Elisa system

Interferon (IFN)-γ tumor necrosis factor (TNF)-α, and interleukin (IL)-2 were measured in the supernatant of cell-cultured medium or the plasma using the ELISA system (BioLegend). Anti-mouse dsDNA IgG antibodies in the plasma were measured using the ELISA system (Wako Pure Chemical Industries). For the measurement of plasma NP-specific IgG antibodies, a MaxiSorp 96-well plate (Nunc) was coated with 10 μg/ml of NP-BSA in PBS at 4°C overnight and blocked with 2% normal goat serum in Tris-buffered saline with 0.05% Tween 20 (TBS-T). Plasma samples diluted 1: 100 were incubated at room temperature for 2 h, and subsequently HRP-conjugated goat anti-mouse IgG was incubated for 2 h. HRP activity was evaluated with TMB solution and 1 M sulfuric acid (Sigma Aldrich). The total urinary protein (uTP) and urinary creatinine (uCr) concentrations were measured by clinical diagnostic reagents (Wako Pure Chemical Industries).

Inflammatory profiles (TNF-α, IL-1β, IL-4, IL-6 and IL-10) of BALF were evaluated by an enzyme-linked immunosorbent assay (ELISA) system (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The samples were centrifuged (3000 g for 10 min at 4 °C), then stored at –80 °C for ELISA testing. In brief, to assess the level of TNF-α, IL-1β, IL-4, IL-6, and IL-10 in the BALF, 96-well plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4 °C. The plates were then washed with phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBS-T) and blocked with 10% FBS in PBS for 1 h at room temperature. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates, and the plates were incubated for 24 h at room temperature. After the plates were washed, biotin-labeled detection antibody and avidin-conjugated horseradish peroxidase (Av-HRP) were added to the plates, and the plates were incubated for 30 min at room temperature. Finally, the tetramethylbenzidine (TMB) substrate was added to the plates, and, after 30 min of incubation in the dark, TMB Stop Solution was added to stop the reaction. The optical density was measured at 450 nm on an automated ELISA reader. (Versa Max, Molecular Devices, CA, USA) [15 (link)].