Fig 2: Detection of N protein in SARS-CoV-2 S, GH, or GR clade virus particles with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Western blot analyses. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Virus titers were measured by plaque assay. (B) Immunoprecipitation analysis. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Fig 3: 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. (C) Dose-dependent spLUC signals for an anti-N-RBD antibody (Sino Biological, Cat#40588-T62–50) in PBST + 8% FBS. (D) Comparison of assay procedure between the ELISA and the spLUC assay. While the ELISA assay takes > 2 hours and involves multiple wash and incubation steps, the spLUC solution-based assay is simply completed in = 30 minutes without the need for wash steps. (E) The S (L15+S25) sensors are able to detect antibodies in 5/5 COVID-19 recovered patients. At all dilutions tested, all 5 patients generated signal above the background signal of two control serum samples collected before the pandemic. (F) The N (LC+SC) sensors are able to detect antibodies in 4/4 COVID-19 recovered patients. At all dilutions of serum tested, all 4 patients generated signal above the background signal of two control serum samples collected before the pandemic. (G) Patient antibodies for SARS-CoV-2 have various epitopes on the S-RBD (red). C004 and C105 have ACE2-competitive epitopes, while 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 10-fold antibody dilutions from 10 nM to 0.001 nM. For (B, C, E, F, H), the data points represent the average of duplicates from two separate experiments. The error bars represent the standard deviation. (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 twelve outpatient sera samples and recombinant C135 IgG protein. Samples were incubated with either no Fab (blue) or C135 Fab (off-white). Patient 72 (serum source of the C135 antibody) had a decrease in signal in the presence of the C135 Fab. In addition to Patient 72, patient 7, 21, 42, 98, 202 also had a decrease in signal. Bars represent the average of two replicates, error bars represent standard deviation.