VHH antibodies*, or nanobodies, are an exciting, novel class of antibodies that offer functional and time saving benefits to basic and translational researchers. VHH antibodies are structurally distinct from traditional antibodies as they are single chain, single domain molecules that can be readily used in various applications including immunofluorescence, high-resolution microscopy, ELISA and western blotting. Improve your scientific productivity by reducing the workflow burden, preventing cross-reactivity, and pinpoint your target in a diverse set of samples and cell types with nanobodies.

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What is a VHH antibody?

VHH antibodies, or nanobodies, were discovered in camelid species such as llamas and alpacas. Nanobody molecules retain the antigen binding region of the heavy chain domain of a conventional antibody, but do not have the other light variable chain domains (Figure 1). These single domain antibodies have a small molecular weight (15 kDa) and have become popular in research and therapeutic settings, as demonstrated by the recent clinical development of Caplacizumab to treat acquired thrombotic thrombocytopenic purpura (aTTP) (1,2). Their unique structures have provided improved functionality in many ways that makes them useful as both primary and secondary antibodies. 

Key featuresAntibodyVHH antibody
Molecular weight150 kDA 15 kDA
Stability  Lower  Higher
Tissue penetration LowerHigher
Labelling density Lower Higher
Lot-to-lot consistency Variable High



Figure 1. Depiction of conventional antibody vs nanobody. A conventional IgG antibody (left) consists of both heavy and light chain regions, with constant (CH, CL) and variable domain (VH, VL). The single domain variable region of the heavy chain makes up the nanobody molecule (VHH). These single domain nanobody molecules can be generated as recombinant antibodies.

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VHH antibodies vs conventional antibodies

Recombinant VHH antibodies are roughly 1/10th the size of a conventional IgG antibody. Due to their high affinity and minimal cross-reactivity, nanobodies can have spatial advantages when binding difficult to access epitopes in complex protein structures, providing more subcellular resolution. 

Benefits of VHH antibodies compared to conventional antibodies:

  • Smaller size: 
    • 2 nm epitope label displacement providing better image resolution
    • Excellent epitope-access in congested cellular and tissue environments, enabling higher labelling density (Figure 2)
  • Stability: Greater chemical and thermal stability than whole immunoglobulins
  • Consistency: Recombinantly produced antibodies provide exceptional lot-to-lot consistency
  • No clustering: Monovalent binders do not cluster
  • Single band purifications: No contamination of the heavy and light chain from degraded IgG
  • Reduce workflow burden: Co-incubate primary and secondary nanobodies to reduce your immunocytochemistry workflow by 50% (Figure 3)
  • Improved secondaries cross-reactivity: No cross-reactivity to other IgGs of rabbit, rat, sheep, goat, guinea pig, human, and macaque serum
  • Super resolution: Validated for super resolution microscopy, including STED and STORM


More secondary VHH antibodies molecules can bind to their target

Nanobodies conjugated to fluorescent molecules binding their primary target

Figure 2.Size comparison of a single domain secondary VHH antibody binding its target versus a conventional IgG secondary antibody. Fluorescently conjugated secondary nanobodies (VHH), such as Alpaca anti-Rabbit IgG Nano (VHH) Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. SA510323), binding both Fab and Fc portions of an IgG primary antibody (left). Traditional fluorescently conjugated secondary antibodies binding to the same target (right) demonstrate that fewer secondary antibody molecules can bind due to their size, when compared to the secondary VHH antibodies.


Reduce immunocytochemistry (ICC) workflow by 50% with Alexa Fluor conjugated secondary VHH antibodies

Nanobodies reduce the immunocytochemistry workflow by 50%

Figure 3.ICC workflow comparison using secondary VHH antibodies versus conventional secondary antibodies. Co-incubation with Alexa Fluor conjugated Alpaca recombinant VHH antibodies eliminates washing and the need for separate incubation in an ICC protocol, which ultimately cuts workflow and sample processing time by 50%.


Image gallery of VHH antibodies data

Multiplexed immunostaining with Alexa Fluor 488 Alpaca Nano secondary antibodies, DAPI, and Phalloidin. (A)Ku80 Monoclonal Antibody (111) (Cat. No. MA512933) was labeled with Alpaca anti-mouse IgG1, VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. SMS1AF488-1-100). (B) Stained with DAPI for nucleus (blue) and mounted in ProLong Diamond, while (C) was stained for F-Actin using Alexa Fluor 594 Phalloidin (Cat. No. A12381). (D) Composite image showing an overlay of all staining.

Multiplex immunostaining of HeLa cells with Alexa Fluor 568 Alpaca Nano secondary antibodies, DAPI, and Phalloidin.  (A) Lamin B1 Polyclonal Antibody(Cat. No. PA5-79606) was labeled Alpaca anti-mouse IgG1, VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. SMS1AF488-1-100). (B) Stained with DAPI for nucleus (blue) and mounted in ProLong Diamond, while (C) was stained for F-Actin using Alexa Fluor 594 Phalloidin (Cat. No. A12381). (D) Composite image showing an overlay of all staining.

One-step immunostaining (top row) vs. sequential immunostaining (bottom row) of HeLa cells. Beta Actin Polyclonal Antibody (Cat. No. PA1-183) (left column) and Lamin B1 Polyclonal Antibody (Cat. No. PA5-79606) (right column) primary rabbit antibodies labeled with Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 647 (Cat. No. SRBAF647-1-100). Scale bar, 20 μm.

Multiplex imaging of Hela cells with three Alexa Fluor Alpaca Nano Secondary antibodies. COX4 Monoclonal Antibody (GT6310) (Cat. No. MA5-17279) labeled with Alpaca anti-mouse IgG1, VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. SMS1AF488-1-100) (Green). beta-3 Tubulin Monoclonal Antibody (2G10) (Cat. No. MA1-118) with Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 647 (Cat. No. SRBAF647-1-100). Primary rabbit anti-Lamin labeled with Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 568 (Cat. No. SRBAF568-1-100). Scale bar, 10 μm.

Immunostaining of HeLa cells. Stably expressing Tubulin-GFP cells were stained with rabbit anti-GFP PABG1 antibody and Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 647 (Cat. No. SRBAF647-1-100). Scale bar, 10 μm.

Confocal and gated STED images of immunostained HeLa cells. Cells were stained with Lamin B1 Polyclonal Antibody (Cat. No. PA5-19468) and labeled with Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 568 (Cat. No. SRBAF568-1-100). Images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser.

Western blot analysis of endogenous ß-Tubulin in HEK293T cell lysate. HEK293T cell lysates probed with beta Tubulin Loading Control Monoclonal Antibody (BT7R) (Cat. No. MA5-16308). Alpaca anti-Mouse IgG2b VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 658 (Cat. No. SMS2BAF568-1-1) was used for labeling.

Multiple Alexa Fluor Alpaca Nano Secondary Antibodies applied for multiplex fluorescent Western blotting. Multiple targets analyzed simultaneously on the same blot. Multiplex fluorescent western blot of GFP-TOM70, ß-Tubulin, and GFP in HEK293T cell lysate. Western blot membrane was simultaneously incubated with primary antibodies and Nano-Secondaries. Green:GFP Polyclonal Antibody (Cat. No. A-11122) labeled with Alpaca anti-Human IgG/Rabbit IgG VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. SRBAF488-1-100) Magenta:beta Tubulin Loading Control Monoclonal Antibody (BT7R) (Cat. No. MA5-16308) labeled with Alpaca anti-Mouse IgG2b VHH, Nano-Secondaries Recombinant Secondary Antibody, Alexa Fluor 658 (Cat. No. SMS2BAF568-1-1).

References

  1. Duggan Shaun. Caplacizumab: First Global Approval. Drugs. 2018; 78(15): 1639-1642. doi: 10.1007/s40265-018-0989-0.
  2. Blair, H.A., Lyseng-Williamson, K.A. Caplacizumab in acquired thrombotic thrombocytopenic purpura: a profile of its use. Drugs Ther Perspect 35, 263–270 (2019). https://doi.org/10.1007/s40267-019-00632-w


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