Selection of a Nanobody Specific to Human Interferon Inducible Protein 10 (IP-10) Using a Combination of Phage Display and the FLI-TRAP Technique

Tuberculosis (TB) is an infectious disease that is considered a major public health problem worldwide. The current diagnostic technology for TB detection is not efficient. Interferon-γ inducible protein 10 (IP-10) is a highly investigated biomarker for TB diagnosis. Nanobody (Nb) is a novel type of antibody fragment which can be produced in microorganisms at a low cost and could be used to replace mAb in diagnostics. This study focused on selection of a nanobody specific to IP-10. Phage display was used for the initial screening anti-IP-10 Nb from a synthetic library. Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP) technique was then used for Nb isolation from the 1^st round of phage biopanning. After FLI-TRAP selection, 11 out of 20 (55%) randomly samples showed CB resistance at a single colony level representing potential anti-IP-10 Nbs. These candidates were sequence verified and 5 out of 11 (45%) showed no stop codon within the Nb genes. With the new proposed method for verification of cell resistance due to Tat transport of Nb-IP-10 complex proposed in this study, it may be possible to obtain 100% true positive identification. Successful identification of one anti-IP-10 Nb from this large size of a synthetic Nb is the first step. In the future, we could generate mutated library of this full-length Nb and use FLI-TRAP to identify other enhanced variants, which maybe suitable to be used in the development of a TB diagnostic kit that is more affordable, easy to store, and has a higher sensitivity and specificity.