Rainbow reader

The ELISA which specifically detects soluble as well as full length human IL-6R was described previously38 (link), and performed with streptavidin-horseradish peroxidase (R&D Systems, Minneapolis, MN, USA) and the peroxidase substrate BM blue POD (Roche, Mannheim, Germany). The enzymatic reaction was stopped by addition of 1.8 M sulfuric acid and the absorbance read at 450 nm on a Tecan rainbow reader (Tecan, Crailsheim, Germany).

For the detection of IL-6 in serum from control patients and sepsis patients and in cell culture supernatants the Human IL-6 ELISA kit from Immunotools (Friesoythe, Germany; Cat.-No. 31670069) was used and for the detection of sIL-6R the DuoSet Human IL-6Rα ELISA kit from R&D systems (Minneapolis, MN, USA; Cat.-No. DY227) was used. Both ELISAs were performed according to the manufacturer’s instructions. For the detection of sIL-6R in sera the samples were diluted 1:100 in blocking buffer, cell culture supernatants were not diluted. For the detection of IL-6 both sera and supernatants were used undiluted. Streptavidin-horseradish peroxidase (R&D Systems, Minneapolis, MN, USA) and the peroxidase substrate BM blue POD (Roche, Mannheim, Germany) were used for the enzymatic reaction which was stopped by addition of 1.8 M sulfuric acid. The absorbance was read at 450 nm on a Tecan rainbow reader (Tecan, Crailsheim, Germany).

The sandwich ELISA, which detects sIL-6R as well as ds-sIL-6R (4-11/Baf227 antibodies) and can be used to quantify total sIL-6R serum levels, was described previously [21 (link), 31 (link)]. To specifically detect ds-sIL-6R (either recombinant or in human serum), a similar approach was used (ds6R/Baf227). Both sandwich ELISAs were performed with streptavidin-horseradish peroxidase (R&D Systems, Minneapolis, Minnesota, US) and the peroxidase substrate BM blue POD (Roche, Mannheim, Germany). The enzymatic reaction was stopped by addition of 1.8 M sulfuric acid, and the absorbance read at 450 nm on a Tecan rainbow reader (Tecan, Crailsheim, Germany).