Aptamers – the ‘animal free’ alternative to antibodies

Aptasol are seeking partners interested in adopting or helping to develop their aptamers to replace the use of traditional animal-derived antibodies.

Summary | Background | Current state of the art
What could your solution be used for? | Collaboration | 3Rs impact


Summary

Aptamers are an alternative to antibodies and can be deployed in every application in which you might use an antibody. Because no animals are used in aptamer selection, molecules that do not illicit an immune response are also good targets for selection purposes. In addition, aptamers are selected in a process that can be carried out under conditions closely matched to those in which the resultant aptamer is required to work. This ensures that the aptamers are fit for purpose and optimized to work in the downstream assay conditions. This kind of selection is not possible for antibodies, which often require extensive re-optimisation before they can be used - and even then they often do not work under the desired conditions.


Background

Aptamers are synthetic single stranded oligonucleotides (ssDNA or ssRNA) which are selected from expertly designed libraries (of approximately 1015 different oligonucleotide sequences) according to their ability to bind to the target of interest.

Typically, we conjugate the target molecule to a carrier which can be physically manipulated by our robotic platform and which does not need to be injected into an animal to stimulate an immune response. Instead, the target is allowed to form complexes with our aptamer library in vitro. The complexes are separated and we recover aptamers with high-affinity and that are highly specific - these aptamers then form the library for the next round of selection. This process is repeated, generating an enriched library at each stage, until the aptamer library predominantly contains aptamers with the desired properties.

After several rounds of selection we have a pool of mixed population aptamers (MPAs), which is similar in concept to polyclonal antibodies. It is at this point that we screen the pool and identify individual or single population aptamers (SPAs) by next generation sequencing or high throughput screening. This process is similar to the identification of monoclonal antibodies from a polyclonal pool and results in monoclonal aptamers. With aptamers there is no need to create a hybridoma line and the resultant product can be chemically synthesised, avoiding a number of issues associated with traditional hybridoma approaches [1].

Once identified, aptamers can be used in exactly the same way as antibodies and are a viable replacement or adjunct. They can be detected through a variety of approaches, can be created from start to finish in less than 12 weeks using our automated platform, and require the use of no animals at any stage. 


Current state of the art

At Aptamer Solutions, we have cutting edge robotic platforms for the identification and characterisation of aptamers against a wide range of targets, including peptides, proteins, whole cells, membrane fragments, whole viruses, microorganisms and small molecules.

Our systems are designed to develop aptamers with the selection conditions as closely matched to the end application as possible. Due to our automated system a typical aptamer selection process with high levels of quality control can be completed in a matter of weeks. In stark contrast, monoclonal antibodies usually take several months to generate, require more target for immunisation and purification, are optimized for binding in the host animal rather than the required experimental conditions and are more limited in their application.

Peptide aptamers offer an alternative type of aptamer, but there are a number of key points where peptide aptamers do not perform as well as our nucleic acid aptamers. Peptide aptamers are created using several expression constructs (for both target and aptamer library) and therefore rely on the physiology of the expressing microorganism. The peptide aptamer itself is often created as part of a structurally constrained scaffold which also limits the use of peptide aptamers in many applications.

A comparison of aptamers with antibodies is summarised on our website.


What could your Solution be used for?

The solution we provide can be used to identify aptamers against almost any target, under almost any assay condition and for virtually any application. Our aptamers are increasingly being perceived as the reagent of choice for a wide range of applications including:

  • Imaging experiments: fluorescence [2,3], bright field and phase contrast, IHC [4,5], ICC
  • Quantification: ELISA (or ELONA) [6], homogeneous assays etc. [7]
  • Rapid diagnostics: Lateral flow devices [8], biosensors, electrochemical detection, nano-wires [9]
  • Binding studies: Dot blots [10], western blots, label free detection, SPR, Biolayer Interferometry
  • Purification: affinity chromatography [11,12]

Need for collaboration

We are seeking partners in three main areas:

1. Partners who want to try aptamers in traditional assay platforms

We aim to replace antibodies in a wide range of applications and assays where they are ordinarily employed. We are seeking collaborators to demonstrate the use of aptamers in these applications. Our key areas of interest currently lie in the use of aptamers for the detection or purification of proteins and peptides so we are seeking other parties with interests in this area for whom antibodies have been unobtainable or otherwise unsuitable.

2. Partners to help develop novel applications for aptamers

We are also developing new aptamer selection processes to expand our customer offering. A key area of interest here is the development of aptamer reporter molecules for important proteins, significant co-factors as well as metabolites and other small molecules including drugs. We are especially keen to demonstrate their application in cutting edge research such as live-cell imaging as well as point-of-care diagnostics or in field environmental testing.

3. Partners who want to produce relevant proof of concept data

We offer our partners access to a range of proprietary libraries based on both ssDNA and ssRNA. Our aptamers offer improved performance to enhance the resulting assay, purification or other processes they are used in; while our library designs greatly improve the chance of success against virtually any target.


3Rs impact assessment

Aptamers can be used in all applications where traditional animal-derived antibodies have been used.

In 2013 in Great Britain alone, nearly 10,000 procedures were carried out in animals, including rodents, rabbits, ferrets, birds and non-human primates, to create monoclonal and polyclonal antibodies [13]. These ‘procedures’ often require the sacrifice of animals (predominantly mice and rabbits). Whilst some antibody generation projects use a single animal (goats or sheep) most others use at least two. It is not unusual for tens of animals to be sacrificed for each selected antibody; for example, multiple mice are often immunised in order to ensure success. In addition to this, the failure rate of antibody projects for certain targets is high, leading to multiple attempts when antibodies are not found. In many cases, even when antibodies are found, they are not fit for their intended purpose: often because the target cannot be presented in the same form as in the end assay or because the target is not sufficiently immunogenic or stable enough to elicit an immune response [13]. This again can lead to multiple attempts to generate the required antibody.

By replacing antibodies with aptamers we can replace the animal requirement altogether. In addition, the resulting synthetic nucleic acid aptamer will give improved assay consistency, stricter quality control and indefinite supply without ever needing to resort to an animal system.


References

  1. Kohler G, Howe SC and Milstein C  (1975). Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur. J. Immunol. 6: 292-5.
  2. Shigdar S et al. (2011). RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule. Cancer Science, 102: 991–998.
  3. Shigdar S et al. (2013). RNA aptamers targeting cancer stem cell marker CD133. Cancer Letters 330 84–95.
  4. Simmons SC et al. (2012). Development of Novel Single-Stranded Nucleic Acid Aptamers against the Pro-Angiogenic and Metastatic Enzyme Heparanase (HPSE1). PLoS ONE 7(6): e37938.doi:10.1371/journal.pone.0037938.
  5. Shigdar S et al. (2013). The Use of Sensitive Chemical Antibodies for Diagnosis: Detection of Low Levels of Epcam in Breast Cancer. PLoS ONE 8(2): e57613. doi:10.1371/journal.pone.0057613.
  6. Drolet DW, Moon-McDermott L, Romig TS (1996). An enzyme-linked oligonucleotide assay.Nat Biotechnol.14(8):1021-5.
  7. Derbyshire N et al. (2012). Toggled RNA aptamers against aminoglycosides allowing facile detection of antibiotics using gold nanoparticle assays. Anal Chem. 84(15):6595-602.
  8. Mao X et al. (2009). Aptamer-Nanoparticle Strip Biosensor for Rapid and Sensitive Detection of Cancer Cells Anal Chem. 81(24): 10013.
  9. Luo X et al. (2011). Ultrasensitive protein detection using an aptamer-functionalized single polyaniline nanowire. Chem. Commun. 47, 6368–6370.
  10. Bunka DHJ et al. (2007). Production and characterization of RNA aptamers specific for amyloid fibril epitopes. Journal of Biological Chemistry 282, 34500-34509.
  11. Romig TS, Bell C, Drolet DW (1999). Aptamer affinity chromatography: combinatorial chemistry applied to protein purification. J Chromatogr B Biomed Sci Appl. 731(2):275-84.
  12. Kökpinar O et al. (2011). Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads. Biotechnol Bioeng. doi: 10.1002/bit.23191.
  13. Annual Statistics of Scientific Procedures on Living Animals Great Britain 2013. (2014) Home Office HC372 ISBN: 9781474103237.

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