## 3 Conclusions  
Nbs hold promise across various fields such as basic research, therapeutics, diagnostics, and biotechnology [30; 31; 32]. Several selection technologies that have been used for mAbs or other types of antibody fragments have been applied to identify Nbs specific to a desired target from a library [33]. The aim of this study is to present a combination of methods that can be utilized to alter an existing antigen-specific Nb to bind with the target harboring mutations. In our study, we were able to redirect the binding affinity of the 1.29 Nb toward the RBD of BA.5 variant by employing error-prone PCR and FLI-TRAP techniques. The protein sequence of SARS-CoV-2 spike RBD (amino acids R319-F541 of the S protein) from the Omicron subvariant BA.5 is based on wild-type SARS-CoV-2 with 17 mutations (see Figure S9), the same mutations identified in the RBD domain of the BA.4 and BA.5.2 variants. Unsurprisingly, even the broad-spectrum 1.29 Nb, which was reported to bind some earlier VOCs, such as the Alpha, Gamma, Beta, and Delta variants (containing 1, 3, 3, and 2 mutations in the RBD region, respectively), was unable to bind BA.5 variant. Our methods identified 2 Nbs, C11 and K9, that demonstrated significantly improved binding to the RBD of the Omicron subvariant BA.5 compared to the prototype 1.29 Nb. In the case of the K9 Nb, we reduced the amount of template from 5 ng to 1 ng, hoping to induce a higher mutation rate. Interestingly, FLI-TRAP was able to identify such an Nb with a unique sequence. Instead of adding point mutations to the template, the C11 Nb, the K9 Nb contains very different CDR1 and CRD3 sequences, as well as elongation in the CDR3. We hypothesize that replication slippage occurred during the mutation process, which could have caused insertion or deletion. Replication slippage refers to occasional slips or stutters of DNA polymerase during replication, leading to the insertion or deletion of nucleotides in the newly synthesized DNA strand. Hence, this phenomenon is a natural source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. An in vitro study of this phenomenon was conducted on different thermostable DNA polymerases, such as those from _Thermus aquatics (Taq_ pol), _Thermococcus litoralis_ (Vent(r) pol), and _Pyrococcus furiosus (Pfu_ pol) [17]. The manufacturer's manual for the GeneMorph II Random Mutagenesis Kit indicates that it employs Mutazyme II polymerase, which consists of 2 error-prone DNA polymerases, i.e., Mutazyme I DNA polymerase and an engineered _Taq_ DNA polymerase variant. In fact, the manual also reported 0.7% insertion and 4.8% deletion rates when using Mutazyme II polymerase. When designing mutations or generating diversity in the Nb sequence, especially through techniques like error-prone PCR or site-directed mutagenesis, DNA polymerase may occasionally insert or delete nucleotides, resulting in sequences with altered lengths or structures [34; 35]. CDRs often exhibit higher variability and sometimes possess repetitive motifs or sequences prone to replication slippage [34; 35]. Antigen-binding assays, ELISA and MST, indicated significantly higher affinities of both the C11 and K9 Nbs to the Omicron BA.5 variant compared to the 1.29 Nb. It should be noted that RBD variants used for these assays were commercially available and produced in mammalian cells so they should possess native structure. A previous study demonstrated that RBD produced in _E. coli_ produced significantly lower ELISA signals if the antibody binds near a glycosite [36]. Molecular docking was utilized to predict the binding patterns and residues involved in these interactions and to elucidate the structural modifications induced by mutations on Nbs and their effects on binding affinity, supporting the experimental findings: 1) Nb-antigen binding through FLI-TRAP resulting in cell resistance to carbenicillin as shown by the spot-plate method, 2) in vitro binding activity as shown by ELISA, and 3) determination of Nb's affinity for RBDs using MST.