Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

It is well known that antibody dependent, cell mediated cytotoxicity is an important mechanism by which antibody exert immune mediated certain effects. So we want to find a better method to test the efficacy of an antibody drug in cancer treatment, by using ADCCSA. The protocol is rapid and straightforward.

By using simple mix and read protocol, we can obtain the result within one day, thereby expediting the research. By using ADCC bioassay, we can perform mass screening of therapeutic antibody drug more accurately. The signal to noise ratio of the assay is high, which can help in accurate evaluation of the therapeutic antibody.

To begin, culture, the target T cells BHT 101 and SW 1736 cells until they reach 80%confluency in T75 flasks. After removing the media, wash the cells once with PBS and incubate them with one milliliter of non-enzymatic cell dissociation buffer for five minutes. Add PBS to stop the reaction.

Spin down the cells at 135G for five minutes, and then add five milliliters of PBS. Determine the cell count and seed the cells at 15, 000 cells per well in 96 well, white polystyrene microplates with a clear, flat bottom. To begin, prepare the target T cells BHT 101 and SW 1736 cells for the assay.

Then prepare Cetuximab to bind to the epidermal growth factor receptor, and Bevacizumab as a negative control. For making the ADCC bioassay buffer, add 1.4 milliliters of low immunoglobulin G serum into 33.6 milliliters of RPMI 1640, as supplied in the kit. Using the buffer, dilute the antibodies to prepare 400 microliters of each antibody in three different concentrations.

Preheat the ADCC bioassay buffer in a 37 degrees Celsius water bath for at least 30 minutes. Remove the ADCC bioassay effector cells from minus 80 degrees Celsius cold storage, and place them in a 37 degrees Celsius water bath for approximately two to three minutes. Gently rock and visually inspect the vial without inverting it during the thawing process.

Then transfer 630 microliters of effector cells to a 15 milliliter tube, containing 3.6 milliliters of ADCCSA buffer. Gently invert the tube twice to mix the cells with the buffer. To begin, prepare the target cells, effector cells, and the desired antibodies for the ADCC assay.

After overnight incubation, remove the media from the target cells and add 25 microliters of ADCC bio acid buffer followed by 25 microliters of antagonists to appropriate wells. Then add 75, 000 defector cells in 25 microliters of ADCC bioassay buffer per well into the 96 well microplate containing target cells, and incubate the plate for six hours at 37 degrees Celsius. To prepare the luciferase assay reagent, add luciferase assay buffer to the lyophilized luciferase assay substrate.

Then add 75 microliters of the luciferase assay reagent to each well of the plate containing target cells, antibody and effector cells and incubate the plate for 30 minutes at room temperature. Using a luminometer, measure the luminescence signal in each well to provide a quantitative readout of ADCC activity. For data analysis, calculate the fold induction, using the formula.

Cetuximab induced higher ADCC activity at all tested concentrations, but no ADCC activity was detected with Bevacizumab in either BHT 101 and SW 11736 cell lines, demonstrating the ADCC effect of targeting EGFR extracellular membrane antigens for both cell lines.