Benayas Beatriz, Morales Joaquín, Gori Alessandro, Strada Alessandro, Gagni Paola, Frigerio Roberto, Egea Carolina, Armisén Pilar, Cretich Marina, Yáñez-Mó María
Agarose Bead Technologies (ABT), Torrejon de Ardoz, Spain.
Department Biología Molecular, Universidad Autónoma de Madrid, IUBM, Centro de Biología Molecular Severo Ochoa, IIS-IP, Madrid, Spain.
Front Bioeng Biotechnol. 2023 Aug 11;11:1238898. doi: 10.3389/fbioe.2023.1238898. eCollection 2023.
One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.
使用细胞外囊泡（EVs）进行生物标志物研究的一个主要限制是缺乏一种合适的分离方法，该方法无法在快速且易于标准化的程序中获得高产量和高纯度的样本。在此，我们报告一种基于源自缓激肽的膜传感肽（MSP）的亲和分离方法。我们设计了一种基于携带阳离子螯合物的琼脂糖珠的方案，使其特异性结合带有6His标签的膜传感肽。这种方法具有几个优点：1）携带阳离子的琼脂糖广泛用于His标签蛋白的分离且已标准化；2）亲和方案可在小体积中进行，对临床常规操作可行且易于管理；3）用咪唑或乙二胺四乙酸（EDTA）洗脱可实现温和且容易的回收，而不会损害细胞外囊泡，便于后续的表征和功能分析。优化后的最终程序是，在与浓缩的细胞条件培养基过夜孵育之前，将0.5毫克肽与10微升长臂钴琼脂糖孵育10分钟。通过添加裂解或核酸提取缓冲液，可直接在琼脂糖珠上进行细胞外囊泡的下游分析，或者用咪唑或EDTA轻轻洗脱，得到完全合格的细胞外囊泡制剂。这种新的分离方法基于对一般膜特征的识别，而不依赖于表面标志物。因此，它是无偏倚的，可用于任何物种的细胞外囊泡样本，即使是来自没有合适抗体的动物或植物物种的样本。作为一种亲和方法，样本处理方案非常简单，耗时少，不需要专门设备，并且可以很容易地引入临床自动化常规操作中。与其他市售试剂盒相比，我们证明了该方法具有高纯度和高产量。这种方法还可以扩大或缩小规模，能够分析极少量的样本，并且由于洗脱过程温和，它与任何下游分析都兼容。
