Immulon 4 hbx elisa plates

Immulon 4 HBX ELISA plates (Thermo Scientific) were coated with recombinant ZIKV PRVABC59 NS1 protein (produced in-house) or recombinant envelope protein (MyBioSource accession number MBS319787) at 2 μg/ml in pH 9.41 carbonate buffer overnight at 4°C. Plates were washed three times with PBS between each step. After being blocked with 5% nonfat (NF) milk for 1 h, mouse sera were incubated at a starting concentration of 1:50, serially diluted 4-fold, and incubated for 2 h at room temperature. For experiments using human sera, a starting concentration of 1:40 was used. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (AP504P; Millipore Sigma) or anti-mouse IgG antibody (AP503P; Millipore Sigma) was used to detect binding of IgG antibodies, followed by development with the HRP substrate (SigmaFast OPD; Sigma-Aldrich). Reactions were stopped by the addition of 3 M HCl, and absorbance was measured at 490 nm on a microplate spectrophotometer (Bio-Rad). Experiments were performed in duplicate. A nonparametric multiple-comparison Kruskal-Wallis test was utilized to examine significance between groups. GraphPad Prism 6 was used to calculate area under the curve (AUC) values.

Immulon 4HBX ELISA plates (Thermo Scientific) were coated with purified CrAra h 1‐core (5 μg/mL), Ara h 1 (5 μg/mL), CrAra h 2 (1 μg/mL) or Ara h 2 (1 μg/mL) overnight at 4 °C. Plates were blocked with PBS containing 0.5% Tween and 1% BSA, washed with PBS‐T and then incubated overnight at 4 °C with serum from peanut‐allergic patients diluted in PBS at 1:5 or 1:10 for Ara h 1 or Ara h 2, respectively. IgE binding was detected with alkaline phosphatase‐conjugated anti‐human IgE (Sigma A3525) and visualized with para‐nitrophenyl phosphate (PNPP) at 1 mg/mL in PNPP substrate buffer (Invitrogen, Carlsbad, CA). Absorbances were measured at 405 nm using a Polarstar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany). For competition assays, ELISA plates were coated with 5 μg/mL Ara h 1 or Ara h 2 as above. Equal volumes of serum from each patient were pooled and diluted 1:10. Pooled serum was incubated overnight at 4 °C with 0, 1, 2, 4 or 8 μg/mL of Ara h 1 or the molar equivalent of CrAra h 1‐core. For Ara h 2, pooled serum was incubated overnight at 4 °C with 0, 0.625, 1.25, 2.5 or 5 μg/mL or the molar equivalent of CrAra h 2. Allergen/pooled serum mixes were added to Ara h 1 or Ara h 2 coated plates in triplicate and incubated overnight at 4 °C. Plates were washed and IgE binding was detected as described above.