Assay principle

Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Revvity low-volume 96-well plate in 20 µl). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.

Standard curve

The IL-15 standard curve is generated in the assay Diluent 5, provided in the kit.

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Inter-assay (n=4)

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample.

The excellent recovery percentages obtained from these experiments show the good dilution linearity of the assay. Samples were HUT-102 supernatants serially diluted in RPMI medium.

Recombinant cytokine was added to HUT-102 supernatant samples, and the measured response was compared to the calculated expected values. The 100% of recovery indicates similar measurements of cytokine from sample and the kit standard.

Cross reactivities were assessed using recombinant proteins from the IL2 cytokine family, and tested at 100 ng/mL. Signals were interpolated on the assay standard curve to interpolate concentrations. The assay is human specific, as mouse IL15 cytokine was not detected using this assay.

The possible interference from human Interleukin 15 Receptor alpha / Fc Chimera (IL15 Rα) was investigated. No interference was observed up to 50 ng/mL for IL15 Rα.

Modulation of secretion of human IL-15 in NCI-H2172 cell line

NCI-H2172 cells plated at 50 kcells/well were stimulated for 48 h with PMA 10 ng/mL, IFN Ɣ 10 ng/mL, or with both compounds for 48H. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed with the Human IL-15 Assay. As expected, treatment with PMA and IFNƔ induced IL-15 production and secretion (IFN-γ Induces IL-15 Trans-Presentation by Epithelial Cells via IRF1 J Immunol (2022) 208 (2): 338–346.)

Validation of HTRF human IL-15 kit on HUT-102 cell line

HUT-102 human cutaneous T cell lymphocyte cells were cultured at a cell density of 2x106 cells/mL in RPMI medium with 10% FBS for 24H at 37°C for supernatant generation to be analyzed by the Human IL-15 HTRF assay. The IL-15 concentration in HUT-102 cell supernatant was 153 pg/mL. These results are in accordance with the literature (Blood (1998) 91 (11): 4265–4272).

Interference with IL-15 receptor

The HTRF human IL-15 assay is able to detect monomeric IL-15 in presence of a large excess of IL-15 Receptor.