a Department of Mechanical Engineering , The University of Akron , Akron , Ohio , United States.
b Department of Biomedical Engineering , The University of Akron , Akron , Ohio , United States.
Organogenesis. 2018;14(2):67-81. doi: 10.1080/15476278.2018.1461306. Epub 2018 Jun 8.
We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.
我们报告了一种使用竞争性免疫聚集和微库尔特计数器的高灵敏度细胞分泌谱检测方法。靶细胞分泌蛋白与抗生物素包被的微球（MPs）竞争与生物素化抗体（Ab）结合，导致功能化 MPs 的聚集减少，形成 MPs 和聚集体的混合物。相比之下，没有靶细胞分泌蛋白时，更多的微球被功能化，更多的聚集体形成。因此，功能化 MPs/聚集体的平均体积减少表明细胞分泌谱浓度增加。这种体积变化由微库尔特计数器测量，用于定量估计细胞分泌谱浓度。血管内皮生长因子（VEGF）是调节血管生成和血管通透性的关键细胞分泌蛋白之一，被用作靶蛋白来证明传感原理。通过测试具有不同 VEGF 浓度的样品生成了标准校准曲线。实现了从 0.01ng/mL 到 100.00ng/mL 的检测范围。我们进一步证明了在不同孵育时间从人间充质干细胞（hMSC）分泌的外源性样本中定量检测 VEGF 浓度的能力。该测定的结果与平行酶联免疫吸附测定（ELISA）测试的结果非常吻合，表明竞争性免疫聚集测定具有特异性和可靠性。该测定具有结构简单、样品制备简单的特点，不仅能够实现 VEGF 的高灵敏度检测，而且只要知道特定的 Ab，就可以很容易地扩展到其他类型的细胞分泌谱分析。
