Immunizations are generally carried out in six-week-old Balb/c female mice. The Hybridoma Core facility has performed fusions with immunized “gene knockout” mice, rats and hamsters as well. Soluble proteins, fusion proteins, peptide conjugates, whole cells and plasmid DNA vectors expressing the antigenic protein can be used as antigens in immunization protocols. Protein antigens are generally mixed with complete Freund's adjuvant or incomplete Freund's adjuvant; when cells are used as the antigen they are injected as a suspension in PBS. These antigens are administered intraperitoneally at two-three week intervals. Plasmid DNA vectors are solubilized in PBS and injected intramuscularly at biweekly intervals. Serum from immunized animals is provided to the investigator’s laboratory for testing to monitor production of antibody in adequate titer for fusion. A final injection of antigen is administered intraperitoneally three to four days prior to fusion.
Conventional somatic cell hybridization techniques are used (1,2). The fusion partner used for murine hybridomas is the P3X63-Ag8.653 cell line developed by Kearney et al (3). Hybridomas are plated in a highly enriched selection medium and macroscopic colonies appear within seven to 10 days. In approximately two weeks, the medium is acidified and ready for screening by the investigator’s laboratory. Immunizations of rats or hamsters followed by fusion of splenocytes with mouse myeloma cells also have been successfully performed (4,5,6).
Laboratories are responsible for the screening of hybridoma culture supernatants at all stages of monoclonal antibody production. The Core Director, David Fox, M.D., is able to assist investigators in devising useful screening strategies, such as flow cytometry or ELISA assay.
Human hybridoma cell lines have been successfully produced in this facility using methods described by Rauch (7). These methods, which use the human B lymphoblastoid line GM4672 as the fusion partner, as well as other methods, will be available on an ongoing basis in this core facility. Please inquire (make inqurie link to contact info).
Sub-Cloning and Cryopreservation
Hybridomas assaying positive for desired antibody secretion are grown in successively larger wells and then subcloned by limiting dilution in the presence of feeder cells or supportive growth factor(s). Monoclonal cultures are selected and the supernatants screened by the investigator’s laboratory. Selected subclones are then expanded and cryopreserved in liquid nitrogen.
The Hybridoma Core can perform rat or mouse isotype determinations from hybridoma culture supernatants.
In Vitro Antibody Production
Milligram amounts of monoclonal antibody can be harvested from high-density cultures of hybridomas using the membrane-based technology of CELLine flasks (Integra Biosciences, 8). The Hybridoma Core can assist laboratory staff in establishing, maintaining and harvesting hybridomas using these culture devices.
Production of Ascites
Ascites productions are limited to those that qualify under ICUCA guidelines for the humane care and justified use of animals. The Hybridoma Core can produce milligram amounts of monoclonal antibody, in vivo, as ascites fluid, from hybridomas established in the Core or from other sources such as the ATCC. Pristane-primed retired breeder female Balb/c mice will develop ascites from most murine (Balb/c) hybridomas. Ascites can also be produced from rat-mouse or hamster-mouse hybridomas in pristane-primed athymic nude mice or normal Balb/c mice immuno-suppressed by gamma irradiation.