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.

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.

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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.

Rabbit Hybridoma Workflow Chart

StepsTimeline (weeks)Deliverables/Internal testing milestoneQC 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 weeks100 ul anti-serumELISA 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 weeksSupernatant from 96 wellsELISA from supernatant
Phase IV: Expansion and antibody purification

(customer choosing one clone; expanding clone and purifying antibodies with affinity chromatography)

 

6-8 weeksOne 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

I have tested the rat polyclonal IgGs to ABCD1 by immunoflourescence on cells overexpressing ABCD1. The antibodies successfully detected the protein (either untagged or tagged with GFP) at a 1:500 dilution and there was little background. The antibodies did not detect ABCD2. So this is very good news, and you may now go ahead and clone out a monoclonal!

Annette Ehrhardt, PhD

Dept. of Pediatrics | Emory University

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