What is a Nanobody?
A complete guide to understanding nanobodies
Quick overview:
A Nanobody, also called single domain antibody (sdAb) or VHH (variable heavy domain of heavy chain), is the small (~15 kDa / 2 nm) antigen binding fragment / variable domain of heavy chain antibodies (HCAbs) that are part of the immune response of camelids (most famously in alpacas, but also llamas, dromedaries, and camels). Interestingly, similar antibodies have also been found in sharks. Cultivation and immunization of alpacas, however, has proven to be less dangerous.
Conventional antibodies like mouse, rabbit, or rat IgGs, which are widely used in many applications in research and medicine, are heterodimeric complexes that consist of two light and two heavy chains. Their antigen binding site requires the interplay of two elements - the variable domains of both the upper light and the upper heavy chains of the antibody’s Fab fragments. As part of their immune response, camelids also produce heavy chain antibodies (HCAbs; subclasses IgG2 and IgG3). In contrast to IgG1, these HCAbs lack the light chains and the CH1 domain of the heavy chains. Hence, they rely on only one variable domain for antigen recognition (Figure 1).
Figure 1: Alpaca Immune Response / Antibody overview: The immune response of alpacas generates three IgG subclasses. IgG1 antibodies contain heavy and light chains. Subclasses 2 and 3 are heavy chain antibodies that lack the light chains as well as the CH1 domain of the heavy chains. The variable domain of HCAbs is called VHH, or in its recombinant separate form, also called nanobody.
The reduced complexity of the antigen binding site allows for efficient recombinant production, enabling manufacturers to produce nanobodies animal free and with zero lot-to-lot variation. Additionally, their small size and compact structure make them highly thermostable, resistant to harsh buffer conditions, and allows for fast and easy tissue penetration.
Nanobodies can be modified, conjugated, and adsorbed onto different matrices for use in diverse assays, ranging from immunoprecipitation to immunofluorescence, immunohistochemistry, protein purification, western blot, and most applications in which conventional antibodies are used.
Proteintech offers a variety of ChromoTek Nanobody-based products for different applications. If you are interested in using our Nanobody products for your own experiments, please refer to Table 1 below or check out our ChromoTek Nanobody product catalog.
You can also contact us if you need technical support or a custom order for any of the available products.
We’re happy to help!
ChromoTek Nanobody products in different applications
Table 1: Overview of applications and suitable ChromoTek product groups
|
Application |
Nanobody based products |
How are Nanobodies produced?
Initial production of Nanobodies against the target antigen is done by immunization of young adult camelids. As explained above, the desired heavy chain antibodies against the target antigen are produced as part of the immune response of the animals. Eventually, B-cells are obtained from a small blood sample from the immunized camelids and used to create recombinant systems for production of nanobodies.
In a multi-step nested PCR, VHH sequences are amplified from the B-Cell cDNA pool. After restriction digests with suitable enzymes, the VHH sequences are ligated into expression vectors, which can then be used for the transformation of E. coli.
A Nanobody / VHH immune - library has been created!
Monoclonal high affinity nanobodies against the target antigen are identified and isolated via Phage display. Sequencing of each respective phage display vector then reveals the sequence of the high affinity binders and these Nanobodies can then be expressed and purified from E. coli.
(see references for a more detailed description of the process, as well as on phage display clone selection and synthetic / naïve VHH libraries)
Nanobody (VHH) structure
Figure 2: Nanobody-Antigen binding modes. A) Binding of the 12 amino acid Spot-tag by the ChromoTek anti-Spot nanobody. The tag wraps around the upper part and merges with the beta sheets of the nanobody. A clamp mechanism encloses the peptide, increasing its binding affinity. (ChromoTek Spot-Tag System). B) Mode of binding of an anti-GFPuv nanobody that involves all three CDRs (adapted from Zhang et al. 2020)
Nanobodies are the variable domain (VHH) of heavy chain antibodies. Their compact structure is composed of a framework of beta sheet stacks that form on the “sides” of the nanobody. On top, three complementary determining regions (CDR1 / CDR2 / CDR3) define the flexible structure of the antigen binding site of the VHH (Figure 2). The lengths and amino acid sequences of the CDRs are variable, which allows for specific binding of a vast range of target epitopes.
Depending on the epitope that is recognized, different residues of the three domains are involved in binding. The mode of interaction between the CDRs and the epitope affects the overall confirmation of the antigen binding site and the relative positions of the CDRs to each other.
Figure 2 shows two binding modes that utilize the CDRs in different ways. In the case of the ChromoTek Spot-Trap™, the Spot-tag peptide wraps around the upper part of the nanobody, aligns with beta sheets of the nanobody, and is enclosed by a clamp mechanism (Figure 3 A), resulting in a very high affinity binding. Binding of GFPuv (a brighter GFP variant) relies on all three CDRs to engage in lateral contacts with the beta-barrel of GFP (Figure 2 B).
The C terminus of the Nanobody is not involved in antigen binding, as it is facing towards the rest of the heavy chain in context of the whole HCAb. This region is often utilized for conjugation of dyes or beads or fusion to other proteins.
Advantages of Nanobody vs. Antibody
Nanobody Size
With a molecular weight of roughly 15 kDa, nanobodies can penetrate tissue samples / organs / animals faster and more easily, which is a big advantage for applications like immunofluorescence, immunohistochemistry etc. Also, higher resolutions can be achieved due to a small epitope-label displacement (Figure 3), especially useful for applications like super-resolution microscopy and confocal microscopy. Furthermore, nanobodies might reach certain epitopes that are inaccessible for conventional antibodies, which are ~10x larger, resulting in more homogenous staining, especially in crowded cellular environments like the nucleus.
Figure 3: Epitope Label Displacement. Left to right: direct labeling via VHH results in the lowest displacement (~ 2 nm), compared to labeling with Nano-Secondary reagents (up to 15 nm), and labeling with conventional polyclonal secondary antibodies (up to 30 nm). Nanobodies can help increase the resolution in imaging applications.
Nanobody Production / Expression in E. coli / Reproducibility
Production of nanobodies only involves an animal in the first step, the initial production of a VHH immune library by immunization of camelids. The animals are not sacrificed, but only a small amount of blood is taken to isolate B Cells and generate immune libraries. From here on, every step is completely animal free. Their smaller size also results in less complex folding, which makes expression in and purification from E. coli possible, lowering time and costs of production and eliminating lot-to-lot variation.
Nanobody Stability
Nanobodies have a very condensed structure with few flexible elements. This comes with an increased thermostability, shelf life, and resistance to harsh buffer conditions (up to: 2 M NaCl / pH 4 – 10 / 4 M UREA). Further, due to this increased thermostability, compact structure, and high affinity binding, nanobodies have proven to be valuable tools in structure determination, for example as chaperones in X-ray crystallography.
Immunoprecipitation and purification with Nanobodies
Nanobodies can be used in any type of Immunoprecipitation (IP / Co-IP / ChIP / RIPas well as in affinity purification. Compared to conventional antibodies, they provide several advantages. The most important being their high specificity, which results in very low background and less nonspecific binding (Figure 4 - GFP-Trap® Agarose). Additionally, when digesting, reducing, or eluting the IP samples, the product is not contaminated with heavy and light chain fragments, as shown in Figure 4. The high binding affinity of nanobodies (1 picomolar – 100 nanomolar range for ChromoTek products) and their compact and robust structure also allows for harsh washing conditions during pulldown or purification.
Ready-to-use products like Agarose, Magnetic Agarose, or M-270 Magnetic Particle coupled Nanobodies are available for many targets. Refer to our Nano-Trap catalog for further information.
Figure 4: Nanobodies in pulldown and protein purification. A) Comparison of the ChromoTek GFP-Trap vs. a conventional anti-GFP antibody with heavy and light chains. The final product of the GFP-Trap shows a single band, while heavy and light chain fragments contaminate the final fraction of the IgG alternative. B) anti-GFP Spot-Cap used for protein purification. A clean one-step purification of GFP is possible due to high specificity and high affinity of the nano-cap.
Same species Multiplexing
Nanobodies are highly epitope specific and our ChromoTek Nano-Secondary reagents against different IgG subtypes make same species multiplexing possible (IF / IHC / SRM / FC / WB). Figure 6 shows multiplexing in HeLa cells with 3 mouse IgG subclass specific ChromoTek Nano-Secondaries (IgG1 / IgG2b / IgG3). With their specificity comes low cross reactivity. This can result in lower background and better signal to noise ratios compared to conventional secondary antibodies.
Figure 4: Multiplexed immunostaining of HeLa cells with 3 subclass specific alpaca anti mouse Nano-secondaries. Grey: Mouse IgG1 anti-Vimentin + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + alpaca anti-mouse IgG3 VHH Alexa Fluor® 568. Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Limitations of Nanobodies
You can’t have one without the other - Despite the many advantages that nanobodies have, they also come with some disadvantages …
Brightness / Degree of labeling (DOL)
ChromoTek Nano-Secondaries typically contain two covalently bound dyes. With two to four nanobodies binding to the Fc fragment (one per chain), 4 – 8 dyes are attached to each primary antibody. Consequently, primary nanobodies (Nano-Boosters & -Labels) have similar limitations in regards to signal intensity.
The classical approach would be to use a polyclonal secondary antibody against the species of the primary antibody. With conventional antibodies being 10x larger than Nanobodies, a higher DOL and a brighter signal per secondary antibody can be achieved. This effect is further amplified through the binding of multiple epitopes of the primary antibody (polyclonality).
So why would you choose Nanobodies? Well, not all assays require high brightness, but rather high resolution (e.g. SRM), which can, due to the minimal epitope-label displacement, make nanobodies the better choice. Their size advantage additionally facilitates more homogenous staining throughout the cell, especially in crowded environments like the nucleus, and better tissue penetration.
Other assays may require strong signal intensity or amplification of signal, in which case conventional IgGs may be the better choice. It really depends on what you want to do and how the limitations of nanobodies or antibodies affect your experiment.
Note: ChromoTek is currently working on different solutions to increase the DOL and brightness of nanobody-based products.
Conclusion
We hope this blog has demonstrated that nanobody-based reagents are not complex, futuristic niche products. Instead, they are accessible, practical, and versatile tools that offer numerous benefits, making them a valuable addition to any laboratory.
Their smaller size compared to conventional IgG antibodies provides distinct advantages, such as enhanced tissue and cell penetration and higher resolution - especially beneficial for imaging applications. Furthermore, their high specificity, strong affinity, and the absence of heavy and light chains make them ideal for various immunoprecipitation assays.
In summary, nanobodies are more than just an alternative to traditional antibodies; they represent a significant advancement in the field. Their numerous advantages can greatly enhance both the efficiency and effectiveness of your research. Regardless of your research area, nanobodies offer a cutting-edge solution that’s ready to be integrated into your lab-work.
References
A guide to: generation and design of nanobodies - PubMed (nih.gov)
Antibody phage display: technique and applications - PubMed (nih.gov)
products/GFP-Trap-Agarose-gta.htm
Written by Philipp Becker, PhD – Product Manager at Proteintech / ChromoTek July 2024
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