The overall goal of this protocol is to assess the ability of an antibody or a serum sample to activate Fc-mediated effector function. This method can help answer key questions in the immunology and vaccinology fields by measuring the activation of antibody-dependent Fc-mediated immunity. The main advantage of this technique is that the experiment can be done within 24 hours from the material preparation to the data analysis.
Demonstrating this procedure will be Mark Bailey, graduate student from the laboratory of Peter Palese. To induce influenza virus hemagglutinin expression via transfection, first plate human embryonic kidney 293 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate. After four hours at 37 degrees Celsius and 5%CO2, transfect the cells with 100 nanograms of DNA coding for viral hemagglutinin and 0.2 microliters of transfection reagent per well and a total volume of 50 microliters of 1X optimum-reduced serum medium.
Add DNA liposome complexes to each well to transfect and return the plate to the incubator for another 18 hours. To induce influenza virus hemagglutinin expression via infection, plate A549 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate for an at least 18-hour incubation at 37 degrees Celsius and 5%CO2. At the end of the incubation, dilute the influenza virus to a multiplicity infection of three in 100 microliters of 1X MEM medium per well and infect the A459 cells for one hour in the cell culture incubator.
At the end of the incubation, replace the inoculum with 100 microliters of fresh 1X MEM medium without virus and return the cells to the incubator for another 18 to 24 hours. To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally-affected cell cultures with 25 microliters of Antibody-Dependent Cell-mediated Cytotoxicity or ADCC assay medium. Next, in a separate 96-well plate, perform four full dilutions of the antibodies of interest in triplicate in fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic and taking care to include the background and no antibody controls.
When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the fourth modified effector Jurkat cells expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well. Return the plate to the cell culture incubator for six hours thawing luciferase substrate buffer in a 37 degree Celsius water bath during the last hour of the incubation.
When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated. In this experiment, a stock-specific monoclonal antibody robustly activated and modified Jurkat cells expressing murine Fc gamma receptor four by approximately 14-fold compared to head-specific monoclonal and control IgG antibodies.
After watching this video, you should have a good understanding of how to set up an experiment for assessing the ability of specific antibodies of interest to activate Fc-mediated immunity.
