Elast elisa amplification system

The EphB4 immune-capture antibodies (Santa Cruz) used for coating (0.3 µg/ml) was adsorbed onto 96 well plates (Maxisorb™, Nunc Millipore Sigma) in H₂CO₃/HCO₃- buffer, pH 9.6 o/n at 4 °C followed by non-site blocking in 20 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween20, 1% bovine serum albumin at 56 °C for 30 min. 10–40 µg cell lysates were incubated for 2 h at 4 °C in presence of protease/phosphatase inhibitors. Primary antibodies incubation was carried out in 50 mM Hepes pH 7.5, 150 mM NaCl, 0.05% Tween20, 1% Bovine Serum Albumin with protease/phosphatase inhibitors incubating for 2 h at 22 °C. Primary monoclonal antibodies raised against either total ubiquitin (Santa Cruz, P4D1 clone, Dallas TX) at 1:200 dilution or K-48 and K-63 branched ubiquitin chains (Cell Signaling, Danvers MA) at 1:100 dilution were used. The ELAST Elisa Amplification System (Perkin Elmer, Waltham MA) was used depending upon the resulting signal intensity. Either OPD (Millipore Sigma, St. Louis MO) or TMB (Thermo Scientific, Waltham, MA) were ELAST Elisa Amplification System (Perkin Elmer, Waltham MA) used as colorimetric substrates for ELISA and reactions ended by adding 2 M sulphuric acid. Optic Density was quantified by spectrophotometric reading at 492 (OPD) or 450 (TMB) wavelengths in a BIOTEK reader (uQuant). All conditions were carried out in triplicates.

Human IgE-binding of the samples was assessed by inhibition ELISA as previously reported [23 ], using a pool of 8 different sera from egg allergic children with IgE specific to OVA, LYS and OM ranging between 35.2–1326.5 kU/L, 2.3–36.6 kU/L and 19.3–120.0 kU/L, respectively. Briefly, OVA, LYS and OM were used as coating antigens (0.5, 2.5 and 1.75 μg, respectively). Serial dilutions of the hydrolysates were incubated with the pooled sera (1:1, v/v) and, after 2 h, added to the appropriate coated plate. Polyclonal rabbit anti-human IgE (Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulin labelled with horseradish peroxidase (HRP) (Dako), diluted 1:1000 and 1:2000 (v/v), respectively, were used for detection. A tyramide-biotin and streptavidin-HRP amplification system was employed, following the instructions of the manufacturer (ELAST ELISA amplification system, Perkin-Elmer Life Sciences, Waltham, MA, USA). The reactions were developed using TMB as substrate, stopped with 0.5 M sulfuric acid and the absorbance read at 450 nm in a plate reader (Multiskan FC, Thermo Scientific).