Polyclonal, monoclonal or recombinant antibodies

Antibodies are a valuable tool in biomedical research. The most commonly used are polyclonal and monoclonal antibodies of animal origin (mAb) and recombinant mAbs of non-animal origin.

polyclonal antibodies

According to Simon Cooper PhD, senior scientist at Abcam, polyclonal antibodies can be produced relatively quickly and inexpensively, are often the first commercially available antibodies against a specific target, and may therefore have the longest publication record.

Since polyclonal antibodies contain a mix of antibody sequences produced in response to an antigen, they can recognize a number of different parts of the same antigen molecule (epitopes), potentially offering greater sensitivity and overall antibody affinity, as well as increasing the signal against targets with low levels of expression. These antibodies may also be suitable for proteins with post-translational modifications or heterogeneity in structure or sequence.1

monoclonal antibodies

MAbs are created using hybridoma technology which involves the fusion of single B lymphocytes generated as part of an immune response from an animal host to an antigen, with immortalized cells. “The mergers create hybridomas capable of continuous cultural growth; each clone expresses antibodies with a single specific sequence for a single epitope on the antigen, “says Cooper. This is advantageous over polyclonal antibodies that have lot-to-lot variability.

The increased specificity of mAb epitopes also lends them to use against epitopes defined in immunoassays for diagnostic and therapeutic applications.

Recombinant antibodies

“Recombinant monoclonal antibodies can be generated from sequences identified using platforms such as yeast or phage visualization technologies,” says Cooper. Huge libraries of structurally and genetically diverse recombinant candidate antibodies can be generated by targeted mutation strategies or by creating synthetic diversity similar to or greater than that of the natural immune system.2

Genes encoding a specific antibody of interest (derived from a synthetic library or sequenced mAb) are cloned into an expression vector allowing for continuous large-scale production with even more batch consistency than mAbs. Using a defined sequence for the recombinant antibody avoids the possibility of mutations or genetic drift in the antibody sequence, Cooper says, which can lead to changes in batch specificity and variability over time with mAbs.

“Recombinants and mAbs share advantages in applications where antigen specificity is required,” Cooper continues. “However, since the sequence is known, recombinant mAbs are more susceptible to broadening of applications, for example against denatured or discontinuous epitopes or in immunohistochemistry.”

During production, highly specific antibodies can be generated against a range of difficult targets not available in an immunization-based approach, including membrane proteins, toxins and even nucleotides. They can also be designed to bind an epitope of choice with a much higher affinity than that obtained in vivo. Because large libraries can be screened in a high-throughput way, it is possible to generate antibodies that distinguish similar compounds and bind their ligands only under desired conditions, such as a specific pH.1

Recombinant antibodies have yet to be widely adopted for multiple reasons, including availability. Yet these non-animal antibodies are heralding a new era of reproducibility and simply represent the tip of a vast expanse of additional molecules that can be accessed with advanced recombinant methods.3

How to choose?

Cooper states that antibody choice often depends on application requirements: inter-batch consistency in a diagnostic process, epitope specificity with respect to a known target, or identification of the presence of an antigen with unknown epitopes and possibly low-level expression. .

However, as more and more recombinant mAbs are generated, there is likely to be a highly specific one available against a particular target, accompanied by application performance data. According to Cooper, Abcam currently offers around 30,000 recombinant mAbs.

Additionally, Cooper says, it is possible to combine defined amounts of different recombinant mAbs, each optimized for a different specific application for the same antigen, to generate a cocktail of antibodies. These oligoclonal antibodies combine the batch-to-batch consistency of a recombinant mAb with the ability to recognize different epitopes on the same antigen, expanding the range of applicable applications. “An oligoclonal approach can replace traditional polyclonal antibodies by taking advantage of the specificity and reproducibility available only from a recombinant mAb,” says Cooper.

Other factors to consider when choosing antibodies are: comparison of antibodies from different suppliers, availability of technical support, application, validation data for the application of interest, interference of additives used to extend shelf life preservation and chronology of high quality publications.1

References

  1. Acharya P, Quinlan A, and Neumeister V. The ABCs of Finding a Good Antibody: How to Find a Good Antibody, Validate It, and Publish Meaningful Data. F1000 Search 2017, 6: 851 doi: 10.12688 / f1000research.11774.1
  2. European Commission, Joint Research Center, Barroso, J., Halder, M., Whelan, M., EURL ECVAM Recommendation on Non-Animal Antibodies, Publications Office, 2020, https://data.europa.eu / doi / 10.2760 / 091625
  3. Gray A, Bradbury ARM, Knappik A, Plückthun A, Borrebaeck CAK, and Dübel S. Animal-free alternatives and antibody icebergs. Biotechnology of nature | COMPLETE 38 | November 2020 | 1234-1241 | https://doi.org/10.1038/s41587-020-0687-9

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