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Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1/20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto VERO E6/VERO E6 TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using four-parameter logistic regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against WHO anti-SARS-CoV-2 Immunoglobulin Standards. Using WHO Standards with low, medium or high anti-SARS-CoV-2 IgG, both Micro-NT and PRNT achieved comparable NT50 values. Micro-NT was found to be highly reproducible (inter-assay CV of 11.64%). Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s 1/5000. We could also characterise immune-escape VOC observing up to 10-fold reduction in NT50 against SARS-CoV-2 Beta variant. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials. It has higher throughput (96 well format versus 12 well) and reduced infection time (18h vs 48-96h) compared to the gold standard PRNT.

Measurement of quantitative antibody responses are increasingly important in evaluating the immune response to infection and vaccination. In this study we describe the validation of a quantitative, multiplex serologic assay utilising an electrochemiluminescence platform, which measures IgG against the receptor binding domain (RBD), spike S1 and S2 subunits and nucleocapsid antigens of SARS-CoV-2. The assay displayed a sensitivity ranging from 73-91% and specificity from 90 to 96% in detecting previous infection with SARS-CoV-2 depending on antigenic target and time since infection, and this assay highly correlated with commercially available assays. The within-plate coefficient of variation ranged from 3.8-3.9% and the inter-plate coefficient of variation from 11-13% for each antigen.