Fig. 3: Clinical validation of UCAD.
a Schematic illustration of the workflow for clinical validation of UCAD. b, c Kinetic curves for the detection of anti-RBD IgG (b) and IgM (c) in 65 anti-RBD positive sera collected from healthy individuals who received two shots of inactivated COVID-19 vaccine and 77 pre-pandemic sera. d, e Endpoint fluorescence signals at 40 min of three clinical cohorts, including 77 pre-pandemic samples, 65 positive samples, and 55 presumptive negative sera collected during the pandemic. Both the anti-RBD IgG and IgM levels of the positive cohort (n = 65) were significantly higher than those of the pre-pandemic (p = 6.76e−27 for IgG and p = 9.15e−15 for IgM) and in-pandemic negative cohorts (p = 1.44e−20 for IgG and p = 2.59e−18 for IgM). But the pre-pandemic and in-pandemic negative cohorts had no significant difference (p = 0.2595 for IgG and p = 0.0607 for IgM) and were thus combined as one negative group (n = 132) to determine cutoffs for distinguishing positive and negative results in UCAD. Unpaired two-tailed t-tests were used to evaluate statistical differences between cohorts. f ROC curves of the UCAD assay for detecting anti-RBD IgG and IgM in 197 human serum samples (positive: n = 65, negative: n = 132). Optimal cutoff fluorescence values were selected through ROC analysis: 3007 n.u. at 40 min for anti-RBD IgG (specificity = 100%, sensitivity = 100%) and 1973 n.u. at 40 min for anti-RBD IgM (specificity = 87.69%, sensitivity = 100%). g Evaluation of UCAD sensitivity and specificity compared to standard CLIA test using a confusion matrix (n = 132 for negative sera and n = 65 for positive sera). The sensitivity of UCAD was 100% (95% confidence interval: 93.0–100%) and its specificity was 98.5% (95% confidence interval: 94.1–99.7%). Source data are available in the Source Data file. ns: p > 0.05, ****p ≤ 0.0001.
