We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides’ ability to inhibit binding of a biotinylated “knottin”-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca²⁺ and Mg²⁺ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for α_vβ₃ but also detectable for both α_vβ₅ and α₅β₁, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for α_vβ₃. For assay validation, we finally determined IC₅₀ values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (α_vβ₃, α_vβ₅, α₅β₁). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.

中文翻译：

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高亲和力RGD-打结素肽作为新工具未标记的RGD肽的α到粘结强度的快速评价_v β ₃，α _v β ₅，α ₅ β _1个整联蛋白受体

我们描述了一种高度敏感的竞争ELISA，无需使用细胞，ECM蛋白或抗体即可测量高通量的RGD肽的整联蛋白结合。该测定法测量（未标记的）RGD肽抑制生物素化的“结蛋白” -RGD肽与表面固定的整联蛋白结合的能力，因此可以定量高，中和低亲和力RGD的结合强度-黏合剂。我们介绍了生物素化的结蛋白-RGD肽，而不是生物素化的环[RGDfK]（如Piras等人所报道），因为对于所有三种整联蛋白而言，整联蛋白的结合都更强并且可以清楚地检测到。为了最大程度地提高灵敏度和成本效益，我们首先优化了几个参数，例如整联蛋白固定化水平，结蛋白-RGD浓度，缓冲液组成，检测标签的类型（生物素，His或cMyc标签）和间隔物长度。因此，我们确定了两个关键因素，即（i）关键间隔区长度（比Gly长）和（ii）在所有孵育和洗涤缓冲液中均存在Ca ²⁺和Mg ²⁺。打结素-RGD肽的结合最强对于α _v β ₃，但也可检测到两个α _v β ₅和α ₅ β ₁，生物素化的同时，结合环[RGDfK]非常弱并且仅用于检测α _v β ₃。为了进行分析验证，我们最终确定了三种未标记肽的IC ₅₀值，即：（i）线性GRGDS，（ii）环[RGDfK]和（iii）结蛋白-RGD自身与三种不同整合素受体（α _v β ₃，α _v β ₅，α ₅ β ₁）。在新的分析主要好处是（我）整合素的消耗极低（50 ng /肽），（ii）检测不需要抗体/ ECM蛋白或表达整合素的细胞，以及（iii）适用于（RGD）高通量筛选的事实-）肽库。