The ALFA system

One tag – Three sdAbs – Unlimited applications

A unique, patented technology based on three single-domain antibodies recognizing a rationally designed epitope tag of only 14 amino acids.
Covering a range from pM to nM affinities, this highly specific sdAb ensemble allows for an unmatched spectrum of applications.
High-resolution imaging in life or fixed cells, immobilization and affinity purification of functional proteins and protein complexes, and even therapeutic applications – everything is possible!


The most versatile system for biomedical applications

The ALFA system centers around the ALFA-tag, a novel, rationally designed epitope tag. In contrast to most conventional epitope tags, the ALFA-tag is not an epitope tag that has been found “by chance” as a minimal linear sequence recognized by a monoclonal antibody. Instead, we developed the ALFA-tag based on rational considerations, which enabled us to equip it with desired features defined beforehand. Accordingly, the ALFA-tag fulfills the following criteria:

  • a) The ALFA-tag is small, well-soluble, hydrophilic, and features balanced charges. The tag, therefore, is predicted to have minimal impact on the physiological function of the protein of interest it is fused to.
  • b) The ALFA-tag sequence is absent from common model organisms. Therefore, any reagents binding the ALFA-tag have a high probability to be highly specific. This is in contrast to common epitope tags like, e.g., the c-myc tag, which has a well-defined target present in mammalian cells.
  • c) The ALFA-tag does not contain any residues that are targets for aldehyde-based fixatives. It is therefore by design resistant to fixatives and can be detected even after harsh fixation with PFA or even glutaraldehyde.

NanoTag developed a high-affinity single-domain antibody (NbALFA), binding the ALFA-tag with low picomolar affinity (KD= 27pM) and extremely low dissociation with a half-life of ~15 hours. In experimental setups allowing for re-binding, immobilization may be stable for up to several days.

Crystal structure (PDB: 6I2G) of ALFA-tag (blue) bound to NbALFA (bronze)
Crystal structure (PDB: 6I2G) of ALFA-tag (blue) bound to NbALFA (bronze)

The high-affinity NbALFA is available in various formats: Conjugated to fluorophores (FluoTag-Q and FluoTag-X2 anti-ALFA) or enzymes (HRP) or immobilized on beads (ALFA SelectorST). This set of tools can be used for a wealth of different applications: Imaging of ALFA-tagged target proteins in live or fixed cells, in-vivo manipulation of ALFA-fusion proteins, highly sensitive detection in ELISA or Western-blot assays, immobilization or purification of ALFA-tagged molecules on solid supports or even for therapeutic applications requiring a stable, high-affinity adaptor system. All this using a single tag.

As the sequence of NbALFA (and the ALFA-tag itself) is published, there are no limits for academic researchers to freely use the system and develop new applications based on NbALFA.

In addition to the high-affinity NbALFA, NanoTag developed a set of two NbALFA derivatives, NbALFAPE (for peptide-elutable) and NbALFACE (for cold-elutable). These NbALFA derivatives feature carefully adapted off-rates, which are ideal, e.g., for biochemical applications. NanoTag’s affinity resins featuring these sdAbs efficiently capture ALFA-tagged target proteins even from dilute samples while allowing for a rapid peptide-triggered elution in physiological buffer at room temperature (ALFA SelectorPE) or even at 4°C (ALFA SelectorCE). Together, the set of three sdAbs covers a >1000-fold range of affinities and therefore allows for an unlimited number of applications based on a unique tag.

Additionally, NanoTag is actively developing new ALFA-based tools to broaden the system’s applicability. For example, we have generated polyclonal anti-ALFA antibodies, recombinant NbALFA fused to various immunoglobulin Fc domains (ALFA-Minibodies), or bispecific nanobodies that can be used as adaptors.

References

  • Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat Commun10, 4403 (2019).
  • Kilisch, M. et al. Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer. Biomol 11, 269 (2021).

Celline
Rapid and efficient affinity selection of target-specific B lymphocytes

NanoTag’s proprietary technology called “Celline” allows for the straightforward discovery of single-domain antibodies by affinity purification of target-specific B lymphocytes from a pool of PBMCs.
Using a single selection step, Celline drastically speeds up sdAb candidate selection while at the same time allowing to set avidity/affinity boundaries.
This method has a proven record for the fast generation of high affinity and low-affinity binders.

The ALFA system
One tag – Three sdAbs – Unlimited applications

A unique, patented technology based on three single-domain antibodies recognizing a rationally designed epitope tag of only 14 amino acids.
Covering a range from pM to nM affinities, this highly specific sdAb ensemble allows for an unmatched spectrum of applications.
High-resolution imaging in life or fixed cells, immobilization and affinity purification of functional proteins and protein complexes, and even therapeutic applications – everything is possible!

Secondary nanobodies
Exploit the full potential of your primary antibodies!

A set of single-domain antibodies recognizing conventional immunoglobulins with exquisite species and isotype specificity is the core of our patented technology.
Our secondary tools provide unique benefits for all antibody-based assays in research and diagnostics by combining reproducible specificity and production with small and high-affinity monovalent binding.
In addition, our technology offers surprising new possibilities like one-step indirect immunofluorescence or easy multiplexing.

FluoTags®
Fully defined ready-to-go single-domain antibody conjugates

FluoTag® is NanoTag’s trademark for camelid single-domain antibodies conjugated to chemical fluorophores.
All FluoTags® carry a defined number of fluorophores at known positions. Accordingly, they are available as FluoTag®-Q (one fluorophore; Quantitative) or coupled to multiple fluorophores (FluoTag®-X2 or FluoTag®-X4).
Together with their monovalent binding mode, the defined fluorophore conjugation of FluoTags® results in a more quantitative and reliable readout in all fluorescence-based analyses.

FX technology
FX technology for reliable post-fixation and expansion microscopy

Our sdAbs can be equipped with fixation (FX) cassettes, short amino acid stretches featuring multiple primary amines.
FX cassettes significantly improve the post-fixation properties of sdAbs, which is beneficial for extended experimental procedures, e.g. STORM and DNA-PAINT microscopy.
In addition, FX cassettes provide multiple anchor points in a ProteinaseK-resistant environment and therefore improve the retention of fluorophores in expansion microscopy (ExM) applications.

Selector resins
Affinity purification made easy!

Selector resins are affinity resins based on controlled immobilization of single-domain antibodies.
For production, high-quality agarose resins are functionalized in-house using propriety chemistry. This results in both a highly specific, stable, and oriented sdAb attachment and an ultra-low non-specific background binding.
The precise and oriented chemistry also ensures optimal functionality and accessibility of the sdAbs and therefore results in beads with a high capacity.

Minibodies
Single-domain antibodies fused to immunoglobulin Fc domains

A fusion of a single-domain antibody to an immunoglobulin Fc domain results in a Minibody. Such divalent recombinant antibodies provide maximal flexibility for technologies based on conventional Fc domains, like, e.g., classical secondary amplification systems.
Minibodies can, however, also be detected using our Secondary nanobodies.

One-step indirect immunofluorescence
One-step indirect immunofluorescence: Combine higher resolution with experimental speed!

Conventional secondary antibodies are multivalent and therefore form large clusters with primary antibodies.
In contrast, our species-specific secondary nanobodes are monovalent binders and therefore form defined stoichiometric complexes with primary antibodies in solution.
This feature efficiently prevents cluster formation. Primary antibodies and secondary nanbodies can therefore be pre-mixed or even co-incubated on the sample and thus enable convenient one-step immunofluorescence or Western assays.

Multiplexing
One-step multi-color detection breaking species boundaries

Due to their monovalent high-affinity binding, NanoTag’s species and isotype-specific secondary nanobodies allow multi-color fluorescent assays using multiple primary antibodies raised in the same species.
Multiplexing thereby drastically expands the possible combinations of primary antibodies and at the same time comes with extra features like a time-saving one-step protocol and an increased imaging resolution.
Discover the new freedom in all multi-color fluorescent assays!

Bi-specific sdAb fusions
Freely combine sdAbs to create new adaptor entities!

Bi-specific sdAb fusions (diabodies) are often used as adaptor molecules bringing two individual target molecules in tight contact.
A fusion of two sdAbs targeting the same molecule at different sites may also be used to increase the apparent binding affinity.
NanoTag has first-hand experience in the design, cloning, and expression of bi-specific sdAb fusions in prokaryotic- and eukaryotic systems and provides ready-to-to vector systems.

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