Monocyte
Inflammation Assays
- Inflammasome/LPS cytokine release
- Predictive immunotox CRA
Inflammasomes are key signaling platforms within the immune system. Inflammasome complexes form in response to infection, tissue damage or metabolic imbalances. Once formed the inflammasomes activate Caspase 1 which in turn activated the pro-inflammatory cytokines IL-1β and IL-18. Targeting the inflammasome and the resulting signaling pathways is a useful tool in modulating the immune system.
Effect of MCC950 on Inflammasome activation – IL-1β and IL-18.
PBMC from three healthy donors were pretreated with MCC950, vehicle (DMSO) or media for 1 hours prior to stimulation with LPS for 4 hours. Nigericin was then added for a further 45 minutes prior to harvest of the cell culture supernatant for quantification of (A) IL-1β and (B) IL-18 by TR-FRET and ELISA respectively. Data points show mean +/- SEM of technical replicates. One-way ANOVA with Dunnett’s multiple comparisons test comparing stimulated (media) to unstimulated, LPS alone, vehicle and MCC950 to vehicle; *<0.05, **<0.01, ***<0.001.
Other formats: PBMC, macrophage subsets, microglia
Cytokine release assay can be used to “de-risk” any potential unwanted effects of novel biotherapeutics in accordance with FDA and EMA recommended guidelines.
Preclinical safety PBMC cytokine release assay (CRA)
PBMC solid phase CRA: release of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-α in response to a panel of antibodies coated onto plastic or TLR stimuli or polyclonal T cell stimuli (PHA).
PBMC isolated from buffy coats were added to wells coated with a-CD28 (TGN1412), a-CD3 (Muromonab), a-CD52 (Alemtuzumab) or with isotype control antibodies, or were stimulated with PHA, LPS, or a cocktail of TLR3/7/8/9 agonists. Cytokine release was measured at 48hr post stimulation. NS = Non-stimulated; Graphs show log10 transformed data with mean of 10 donors indicated. One way ANOVA with Sidak’s multiple comparisons test performed on log10 transformed data with significance indicted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Note for IL-8 where values were above the ULOD these were assigned ULOD value.
Other readouts: frozen disease PBMC
Other formats: Whole Blood CRA
Other immuno-tox assays: include ADCC/ CDC/ ADCP
Preclinical safety whole blood cytokine release assay (CRA)
Whole blood soluble CRA; release of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-α in response to a panel of antibodies or polyclonal T cell stimuli (PHA).
Whole blood was added to wells containing a-CD28 (TGN1412), a-CD3 (Muromonab), a-CD52 (Alemtuzumab) or with isotype control antibodies or were stimulated with PHA. Cytokine release was measured at 48hr post stimulation by MSD. NS = Not stimulated. Graphs show log10 transformed data with mean of 10 donors indicated. One way ANOVA with Sidak’s multiple comparisons test performed on log10 transformed data with significance indicted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Note for IL-8 where values were above the ULOD these were assigned the ULOD value.
Drug Discovery Tool
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