Department of Rheumatology, Aarhus University Hospital, Aarhus, Denmark; Department of Biomedicine, Aarhus University, Aarhus, Denmark; Institute of Clinical Medicine, Aarhus University, Aarhus, Denmark.
Department of Biomedicine, Aarhus University, Aarhus, Denmark; Institute of Clinical Medicine, Aarhus University, Aarhus, Denmark.
J Immunol Methods. 2020 Nov;486:112866. doi: 10.1016/j.jim.2020.112866. Epub 2020 Sep 15.
In human autoimmune diseases, low plasma levels of complement factors C3 and C4 are commonly used as a proxy for complement activation. The measurements of C3 and C4 concentrations (the result of synthesis and consumption) however, show low sensitivity in patient follow-up. We find that the estimation of the C3dg fragment released during complement activation is a better parameter for complement activation. Available techniques for measuring the activation fragment C3dg, e.g. immune-electrophoresis or involving PEG-precipitation, are time-consuming and difficult to standardize. Here we examine the specificity and use of an antibody with mono-specificity for a neoepitope at the N-terminus of C3dg, which is only exposed after cleavage of C3. We present a stable, reproducible, and easy-to-use, time-resolved immunoassay with specificity for C3dg that can be used to directly evaluate ongoing complement activation. We demonstrate that the assay can be applied to clinical samples with a high specificity (95%) and a positive likelihood ratio of 10. It can also differentiate the complement related disease Systemic Lupus Erythematosus from controls and other immune-mediatedimmune mediated diseases like Rheumatoid Arthritis (86% specificity) and Spondyloarthritis (91% specificity). Further, we establish how the assay may also be used for experimental research in in vivo mouse models.
在人类自身免疫性疾病中，血浆补体因子 C3 和 C4 水平降低通常被用作补体激活的替代指标。然而，C3 和 C4 浓度的测量（合成和消耗的结果）在患者随访中灵敏度较低。我们发现，估计补体激活过程中释放的 C3dg 片段是更好的补体激活参数。目前用于测量激活片段 C3dg 的技术，例如免疫电泳或涉及 PEG 沉淀，既耗时又难以标准化。在这里，我们研究了一种针对 C3dg N 端新表位的单特异性抗体的特异性和用途，该表位仅在 C3 切割后才暴露。我们提出了一种稳定、可重现且易于使用的时间分辨免疫分析方法，该方法特异性针对 C3dg，可用于直接评估正在进行的补体激活。我们证明该检测方法可应用于临床样本，具有高特异性（95%）和阳性似然比为 10。它还可以区分与补体相关的疾病系统性红斑狼疮与对照以及其他免疫介导的疾病，如类风湿关节炎（86%的特异性）和脊柱关节炎（91%的特异性）。此外，我们还确定了该检测方法如何也可用于体内小鼠模型的实验研究。
