Antibody Development - Structural Characterization of Antibodies with Cryo-EM

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July 7, 2022

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The advantage of using Transmission Electron Microscopy (TEM)

Due to their intrinsic flexibility, antibodies can be difficult to study by X-ray crystallography and are outside of the acceptable mass range for NMR studies. Transmission electron microscopy (TEM) imaging of either negative stained (NS) or vitrified samples is much more tolerant of both antibody flexibility as well as disordered protein termini. These approaches are well suited for structural characterization of antibodies, nanobodies and their complexes. Even low-resolution negative stain images can provide a wealth of information, particularly when combined with 2D classification. Requiring very little sample, this technique can be useful in providing a quick assessment of the epitope/paratope relationship between novel high affinity mAbs (identified during an antibody campaign) and the target antigen.

High Resolution Epitope Mapping by Cryo-EM

As described in more detail in a prior post, single particle analysis workflows enable rapid, high resolution epitope mapping by cryo-EM. The higher working sample concentrations relative to negative stain workflows and similar tolerance for flexibility make cryo-EM ideally suited to return resolutions at the Ab-Ag interface in which clear side chain density can be observed. Full length mAbs and multiplex antibody binding can also be readily accommodated when generation of Fab fragments would be challenging or time-consuming.

Antibodies in Vaccine Development

In the case of highly antigenically variable pathogens, isolating pathogen functional antibodies, studying the interaction of these antibodies with their targets, and designing vaccine candidates can be a powerful alternative approach to traditional vaccines (e.g., 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 with activity. High resolution structures of neutralizing antibody fragments (FABs) bound to targets can also help to address questions of cross-specificity and potency.

Cryo-EM image of negative stain screening | NanoImaging Services
Example cryo-EM image of Negative Stain transmission electron microscopy analysis.

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  • 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.
  • 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.
  • Barnes, C. O. (2020). Structures of Natively-Glycosylated HIV-1 Envelope Trimers Define Antibody-Mediated Neutralization of HIV-1. Biophysical Journal, 118(3), 3a.

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Structural characterization of antibodies and interactions with antigens has many applications in antibody development and biologics discovery | Nano Imaging

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