###### Abstract  
The shark-derived single-domain antibody VNAR (variable domain of new antigen receptor) has many advantageous features that make the VNAR suitable for improving current monoclonal antibody therapy deficiencies or disease diagnosis methods. In order to discover more VNARs, it is necessary to improve the efficiency of the isolation process. This research aims to enhance the VNAR discovery platform by dual displaying the semi-synthetic VNAR library and green fluorescent protein tag on the yeast surface. The GFP tag can be used to determine the degree of VNAR expression. The diversity of the semi-synthetic VNAR library constructed in this study is verified to be 1.97 \(\times\) 10\({}^{9}\) by next-generation sequencing (NGS). We conveniently screened VNARs against the feline neonatal Fc receptor or feline infectious peritonitis virus nucleocapsid protein by sequential MACS and FACS. To find more diverse VNARs, we analyzed the NGS data of VNAR CDR3 genes before and after biopanning. By comparing the frequency change of each sequence, we found that the amplification factor of sequences was increased by biopanning. Four VNAR candidates selected by the high-frequency and high-amplification factor criteria showed an antigen-binding ability. The results demonstrate that biopanning from a yeast surface displaying a semi-synthetic VNAR library followed by the NGS assay can generate antigen binders rapidly without the need for shark rearing and long-term immunization. single-domain antibody; variable domain of new antigen receptor; yeast surface display; green fluorescent protein; semi-synthetic library; next-generation sequencing; neonatal Fc receptor; feline infectious peritonitis virus +
Footnote †: journal: Medical  
are recombinantly produced. However, the affinity of those non-natural and incomplete mAb fragments is lower than the complete antibody. Furthermore, the cost of production is also higher than conventional antibodies because of the unstable molecular structure [5; 6]. In 1993, the [PERSON] team found the heavy-chain-only antibodies (HCAbs) in camelid animals (camelids and alpacas) which lack the light-chain and the heavy-chain constant region 1. The variable domain of the heavy-chain-only antibody (VHH) is recombinantly expressed as the single-domain antibody or Nanobody [7]. VHH is small (15 kDa) and has a long CDR3 that can recognize the structure of the antigen. Therefore, the VHH is often conformation-dependent when isolated from the antibody library [8]. Nevertheless, VHH has other advantages against a conventional antibody, as it is more resistant to proteolytic enzymes, chemical denaturants, extreme pH, and temperature changes due to its natural molecular composition [9]. Many hydrophilic amino acids and intramolecular disulfide bonds in VHH also provide excellent solubility and thermostability [9; 10; 11]. Other HCAbs also found in cartilaginous fishes are called immunoglobulin new antigen receptors (IgNARs) [12]. Although the molecular weight of the variable domain of new antigen receptors (VNAR) is only 12 kDa, its CDR3 is longer than VHH, and the molecule has advantageous features similar to VHH [13; 14; 15; 16]. Various techniques for screening mAbs were also applied to isolate antigen-specific single-domain antibodies. The phage display, ribosome display, and yeast surface display were used to build the VNAR antibody library [17; 18; 19]. Before the screening, constructing an immune antibody library or non-immune antibody libraries, such as a naive antibody library or synthetic antibody library, is necessary. High-affinity VNAR can be extracted from the immune antibody library, which is generated from immunization against the infectious agent as the shark's in vivo immune mechanisms mature the antibody repertoire to yield high-affinity antibodies. But the cost of rearing animals and immunization is far greater than constructing the VHH immune antibody library [20].