Applying the E. coli's twin-arginine translocation pathway to isolation of biomarker-specific nanobodies from a synthetic camelized human nanobody library

Monoclonal antibodies (mAbs) have been used extensively both for treatment and diagnostics. Phage display has been successfully used for isolation of many mAbs currently sold in the market. However, the main drawback is that it could result in a large number of false positives. In this study, we explored the feasibility of combination of two powerful antibody isolation techniques, phage display and Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP), to identify nanobodies (Nbs) that are specific to HBsAg, an antigen commonly used for hepatitis B infection diagnostics. A synthetic camelized human nanobody library was subjected to 2 rounds of biopanning against HBsAg adr subtype, commonly found in southeast Asia. As expected, sequencing analysis of all 12 randomly selected clones from biopanning showed truncated Nbs, representing false positive. Full-length Nb genes were amplified from the phage eluted during the 2nd round of biopanning was subcloned into FLI-TRAP system for isolation. For evaluation, 16 clones were also randomly picked and submitted for sequencing analysis. Interestingly, 15 out of 16 clones had the same sequence and were full-length Nb, so C1 was used to represent these clones. C10, however, was truncated at framework 3. ELISA result of crude extract showed that C1 showed binding activity approximate to 4.5 fold higher than reference Nb and approximate to 1.46 fold lower than commercial purified monoclonal antibodies while its WB result showed that C1 had a higher protein yield than the reference Nb. C10 did not show ELISA signal nor was detected in WB, thus truncation was confirmed since the detection was performed using anti-FLAG antibody specific to FLAG epitope tag fused to the C-terminus of Nb. Nonetheless, our study demonstrated the feasibility to use FLI-TRAP after initial phage display screening to easily identify full-length Nbs. This combined platform would be powerful tool for easy isolation of Nb against new target as well as for affinity maturation.