Services
Immunization
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.
Fusion
Standard somatic cell hybridization procedures 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).
Sub-Cloning and Cryopreservation
Hybridomas assaying positive for desired antibody secretion are grown in successively larger wells and then subcloned in the presence of thymocyte feeder cells. After growth of subclones the supernatants are rescreened. Selected subclones are then expanded and cryopreserved in liquid nitrogen.
Production of Ascites
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.
Hybridomas Available
The Hybridoma Core has a database identifying many of the hybridomas that University of Michigan investigators have deposited for storage in the Core. In some cases, antibody can be produced from these established hybridomas with permission of the depositor. If you are looking for a particular hybridoma cell line or monoclonal antibody, please inquire at hybridoma@umich.edu.
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, 7). The Hybridoma Core can assist laboratory staff in establishing, maintaining and harvesting hybridomas using these culture devices.
Screening Systems
Laboratories are responsible for the screening of hybridoma culture supernatants at all stages of monoclonal antibody production. The Core Directors, David Fox, M.D., and James Baker, M.D., are able to assist investigators in devising useful screening strategies, such as flow cytometry or ELISA assay.
Isotyping ELISA
The Hybridoma Core performs an ELISA assay for determining murine isotypes/subtypes from hybridoma tissue culture supernatants.
Human-Human Hybridomas
These have been successfully produced in this facility using methods described by Rauch (8). 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.
Rabbit Hybridomas
The Hybridoma Core is acquiring the rabbit plastocytoma cell line 240E1. Fusions of splenocytes from immunized rabbits with this cell line have successfully generated stable hybridomas secreting monoclonal rabbit antibodies (9). This procedure may be introduced in the Hybridoma Core on an experimental basis in the future; please inquire regarding the status of the technology.
