Pharmingen human elisa set

After final CP001 administration, whole blood samples were collected by cardiac puncture for measurement of blood IgE level. The blood was placed in Vacutainer tubes containing EDTA (BD science, NJ, USA) and blood plasma was isolated. Total IgE levels in plasma were determined by sandwich ELISA using the BD PharMingen mouse IgE ELISA set. Briefly, plates were coated with capture antibody in ELISA coating buffer (Sigma-Aldrich) and incubated overnight at 4°C. Plates were washed with PBS-Tween 20 (0.05%) and subsequently blocked (10% FBS in PBS) for 1 h at 20°C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates and plates were incubated for 2 h at 20°C. After washing, biotin-conjugated anti-mouse IgE and SAv-HRP (streptavidin-horseradish peroxidase conjugate) were added to the plates and plates were incubated for 1 h at 20°C. Finally, tetramethylbenzidine (TMB) substrate solution was added to the plates and after 15 min incubation in the dark, a 2 N H₂SO₄ solution was added to stop the reaction. Optical densities were measured at 450 nm on an automated ELISA reader (Versa Max, Molecular Devices, CA, USA). IL-6 and IL-8 levels were measured in HMC-1 supernatant by sandwich ELISA using BD Pharmingen human ELISA set. The sandwich ELISA procedures were performed by following the same protocols described above.