Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.

"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Affimer sandwich probes for stable and robust lateral flow assaying.

Lateral flow assays (LFAs) widely deployed for on-site diagnosis have predominantly utilized antibodies as recognition molecules. Antibodies with limited thermal stability deteriorate the performance of the LFA over time. Herein, we demonstrate a stable and robust LFA by utilizing thermally stable peptide-based 12-14 kDa affimers as recognition molecules, in lieu of conventional protein-based antibodies to analyze complex samples with a significantly improved shelf life at room temperature. The model system studied here is that of interleukin-8 (IL8) biomarker for validating the efficacy of the proposed approach, using a pair of affimer probes that demonstrates dual functionality of capturing and reporting. Affimers immobilized on the test zone of LFA serve as capture probes for IL8-affimer-MB complexes. Whereas affimers conjugated with the MBs that enable extraction of IL8 from the sample matrix serve as reporters for visual detection. The MB complexes captured at the test zone resulted in brownish test bands that enable concentration-dependent detection of IL8. The assay yielded sensitive visual detection of IL8 at ng/mL levels (~ 0.1 ng/mL and 1 ng/mL in buffer and human plasma, respectively), within 20 min, using sample volumes of ~ 100 µL. Importantly, the stability of affimer-incorporated LFA improved significantly in contrast to antibody-incorporated LFA over time, even when stored at 4 °C. Therefore, the proposed affimer-based LFA in conjunction with MBs offer stable and reliable detection of biomarkers at clinically relevant concentration ranges in complicated matrices, even without requiring cold storage, hence, offering a promising avenue for on-site diagnosis in resource-limited settings.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Affimer-Based Europium Chelates Allow Sensitive Optical Biosensing in a Range of Human Disease Biomarkers.

The protein biomarker measurement has been well-established using ELISA (enzyme-linked immunosorbent assay), which offers good sensitivity and specificity, but remains slow and expensive. Certain clinical conditions, where rapid measurement or immediate confirmation of a biomarker is paramount for treatment, necessitate more rapid analysis. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient's bedside. Here, we report a platform for biosensing based on chelated Eu3+ against a range of proteins including biomarkers of cardiac injury (human myoglobin), stroke (glial fibrillary acidic protein (GFAP)), inflammation (C-reactive protein (CRP)) and colorectal cancer (carcinoembryonic antigen (CEA)). The Eu3+ ions are chelated by modified synthetic binding proteins (Affimers), which offer an alternative targeting strategy to existing antibodies. The fluorescence characteristics of the Eu3+ complex with modified Affimers against human myoglobin, GFAP, CRP and CEA were measured in human serum using λex = 395 nm, λem = 590 and 615 nm. The Eu3+-Affimer based complex allowed sensitive detection of human myoglobin, GFAP, CRP and CEA proteins as low as 100 fM in (100-fold) diluted human serum samples. The unique dependence on Eu3+ fluorescence in the visible region (590 and 615 nm) was exploited in this study to allow rapid measurement of the analyte concentration, with measurements in 2 to 3 min. These data demonstrate that the Affimer based Eu3+ complexes can function as nanobiosensors with potential analytical and diagnostic applications.

Affimers as an alternative to antibodies for protein biomarker enrichment.

INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.

Exploiting the Affimer platform against influenza A virus.

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents. IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies.

Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a ß-lactamase - ß-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Quantification of Proteins in Blood by Absorptive Microtiter Plate-Based Affinity Purification Coupled to Liquid Chromatography-Mass Spectrometry.

Liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly used for quantification of proteins in blood. This development is prompted by ongoing improvements in detection sensitivities of LC-MS instruments and corresponding sample preparation workflows. The combination of immunoaffinity enrichment and targeted LC-MS detection is a notable analytical platform in this regard as it allows for the quantification of low abundance proteins in biological matrices like plasma and serum. Here, we describe such hybrid methods which are based on the enrichment of proteins with antibodies or affimers coupled to adsorptive microtiter plates, the proteolytic digestion of enriched proteins to release protein-specific peptides, and the detection of these peptides by microflow LC coupled to selected reaction monitoring MS.

Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer.

Intact Transition Epitope Mapping-One-step Non-covalent force Exploitation (ITEM-ONE) analysis reveals an assembled epitope on the surface of Pertuzumab, which is recognized by the anti-Pertuzumab affimer 00557_709097. It encompasses amino acid residues NSGGSIYNQRFKGR, which are part of CDR2, as well as residues FTLSVDR, which are located on the variable region of Pertuzumab's heavy chain and together form a surface area of 1381.46 Å2. Despite not being part of Pertuzumab's CDR2, the partial sequence FTLSVDR marks a unique proteotypic Pertuzumab peptide. Binding between intact Pertuzumab and the anti-Pertuzumab affimer was further investigated using the Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) approach. Quantitative analysis of the complex dissociation reaction in the gas phase afforded a quasi-equilibrium constant (KD m0g#) of 3.07 × 10-12. The experimentally determined apparent enthalpy (ΔHm0g#) and apparent free energy (ΔGm0g#) of the complex dissociation reaction indicate that the opposite reaction-complex formation-is spontaneous at room temperature. Due to strong binding to Pertuzumab and because of recognizing Pertuzumab's unique partial amino acid sequences, the anti-Pertuzumab affimer 00557_709097 is considered excellently suitable for implementation in Pertuzumab quantitation assays as well as for the accurate therapeutic drug monitoring of Pertuzumab in biological fluids.

Affinity purification of fibrinogen using an Affimer column.

BACKGROUND: Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. METHODS: Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. RESULTS: Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. CONCLUSIONS: The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. GENERAL SIGNIFICANCE: Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.

Protein-conjugated microbubbles for the selective targeting of S. aureus biofilms.

Staphylococcus aureus (S. aureus) is an important human pathogen and a common cause of bloodstream infection. The ability of S. aureus to form biofilms, particularly on medical devices, makes treatment difficult, as does its tendency to spread within the body and cause secondary foci of infection. Prolonged courses of intravenous antimicrobial treatment are usually required for serious S. aureus infections. This work investigates the in vitro attachment of microbubbles to S. aureus biofilms via a novel Affimer protein, AClfA1, which targets the clumping factor A (ClfA) virulence factor - a cell-wall anchored protein associated with surface attachment. Microbubbles (MBs) are micron-sized gas-filled bubbles encapsulated by a lipid, polymer, or protein monolayer or other surfactant-based material. Affimers are small (∼12 kDa) heat-stable binding proteins developed as replacements for antibodies. The binding kinetics of AClfA1 against S. aureus ClfA showed strong binding affinity (KD = 62 ± 3 nM). AClfA1 was then shown to bind S. aureus biofilms under flow conditions both as a free ligand and when bound to microparticles (polymer beads or microbubbles). Microbubbles functionalized with AClfA1 demonstrated an 8-fold increase in binding compared to microbubbles functionalized with an identical Affimer scaffold but lacking the recognition groups. Bound MBs were able to withstand flow rates of 250 µL/min. Finally, ultrasound was applied to burst the biofilm bound MBs to determine whether this would lead to biofilm biomass loss or cell death. Application of a 2.25 MHz ultrasound profile (with a peak negative pressure of 0.8 MPa and consisting of a 22-cycle sine wave, at a pulse repetition rate of 10 kHz) for 2 s to a biofilm decorated with targeted MBs, led to a 25% increase in biomass loss and a concomitant 8% increase in dead cell count. The results of this work show that Affimers can be developed to target S. aureus biofilms and that such Affimers can be attached to contrast agents such as microbubbles or polymer beads and offer potential, with some optimization, for drug-free biofilm treatment.

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry.

There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Reagentless Affimer- and antibody-based impedimetric biosensors for CEA-detection using a novel non-conducting polymer.

Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1-100 fM vs. 1 fM - 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 µl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection.

Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology.

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.

Affimers as anti-idiotypic affinity reagents for pharmacokinetic analysis of biotherapeutics.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.