Epitope Mapping

Scientific rendering of 3D protein structure, structural biology | Nano Imaging
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To develop antibody therapeutics and vaccines, researchers need to thoroughly understand how antibodies interact with their target antigens. Epitope mapping, a process that identifies the precise region where antibodies bind to antigens, is essential for unraveling immune responses and designing targeted therapeutic antibodies. By deeply understanding the structure of the epitope, we can uncover atomic interactions and conformational changes, and their impact on the mechanism of action (MOA) of these therapeutic agents.

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool, providing unparalleled resolution and structural details for epitope mapping. By combining cryo-EM imaging with single particle analysis workflows, scientists can confidently solve structures, contributing to our understanding of complex biological systems.

Negative stain transmission electron microscopy (TEM) workflows offer additional analysis of antibody antigen complexes for epitope mapping. This technique involves staining the sample with heavy metals to enhance contrast for visual analysis. However, negative stain TEM provides only nanometer-range resolution, which is lower than typical cryo-EM structures. Additionally, the heavy metal stain can often introduce structural artifacts by dehydrating the sample. Despite these limitations, negative stain TEM still plays a vital role in validating the integrity of antigen-antibody complexes and identifying the specific region of the antigen recognized by antibodies. Moreover, it often yields faster results than cryo-EM imaging.

By combining 2D classifications gained through negative stain TEM imaging and high resolution 3D models and maps from cryo-EM imaging on ThermoFisher Krios microscopes, researchers gain a wealth of information about their antibody-antigen complexes. This multi-faceted approach provides a comprehensive understanding of the intricate structure of these critical biological interactions.

Learn More About Antibody-Antigen Complexes:

  • Antibody-Antigen Complexes
  • Antibody-Antigen Complexes

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Frequently Asked Questions

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Why should I use cryo-EM for Epitope Mapping instead of Hydrogen-Deuterium Exchange (HDX)?

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HDX can offer faster initial results compared to cryo-EM, particularly when preliminary work involving vitrification conditions and grid screening needs to be done. However, once these conditions are established, the turnaround times for both techniques can become similar.

Cryo-EM can still be considered a “fast” technique, as we are able to provide results in 2 weeks or less from sample receipt.

It's important to note that HDX provides "low-resolution" details, primarily revealing the general area of interactions. In contrast, cryo-EM has the capability to provide specific atomic interactions through a ~3.5 Angstrom resolution map.

When it comes to FABs binding to the same antigen simultaneously, the more, the better. We can get epitopes for all FABs in one experiment. Additionally, the added mass will make the particles easier to pick and to align, thus increasing the possibility of achieving higher resolution. Learn more about the differences between techniques here.

Does NIS have any experience with Epitope Mapping?

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Yes, in this paper published in Science, you can read more about NIS’s cryo-EM work done to solve an epitope structure. In this project, NIS was able to use low resolution maps to identify the FAB binding areas and provide expert suggestions for the inactivation mechanism, and higher resolution maps to fully define the epitopes. We were able to overcome preferred orientation by using different grid types and data collection strategies.

Pavel A. Nikitin et al. ,IMM-BCP-01, a patient-derived anti–SARS-CoV-2 antibody cocktail, is active across variants of concern including Omicron BA.1 and BA.2.Sci. Immunol.7,eabl9943(2022).DOI:10.1126/sciimmunol.abl9943

What sample requirements are there to gain a high resolution Epitope Map?

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At this time, NIS is able to work with soluble antigens/FAB complexes with a minimum size of 100 kDa. Epitope mapping can be applied to analyze nanobody or VHH complexes if the minimum size requirements are met. The epitope should not be on a loop or other flexible parts of the antigen, and the complex should not show extreme behaviors (e.g., severe preferred orientation).

How long will Epitope Mapping with NIS take?

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You can expect to receive a fully refined, 3D map within 2 weeks of sample receipt, assuming your sample meets certain criteria, such as being well-behaved in ice or the epitope not being located on a flexible loop. We understand the challenges associated with samples behaving poorly in ice, and we have developed strategies to overcome these issues. To learn more about our approach to addressing such challenges, we invite you to read our blog post here.

How much does Epitope Mapping with NIS cost?

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The price to obtain a high-resolution structure, which includes the Initial Starter Pack and additional data collection for mapping, is $35,000. Optional model building is available for $8,800.

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