Rabbit Monoclonal Antibodies: Development & Production
Recombinant rabbit monoclonal antibodies are excellent tools for challenging targets such as small molecules or post-translational modifications. They have high affinity due to the rabbit’s robust immune system. Several methods are used to develop custom rabbit monoclonal antibodies, such as hybridoma generation, phage display, or B-cell cloning. ProSci’s B-cell development platform allows for careful selection of the ideal antibodies and preservation and immortalization through conversion to a recombinant. Recombinant antibodies provide the specificity of a monoclonal antibody but, more importantly, eliminate the potential loss of a hybridoma clone through preservation of the heavy and light chain sequences which minimizes lot-to-lot production variability through production as recombinant proteins.
Rabbits can provide benefits over mice when developing monoclonal antibodies. Rabbit spleens contain more B cells, increasing the probability of finding the right clone for your needs. Rabbits have a larger CDR-3 repertoire which can offer better selectivity and specificity to a desired antigen due to the higher incidence of longer CDR-3 loops in rabbits when compared to mice.
Longer CDR-3s can improve affinities to targets by 10 to 100-fold over murine antibodies. With a unique immune system that reduces immunodominance, they are the ideal host to develop antibodies against a wider range of epitopes and are preferred for developing antibodies against small molecules.
Outsourcing recombinant rabbit monoclonal antibody development to ProSci’s antibody experts allows you to focus on your core research.
Single B-Cell Cloning
ProSci’s recombinant rabbit monoclonal antibody development platform sorts and isolates antigen-specific B-cells, preserving native heavy and light chain pairs. The preservation of native pairs minimizes the amount of screening and sequence bias found in phage display development of conventional antibodies. It eliminates the chromosomal recombination found during fusion to generate a hybridoma. The antibody-secreting B-cells are cultured individually and screened after two weeks for antibodies. Heavy and light chains from the desired B-cells are then cloned and transiently transfected. The resulting supernatant is screened again to ensure the selection of antibodies that meet our clients’ needs.
Monoclonal Technology Comparison
| Hybridoma | Phage Display | Single B Cell Cloning |
|
|---|---|---|---|
| Starting Material | B Cells | B Cells | B Cells |
| Antibody Diversity | Medium | Low | High |
Schedule
Phase
Detailed Procedure
Procedure
I
Immunization
- Immunization of 3 rabbits on 10-week protocol
- ELISA testing of serum
- Final boost and spleen isolation
II
Single-cell Sorting, Culturing, and Screening
- Sorting and culturing antibody secreting B-cells
- Screening of supernatants by indirect ELISA
- Supernatants from ELISA-positive wells
III
Antibody Cloning and Small-Scale Expression
- Cloning variable regions from B-cells
- Transient expression of recombinant antibodies
- Screening of supernatants by indirect ELISA
- Supernatants from ELISA-positive wells
IV
Sequencing, Expression, and Purification
- Sequencing heavy and light chains
- Expression and purification of up to 5 antibodies
- Delivery of sequences and plasmid DNA of up to 5 antibodies
Antigen Requirements
Antigen Purity
- >90% antigen purity is recommended to ensure effective conjugation, specificity, and higher affinity.
Protein Antigen Specifications
- Quantity 5 mg without tag at concentrations of 1 mg/ml or higher.
- Size At least 10 kDa
- Production method Use of native protein is preferred over recombinant protein; if a recombinant protein is provided, use an expression system similar to the origin of protein (e.g., use a mammalian expression system for human or other mammalian proteins).
- Tags Tags are not recommended for immunization, and large fusion proteins such as GST or MBP must be cleaved as antibodies will be generated against these in addition to the protein of interest.
Protein Antigens in Solution
- Concentration Antigens should be submitted at a concentration of 1 mg/ml or higher.
- Buffers such as PBS, Tris, <4M urea are preferred; SDS and citrate buffers are also acceptable. Please note that for services such as ELISA and immuno-affinity purification, the antigen must be soluble.
- Information required
- buffer
- concentration
- total volume
Please submit an antigen submission form with the information above prior to sending your antigen.
Peptide Antigen Specifications
- Quantity
- 5 mg for us to conjugate, or
- 1 mg conjugated to a carrier protein (KLH preferred), and
- 2-3 mg unconjugated for screening
Other Antigen Types
Protein antigens are preferred for rabbit monoclonal antibody development; for other antigen types refer to our antigen requirements guide or contact us.
Premier Package
Rabbit monoclonal antibody development using client-provided splenocytes
- B-cell isolation and sorting
- Cloning and final expression
Pro Package
Rabbit monoclonal antibody development
- 3 rabbit immunization protocol
- B-cell isolation and sorting
- Cloning and final expression
Add-ons
Additional Small-Scale Purified Antibody If additional purified antibodies are needed, we offer purified antibodies from 30 ml culture for additional available clones during the selection process.
Additional Final Clones If you want more final clones than the 5 offered in the packages, we offer additional clones, including the antibody sequence and DNA.
Customized Sorting and Screening If you need additional controls during screening, contact our antibody experts to design the optimal screening and sorting strategies to develop the desired antibodies.
Our Assurance
Because of the nature of developing antibodies from immunization to final clones, we implement project milestones consisting of clear, actionable go/no-go decision points during the development process. Our experts review the antibody development progress at each milestone to ensure your antibody success.
Custom Rabbit Monoclonal Antibodies
Once antibody development is complete, production can start. We offer transient expression of rabbit monoclonal antibodies in a mammalian expression system to produce larger quantities of purified rabbit monoclonal antibodies.
ProSci’s team of antibody specialists will help accelerate your research, therapeutic, or diagnostic project from idea to reality. Take advantage of decades of experience and contact our Team today for a free consultation on your project.
Order Recombinant Rabbit Monoclonal Antibody Development Services
