Antibody Development - Structural Characterization of Antibodies

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Structural characterization of antibodies and their interactions with antigens has many applications in antibody development and biologics discovery, including identification of paratopes/epitopes, correlation between structures and activity, identification of cross-specificity, and monoclonal antibody (MAB) optimization. Structural data can also be used in IP protection, and in the formulation and delivery space.


The advantage of using TEM

Owing to their intrinsic flexibility, however, antibodies can be difficult to study structurally by X-ray crystallography and are outside of the acceptable mass range for NMR studies. Transmission electron microscopy imaging of either negative stained or vitrified samples is much more tolerant of flexibility and/or disordered protein termini, making the approach well suited for structural characterization of antibodies, nanobodies and their complexes. Even low-resolution negative stain  images can provide a wealth of information not otherwise achievable, particularly when combined with 2D classification


Related applications

Epitope mapping

Negative stain, especially if coupled with 2D class averaging, is a quick way to assess (at low resolution) the epitope/paratope relationship between novel MABs identified during an antibody campaign and the target antigen. The technique also requires very little sample. Once antibodies of interest have been identified, the antigen-antibody fragment complex can be used for high resolution structure determination using single particle analysis.


Antibodies in Vaccine Development                

In the case of highly antigenically variable pathogens, isolating pathogen functional antibodies, studying their interaction with their targets and then designing vaccine candidates could be a useful alternative to the traditional vaccines (killed or attenuated pathogens or protein subunits). Negative stain TEM can be used to quickly characterize polyclonal antibodies, identify the different epitopes and correlate structures and activity. High resolution structures of neutralizing antibody fragments (FABs) bound to targets may provide useful information with regard to cross-specificity and potency. 

Citations

  • Pan, J., Peng, H., Chen, B., & Harrison, S. C. (2020). Cryo-EM Structure of Full-length HIV-1 Env Bound with the Fab of Antibody PG16. Journal of Molecular Biology, 432(4), 1158–1168. https://doi.org/10.1016/j.jmb.2019.11.028 ​
  • Campbell, M. G., Cormier, A., Ito, S., Seed, R. I., Bondesson, A. J., Lou, J., Marks, J. D., Baron, J. L., Cheng, Y., & Nishimura, S. L. (2020). Cryo-EM Reveals Integrin-Mediated TGF-β Activation without Release from Latent TGF-β. Cell, 180(3), 490-501.e16. https://doi.org/10.1016/j.cell.2019.12.030 ​
  • Barnes, C. O. (2020). Structures of Natively-Glycosylated HIV-1 Envelope Trimers Define Antibody-Mediated Neutralization of HIV-1. Biophysical Journal, 118(3), 3a. https://doi.org/10.1016/j.bpj.2019.11.211

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