During the early stages of disease development, protein biomarkers can leak into the blood creating opportunities for early diagnosis of disease with minimally invasive sampling. These proteins biomarkers however are often masked by the presence of more abundant functional blood proteins, making specific detection a challenge with most current immunoassays. We we report on the development of a magnetic bead based solid-phase PEA (SP-PEA) for sensitive detection of proteins in plasma and serum samples. Antibody functionalized magnetic beads are used to capture the target of interest. Following capture, non-specifically bound proteins are washed off before PEA probes are added for detection of the bound proteins. Compared to hoogenous PEA, SP-PEA admits the use of larger sample volumes to increase available target molecules, higher concentration of detection reagents for more efficient formation of detection complexes and washes for removal of nonspecific background. We compared SP-PEA to solution phase PEA for the detection of cytokines: interlukin-6, interlukin-2, interlukin-4, interlukin-10 and Tumor Necrosis Factor-alpha, and we demonstrated an increased sensitivity by 15 to 60 fold in buffer and chicken serum. We further expanded SP-PEA to detect extracellular vesicles (EVs) through combinations of proteins on the surface of specific EV populations.

中文翻译：

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用于敏感蛋白质和细胞外囊泡检测的基于磁珠的邻近延伸测定

在疾病发展的早期阶段，蛋白质生物标志物可能会泄漏到血液中，从而为通过微创取样进行疾病早期诊断创造了机会。然而，这些蛋白质生物标志物通常被更丰富的功能性血液蛋白质的存在所掩盖，这使得特异性检测成为大多数当前免疫测定的挑战。我们报告了基于磁珠的固相 PEA (SP-PEA) 的开发，用于灵敏检测血浆和血清样品中的蛋白质。抗体功能化磁珠用于捕获感兴趣的目标。捕获后，在添加 PEA 探针以检测结合的蛋白质之前，洗掉非特异性结合的蛋白质。与均质 PEA 相比，SP-PEA 允许使用更大的样品体积来增加可用的目标分子，更高浓度的检测试剂可更有效地形成检测复合物和洗涤以去除非特异性背景。我们比较了 SP-PEA 与溶液相 PEA 检测细胞因子：interlukin-6、interlukin-2、interlukin-4、interlukin-10 和肿瘤坏死因子-α，我们证明在缓冲液中灵敏度提高了 15 到 60 倍和鸡血清。我们进一步扩展了 SP-PEA，通过特定 EV 群体表面的蛋白质组合来检测细胞外囊泡 (EV)。我们证明在缓冲液和鸡血清中灵敏度提高了 15 到 60 倍。我们进一步扩展了 SP-PEA，通过特定 EV 群体表面的蛋白质组合来检测细胞外囊泡 (EV)。我们证明在缓冲液和鸡血清中灵敏度提高了 15 到 60 倍。我们进一步扩展了 SP-PEA，通过特定 EV 群体表面的蛋白质组合来检测细胞外囊泡 (EV)。