Human il 2 quantikine elisa kit

Interleukin-2 was measured in serum using a commercial ELISA kit (Quantikine Human IL-2 ELISA kit; R&D System Inc.) The absorbance was read at 450 nm using Spectra Max 190 micro-plate reader (Molecular Devices, Sunnyvale, CA).

The measurement of serum cytokines concentrations was determined with the usage of a quantitative commercial enzyme-linked immunosorbent assay (ELISA) technique, following the manufacturers’ recommendations. In brief, the serum concentration of IL-2 was measured using a Human IL-2 Quantikine ELISA Kit (sensitivity, 7 pg/mL; R&D Systems, Minneapolis, MN, USA); the serum concentration of IL-4 was determined using the Human IL-4 Quantikine HS ELISA Kit (sensitivity, 0.22 pg/mL; R&D Systems, USA); the concentration of serum IL-6 was measured using the Human IL-6 Quantikine HS ELISA Kit (sensitivity, 0.11 pg/mL; R&D Systems, USA); the serum concentration of IL-10 was determined using the Human IL-10 Quantikine HS ELISA Kit (sensitivity, 0.17 pg/mL; R&D Systems, USA), and the concentration of serum IFN-gamma was determined using the Human IFN-γ Quantikine ELISA Kit (sensitivity, 8 pg/mL; R&D Systems, USA). The results were measured with an automatic reader VICTOR3 (Perkin Elmer, Waltham, MA, USA); that action is based on the measurements of the light absorbance of the tested material and its comparison with control samples of known concentration. The WorkOut computer program, working with the reader on the basis of known concentrations, plotted linear curves on the basis of which the concentration of cytokines in the tested samples was calculated.

IFN-γ (Human IFN-gamma Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA) and IL-2 (Human IL-2 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA) concentrations in the plasma of subjects were measured using sandwich ELISA on the day of first ingestion (week 0) and after 4, 8, 12, and 16 weeks.

IL-2 secreted into the media by Jurkat cells was quantified using a human IL-2 Quantikine ELISA kit (R&D Systems).

Female BALB/c mice (18–20 g, n = 3) received a single intravenous injection of (0.8 mg/kg) wtIL-2, mIL-2, or VGRmIL2-IC in a volume of 0.2 ml. Blood samples were collected at different time intervals (0, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h), and the sera were stored at − 20 °C in triplicate. Finally, the concentrations of IL-2 and VGRmIL2-IC were evaluated using the human IL-2 Quantikine ELISA Kit (R & D Systems, U.S). The pharmacokinetic parameters, including the area under the curve (AUC) from time zero to the time that protein is detectable in the circulation (AUC 0–t), AUC 0–∞, terminal half-life (t½), elimination rate constant, mean residence time (MRT), and apparent total clearance rate (CL/F), were calculated for wtIL-2, mIL-2, and VGRmIL2-IC.

AIM V medium was used for the dilution of cells, antibodies, and peptides. Fifty μL of the HLA-DQ2.5 + IHW09023 mixture (4.0 × 10⁵ cells/well) with anti-human MHC Class I antibody (Clone: W6/32, Bio X Cell, Inc), anti-HLA-DR antibody (Clone: L243, Bio X Cell, Inc), and an equal amount of 33mer gliadin peptides (final concentration: 1 μM) or AIM V medium control were distributed in 96 well round-bottom plates. Fifty μL of serially diluted antibodies were then applied in triplicate, and an equal amount of DQ2.5-α2 glia specific TCR-transduced human CD4⁺ T cells (1.0 × 10⁵ cells / well) were finally distributed. The mixture was incubated at 37 °C, at 5% CO₂ overnight. After overnight culture, supernatants were collected to measure IL-2 concentration using a Human IL-2 Quantikine ELISA Kit (R&D Systems) by using VersaMax Microplate Reader (Molecular Devices, LLC) and SoftMax Pro 6.4 (Molecular Devices, LLC).