Rabbit Hybridoma Development

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. Finally, rabbit monoclonal antibodies have higher affinity and lower background than mouse or rat monoclonal antibodies when used in various diagnostic assays, such as IHC-FFPE, ELISA and Western blot.

Rabbit hybridomas, however, are difficult to make. Either rabbit-mouse hetero-hybridomas, derived from fusion of rabbit splenocytes with mouse myeloma cells, or rabbit homo-hybridomas, derived from fusion of rabbit splenocytes with rabbit plasmacytoma cells, showed cellular senescence and extremely low growth rates, which often led to a very small number of stable clones being established (<50 clones per rabbit spleen), largely owing to chromosomal instability of rabbit B cells after cell fusion and genetic recombination.

BiCell Scientific Inc has developed a cell fusion technique and a serum-free culturing method to support the the rapid growth of rabbit hybridomas and the stable production of rabbit monoclonal antibodies. Our fusion technique can generate 1,000-5,000 stable clones from each rabbit spleen. Our rabbit hybridoma culturing approach is included into rabbit monoclonal antibody service in Custom Antibodies.

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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.

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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.

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Hybridoma cell enrichment. Antibody secreting rabbit hybridoma cells were enriched by antigen based labeling, followed by magnetic bead sorting and plating into 96 wells. The sorted hybridoma cells were allowed to form colonies in serum-free medium and their supernatants were screened by ELISA and positive signals were shown in blue color.

rabbit-mab-clone-morphology

Rabbit hybridoma clone morphology. Rabbit hybridomas appear very differently from mouse or rat hybridomas in that they don't form dome-like highly-packed cell aggregation but instead spread over a wider area. Rabbit hybridoma clones tend to be bigger and flatter but show less cell-cell contact than mouse or rat hybridoma clones.

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Antigen labeling of rabbit hybridoma cells. Rabbit hybridoma cells are transiently crosslinked to immobilize antibody proteins on the cell surface. FITC-labeled antigen binds to and decorates the hybridoma cells that secrete the correct antibody. The decorated hybridoma cells can then be sorted with FACS or magnetic beads coated with anti-FITC antibody.

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

"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

Contact us for questions or custom requests!