Abstract 5595: Rapid and sensitive determination of cytokine release from cells without the need for sample transfer
Authors: Lazar Dan F. , Kupcho Kevin R. , Sondgeroth Casey A. , Thompson David V. , O'Brien Martha A. , Gilden Julia K. , Hsiao Kevin , Cali James J. 
Published: September 2020

Abstract
Cytokines play a major role in cancer biology as inflammatory and immunomodulatory agents and are frequently measured in cell culture models and other biological samples. Various methods are available for in vitro measurement of cytokines, but they typically require sample transfer, sample dilutions, multiple wash steps, time-consuming protocols, and/or specialized instrumentation. We have utilized NanoLuc® Binary Technology (NanoBiT®) to develop a completely homogeneous and rapid assay method (&lt; 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 approach, separate antibodies to a specific cytokine are individually labeled with either 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 subunits reconstitutes a bright luciferase that produces light proportional to analyte levels when the substrate furimazine is present. Utilizing this technology, homogeneous bioluminescent immunoassays have been developed for several cytokines, including IL-1β, IL-2, IL-6 and IFN-γ. These assays share excellent sensitivities (LODs typically &lt; 10 pg/ml) and broad linear ranges extending over three or more logs of analyte concentration, significantly mitigating the need for sample dilutions. Following 24-hour treatment of human PBMCs in 96-well plate format 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 cell stimulus, maximal signal to background ratios (S/B) achieved for the various cytokines assayed were 347-, 450-, 580- and 655-fold for IL-1β, IL-2, IL-6 and IFN-γ, respectively. In a separate cell model comprised of activated T cells and target Raji B cells induced for 20 hours with increasing concentrations of the bispecific T-cell engager Blincyto®, dose-dependent 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 release of 82- and 168-fold, respectively. In all cases, a calibration curve of recombinant cytokine enabled straightforward conversion of relative light units (RLU) to concentration of released cytokines. The implementation of this novel detection chemistry will enable rapid “add-and-read” assays for cytokine detection amenable for both low- and high-throughput screening applications.
Citation Format: Dan F. Lazar, Kevin R. Kupcho, Casey A. Sondgeroth, David V. Thompson, Martha A. O'Brien, Julia K. Gilden, Kevin Hsiao, James J. Cali. Rapid and sensitive determination of cytokine release from cells without the need for sample transfer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5595.