Vaccine, Immunity, and Cancer Directorate, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.
mSphere. 2024 Nov 21;9(11):e0039324. doi: 10.1128/msphere.00393-24. Epub 2024 Oct 31.
Serology testing is commonly used to evaluate the immunogenicity of COVID-19 vaccines and measure antibodies as a marker of previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, four laboratory-developed serology enzyme-linked immunosorbent assays (SARS-CoV-2 anti-Spike and anti-Nucleocapsid immunoglobin G [IgG] and immunoglobin M [IgM]) calibrated to the WHO International Standard 20/136 were validated via analytical measuring interval (limit of blank [LOB], limit of detection [LOD], and limit of quantification [LOQ]), linearity, and precision according to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP17-A2, EP06 2nd Edition, and EP05-A3. For Spike IgG, LOB was 3.0 binding antibody units per milliliter (BAU/mL), LOD was 4.1 BAU/mL, and LOQ was 27.1 BAU/mL. For Nucleocapsid IgG, LOB was 1.9 BAU/mL, LOD was 3.2 BAU/mL, and LOQ was 24.6 BAU/mL. For Spike IgM, LOB was 57.1 BAU/mL, LOD was 69.0 BAU/mL, and LOQ was 113.5 BAU/mL. For Nucleocapsid IgM, LOD was 242.2 BAU/mL, LOD was 289.9 BAU/mL, and LOQ was 572.4 BAU/mL. Each assay displayed good linearity (max % deviation from linearity (≥LOQ) = 10.7%). The result of within-run repeatability evaluation for medium positive samples was 7.7% for Spike IgG, 4.6% for Nucleocapsid IgG, 7.5% for Spike IgM, and 10.1% for Nucleocapsid IgM. The total precision, including medium positive sample variability across 20 days, three reagent kits, and two operators, was 13.5% for Spike IgG, 14.5% for Nucleocapsid IgG, 17.6% for Spike IgM, and 16.2% for Nucleocapsid IgM. The assays were successfully validated following the applicable CLSI guidelines. All assays met the ±20% deviation from linearity and the ±20% coefficient of variation specification for precision and repeatability.
Reliable and validated serology assays are of increasing importance as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to evolve and cause outbreaks. Validation of serology assays along with calibration to the International and National Standards (such as anti-SARS-CoV-2 Immunoglobulin WHO International Standard 20/136 or Frederick National Laboratory for Cancer Research's National Serology Standard COVID-NS01097) is critical to ensuring that results from clinical studies are reliable and comparable among various assays and laboratories. We describe the design and execution of a comprehensive study that established the analytical measuring intervals, linearity, precision, and repeatability of four in-house developed serology enzyme-linked immunosorbent assays (SARS-CoV-2 anti-Spike immunoglobin G [IgG] and immunoglobin M [IgM] and anti-Nucleocapsid IgG and IgM) following applicable Clinical and Laboratory Standards Institute (CLSI) guidelines. Overall, this study provides practical guidance on experimental design strategies and data analysis techniques, pertaining to the validation of COVID-19 serology assays according to CLSI guidelines, for use in clinical research studies.
本研究中，我们对四种实验室开发的酶联免疫吸附测定法（SARS-CoV-2 抗刺突和抗核衣壳免疫球蛋白 G [IgG]和免疫球蛋白 M [IgM]）进行了验证，这些方法均经过校准，符合世界卫生组织国际标准 20/136，并根据临床和实验室标准化协会（CLSI）指南 EP17-A2、EP06 第二版和 EP05-A3 进行了分析测量区间（空白限 [LOB]、检测限 [LOD] 和定量限 [LOQ]）、线性度和精密度验证。对于 Spike IgG，LOB 为 3.0 个结合抗体单位/毫升（BAU/mL），LOD 为 4.1 BAU/mL，LOQ 为 27.1 BAU/mL。对于 Nucleocapsid IgG，LOB 为 1.9 BAU/mL，LOD 为 3.2 BAU/mL，LOQ 为 24.6 BAU/mL。对于 Spike IgM，LOB 为 57.1 BAU/mL，LOD 为 69.0 BAU/mL，LOQ 为 113.5 BAU/mL。对于 Nucleocapsid IgM，LOD 为 242.2 BAU/mL，LOD 为 289.9 BAU/mL，LOQ 为 572.4 BAU/mL。每个检测都显示出良好的线性度（≥LOQ 时最大 %偏离线性度（≥LOQ）为 10.7%）。在对中阳性样本进行重复性评估时， Spike IgG 的日内重复性为 7.7%，Nucleocapsid IgG 为 4.6%，Spike IgM 为 7.5%，Nucleocapsid IgM 为 10.1%。包括 20 天、三个试剂试剂盒和两个操作人员在内的总精密度，Spike IgG 为 13.5%，Nucleocapsid IgG 为 14.5%，Spike IgM 为 17.6%，Nucleocapsid IgM 为 16.2%。所有检测均按照适用的 CLSI 指南成功验证。所有检测均符合线性度偏差±20%和精密度及重复性变异系数±20%的规格。
随着严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）病毒不断演变并引发疫情，可靠且经过验证的血清学检测变得越来越重要。对血清学检测进行验证，并与国际和国家标准（如抗 SARS-CoV-2 免疫球蛋白世界卫生组织国际标准 20/136 或弗雷德里克国家癌症研究实验室国家血清学标准 COVID-NS01097）进行校准，对于确保临床研究中的结果可靠且在各种检测和实验室之间具有可比性至关重要。我们描述了一项综合研究的设计和实施情况，该研究根据适用的临床和实验室标准化协会（CLSI）指南，确定了四种内部开发的血清学酶联免疫吸附测定法（SARS-CoV-2 抗刺突免疫球蛋白 G [IgG]和免疫球蛋白 M [IgM]以及抗核衣壳 IgG 和 IgM）的分析测量区间、线性度、精密度和重复性。总体而言，本研究为根据 CLSI 指南验证 COVID-19 血清学检测提供了实用的实验设计策略和数据分析技术方面的指导，可用于临床研究。
