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 up to a 1536-well format by simply resizing each addition volume proportionally.

Human IL-1β standard curve

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 4 different experiments, and the % CV was calculated for each sample.

The excellent % of recovery obtained from these experiments show the good dilution linearity of the assay. Samples are PBMC supernatants serially diluted with the kit DIL5 diluent.

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

Cross reactivities were assessed using recombinant proteins from the IL-1 cytokine family. Proteins were tested up to 6 500 pg/mL, and standard curves were generated for each. Signals were interpolated on the assay standard curve to interpolate concentrations. The assay is human specific, as mouse IL-1β cytokines were not detected.

Modulation of secretion of human IL-1β in THP1 cell line

THP1 cells plated at 25, 50, and 100 kcells/well were stimulated for 18h with PMA and LPS, respectively used at 50 ng/mL and 1 µg/mL. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed with the Human IL-1ß Assay.

IL-1ß secretion in PBMC stimulated with LPS

PBMC plated at 50, 100, and 200 kcells/well were stimulated for 18 h with increasing concentrations of LPS ranging from 0.02 to 2 µg/mL. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the Human IL-1ß assay kit.

Effect of dexamethasone on PBMC induced IL-1ß release

PBMC plated at 50 and 100 kcells/well were stimulated for 18h with 0.2 µg/mL of LPS and a co-treatment of increasing concentrations of dexamethasone, ranging from 0.05 to 50 µg/mL. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the Human IL-1ß Assay Kit.