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Development, Validation, and Utilization of a Luminex-Based SARS-CoV-2 Multiplex Serology Assay
Daisy R Roy1, Troy J Kemp1, Katarzyna Haynesworth1
1Vaccine, Immunity, and Cancer Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
Microbiology Spectrum
|March 17, 2023
Summary
A new Luminex multiplex assay accurately measures SARS-CoV-2 IgG antibodies against various viral variants and other coronaviruses. This validated test is crucial for seroprevalence studies and evaluating vaccine responses.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- The COVID-19 pandemic necessitated robust serological assays for tracking disease spread and vaccine efficacy.
- Existing SARS-CoV-2 antibody tests often focus on single antigens or variants, limiting comprehensive immune response evaluation.
- There is a need for versatile assays capable of detecting antibodies against multiple SARS-CoV-2 variants and related coronaviruses.
Purpose of the Study:
- To develop and validate a Luminex bead-based multiplex serology assay for quantifying SARS-CoV-2 specific IgG antibodies.
- To assess the assay's performance against wild-type and mutant SARS-CoV-2 proteins, as well as other human coronaviruses.
- To establish the assay's sensitivity, specificity, and reproducibility for use in clinical and research settings.
Main Methods:
- Developed a Luminex multiplex assay targeting full-length spike (S), nucleocapsid (N), and receptor-binding domains (RBDs) of SARS-CoV-2 (wild-type and mutants), SARS-CoV-1, MERS-CoV, and common human coronaviruses.
- Validated the assay using 160 negative controls and 60 PCR-confirmed SARS-CoV-2 infected individuals.
- Determined assay performance metrics including sensitivity, specificity, accuracy, precision, and dilutional linearity, using WHO international standards for quantification.
Main Results:
- The assay demonstrated high sensitivity (95-100%) and specificity (98.8-100%) for anti-S, -N, and -RBD IgG antibodies.
- Results are reported in BAU/mL, traceable to the WHO international standard.
- The multiplex assay showed excellent agreement with singleplex assays, with no observed antigen interference and high reproducibility (CV ≤20%).
Conclusions:
- The validated Luminex multiplex serology assay is highly sensitive and specific for detecting SARS-CoV-2 IgG antibodies.
- This assay is suitable for assessing immune responses in infected and vaccinated individuals, including against various SARS-CoV-2 variants.
- The multiplex format allows for simultaneous detection of antibodies against multiple coronaviruses, enhancing understanding of cross-protective immunity.
