Il 4 elisa kit

Cytokines were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions. CCL11 (Eotaxin-1) and CCL24 (Eotaxin-2) ELISA kits were purchased from R&D Systems (Minneapolis, MN). IL-4 ELISA kit was purchased from BioLegend (San Diego, CA). CCL2 was obtained from Peprotech (Rechovot, IL) and Relm-α was detected as previously described^{59 (link)}. Lower detection limits for these assays were 15.625 pg/ml (CCL11) and 31.25 pg/ml (CCL24, CCL2 and Relm-α).

Fluorochrome-conjugated monoclonal antibodies to mouse CD4, KJ1.26, IL-4, and Foxp3 were obtained from Biolegned. IL-4 ELISA kit was purchased from Biolegend. The lunasin peptide with 43-amino acid was chemically synthesized with 97% purity by LifeTein (South Plainfield, NJ) [16 (link)]. Ovalbumin (OVA, chromatographically purified) was from Worthington Biochemical Corp (Lakewood, NJ).

The concentration of mouse IL-4 was measured in both BALF and serum using an IL-4 ELISA kit (BioLegend, San Diego, CA, USA) following the manufacturer’s recommendations. Mixtures of BALF (or serum, 50 µL each) and assay buffer (50 µL) were reacted with anti-IL-4 antibody in a 96-well plate for 2 h at room temperature. After removal of unbound proteins, detection antibody solution (100 µL), and avidin–horseradish peroxidase (HRP) D solution (100 µL) was sequentially added to the wells, after which samples were incubated at room temperature for 30 min. Finally, these mixtures were reacted with substrate solution (100 µL) for 15 min, after which the reaction was stopped with blocking solution (100 µL). The absorbance of the mixture was then read at 450 nm using a Versa-max plate reader (Molecular Devices, San Jose, CA, USA).