## 3 Results
### Selection of VNAR-Displaying Yeats against Antigens  
After establishing the surface display of a GFP-tagged VNAR fusion protein on yeast, we tested this system for selecting a VNAR from the semi-synthetic library by MACS followed by FACS. Two different antigens were chosen, from a purified protein to a non-purified antigen: the feline neonatal Fc receptor (ffcRn), designated purified protein, and the nucleocapsid protein of the feline infectious peritonitis virus (FIPV) from a commercial vaccine representing the non-purified antigen. FcRn is located on antigen-presenting cells and neutrophils and can bind to immunoglobulin IgG and serum albumin. Its primary function is to reduce the degradation and recycling of the IgG protein, thereby prolonging the half-life of IgG. Thus, FcRn can extend the half-life of biologically, and its antagonist is expected to treat autoimmune diseases [40]. Thus, the isolating of ffcRn-binding VNAR could be helpful to antibody therapy in feline species. \begin{table}
\begin{tabular}{l c} \hline \hline  & **After Biopanning** \\ \hline Obtained clones & \(6.4\times 10^{9}\) \\ \hline Total number of read sequences & 375,629 \\ \hline Number of merged sequences & 249,437 (100\%) \\ \hline CDR3 coding sequences fits in-frame mutations & 118,791 (47.6\%) \\ \hline Unique sequences & 76,780 (30.8\%) \\ \hline Default scaffold (HEL-5A7) & 10,092 (4.0\%) \\ \hline Estimated library size & \(1.97\times 10^{9}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Summary of next-generation sequencing results for semi-synthetic VNAR library. Figure 3: The distribution profile for different lengths of mutated CDR3 sequences before and after rounds of biopanning against the ffcRn or FIPV nucleocapsid protein. The frequencies of unique CDR3 sequences were obtained from the NGS data, then processed and portrayed as the stacked bar chart. To identify frcRn-binding VNAR, the biotinylated feline FcRn (ffcRn) was conjugated with streptavidin-coated magnetic microbeads and then incubated with the yeast library. After magnetic cell sorting, yeast cells were grown, induced, and incubated with the biotinylated frcRn. Furthermore, the desired clones were labeled with APC-conjugated streptavidin and isolated by FACS. Each yeast cell was examined and sorted for its VNAR display level (GFP fluorescence) and antigen binding level (APC fluorescence). Yeast cells scoring above a gating threshold in both fluorescence channels were collected, grown, and induced as the sample for the following selection round. In three subsequent selection rounds, it was found that the frequencies of GFP-positive cells increased between rounds (Table 3), indicating that VNAR-expressing yeasts were specifically enriched. The frequency of GFP\({}^{+}\)APC\({}^{+}\) cells in the control group decreased to about 1/10 after first round of panning (Table 3, from 0.00124 to 0.00012), suggesting that MACS-sorted false-positive clones are mostly excluded after FACS sorting. Moreover, the increased frequency of GFP\({}^{+}\)APC\({}^{+}\) cells in the control group after the second round of panning (Table 3, from 0.00012 to 0.00047) indicated the undesired enrichment of VNARs that bind to the fluorescent-conjugated streptavidin. To evaluate the enrichment of targeted sorting between FACS rounds, the normalization procedure by calculating the ratio of the GFP\({}^{+}\)APC\({}^{+}\) cells in all VNAR-expressing cells was completed before the fold enrichment analysis. Table 3 shows the results of the enrichment fold analysis, which compares the ratio change of the target population before and after biopanning. It shows that the enrichment folds of the first, second, and third round of screening are 3.88, 3.37, and 5.86, respectively. The final round showed a more significant increase, indicating that FACS effectively enriches the frcRn-specific yeast.