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VHH Single Domain Antibodies (NANOBODIES®) 

 

Alpaca-derived secondary antibodies from Jackson ImmunoResearch are available as total polyclonal cocktail and as VHH single domain antibodies (nanobodies).

 

Camelid species such as Alpaca and Llama produce a class of antibodies composed only of heavy chains (see Fig. 1) that is a unique class of immunoglobulins among mammals. Three IgG-subclasses are found in their sera: the conventional IgG1 as well as IgG2 and IgG3. The latter lack the light chain and first-constant (CH1) domain (Fig.1). These heavy chain-only antibodies are smaller than normal IgGs, but are robust and have full specificity and affinity to their respective antigens.

These antigen-binding fragments, also known as variable heavy-chain only fragment (VHH) antibodies or NANOBODIES®, are an exciting antibody format .With their small 15kDa size and exceptional specificity and penetration properties, VHH fragments offer an excellent solution for high-quality, high-resolution imaging. Jackson ImmunoResearch's AffiniPure-VHH® Secondaries are specialised detection reagents that utilise the unique properties of VHH fragments to produce high-quality results.

 

 

Fig.1: Schematic of conventionel IgG (left), heavy chain-only Alpaca IgG2/ IgG3 antibody and VHH Single Domain Antibody (right).

 

Single Domain -VHH-Antibodies (NANOBODIES® ) from Jackson ImmunoResearch

 

Jackson ImmunoResearch's AffiniPure™ VHH® Secondaries are polyclonal VHH fragment antibodies (nanobodies®) produced in alpacas. They are available with specificity for human, rabbit or mouse. Being 10 times smaller than conventional whole IgG antibodies, these 15kDa VHH fragment antibodies are ideal for imaging experiments requiring good penetration. Jackson ImmunoResearch AffiniPure-VHH® secondary antibodies are cross-adsorbed to provide exquisite specificity against the target species, with minimal cross-reactivity to other commonly used species. This makes them suitable for use in multiple labelling experiments. Jackson ImmunoResearch Single Domain VHH Antibodies are available conjugated to reporter enzymes and a range of fluorescent dyes, including Alexa Fluor®, providing scope for high-resolution immunohistochemistry and immunofluorescence.

 

Small size offers the means to higher-resolution imaging

 

Although direct labelling with NANOBODIES® offers the smallest possible size for an antibody-antigen complex, this approach is not always possible due to the limited availability of commercially available NANOBODIES® raised against individual target proteins. 

However, researchers can exploit the smaller size of the antibody-nanobody complex by using AffiniPure VHH® secondaries from Jackson ImmunoResearch. Adding an indirect detection step only marginally increases the diameter of the binding complex. This maintains a relatively close distance between the probe and target protein, enabling much finer localisation of target antigens compared to traditional primary-secondary antibody detection complexes. 


 

Schematic comparing the linkage error of direct NANOBODY® staining versus indirect IgG+AffiniPure-VHH® Secondary staining (Image: Jackson ImmunoResearch)

 

Advantage of polyclonal vs monoclonal NANOBODIES®

 

In contrast to many other single domain VHH antibodies that are currently available NANOBODIES® from Jackson ImmunoResearch have the additional advantage of being polyclonal. Polyclonal single domain VHH antibodies allow for an even better signal amplification and have the same reduction in linkage error compared to monoclonal nanobodies.They offer a heterogeneous population of antibodies, each with a different paratope detecting a different portion of the primary antibody.

The unique CDR (Complementarity Determining Regions) of VHH Fragments are reported to enable access to antigen epitopes (Bannas et al 2017) which are otherwise unavailable to conventional antibodies. For example, the long loop structure of the VHH Fragment CDR3 may facilitate binding to protein surface pockets.

 

 

This schematic shows how the number of polyclonal AffiniPure-VHH® Secondaries that can bind to a primary antibody is increased compared to monoclonal NANOBODIES®. By binding to the entire primary antibody rather than a specific site, region or sequence, the polyclonal AffiniPure-VHH® secondary antibody can coat the primary antibody extensively, enabling higher labelling efficiency and a brighter signal. (Image: Jackson Immuno Research).

 

NANOBODIES® have a higher tissue penetration than conventional secondary antibodies

 

NANOBODIES® demonstrate excellent tissue penetration owing to their small size (Chakravarty et al., 2014; Debie et al., 2020; Hernández et al., 2019), enabling them to move freely through tissue with a diffusion coefficient lower than that of conventional antibodies (Jovčevska & Muyldermans, 2019).

In addition non-specific binding via the Fc-region is one of the most common sources of background signal in tissue staining and cell assays.The absence of a fragment crystallizable (Fc) region improves the clearance of the molecule from tissues, making nanobodies an excellent tool for diagnostic imaging techniques such as immuno-PET (Krasniqi et al., 2018). AffiniPure-VHH® secondary antibodies utilise these unique characteristics, enabling excellent tissue penetration and clearance without the need for extended incubations or potentially damaging detergents to remove this background.

Due to their small size, NANOBODIES®, which lack the Fc fragment, can be used for immunostaining of live cells (Lelek et al., 2021). In comparison, conventional immunoglobulins with Fc fragments may affect biological functions.

 

Jackson ImmunoResearch's NANOBODIES® are cross-adsorbed for improved specificity and reduced background

 

JIR AffiniPure-VHH® secondary single-domain antibodies are cross-adsorbed against commonly used species to reduce background noise and enhance specificity (learn more about cross-adsorbtion). Our range of AffiniPure-VHH® Secondaries can be used together to produce highly specific multiple labelling images. 

 

Jackson ImmunoResearch's NANOBODIES® offer a multitude of available conjugates 

 

Jackson ImmunoResearch's NANOBODIES® are available with a wide range of conjugates, including fluorescent dyes across the full spectrum.  Fluorophores include dyes from ultraviolet to far-red allowing researchers maximum flexibility when building experiments, particularly those identifying multiple target proteins.

 

Find Nanobodies ...

 

Find Alpaca VHH Single Domain Antibodies (Nanobodies®)

 

Also available from Jackson ImmunoResearch - polyclonal alpaca secondary antibodies:

 

The polyclonal alpaca secondary antibodies (complete set of IgG1, IgG2 and IgG3) from Jackson ImmunoResearch detect Rabbit IgG (H+L), Mouse IgG (H+L) or Human IgG (H+L). To avoid any unspecific background staining due to BSA blocking, all antibodies are cross-adsorbed against bovine serum proteins. In addition, they have no cross-reactivity with rabbit, mouse or human, depending on your choice of species-reactivity. Here you can find anti-alpaca antibodies.

 

Available from BIOZOL - Alpaca Normal Serum for blocking

 

 

References:

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NANOBODY® and NANOBODIES® are registered trademarks of Ablynx N.V.
AffiniPure-VHH® is a trademark of Jackson ImmunoResearch Laboratories, Inc.