Quantifying Immune Markers in Arthritis

Inflammatory cytokines, chemokines and growth factors in ankles or sera were determined using ELISAs specific for each molecule (DuoSets, R&D). Ankles were homogenized in PBS supplemented with a protease inhibitor cocktail (Sigma). Supernatants were collected using centrifugation at 12,000g for 5 min at 4 °C, and the protein concentration was determined using bicinchoninic acid protein assay reagents (Thermo Scientific)58 (link)60 (link). Immunoglobulins were quantified using ELISA (Southern Biotechnology Association). Antinuclear antibodies (ANA) were determined utilizing a mouse ANA ELISA kit (Alpha Diagnostic International). RF in serum diluted 1:25 was detected employing 1 μg ml⁻¹ rabbit IgG (Dako, X0903)-coated ELISA plates61 (link). ELISA for antiCCP (anti-CCP) antibodies was performed as described with modification62 (link). Briefly, biotin-conjugated CCP peptides were synthesized (ThermoFisher) and bounded (1 μg ml⁻¹) to NeutrAvidin-coated microplates (ThermoFisher). Mouse sera (1:100) were incubated for 1 h at room temperature followed by incubation with HRP-conjugated sheep anti-mouse IgG. The results are presented as the product of OD_450 nm × serum dilution. Previously described methods were employed to generate synovial proteome microarrays, which were probed with mouse serum63 (link).