Affinity reagents are among the most frequently used molecules in biological research. They allow us to examine protein expression, binding and localisation to increase our understanding of the molecular world and develop new medicines. The fields of both life science research and diagnostics have been enabled to a large extent by the use of these reagents. However, as in every other arena, over the past few decades the technology behind affinity reagents has progressed to create molecules that are increasingly fit for purpose and suffer fewer disadvantages. Affinity reagents that offer increased ease of use and confidence in the data obtained are now available to life science researchers.
Antibodies were established as the gold standard affinity reagent, with polyclonal and monoclonal antibodies being used widely in labs across the globe. Providing high affinity binding teamed with a degree of specificity, these molecules were well understood and trusted by researchers. Yet the ever increasing concern within the research community (link is external) regarding the reproducibility of these reagents has highlighted the numerous disadvantages that stem from the use of a hijacked by-product of an animal immune system, which was never intended for use in such applications.
Derived directly from an animal as part of the immune response to infection polyclonal antibodies were superseded by the advent of the hybridoma 40 years ago. The development of the hybridoma cell line allowed for the production of monoclonal antibodies, which were thought to offer users increased specificity and reduce the batch-to-batch variation seen in polyclonals. But these promises have not held true. Many, if not most, antibodies that are now available to the research community show cross-reactivity, and this includes monoclonals. The genetic drift and complications with expression seen in hybridomas mean that batch-to-batch variation has become an issue with antibodies that extends beyond polyclonals. Moreover, problems with the large size of antibody molecules, the high costs and slow production times, the disulphide bridges within their structures that render them unstable within the cell and the difficulties in generating antibodies to cytotoxic targets or targets conserved between species has led to the fall from grace of this once beloved affinity reagent.
First reported 25 years ago, aptamers are short, single strands of DNA or RNA that assume 3-D configurations that facilitate specific binding interactions with other chemical species. Designed from the beginning to act as affinity reagents they offered many improvements over the traditional use of antibodies. They are smaller in size, more easily modified with dyes and labels, have shorter production times of up to 12 weeks and cheaper production costs. As they are produced wholly in vitro they can be theoretically targeted to bind any molecule. Offering high specificity target binding and affinities comparable to that of antibodies, this evolution in affinity reagents seemingly offered a much improved toolkit to biological researchers.
Despite apparently being designed for purpose some of the drawbacks of this technology prevented scientists from adopting aptamers for use universally within the life sciences. The rapid degradation of aptamers (particularly RNA aptamers) by nucleases within biological media and their inherent need for Mg2+ ions to fold mean that they are often incompatible with the experimental environment. Although attempts have been made to adapt the nucleotides in the molecular backbone to increase the chemical diversity of aptamers, the biophysical characteristics of RNA, DNA, 2’F, 2’amine and even 2’-O-Me aptamers is more narrowly confined than that of antibodies which can rely on the full range of twenty naturally occurring amino acids; this limited chemical diversity restricts the range of targets to which aptamers can be developed. Finally, the overall oligonucleotide aptamer structure is negatively charged and very hydrophobic, which makes binding to targets on proteins that are hydrophobic or acidic is particularly challenging.
Affimer technology is the latest technological advancement in affinity reagents. In developing the Affimer we have engineered a new affinity protein combining the best features of the previous affinity reagents whilst excluding previous limitations of such molecules. Affimer technology offers highly selective, high affinity binding to a chosen target. Their protein structure means that they are not limited in the molecules to which they can bind, yet they contain no disulphide bonds or post-translational modifications making them suitable for applications inside the reducing interior of a live cell, whether as inhibitors of protein interactions or as probes for live cell imaging. Affimer proteins are easily modifiable by the addition of specific tags, enzymes and fluorophores, which can expand their capabilities and function in a range of molecular and cellular assays. They are highly stable, offering an increased shelf-life for this affinity reagent whilst also allowing them to function in a range of experimental conditions.