Abstract：
Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.
摘要：
纳米细胞外囊泡（EV）膜上的表面蛋白不仅在细胞间通讯中起着至关重要的作用，但也是EV检测的特异性结合靶标，隔离和跟踪。EV表面的蛋白质生物标志物丰度低，集群的形成和复杂的电动汽车表面网络对电动汽车的研究提出了重大挑战。采用大尺寸的亲和配体，如抗体，在这些囊泡的检测和表征中,通常导致EV表面上的蛋白质的检测灵敏度降低或定量差。由于其体积小、专一性高，Affibody分子在EV研究中作为强大的亲和配体成为其单克隆抗体对应物的潜在替代品。在这项研究中,我们提出了一个关于抗HER2抗体在标记和检测HER2阳性EV方面优于抗HER2抗体的理论框架，其次是证明了HER2Affibody在访问EV表面和通过包括荧光强度在内的多种类型的方法检测EV方面的优势，比色法,和荧光偏振。在访问来自不同人类癌细胞系的HER2阳性EV时，HER2抗体比三个HER2抗体克隆的表现高出10倍。此外，HRP-Affibody分子可以检测来自掺入人血清的癌细胞的EV，其灵敏度比抗体对应物高至少2倍。此外,在荧光偏振测定中，不需要从结合的配体中分离游离，FITC标记的HER2Affibody可以灵敏地检测HER2阳性电动汽车，具有临床相关的检测限，而HER2抗体在相同条件下未能检测到EV.与电动汽车中庞大大小的抗体相比，在接近和检测表面靶标方面具有明显的优势，在电动汽车表面分子结构的精确表征和定量中，Affibody可能成为下一代亲和配体。
