Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
Anal Chem. 2022 Sep 27;94(38):12990-12999. doi: 10.1021/acs.analchem.2c01325. Epub 2022 Sep 12.
Current design of serological tests utilizes conservative immunoassay approaches and is focused on fast and convenient assay development, throughput, straightforward measurements, and affordability. Limitations of common serological assays include semiquantitative measurements, cross-reactivity, lack of reference standards, and no differentiation between human immunoglobulin subclasses. In this study, we suggested that a combination of immunoaffinity enrichments with targeted proteomics would enable rational design and development of serological assays of infectious diseases, such as COVID-19. Immunoprecipitation-targeted proteomic assays allowed for sensitive and specific measurements of NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum and enabled differential quantification of anti-SARS-CoV-2 antibody isotypes (IgG, IgA, IgM, IgD, and IgE) and individual subclasses (IgG1-4 and IgA1-2) in plasma and saliva. Simultaneous evaluation of the numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic performance. Further validation revealed that anti-RBD IgG1, IgG3, IgM, and IgA1 levels were significantly elevated in convalescent plasma, while IgG2, IgG4, and IgA2 were not informative. Anti-RBD IgG1 levels in convalescent (2138 ng/mL) vs negative (95 ng/mL) plasma revealed 385 ng/mL as a cutoff to detect COVID-19 convalescent plasma. Immunoprecipitation-targeted proteomic assays will facilitate improvement and standardization of the existing serological tests, enable rational design of novel tests, and offer tools for the comprehensive investigation of immunoglobulin subclass cooperation in immune response.
目前的血清学检测设计采用保守的免疫分析方法，侧重于快速、便捷的检测开发、通量、简单的测量和可负担性。常见血清学检测的局限性包括半定量测量、交叉反应、缺乏参考标准以及无法区分人类免疫球蛋白亚类。在这项研究中，我们提出，免疫亲和富集与靶向蛋白质组学相结合，将能够合理设计和开发传染病（如 COVID-19）的血清学检测。免疫沉淀靶向蛋白质组学检测可实现 NCAP_SARS2 蛋白的灵敏和特异性测量，血清中的检测限为 313 pg/mL，并能够对 SARS-CoV-2 抗体的同种型（IgG、IgA、IgM、IgD 和 IgE）和个体亚类（IgG1-4 和 IgA1-2）进行差异定量。同时评估众多抗原-抗体亚类组合揭示了受体结合域（RBD）-IgG1 作为具有最高诊断性能的组合。进一步验证表明，恢复期血浆中 RBD-IgG1、IgG3、IgM 和 IgA1 水平显著升高，而 IgG2、IgG4 和 IgA2 则没有信息。恢复期（2138 ng/mL）与阴性（95 ng/mL）血浆中的抗-RBD IgG1 水平揭示 385 ng/mL 作为检测 COVID-19 恢复期血浆的截止值。免疫沉淀靶向蛋白质组学检测将促进现有血清学检测的改进和标准化，能够合理设计新型检测，并为全面研究免疫球蛋白亚类在免疫反应中的合作提供工具。
