Elisa stop solution

Antigen-induced IFN-γ and IL17-A secretion in total lung cells and splenocytes were quantified by ELISA using the IFN-γ Mouse Uncoated ELISA Kit and the IL-17A (homodimer) Mouse Uncoated ELISA Kit (both from Thermo Fisher Scientific). The cytokine analyses were performed following the manufacturer’s instructions using Costar 3590 plates (Corning). Color development was stopped with ELISA Stop Solution (Thermo Fisher Scientific), and the plates were read using a Multiskan^™ FC Microplate Photometer at OD₄₅₀.

Enzyme-linked immunosorbent assay (ELISA) were performed to determine titers of antigen-specific IgA in the lung wash samples. Microtiter plates were coated with 5 μg/ml Ag85B or ESAT-6 (Lionex GmbH, Braunschweig, Germany) and incubated overnight at 4°C. The plates were then washed with the washing buffer (0.1% Tween-20 in PBS) and blocked with PBS/1% BSA. The plates were incubated with the blocking solution for 1 h at 37°C. The plates were washed three times with the HydroSpeed^™ plate washer (TECAN, Männedorf, Switzerland), after which the samples, in two-fold serial dilutions, were applied to the coated plates, followed by incubation for 1 h at 37°C. After incubation, the plates were washed three times with the washing buffer, followed by exposure to HRP-conjugated anti-mouse IgA (1,1,000 dilution; Sigma-Aldrich) in 1% BSA 0.05% Tween-20 in PBS for 1 h at 37°C. The plates were washed five times with PBS/0.1% Tween-20 before addition of the eBioscience^™ TMB Solution (1X; Thermo Fisher Scientific) for color development. The reaction was stopped after 15 min with the ELISA Stop Solution (Thermo Fisher Scientific), and the OD₄₅₀ was measured using a Multiskan^™ FC Microplate Photometer (Thermo Fisher Scientific). PBS samples were included on all plates and used to set the threshold for calculations of the titer in the lung wash samples.

Human TNFα (Gibco, PHC3011) was biotinylated using an EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific, 21925) according to manufacturer’s instructions. Human TNFR2 (Acro Biosystem, TN2-H5227) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455). Plates were incubated at 4°C overnight. Plates were washed with PBST (wash buffer 0.1%) and blocked with 1% BSA (Fisher Scientific, BP1600-100) in PBS for 1 hour at 37°C. AN3025 or human IgG₁, κ (BioxCell, BE0297) was titrated, distributed 50μL per well and incubated for 1 hour at 37°C. 50μL biotinylated human TNFα was added per well to achieve the final TNFα concentration at 100ng/ml. After another 1-hour incubation, plates were washed with PBST (wash buffer 0.1%). Streptavidin-HRP conjugate (Thermo Scientific, N504) was distributed to detect bound biotinylated human TNFα. After 1-hour incubation of secondary antibody at 37°C, plates were washed with PBST (wash buffer 0.1%). Plates were developed using TMB substrate solution (eBioscience,00-4201-56) and stopped with ELISA stop solution (Invitrogen, SS04). The level of bound biotinylated human TNFα was determined by reading absorbance at 450 nm.