Mendes Maria S M, Rosa Marguerita E, Coutinho João A P, Freire Mara G, E Silva Francisca A
CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro, Portugal.
CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro, Portugal.
Int J Biol Macromol. 2023 Dec 31;253(Pt 8):127540. doi: 10.1016/j.ijbiomac.2023.127540. Epub 2023 Oct 18.
Although pentraxin-3 holds promise as a diagnosis/prognosis biomarker of microbial infections and lung cancer, its analysis in human serum can be constrained by matrix effects caused by high abundance proteins - human serum albumin and immunoglobulin G. Aqueous biphasic systems composed of polymers and citrate buffer are here proposed as a serum pretreatment step to improve the accuracy of pentraxin-3 analysis. Binodal curves were determined to identify the compositions required to form two phases and to correlate the polymers' properties and performance in serum pretreatment and biomarker extraction. Aqueous biphasic systems were evaluated regarding their ability to deplete human serum albumin and immunoglobulin G at the interphase. Polymers of relatively high to intermediate hydrophobicity were unveiled as efficient components to deplete high abundance serum proteins. Considering the possibility to extract pentraxin-3 from human serum into the polymer-rich phase, the system composed of polyethylene glycol with a molecular weight of 1000 g·mol simultaneously achieved >93 % of human serum albumin and immunoglobulin G depletion and complete biomarker extraction. The accuracy of analysis of pretreated human serum by enzyme-linked immunosorbent assays outperformed that of a non-pretreated sample, with a relative error of 0.8 % compared to 14.6 %, contributing to boost pentraxin-3 usefulness as a biomarker.
尽管五聚体-3有望成为微生物感染和肺癌的诊断/预后生物标志物，但其在人血清中的分析可能会受到高丰度蛋白质——人血清白蛋白和免疫球蛋白G所引起的基质效应的限制。本文提出了由聚合物和柠檬酸盐缓冲液组成的双水相系统作为血清预处理步骤，以提高五聚体-3分析的准确性。通过测定双节线曲线来确定形成两相所需的组成，并关联聚合物在血清预处理和生物标志物提取中的性质和性能。对双水相系统在相间耗尽人血清白蛋白和免疫球蛋白G的能力进行了评估。相对较高至中等疏水性的聚合物被发现是耗尽高丰度血清蛋白的有效成分。考虑到从人血清中提取五聚体-3到富含聚合物的相中的可能性，由分子量为1000 g·mol的聚乙二醇组成的系统同时实现了>93%的人血清白蛋白和免疫球蛋白G的耗尽以及生物标志物的完全提取。通过酶联免疫吸附测定法对预处理的人血清进行分析的准确性优于未预处理的样品，相对误差分别为0.8%和14.6%，这有助于提高五聚体-3作为生物标志物的实用性。
