Luminescence-based ELISA for Phl p 5-specific IgG1 and IgG2c

Levels of Phl p 5-specific IgG1 and IgG2c were determined using a luminescence-based ELISA assay as previously described (Weinberger et al., 2013 (link)). In short, 96-well plates for immunoassays (Greiner) were coated for 24 h at 4°C with recombinant Phl p 5 (per well 50 μl of 1 μg/ml Phl p 5 in PBS). Afterwards, plates were washed with 0.1% Tween-20 in PBS (v/v) and incubated with blocking buffer (0.1% (v/v) Tween 20 and 2% (w/v) skim milk in PBS, pH 7.5) for 1 h at RT, before washing the plates again. Then, the plates were incubated with serum diluted (1:10,000) in blocking buffer for 1 h at RT, washed again, before the horse radish peroxidase (HRP)-conjugated antibodies for the detection of IgG1 (Zymed) or IgG2c (Zymed; diluted 1:1000 in blocking buffer) were added to the wells for 1 h at RT. After that, the luminometric assay (BM chemiluminescence substrate, Roche) was developed by adding the substrate (luminol diluted 1:2 in H₂O) to each well. After 3 min incubation, chemiluminescence (photon counts/s) was determined using an Infinite M200 Pro Plate Reader (Tecan).