## 4 Discussion
However, this expression system produces recombinant proteins containing glycoforms different from those produced by mammalian cells, which in effect offers no more benefits than a plant-based expression system [56; 57]. The usual approach for the production of glycosylated viral proteins is their expression in mammalian cells, such as HEK or CHO, but this approach is time-consuming and labor-intensive [58]. Here we used transiently transformed _N. benthamiana_ to produce N protein and HEV/RBD fusion protein to set up the iELISA test for serological analysis of serum samples from COVID-19 patients. The time required for the expression and purification of the N and HEV/RBD diagnostic reagents was estimated to be 5-6 weeks, starting with _N. benthamiana_ seeds germination and plant growth until recombinant protein extraction and purification. Plant leaves infiltrated with pEff-HEV/RBD resulted in the expression of an 81 kDa fusion HEV/RBD protein at up to 100 mg/kg of FW. Agorinfiltration of _N. benthamiana_ with pEAQ-HT-N resulted in the expression of a 49.5 kDa N protein at up to 30 mg/kg of FW. The N protein is expressed in a lower, but still satisfactory, amount. In this study, the optimal conditions for iELISA were set at 5 mg/mL for the HEV/RBD coating antigen and 10 mg/mL for the N coating antigen, with a final volume of 100 \(\upmu\)L (equal to 0.5 \(\upmu\)g/well and 1 \(\upmu\)g/well, respectively). Approximately \(\sim\)50 \(\upmu\)g is needed for coating a whole 96-well plate for HEV/RBD and \(\sim\)100 \(\upmu\)g for N protein. In _E. coli_ expression, it has been reported that 1.5 mg of RBD protein can be produced per liter of culture [59]. Using the same protocol, 1 L of culture would produce enough protein to process 3000 samples, while 1 kg of FW plant leaves could produce enough protein to process 200,000 samples at the HEV/RBD fusion yield described herein. The IMAC purification steps allow for the obtaining of recombinant proteins (N and HEV/RBD) with very high purity (Figures 3a and 4a). The HEV/RBD and N recombinant proteins were proven to be recognized by commercially available anti-RBD mAb and anti-histag mAb (Figures 3a and 4a), respectively. Furthermore, the recombinant proteins were identified by MALDI-TOF and analyzed by mass photometry. As previously shown, we demonstrated that the plant-derived N protein forms tetramers. The tetramers are the results of disulfide cross-links of noncovalent N dimers [37]. Dimerization of SARS-CoV-2 nucleoprotein affects the sensitivity of ELISA based on N protein for serological diagnostics of COVID-19 [60]. Previously, we demonstrated that HEV/RBD can assemble into high-order structures [30]. Here, the mass photometry analysis shows that the HEV/RBD fusion protein is largely monomeric in PBS buffer and does not form virus-like particles (VLPs). We demonstrated that the assembly and stability of HEV capsid VLPs are lower and depend on the purification method, buffer conditions, and the position of the inserted foreign protein [47; 61]. A panel of 84 sera from patients diagnosed with COVID-19 was used to assess the recognition between plant-derived antigens and serum antibodies. The results from HEV/RBD-based iELISA and double recognition ELISA (based on N and HEV/RBD) were compared to those obtained by a commercially available ELISA COVID-19 S1-S2 IgG kit (Dia.Pro D. B., Italy). The agreement between the HEV/RBD-based iELISA and commercial ELISA test was 91.66%, with [PERSON]'s k index 0.8316 [0.710-0.951]. We established a cut-off value of A492 nm = 0.175 for the HEV/RBD iELISA, based on ROC curve analysis (Figure 8). The agreement between the double recognition iELISA and commercial ELISA test was 89.29% with [PERSON]'s k index 0.7850 [0.653-0.917] with cut-off = 0.154. Using our ELISA test, we detected RBD-specific IgG in 51.2% of patients who had tested positive for COVID-19 by rapid antigenic test; 58.3% of patients were positive according to commercial ELISA.