01 July 2016

There is a current crisis in the life sciences. Reproducibility of results is at a reported all time low, with only 18% of data able to be verified by independent groups (Baker, 2015). As a consequence, the National Institute of Health (NIH) has urged the scientific community to discover new ways to increase reproducibility, robust, and transparent research (McNutt, 2014).

Commercially available antibodies are commonly used in the life sciences as a direct agonist / antagonist or to investigate the mechanism of action of a particular treatment. Despite their acceptance within the scientific community, antibodies are reported to be one of the largest sources of variation within experiments. Researchers commonly report differences in binding affinities and specificities between batches, leading to frustration and wasted money. It is estimated that ~50% of the $1.6 billion spent globally on protein-binding reagents is lost on unreliable antibodies (Baker, 2015 and Bradbury and Plückthun, 2015).

Whilst antibodies have many merits, the problem with antibody variability has driven scientists to find alternatives affinity reagents, including aptamers, peptide scaffolds, and recombinant antibodies. With a need for more reliable, specific, and versatile affinity reagents, aptamers offer a significant alternative allowing researchers to collaborate and corroborate, advancing scientific progress.

Causes of antibody variability

Despite the advances in antibody research, the isolation and production of novel antibodies in a reproducible fashion remains a challenge. The reliance on a host animal’s immune system means that many variables can affect the overall antibody population including animal species, conditions, and other external factors. As a result, it has been shown that over 50% exhibit no specific binding or inability to recognise their target, despite being sold to researchers (Berglund et al., 2008). It is a known problem that the same antigen when injected into two different hosts from the same species can produce different antibody titres, possess different specificities, and different affinities. This means that “batch-to-batch” variability is an issue when re-launching a polyclonal antibody production. Batch to batch variability may also variably depend on the quality of the animal host and also to a large extent on the animal housing and treatment conditions which may all prove unfavourable toward the end product.

Once sera have been extracted from the animal, isolation and storage of the antibody can also yield problems with reproducibility. Antibody degradation can result in fragments binding to other unrelated targets and rendering interpretation difficult. The limited stability of human antibodies negatively impacts many processes including expression, purification, and formulation of antibodies for use (Rouet et al. 2014). This is compounded further in the case of monoclonal antibodies, in which further processing increase the likelihood of contamination and the need for freeze/thaw cycles of cell lines.

 The advantages of aptamers for reproducible research

The use of aptamers can help negate some of the caveats associated with antibodies. Aptamer technology possesses several traits that can help improve reproducibility:

  • Aptamer stability

Aptamers prove much more stable at ambient temperatures than antibodies, yielding a much higher shelf life and eliminating the need for a continuous cold chain. Aptamers can withstand repeated round of denaturation without loss of function. They are also more flexible than internally disulphide-linked antibodies and are easily modified during chemical synthesis. Aptamers can therefore fold into a flexible but well defined 3D structure to their target molecules. This reduction in degradation products means that it is less likely to bind to other unrelated targets.

  • Versatility in conjugation chemistry

Antibodies may have several requirements, including use as an inhibitor, western blotting tool and fluorescence tool. Previously, researchers would be limited by the types of conjugation available and would often have to use different clones for different assays, potentially offering confusing data. The ease of conjugation chemistry with aptamers means that one aptamer can be potentially used for all assays, reducing variability and the costs associated of finding and purchasing multiple antibody clones.

  • Chemical manufacturing processes

Moreover, aptamers are chemically synthesised using solid-phase synthesis, resulting in minimal batch-to-batch variation (Kong and Byun, 2013). Like primers, aptamers can be produced with high yield in a consistent fashion and permit the use of synthetic modified nucleotides / inverted caps to further increase stability and reduce degradation by nucleases.

Improved data quality

The properties of aptamers are ideal to aid safety and efficacy testing for regulatory purposes. A study conducted by the US FDA demonstrated the enhanced applicability of aptamers in quality control testing of therapeutic proteins by detecting tiny differences among protein products that were undetected by animal based monoclonal antibodies (Groff et al. 2015). Moreover, the UK’s Department of environment food and rural affairs (DEFRA) carried out a project to develop aptamers for rabies batch potency testing with the ultimate goal of replacing mice used to generate antibodies for the detection of contaminants in vaccine preparations  (Department for Environment Food and Rural Affairs, 2014).

Conclusions

The lack of reproducibility with many commercial antibodies may ultimately have long-lasting consequences. Studies are being funded based on invalid data published in the public domain and years of collective research time are being spent trying to recreate flawed data. The problem with reproducibility has resulted in a growing shift towards other affinity reagents. Aptamers serve to fill the gap in offering a generally higher performing alternative to provide scientific, economic, and time saving advantages.

Aptamer Group

The Aptamer Group takes a high-throughput approach using liquid handling robotics and dedicated researchers to identify aptamers against novel targets. We are committed to finding the perfect aptamers to your target and use a proprietary selection technique to identify high affinity aptamers with specificity in as short as 3 months.

Aptamer Group’s biomarker discovery, diagnostic and therapeutic divisions present a wide application for aptamers as affinity reagents. Through our know-how and key collaborators, we are able to help facilitate the development of aptamers as affinity reagents for many targets of interest in the research and commercial settings with guaranteed consistency in results and reproducibility. For any further information, please contact info@aptamergroup.co.uk

References

Baker, M., 2015. Reproducibility crisis: blame it on the antibodies. Nature 521, 274–276.

Berglund, L., Bjorling, E., Oksvold, P., Fagerberg, L., Asplund, A., Al-Khalili Sziguarto, C., et
al., 2008. A genecentric human protein atlas for expression profiles based on antibodies. Mol. Cell. Proteomics 7, 2019–2027.

Bunka, D.H.J., Platonova, O., Stockley, P.G., 2010. Development of aptamer therapeutics.
Curr. Opin. Pharmacol. 10, 557–562.

Bradbury, A., Plückthun, A., 2015. Reproducibility: standardize antibodies used in research. Nature 518, 27–29.

Department for Environment Food and Rural Affairs. Development of aptamer-based
technology to detect residues of veterinary drugs, and constituents of vaccines —
VM02162. http://randd.defra.gov.uk/Default.aspx?Menu=Menu&Module=
More&Location=None&Completed=2&ProjectID=16304.

Groff, K.; Brown, J.; Clippinger, A.J. Modern affinity reagents: Recombinant antibodies and aptamers. Biotechnology Advances 2015, 33, 1787. http://dx.doi.org/10.1016/j.biotechadv.2015.10.004

JAYASENA, S. D., (1999), Aptamers: An emerging class of molecules that rival antibodies in diagnostics: Clinical Chemistry, v. 45,p. 1628-1650.

Kong, H. Y., & Byun, J. (2013). Nucleic Acid Aptamers: New Methods for Selection, Stabilization, and Application in Biomedical Science. Biomolecules & Therapeutics, 21(6), 423–434. http://doi.org/10.4062/biomolther.2013.085

McNutt, M., 2014. Journals unite for reproducibility. Science 346, 679

Shin, S., Kim, I.H., Kang, W., Yang, J.K., Hah, S.S., 2010. An alternative to Western blot analysis using RNA aptamer-functionalized quantum dots. Bioorg. Med. Chem. Lett. 20,
3322–3325.

Toh, S.Y., Citartan, M., Gopinath, S.C.B., Tang, T.-H., 2015. Aptamers as a replacement for
antibodies in enzyme-linked immunosorbent assay. Biosens. Bioelectron. 64C,
392–403.