Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus, Denmark.
Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark.
Front Immunol. 2020 May 5;11:774. doi: 10.3389/fimmu.2020.00774. eCollection 2020.
We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) ( = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals ( = 144) ( < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system.
我们旨在建立一种灵敏而稳健的测定方法，用于估计小鼠和人血浆样品中补体成分 C3 水平的系统补体激活。为了以特定且生理相关的方式捕获激活产物 iC3b 和 C3dg，我们利用了一种构建体，该构建体由人补体受体 2（CR2）的 iC3b/C3dg 结合位点与小鼠 IgG 的 Fc 部分连接而成。该构建体可结合来自小鼠和人类的 C3dg 和 iC3b。我们通过蛋白 G 亲和层析从小鼠 B 骨髓瘤细胞系上清液中纯化 CR2-IgG 构建体，即 J558L-CR2-IgG。CR2-IgG 构建体用于在微孔板中捕获 C3 片段，并用抗小鼠或抗人-C3 抗体检测结合的 C3 片段。最初，我们使用纯化的 C3 片段测试了测定方法的特异性。此外，使用基于 CR2 的测定方法，我们在激活的小鼠血清中测量到高达三倍的信号，而在未激活的小鼠血清中则没有信号，而在 C3 敲除小鼠的激活血清中则没有信号。我们测试了从小鼠实验中生成的样本；通过向 C57bl6 小鼠注射眼镜蛇毒因子或热聚集 IgG 来诱导补体激活，然后在不同时间点从 EDTA 血液样本中提取并测量 iC3b/C3dg。我们观察到在预期高和低补体激活的样本之间信号有明显的时间依赖性差异。此外，使用该测定方法测量人 C3 片段，我们发现系统性红斑狼疮（SLE）患者（n=144）的 iC3b/C3dg 水平明显高于健康个体（n=144）（<0.0001）。我们提出了两种功能免疫测定法，能够在小鼠和人类中测量系统水平的 C3 激活产物 iC3b 和 C3dg。据我们所知，这些是首次使用生理相关的捕获构建体（如 CR2）的补体激活测定方法。当研究涉及补体系统的小鼠模型和人类疾病时，这些测定方法将是一个相关的工具。
