Human PD-1 QuicKit ELISA

Human peripheral blood mononuclear cells (PBMCs) were cultured at 3 x 106 cells/mL in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and were either left untreated or treated with 10 ug/mL PHA-L and 20 ng/mL of recombinant human (rh) IL-2 for 5 days. After 5 days, cells were then treated with 10 ng/mL PMA and 500 ng/mL Ionomycin for an additional 24 hours before harvesting conditioned media. CD4+ T cells were isolated from PBMCs using MagCellect™ Human CD4+ T Cell Isolation Kit (R&D Systems®, Catalog # MAGH102). For Cloudz™ stimulation, CD4+ T cells were cultured at 5 x 105 /mL using ExCellerate™ Human T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030) for the duration. T cells were treated with 25 μL of Cloudz CD3/28 particles per mL of culture media for 5 days. Cell conditioned media was collected by centrifugation and stored at ≤ -20 °C until assayed. After centrifugation, the remaining cells and Cloudz particles were resuspended with 1X Release Buffer. After dissolution of Cloudz particles, cells were lysed as indicated on the next page. 

Natural and recombinant human PD-1.

Intra-Assay Precision (Precision within an assay) Two samples of known concentration were tested twenty times on one plate to assess intra-assay precision

Inter-Assay Precision (Precision between assays) Two samples of known concentration were tested in ten separate assays to assess inter-assay precision. Assays were performed by at least three technicians

The recovery of human PD-1 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human PD-1 were diluted with calibrator diluent to produce samples with values within the dynamic range of the assay. 

Scientific Data