## 3 Results
### Selection of VNAR-Displaying Yeats against Antigens
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multicolumn{2}{c}{**Sorting Round**} & \multicolumn{3}{c}{**1 st**} & \multicolumn{3}{c}{**2 nd**} \\ \hline  & & **Control** & **Treatment** & **Fold** & **Control** & **Treatment** & **Fold** \\ \hline \multirow{3}{*}{fFcRn} & GFP\({}^{+}\) (\%) & 24.19 & 23.75 & & 34.15 & 31.58 & \\ \cline{2-7}  & GFP\({}^{+}\)APC\({}^{+}\) (normalized) & 0.00124 & 0.00479 & 2.88 & 0.00012 & 0.00040 & 3.37 \\ \hline \multirow{3}{*}{FIPV nucleocapsid} & GFP\({}^{+}\) (\%) & 37.15 & 38.44 & & 39.18 & 40.58 & \\ \cline{2-7}  & GFP\({}^{+}\)APC\({}^{+}\) (normalized) & 0.00242 & 0.00333 & 1.38 & 0.00042 & 0.00086 & 2.07 \\ \hline \multicolumn{2}{c}{**Sorting Round**} & \multicolumn{3}{c}{**3 rd**} & \multicolumn{3}{c}{**4 th**} \\ \hline  & & **Control** & **Treatment** & **Fold** & **Control** & **Treatment** & **Fold** \\ \hline \multirow{3}{*}{ffcRn} & GFP\({}^{+}\) (\%) & 47.71 & 47.12 & & & & \\ \cline{2-7}  & GFP\({}^{+}\)APC\({}^{+}\) (normalized) & 0.00047 & 0.00276 & 5.86 & ND 1 & ND 1 & ND 1 \\ \hline \multirow{3}{*}{FIPV nucleocapsid} & GFP\({}^{+}\) (\%) & 58.25 & 57.34 & & 60.04 & 60.74 & \\ \cline{2-7}  & GFP\({}^{+}\)APC\({}^{+}\) (normalized) & 0.00008 & 0.00052 & 6.44 & 0.00016 & 0.00141 & 8.80 \\ \hline \hline \end{tabular} \({}^{1}\) ND, not done.  
\end{table} 
Table 3: The enrichment fold of antigen-specific FACS sorting. resented by the sequence GWKGMRCKKHMCGGGGHV, RQRESTSCRKRRRCRVIST, and KSRRKLERNRCKTRRPCPIRSSR in the 18-, 20-, or 23-mer CDR3 region, respectively (Figure 5A). Another target for isolating antigen-specific VNAR in this study is the FIPV nucleocapsid. FIPV is the pathogen of feline infectious peritonitis (FIP). After feline coronavirus infects cats, it mutates to FIPV and causes a fatal hyper-inflammatory response [42]. Although there is a commercial live feline infectious peritonitis virus vaccine (FELOCELL(r) FIP) from Zoetis, its preventive effect is controversial [43]. The success of mAb treatment in COVID-19 sheds some light on saving cats from FIP, but only a few studies on feline mAb treatment against viral diseases worked [44; 45]. The nucleocapsid of the FIPV has been developed as a subunit vaccine and showed a positive result in a vaccination/challenge experiment [46]. Therefore, identifying FIPV nucleocapsid-specific binders may be valuable for developing FIPV diagnostic kits and vaccines. To identify FIPV nucleocapsid-binding VNARs, one round of MACS followed by four rounds of FACS were performed essentially as described above. First, the feline coronavirus nucleocapsid mAb conjugated magnetic beads were incubated with the reconstituted FIPV vaccine mixture to capture FIPV nucleocapsid. After the FIPV nucleocapsid was presented by magnetic beads, the VNAR library was added to initiate MACS screening. Bound yeast cells are isolated by magnet-based separation and subjected to four rounds of FACS selection. The MACS-sorted yeasts were grown, induced, and incubated with the reconstituted FIPV vaccine mixture, then labeled with the biotinylated feline coronavirus nucleocapsid mAb. Finally, the bound yeasts were detected by APC-conjugated streptavidin and isolated by FACS. After four selection rounds, the yeast cells with a high display density and high capacity to bind the FIPV nucleocapsid were enriched for the NGS assay. The enrichment fold and amplification factor were calculated as described above. Table 3 shows that the enrichment folds of the first, second, third, and fourth round of screening are 1.38, 2.07, 6.44, and 8.80, respectively. The enrichment fold change between FIPV nucleocapsid sorting rounds reveals a noticeable increasing tendency compared to the result of fFcRn FACS sorting (from 1.38 to 8.80 versus 2.88 to 5.86). The frequency of GFP+APC+ cells in the control group changed from 0.00242 to 0.00042, then 0.00008, and rebound to 0.00016 after the third panning. The NGS data revealed that the number of unique FIPV nucleocapsid-binding VNARs decreased from 2649 to 981 after sorting.