一种用于高灵敏度细胞分泌组检测的微流控竞争性免疫聚集试验。

IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Organogenesis Pub Date : 2018-01-01 Epub Date: 2018-06-08 DOI:10.1080/15476278.2018.1461306
Fan Liu, Pawan Kc, Liwei Ni, Ge Zhang, Jiang Zhe
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引用次数: 5

摘要

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组检测方法。靶细胞分泌组蛋白与抗生物素包被的微颗粒(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,没有靶细胞分泌组蛋白,更多的微粒被功能化,形成更多的聚集体。因此,功能化微粒/聚集体平均体积的减少表明细胞分泌组浓度的增加。这种体积变化是通过微库尔特计数器测量的,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(Vascular endothelial growth factor, VEGF)是调节血管生成和血管通透性的关键细胞分泌组蛋白之一,作为靶蛋白来论证其传感原理。通过检测不同VEGF浓度的样品,生成标准校准曲线。检测范围为0.01 ng/mL ~ 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)分泌组中采集的外源性样品中VEGF浓度的定量。实验结果与平行酶联免疫吸附试验(ELISA)的结果一致,表明竞争性免疫聚集试验的特异性和可靠性。该方法结构简单,样品制备方便,不仅能对VEGF进行高灵敏度的检测,而且只要知道特异性的Ab,就可以很容易地扩展到其他类型的细胞分泌组分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

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.

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来源期刊
Organogenesis
Organogenesis BIOCHEMISTRY & MOLECULAR BIOLOGY-DEVELOPMENTAL BIOLOGY
CiteScore
4.10
自引率
4.30%
发文量
6
审稿时长
>12 weeks
期刊介绍: Organogenesis is a peer-reviewed journal, available in print and online, that publishes significant advances on all aspects of organ development. The journal covers organogenesis in all multi-cellular organisms and also includes research into tissue engineering, artificial organs and organ substitutes. The overriding criteria for publication in Organogenesis are originality, scientific merit and general interest. The audience of the journal consists primarily of researchers and advanced students of anatomy, developmental biology and tissue engineering. The emphasis of the journal is on experimental papers (full-length and brief communications), but it will also publish reviews, hypotheses and commentaries. The Editors encourage the submission of addenda, which are essentially auto-commentaries on significant research recently published elsewhere with additional insights, new interpretations or speculations on a relevant topic. If you have interesting data or an original hypothesis about organ development or artificial organs, please send a pre-submission inquiry to the Editor-in-Chief. You will normally receive a reply within days. All manuscripts will be subjected to peer review, and accepted manuscripts will be posted to the electronic site of the journal immediately and will appear in print at the earliest opportunity thereafter.
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