Immune safety assays

Cytokine release assays (CRA)

CLS-logo.png Concept Life Sciences offers a science-led optimization approach for your test substance in determining the most suitable CRA format before performing a screen in a larger donor cohort. 

  • New molecular entities may induce infusion reactions and in turn, lead to cytokine release syndrome (CRS) characterized by a broad inflammatory response and adverse reactions.
  • Following the TeGenero TGN1412 FIH trial that led to 6 volunteers suffering multi-organ failure1, the FDA issued draft guidance on Nonclinical Safety Evaluation of the Immunotoxic Potential of Drugs and Biologics (Feb 2020).
  • There are multiple protocols to test Human cell-based in vitro assays to detect harmful cytokine release and each needs optimizing for each Test Substance2
  • Need to identify the most sensitive model of detection of cytokine release and optimize protocol.


  1. BMJ 2006;332:677 - Learning from the TGN1412 trial 
  2. Findlay, Eastwood, Ball et al. 2011; Comparison of novel methods for predicting the risk of pro-inflammatory clinical infusion reactions during monoclonal antibody therapy. Journal of Immunological Methods, 371, 134-142

Science-led approach - optimization

Our tailored and customized approach ensures a robust experimental design to provide a strong data package and address your scientific questions.

  • Two methods of Test Substance exposure to the biological sample (PBMCs and Whole Blood) are assessed – wet coated onto a plate or applied in aqueous phase.
  • 44 groups for optimization including reference controls Lemtrada® (Alemtuzumab) and Lipopolysaccharide (LPS) known to cause cytokine storm in patients.
  • Th1/Th2 cytokine release profiles are measured using the Multiplex Luminex platform. An exemplar cytokine panel includes GM-CSF, IFN-γ, IL-β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNFα.

Methods of data analysis

For each sample replicate the fold change over vehicle control is calculated, a 2-fold change over vehicle control is considered as relevant. The percentage of responding donors can also be calculated and compared to relevant reference controls.

Using standard assay reference control, Lemtrada® and LPS induce a consistent and widespread cytokine release effect across the donor cohort.

How our cytokine release assay is performed

1. Peripheral blood samples from multiple donors are used to ensure genetic diversity.2. Test and control compounds are prepared to required concentrations and applied wet coated onto assay plates or used in aqueous phase; diluted blood is then added and plates are incubated for 1, 6 or 24 hrs.

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3. Test substances interact with their specific cellular targets leading to the release of cytokines from activated immune cells.4. Assay plates are spun down and supernatants are used to measure a panel of cytokines such as GMCSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNFα using the MSD technology.

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5. Data are analyzed and displayed in a series of convenient formats such as heatmaps, bar graphs and dot plots to provide a quick overview of cytokine responses across different donors or to determine the population of responders vs non-responders for individual treatments and cytokine. CRA flow image 5.png