Rabbit Hybridoma Development
Rabbits belong to the order Lagomorpha, which is evolutionary distinct from the order Rodentia, to which mice and rats belong. Rabbit monoclonal antibodies possess several key advantages over rodent monoclonal antibodies. First, rabbit monoclonal antibodies are able to recognize epitopes on human antigens that are not immunogenic in rodents, therefore increasing the total number of targetable epitopes. Second, owing to their larger body size, rabbits can provide 50 times more spleen B cells than mice. Higher total B cell number warrants higher antibody diversity. Third, rabbit monoclonal antibodies can be more readily humanized by grafting into human antibody framework or Fc domain.
Rabbit hybridomas, however, are difficult to make. Both rabbit-mouse hetero-hybridomas, derived from fusion of rabbit splenocytes with mouse myeloma cells, and rabbit homo-hybridomas, derived from fusion of rabbit splenocytes with rabbit plasmacytoma cells, showed low growth rates, low antibody yields, and gradual losses of antibody secretion.
BiCell Scientific Inc has developed a serum-free culturing method that supports the rapid growth of rabbit hybridomas and the stable production of rabbit monoclonal antibodies. Our rabbit hybridoma culturing approach is included into rabbit monoclonal antibody service in Custom Antibodies.
Rabbit-mouse hetero-hybridoma development. Rabbit splenocytes were fused with mouse myeloma cells and cultured in HAT containing medium. Stable hybridoma clones started to show on Day 7.
Hybridoma cell plating and screening. Antibody secreting rabbit hybridoma cells were cultured in serum-free medium and their supernatants screened by ELISA in 96-well plates.
Rabbit Hybridoma Workflow Chart
| Steps | Timeline (weeks) | Deliverables/Internal testing milestone | QC standard |
| Phase I: Antigen preparation (peptide antigen, 13-19 aa length, will be designed against target protein and its region of interest, synthesized and conjugated to KLH)
| 2 weeks | / | COA
|
| Phase II: Animal immunization (two New Zealand rabbits will be immunized with antigen; conventional protocol – three injections)
| 8-10 weeks | 100 ul anti-serum | ELISA after 3rd injection |
| Phase III: Cell fusion and screening (fusion of BiCell's proprietary mouse myeloma cells with splenocytes from one best responder animal to generate hybridoma and perform antigen based hybridoma selection, enriching, and plating)
| 4-6 weeks | Supernatant from 96 wells | ELISA from supernatant |
| Phase IV: Expansion and antibody purification (customer choosing one clone; expanding clone and purifying antibodies with affinity chromatography)
| 6-8 weeks | One hybridoma cell line (to customer); purified monoclonal antibody from the selected hybridoma cell line (1mg) (BiCell Scientific will bank all remaining clones; customer can request additional clones by paying a service fee for hybridoma recovery) | ELISA from supernatant |