## 3 Results
### iELISA Optimization Using the Plant-Derived HEV/RBD and N  
The plant-derived recombinant proteins were used to develop an iELISA to test anti-N or anti-RBD antibodies in human sera. The optimal concentration of the recombinant proteins to coat the iELISA plate was 5 \(\mathrm{\SIUnitSymbolMicro}\)g/mL for HEV/RBD protein and 10 \(\mathrm{\SIUnitSymbolMicro}\)g/mL for N protein; serum dilution was 1:40, and II Ab (anti-IgG) was 1:10,000. The optimal conditions were determined based on the optimal relation between the positive and the negative samples (P/N) in the different coating antigen concentrations (Figures S2 and S3, Supplementary File). Figure 5: Determination of mass distribution by MP of N protein expressed and purified from plants. MP displayed as histograms of the masses associated with single-molecule surface adsorption events of the N protein in PBS buffer. Figure 6: Determination of mass distribution by MP of HEV/RBD protein expressed and purified from plants. The sensitivity, specificity, and cut-off value of the iELISA were determined by testing 84 serum samples from hospitalized patients with proven SARS-CoV-2 diagnosis with the fast COVID-19 Ag test and 11 serum samples collected before the COVID-19 pandemic. The assay cut-off value (A492 nm = 0.139) was determined as a 3\(\times\) standard deviation (SD) above the mean optical density of pre-pandemic sera (Figure 7). Furthermore, the HEV/RBD coating Ag was analyzed by serum samples (\(n\) = 6) negative for anti-S IgG and positive for anti-HEV IgG. The results from the iELISA test show that anti-HEV-specific antibodies recognized the HEV/RBD Ag (Figure S4 supplementary). The retained antigenicity of HEV capsid protein fused with RBD can be an obstacle to the correct evaluation of the anti-RBD iELISA specificity. Receiver Operating Characteristic (ROC), Specificity, and Sensitivity of In-House ELISA with Plant-Derived HEV/RBD Protein  
The Receiver Operating Characteristic (ROC) curve was generated using the results obtained by analyzing 36 negative and 48 positive human serum samples (\(n\) = 84), previously tested by a commercial anti-Spike 1(RBD)/2 IgG ELISA Kit (Dia.Pro D. B., Italy). Notably, a comparison between iELISA (HEV/RBD) and a validated, high-sensitivity commercial ELISA kit showed a sensitivity of 89.58% (95% Cl: 77.34-96.53) and a specificity of 94.44% (95% Cl: 81.34-99.32). The assay cut-off value (A492 nm = 0.175) was determined as the optimal value of sensitivity and specificity (Figure 8). The area under the curve (AUC) is 0.943. The raw data are presented in Table S1, Supplementary file. Figure 7: Plant-derived SARS-CoV-2 N and HEV/RBD protein detect IgG in serum samples from COVID-19 hospitalized patients (\(n\) = 84), indicated in blue and green. Results are presented as the optical density value (OD) of analyzed serums. A cut-off for positivity was determined as 3 \(\times\) SD (standard deviation) above the mean optical density of pre-pandemic sera (\(n\) = 11) indicated in orange. The comparison between the two variants of cut-off and a summary of the performance of the HEV/RBD-based iELISA are presented in Table 1.  
The comparison between the anti-Spike 1(RBD)/2 IgG ELISA Kit (Dia.Pro D. B., Italy) commercial kit and the in-house ELISA is presented in Table 2.  
Comparison between the commercial anti-Spike 1(RBD)/2 IgG ELISA Kit (Dia.Pro D. B., Italy) commercial kit and iELISA are characterized by a high agreement of 91.66% and Cohen's k: 0.8316 between the results of the two methods. Of the 84 tested serums, the commercial kit identified 49 positive and 36 negative samples. Of the 48 positive samples,