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The rabbit immune system has been documented as a vehicle for developing antibodies with higher affinity and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic in mouse. However, until recently, the type of antibodies available from rabbit had been limited in scope to polyclonal antibodies. Several efforts were made to generate rabbit monoclonal antibodies after the development of mouse hybridoma technology in the 1970s. Research was conducted into mouse-rabbit hetero-hybridomas to make rabbit monoclonal antibodies. However, these hetero-hybridomas were ultimately difficult to clone, and the clones, generally unstable, and did not secrete antibody over a prolonged period of time.
Initial fusion partner
In 1995, Katherine Knight and her colleagues, at Loyola University of Chicago, succeeded in developing a double transgenic rabbit over-expressing the oncogenes v-abl and c-myc under the control of the immunoglobulin heavy and light chain enhancers. The rabbit formed a myeloma-like tumor, allowing the isolation of a plasmacytoma cell line, named 240E-1. Fusion of 240E-1 cells with rabbit lymphocytes produced hybridomas that secreted rabbit monoclonal antibodies in a consistent manner. However, like the early mouse myeloma lines developed in the 1970s, stability was a concern. A number of laboratories which had received the 240E-1 cell line from Dr. Knight’s laboratory reported stability problems with the fusion cell line 240E-1.
Improved fusion partner
In 1996, Weimin Zhu and Robert Pytela, at the University of California San Francisco (UCSF), obtained 240E-1 from Dr. Knight’s laboratory and attempted to develop an improved rabbit hybridoma. Improvements in the characteristics of 240E-1 were accomplished by repeated subcloning, selection for high fusion efficiency, robust growth, and morphological characteristics such as a bright appearance under a phase contrast microscope. Selected subclones were further tested for their ability to produce a stable hybridoma and monoclonal antibody secretion. After multiple rounds of subcloning and selection processes, a new cell line named 240E-W, was identified and which expressed better fusion efficiency and stability. Cell line 240E-W has since been further developed and optimized for production of rabbit monoclonal antibodies for research and commercial applications.
The process of hybridoma formation in a rabbit first entails obtaining B-cells from a rabbit that has been immunized. There are numerous immunization protocols for rabbit, notably for the generation of polyclonal antibodies. After immunization, B-cells are fused with a candidate rabbit fusion partner cell line to form hybridomas. Resulting antibodies from hybridomas are screened for an antigen which meets criteria of interest by diagnostic tests such as ELISA, Western blot, Immunohistochemistry and FACS. The resulting hybrdomas may be subcloned to ensure monoclonal characteristics.
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- Rabbit Monoclonal Antibodies Available for Use in Cancer-Related Research.
- Cellosaurus entry for 240E-1
- Cellosaurus entry for 240E-W