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. In this Lumit™ immunoassay approach, 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, Lumit immunoassays have been developed for several cytokines, including human IL-1b, IL-2, IL-4, IL-6, IFN-g, and TNF-a. These assays typically exhibit a lower limit of detection (LLOD) less than 10 pg/ml and linearity over more than three logs of analyte concentration, with maximal signal-to-background ratios (S/B) greater than 1000.
Following treatment of human peripheral blood mononuclear cells (PBMC) in 96-well plates with vehicle, LPS, R848, or a combination of PMA and ionomycin (Cell Stimulation Cocktail, CSC), the non-lytic cytokine detection reagents were added directly to the culture wells containing cells and medium. Dose- and time-dependent release of cytokines were observed over a wide range of response levels without the need for sample dilution. Alternatively, replicate aliquots of medium from each cell treatment well were transferred to a separate assay plate for simultaneous determination of multiple cytokines released from individual cell wells. In a cell model comprised of purified CD8+ T cells and target Raji B cells treated with increasing bispecific T-cell engager Blincyto®, release of IL-2 and IFN-g was observed with an EC50 of ~0.2 ng/ml and maximal S/B for IL-2 and IFN-g of 82- and 168-fold, respectively. In addition, differentiation of purified CD4+ T cells into the Th2 phenotype resulted in significant IL-4 release in response to subsequent CSC treatment. Following reagent addition, assay signal exhibited glow kinetics with a half-life of approximately two hours. In addition, 384-well, assay performance for determination of cytokine release from treated human PBMC resulted in Z’ factors greater than 0.7, indicating the assay format is amenable to screening applications.
The implementation of this novel detection chemistry will enable rapid “add-and-read” determinations of cytokine release from cells for both low- and high-throughput applications, including quantitative assessments of T cell activation and differentiation.