Since the development of hybridoma technology by Köhler and Milstein in the 1970s, monoclonal antibodies have become essential tools in biochemistry, molecular biology, and medical research. Over the years, the mouse has been the predominant animal host for antibody production. However, in recent years, rabbit monoclonal antibodies have been sought after by researchers and industry leaders around the world as a suitable alternative to rodent-derived antibodies.
Despite the reproducibility and specificity that mouse antibodies deliver, several intrinsic disadvantages limit their applications. It wasn’t until 1995 that Katherine Knight and colleagues developed the hybridoma-based technology for rabbit antibody production and proposed them as an improved alternative to mouse antibodies for the scientific community.
Rabbits are characterized by a variety of natural features that make them very attractive for monoclonal antibody development. Their larger overall body size (spleen, bone marrow, blood volume), when compared to rodents, indicates that more lymphocytes (B cells) are available for producing antibodies. The scarcity of inbred rabbit laboratory strains is also advantageous as inbreeding decreases the diversity of immune responses and the overall sensitivity of the antibodies produced.
As rabbits are evolutionarily closer to humans compared to rodents, their antibodies afford wider highly distinctive antibody repertoires that can recognize a greater variety of epitopes on human antigens that are normally non-immunogenic in rodents (small molecules, peptides, etc.) with great affinity and specificity. Rabbits are also well-known for mounting a strong immune response against foreign antigens when compared to other laboratory species. These properties reduce the likelihood of cross-reactivity with non-targeted peptide sequences for better, more accurate results with decreased background staining and false positive rates in assays.
With the continued demand from researchers for better antibodies and reagents, rabbit monoclonal antibodies hold the potential to outperform antibodies raised from other species for various applications, as further outlined below.
Western Blot (WB)
Choosing the right antibodies is essential for cleaner, more reliable, and reproducible WB results. Most primary research antibodies for WB are developed in mice and rabbits—although theoretically any host species can be used. However, the greater repertoire of rabbit monoclonal antibodies allows for more successful screening, isolation, and cloning of high-affinity antibodies—which is especially important when aiming to make WB antibodies to more challenging epitopes.
Western blot analysis of extracts of Human cerebellum lysate, using rabbit monoclonal BDNF antibody (Image Source: Boster).
Immunohistochemistry (IHC)
For IHC, antibodies need to be able to identify epitopes in the background with more noise (i.e. FFPE tissues). In this case, rabbit monoclonal antibodies are particularly useful because they can be highly sensitive while retaining specificity even in strict applications like IHC. Correspondingly, in many IHC studies that compared rabbit and mouse monoclonal antibodies to the same human antigens, rabbit antibodies consistently demonstrated higher sensitivity. IHC with FDA-approved rabbit antibodies that aid treatment decisions are compiled in this article.
Immunoprecipitation (IP)
For IP applications, high-affinity monoclonal antibodies should be used because low-affinity antibodies may not form an antigen-antibody complex in the solution. Rabbit monoclonal antibodies can have extremely low equilibrium dissociation constants in the picomolar range (KD = 10-12M), with a 10-100x higher affinity for target antigen than mouse antibodies.
ELISA
As rabbit monoclonal antibodies have inherent monospecificity toward a single epitope and are better at identifying subtle differences (epitope variations, mutations, conformational changes, etc.) between a wide range of antigens, with a lower chance of cross-reacting with other proteins. This allows fine detection and quantitation of small differences in antigen during ELISA. Rabbit monoclonal antibodies also bind to the target antigen with greater affinity. They prove to have a higher signal-to-noise ratio and fewer problems of cross-reactivity often observed with mouse monoclonal antibodies.
Post-Translational Modification (PTM) Detection
The rabbit immune system can produce antibodies against small epitopes existing in small molecules, lipids, and polymers, which permit rabbit monoclonal antibodies to identify subtle changes from PTMs or single amino acid substitutions.
Therapeutic uses
Over the past two decades, therapeutic monoclonal antibodies have become clinically and commercially successful drugs. Most FDA-approved therapeutic monoclonal antibodies are either murine, humanized murine, or human variable domain amino acid sequences. Although no therapeutic rabbit mAbs have been approved by the FDA thus far, several rabbit mAbs have currently entered various clinical trials—as registered at ClinicalTrials.gov. Along with an account of successful humanization strategies, there is no current evidence that rabbit or humanized rabbit mAbs are more immunogenic than their murine counterparts.
Virus particles interacting with antibody molecules (Image Source: Freepik)
One disadvantage specific to rabbit monoclonal antibody development is that the technology is quite time-consuming and costly, and the success rate is relatively low. This often translates to rabbit monoclonals often being priced higher than alternatives raised in mice.
Antibodies generated through the use of recombinant technology overcome those issues and have made rabbit antibody production more achievable than the traditional method. In addition, recombinant antibodies also have the following benefits:
- Highest level of stability and consistency between batches
- Ease of scalability and continuity of supply
- High-throughput in vitro manufacture
- The sequence is available for downstream development (humanization, etc.)
- Improved sensitivity and confirmed specificity via antibody engineering
If you cannot find a suitable rabbit monoclonal antibody for your particular application, Boster offers a custom rabbit monoclonal antibodies production service using a proprietary plasma cell discovery (PCD) platform that delivers application-specific antibodies with guaranteed affinity and specificity—even for difficult targets.
Leveraging 25+ years of expertise in antibody production, the company offers three standard protocol packages and several extension options. Apart from the ability to produce customized antibodies, Boster also offers highly validated, ready-to-use catalog antibodies for your research. Get in touch today to discuss your project requirements and save time to focus on your research!
References:
Leenaars, M., & Hendriksen, C. F. M. (2005). Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR Journal, 46(3), 269-279. doi: https://doi.org/10.1093/ilar.46.3.269
Moure, J. G. V., & Vara, J. A. R. (2005). Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues. Journal of Veterinary Diagnostic Investigation, 17(4), 346-350. doi: https://doi.org/10.1177/104063870501700407
Rossi, S., Laurino, L., Furlanetto, A., Chinellato, S., Orvieto, E., Canal, F., … & Tos, A. P. D. (2005). Rabbit monoclonal antibodies: a comparative study between a novel category of Immunoreagents and the corresponding mouse monoclonal antibodies. American Society for Clinical Pathology, 124, 295-302. doi: https://doi.org/10.1309/NR8HN08GDPVEMU08
Weber, J., Peng, H., & Rader, C. (2017). From rabbit antibody repertoires to rabbit monoclonal antibodies. Experimental & Molecular Medicine, 49, e305. doi: https://doi.org/10.1038/emm.2017.23
Zhang, Z., Liu, H., Guan, Q., Wang, L., & Yuan, H. (2017). Advances in the isolation of specific monoclonal rabbit antibodies. Frontiers in Immunology, 8, 494. doi: https://doi.org/10.3389/fimmu.2017.00494