Legend max human il 2 elisa kit

PBMCs (2 × 10⁵) activated with 100 ng/ml anti-CD3 antibody and 10 ng/ml IL-2 or with Dynabeads Human T-Activator CD3/CD28 (#11161D, Gibco) at a bead-to-cell ratio of 10:1 were incubated with the test compounds for 3 days. Granzyme B levels in the collected supernatants were determined using a LEGEND MAX™ Human Granzyme B ELISA Kit (#439207, BioLegend) in accordance with the manufacturer’s protocol. Jurkat T cells (5 × 10⁴) were activated by Dynabeads Human T-Activator CD3/CD28 (#11161D, Gibco) at different bead-to-cell ratios (0:1, 5:1, and 10:1) for 48 h. The IL-2 concentration in the supernatants was measured by a LEGEND MAX™ Human IL-2 ELISA Kit (#431807, BioLegend) following the manufacturer’s protocol. The levels of 12 molecular species in the culture supernatant were quantified using a Luminex platform (Human Th Cytokine Panel, BioLegend) for the simultaneous detection of the following molecules: IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 22, IFN-γ, and TNF-α, according to the manufacturer’s instructions.

K562 cells stably expressing membrane-anchored anti-CD3 single chain Fv (clone OKT3) and/or PD-L1 were plated at 12,500 cells/well in 96-well plate. 1 × 10⁵ primary T cells (Bloodworks Northwest) and serial dilutions of each test article were added to each well. In some cases, titrations of ALPN-202 or monomeric CD80 vIgD were combined with 100 nM blocking anti-PD-L1 or anti-CD28. Plates were incubated 24 h at 37 °C. Secreted IL-2 was measured using a LEGEND MAX™ Human IL-2 ELISA kit (BioLegend).
In separate experiments, 1 × 10⁵ primary T cells were co-cultured with 2 × 10⁴ K562/PD-L1 cells and incubated with a 100 nM–0.02 nM ALPN-202, agonist anti-CD28 (clone 28.2, BioLegend), Fc control, anti-PD-L1 (atezolizumab), anti-CTLA-4 (ipilimumab), or a combination of anti-PD-L1 and anti-CTLA-4. Submaximal TCR stimulation was provided by 12.5, 2.5, 0.5, or 0 nM anti-CD3 (clone OKT3, BioLegend). After 16 h supernatants were collected and analyzed for secreted IL-2. Cells were then treated with brefeldin A/monensin for 6 h and characterized by flow cytometry for intracellular IL-2 and CD25 upregulation. Four donors were tested in two separate experiments with all samples run in triplicate.

IL-2 was measured with the LEGEND MAX™ Human IL-2 ELISA Kit (BioLegend, 8899 BioLegend Way, San Diego, CA 92121m USA) according to the manufacturer’s description (Product Cat. No: 431807) with SPECTROstar Nano (BMG Labtech GmbH, 77799 Ostenberg, Germany). The Il-2 detection range was 15.6–1000 pg/mL. The optical density (OD) was measured at 450 nm wavelength.