3 3 5 5 tetramethylbenzidine (tmb)

The protein binding sites in wells of a high protein binding 96-well microtiter plates were saturated by coating with 100 μg Sen. The plate was incubated overnight at 4°C, after which the wells were washed 2x with PBS and incubated with hPg (0–1.6 mM) at 25°C for 1 h. Two additional washes with PBS were made, followed by 30 min incubations with monoclonal mouse-anti hPg conjugated to HRP (Santa Cruz Biotechnology). HRP activity was detected by a 10 min incubation with a 100 ml mixture of H₂O₂ and the chromogenic substrate, 3,3′,5,5′-tetramethylbenzidine (TMB; R&D Systems). The reaction was terminated by adding 2 N H₂SO₄ and the A_{450 nm} was determined. The saturation curve for the binding of hPg to Sen was generated using GraphPad Prism 9.0.

ELISA plates (Maxisorp, Nunc) were coated with 5 μg/mL of recombinant Phl p 7 (MRC/Asthma UK Protein Production Facility, King's College London, UK). Plates were blocked with PBS/1% BSA and washed throughout with PBS/0.05% Tween-20. 1 μg/mL purified 102.1F10 IgG₄/IgE, negative isotype controls human myeloma IgG₄ (Calbiochem), MOv18 IgE34 (link) and positive control Phl p 7 reactive patient serum12 (link) were incubated in triplicate for 2 hours at room temperature, followed by incubation with isotype specific antibodies. IgG₄ antibodies were detected with mouse anti-human IgG₄ biotin-conjugated antibody (clone G17-4, BD Biosciences), incubated at 1 μg/mL, followed by streptavidin–horseradish peroxidase (R&D Systems). IgE antibodies were detected with polyclonal goat anti-human IgE (Sigma-Aldrich) directly conjugated with horseradish peroxidase using the manufacturer's recommended dilutions. ELISA plates were developed using TMB (R&D Systems) by measuring absorbance at 450 nm.

For IL-6 and TNF-α measurement, a sandwich ELISA was employed [26 (link)]. In brief, purified (goat) anti-human IL-6 or (mouse) anti-human TNF-α antibody (both from R&D Systems, Minneapolis, MN) were used to coat a microtiter plate overnight. Whole blood assay supernates were incubated in the wells at room temperature followed by (rabbit) anti-human IL-6 antibody (Sigma-Aldrich, St. Louis, MO) or biotinylated (goat) anti-human TNF-α (R&D Systems). Human serum-absorbed peroxidase conjugated (goat) anti-rabbit IgG (Rockland Immunochemicals, Pottstown, PA) or streptavidin-HRP for TNF-α (R&D Systems) were used for detection. The colorimetric conversion of substrate (TMB, R&D systems) was quantified at 450 nm using the VERSAmax microplate reader (Molecular Devices, San Jose, CA). Cytokine concentrations were interpolated from an integrated 8-point standard curve created using purified recombinant human proteins. The limits of detectability for the human cytokine assays were: IL-6, 60 pg/mL and TNF-α, 15 pg/mL. Human IL-10 was measured using a commercially available kit with limits of detection at 31.3 pg/mL (human IL-10 Duoset, R&D Systems).

Four cytokines, IL8, IL10, TNFα, mCSF (all from R&D Systems), chosen according to the results of the cytokine profiler, were further validated using ELISA. Samples were analyzed as individual values for each patient. In brief, ELISA plates were coated with capture antibody overnight at RT. The plates were then washed with wash buffer (0.05% tween diluted 20 x in PBS) and blocked with 1% bovine serum albumin-PBS) for 1 hr at RT. The plates were then washed, and samples and standards were added for a further 2 hrs incubation at RT. Secondary antibodies, followed by streptavidin-HRP and substrate solution (TMB, R&D systems) were then used and the reaction was terminated by the addition of 2N H₂SO₄. The results were analyzed with a spectrophotometer (ELISA reader) at 540nm wavelength.

Recombinant murine and chimaeric SPE-7 IgE (rmSPE-7 and rchSPE-7) protein expression and DNP-specificity was confirmed using ELISA plates (Maxisorp; Nunc) coated overnight with 5 mg/ml DNP-HSA (Sigma-Aldrich), blocked with PBS-1% BSA, and incubated with cell-free culture supernatants for 2 hours. This was followed by addition of HRP-conjugated anti-human IgE detection antibody diluted 1/5000 (Sigma), or anti-mouse IgE detection antibody diluted 1/2000, and then avidin-HRP diluted 1/1000 (both R&D Systems). IgE was detected with TMB (R&D Systems) by measuring absorbance at 450 nm.