Mouse anti human ige hrp

The rehydration buffer in which TOT and SPP pollen fraction samples were initially dissolved was exchanged with 0.1 M carbonate buffer, pH 9.6, and solutions were filtered with Amicon Ultra-0.5 Centrifugal filters with a cutoff of 3 kDa (Millipore). Analytical 12% 1D SDS-PAGE profiles under reducing conditions before and after this buffer exchange were recorded and the resulting profile showed no major differences (data not shown). Individual serum IgEreactivity to the commercial short ragweed caps (ImmunoCAP, w1) was determined on the ImmunoCAP System (Phadia AB/Thermo Fisher Scientific) according to the manufacturer's instructions. The results are presented as kUA/L, where the cut-off for allergen-specific IgE was ≥0.10 kUA/L (Table 1). Quantitative ELISA measurements against 3 pollen fractions were performed using methods described by Apostolovic et al. [26] (link). After incubation with 50 µL mouse-anti-human IgE-HRP (Abcam, diluted 1/2000) for 1 h at RT, TMB substrate was added and the reaction was stopped with 1 M H2SO4. Results were expressed as kUA/L and were considered positive when the IgE responses exceeded the mean + 3 SD of the 2 healthy controls (kUA/L ≥0.10). More details are available in Supplementary Information.