Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

Area of Science:

• Immunology
• Virology
• Biotechnology

Background:

• The COVID-19 pandemic necessitates rapid, safe detection of neutralizing antibodies against SARS-CoV-2.
• Existing methods often require live viruses, posing safety and logistical challenges.
• Detecting antibodies targeting the SARS-CoV-2 receptor-binding domain (RBD) is crucial for assessing immunity.

Purpose of the Study:

• To develop and validate a safe, rapid, and easy enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against the SARS-CoV-2 RBD.
• To evaluate the assay's performance using clinical samples and compare it to a gold standard.

Main Methods:

• A competitive ELISA was designed to mimic virus-host interaction using purified SARS-CoV-2 RBD and human ACE2.
• The assay was validated using known concentrations of standards.
• Performance was assessed using 50 serum samples from COVID-19 convalescent individuals and 50 from healthy controls, compared against a commercial total IgG assay.

Main Results:

• The developed ELISA demonstrated a sensitivity of 88% and a specificity of 92%.
• A strong negative correlation (r² = 0.9539) was observed between optical density and nAb concentration.
• The assay provides qualitative and quantitative detection of anti-RBD nAbs.

Conclusions:

• The in-house developed neutralizing ELISA is a high-throughput, reliable tool for detecting anti-RBD nAbs.
• This assay can be implemented in standard laboratories without special biosafety requirements.
• It is suitable for seroprevalence studies, vaccine evaluation, and selecting convalescent plasma donors.