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

机译：了解基于聚集的测定：蛋白质电晕的性质和抗原物质上表位的数量

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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：已经开发了利用金纳米颗粒（NPs）聚集的检测方法的开发，用于检测诊断中的生物分子。这些测定通常基于抗体-抗原相互作用来介导NP的聚集。然而，对于这些测定的执行基础的蛋白质参数还不是很了解。在这项研究中，我们系统地检查了由抗体或抗原形成的NPs上蛋白电晕的性质，以及抗原上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：具有多组氨酸标签的13 kDa重组登革热病毒包膜结构域III蛋白（DIII-His）和19 kDa血管内皮生长因子A（VEGFA），以在NPs周围形成蛋白电晕并研究由NPs诱导的聚集它们的单克隆和多克隆抗体。然后，我们使系统往复运动以与抗体形成蛋白质电晕，并比较了抗原诱导的聚集。我们证明了蛋白质电晕的性质很重要，因为与由抗体形成的电晕相比，由抗原形成的电晕具有较低的检测限，并引起更大程度的NP聚集。此外，抗原上的表位数目很重要，因为靶向抗原上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，靶向抗原上单个表位的单克隆抗体仅诱导抗原-电晕系统聚集。我们的结果表明，对抗体-抗原系统的理解对于建立合理选择蛋白质的指导原则至关重要，该指导原则用于设计基于NP的基于聚集的分析。

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