The production of monoclonal antibodies in targeting specific antigens to diagnose and treat certain diseases has been in existence for many years dating back to 1975. And this was the first time that Köhler and Milstein gave a description of the fusion of immunized mouse spleen B cells together with cancerous myeloma cells which then results in hybridoma cells that are immortal and secrete a specific antibody. Despite the several uses of mice in researching monoclonal antibodies, their reproducibility, specificity, their sensitivity is limited, and are also liable to antigens heterogeneity and conditional changes in the experiment.
In recent years through technology and more research, rabbit monoclonal antibodies arise which serve as an integral part of research antibodies. For therapeutic applications of monoclonal antibodies, rabbit monoclonal antibodies have to gain more stand in use to carry out research even in life sciences. And this is due to the recognition that scientists have found in the uniqueness of rabbits’ antibodies such as having broader reactivity with commonly studied animal tissues and also the stronger and more diverse immune responses that are gotten from rabbit antibodies.
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Why rabbits are used for monoclonal antibodies
The reason rabbits are gaining more use for monoclonal antibodies in research is as a result of unique traits that are present in rabbits making them ideal for monoclonal antibody production. Some of these traits in rabbit monoclonal use can be seen in the following instances.
- Antibody repertoire: this is the complete set of antibodies that are produced altogether. The majority of the primary antibody repertoire in most species is being generated through the rearrangement of V, D, and J genes segments of immunoglobulin (lgG) genes which are within the developing lymphocytes. The VDJ gene combinations can be further varied through somatic diversification, leading to the development of a secondary antibody repertoire. However, for rabbits, they are in their ontogeny and their genetic makeup facilitates diversity in antibodies. The immunoglobulin in heavy chain locus in rabbits comprises more than fifty functional V genes segments having one that is predominantly rearranged. Much of the antibody diversity is generated by somatic gene conversions and hypermutation mechanisms. With these traits, rabbits could elicit a robust response.
- On the recovery of B cells from the spleen, bone marrow, or blood, the generation of monoclonal antibodies is predicated. These cells are of high quality in rabbits and this makes them more advantageous.
- When rabbits are compared with other species, in the laboratory, they show more heightened immune responses. The immune response of rabbits is more sensitive in that it elicits vigorous responses to small molecules without the aid of carrier proteins such as KLH.
- The laboratory strains of rabbits are outbred, serving lots of benefits as inbred are capable of decreasing the diversity of immune responses and the general sensitivity of the antibodies that are produced.
- The increased evolutionary distance between rabbits and humans gives room for rabbits to be uniquely immunogenic to certain antigen epitopes and gives rabbits’ total pool of specific antibodies to be larger and more diverse. The diverse range also facilitates the production of antibodies that cross-react with rodent and human antigens and this makes rabbit monoclonal antibody to be suitable for the study of human tumor xenografts and mouse models of human diseases.
- Complementarity-determining regions (CDRs): these are the segment variable regions of the antibody that is the heavy and light chains where antigen-binding interactions occur. The heavy chains of rabbit monoclonal antibodies have a longer third CDR (CDR H3) and this is the reason rabbit monoclonals naturally have a higher affinity that is 10-100 times.
- Because of extra disulfide bonds, rabbit monoclonal antibodies have longer shelf life and stability
Recombinant rabbit monoclonal antibodies
Recombinant antibodies are monoclonal antibodies that are generated in an artificial environment outside the living organism from the cloned heavy and light chain antibody genes. Any species of antibody-producing animal can be cloned for their antibody genes and recombinant antibody production once enough genomic sequence is known for the design of primers or hybridization probes. To drive the results of the cloned heavy and light chain genes as separate proteins, high-yield plasmid DNA vectors that are transfected into hosts such as bacteria, yeast, or mammalian cell lines are used. These are then assembled into natural IgG molecules within the hosts. The engineered cell lines are stored for long periods or used to precipitate the mass production of monoclonal antibodies once it has been probed and separated.
The recombinant antibody protocol is involved in the construction of antibody libraries. These libraries vary in the source of the cloned heavy and light chain cDNA sequences. The sources according to which they are varied are naïve, immune, semi-synthetic, or fully synthetic.
The naïve and immune libraries are the animal-derived sequences. And this is because they are directly from cloning lgG genes from immunized and non-immunized animals. For semi-synthetic and fully synthetic libraries, they are derived from IgG sequences from known antibody constructs.
Rabbit monoclonal antibodies have the propensity of outperforming the traditional monoclonal antibodies while also providing better and more accurate results with decreased background staining and false-positive rates in assays.
With the combination of monoclonal antibody technology and recombinant DNA and protein expression, there will be a construction of a direct link between antibody genotype and the phenotype. To have the actual power of the recombinant technology is to have the ability to select and screen for the specific characteristics of the binding kinetics.