Affimers as an Alternative to Antibodies in an Affinity LC–MS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum

Antibodies are indispensable tools in biomedical research, but their size, complexity, and sometimes lack of reproducibility created a need for the development of alternative binders to overcome these limitations. Affimers are a novel class of affinity binders based on a structurally robust protease inhibitor scaffold (i.e., Cystatin A), which are selected by phage display and produced in a rapid and simple <i>E. coli</i> protein expression system. These binders have a defined amino acid sequence with defined binding regions and are versatile, thereby allowing for easy engineering. Here we present an affimer-based liquid chromatography–mass spectrometry (LC–MS) method for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker for chronic obstructive pulmonary disease. The method was validated according to European Medicines Agency and U.S. Food and Drug Administration guidelines and enabled quantitation of serum sRAGE between 0.2 and 10 ng/mL. Comparison between the affimer-based method and a previously developed, validated antibody-based method showed good correlation (<i>R</i>² = 0.88) and indicated that 25% lower sRAGE levels are reported by the affimer-based assay. In conclusion, we show the first-time application of affimers in a quantitative LC–MS method, which supports the potential of affimers as robust alternatives to antibodies

Positive correlation between the number of IL-17⁺ cells and neutrophils in the submucosa of bronchial biopsies from atopic (r_s = 0.44; p<0.001) and nonatopic (r_s = 0.45, p = 0.009) asthmatics (A), or from asthmatics who are inhaled corticosteroid (ICS) (r_s = 0.35; p = 0.01) and non-ICS (r_s = 0.48; p<0.0001) users (B).

<p>Positive correlation between the number of IL-17⁺ cells and neutrophils in the submucosa of bronchial biopsies from atopic (r_s = 0.44; p<0.001) and nonatopic (r_s = 0.45, p = 0.009) asthmatics (A), or from asthmatics who are inhaled corticosteroid (ICS) (r_s = 0.35; p = 0.01) and non-ICS (r_s = 0.48; p<0.0001) users (B).</p

IL-17 expression in the submucosa of bronchial biopsies of 4 groups of studied population.

<p>atopic inhaled corticosteroid (ICS) user (frame A), nonatopic ICS user (frame B), atopic non-ICS user (frame C), nonatopic non-ICS user (frame D). Single staining for IL-17 (frame E; blue) and MPO (frame F; red) and double staining for IL-17 and MPO (frame G; purple) in adjacent sections of a nonatopic non-ICS user asthmatic patient. Single staining for IL-17 (frame H; blue) and EPX (frame I; red) and double staining for IL-17 and EPX (frame J; purple) in adjacent sections of an atopic non-ICS user asthmatic patient.</p

Inflammation in atopic and nonatopic asthmatics.

<p>Inflammation in atopic and nonatopic asthmatics.</p

Number of IL-17⁺ cells in submucosa in bronchial biopsies from atopic and nonatopic asthmatics who are inhaled corticosteroid (ICS) users or non-ICS users.

<p>Number of IL-17⁺ cells in submucosa in bronchial biopsies from atopic and nonatopic asthmatics who are inhaled corticosteroid (ICS) users or non-ICS users.</p

Patient characteristics.

<p>Patient characteristics.</p

Negative correlation between the number of IL-17⁺ cells in the submucosa of bronchial biopsies and serum specific IgE (Phadiatop) from asthmatics (rs = -0.37; P<0.001).

<p>Negative correlation between the number of IL-17⁺ cells in the submucosa of bronchial biopsies and serum specific IgE (Phadiatop) from asthmatics (rs = -0.37; P<0.001).</p

Patient characteristics.^*

*<p>Patients were selected from the GLUCOLD cohort <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087443#pone.0087443-Lapperre2" target="_blank">[8]</a>; only persistent smokers and ex-smokers were included. Values before and after treatment are presented of short-term ICS treatment (baseline and after 6 months), and long-term ICS treatment (6 months to 30 months).</p><p>Data are expressed as medians [Interquartile Ranges]; n = number; FEV₁, post, L = FEV₁ after salbutamol expressed in liters; PC₂₀ = provocative concentration of metacholine causing a fall in FEV₁ of >20%;</p>‡<p>Cell counts/10⁻⁷ per m² of subepithelium;</p>§<p>Cell counts×10⁴ per mL.</p

Multiple regression analyses: effects of ICS treatment in smokers and ex-smokers, and the interaction between ICS treatment and smoking (smoking×ICS) on changes in lung function, hyperresponsiveness and inflammatory cells in biopsies and sputum.^*

*<p>Patients were selected from the GLUCOLD cohort <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087443#pone.0087443-Lapperre2" target="_blank">[8]</a>; only persistent smokers and ex-smokers were included. All analyses are adjusted for age and sex. Data are expressed as B, p (regression coefficient, p-value); significant data (p<0.05) are presented in bold; FEV₁, post, L = FEV₁ after salbutamol expressed in liters; PC₂₀ = provocative concentration of metacholine causing a fall in FEV₁ of >20%;</p>‡<p>Cell counts/10⁻⁷ per m² of subepithelium;</p>§<p>Cell counts×10⁴ per mL. The absolute changes in bronchial cell counts and sputum cell counts were normalized by Ln-transformation.</p

Dose-response curves of budesonide in healthy controls and COPD patients GOLD stages I–IV.

<p>Values are expressed as mean ± SEM.</p