Our Nanobody® Solutions

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

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

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

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

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

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

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

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.