Opteia tmb substrate set

Supernatant and BAL cytokine measurements were performed using commercial IL-13 (eBioscience), IL-17 (BioLegend), IFNγ (BioLegend), and IL-5 (BioLegend) ELISA kits. All commercial ELISA assays were performed according to the manufacturer’s supplied instructions. To measure serum HDM-specific IgG1, a 96-well plate (Corning Costar 3361) was first coated overnight at 4°C with 25 mg/ml HDM in PBS. After plate washing, serum samples and controls were added and incubated at 23°C for 2 hours followed by washing and incubation with a detection biotinylated anti-mouse IgG1 (Biolegend, RMG1-1) at 1:750 dilution for 60 minutes. After washing, streptavidin-HRP (Biolegend) was added for 30 minutes. The plate was washed again, and the ELISA was developed with BD OptEIA TMB substrate set (BD Biosciences). OD measurements were recorded at 450–570 nm using a CLARIOstar (BMG Labtech) plate reader. A reference standard for HDM-specific IgG1 ELISAs was generated by pooling serum samples from HDM-sensitized mice, and varied dilutions of this standard were used to generate a standard curve. ELISA results were scaled such that this reference sample has a relative abundance of HDM-specific IgG1 of 1, and the same pooled reference standard was used for all HDM-specific IgG1 ELISAs reported in this manuscript.

MPyV VP1-specific ELISAs were conducted as previously described [25 (link)]. Briefly, recombinant VP1 protein produced in E. coli and highly purified (kind gift from Dr. Robert Garcea, University of Colorado, Boulder) was used to coat 96-well plates (at 0.1 μg/mL in carbonate buffer, 50 ng/well). Purified recombinant GST-GFP fusion was used to coat wells used to assay responses to GFP. Bound antibody was detected using biotin-conjugated goat antibodies specific for mouse IgG and streptavidin-conjugated HRP (Vector Laboratories). ELISA plates were developed using BD OptEIA TMB Substrate Set (BD Pharmingen), and reactions were stopped with 2 N sulfuric acid. Optical densities were read at 450 nm using a Molecular Probes microplate reader and analyzed using Softmax software. ApoA1-specific, GFP-specific, and yeast protein-specific assays used plates coated with 0.1 μg/well ApoA1 or GFP or 1 μg/well total yeast proteins from strain PAP1502 cells, respectively. IgG titers are expressed as reciprocals of the highest serum dilutions that gave responses twofold above the negative controls plus standard deviation.