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 E. coli 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 ( R2 = 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.

中文翻译：

---

Affimers作为亲和力LC-MS分析中抗体的替代品，用于定量测定人血清中高级糖基化终产物（sRAGE）的可溶性受体。

抗体是生物医学研究中必不可少的工具，但是它们的大小，复杂性以及有时缺乏可重复性，因此需要开发替代性的粘合剂来克服这些局限性。亲和剂是基于结构坚固的蛋白酶抑制剂支架（即胱抑素A）的新型亲和结合剂，其通过噬菌体展示选择并在快速和简单的大肠杆菌蛋白质表达系统中产生。这些结合剂具有限定的氨基酸序列和限定的结合区，并且用途广泛，从而易于工程化。在这里，我们介绍了一种基于亲和素的液相色谱-质谱（LC-MS）方法，用于定量分析晚期糖化终末产物（sRAGE）的可溶性受体，该药物是慢性阻塞性肺疾病的有前途的生物标志物。该方法已根据欧洲药品管理局和美国食品药品监督管理局的要求进行了验证，并能够在0.2至10 ng / mL之间定量测定血清sRAGE。基于亲和物的方法与先前开发的，经过验证的基于抗体的方法之间的比较显示出良好的相关性（R2 = 0.88），并表明基于亲和物的测定报告的sRAGE水平降低了25％。总之，我们显示了亲和体在定量LC-MS方法中的首次应用，它支持了亲和体作为抗体的强大替代品的潜力。88），并指出基于依恋者的分析报告的sRAGE水平降低了25％。总之，我们显示了亲和体在定量LC-MS方法中的首次应用，它支持了亲和体作为抗体的强大替代品的潜力。88），并指出基于亲和分析的sRAGE水平降低了25％。总之，我们显示了亲和体在定量LC-MS方法中的首次应用，它支持了亲和体作为抗体的强大替代品的潜力。