A validated clinical Affimer-assay for the quantification of sRAGE in COPD

Scientists from the University of Groningen, Netherlands, led by Professor Rainer Bischoff have published a new study in the Journal of Proteome Research, outlining an Affimer-based LC-MS assay, validated to regulatory guidelines, for the quantification of the clinical biomarker sRAGE.

Soluble Receptor for Advanced Glycation End-products (sRAGE) has arisen as a promising biomarker for chronic obstructive pulmonary disease (COPD) from a number of large-scale clinical studies. This anti-inflammatory receptor acts as a decoy receptor for pro-inflammatory ligands in the lungs, linking low levels of sRAGE to COPD, in particular diseases such as emphysema. Consequently, the quantification of this biomarker could offer valuable clinical information in diagnosis and treatment of COPD. Diabetes mellitus, autoimmune diseases and neurological conditions have also been associated with sRAGE.  A validated and accurate assay for sRAGE quantification is essential to allow further research into the biological function and clinical application of this biomarker.

Following concerns over the accuracy and batch-to-batch variability of the single available validated antibody-based immunoassay for sRAGE, Bischoff’s group investigated the possibility of developing an alternative Affimer-based assay. Affimer reagents are highly specific and sensitive antibody alternatives, where orientation on a solid surface can be controlled for increased capture. Their manufacture is simple and highly reproducible in E.coli, ensuring consistent batch-to-batch performance for immunoassay reagents and a completely animal-free production.

Bischoff and his team developed an Affimer-based liquid chromatography mass spectrometry method for the quantification of sRAGE within the clinically relevant range 0.2-10 ng/mL. A combination of two sRAGE-specific Affimer reagents were adsorbed onto microtiter plates to capture endogenous sRAGE from serum samples. Accurate quantification of sRAGE by LC-MS was shown for a 1/x-weighted linear calibration model for standards across the calibration curve, with both accuracy and precision within the acceptable limits for both EMA and FDA guidelines (± 15%).

Recovery of the Affimer-based enrichment process for sRAGE was high (>90%) and precise (CV<10%) both for duplicate and individual measurements, showing the reliability and sensitivity achievable with Affimer-based assays. The selectivity of the Affimer reagents in this assay was demonstrated by performing both spike recovery and ligand challenge testing using the LC-MS assay for sRAGE. Spike recovery exploited six different sources of serum, a lipemic sample and a haemolytic sample, all of which showed acceptable biases within ± 15%, meeting the EMA and FDA criteria for bioanalytical assays. For ligand challenge assays 0.2 ng/mL sRAGE calibrants were fortified with over 10,000 molar excess of a number of sRAGE ligands (HMGB1 protein, S100A12, SAA1 and CML-BSA), none of which affected the accurate quantification of sRAGE in the Affimer-based assay.

Forty clinical samples were compared across the available antibody-based assay and the developed Affimer-based assay for sRAGE, revealing a good agreement between the two methods (R2=0.88).

This study highlights the robust nature of Affimer reagents, offering high target specificity and selectivity within a variety of matrices, with consistent performance to meet the validation criteria for regulatory approval. The lot-to-lot reproducibility, easy manufacture and rapid isolation of quality binders from the Affimer library means that these binders can overcome some of the major limitations associated with antibodies and as such present a real alternative to traditional antibody reagents.