You struggle to access to your hidden target epitope? With our VHH screening service, get the maximum number of VHH binders with the highest binding affinity, thanks to our wide VHH antibody phage display libraries (1.51×1010 different clones). Choose your antigen of interest (peptide, protein, cell…) and we make sure you receive at least 3 highly specific binders within maximum 7 weeks.

Why choose ProteoGenix
for your VHH screening?

Binders guaranteed

We guarantee at least
3 binders to your
antigen of interest.

Your VHH in 7 weeks

Get your full VHH
antibody sequence within
maximum 7 weeks.

IP free

Be the owner of the VHH
antibody sequence that we
generate for you.

High diversity

Maximize the number of relevant
binders using our high diversity
VHH library with 1.51×1010
different clones.

Several possible antigen targets

Our library is tested against a
wide variety of antigens: proteins,
peptides, small molecules, whole
cells…

PhD project manager experts

Benefit from our over 20
years mAb development and
phage display expertise.

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Overview of our VHH antibody screening process

In Silico epitope mapping is a fully software-based technique used to computationally predict the binding site of the
antibody of interest with its antigen, or for preliminary epitope binding investigation.

Antigen design

  • Designed by us: hapten, peptide, protein, whole cell…
  • Provided by customer (you)

VHH library screening and Biopanning

  • Screening of naive camelid VHH library against your antigen
  • 4-6 rounds of biopanning to get the pool of binders

Screening and validation by ELISA

  • At least 96 single phage binders screening, until at least
  • 3 to 10 desired binders are identified

Phage DNA extraction + antibody screening

VHH sequence delivery to the customer

Antibody
Maturation

VHH
Humanization

Antibody
Conjugation

Bispecific
Antibody
Development

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Case study

Example Of VHH Antibody Screening Using A Naïve Library

Service performed

We were asked by a customer to identify at least 10 different binder sequences against an antigen (recombinant protein) that he provided us. Biopanning was performed using our VHH camelid naive library of a very high diversity. A total of 30 positive clones was screened and 14 different unique sequences with strong and specific binding were delivered to the customer.

Biopanning results

 

Rounds Antigen concentration Dae Number Of Washings Phage Quantity(pfu) Phage Quantity(pfu)
Phage Quantity(pfu) Input Phage Quantity(pfu)
1 50ug/ml 5 2.0*1010 2.5*106
2 50ug/ml 5 2.0*1010 5.2*108
3 50ug/ml 6 5.0*1011 6.0*108

Data show significant enrichment over the rounds (bold). As round 3 already shows excellent enrichment, it was decided to stop to prevent decrease in phage diversity.

Polyclonal phage ELISA

Phage Quantity(pfu)/well Coating with antigen Coating with antigen Coating with antigen Coating with buffer(negative control) Coating with buffer(negative control) Coating with buffer(negative control)
R1 R2 R3 R3 R2 R3
3.16*1011 2.85 2.35 2.91 0.04 0.03 0.03
1*1011 2.46 2.34 2.27 0.02 0.02 0.02
3.16*1010 1.84 2.75 2.29 0.02 0.02 0.02
1*1010 1.85 2.28 2.52 0.01 0.02 0.01
3.16*109 0.52 1.78 2.42 0.01 0.02 0.01
1*109 0.04 1.03 1.9 0.01 0.01 0.01
3.16*108 0.08 0.17 1.04 0.01 0.01 0.01
0 0.01 0.01 0.02 0.01 0.01 0.01

Data confirm significant enrichment as indicated by good specific binding + very low background (bold).

Monoclonal phage ELISA of the 14 unique antibodies identified
(unique means unique sequence)

Positive signals (coating with antigen – coating with buffer)

1 2 3 4 5 6 7 8 9 10 11
A 1.9 0.13 0.1 1.33 1.96 0 0.23 0.02 0.18 1.64 0
B 1.43 0 0 0 0 -0.01 1.78 1.27 1.82 0.07 1.65
C 0 0 0 1.82 2.07 -0.01 0.1 1.81 0.03 0 0
D 0 0 0 0 0 0 1.7 0.01 2.19 0.03 0
E 0 0.02 0 0 1.74 0 0.05 0.04 0 1.29 0
F 1.5 0.96 1.03 0.44 0 2.27 0.17 1.46 0 0.01 0
G 1.74 0 1.15 0 -0.01 0 1.78 0.02 0.01 1.27 0
H 0.01 0 0.01 0 1.11 -0.01 0.21 1.48 1.29 1.04 0

30 clones are clearly positive (bold) and were sequenced, resulting in a total of 14 different unique sequences identified. All 14 sequences were delivered to customer. Our customer expressed the 14 antibodies as recombinant VHHs and tested them against cells overexpressing the protein of interest at their surface. Ten of them bound well to the cells with a clear shift,compared to control cell, of which 5 even had blocking activity.

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What is a VHH single domain antibody?

Conventionally we use IgG antibodies from mice, rat, rabbit etc. They have a size of 150kDa and are composed of 2 heavy and 2 light chains.
A group of researcher found that all members of the Camelidae family possess a special subclass IgG devoid of light chains; these antibodies consist of two heavy chains attached to variable domains (VHH).
Due to this, camelidae antibodies are much smaller, about 95kDa. The VHH alone, which is 15kDa, is also known as single domain antibody.
It is considered as the smallest naturally derived antigen-binding fragment, which is important for the development of several antibody drugs or diagnostic tools.

Conventional antibody VS Camelidae antibody : Camelidae antibody doesn't have light chain and have on its heavy chain VHH single domain antibody
Conventional antibody VS Camelidae antibody

What are the key features of VHH antibodies?

VHH antibodies present several characteristics and advantages making them a valuable tool that can be exploited in the research field
as well as in therapeutics and diagnostic applications. Some of these characteristics include:

  • High stability – These antibodies can resist to both high acidity and temperature conditions. They also have a high ability to fold back into functional conformation after denaturation.
  • Very small molecular size (15 kDa) – The small size of these antibodies helps them penetrating easily cells and tissues and readily crossing the blood-brain barrier, and recognizing cryptic antigenic sites that are not accessible to conventional antibodies.
  • Easy expression – These antibodies are easy to express in E. coli and to be labeled with a fluorescent marker or to be conjugate with an enzyme for research applications
  • Very high solubility – Due to their increased hydrophilic function
  • Very High binding affinity – To their target antigens which lowers the off-target effects and facilitates the prediction of the mode of action.
  • High sequence homology to human antibody heavy chain (HV) – Which is synonymous of less immunogenicity and reactivity.
  • Very simple production – Which is lowering the costs and eases the manufacturing and technology transfer

VHH applications in research

VHH antibodies, also called “nanobodies“, are single domain antibodies corresponding to the variable region of heavy chain of camelid anti-body. Thanks to their small size, 15 kDa, these molecules are featured by their ability to access to hidden epitopes and to easily associate with concave shaped proteins, like catalytic sites of enzymes, which are usually cryptic and not accessible to conventional antibodies.

Their small size also facilitates their penetration and clearance from tissues. VHH antibodies are also known as single domain structures which enables them to be expressed in cells without a need for supramolecular assembly and folding as it’s the case for immunoglobulins. Their small size, robustness and high stability make them effective tools for intracellular protein manipulation and analysis. In addition to their application in clinical and diagnosis fields, VHH have also paved the way for highly valuable applications in research and agriculture.

VHH in research

Development Of Chromobodies: Vhh Gfp-binding Protein (GBP)

  • Used as cellular tracers
    Conventional antibodies are usually used for the protein visualization through immunolabelling in fixed cells.However, their use in living cells is limited by their folding defects or the fluorochrome interference. This problem was overcome thanks to using chromobodies, also called fluobodies, that are molecules comprising a VHH fused to a fluorescent protein able to fold into functional antigen-binding proteins,even in the reducing intracellular environment within the cells. Chromobodies serve as tracers for in vivo intracellular target and organelle localization and for direct visualization of native, endogenous proteins.
  • Used in super-resolution microscopy
    VHH GFP-binding proteins, or GBPs, are highly suitable in the super-resolution imaging techniques, as they facilitate the visualization of GFP fusion proteins. Indeed, thanks to their small size, coupling a fluorochrome to GBP improves labeling with minimal linkage errors, and thus leads to a best imaging quality. Similarly, GBP can be fused to gold nanoparticle and helps in tracking GFP tagged membrane proteins or intracellular proteins within living cells.
  • Used in protein-protein interaction studies
    A GBP-based fluorescent-three-hybrid approach is an approach developed to study in vivo protein-protein interactions. Its principle is based on fixing a GBP at a particular subcellular compartment and to fuse it to an anchoring protein, whereas the two target proteins are fused either to RFP or GFP. If the proteins interact, this leads to protein clustering and to strong colocalization of GFP-RFP signals at the location of the GBP.

Purification And Immunoprecipitation

  • Protein purification ligands
    Thanks to their small size, monomeric nature and single-domain format, VHH antibodies have a high capacity of binding to solid supports with low nonspecific background binding. This makes VHH antibodies a suitable affinity capture ligands and best protein purification ligands compared to larger antibodies. Their very high stability and refolding characteristics allow them to resist to stringent conditions. These proteins might be used, for instance, for IgG depletion from blood, scavenging proteins from plasma and to perform protein-A-based purification.
  • Immunoprecipitation
    VHH antibodies can also be used in the immunoprecipitation studies and for chromatin immunoprecipitation, thanks to their above-mentioned properties.

Targeted Functional Inactivation Of Proteins With Vhhs

  • Immunomodulators (intrabodies)
    VHH antibodies have the ability to bind to catalytic sites, even those with concave structure, usually inaccessible to bigger antibodies.This makes them an ideal in vivo immunomodulators as they can interfere with the natural conformation, localization or function of proteins,leading to functional knockdown/knockouts.

VHH in plant science and
biotechnology/ agriculture

Protein Tracking

VHH-based tracing technology is highly suitable for tracking endogenous proteins of plants, allowing the visualization of endogenous cellular activities in plant cells. For instance, actin filaments can be visualized in tobacco cells thanks to actin chromobodies that label actin cytoskeleton.

Protein Mislocalization And Protein Purification

Some knockdown strategies in plants are based on using VHH antibodies that can be targeted to a target protein to artificially alter its natural localization and thus, inhibit its function. VHH antibodies can also be used as intracellular “nanotraps”. For instance, GFP targeting VHH antibodies can be anchored at a distinct cellular compartment to trap GFP-tagged target protein.

Targeted Degradation

VHH antibodies can be used for immunomodulation through ubiquitin-proteasome system (UPS), by fusing them to degrons, thus leading to targeting the protein of interest for ubiquitylation and proteasome-mediated degradation.

Nanobody Mediated Resistance Against Plant Pathogens

VHH antibodies are promising tools in plant resistance to invading and deadly pathogens. Ectopic expression of antigen specific VHH can establish resistance to some pathogens. The ability of VHH to bind some key adhesion proteins of pathogens prevent them from reproducing, moving and attaching to plants, and thus block their life cycle. VHH reduce also plant damage by neutralizing toxins secreted by some pathogens.