Quanta lite ana elisa

Autoreactivity against nuclear and cytoplasmic self-antigens was determined with QUANTA Lite ANA ELISA (Inova Diagnostics) as described (52 (link)). Polyreactivity against ssDNA, dsDNA, Keyhole limpet hemocyanin (KLH), human insulin and lipopolysaccharide (LPS) was determined as described (22 (link)). To measure NP binding, high-binding 96-well plates (Corning) were coated overnight with 50 μl of PBS containing 10 μg/ml of NP₄-BSA or NP₂₅-BSA (Biosearch Technologies). After washing with PBS containing 0.05% of Tween 20 (Sigma), wells were blocked with PBS containing 1% of BSA for 2 h at room temperature. Monoclonal antibodies were incubated at 4 μg/ml or 7 consecutive 1:4 dilutions in PBS for 2 h at room temperature. After washing, HRP-conjugated goat α-human IgG (Jackson ImmunoResearch) was added at 0.16 μg/ml for 1 h at room temperature. After additional washing, HRP was revealed with 1-Step ABTS Substrate Solution (ThermoFisher Scientific). Absorbance was measured at 405 nm after incubation for 20 min at room temperature.

Single B cells were cultured in the presence of NB-21.2D9 feeder cells, kindly provided by Dr. Garnett Kelsoe from Duke University (46 (link)). NB-21.2D9 cells were seeded into 96-well plates at a density of 1000 cells per 100 μL/well in B cell medium (RPMI-1640 supplemented with 10% FBS, 55 μM 2-mercaptoethanol, 100 Units/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM nonessential amino acid). Next day (day 0), recombinant mouse IL-4 (4 ng/mL, 100 μL/well; Peprotech) was added to the cultures, and single B cells were then sorted directly into each well using a BD FACSAria cell sorter. On day 2, 100 μL of culture medium were removed from the cultures and 200 μL of fresh B cell medium were added. From days 3 to 8, two-thirds of the culture media (200 μL) were replaced with fresh B cell medium every day. On day 10, the culture supernatants were harvested and total IgG levels were determined by ELISA. Additionally, autoreactivity against nuclear and cytoplasmic self-antigens was determined by QUANTA Lite ANA ELISA (Inova Diagnostics).