Our in vitro cell-based pharmacology services can be broadly divided into primary cell-based assays and cultured cell-based assays.
Primary cell-based assays include agonist-mediated T cell activation, mixed lymphocyte reaction (MLR), antigen recall, T cell and NK cell toxicity, DC activation, macrophage-based phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and binding of test articles. Multiplexed cytokine release quantitation (using ELISA or MSD) and flow cytometry-based cellular marker detection are frequently used for these assays. Cultured cell-based assays include the evaluation of cell surface marker expression and binding of test articles, fluorescence or luminescence reporter assays, and multiple readouts of cell proliferation and cell death. Biocytogen also performs biochemical binding assays and affinity determinations using surface plasmon resonance (SPR).
List of our in vitro services:
- Drug-Cell binding assays
- Drug blocking assays
- In vitro stimulation assays
- Mixed lymphocyte reaction (MLR)-based in vitro stimulation assays
- In vitro killing assays (ADCC, CDC)
- Hemolysis/coagulation tests
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Antibody-dependent Cellular Cytotoxicity (ADCC) Assay
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Fig 1. ADCC with anti-human CTLA4 antibody
In this assay, human PBMCs were used as effector cells. Jurkat- hCTLA4 cells were labeled with high concentration of CFSE as the target cells, and Jurkat WT cells labeled with low concentration of CFSE as internal controls. Effector cells, target and internal control cells were incubated for 18 hours in the presence of varied concentrations of anti-human CTLA-4. Specific Cytotoxicity=[1-(No-drug control Ratio/Experimental Ratio)]*100%; Ratio=(CFSElow cells /CFSEhigh cells)
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Complement-dependent cytotoxicity (CDC) assay
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Rituximab-mediated CDC assay. Daudi cells were mixed with varied concentrations of rituximab or IgG1 control in the presence of complement-containing media and incubated for 2 hours. Cytotoxicity was measured by flow cytometry using Calcein AM.
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T Cell Activation Assay
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Human PBMCs (0.1 million) were added into wells of a round bottom 96-well culture plate with SEB (10 ng/mL) and varied amount of Ipilimumab or isotype control, and incubated for 48 hours. Release of IL-2 was determined by ELISA. The results show that ipilimumab dose-dependently potentiated SEB-stimulated IL-2 production by PBMCs.
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Mixed Lymphocyte Reaction (MLR) Assay
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Human T cells and allogeneic DCs were mixed in the ratio of 5:1 in wells of a round bottom 96-well culture plate in the presence of varied concentration of nivolumab or isotype control antibody and incubated for 72 hours. Release of IFN-γ was determined by ELISA. The results show that nivolumab dose-dependently potentiated IFN-γ production in mixed lymphocyte reaction (MLR).
