Single‐reaction multi‐antigen serological test for comprehensive evaluation of SARS‐CoV‐2 patients by flow cytometry

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen‐based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS‐CoV‐2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine‐like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory‐confirmed patients no SARS‐CoV‐2‐specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS‐CoV‐2 virus might provide insight into the wide array of clinical presentations of COVID‐19. Magnetic fluorescent beads coated with different SARS‐CoV‐2 antigens can be mixed to test simultaneously for antibodies present in patient plasma in highly multiplexed flow cytometry‐based assays. This sensitive method allows identification, in a single reaction, of several Ig‐isotypes and specificities, revealing differential responses between COVID‐19 patients.