Abstract
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. IMPORTANCE: 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.