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.

This article was corrected on 26 June 2021. See the end of the full text for details.

Basic Protocol 1: Total RNA isolation from camelid leukocytes

Basic Protocol 2: First-strand cDNA synthesis; V_HH 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