Menu

Close

Circumvention of common labeling artifacts using secondary nanobodies

Shama Sograte-Idrissi, Thomas Schlichthaerle, Carlos J. Duque-Afonso, Mihai Alevra, Sebastian Strauss, Tobias Moser, Ralf Jungmann, Silvio Rizzoli, Felipe Opazo

Preprint posted on October 25, 2019 https://www.biorxiv.org/content/10.1101/818351v1

A novel, small, and smart tool in fluorescence microscopy: nanobodies.

Selected by Mariana De Niz

Categories: cell biology, immunology

Background

The most common procedure to reveal the location of specific subcellular elements in biological samples is via immunostaining followed by optical imaging. Standard immunodetection approaches use typically a primary antibody which binds the protein of interest  and a secondary antibody that binds to the primary antibody and carries a detection element which can be either a fluorophore or a single strand of DNA (used in DNA-PAINT). Although this classical immunostaining procedure is widely used, important limitations are size (leading to displacement errors incompatible with super-resolution microscopy) and the ability of secondary antibodies to bind more than one epitope. An alternative to secondary antibodies that addresses these limitations are monovalent recombinant secondary nanobodies. Moreover, nanobodies are more suitable for staining thick biological samples, greatly reducing the incubation times usually needed when using secondary antibodies. Finally, nanobodies overcome the issues faced when performing classical multiplexed immunostaining, which requires primary antibodies to be raised in different species. Nanobodies can be pre-mixed with primary antibodies prior to staining, circumventing the species limitation. In this preprint, Sograte-Idrissi et al (1) explore the use and advantages of secondary nanobodies over antibodies.

 

Key findings and developments

General findings

  • In their work, the authors explored the use of secondary nanobodies for several microscopy applications.
  • They confirmed that usage of secondary nanobodies decreases linkage error in STED microscopy and DNA-PAINT and increases localization accuracy compared to secondary antibodies.
  • They showed that pre-mixing of secondary nanobodies with primary antibodies before staining is successful- leading to reduced experimental time, and allowing the use of multiple primary antibodies from the same animal species.
  • They demonstrate that secondary nanobodies are better able to penetrate thick tissues by using them in optically cleared samples,
  • They show that secondary nanobodies avoid artificial clustering sometimes observed when using secondary antibodies.
Figure 1. Advantages of secondary nanobody use. a) Nanobody use increases localisation accuracy. b) Pre-mixing of primary Ab and secondary Nb allows use of primary antibodies from the same animal species. c) Use of nanobodies avoids artificial clustering.

Specific findings

Secondary nanobodies provide higher staining accuracy than secondary antibodies

  • Using STED microscopy, the authors imaged COS-7 cells stained with a monoclonal primary anti-tubulin antibody directly conjugated to Abberior Star635P and further recognized by either a polyclonal secondary antibody or a monovalent secondary nanobody carrying AbberiorStar580.
  • An autocorrelation analysis was performed on these images to evaluate the staining accuracy of the secondary probes. Analysis of the secondary nanobody showed greater accuracy. This was confirmed upon staining peroxisomes within primary hippocampal neurons.
  • The authors then explored secondary nanobody accuracy in the context of DNA-PAINT. The microtubule network of a fibroblast cell line was stained with monoclonal primary antibodies against alpha-tubulin, and detected with a secondary nanobody coupled to a docking strand. Greater accuracy on the apparent diameter of microtubule filaments was achieved.

Bypassing the primary antibody animal species limitations

  • Pre-mixing the primary and secondary antibodies prior to incubating them with the sample would save experimental time and costs, however, this is currently not possible due to the polyclonality and the bivalency of secondary antibodies that result in aggregation of the primary-secondary antibody complex and failure to stain the intended target in the sample.
  • Bypassing the pre-mixing limitation with monovalent secondary probes would allow using several primary antibodies raised in the same species.
  • In their work, the authors used monoclonal antibodies raised in mice against different components (tubulin, Golgi and nuclear pore complex), and pre-mixed each with secondary nanobodies anti-mouse carrying different fluorophores. Upon staining COS7 cells, minimal background and negligible cross-talk was observed between the channels.
  • Using Exchange-PAINT they obtained a super-resolved view of a single neuronal synapse in 3D using two primary antibodies from the same species.

Secondary nanobodies enhance sample penetration in shorter incubation time

  • The authors pre-mixed primary antibodies with secondary nanobodies for use in a complex thick sample: cochleae extracted from mice. They then compared how long the primary/secondary antibody and the primary antibody/secondary nanobody combinations need, to achieve homogeneous staining of the sample. They then imaged the sample after decalcification and optical clearing.
  • They observed faster homogeneous staining upon use of the primary antibody-secondary nanobody mix.

Secondary nanobodies reduce probe-induced clusters of target proteins on living cells

  • To test if the 2-nanobodies reduce probe-induced clustering of target molecules, the authors analysed the surface distribution of IgM containing B cell receptors (IgM-BCRs) on a human B cell line.
  • Cells were stained and chemically fixed with aldehydes to be imaged under stimulation emission depletion (STED) microscopy.
  • They found that the use of secondary nanobodies reduces probe-induced clusters, still observed using primary/secondary antibody combinations.

Probe-induced clusters of target proteins in aldehyde-fixed cells

  • Conventional fixations times with 4% PFA do not necessarily prevent protein movement. A more efficient fixative such as glutaraldehyde (GLU) could be used, but it generates unwanted autofluorescence and only few affinity molecules find their target epitopes after GLU crosslinking. An alternative is glyoxal, although its implementation is very recent.
  • Using primary/secondary antibody mixes, artefactual clustering formation was observed at most times of incubation with 4% PFA, except 30 minute incubation. Conversely, using secondary nanobodies had no significant change between live, 10 or 30 minutes of fixation with 4%PFA.

 

What I like about this preprint

I like that the authors explored and validated a relatively novel tool. Often, introducing new technologies to research can be a challenge, and thorough validation helps doing this.

 

Open questions

  1. You explored in great detail the comparison to secondary antibodies. Are there any limitations the science community should be aware of upon using nanobodies?
  2. Why in your opinion, is it still not a widely used tool?
  3. Are there any advantages in using nanobodies beyond fluorescence microscopy? Namely in other assays that also depend on immune complex formation?
  4. What are the characteristics of nanobodies in the context of photobleaching and blead-through?

 

References

  1. Sograte-Idrissi S, et al., Circumvention of common labelling artefacts using secondary nanobodies, 2019, bioRxiv,doi:10.1101/818351.

 

Posted on: 3rd April 2020

doi: https://doi.org/10.1242/prelights.18097

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    preLists in the cell biology category:

    FENS 2020

    A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

     



    List by Ana Dorrego-Rivas

    Planar Cell Polarity – PCP

    This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

     



    List by Ana Dorrego-Rivas

    BioMalPar XVI: Biology and Pathology of the Malaria Parasite

    [under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

     



    List by Gautam Dey, Samantha Seah

    1

    Cell Polarity

    Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

     



    List by Yamini Ravichandran

    TAGC 2020

    Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

     



    List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Gautam Dey

    3D Gastruloids

    A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells)

     



    List by Paul Gerald L. Sanchez and Stefano Vianello

    ECFG15 – Fungal biology

    Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

     



    List by Hiral Shah

    ASCB EMBO Annual Meeting 2019

    A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

     



    List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

    EMBL Seeing is Believing – Imaging the Molecular Processes of Life

    Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

     



    List by Gautam Dey

    Autophagy

    Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

     



    List by Sandra Malmgren Hill

    Lung Disease and Regeneration

    This preprint list compiles highlights from the field of lung biology.

     



    List by Rob Hynds

    Cellular metabolism

    A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

     



    List by Pablo Ranea Robles

    BSCB/BSDB Annual Meeting 2019

    Preprints presented at the BSCB/BSDB Annual Meeting 2019

     



    List by Gautam Dey

    MitoList

    This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

     



    List by Sandra Franco Iborra

    Biophysical Society Annual Meeting 2019

    Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

     



    List by Joseph Jose Thottacherry

    ASCB/EMBO Annual Meeting 2018

    This list relates to preprints that were discussed at the recent ASCB conference.

     



    List by Gautam Dey, Amanda Haage
    Close