Diluted serum or plasma specimens are added to wells coated with SARS-CoV-2-derived antigens. If IgG antibodies specific for SARS-CoV-2 are present in the specimen, they bind to the SARS-CoV-2 antigens attached to the well, forming stable antigen/antibody complexes. The microplate wells are then washed and an anti-human IgG antibody-horseradish peroxidase (HRP) conjugate is added. If the antigen/antibody complex is present, the HRP conjugate binds to the complex and remains in the well.
After a second wash step, the TMB substrate is added, which reacts with the HRP conjugate to produce a blue colour that is more intense than the colour produced in samples that do not contain any anti-SARS-CoV-2 IgG antibodies.
The stopping solution is added to each well to terminate the reaction between the HRP-conjugate and the TMB substrate. The optical density (OD) in the wells is measured with an absorbance microplate reader at 450 nm.