Rapid and sensitive determination of cytokine release from cells without the need for sample transfer

Abstract

We have utilized NanoLuc® Binary Technology (NanoBiT®) to develop a homogeneous and rapid assay method (≤ 70 min completion time) to measure cytokines released from cells in culture without the need for sample transfer and requiring only a standard, plate-reading luminometer for signal acquisition. Separate antibodies to a specific cytokine are labeled with the small, 11-amino acid subunit of NanoBiT luciferase (SmBiT) or its 17.6 kDa complementary subunit (LgBiT). When SmBiT- and LgBiT-labeled antibodies converge on the target cytokine, the resultant proximity of SmBiT and LgBiT reconstitutes a bright luciferase that produces light proportional to analyte levels. In this manner, immunoassays have been developed for several cytokines, including IL-1β, IL-2, IL-6 and IFN-γ. In general, these assays share LLODs < 10 pg/ml and linearity over three logs of analyte concentration, mitigating the need for sample dilution. Following 24 h treatment of human PBMCs in 96-well plates with vehicle, LPS, R848, or a combination of PMA and ionomycin, cytokine detection reagents were added directly to the culture wells containing cells and medium. Depending on stimulus, maximal signal to background ratios (S/B) achieved for the cytokines were 347-, 450-, 580- and 655-fold for IL-1β, IL-2, IL-6 and IFN-γ, respectively. In a cell model comprised of activated T cells and target Raji B cells induced for 20 h with increasing bispecific T-cell engager Blincyto®, release of IL-2 and IFN-γ were observed with an EC50 of ~0.2 ng/ml and maximal S/B for IL-2 and IFN-γ of 82- and 168-fold, respectively. The implementation of this novel detection chemistry will enable rapid “add-and-read” assays for cytokine detection for both low- and high-throughput applications.