UltraTek Anti-Maus

Polyclonal anti-immunoglobulin G (anti-IgG) secondary antibodies are indispensable tools for many molecular biology techniques and diagnostic tests. However, their animal production is a major ethical problem. Here we present a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced on a large scale in  Escherichia coli and could make secondary antibody production in animals obsolete. Their recombinant nature allows for fusion with affinity tags or reporter enzymes, as well as efficient maleimide chemistry for fluorophore coupling. 

We demonstrate their superior performance in Western blotting in both peroxidase- and fluorophore-bound forms. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and high-resolution microscopy with much less label shift than traditional secondary antibodies. They also allow for simpler and faster immunostaining protocols and enable multi-target localization using primary IgGs of the same species and class.

introduction

Mouse and rabbit antibodies are fundamental tools for many basic research techniques and medical diagnostic assays. These primary antibodies are usually detected or immobilized indirectly via polyclonal anti-IgG secondary antibodies.

•  The need for a continuous supply of anti-IgG sera necessitates the maintenance, immunization, exsanguination and ultimately culling of large numbers of goats, sheep, rabbits and donkeys, which is not only costly but also poses a major welfare and ethical issue (Shen, 2013; Reardon, 2016). 
• In addition, each new batch of serum contains another heterogeneous mixture of antibodies that have to be affinity-purified on IgG columns and then freed (by pre-adsorption) of non-specific and cross-reacting antibodies. In addition, the success of this process must be quality checked each time. 
• The large size of secondary antibodies (∼10–15 nm; 150 kD) is also a disadvantage as it limits tissue penetration and introduces considerable label displacement, reducing the image resolution achievable by super-resolution fluorescence microscopy methods (Ries et al., 2012; Szymborska et al., 2013; Pleiner et al., 2015). Furthermore, their non-recombinant nature precludes genetic engineering (tagging or fusion with reporter enzymes).
• Why then have recombinant anti-IgG detection reagents not replaced secondary polyclonal antibodies? The main problem is the signal strength. For example, the signal in traditional immunofluorescence is enhanced by (a) multiple secondary IgG molecules binding to different epitopes of a primary antibody; (b) a large IgG that tolerates many labels per molecule; and (c) their bivalent binding mode, which exploits avidity for high-affinity target recognition. Given these facts, it seems very challenging to achieve comparable signal levels with a small, monovalent, monoclonal reagent.

We considered nanobodies, single domain antibodies derived from camelid heavy chain antibodies (Hamers-Casterman et al., 1993; Arbabi Ghahroudi et al., 1997; Muyldermans, 2013), as perhaps the best candidates for such reagents. 

Because of their small size (∼3 × 4 nm; 13 kD), possibility of their renewable production as recombinant fusion proteins, and favorable biophysical properties, nanobodies have attracted considerable attention as powerful tools in cell biology ( Helma et al., 2015 ) and structural biology (Desmyter et al., 2015) and as future therapeutics (  Van Bockstaele et al., 2009; Kijanka et al., 2015). ). They are particularly useful for high-resolution imaging(Ries et al., 2012; Szymborska et al., 2013; Pleiner et al., 2015; Göttfert et al., 2017; Traenkle and Rothbauer, 2017).

The resolving power of some of the best microscopes reported so far (e.g. ∼6 nm by Balzarotti et al. [2017] ; ∼10–20 nm by Xu et al. [2012] or Huang et al. [2016]) can be seen as a result of the offset between fluorescent label and target introduced by primary and secondary antibodies (20-30 nm). Site-specifically labeled nanobodies represent a promising solution to this problem, as they can place fluorophores closer than 2 nm to their antigen and, despite their small size, even tolerate up to three dyes (Pleiner et al., 2015).

In this study, we describe the generation of a comprehensive toolbox of nanobodies against all mouse IgG subclasses and rabbit IgG. This work required very extensive optimizations to our routine nanobody selection efforts, such as B. a time-extended and thus affinity-enhancing immunization scheme, subsequent affinity maturation including off-rate selections, and testing and improvement of approximately 200 initial candidates. When site-specifically labeled with fluorophores, the resulting nanobodies performed remarkably well in Western blotting and immunofluorescence. In contrast to polyclonal secondary antibodies, they even enable multicolored labeling and colocalization in one step. 

In stochastic optical reconstruction microscopy (STORM; Rust et al., 2006) of microtubules, an anti-mouse κ-light chain nanobody showed greatly reduced fluorophore offset distances, suggesting its use as a superior alternative to traditional anti-mouse secondary antibodies suggests. Furthermore, we show that anti-IgG nanobodies conjugated to HRP or expressed as fusions with ascorbate peroxidase (APEX2; Lam et al., 2015) and thus can be used for enhanced chemiluminescence Western blotting, colorimetric ELISAs, or immuno-EM detection be able. These monoclonal recombinant nanobodies are therefore a perfect replacement for conventional polyclonal secondary antibodies of animal origin. We imagine that they can be constructed in such a way