On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination

The Covid-19 pandemic, caused by SARS-CoV-2, resulted in more than 5 million deaths being one of the biggest challenges the world faces today. Here we present optimizations to all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine the specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay in a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals; and 103 individuals without confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals of our cohort before and after receive ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It also represents a valuable tool for elucidating the immunological consequences of new viral strains and for monitoring vaccination coverage and the duration of response to different immunization regimens.