High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers at early phases of disease development. Proximity extension assays (PEA) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here we explore nanobodies (Nb) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

中文翻译：

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使用位点特异性寡核苷酸缀合的纳米抗体进行灵敏的蛋白质检测

高质量的亲和探针对于灵敏和特异的蛋白质检测至关重要，特别是在疾病发展的早期阶段检测蛋白质生物标志物。邻近延伸分析 (PEA) 已用于高通量多重蛋白质检测，在一或几微升血浆中多达几千种不同的蛋白质。就此类测定的重现性和标准化而言，克隆亲和试剂可以提供优于常用多克隆抗体 (pAb) 的优势。在这里，我们探索纳米抗体 (Nb) 作为 pAb 的替代品，作为 PEA 的亲和试剂。我们描述了一种有效的位点特异性方法，用于通过使用 Sortase A (SrtA) 的酶偶联制备高质量的寡聚 Nb 探针。该程序允许在结合后方便地去除未结合的亲和试剂。纯化的高级 Nb 探针用于 PEA，反应提供了一种从一组亲和试剂中选择最佳结合试剂对的有效方法。我们证明，与使用 pAb 探针相比，用于白细胞介素 6 (IL6) 检测的基于 Nb 的 PEA (nano-PEA) 可以提高检测性能。我们鉴定并验证了能够成对结合而不竞争 PEA 检测 IL6 抗原的 Nb 组合。