Monoclonal antibodies are used as primary antibodies and they are produced in the lab as artificial antibodies from a single B cell clone and have the capability of binding to a single antigen. Monoclonal antibodies are homogeneous and they cannot form lattices with monomeric proteins as they can only bind to a single epitope on the antigens. Monoclonal antibodies can be produced with any other given substances and this makes them useful in detecting and purifying other substances of interest. Rabbits’ monoclonal antibodies are antibodies developed in rabbits and are commonly used to generate antibodies. And the need for rabbit monoclonal antibody development arises due to the unique features it has in the production of monoclonal antibodies. They are capable of producing a large variety of B cells and antibody structures. They also have the potential of producing antibodies 10-100 times higher in affinity for the target antigens.
The advantages of Rabbit monoclonal antibodies
When it comes to producing antibodies, custom rabbit monoclonal antibodies provide more strategic benefits.
- High Affinity: rabbit customs have high-affinity monoclonal antibodies which are related to its unique immunoaffinity maturation process. With the high affinity of rabbits, the dissociation factor can reach picomolar levels (kd=10-12 mol/l). L). Rabbit monoclonal antibodies have great potential for applications in the development of more sensitive detection reagents and more effective drugs because of the high affinity that they possess.
- Superior specificity and low background: the binding specificities of rabbit monoclonal antibodies are very strong and approximately 1000 different monoclonal antibodies were discovered using the western blot experiments methods. The immunohistochemical experiments also confirm the superior specificity of rabbit monoclonal antibodies. Rabbit mAbs have a clearer image of the immunohistochemistry making background staining on the paraffin section to be greatly reduced.
- More novel epitopes can be identified: rabbit antibodies can recognize more epitopes. When rabbit-specific antibodies are used, it increases the recognition of certain epitopes such as phosphorylation sites and other post-translationally modified protein sites. Rabbits can generate higher affinity against difficult epitopes such as protein modifications, enzyme cleavage sites, or conformational epitopes.
- Hybridoma clones more: the clonal hybridoma has larger spleens and the B lymphocytes can be fused nearly 50 times. In each of these fusions, hybridoma clones production can be many times larger. Rabbit hybridoma cells are also suitable for a high-density culture having the interval between cell passages shortened.
- Essential in detecting Post-Translational Modifications: rabbit immune system has the propensity of producing antibodies against small epitopes that exist in small molecules, lipids, and polymers. This permits rabbit monoclonal antibodies to be able to identify subtle changes from the Post-Translational Modifications ( PTMs) or single amino acid substitutions.
Rabbit monoclonal antibody techniques
Some techniques are most commonly used in the development of rabbit monoclonal antibodies which are rabbit hybridoma, phage display, B-cell cloning, and plasma cell cloning.
- Rabbit hybridoma: this technique uses in rabbit hybridoma has a resemblance to that of mouse hybridoma technology. And this works by immunizing the animal, and when the titers are at the highest, B-cells are collected from the spleen of the host. With myeloma cells, the B-cells harvested will be hybridized to generate an immortal cell line that will have continuous production of specific monoclonal antibodies.
- Phage display: this technique is a higher recombinant DNA technique used in generating rabbit monoclonal antibodies and also for single-domain antibodies. Once the animal has been immunized, blood will be drawn out and the peripheral blood mononuclear cells or other B-cell producing materials are extracted. From these cells, RNA is isolated and then used to generate cDNA. The cDNA generated is used to construct large libraries of antibodies that can be expressed on the surface of the phage and selected for binding on the specifically targeted antigens. The specific DNA sequence of antibodies becomes easily determined and the antibodies are effortlessly expressed and purified.
- B-cell cloning: this technique is also a recombinant process used in the lab for rabbit monoclonal antibodies. The starting of this method is like that of a phage display. Here, PBMCs or other B-cells that produce tissues are isolated. Different sorting techniques are used to specifically target the B-cells in the mixture and separate them into colonies. After the colonies have been grown and screened, it is then used to select the cells which express the desired monoclonal antibodies. The variable regions of these antibodies that comprise the heavy and light chain are cloned and expressed in HEK 293 cells.
- Plasma cell cloning: in the method of monoclonal antibody development, this plasma cell cloning is almost identical on the surface to the B-cell cloning. What makes the difference between the two is that the plasma cells are fully isolated instead of isolating all B-cells for screening. Through the isolation of plasma cells from less developed B-cells, antibodies are obtained from the most matured cells. The variable of heavy and light chains is collected through PCR and expressed in HEK 293 cells.
Antibodies are essential tools in carrying out laboratory research and it has been in existence for many years. The scope of antibodies has also been expanded having artificial antibodies that can target specific antigens and the laboratory development of antibodies is the monoclonal antibodies which have diverse uses in diagnosing and treatment of certain diseases. With technology, rabbit monoclonal antibodies have been chosen to be of better results than that of other rodents as the spleen contains more B cells for more rabbit antibody production than that of mice which were once common in research and development of antibodies.