A toolbox of anti-mouse and rabbit IgG secondary nanobodies

Abstract

Polyclonal anti-IgG secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in E. coli and could thus make secondary antibody-production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore-coupling. We demonstrate their superior performance in Western Blotting, both in peroxidase-and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and super-resolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols and even allow multi-target localization with primary IgGs from the same species and of the same class.not peer-reviewed)

Circumvention of common labelling artefacts using secondary nanobodies

“…A similar gain in precision was observed previously using dSTORM. 12 Additionally, we performed an autocorrelation analysis on single microtubules to corroborate their difference in size, and we observed a significantly faster loss in autocorrelation for microtubules stained with 2.Nb (Fig. 2H).…”

“…Although it has been already demonstrated that small primary affinity probes are able to minimize the linkage error, 4,8,12 there was just one indication performed in dSTORM that secondary nanobodies can increase the labelling precision. 12 Here we show, in addition to the resolution improvement by using 2.Nbs on DNA-PAINT and STED microscopy, that bulky secondary antibodies not accurately represent the distribution of the primaries, due to a combination of their polyclonal nature and large size (Fig. 1A).…”

“…1 †), however, pre-mixing has been shown to work when using monovalent binders against 1.Abs. 12 This feature eliminates the animalspecies limitation of the primaries when detecting two or more POIs. We first showed that it is possible to use 2 mouse 1.Abs in a simpler Western-blot assay (ESI Fig.…”

“…Nbs). 12 Secondly, the large size of the 1.Ab-2.Ab complex makes them to perform poorly in crowded cellular environments or when the epitopes are abundant and densely arranged. In this respect, smaller probes such as aptamers or nanobodies are more efficient in the detection of the POI.…”

“…It has been shown that by pre-mixing 1.Abs with 2.Nbs in a tube prior staining, one could circumvent this species limitation and use on a sample 1.Abs from the same species. 12 Finally, conventional antibodies used commonly for immunodetections are bivalent binders, i.e. each antibody molecule can bind two POIs/epitopes simultaneously.…”