## 4 Discussion  
The availability of efficient expression systems for the rapid and efficient production of immunogenic proteins is critical in the fight against emerging infectious diseases. Using sensitive and affordable diagnostic kits makes it possible to carry out large-scale seroepidemiological studies in humans and animal species susceptible to emerging infections. Serological tests can be useful to identify highly reactive donors for convalescent plasma therapy or to measure vaccine efficacy. In this study, we report the usefulness of the plant expression system in large-scale and cost-effective production of SARS-CoV-2 nucleoprotein and fusion HEV/RBD protein and their application in serological tests for detecting antibody responses in COVID-19 patients. We describe the development of an indirect in-house ELISA using the fusion HEV/RBD protein as a coating antigen. Further, we report the establishment of a double recognition immunoassay based on two antigens (N and HEV/RBD) of SARS-CoV-2. Finally, we compared the specificity and sensitivity of the iELISA tests with the validated commercial ELISA COVID-19 Si-S2 IgG kit (Dia.Pro D. B., Italy). Nucleoprotein was selected because it is shown to be the most abundantly expressed protein during SARS-CoV-2 infection and highly immunogenic [38; 39]. The antibodies produced against N are unlikely to neutralize SARS-CoV-2 directly [40]. RBD, on the other hand, is the primary target for neutralizing antibodies, and the demonstration of such  
Figure 9: ROC analysis of double recognition immunoassay (HEV/RBD 5 \(\upmu\)g/mL + N 5 \(\upmu\)g/mL). The blue line shows the mean area under the curve (AUC) plot. The area under the curve (AUC) is 0.917.  
antibodies indicates the presence of a protective immune response [41]. Since each of these proteins has advantages and disadvantages as diagnostic antigens, using them all together will most likely yield an overall picture. Additionally, people who receive S-based vaccinations do not have antibodies to N protein [42]. There are many different heterologous expression systems, including _E. coli_, yeast, insect cells, mammalian cells, and plants, for the expression of highly specific detection probes in research and diagnostics, each with specific benefits and drawbacks [43; 44; 45; 46; 47]. Theoretically, plant expression systems have several advantages: plants can be used for the production of large amounts of high-quality recombinant proteins, with similar post-translational modifications (PTMs) as mammalian cells, which can then be utilized for the detection and treatment of several viral diseases [48]. Leafy crops like _Nicotiana benthumiana_ can be cultivated under controlled conditions and grown to agricultural scale, generating significant amounts of biomass. _N. benthamiana_ has been used for transient expression, providing a higher yield of recombinant proteins within a shorter timeline and successfully applied as a rapid response platform addressing emerging viral diseases [49; 50]. However, there are several challenges in producing diagnostic Ag in plants. Various techniques have been used to overcome the glycosylation pattern-induced product heterogeneity and proteolytic degradation of recombinant proteins [51; 52]. PTMs play a significant role in the efficacy of plant-derived diagnostic reagents and affect antigen recognition and sensitivity of diagnostic tests. The SARS-CoV-2 structural proteins are extensively glycosylated [53]. They are primarily complex-type N- and high-mannose-type glycans [29; 54]. Nonetheless, high-mannose glycan' structure is universal throughout eukaryotes, which may lessen the variations between RBD generated by plants and mammals [55]. The use of the baculovirus-insect expression methods can be employed easily for the generation of SARS-CoV-2 viral glycoproteins for research purposes or vaccine development.