MSD ECL binding assays

Plasma samples from vaccinees and COVID-19 patients were heat-inactivated at 56°C for 30 minutes and tested using multiplexed ECL detection in a 96-well plate format with MSD® V-PLEX® serology panels and instrumentation according to the manufacturer’s instructions. V-PLEX COVID-19 Coronavirus Panel 2 kits were used to detect IgM, IgG, and IgA antibodies to SARS-CoV-2 N, S1 NTD, RBD, and spike antigens and to spike proteins of SARS-CoV-1 and other HCoVs including HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E. V-PLEX SARS-CoV-2 Panel 9 and 11 kits were used to determine IgG antibody concentrations and RBD-ACE2 blocking antibody percentages to different SARS-CoV-2 variant RBDs, with Alpha, Beta, Gamma, Epsilon, Kappa, Eta/Iota, B.1.526.2, P.3 and Wuhan-Hu-1 present in both panel 9 and 11, and with B.1.214.2 in panel 9 and Delta in panel 11. V-PLEX SARS-CoV-2 Panel 20 kits were used to determine IgG antibody concentrations to Alpha, Beta, Gamma, Delta, and Wuhan-Hu-1 SARS-CoV-2 variant spike proteins. Plasma samples were analyzed in duplicate at a 1:5,000 (for IgG binding assays) or a 1:10 (for RBD-ACE2 blocking assays) dilution in MSD diluent. Coronavirus-specific antibodies were detected with anti-human IgM, IgG, or IgA antibodies, or indirectly with human ACE2 protein (for RBD-ACE2 blocking assays) conjugated to SULFO-TAG™ ECL labels and read with a MESO® QuickPlex® SQ 120 instrument. Cutoff values for positive antibody test results for Wuhan-Hu-1 antigens were determined by the manufacturer based on sera from 200 pre-pandemic healthy adults and 214 PCR-confirmed COVID-19 patients. We tested an additional 37 healthy adult pre-pandemic plasma specimens to evaluate the manufacturer’s cutoff values, and to determine cutoffs for positive binding to variant virus antigens, defined as the mean plus three standard deviations of the results from the pre-pandemic specimens. Antibody binding ratios for Wuhan-Hu-1 and viral variant antigens were only calculated for specimens that were above the cutoff values for positive results. Saliva samples were analyzed in duplicate at a 1:5 dilution in MSD diluent 2. Each plate contained duplicates of a 7-point calibration curve with serial dilution of a reference standard, a blank well and three positive control samples. Calibration curves were used to calculate antibody unit concentrations by backfitting ECL signals measured for each sample to the curve.