Htrf® Measurement of Cytokine Release from Fresh Blood Samples

Cisbio offers a comprehensive line of HTRF cytokine assays to investigate the functional response of immune cells. These versatile, homogeneous assays are suitable for a variety of sample types, such as cell culture supernatants, using immortalized cell lines or more physiological cell models such as PBMC. Blood Assay Solutions is specialized in the development of assays performed in whole blood. Investigating cytokine release in whole blood gives access to a complex biological environment where all blood components are represented (PBMC and granulocytes including neutrophils, autologous plasma, red blood cells, platelets) thus allowing the investigation of important physiological immune responses like cytokine storm, inflammation and immunogenicity. This application note, in collaboration with Blood Assay Solutions, illustrates the use of Cisbio’s HTRF cytokine assays for the analysis of immunological response in samples directly obtained from donors’ whole blood. It is clear today that the evaluation of potential adverse effects of drugs on the human immune system should be incorporated throughout their standard development. Drug immunotoxicity can be defined as unintended immunosuppression or immunomodulation. Whereas immunosuppression can lead to decreased host resistance to infectious agents or tumor cells, enhancement of the immune system can exaggerate autoimmune diseases or allergic reactions. Drug-induced systemic immune activation can lead to severe reactions associated with an excessive cytokine release, also termed cytokine storm. Because the function of the immune system is highly species-specific, cytokine release in humans is hard to predict through animal studies. Whole blood assays have been described in drug development as an easy-to-do in vitro solution that could mimic the human situation. Such assays represent a fast and easy solution to monitor cytokine release in humans, enabling a prediction of efficacy as well as safety of the drug. This approach is faster and cheaper than the more established PBMC based assay, since no purification and isolation of cells is required. In addition, small volumes of blood (i.e. <5 mL) are sufficient to test multiple experimental conditions. In order to further improve both sample studies and and time savings, along with precise and accurate interpretation of biological responses, HTRF cytokine immunoassays were used for cytokine quantification. This study demonstrates the capability of HTRF cytokine assays to measure cytokine release in whole blood samples. Relevant immune pathways and related agonists were selected to target either the Toll-Like receptor pathways, the STING pathway (scheme 1) or the TCR activation pathway (scheme 2). Finally human IL1β, IL2, IL6, IL8, TNFα and IFNγ were quantified by HTRF. Furthermore, HTRF data were compared with data obtained with ELISA reference kits to evaluate the correlation between the two techniques.