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Camelid single-domain Antibodies (sdAb)

A serendipitous discovery in the early 90s at the Free University of Brussels resulted in Dr. Hamers-Casterman finding a new class of antibodies lacking the light chains in the blood of camels (1). These heavy-chain-only antibodies (HcAbs) can be found in camels, dromedaries, and all the new world camels (llamas, alpacas, guanacos & vicuñas).

The minimal domain necessary to bind an epitope/antigen is what is today known as a single-domain antibody (sdAb), variable domain of the heavy-chain-only antibody (VHH), and commercially as Nanobody® (a trademark of Ablynx, now Sanofi).

NanoTag Biotechnologies GmbH has extensive experience and proprietary technologies to screen, select, engineer, validate, and produce specific nanobodies for various applications.

SdAbs keep gaining popularity in multiple applications, from super-resolution microscopy (2–6), affinity purifications (7–10), crystallography (9,11–13), biosensors (14–16), cryo-EM (8,17,18) to medical diagnostics (19,20), clinical imaging (21,22), theragnostic (23), monoclonal and cell therapies (24).

This broad spectrum of applications is due to several unique features that make them attractive when compared to conventional antibodies.

Below you can find more information about the principal benefits and features of sdAbs, FluoTags, and Selectors.
Recombinantly Produced
SdAbs are recombinantly produced in bacteria, yeast, plants, or mammalian cells.
1. No need for animals for production.
2. High reproducible results, minimal to no batch effect, friendly for GMP production.
3. Highly modifiable, easy incorporation of genetically added tags (e.g., minibodies).
4. Production is easily scalable, from a petri-dish, lab-size flasks to large bioreactors.
Highly Stable
SdAbs are highly stable molecules, that resist a wide variety of pH and temperatures. They are typically highly soluble and tolerate lyophilization well. Their robust stability allows the administration of nanobodies intravenously, subcutaneously, by inhalation, and even oral administration. In addition, sdAbs are very poor immunogenic in humans, and efforts to humanize them are possible if necessary.
Monovalent Binders
SdAbs are by default monovalent (however, it is possible to dimerize or trimerize them).
This feature of sdAbs results in the following advantages:
1. No probe-induced clusters (possible artifact in divalent and polyclonal antibodies).
2. A more linear response between the target molecule and readout (e.g., fluorescence microscopy, western blot, ELISA, etc.).
10x Smaller than IgG
SdAbs are only ~15 kDa and ~2x3 nm (length-x-high), while IgGs are ~150 kDa 10x15 nm (length-x-high). Due to their small size, sdAbs have a superior sample penetration (diffusion) in complex biological specimens. (4, 25)
1. This feature improves stainings on tissue or complex samples.
2. Biodistribution in organisms is facilitated, especially in remote regions, due to their small size and absence of the Fc domain, which is regularly being “checked” at Fc receptors.
Superior for Microscopy
NanoTag has developed sdAbs specifically for microscopy (FluoTags®).
FluoTags® are coupled directly to fluorophores (1 = Q; 2 = X2; 2x2 = X4).
Their small size and monovalency bring fluorophores closer to the target of interest (no need for secondary Ab detection). This increased precision is vital for super-resolution microscopy. FluoTag®'s size also allows to find more epitopes than conventional antibodies, particularly in crowded regions. (2-6)
Site-Specific Conjugation
NanoTag can add specific chemistries to conjugate molecules in a stoichiometric and site-specific manner. This allows a high versatility in conjugations of small molecules like drugs, fluorophores, biotin, HRP, DBCO, and even DNA strands (site-directed to avoid losing epitope binding). Importantly, we provide site-specific immobilization on our affinity Selector resins, which are decorated with oriented sdAbs, which results in a higher capacity per μl of resin and improved reproducibility.
Custom Conjugation
NanoTag ́s sdAbs can be site-specific conjugated to different molecules. Our shop offers a wide selection of sdAbs conjugated to biotin, HRP, click chemistries, and many types of fluorophores. Please contact us if you need any other molecules to be on our or your nanobodies!
Cleaner Immunoprecipitations (IP)
Our Selector resins are generated by site-specific covalent immobilization of sdAbs on non-magnetic or magnetic agarose beads. In contrast to conventional pull-down assays suffering from contaminations with antibody heavy and light chains co-eluting with the target protein, our technology largely prevents antibody-derived contaminations and thereby gives ultra-pure results.
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Bibliography & References
  1. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).
  2. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. a simple, versatile method for GfP-based super-resolution microscopy via nanobodies. Nature methods 1–6 (2012) doi:10.1038/nmeth.1991.
  3. Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun 6, 7933 (2015).
  4. Sograte-Idrissi, S. et al. Circumvention of common labelling artefacts using secondary nanobodies. Nanoscale 12, 10226–10239 (2020).
  5. Ganji, M., Schlichthaerle, T., Eklund, A. S., Strauss, S. & Jungmann, R. Quantitative Assessment of Labeling Probes for Super‐Resolution Microscopy Using Designer DNA Nanostructures. Chemphyschem 22, 911–914 (2021).
  6. Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat Methods 16, 1045–1053 (2019).
  7. Jin, F. et al. Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins. Commun Biology 4, 366 (2021).
  8. Zhao, J. et al. Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins. Proc National Acad Sci 119, e2207200119 (2022).
  9. Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat Commun 10, 4403 (2019).
  10. 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).
  11. Che, T. et al. Nanobody-enabled monitoring of kappa opioid receptor states. Nat Commun 11, 1145 (2020).
  12. Koenig, P.-A. et al. Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371, eabe6230 (2021).
  13. Rasmussen, S. G. F. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature469, 175–180 (2011).
  14. Mann, F. A., Lv, Z., Großhans, J., Opazo, F. & Kruss, S. Nanoröhren‐Nanobody‐Konjugate als zielgerichtete Sonden und Marker für die In‐vivo‐Nahinfrarot‐Bildgebung. Angew Chem-ger Edit 131, 11591–11596 (2019).
  15. Vigano, M. A. et al. Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster. Development dev.191700 (2021) doi:10.1242/dev.191700.
  16. Gerdes, C. et al. A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol. Nat Commun 11, 2729 (2020).
  17. Uchański, T. et al. Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. Biorxiv 18, 812230 (2019).
  18. Masiulis, S. et al. GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature565, 454–459 (2019).
  19. Hosseindokht, M., Bakherad, H. & Zare, H. Nanobodies: a tool to open new horizons in diagnosis and treatment of prostate cancer. Cancer Cell Int 21, 580 (2021).
  20. Salvador, J.-P., Vilaplana, L. & Marco, M.-P. Nanobody: outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem 411, 1703–1713 (2019).
  21. Rosenfeld, L. et al. Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer. J Med Chem 63, 7601–7615 (2020).
  22. Berland, L. et al. Nanobodies for Medical Imaging: About Ready for Prime Time? Biomol 11, 637 (2021).
  23. D’Huyvetter, M. et al. Radiolabeled nanobodies as theranostic tools in targeted radionuclide therapy of cancer. Expert Opin Drug Del 11, 1939–1954 (2014).
  24. Wang, J. et al. Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment. Front Immunol 12, 838082 (2022).
  25. Fang, T. et al. Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure. Nat Methods 15, 1029–1032 (2018).

What we can offer you

At NanoTag Biotechnologies, we offer tailored sdAb discovery services for your protein of interest, custom sdAb conjugation, and a validated off-the-shelf product portfolio. The sdAbs that are directly coupled to fluorophores can be found as FluoTags®, or if immobilized on agarose and magnetic agarose beads, they can be found as Selectors.

In addition, our sdAbs are also available in an unconjugated version or as sdAbs bearing biotin, cup-per-free click chemistry DBCO, enzymes like the Horse Radish Peroxidase (HRP), or even Fc domains from different species (Minibodies).

If you need a different functional molecule on them, please contact us with your idea.

Have a look in our webshop.
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