Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
J Immunol Methods. 2022 Jun;505:113277. doi: 10.1016/j.jim.2022.113277. Epub 2022 Apr 27.
The mass concentration of specific proteins is often used as a biomarker and play an important part in diagnostics of inflammatory diseases. Monodisperse proteins are robustly measured in immunoassays, but it is considerably more complicated to measure polydisperse oligomeric proteins. The degree of protein oligomerization is critical for functional aspects. For such proteins, information on both the mass concentration as well as the degree of oligomerization is important. Here, a time-resolved immunofluorometric assay (TRIFMA) with sensitivity for protein structure to detect homo-oligomeric and polydisperse proteins is presented. An established TRIFMA for mannan-binding lectin (MBL) was modified by implementing an additional blocking step prior to coating with capture antibodies, leading to a decrease in coating density. Recombinant human MBL was sorted into small, intermediate, and large complexes, using gel permeation chromatography. Small MBL complexes were poorly detectable by TRIFMA with a sparse antibody coating, while larger complexes produced a strong response. From comparison of molecular dimensions, this difference can be related to the size of oligomers. In conclusion, it is possible to design oligomer-size-sensitive immunoassays by regulating the inter-molecular distance of capture antibodies on a scale comparable to the size of the oligomers.
特定蛋白质的质量浓度通常用作生物标志物，并在炎症性疾病的诊断中发挥重要作用。单分散蛋白质在免疫测定中得到了稳健的测量，但测量多分散的寡聚蛋白质要复杂得多。蛋白质寡聚化的程度对于功能方面至关重要。对于这类蛋白质，质量浓度和寡聚化程度的信息都很重要。本文提出了一种具有蛋白质结构敏感性的时间分辨免疫荧光分析（TRIFMA），用于检测同聚寡聚体和多分散的蛋白质。通过在与捕获抗体包被之前实施额外的封闭步骤，对用于甘露聚糖结合凝集素（MBL）的现有 TRIFMA 进行了修改，从而降低了涂层密度。使用凝胶渗透色谱将重组人 MBL 分拣成小、中和大复合物。小 MBL 复合物用稀疏的抗体涂层难以检测到 TRIFMA，而较大的复合物则产生强烈的反应。通过比较分子尺寸，可以将这种差异与寡聚物的大小联系起来。总之，可以通过调节捕获抗体在与寡聚物大小可比的范围内的分子间距离，设计出对寡聚体大小敏感的免疫分析。
