**4. Concluding remarks**

Nanobodies are highly versatile tools with interesting biochemical properties, which result in their application in various fields ranging from basic research and diagnostics to therapy. In this chapter, we aim to shed light on their multifunctionality and in this way encourage other researchers to include this technology in their future projects. Since their discovery in 1993, the numbers of publications wherein nanobodies are employed are gradually increasing which indicate that their merit has been proved. Here, we have shown that nanobodies have a high therapeutic potential and form an ideal stepping stone to drug development. Despite isolated cases, nanobodies are not capable of traversing the cellular membrane, preventing their direct use as a therapeutic. The effects observed with nanobody treatment are established through multiple mechanisms. Nanobodies can act as an inhibitor of enzymatic activity, interfere with specific protein-protein interactions, and shield a protein of interest from aberrant cleavage, or they can be used as a tool to target proteins for proteasomal degradation. We believe that effects triggered by nanobodies in vitro or in vivo are a faithful representation of what to expect with conventional pharmacological drugs, since both compounds directly target the resident endogenous protein. However, since current experiments are often limited to cell-based assays, animal experiments are warranted to confirm their effectiveness. Furthermore, nanobodies have a lot to offer as a research tool. They can help researchers to elucidate protein functions and thereby gain insight in biological pathways. Several strategies are possible, ranging from subcellular delocalization to the induction of protein knockouts. Last but not least, nanobodies may represent an adequate answer to problems encountered with (conventional) antibody reproducibility [86, 87]. Indeed, particularly polyclonal antibodies run out of stock at some point in the future, making experimental verification impossible. Because nanobody cDNAs are readily obtained and researchers all over the world can use exactly the same nanobody in their experiments, problems of reproducibility can be reduced. In the future, we hope to stimulate a closer consultation within the nanobody field and by doing so taking the research to the next level.
