Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.

No abstract

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.

BackgroundThe 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored.Methodology/Principal FindingsTo identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates.Conclusions/SignificanceThe concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.