Fig. 1: Engineering luminescent biosensors for rapid and quantitative detection of SARS-CoV-2 antibodies.

a, Schematic of the solution-based serology assay. Patient antibodies are incubated with SARS-CoV-2 S or N proteins fused to LgBiT/SmBiT. For the population of antibodies with one arm bound to the LgBiT sensor and the other arm bound to the SmBiT sensor, the NanoBiT luciferase enzyme is reconstituted and, thus, can produce active luciferase signal. b, Dose-dependent spLUC signals for the recombinant anti-S-RBD antibody C004 in PBST + 10% FBS. Two technical replicates are plotted from n = 1 independent experiment. c, Dose-dependent spLUC signals for an anti-N-RBD antibody (Sino Biological, cat. no. 40588-T62–50) in PBST + 8% FBS. Two technical replicates are plotted from n = 1 independent experiment. d, Comparison of assay procedures between the ELISA and the spLUC assay. While ELISA takes more than 2 h and involves multiple wash and incubation steps, the spLUC solution-based assay is simply completed in 30 min or less without the need for wash steps. e, The S (L15 + S25) sensors are able to detect antibodies in 5/5 recovered patients with COVID-19. At all dilutions tested, all five patients generated signal above the background signal of two control serum samples collected before the pandemic. Each dot represents a technical replicate. n = 2 independent experiments with three replicates each are plotted for all samples, except for those of Patient 1, Patient 7 and Control 2, which have n = 1 independent experiment plotted owing to limited reagents. f, The N (LC + SC) sensors are able to detect antibodies in 4/4 recovered patients with COVID-19. At all dilutions of serum tested, all four patients generated signal above the background signal of two control serum samples collected before the pandemic. Two technical replicates are plotted from n = 1 independent experiment. g, Patient antibodies for SARS-CoV-2 have various epitopes on the S-RBD (red). C004 and C105 have ACE2-competitive epitopes, whereas C135 and CR3022 (blue) have non-ACE2-competitive epitopes. h, S sensors can detect patient antibodies of various epitopes with similar sensitivity. C004, C105, C135 and CR3022 patient antibodies were incubated with the S sensors at ten-fold antibody dilutions from 10 nM to 0.001 nM. The average of three technical replicates from n = 2 independent experiments are plotted. i, Schematic of antibody epitope competition assay with patient serum samples. Direct signal is compared to signal generated in the presence of the pre-incubated 1 µM Fab +1 nM sensor. j, Competition assay performed with C135 Fab on 12 outpatient sera samples and recombinant C135 IgG protein. Samples were incubated with either no Fab (blue) or C135 Fab (off-white). Sera 7, 42 and 98 showed more than 50% decreases in luminescence signal, suggesting the presence of antibodies with the C135 epitope. Each dot represents a technical replicate from n = 1 independent experiment. The center of the bar represents the mean of the measurements. Direct signals (−Fab) were measured in technical duplicates, and competition signals (+C135 Fab) samples were done in technical triplicate. For b, c, e, f and h, the center of the line represents the mean of all measurements. Lines connecting the means of the samples are plotted. RLU, relative luminescence unit; RT, room temperature; NFM, non-fat milk; O/N, overnight.

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