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组检测方法。靶细胞分泌蛋白质组蛋白与抗生物素涂层微粒（MPs）竞争以与生物素化抗体（Ab）结合，从而导致功能化MP的聚集减少并形成MP和聚集体的混合物。相比之下，如果没有靶细胞分泌蛋白质组蛋白，则会有更多的微粒被功能化，并且会形成更多的聚集体。因此，功能化的微粒/聚集体的平均体积的减少表明细胞分泌组浓度的增加。通过微型库尔特计数器测量该体积变化，该计数器用于定量估计细胞分泌组浓度。血管内皮生长因子（VEGF），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，以证明其传感原理。通过测试具有各种VEGF浓度的样品可生成标准校准曲线。检测范围为0.01 ng / mL至100.00 ng / mL。我们进一步证明了在不同孵育时间从人间充质干细胞（hMSCs）分泌组收集的外源样品中VEGF浓度的定量。该测定的结果与平行酶联免疫吸附测定（ELISA）试验的结果非常吻合，表明竞争性免疫聚集测定的特异性和可靠性。结构简单，样品制备简单，