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Understanding aggregation-based assays nature of protein corona and number of epitopes on antigen matters

机译：了解基于聚集的蛋白质电晕的分析性质和抗原事项的表位数

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The development of assays that exploit aggregation of gold nanoparticles (NPs) has been widely studied for detection of biomolecules in diagnostics. These assays are often based on antibody–antigen interactions to mediate aggregation of NPs. However, the protein parameters underlying the performance of these assays are not well understood. In this study, we systematically examine how the nature of the protein corona on the NPs, formed from either antibody or antigen, and how the number of binding sites or epitopes on the antigen affect aggregation. We selected two small antigen proteins: 13 kDa recombinant dengue viral envelope domain III protein with a polyhistidine tag (DIII-His), and 19 kDa vascular endothelial growth factor A (VEGFA), to form protein corona around NPs and study the aggregation induced by their monoclonal and polyclonal antibodies. We then reciprocated the systems to form protein corona with the antibodies and compared the aggregation induced by the antigens. We showed that the nature of the protein corona matters, as the corona formed from antigens had lower limits of detection and elicited greater degrees of NP aggregation compared to the corona formed from antibodies. Furthermore, the number of epitopes on the antigen matters, as polyclonal antibodies, which target multiple epitopes on the antigen, were able to induce aggregation for both antigen- and antibody-corona systems. In contrast, monoclonal antibodies that target a single epitope on the antigens induced aggregation for the antigen-corona system only. Our results showed that an understanding of the antibody–antigen system is crucial for establishing guidelines for rational selection of proteins in the design of aggregation-based assays with NPs.
机译：利用金纳米颗粒（NPS）聚集的测定的发展已被广泛研究用于检测诊断中的生物分子。这些测定通常基于抗体 - 抗原相互作用以介导NPS的聚集。然而，这些测定性能下面的蛋白质参数尚不清楚。在这项研究中，我们系统地检查了蛋白质电晕的性质如何在NPS上形成的，由抗体或抗原形成，以及抗原的结合位点或表位的数量如何影响聚集。我们选择了两种小抗原蛋白：13kDa重组登革热病毒包络结构域III蛋白，具有多亚氨酸标签（DIII-HIS）和19kDA血管内皮生长因子A（VEGFA），形成蛋白质电晕，并研究诱导的聚集它们的单克隆和多克隆抗体。然后，我们将系统与抗体形成蛋白质电晕，并比较抗原诱导的聚集。我们表明，与由抗原形成的电晕具有较低的检测限，与由抗体形成的电晕相比，蛋白质电晕物质的性质具有较低的检测限，并且引发了更多程度的NP聚集。此外，抗原物质的表位数作为靶向抗原上的多个表位的多克隆抗体，能够诱导抗原和抗体 - 电晕系统的聚集。相反，靶向抗原诱导抗原 - 电晕系统的单个表位的单克隆抗体。我们的研究结果表明，对抗体 - 抗原系统的理解对于建立理性选择蛋白质的结构指导至关重要，该指导是以NPS为基于聚集的测定。

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