## 4 Discussion  
We developed a rapid and improved platform for screening affinity VNARs by MACS and FACS, followed by next-generation sequencing analysis. By the property of GFP, our system allows us to monitor the VNAR display level on each yeast clone. We validated this platform by selecting affinity VNARs against purified and non-purified proteins. The entire process does not involve animal immunization, which is the crucial step of antibody isolation. Generally, the immunization process for preparing immune antibody libraries takes 2 to 4 months. Milligrams of highly purified antigens are required for immunization [8], and  
Figure 6: Validation of FACS-sorted VNAR-expressing yeast binding to its ligand. VNAR-expressing yeasts were incubated with biotinylated fFcRn, reconstituted FIPV vaccine, or biotinylated lysozyme. The fFcRn-bound yeast was detected by PerCP-conjugated streptavidin, and the FIPV nucleocapsid-bound yeast was detected by biotinylated mouse anti-feline coronavirus nucleocapsid mAb and PerCP-conjugated streptavidin. The fluorescence intensity of GFP and PerCP was measured by fluorescent plate reader. (**A**) The binding of fFcRn by V6 or V5 VNAR-expressing yeasts. (**B**) The binding of FIPV nucleocapsid by V3 or V2 VNAR-expressing yeasts. Data are mean \(\pm\) SE (\(n\) = 3). Means with different letters among groups are significantly different (\(p\) < 0.05) by [PERSON]’s multiple range test. it is necessary to consider whether the antigen is immunogenic or toxic to animals. The in vitro antibody discovery process can strictly control the screening environment, such as screening antibodies under different pH environments [47] or screening for antibodies that can bind to different conformations of antigens without re-immunization. The yeast clones evaluated by NGS analysis can avoid losing potential VNAR candidates. The frequency and amplification factor analysis based on NGS data can also help exclude many non-target clones in the last screening stage to accurately select the functional VNAR sequence. The pLogo analysis can be used to extract consensus antigen-binding CDR3 motifs which may be helpful to design the in vitro affinity maturation experiment. The yeast agglutinin display expression system can be divided into \(\alpha\)-agglutin and a-agglutin display expression systems. The C-terminus of the anchoring protein in an \(\alpha\)-agglutin display expression system is used for connecting to the yeast surface [48]. Therefore, only the N-terminus of the anchoring protein is available for expressing the heterologous protein. In contrast, the anchoring protein Aga2p of the a-agglutin display expression system uses disulfide bonds to connect with the yeast surface, so the N-terminus and C-terminus of Aga2p are unrestricted to express heterologous proteins [49]. Thus, a dual display of proteins on the yeast cell surface was established to quantify binding reactions or enzymatic bioconjugation reactions between both ends of Aga2p [49]. In this study, the GFP protein was expressed at the C-terminus of Aga2p, and the VNAR antibody library was placed at the N-terminus, since the orientation of protein expression in a eukaryotic cell is from the N-terminus to the C-terminus. If the GFP protein is functionally expressed, the VNAR antibody is also well expressed. Moreover, the cell-based ELISA results prove that this study's co-expressed GFP and VNAR are functional. Generally, before the FACS sorting of yeast, it is necessary to fluorescently specify the surface-expressed protein and the antigen bound to it. In this dual display system, an anchored VNAR fusion protein can emit GFP fluorescence spontaneously and then label the bound antigen for screening. After that, the dual fluorescence can be detected by a fluorescent ELISA reader or analyzed and sorted by a flow cytometer. The VNAR antibody library established in this study retains two conserved cysteines and four atypical cysteines in the HEL-5A7 gene scaffold.