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.

Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions

“…A high bind ELISA plate (Sarstedt, Nümbrecht, Germany) was coated with 60 μL of 5 μg mL −1 anti-αSO nanobody (the nanobody was produced by standard llama immunization with αSOs followed by cloning of B-cells and selection by bacteriophage display 41 (J. N. and D. E. O., unpublished results)), incubated overnight at 4 °C, emptied by inversion and gentle tapping on a table and blocked with 75 μL 2% BSA in 1× PBS for 30 min at 37 °C. Then the plate was washed on an Intelispeed Washer IW-8 (BioSan, Riga, Latvia) with 0.05% Tween 20 in 1× PBS three times and incubated with 50 μL 2 μg mL −1 αSO for 1 hour at 37 °C.…”

Drug repurposing screens identifies compounds that inhibit α-synuclein oligomers’ membrane disruption and block antibody interactions

“…A High Bind ELISA plate (Sarstedt, Nümbrecht, Germany) was coated with 60 μL of 5 μg/mL anti-αSO nanobody (produced by standard llama immunization with αSOs followed by cloning of B-cells and selection by bacteriophage display 34 (J.N. and D.E.O., unpublished results), incubated overnight at 4℃, emptied by inversion and gentle tapping on a table and blocked with 75 μL 2% BSA in 1x PBS for 30 min at 37℃.…”