Goat anti mouse igg fc

ELISAs were performed as previously described⁷ . Briefly, 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 200 ng of recombinant IL-1RA protein (Biolegend #553906) in PBS and incubated overnight at 4 C. Plates were dumped out and incubated with 2% Human Serum Albumin (HSA) (Celprogen #HSA2001-25-2) in PBS for 2 h at RT. Plates were washed 3× with 200 μl wash buffer (PBS 0.05% Tween). Samples were diluted in 2% HSA and added to the plate to incubate for 2 h at RT. Mouse anti-human IL-1RA (Prospec #ant-238) was used as a positive control. Plates were washed 6× with wash buffer. Goat anti-human IgG Fc (Sigma Aldrich, #AP113P) diluted 1:10000 in 2% HSA was added to the plates and incubated for 1 h at RT. For the positive control, 1:5000 Goat anti-mouse IgG Fc (Thermo Fisher Scientific, #A16088) in 2% HSA was used. Plates were washed 6x. Plates were developed with 100 μl of TMB Substrate Reagent Set (BD Biosciences #555214) and the reaction was stopped after 5 min by the addition of 2 N sulfuric acid. Plates were then read at a wavelength of 450 nm.