An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.
The overall goal of this procedure is to generate antibody producing hybridoma cell lines for the biotechnological production of monoclonal antibodies. This method can be used to generate specific binders for the detection and visualization of biomolecules and for the development of effective detection systems, like ELISA, flow cytometry, or magnetic cell sorting. The main advantage of this technique is that it facilitates the generation of monoclonal antibody producing cell lines with a predefined specificity and a continuous availability.
To prepare the myeloma cells, thaw a vial of murine SP2/0 cells in warm water. Then transfer the cells into 10 mL of culture medium and collect them by centrifugation. Re-suspend the pellet in 10 mL of fresh culture medium and seed the cells into a 25 square centimeter flask for incubation at 37 degrees celsius, and 5%CO2, preserving aliquots of the cells for longterm storage after the appropriate number of expansions.
To prepare feeder cells, remove the fur from a 12 week old NMRI mouse and disinfect the abdomen with 70%ethanol. Next, inject 5 mL of ice cold RPMI medium into the abdominal cavity and use tweezers to massage the abdomen. Then withdraw the feeder cell suspension and dispense the cells into a new 50 mL tube.
After transferring a second feeder cell harvest into the same 50 mL tube, as just demonstrated, bring the total volume in the tube to 50 mL with fresh full cell culture medium. Then transfer the suspension to a new 100 mL reservoir, and repeat the addition. Then mix the cell suspensions well and seed 100 mcL of cells per well into 10 96-well cell culture plates for overnight incubation at 37 degrees celsius and 5%CO2.
For cell fusion, use the front end of a syringe plunger to press a freshly harvested spleen through a 40 micron strainer into a 50 mL tube. Suspend the cells in 20 mL of full cell culture medium, and then centrifuge. Next, use a 5 mL pipet to flush the myeloma cells with the culture supernatant and transfer the floating cells to a 50 mL conical tube for centrifugation, followed by four washes in 20 mL of balanced salt buffer.
After the last wash, add three times the volume of splenocytes to one part volume of myeloma cells and obtain a cell pellet by centrifugation. Re-suspend the cell mixture in 1 mL of fresh balanced salt buffer and mix 200 mcL of cells with 200 mcL of PEG 8000. Transfer the cell mixture into an electroporation cuvette with a 2 mm diameter and pulse the cells at 600 to 650 V for 25 ms, then rest the cells at room temperature for 3 minutes, before seeding them drop-wise into an Erlenmeyer flask containing 100 mL of selection medium.
After fusing the last 200 mcL of cells, incubate the pooled fusion products for three hours in a cell culture incubator. At the end of the incubation, supplement the cell culture with fresh hypoxanthine as a serine, and thymidine. Transfer the suspension to a reservoir and then add 100 mcL of the electroporated cell mixture to each well of feeder cells.
Finally, return the plates to the cell culture incubator. After seven days, check the wells under a 10x magnification for the presence of mono-or polyclonal cells. The monoclonal cells will appear as one cluster of cells that originate from a single cell.
The polyclonal cells will appear as multiple clusters of cells in one well. Then replace the supernatant with fresh selection medium, and return the cells to the incubator for another seven days. At the end of the second incubation, collect the supernatant for ELISA and add fresh full cell culture medium to the cells, for expansion of the hybridomas, into 24-well culture plates.
To cryoconserve the positive mono-and polyclonal hybridomas for longterm storage, harvest the cells from the 24-well plate when they reach an at least 60%confluency. Centrifuge the collected cells and re-suspend the pellet in 500 mcL of full cell culture medium. Transfer the cells into cryotubes.
Then mix the suspensions with 500 mcL of freeze medium and store the hybridomas at minus 80 degrees celsius for three days. For limiting dilution of the polyclonal hybridomas, after the first seven days of culture, perform an ELISA on the culture supernatants to identify the appropriate monoclonal hybridomas for mass culture. Next, transfer the monoclonal hybridomas that exhibited the appropriate ELISA signals to a cell culture flask.
Add medium and then return the culture to the incubator. Cryopreserving three to five aliquots in total during mass culture is sufficient. After two to four weeks, filter the supernatant through a 0.45 micron cell strainer and mix the supernatant with binding buffer at a 3:1 ratio.
Then wash a Protein A column with washing buffer and load the supernatant onto the column, collecting the flow-through. Elute the bound antibody with 0.1 molar citrate followed by the immediate neutralization of the pH with 500 mcL of TrisHCl. In this representative experiment, immune sera were titrated in comparison to serum from a naive mouse.
Both sera from the immunized mice exhibited a significantly higher antigen-specific titer compared to the naive animal, with a dilution of 1:5000 sufficient for cell fusion. Here a standard elution profile of a monoclonal antibody with an IgG1 isotype, purified via Protein A mediated affinity chromatography as just demonstrated, is shown. Further characterization of the eluate by SDS-PAGE revealed clearly visible proteins at 50 and 25 kDa, weights consistent with that expected for heavy-and light-chain antibody proteins respectively, indicating a successful purification of the isolated antibody molecule.
Once mastered, this technique can be completed within three and a half hours if performed properly. After watching this video, you should have a good understanding of how to generate monoclonal antibody producing cell lines.
