Validation of VHHs

How does ChromoTek validate their alpaca single domain antibodies?

ChromoTek utilizes genetic strategies and comparison with independent antibodies for the validation of our VHHs against fluorescent proteins and peptide tags:

  • In the genetic approach, VHHs are tested in their target application (immunoprecipitation, immunofluorescence and/or Western blot) both on cell lines that express and do not express their cognate fluorescent protein or peptide tag.
  • In addition, our Nanobodies are benchmarked with established conventional antibodies.

Our Nanobodies are always sequenced; in several cases, we even know their crystal structure. Furthermore, we thoroughly characterize and validate our monoclonal Nanobodies. Their recombinant production in combination with high QC standards ensures reliable and stable alpaca single domain antibody products virtually without lot-to-lot variations.

Discussion of antibody validation

The validation and reliable production of antibodies are intensively discussed in life sciences both in academia and pharmaceutical industry (Bradbury and Plückthun 2015; Baker 2015). This debate complains about insufficient antibody quality and, consequently, wasted research time and funds and seeks options to resolve these issues by standardizing antibodies and their production.
A comprehensive set of guidelines for the validation of antibodies was recently published in “A proposal for validation of antibodies” by the International Working Group for Antibody Validation (M. Uhlen et al. 2016). The working groups suggests:

Five conceptual pillars for the validation of antibodies

  • genetic strategies
  • orthogonal approaches
  • comparison with an independent antibody
  • tagged protein expression
  • IP-MS

Of these, at least one, better more approaches should be used to validate an antibody properly. ChromoTek utilizes in fact two approaches for validation: genetic strategies and comparison with independent antibodies for the validation of our VHHs against fluorescent proteins and peptide tags.

 

Read more about the ongoing antibody validation discussion below

Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents
Andrew R. M. Bradbury and Andreas Plückthun 2015 in Protein Engineering, Design & Selection, vol. 28 no. 10, pp. 303–305

Reproducibility crisis: Blame it on the antibodies Antibodies are the workhorses of biological experiments, but they are littering the field with false findings. A few evangelists are pushing for change.
Monya Baker 2015 in Nature 521, 274–276

A proposal for validation of antibodies
Mathias Uhlen, Anita Bandrowski, Steven Carr, Aled Edwards, Jan Ellenberg, Emma Lundberg, David L Rimm, Henry Rodriguez, Tara Hiltke, Michael Snyder & Tadashi Yamamoto 2016 in Nature Methods 13, 823–827)