Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.
The Center for Translational Medicine, The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou, Jiangsu, China.
Front Immunol. 2023 Jan 24;14:1081793. doi: 10.3389/fimmu.2023.1081793. eCollection 2023.
C1s activation is associated with the pathogenesis of various diseases, indicating the potential value of C1s activation detection in clinic. Here we aimed to establish fluorescence resonance energy transfer (FRET)-based immunoassay for the quantitative detection of activated C1s in serum.
FRET-based fluorogenic peptides, sensitive to the enzymatic activity of activated C1s, were prepared and labeled with the fluorophore ortho-aminobenzoic acid (Abz) and quencher 2,4-dinitrophenyl (Dnp), and then were further selected depending on its Kcat/Km value. C1s in the samples was captured and separated using anti-C1s-conjugated magnetic microbeads. Next, enzymatic activity of activated C1s in samples and standards was examined using fluorescent quenched substrate assays. Limit of detection (LOD), accuracy, precision, and specificity of FRET-based immunoassay were also investigated.
This method presented a linear quantification range for the enzymatic activity of activated C1s up to 10 μmol min mL and LOD of 0.096 μmol·min·mL for serum samples. The recovery of the method was in the range of 90% ~ 110%. All CV values of the intra-analysis and inter-analysis of three levels in samples were less than 10%. The cross-reaction rates with C1r enzyme, MASP1, and MASP2 were less than 0.5%. No significant interferences were found with bilirubin (0.2 mg mL), Chyle (2000 FTU), and haemoglobin (5 mg mL), but anticoagulants (EDTA, citrate and heparin) inhibited the enzymatic ability of activated C1s. Thus, this established method can be used for the determination of active C1s in human serum samples in the concentration interval of 0.096-10.000 μmol min mL.
One anti-C1s-based FRET immunoassay for activated C1s detection in serum samples were established, and it will be useful to explore the role of C1s activation in the pathogenesis, diagnosis and treatment in complement-related diseases.
C1s 的激活与各种疾病的发病机制有关，这表明 C1s 激活检测在临床上具有潜在价值。本研究旨在建立基于荧光共振能量转移（FRET）的免疫分析法，用于定量检测血清中活化的 C1s。
制备对活化 C1s 的酶活性敏感的基于 FRET 的荧光肽，并分别用荧光团邻氨基苯甲酸（Abz）和猝灭剂 2,4-二硝基苯（Dnp）标记，然后根据其 kcat/Km 值进一步选择。使用抗-C1s 偶联的磁性微球捕获和分离样品中的 C1s。接下来，使用荧光猝灭底物测定法检查样品和标准品中活化 C1s 的酶活性。还研究了基于 FRET 的免疫分析的检测限（LOD）、准确性、精密度和特异性。
该方法在 0.096-10.000 μmol min mL 的浓度范围内对活化 C1s 的酶活性呈现线性定量范围，血清样品的 LOD 为 0.096 μmol·min·mL。该方法的回收率在 90%-110%范围内。在样品的三个水平的内分析和间分析中，所有 CV 值均小于 10%。与 C1r 酶、MASP1 和 MASP2 的交叉反应率均小于 0.5%。胆红素（0.2 mg mL）、乳糜（2000 FTU）和血红蛋白（5 mg mL）无明显干扰，但抗凝剂（EDTA、柠檬酸盐和肝素）抑制了活化 C1s 的酶活性。因此，该方法可用于测定人血清样品中 0.096-10.000 μmol min mL 浓度范围内的活性 C1s。
建立了一种基于抗-C1s 的 FRET 免疫分析法，用于检测血清样品中活化的 C1s，这将有助于探索 C1s 激活在补体相关疾病的发病机制、诊断和治疗中的作用。
Zotero 插件