Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Plasma using a Coronavirus Antigen Microarray

Rafael R. de Assis; Aarti Jain; Rie Nakajima; Algis Jasinskas; Jiin Felgner; Joshua M. Obiero; Oluwasanmi Adenaiye; Sheldon Tai; Filbert Hong; Philip Norris; Mars Stone; Graham Simmons; Anil Bagri; Martin Schreiber; Andreas Buser; Andreas Holbro; Manuel Battegay; Donald K. Milton; Huw Davies; Laurence M. Corash; Michael P. Busch; Philip L. Felgner; Saahir Khan

The coronavirus antigen microarray used in this investigation includes 67 antigens across subtypes expressed in either baculovirus or HEK-293 cells (Table 1) . These antigens were provided by Sino Inc. (Wayne, PA) as either catalog products or custome synthesis service products. The antigens were printed onto microarrays, probed with human sera, and analyzed as previously described 9, 13, 14 . Briefly, lyophilized antigens were reconstituted to a concentration of 0.1 mg/mL in phosphate-buffered saline (PBS) with 0.001% Tween-20 (T-PBS) and then printed onto nitrocellulose-coated slides from Grace Bio Labs (GBL, Bend, OR) using an OmniGrid 100 microarray printer (GeneMachines). The microarray slides were probed with human sera diluted 1:100 in 1x GVS Fast Blocking Buffer (Fischer Scientific) overnight at 4°C, washed with T-TBS buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 in ddH2O adjusted to pH 7.5 and filtered) 3 times for 5 minutes each, labeled with secondary antibodies to human IgA and IgG conjugated to quantum dot fluorophores for 2 hours at room temperature, and then washed with T-TBS 3 times for 5 minutes each and dried. The slides were imaged using ArrayCam imager (Grace Bio Labs, Bend, OR) to measure background-subtracted median spot fluorescence. Non-specific binding of secondary antibodies was subtracted using saline control. Mean fluorescence of the 4 replicate spots for each antigen was used for analysis.