Antibodies are a stalwart tool in biomedical research. Most commonly used are animal-derived polyclonal and monoclonal antibodies (mAbs), and nonanimal-derived recombinant mAbs.
According to Simon Cooper PhD, senior scientist at Abcam, polyclonal antibodies can be produced relatively quickly and inexpensively, are often the first antibodies available commercially against a specific target, and may therefore have the longest publication track record.
Since polyclonal antibodies contain a mix of antibody sequences produced in response to an antigen, they may recognize a number of different parts of the same antigen molecule (epitopes), potentially offering higher sensitivity and overall antibody affinity as well as increasing the signal against targets with low expression levels. These antibodies may also be well-suited for proteins with post-translational modifications or heterogeneity in structure or sequence.1
mAbs are created using hybridoma technology that involves fusing individual B cells that are generated as part of an immune response from an animal host to an antigen, with immortalized cells. “The fusions create hybridomas capable of continuous growth in culture; each clone expresses antibodies with a single sequence specific to a single epitope on the antigen,” says Cooper. This is advantageous compared to polyclonal antibodies that have batch-to-batch variability.
The increased epitope specificity of mAbs also lends them to be used against defined epitopes in immunoassays for diagnostic applications and in therapeutics.
“Recombinant monoclonal antibodies can be generated from sequences identified using platforms such as yeast or phage display technologies,” says Cooper. Huge libraries of structurally and genetically different recombinant candidate antibodies can be generated either by targeted mutation strategies or by creating synthetic diversity similar to, or greater than that of the natural immune system.2
The genes encoding a specific antibody of interest (derived from either a synthetic library or sequenced mAb) are cloned into an expression vector allowing continuous production at scale with even more batch-to-batch consistency than mAbs. Using a defined sequence for the recombinant antibody avoids the possibility of mutations or genetic drift in the antibody sequence, says Cooper, which can lead to changes in specificity and batch variability over time with mAbs.
“Recombinants and mAbs share advantages in applications where antigen specificity is required,” continues Cooper. “However, because the sequence is known, recombinant mAbs are more amenable to broadening of applications, for example, against denatured or discontinuous epitopes or in immunohistochemistry.”
During manufacture, highly-specific antibodies can be generated against a range of difficult targets unavailable in an immunization-based approach, including membrane-proteins, toxins, and even nucleotides. They can also be engineered to bind an epitope of choice with much higher affinity than that obtained in vivo. Because large libraries can be screened in a high-throughput manner, antibodies can be generated 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 embraced for multiple reasons including availability. Yet these nonanimal-derived antibodies are heralding in a new era of reproducibility and merely represent the tip of a vast expanse of additional molecules that can be accessed by advanced recombinant methods.3
How to choose?
Cooper says the choice of antibody often depends upon the requirements of the application: inter-batch consistency in a diagnostic process, epitope specificity to a known target, or identifying the presence of an antigen with unknown epitopes and possibly low-level expression.
However, as more and more recombinant mAbs are generated the chances are good that there will be a highly-specific one available against a particular target, accompanied by application performance data. According to Cooper, Abcam currently offers approximately 30,000 recombinant mAbs.
Furthermore, says Cooper, it is possible to combine defined amounts of several recombinant mAbs—each optimized for a different specific application for the same antigen—to generate an antibody cocktail. These oligoclonal antibodies combine the batch-to-batch consistency of a recombinant mAb with the ability to recognize different epitopes on the same antigen, broadening the applicable range of applications. “An oligoclonal approach can replace traditional polyclonal antibodies while taking advantage of the specificity and reproducibility only available from a recombinant mAb,” says Cooper.
Other factors to consider when choosing antibodies are: comparisons of antibodies from different vendors, availability of technical support, the application, validation data for the application of interest, interference of additives used to extend shelf life, and high-quality publication history.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. F1000Research 2017, 6:851 doi: 10.12688/f1000research.11774.1
- European Commission, Joint Research Centre, Barroso, J., Halder, M., Whelan, M., EURL ECVAM recommendation on non-animal-derived antibodies, Publications Office, 2020
- Gray A, Bradbury ARM, Knappik A, Plückthun A, Borrebaeck CAK, and Dübel S. Animal-free alternatives and the antibody iceberg. Nature Biotechnology | VOL 38 | November 2020 | 1234–1241