New Complement Dependent Cytotoxicity Assay and its application to COVID-19 patient sera harboring anti-lymphocyte antibodies.

Abstract Complement dependent cytotoxicity (CDC) is one of the ways IgM and IgG antibodies can mediate lysis target cells, being antibody (Ab) isotype with highest CDC activity. In a cohort hospitalized COVID-19 patients we identified that 27% carried anti-lymphocyte Ab in their sera. To quantify sera potential lytic activity against healthy donor (HD) PBMC developed flow-based assay an internal negative control. The allows for normalization results therefore inter-experiment data comparison without need using counting beads. short, after CFSE labelling, 2 million are incubated either complete media (CM) or different experimental conditions (Ab, sera, plasma), then they washed thoroughly before adding them to equal numbers unlabeled (internal control). This mixture hot CFSE+ cold CFSE-targets rabbit complement hour, stained flow. A “ratio tube” containing CM left at 4 degrees used calculate initial CFSE+/CFSE− ratio. We percentage based on ratio each tube normalizing it tube. Deploying this assay, found B T cells five two patients’ respectively, out 23 tested. improved shows high reproducibility quick, reliable, easy test multiple conditions. work was supported by Divisions Intramural Research NIAID, NIH