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本研究针对病毒多抗原、多抗体亚型检测的需求，基于悬浮芯片技术建立了一种可同时检测多种抗原蛋白多种抗体亚型的多重检测方法，为血清抗体特征的评估提供了一种高通量、快速的技术手段。以新冠病毒为研究模型，选用荧光编码微球作为反应载体，通过化学偶联技术将新冠病毒的核衣壳蛋白（Nucleocapsid, N）、刺突蛋白S2亚基（spike S2, S2）、受体结合域（Receptor binding domain, RBD）以及N端结构域（N-terminal domain, NTD）固定于微球表面。优化血液样本的稀释倍数和与微球的孵育时间后，采用生物素标记的小鼠抗人IgG1、IgG2、IgG3和IgG4亚型抗体作为检测二抗，建立针对各IgG亚型的标准品体系，并根据中位荧光强度（Median FluorescenceIntensity, MFI）对血样中的抗体亚型进行定量分析。所建立的检测体系可同时实现对SARS-CoV-2的N、NTD、RBD及S2四种抗原IgG亚型抗体的检测。其中，N-IgG1、NTD-IgG1、RBD-IgG1和S2-IgG1的检测灵敏度分别为84.38%、93.75%、90.63%和96.88%，特异度分别为100%、94.44%、100%和100%。本研究成功构建了一种基于悬浮芯片的多重检测体系，能够高效检测新冠病毒N、NTD、RBD与S2四种抗原对应的IgG亚型，适用于血样中抗体亚型分布特征的通量分析。
Abstract：To address the need for detecting multiple viral antigens and antibody subtypes simultaneously, thisstudy established a multiplex detection method based on suspension chip technology. This method enables high-throughput and rapid profiling of serum antibody responses against various antigenic proteins and theircorresponding IgG subtypes. Using SARS-CoV-2 as a model, fluorescently encoded microspheres wereemployed as the solid-phase carriers. Four SARS-CoV-2 antigens—including the nucleocapsid(N) protein,spike S2 subunit(S2), receptor binding domain(RBD), and N-terminal domain(NTD)—were covalentlyimmobilized on the surface of the microspheres via chemical coupling. The assay conditions, including plasmadilution and incubation time with microspheres, were optimized. Biotin-labeled mouse anti-human IgG1, IgG2,IgG3, and IgG4 antibodies were used as secondary antibodies for isotype-specific detection. Standard referencecurves were established for each IgG isotype, and quantitative analysis of antibody levels in plasma samples wasperformed based on the median fluorescence intensity(MFI). The developed assay enables simultaneousdetection of four SARS-CoV-2 antigens(N, NTD, RBD, and S2) and their corresponding IgG isotypes. Thedetection sensitivities for N-IgG1, NTD-IgG1, RBD-IgG1, and S2-IgG1 were 84.38%, 93.75%, 90.63%,and 96.88%, respectively, with specificities of 100%, 94.44%, 100%, and 100%. In conclusion, this studysuccessfully developed a suspension chip – based multiplex immunoassay capable of efficiently detecting IgGisotypes against four SARS-CoV-2 antigens, offering a robust platform for high-throughput analysis of antibodyisotype distributions in plasma samples.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250136
中图分类号:R511;R446.6
引用信息:
[1]房美钰,张乔,郭丽等.基于悬浮芯片技术的新冠病毒多抗原多亚型抗体快速检测方法的建立及应用[J].病毒学报,2025,41(03):668-675.DOI:10.13242/j.cnki.bingduxuebao.250136.
基金信息:
北京市自然科学基金（项目号：L222055）,题目：新冠肺炎康复者接种疫苗后的获得性免疫特征研究~~