6 ng/mL) was used as cut-off; this corresponded to the 98th percentile among the healthy controls.