Why go recombinant?
Advantages of sequence defined antibodies.
Scientific progress as well as life-saving diagnostic decisions are based on assays built on the fundamental principle of reproducibility of experimental results.
A second essential requirement is utmost capability to discriminate true from false-positive and false-negative results.
Despite all care we built into our sophisticated biological, biochemical or immunological assays, today we still depend on one component which is neither available in unlimited amounts to provide reproducibility, nor is it defined well enough to assure minimal false-positive and false-negative results.
These are animal derived serum products, typically used as first and/or secondary antibodies.
In a PCR-based assay, would you use a primer set without knowing the exact sequence of these primers and without the possibility to sequence the generated PCR amplicon for confirmation?
Nowadays, when using antibodies for detection of a specific protein, we have to do exactly that, since typical diagnostic or research antibodies are not defined by their sequence, often contain many undefined additional components and other IgGs present in the animal which produced them, and vary in their composition over time1.
Most antibodies used for research and diagnostics today are still polyclonal serum products obtained after animal immunization – mainly due to the low prices. On the other hand, this animal origin is responsible for significant batch to batch variations and product discontinuity.
Also, animal derived antisera always contain many other Immunglobulins of unknown specificity, causing an intrinsic risk of exhibiting unwanted reactivities which can never be completely excluded by affinity purification steps2-7.
While also still depending on animal immunization, the hybridoma technology substantially improved this scenario, as it allowed the production of monoclonal antibodies for the first time.
Yet, contrary to widespread belief, these antibodies are not always monospecific. As shown in a multicentric study8 of 185 monoclonal hybridoma sequences, a striking 30 % of these hybridomas expressed additional productive light chains, sometimes even additional heavy chains, resulting in the generation of an oligospecific mixture of antibodies in their supernatant.
So, monoclonals are not as defined as we thought. Furthermore, hybridoma antibodies do not provide the epitope diversity of polyclonals, which can result in lower signals and a more narrow application profile. Finally, storage of hybridoma lines in liquid nitrogen constitutes a quite expensive and not flawless system, that already caused the loss of countless hybridoma clones, making a future reproduction of results with these antibodies impossible.
As a result, the only unequivocally monoclonal antibodies are sequence defined antibodies generated and produced with recombinant technologies, typically antibody phage display. Only these antibodies provide infinite reproducibility, since they can always be reconstituted from their electronically stored or printed amino acid sequence.
The creation of MULTICLONALS9, target specific and multi-epitope binding defined mix of sequence defined recombinant antibodies, allows to combine the benefits of polyclonal antibodies with monoclonals plus while adding the benefits of sequence defined reagents.
Since the DNA encoding Abcalis® antibodies is always available from the start, we can fuse the antigen binding end to many other protein domains. Typically, Fc from other species than human can be used, to combine e.g. three different Abcalis® antibodies in a three colour staining, using three different secondary antibodies. An example of a triple colour immunfluorescence application is given by:
- Moutel et al. (2009). A multi-Fc-species system for recombinant antibody production. BMC Biotechnology 9:14
Versatile recombinant format: “choose your species”! ScFv-Fragments from human libraries can be detected by anti-mouse Fc, anti-rabbit Fc or other secondary antibodies if they have been fused accordingly. While we may not always have every possible combination on stock, we do produce respective versions on request – or even fuse other Fc parts or detection domains you may need for your particular experiment. Just contact us and ask for a quote.
1Bradbury A, Plückthun A. (2015). Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Protein Eng Des Sel. 28(10):303–305.
2Berglund L et al. (2008). A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol Cell Proteomics 7(10):2019-2027.
3Bradbury A, Plückthun A. (2015). Reproducibility: Standardize antibodies used in research. Nature 518(7537):27-29.
4Dove A. (2017). Technology Feature | Agreeable antibodies: Antibody validation challenges and solutions. Science 357(6356):1165-1167.
5Goodman, 2018. The antibody horror show: an introductory guide for the perplexed. New Biotechnol 45:9-13.
6Bradbury A, Plückthun A. (2015). Antibodies: Validate recombinants once. Nature 520(7547):295.
7Taussig MJ et al. (2018). Antibody validation: a view from the mountains. N Biotechnol 45:1-8.
8Bradbury et al. (2018). When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. MAbs 10(4):539-546.