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Functional Assays
NJ Bio utilizes plate-based assays to characterize the ability of compounds to promote or inhibit cell proliferation, establish the cytotoxicity of compounds by measuring live/dead cells. Assay optimization would include a time course of incubation and compound concentration analysis. Client will supply proteins for analysis.
Services include:
- 2D and 3D cytotoxicity assays
- ADCC (antibody-dependent cell-mediated cytotoxicity)
- Immunoassays (by ELISA)
2D & 3D Cytotoxicity Assays
NJ Bio utilizes in vitro cell cytotoxicity assays to analyze the potency of toxins or antibody-drug conjugates (ADCs). Each ADC will be tested against a variety of cancer cell lines expressing the targeted surface antigens and normal cell lines to evaluate in vitro safety. These assays quantitate ATP released by surviving cells at the end of the experiment, which directly reflects cell viability. NJ Bio will work closely with you to design custom assays for your molecules.
Cell Lines: NJ Bio acquires the required cell lines mainly from ATCC and DSMZ. Depending on the targeted antigen, cell panels specific to your antibody will be designed.
NJ Bio has experience with the following cancer cell lines:
- SKBR3
- BT474
- NCIN87
- PC3
- JIMT-1
NJ Bio offers classic 2D and novel 3D cytotoxicity assays, which provide comprehensive in vitro analysis of your ADC. The 3D assays utilize spheroids to mimic the physiological tumor microenvironment in comparison to the 2D assays where the cells adhere to the bottom of the well. Timelines for 2D assays are about five days and for 3D assays about ten days.
Process:
- Establish new cell line
- Plate 5,000 cells per well into the 96-well plates
- Flat bottom tissue culture 96-well plates are used for 2D
- Round bottom low adhesion Matrigel coated 96-well plates are used for 3D
- Add 10-fold dilutions of ADC or drugs
- Dilutions start at 1 mM for drugs and 1 µM for ADCs
- Use Cell Titer Glow to lyse remaining live cells
- Read ATP concentration using plate reader
ADCC (antibody-dependent cell-mediated cytotoxicity)
NJ Bio has the capability of performing assays that will examine the ADCC activity of your therapeutic antibody. A bioluminescent ADCC reporter assay is used to determine whether your antibody will illicit an immune mediated cancer cell killing. This assay measures the expression of luciferase and is not cytotoxic. We will work together to determine the optimum Effector:Target ratio and dose-dependent kinetic response in a series of antibody concentrations. Testing will be performed against a variety of cell lines expressing the target antigen. Client will supply the antibody for this ADCC assay.
Figure 1: (A) SKBR3 cells were used to test a specific IgG and a non-specific IgG; (B) The ratios of effector to target cells were optimized at different concentrations of antibody.
ELISA Assay:
NJ Bio offers ELISA assays for quantification of analytes in a sample. Depending on your analyte, certain antibody combinations and assays can be designed to meet your needs. We will work with you directly to design assays specific to your analyte(s), whether it be an antibody or a more complex protein.
