Unraveling the Molecular Basis of Antibody-Antigen Complexes with Cryo-EM and Negative Stain TEM Imaging
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.
Cryo-EM: A Revolution in Structural Biology
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 TEM: A Vital Complementary Imaging Technique
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.
Integrative Imaging: A Comprehensive Look at Antibody-Antigen Interactions
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.
Within 2 Weeks, Researchers Can Obtain Crucial Information About Antibody-Antigen Binding.
Traditionally, scientists have regarded X-ray crystallography (XRC) as the gold standard approach for epitope mapping. While XRC has successfully provided atomic-level details of antibody-antigen complexes, it often encounters significant challenges when dealing with flexible antigens, large complexes, low concentrations, or difficult-to-crystallize samples. This is where cryo-EM becomes important, offering a cutting-edge solution to overcome these limitations.
At NIS, we offer an Epitope Mapping service that uses cryo-EM imaging and data collection to deliver a comprehensive understanding of your antibody-antigen complex. This comprehensive approach enables us to deeply understand the molecular interactions between antibodies and antigens. For suitable samples, we can provide a a 3D reconstruction to a resolution at the epitope/paratope interface suitable for tracing the chains and assigning 75% of the side chains, within 2 weeks of sample receipt. Gain an in-depth understanding of NIS's rapid cryo-EM Epitope Mapping service in our blog here.
An ideal sample is represented by a antigen:FAB complex with a minimum size of 100 kDa, and the epitope should not be located on a loop or other flexible segments of the antigen, and the complex should not exhibit extreme behavior such as severe preferred orientation. If your sample does not fall within these guidelines, we will work closely with you to determine next steps for solving your antibody-antigen complex.
The use of cryo-EM and negative stain TEM imaging for epitope mapping opens up exciting possibilities for unraveling antibody-antigen interactions. These state-of-the-art techniques empower researchers to gain valuable insights into the functional implications of these vital molecular interactions.
Learn more about different techniques for epitope mapping:
Contact us to begin mapping your antibody-antigen complexes.
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Frequently Asked Questions
What are the deliverables for NIS's rapid Epitope Mapping service?
Does NIS have any experience with Epitope Mapping?
How long will Epitope Mapping with NIS take?
How much does Epitope Mapping with NIS cost?
Why should I use cryo-EM for Epitope Mapping instead of Hydrogen-Deuterium Exchange (HDX)?
Why should I use cryo-EM for Epitope Mapping instead of X-ray Crystallography (XRC)?
What are obstacles you see in epitope mapping with cryo-EM?
What sample requirements are there to gain a high resolution Epitope Map?
Is a full length mAb required? Can I use a Fab?
Have you been able to work with unstructured antigens, such as tau or a-synuclein?
Is glycosylation a problem for cryo-EM and antibody complexes? Is it necessary for cryo-EM reconstructions?
Is there a limit to the number of antibodies bound to a single antigen that can be visualized at the same time?
Does NIS provide antibody purification or digestion services?
