Phadia immunocap 250 analyzer

Peripheral blood mononuclear cells (PBMCs) were isolated and cultured as described before (15 (link)). In short, for 79 GPA patients PBMCs were available and were cultured for 12 days with or without 3.2 μg/mL CpG-ODN 2006 (Hycult Biotech, Uden, the Netherlands), 100 ng/mL IL-21 (Immunotools, Friesoythe, Germany) and 100 ng/mL BAFF (PeproTech Inc., Rocky Hill, CT, USA). Stimulated and spontaneous PR3-ANCA (RU/mL) production was determined in the supernatant by Phadia ImmunoCAP® 250 analyzer using EliA PR3^S (Thermo Fisher Scientific) and total spontaneous and stimulated IgG production was assessed by ELISA. Five samples (2 F-R and 3 N-R) were excluded because of a culture infection.

ANCA detection was performed by indirect immunofluorescence, as described previously (14 (link)). ANCA titers of 1:40 or higher were considered positive. Serum PR3-ANCA levels could be determined in samples of 51 patients by Phadia ImmunoCAP® 250 analyzer using EliA PR3^S (Thermo Fisher Scientific, Waltham, MA, USA).

Cell culture and quantification of total and PR3-ANCA IgG was performed as previously described by our group (22 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from GPA patients, who produce ANCA upon in vitro induction (20 (link)), and stored in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 50 µg/mL gentamycin (GIBCO, Life Technologies, Grand Island, NY, USA), 10% fetal calf serum (FCS, Lonza), and 10% dimethyl sulfoxide (DMSO). Cryopreserved PBMCs were thawed, and cell suspensions were adjusted to 10⁶ cells/ml in RPMI + 10% FCS. Cells were seeded in 48-well plates (Corning, NY, USA) and stimulated with 3.2 µg/ml CpG-oligodeoxynucleotides (ODN) 2006 (Hycult Biotech, Uden, the Netherlands), 100 ng/ml B cell-activating factor (BAFF; PeproTech Inc., Rocky Hill, NJ, USA), and 100 ng/ml IL-21 (Immunotools, Friesoythe, Germany) at 37°C with 5% CO₂, in the presence and absence of 1 nM ShK-186 (Kineta Inc., Seattle, WA, USA). After 12 days, the culture supernatants were harvested and levels of both total IgG and PR3-ANCA IgG were determined using in-house enzyme-linked immunosorbent assay and Phadia ImmunoCAP 250 analyzer with EliA PR3^S (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Levels of PR3-ANCA IgG are expressed as response units (RU) per milliliter.