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11 Reasons Why You Should Use Recombinant Antibodies (rAbs)

Posted in: Protein Expression and Analysis
11 Reasons Why You Should Use Recombinant Antibodies (rAbs)

Monoclonal antibodies: You’ve probably heard a lot about them. Unsurprisingly, you may have also used them in your research. These antibodies (mAbs) are classically produced by the hybridoma technology pioneered by Köhler and Milstein in 19751: A mouse is immunized with the substance against which you need to produce an antibody. The mouse spleen cells (consisting mainly of immune B-cells) are then fused with special myeloma cells. The result is an immortal hybrid cell, a hybridoma, which secretes the desired mAb. This technology is used to this day to produce mAbs with exquisite specificity and in unlimited quantities.

Despite these attractive features, mAbs suffer from several disadvantages, some or all of which you may have come across. Fortunately for us, recombinant antibodies (rAbs) arrived and revolutionized the world of monoclonal antibody production, overcoming several limitations associated with mAbs. How so? Read on!

First of All, What Are Recombinant Antibodies?

rAbs are constructed in vitro, outside the constraints of the immune system, using recombinant DNA technologies. The antibody genes are isolated and then incorporated into plasmid DNA vectors, and the resulting plasmids are transferred into expression hosts such as bacteria, yeast, or mammalian cell lines. rAbs can be used in all applications where classical mAbs are used.

 How Then, You Ask, Do rAbs Overcome the Drawbacks of mAbs?

  1. They require less purified antigen to produce than classical mAbs.
  2. The production time of rAbs is weeks vs months for mAbs, and rAb production is amenable to high-throughput processing.
  3. Unlike mAbs, rAbs can be constructed against virtually any antigen, including human and highly conserved antigens (not recognized as foreign by the mouse’s immune system), as well as non-immunogenic (do not elicit an immune response) and toxic molecules (cannot be injected into the mouse host).
  4. rAbs circumvent the human immune response elicited by murine mAbs. If administered to humans, a mAb (even a “humanized” one) will appear as foreign to the human immune system, and will cause an adverse hypersensitivity reaction.
  5. rAbs can be produced in several formats: Fab fragments (the antibody’s “arms”), single-chain variable region fragments (scFv, consist solely of the antibody’s binding site), diabodies (dimeric scFvs), and can be expressed in several hosts: coli, mammalian cells, yeast, fungi, insect cells, and even plants. Bacterial expression systems are particularly quick and inexpensive.
  6. rAbs are amenable to fusion with drugs and toxins, and can thus be used in therapeutics.
  7. rAbs circumvent the need to immortalize B-cells in the form of hybridomas. Remember that hybridomas are not normal cells, and have an unusual assortment of chromosomes, the loss of which can occur at any time during the culture process. In fact, anyone who has worked with hybridomas can tell you that these finicky, supposedly immortal cells can die off, not grow when taken out of cryogenic storage, or simply decide to stop secreting the mAb (due to loss of antibody genes). All of these obstacles are bypassed by rAbs, which are constructed by isolation of the respective antibody genes. If you have access to an antibody gene, you will have at your disposal an unlimited source of the antibody itself; all you would need is a protein expression platform.
  8. rAbs are defined by the sequences that encode them, making them more reliable, and providing more reproducible results than mAbs.
  9. rAbs can be readily optimized, as their nucleic acid sequences are defined and readily accessible. Do you need to improve the affinity of your rAb? No problem! Affinity maturation of rAbs, often to levels unattainable by mAbs, is possible through several molecular biology techniques such as error-prone PCR and site-directed mutagenesis.
  10. The selection process for rAbs is highly flexible and can be adjusted to favor the isolation of antibodies with specific properties. With classical mAb production, the level of control you have over the process stops as soon as the immunogen is injected into the animal. From there on, you are at the mercy of the mouse’s immune system, which will dictate how the mAb is built, selected, and optimized for binding. This is why mAbs need to be extensively characterized to ensure that you have a mAb against the intended antigen. Moreover, mAb-producing hybridomas need to undergo single-cell cloning, a laborious, time-consuming, often frustrating process.
  11. Mass production of rAbs does not require the use of animals (as is the case with ascites production of mAbs), and thus overcomes ethical concerns over animal distress, discomfort, and pain. In fact, the production of some types of rAbs, from start to finish, completely bypasses the use of animals.

Now comes the fun part. How are rAbs made?

Methods of rAb generation include phage display, bacterial, yeast, ribosome, and mammalian cell display. For the scope of this article, we will focus on Phage Display, the most widely used of those techniques.

What is Phage Display?

This technology, conceived by George P. Smith in the mid-1980s, uses M13 filamentous bacteriophages, viruses that infect, but do not kill E. coli cells. Smith realized that it was possible to insert a gene encoding a foreign protein into the phage coat protein genes. The foreign peptide is then expressed and displayed on the phage, and is accessible for screening. More importantly, a direct physical linkage is created between the displayed protein and its encoding gene2. In 1990, McCafferty et al. showed that this technique can be used to express antibody fragments on the phage surface, and the concept of antibody libraries was born3.

Production of an Antibody Library

First, the genes encoding the antibody heavy and light fragments are amplified from antibody-producing cells, or synthesized in the lab. Heavy and light fragments are then joined together and cloned into special phage vectors. Because heavy and light chains are combined randomly, each phage has the potential to display on its surface a unique antibody with a specific antigen-binding site, resulting in a large repertoire: an antibody library.

Selection of Antibodies

Now that you have a library containing thousands of phages, each carrying a different antibody, how can you fish out the one displaying your desired antibody? This is achieved by panning, a selection process against the antigen of interest. The simplest panning procedure is a variation of common ELISA procedure: The antibody library is incubated with the target immobilized on a solid phase (e.g. an ELISA plate). Washing removes unbound phage, and specifically bound phages are eluted, amplified by infecting E. coli cells, and the process is repeated 3-4 times. By increasing the stringency of the washing and/or lowering the antigen concentration at each panning round, you end up isolating the phages that are displaying antibodies with the highest affinity and stability. Genes for the selected antibodies are sequenced and if necessary, subjected to affinity maturation. The genes for the best antibodies are then transferred into an appropriate expression system for large-scale production.

There you have it, a primer on recombinant antibodies! I hope you’re intrigued to try them!


  1. Köhler G., Milstein C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-97.
  2. Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315-17.
  3. McCafferty, J. et al. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552-54.
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1 Comment

  1. Recombinant Antibodies on December 30, 2015 at 9:45 am

    You are right about all the reasons.

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