Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS‐CoV‐2 spike protein

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID‐19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer‐friendly non‐adherent Jurkat T‐cell line that stably expresses the full‐length native spike “S” protein of SARS‐CoV‐2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self‐cleaving sequence, allowing to accurately quantify the presence of anti‐S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double‐staining with the test sera and anti‐EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti‐S antibodies with protective neutralizing activity are long‐lasting and can be detected in sera 8 months after infection. Synopsis This study shows the development of a new method for the classification of human blood donors according to the presence of anti‐Spike (S) antibodies of SARS‐CoV‐2 that bind to the native S protein expressed on the surface of the human Jurkat human T cell line. The flow cytometry method provides quantitative data over a wide range of fluorescence intensities. The co‐expression of the hEGFRt marker allows to set a clear positive/negative threshold according to the slope of fluorescence intensities. The method is more sensitive and reliable than serological methods based on the expression of recombinant proteins. The method correlates better with the presence of protective neutralizing antibodies than ELISA methods. The use of the method allows to conclude that the humoral response anti‐SARS‐CoV‐2 is long‐lasting and that the percentage of the population already immune is higher than suspected. Graphical Abstract This study shows the development of a new method for the classification of human blood donors according to the presence of anti‐Spike (S) antibodies of SARS‐CoV‐2 that bind to the native S protein expressed on the surface of the human Jurkat human T cell line.