A look into recombinant rabbit monoclonal antibodies

Nature's InsideView interviews Dr Weimin Zhu, Senior Vice President of Antibody Technology, for a profile feature on the RabMAb® platform

Abcam has been helping scientists since 1998 by providing high-quality biological binders and assays for research around the world. In order to address the changing demands of scientific research, the company has made considerable investments in developing and acquiring new technologies.

More than ten years ago, Weimin Zhu created Abcam's RabMAb® antibody platform. He continues to play a key role in product development and technology innovation. Antibodies derived from this platform are used around the world in research, diagnostic, and therapeutic fields.

Q: How does the RabMAb® platform benefit scientific research, and at what scale?

Q: How does the RabMAb® platform benefit scientific research, and at what scale?

The majority of monoclonal antibodies are generated in mice. However, antibody-producing B cells from mice are unable to generate every kind of antibody required. To overcome the limitations of mouse B cells, we developed a unique method of monoclonal antibody development using rabbits: the RabMAb® platform.
Although the basic methods of making a RabMAb® antibody is similar for mouse monoclonals, RabMAb® antibodies provide several unique benefits. This includes antigen diversity, high affinity, and high specificity - all of which are essential for the reproducibility of long-term and large-scale research.

Today, we have over 9,000 recombinant monoclonal RabMAb® antibodies. We also provide custom RabMAb® services to over 700 universities, institutes, and companies globally, and have developed more than 275 IVD-grade IHC antibody clones.

Our anti-Bcl-XL recombinant rabbit monoclonal antibody - ab178844 (left) being tested on multiple sample types in western blot against a highly-cited competitor anti-Bcl-XL rabbit polyclonal antibody.

Q: Why use the rabbit system to make antibodies?

​​​​Compared with other species, such as mouse, rat, and chicken, the rabbit has a unique immune system. Rabbits have a large B-cell repertoire, which essentially means they can generate a very diverse range of antibodies.

The rabbit immune system optimizes affinity by mechanisms that are more efficient than those of mice and other rodents. Rabbits can be used to produce these high-affinity antibodies, even against antigens that are not immunogenic in mice. On average, rabbit monoclonal antibodies have 10-100 times higher affinity for a target antigen than mouse monoclonal antibodies.

Rabbit antibodies are also better at distinguishing subtle differences. such as epitope variations like modifications, mutations and conformational changes, due to the larger and more diverse B-cell repertoire. RabMAb® antibodies combine the best properties of monoclonal antibodies with the most desirable attributes of rabbit polyclonal antibodies:
  • Diverse epitope recognition
  • High affinity and specificity
  • Reactivity across species to enable research with more common rodent models - eliminating the need for surrogate antibodies in preclinical studies
  • A renewable resource, since we produce them via a recombinant DNA production platform to ensure batch-to-batch consistency, quality, and scalability to the gram level

Q​: Is quality a big problem for researchers who work with antibodies?

Yes, it certainly can be. For example, there are times when a mouse antibody performs well initially but fails to produce the same results in subsequent batches. This can be due to hybridoma cell-line drift, gene loss or mutations. Batch-to-batch differences occur much more frequently with polyclonal antibodies - although polyclonal antibodies continue to play a vital role in life science research.

I think there are two antibody quality issues here: reproducibility and specificity. When we develop RabMAb® antibodies, we take several steps to address both of these issues.

Firstly, the majority of RabMAb® antibodies we offer are recombinant, to avoid hybridoma cell drift and batch-to-batch variation since recombinant antibody production processes are controlled and reliable. The recent addition of AxioMx's phage display-based in vitro​ antibody technology complements the RabMAb® platform and allows the reproducible production of new monoclonal antibodies on a timescale of weeks rather than months.

Secondly, to address the concern around specificity, every RabMAb® antibody is tested in five key applications (western blot, immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, and fluorescence-activated cell sorting), as well as in human, mouse, and rat tissues to check cross-reactivity. We have introduced KO validation for our antibodies using KO cell lines generated using the CRISPR/Cas9 system. The specificity of these KO-validated antibodies is verified by western blot using wild-type and KO cell lines1. This adds another layer of validation to confirm the antibody's specificity to the target.

Q: Are there any drawbacks to using the rabbit system?

When we initially introduced the technology the yield was quite low. Although rabbit-generated antibodies are typically 10-100 times more sensitive than mouse antibodies, they also generate an antibody yield around 10-100 times lower than mouse B cells in culture.

However, in 2005, we cloned the necessary rabbit genes to switch to producing recombinant rabbit antibodies from a cell line. This solved the low-yield issue. Now we can readily provide reproducible rabbit antibodies in large quantities.

Q: In what ways are you still working to improve your platform?

I have a technology team in Burlingame, CA, devoted to this. We're working on developing larger panels of antibodies against the same target, which is useful for the diagnostics side of our business. Making the best quality antibody can be 'numbers game', much like screening for candidate drug molecules: you need a large number of clone candidates to cover the broad spectrum of epitopes in order to select the best ones. You also need to prepare the right immunogen, the best immunization strategy, and a smart screening scheme to achieve this.

On top of that, we're introducing next-generation sequencing (NGS) technology to develop RabMAb® antibodies. We are confident we can characterize all rabbit B cells this way, and by using bioinformatics to develop customized algorithms based on our NGS data to select the best clone(s) for a particular project. This way we hope to generate even larger panels of antibodies against the same antigenic target in the near future.

Click here for the full Nature InsideView profile feature.

1​Abcam validates antibodies using KO cell lines from Horizon Discovery.

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