Extended Data Fig. 8: Quality assurance and calibration of assays for neutralizing antibodies to SARS-CoV-2.
From: Accelerated waning of the humoral response to COVID-19 vaccines in obesity
a-b, Linearity of the assay. Neutralization curves (a) and corresponding expected/obtained NT50s (b) against wildtype SARS-CoV-2 for a dilution series of high titre positive control serum. Infection of reporter cells is quantified as % maximum luminescence at 16 h. Mean values ± SEM are shown for experiments performed in duplicate (a). Solid line, line of best fit (dilutions above the limit of quantification). R2, coefficient of determination (dilutions above the limit of quantification). Dotted line, limit of quantification. c, Precision of the assay. NT50s against wildtype SARS-CoV-2 for a medium titre positive control serum sample from 18 independent experiments are summarised as a Tukey boxplot. CV, inter-assay coefficient of variation. Dotted line, limit of quantification. d, External validation of the assay. Comparison of NT50s against both wildtype (blue) and Omicron (BA.1) variant (orange) SARS-CoV-2 from this paper (reporter cells, Cambridge) with results of Focus Reduction Neutralization Tests (FRNTs, Oxford) for a panel of 28 serum samples from convalescent plasma donors. rho, Spearman’s rank correlation coefficient. Dotted line, limit of quantification. e-f, Calibration of the assay. Neutralization curves (e) and corresponding NT50s (f) against wildtype SARS-CoV-2 for WHO International Standard 20/136 (WHO IS 20/136) from 5 independent experiments. Infection of reporter cells is quantified as % maximum luminescence at 16 h. Mean values ± SEM are shown for experiments performed in duplicate (e). NT50s are summarised as a Tukey boxplot (e Minimum: 654, Maximum: 1550, Median: 1006, 25% percentile: 718. 75% percentile: 1157 and f Minimum: 1315, Maximum: 3634, Median: 1851, 25% percentile: 1485, 75% percentile: 2822, Geometric Mean: 1967). GM, geometric mean. Dotted line, limit of quantification.
