Our Nanobody® Solutions

Benefit from our proprietary synthetic and humanized VHH library for customized Nanobody® selection, validation and affinity maturation projects.

Our Technology

Our  synthetic  single  chain  antibody

VHHs have several unique features including small size (15 kDa), high stability/solubility while retaining high binding affinity to their target antigens. VHHs are composed of 3 hypervariable regions, supporting antigen recognition against diverse targets and separated by 4 framework regions.

Our VHH scaffold is humanized for therapeutic applications reducing the level of immunogenicity. There is no animal use, thus antigens are not processed and VHHs can be developed against non-immunogenic or highly conserved proteins.

Proprietary Nanobody Library

Our  synthetic  humanized VHH  library

Our synthetic antibody library is a naive library with a complexity of 3.109 VHHs enabling to select highly functional conformational nanobodies and intrabodies.

Our Hybribody library possesses more diversity than in a herd of llamas! This is attributed to the rapid advances in synthetic biology and creative scientific efforts of our collaborators Drs. F. Perez and S. Moutel (Curie Institute, Paris) and Dr. A. Olichon (CRCT, Toulouse) (https://elifesciences.org/articles/16228).

 

Hybribody is a completely animal free antibody selection service. Discover here what our Lamas look like.

Our Nanobody® Solutions

Nanobody selection, validation and maturation

Selection of Synthetic VHHs - in vitro
Nanobodies for Cell Surface Antigens
Intrabody Selection and Validation
Optimization : Extracellular Affinity
Maturation by Yeast Display
Optimization : Intracellular Affinity
Maturation by Y2H
Minibody Cloning and Production
VHH for Immunofluorescence
Our Ubiquitin Selectors with Nanotag®
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Our optimized Phage Display selection technology delivers high quality and fully functional humanized recombinant nanobodies recognizing native antigens. Importantly, the structural integrity of the antigen is maintained throughout the entire VHH selection and validation process. Therefore, our nanobodies are more likely to recognize endogenous antigens for in vitro and in vivo applications, such as immunofluorescence, live cell imaging, protein degradation or blocking protein activity.

Key benefits

  • Conformational antibodies
  • Deliverables of the validated VHH binders include DNA sequences and plasmids
  • Customized selection conditions (buffer, temperature, etc)
  • Animal-free technology
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Nanobodies are selected against membrane proteins or cell-surface antigens under native conditions.

The VHH candidates are directly selected on living cells by phage display. In this process, our proprietary library is incubated with the antigen expressed on cells. Subsequently, a washing step with antigen-free cells is performed and 3 to 4 rounds of selection follow.


Selected nanobodies are validated by immunofluorescence (IF) or phage-FACS assays. This selection strategy works very well for microbial antigens, such as expressed by parasites, bacteria or viruses.

Key benefits

  • Variety of conformational nanobodies against a cell-surface target
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Our intrabodies represent functional intracellular nanobodies expressed and properly folded in the cell cytoplasm where they recognize their endogenous target.

To enrich for intrabodies, we combine classical Phage Display Screening with our expertise in Yeast Two-Hybrid screening. These functional nanobodies are expressed inside the cell where they target endogenous antigen.


This combined strategy is useful for VHH selection against soluble antigens that are particularly difficult to express or purify in heterologous expression systems.

Key benefits

  • Enrichment of intracellular targeting nanobodies
  • No batch-to-batch variability
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In vitro affinity maturation of nanobodies and scFv.

We use a combination of mutagenesis and yeast display to greatly improve the binding affinity of your nanobody or scFv. A mutant VHH or scFv library (2-5 x 106) derived from the parental sequence is generated by introducing mutations either across the entire length of the antibody sequence or restricted to the CDR regions and cloned into our proprietary vector for yeast surface expression.

Yeast expressing the mutant library is subjected to several rounds of cell sorting and the concentration of antigen is gradually decreased to select nanobodies with the highest binding affinities. Our yeast display platform controls for the antigen-antibody equilibrium, which is a major advantage compared to phage display.

Key benefits

  • Cost-effective and quick optimization method
  • No drift-effect like in phage display
  • Apparent kD measurements
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Maturation of VHH with our Yeast Two-Hybrid technology to obtain higher binding affinities for specifically intracellular applications (Intrabodization).

This service is divided into two phases: 1) creation of a mutant library generated by mutagenesis of the entire molecule or the CDR regions only 2) perform Y2H selection on the mutant library to identify intrabodies with increased binding affinities. The selection pressure is adjusted during the screening process to enrich for intrabodies with increased binding affinities.

Key benefits

  • Cost-effective and rapid optimization process
  • No-drift effect compared to phage display
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Fuse your VHH with a Fc species of your choice to generate minibodies.

The selected VHH candidates are fused to Fc isotype-specific fragments from either human, mouse, sheep, rabbit, dog and rat to generate a “Minibody". This format is easily produced in CHO or HEK cells and can be used to stain antigens with standard antibody reagents.

Key benefits

  • Versatile & flexible use for a vast array of applications (PLA, ELISA…)
  • No batch-to-batch variability
  • Possible fusion with Fc fragments from human, mouse, sheep, rabbit, dog and rat
  • Enhanced avidity
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To identify VHH candidates for immunofluorence (IF) applications, we express your antigen in cells as a fusion protein to a fluorescent tag which is targeted to a discrete subcellular localization. VHH-derived supernatants from E. coli are tested for co-localization by IF using standard secondary antibody reagents. This method enables the ranking of VHH candidates based on immunofluorescence intensity and co-localization analysis.

Key benefits

  • ELISA & IF-validated VHH nanobody® in a single cDNA clone
  • No variability from batch to batch
  • Versatile tool thanks to the possible fusion with Fc fragments from human, mouse, sheep, rabbit, dog and rat
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Ub-Selectors are the result from our Hybribody technology and are commercially available by Nanotag GmbH. These reagents are a perfect illustration of Hybribody nanobody solutions.

 

In general, ubiquitin is non-immunogenic and ubiquitin-chain specific nanobodies cannot be selected in animals. Therefore, our products represent a new entry point in the ubiquitin world and represent a new class of reagents that now can be developed.

Our Nanobody® Solutions

Nanobody selection, validation and maturation

our publications

Customer Publications

NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

Moutel S, Bery N, Bernard V, Keller L, Lemesre E, de Marco A, Ligat L, Rain JC, Favre G, Olichon A, Perez F. Elife 2016 Jul 19;5. pii: e16228

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Tomoyuki Hatano, Tzer Chyn Lim, Ingrid Billault-Chaumartin, Anubhav Dhar, Ying Gu, Teresa Massam-Wu, William Scott, Sushmitha Adishesha, Bernardo Chapa-y-Lazo, Luke Springall, Lavanya Sivashanmugam, Masanori Mishima, Sophie G. Martin, Snezhana Oliferenko, Saravanan Palani, Mohan K. Balasubramanian Journal of Cell Science, Toole and Resources,| 23 September 2022

Single-domain antibodies for functional targeting of the signaling scaffold Shoc2

Jang H, Wilson PG, Sau M, Chawla U, Rodgers DW, Galperin E. Mol Immunol. 2020 Feb;118:110-116

Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies

Elorza-Vidal X, Xicoy-Espaulella E, Pla-Casillanis A, Alonso-Gardón M, Gaitán-Peñas H, Engel-Pizcueta C, Fernández-Recio J, Estévez RHum. Mol Genet. 2020 May 8;29(7):1107-1120

Development and characterization of single-domain antibodies neutralizing protease nexin-1 as tools to increase thrombin generation

Kawecki C, Aymonnier K, Ferrière S, Venisse L, Arocas V, Boulaftali Y, Christophe OD, Lenting PJ, Bouton MC, Denis CV. J Thromb Haemost. 2020 Sep;18(9):2155-2168

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth.

Wojnacki J, Nola S, Bun P, Cholley B, Filippini F, Pressé MT, Lipecka J, Man Lam S, N'guyen J, Simon A, Ouslimani A, Shui G, Fader CM, Colombo MI, Guerrera IC, Galli T. Cell Rep. 2020 Dec 22;33(12):108536

Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies

Dupré E, Danis C, Arrial A, Hanoulle X, Homa M, Cantrelle FX, Merzougui H, Colin M, Rain JC, Buée L, Landrieu I. ACS Chem Neurosci. 2019 Sep 18;10(9):3997-4006

DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody

Silvia Galli, Larry Melidis, Sean M. Flynn, Dhaval Varshney, Angela Simeone, Jochen Spiegel, Sarah K. Madden, David Tannahill, and Shankar Balasubramanian, J. Am. Chem. Soc. 2022, 144, 50, 23096–23103

Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

Clément Danis , Elian Dupré , Orgeta Zejneli , Raphaëlle Caillierez , Alexis Arrial , Séverine Bégard , Justine Mortelecque , Sabiha Eddarkaoui , Anne Loyens , François-Xavier Cantrelle , Xavier Hanoulle , Jean-Christophe Rain , Morvane Colin , Luc Buée, Isabelle Landrieu Mol Ther. 2022 Apr 6;30(4):1484-1499.

Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

Manel Zeghal, Kevin Matte, Angelica Venes, Shivani Patel, Geneviève Laroche,Sabina Sarvan, Monika Joshi, Jean-Christophe Rain, Jean-François Couture, PatrickM. Giguère Journal of Biological Chemistry 20 July 2023, S0021-9258(23)02135-X

Library publication

Tissue-specific targeting of DNA nanodevices in a multicellular living organism

Kasturi Chakraborty et al. Elife. 2021 Jul 28;10:e67830

Library publication

Novel FGFR4-Targeting Single-Domain Antibodies for Multiple Targeted Therapies against Rhabdomyosarcoma

Nagjie Alijaj et al. Cancers (Basel). 2020 Nov 10;12(11):3313.

Library publication

Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab

Valentina Galli et al. Elife. 2017 Oct 12;6:e25197

Library publication

Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors

Ronan Crepin et al. Biochem Biophys Res Commun. 2017 Dec 2;493(4):1567-1572.

Library publication

Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma patient-derived xenografts

Ronan Crepin et al. Pigment Cell Melanoma Res. 2017 May;30(3):317-327.

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