A High Through-put Assay for Circulating Antibodies Directed against the S Protein of Severe Acute Respiratory Syndrome Corona virus 2

Svenja Weiss; Jeromine Klingler; Catarina Hioe; Fatima Amanat; Ian Baine; Erna Milunka Kojic; Jonathan Stoever; Sean Liu; Denise Jurczyszak; Maria Bermudez-Gonzalez; Viviana Simon; Florian Krammer; Susan Zolla-Pazner

The beads needed for a single run (2,500 beads/well x number of wells) were pelleted and resuspended in assay buffer (PBS, 0.1% BSA, 0.02% Tween-20) to a volume that delivered 2,500 beads to each well in an aliquot of 50 μL/well. Serum was diluted in assay buffer to twice the desired dilution, added as 50 μL/well to the wells containing the beads and incubated at room temperature for 1 hr on a plate shaker at 600 rpm. After two washes in assay buffer, 100 μL/well of biotinylated-antihuman total Ig (Abcam, catalog #ab97158) at 2 μg/ml was added and incubated for 30 min at room temperature on a plate shaker. After two washes, 100 μL/well of Streptavidin-Phycoerythrin (PE) at 1 μg/ml was added (BioLegend Catalog #405204) followed by a 30 min incubation at room temperature on a plate shaker. After two additional washes, 100 μL of assay buffer/well was added and put on a shaker to resuspend the beads. The plate was read with a Luminex Flexmap 3D instrument. Samples were tested in duplicate and the results were recorded as mean fluorescent intensity (MFI). Serum was used for the experiments herein. In previous work using a similar format, both serum and plasma . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.