Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research

Abstract

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; V_H H and V_H repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Keywords: ELISA; immunocytochemistry; immunohistochemistry; intrabodies; nanobodies; phage display; recombinant antibodies.

Figure 1.

Flowchart of the llama leukocyte RNA isolation procedure performed using the LeukoLok™ Total RNA isolation system from Thermo Fisher (Catalog #AM1923).

Figure 2.

Schematic diagrams of the conventional and sandwich panning procedures. A) Conventional panning. Wells of a 96-well microtiter plate are coated directly with the target protein. B) Sandwich panning. Wells of a 96-well plate are first coated with an antibody against the target protein, then incubated with the target protein which binds to the antibody. In both cases the target-containing wells are then incubated with the nAb phage display library. Phage displaying target-specific nAbs on their pili will bind to the attached target protein whereas those displaying non-target specific nAbs will not and remain in solution. After washing away the non-target specific nAbs, the bound target-specific nAbs are eluted and retained for further analysis and use. Steps common to both procedures are in black font.