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.

中文翻译：

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Affimers作为蛋白质生物标志物富集抗体的替代品。

介绍

评估蛋白质结合剂的特异性是蛋白质生物标志物测定开发中必不可少的第一步。Affimer是新型的蛋白质结合剂，可以在多种基于蛋白质捕获的分析中取代抗体。选择亲和性化合物是因为它们对靶蛋白具有高度特异性，并且具有优于抗体的优点，例如批次之间的可重复性，并且在广泛的化学条件下均稳定。在这里，我们模拟了针对两种非相关蛋白IL-37b和胰岛素原的亲和素和市售单克隆抗体的典型初始筛选，以评估亲和素作为抗体替代品的潜力。

方法

通过在人血浆中基于磁珠捕获其重组蛋白靶标，研究了抗IL-37b和抗胰岛素原亲和物和抗体的结合特异性。使用SDS-PAGE，考马斯亮蓝染色，蛋白质印迹和基于LC-MS / MS的蛋白质组学分析捕获的蛋白质。

结果

所有亲和分子和抗体都能够在人血浆中结合其靶蛋白。凝胶和LC-MS / MS分析表明，亲和和基于抗体的捕获均导致共同纯化背景蛋白。然而，基于亲和物的捕获显示IL-37b和胰岛素原的相对富集最高。

结论

对于两种测试的蛋白质，与单克隆抗体相比，亲和素在从人血浆中纯化其靶蛋白质时显示出更高的特异性。这些结果表明，亲信分子是用于蛋白质生物标记物测定开发的有希望的抗体替代工具。