In Vitro Assays

Biocytogen provides a wide spectrum of in vitro cell-based and biochemical assays. In vitro cell-based assays 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 agents. Multiplexed cytokine release quantitation and flow cytometry (FACS)-based cellular marker detection are among the frequently used detection methodologies. Cultured cell-based assays include expression of cell surface markers or targets and binding of test agents, multiple readouts of cell proliferation and cell death, as well as reporter assays using fluorescent and luminescent, and optical detections. Biocytogen also performs biochemical binding assays and affinity determinations using surface plasmon resonance (SPR).

Our Services

  • Cell-binding assays of the drug
  • Humanized mouse splenocyte-binding assays of the drug 
  • Drug blocking tests 
  • In vitro stimulation tests
  • MLR-based in vitro stimulation tests
  • In vitro killing assays (ADCC, CDC)
  • Hemolysis/coagulation tests

Cell Surface Antigen Binding Assay: Detection of Human PD-L1 on Engineered MC38-hPD-L1 Cells

Cell-Surface-Antigen-Binding

MC38 is a mouse colon cancer cell line. MC38-hPD-L1 is an engineered MC38 cell line expressing human PD-L1 in place of mouse PD-L1. Analysis of PD-L1 expression with anti-mouse and anti-human PD-L1 antibodies by flow cytometry demonstrates that human PD-L1 indeed is expressed  only in MC38-hPD-L1, whereas mouse PD-L1 is only expressed in the wild type MC38.

 

Case2: Antibody-dependent Cellular Cytotoxicity (ADCC) Assay

Antibody-dependent-Cellular

Ratio=(CFSElow cell /CFSEhigh cell)

Specific Cytotoxicity=[1-(No-drug control Ratio/Experimental Ratio)]*100%

Specific-Cytotoxicity

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)

Case 3: Complement-dependent cytotoxicity (CDC) assay

Rituximab-mediated CDC assay. Raji cells were mixed with varied concentrations of rituximab in the presence of complement-containing media and incubated for 2 hours. LDH released to media was determined as indication of cytotoxicity. Rituximab demonstrated excellent CDC activity against Raji cells.

Cytotoxicity%=(Experiment-Spontaneous)/(Max release)*100%

Case 4: T Cell Activation Assay

T-Cell-Activation-Assay

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.

Case 5: Mixed Lymphocyte Reaction (MLR) Assay

Mixed-Lymphocyte-Reaction

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).

Case 6: RBC Sediment Test

Sediment-Test

Two percent (2%) red blood cells (RBC) was incubated with anti-human CD47 antibody of varied concentrations from 0 ng/mL to 10 ng/mL. Erythrocyte sediment was detected after 0.5 hours. RBC would normally precipitate to the bottom without toxic or pathological interference. As shown in the panel, toxic anti-CD47 antibody (top) prevented RBC sediment at lower threshold concentration (indicated by the red bars) than the less toxic anti-CD47 antibody (bottom).

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