Streptavidin horseradish peroxidase

In order to disrupt disulfide bonds, samples were treated with β-mercaptoethanol for 3 min at 95°C before electrophoresis. All samples were diluted in SDS-containing buffer and loaded on to 4–20% Novex Tris-Glycine polyacrylamide gel (Life Technologies). SeeBlue Plus2 Pre-Stained Standard ladder (Life Technologies) was used to indicate molecular weights (Mw). Following electrophoresis, the proteins were blotted onto a polyvinylidene difluoride membrane (Biorad, Hercules, CA) by 100 V for 1 h at 4°C. Milk and casein (5% and 1%, respectively) in 0.1% Tween-20 was used to block the membrane (1–2 hrs at room temperature) before incubation overnight at 4°C with biotinylated antibodies followed by incubation with streptavidin-horseradish peroxidase (GE Healthcare). The protein bands were developed by a chemiluminescent peroxidase substrate (Lumigen from GE Healthcare), and images were acquired by image station 2000R (Kodak).

To assess immunoglobulin E (IgE) levels, the mice were bled via cardiac puncture 24 hours after the last HDM challenge. Serum was obtained by centrifugation of blood samples at 9300×g for 5 minutes. The supernatant was removed and stored at −80°C. The total IgE was measured by ELISA. Briefly, ELISA plates (Affymetrix eBioscience, San Diego, CA, US) were coated overnight with 2 µg/mL purified anti-mouse IgE (BD Biosciences, San Jose, CA, US). Plates were washed with 0.05% Tween 20 (Sigma-Aldrich, NSW, AU) in PBS between each step. Plates were blocked with 1% BSA in PBS before addition of purified mouse IgE isotype standard monoclonal antibody (BD Biosciences) or mouse serum. IgE was detected using biotinylated rat anti-mouse IgE (BD Biosciences) at concentration of 2 µg/mL. Streptavidin-horseradish peroxidase (GE Healthcare Life Sciences, NSW, AU) was added followed by 2,2′ –azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (Hoffmann-La Roche, Basel, Switzerland) supplemented with 0.1% hydrogen peroxide (Chem-Supply, Adelaide, AU) to produce a colour reaction. This reaction was stopped by adding 20% wt/vol SDS (Bio-Scientific, Sydney, AU). Plates were read spectrophotometri-cally at an absorbance of 405nm (Microplate AutoReader EL311; BioTek, Winooski, VT, US), and data were analysed using linear regression analysis in AssayZap (Biosoft, Cambridge, UK).

ELISA plates were coated overnight at 4 °C with 10 μg ml⁻¹ of MMRI7 anti-DEC-205 mAb (BD Biosciences). Unbound mAb has washed away (PBS, 0.05% [v/v] Tween-20) before proteins (diluted in PBS 3% [w/v] bovine serum albumin) were added (4 °C, overnight). Captured protein was exposed to various biotinylated CpG oligonucleotide at specified concentrations (2 h, room temperature), detected with streptavidin-horseradish peroxidase (2 h, room temperature) (GE Healthcare) and colorimetric reaction visualized using ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid). Each titration point was performed in duplicate.