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Making Hybridomas Happen - 5 Tips For Creating and Optimizing Your Selection of Hybridoma Clones

Posted on: September 18, 2019

Hybridoma technology has revolutionized how antibodies are created in the laboratory and has been essential to the development of therapeutic antibodies for treatment of cancer, autoimmune disorders and the development of innovative diagnostics. Hybridomas are cell fusions of splenocytes collected from mice immunized with an antigen of interest and immortalized myeloma cells. A single hybridoma multiplies into cell clones and produces a monoclonal antibody against an epitope of the immunizing antigen, and this secreted antibody can be collected and used for a wide variety of applications. You can make hybridomas in your own laboratory with an appropriate animal protocol in place, or you can work with an experienced contract research organization to help you create and develop a panel of hybridomas for your next project.

Contemplate These Five Tips As You Consider Creating Hybridomas

Hybridoma technology at work in a laboratory.

1. Optimize Antibody Immunization

Consider your choice of animal (genetic background), antigen (peptide sequence, formulation, whole recombinant protein, domain, solubilized membrane fraction, etc.), adjuvant, and site of injection as you plan your hybridoma immunization protocol. You can use whole antibodies or portions of antibodies as immunizing proteins in order to create monoclonal antibodies that target specific antibody regions, such as anti-idiotype or anti-isotype antibodies. The frequency of immunizations and health and well-being of the immunized animals also impacts the overall immune response in the spleen, which will be the source of splenocytes for the fusion. You can screen animals for their antibody titers against the immunizing antigen, but titer does not reflect the binding qualities of the antibodies generated in that animal. Don't assume low titer responders will not provide you with a quality antibody.

2. Fusion Situations

Splenocytes and myeloma cells can be fused using different techniques including electrofusion and chemical fusion so consider trying multiple approaches to optimize fusions. Animal titers may give insight into how densely to plate your splenocytes as fusions from low titer responders may yield better results when plated at higher density. Take care when selecting the myeloma fusion partner and make sure these cells are of low passage number (fewer than 15 passages) and are in exponential growth phase.

3. Culture With Care

Fusions are incubated for 10-14 days in specialized HAT medium that will select out unfused myeloma cells. After this initial incubation, remaining fused cells must be serially diluted so that an individual cell fusion per well, that will start to expand into a clone. You want your monoclonals to be fairly rugged from the start so don't baby them along with high serum media as this will only prolong their inevitable demise. No more than 10% high quality, non-heat treated serum is required post fusion. However, the inclusion of ITS supplement (Insulin-Transferrin-Selenium) can make a real difference so should be included for happy cells.

4. Screen Time

The clones can be screened for antibody production by ELISA or flow cytometry. Strong candidates can be further assessed in functional assays and the best clones can be grown up in larger batches or cryopreserved for later use.

5. Long-Term Antibody Production

Once you select an optimal clone, you may need to culture the clone for several weeks to scale up your hybridoma for large scale antibody production. Special additives, such as cytokines, can be used in the culture media to help support clone expansion and sustained monoclonal antibody production.

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