Nuclear Magnetic Resonance Analysis based CreMap™ Epitope Mapping Service

Background Key Features Published Data FAQ Resources

Creative Biolabs has extensive experience in epitope mapping by nuclear magnetic resonance (NMR) analysis with the high degree of sophistication and technical expertise. Technicians in Creative Biolabs are always here to support clients’ projects and try to find the best solution for each project.

Background

The antibody is the protein capable of recognizing antigen with high specificity and affinity. The antigen is recognized by atomic interactions between up to six highly variable loops called epitopes. Identification of epitopes is of great importance for the utilization of antibody, especially in the discovery and development of new therapeutics and vaccines design. A variety of methods for epitope mapping has been established up to now.

Nuclear magnetic resonance is particularly suited for determining the boundaries of epitopes recognized by antibodies. NMR analysis for identifying epitopes is based on the different mobility between the amino acid residues of the antigen that interact tightly with the antibody and peripheral residues that do not directly interact with the antibody. Epitopes interacting with the antibody undergoes significant changes in chemical shift. A detailed structural analysis for antigen-antibody complex is provided. Therefore, the nature of how the epitope is recognized could be accurately explained.

Basically, the NMR-based method for the epitope mapping is derived from the comparison between measurements of the spectrum. One is called HOmonuclear Hartmann-Hahn (HOHAHA), the other is called Rotating frame Overhause Effect Spectroscoty (ROSEY). The HOHAHA spectrum reveals the information about the dynamic states of the protons derived from the epitope where there is a chemical bond. ROESY, also known as CAMELOSIN (Cross-relaxation Appropriate for Minimolecules Emulatesd by Locked SPINs) is useful for determining which signals arise from protons that are close to each other in space. ROESY still could be used even if the protons are not literally bonded. Comparison of spectra between the tightly interacting and indirect residues enables the assignment of the mobile segments of the antigen.

Fig.1 The diagram of the workflow for epitope mapping based on NMR analysis.

Key Features

NMR could be applied to study antibodies in complex exhibiting very weak affinity, which is difficult to study by other mapping methods. The idea that NMR can be used for epitope mapping is that NMR records a spectrum of the free, labeled antigen plus an unlabeled antibody, and records the spectrum of the antigen-antibody complex. Comparison between these spectrums unravels the epitopes.
NMR is very sensitive, especially for the large antigen-antibody complex and provides a complete description of the residue interacting with the antibody, which may not be obtained by other epitope mapping methods.
NMR mapping provides the detailed information for validating the binding footprint of one molecule over another. Results obtained from NMR are quite accurate and as detailed as X-ray crystallization.

NMR has greatly affected the way scientists are characterizing specificity and the affinity of a variety of molecules. Creative Biolabs provides a robust NMR platform regarding epitope mapping. We perform complete toolset comprising trusted solutions for every step we take. We now provide the optimized, validated platform in order to minimize and eliminate the trial-and-error phase. If you are meeting up with difficulties in determining epitopes, please feel free to contact us by sending E-mail. A formal feedback will be sent back as soon as possible. We are always more than ready to reach out.

Other optional CreMap™ B cell epitope mapping services:

Published Data

Unique Epitope–Antibody Interactions Defined by NMR Combined with Yeast Surface Display

Fig. 2 SPR binding studies of the CA-IX mAbs and F(Ab)s to immobilized ePG-1 polypeptide. (Feng Ni, 2023)

The article details the utilization of high-resolution nuclear magnetic resonance (NMR) and yeast surface display (YSD) for characterizing unique epitope-antibody interactions within the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX (CA-IX). The research showcases the identification of binding interactions of two novel antibodies, which are specific to this domain. Through titration NMR spectroscopy and complementary YSD techniques, the study not only identified the binding sites of these antibodies on the CA-IX but also elucidated the complex structural dynamics involved. Epitope Mapping by NMR allowed for a precise determination of how the antibodies interact with specific sequences within the disordered region of CA-IX, highlighting their binding to unique sequences and how these interactions impact the structural configuration of the region. This approach is vital for developing targeted therapies, as it enables the design of antibody-based treatments that can selectively interact with specific parts of a protein involved in disease processes, in this case, potentially offering new avenues for cancer therapy by targeting tumor cell environments.

References

Abbott, W.M., M.M. Damschroder, and D.C. Lowe, Current approaches to fine mapping of antigen-antibody interactions. Immunology, 2014. 142(4): p. 526-35.
Kustanovich, I. and A. Zvi, Epitope mapping antibody-antigen complexes by nuclear magnetic resonance spectroscopy. Methods Mol Biol, 1996. 66: p. 25-37.
Bardelli, M., et al., Epitope mapping by solution NMR spectroscopy. J Mol Recognit, 2015. 28(6): p. 393-400.
Blech, M., et al., One target-two different binding modes: structural insights into gevokizumab and canakinumab interactions to interleukin-1beta. J Mol Biol, 2013. 425(1): p. 94-111.
Ni, Feng, et al. "Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display." Mabs. Vol. 15. No. 1. Taylor & Francis, 2023.

FAQ

What is epitope mapping by NMR analysis?
Epitope mapping by NMR analysis involves identifying the regions of an antigen that interact with antibodies. This technique uses the sensitivity of NMR spectroscopy to monitor changes in the antigen's nuclear magnetic environment upon antibody binding, revealing the structure and dynamics of the epitope.
How does NMR contribute to understanding antigen-antibody interactions?
NMR spectroscopy provides detailed information about the physical interactions and conformational changes occurring in both the antigen and antibody upon binding. It offers a unique insight into the dynamics of the binding process, helping to elucidate how antibodies recognize and bind to specific epitopes on antigens.
What are the advantages of using NMR for epitope mapping?
NMR allows for the precise determination of the boundaries and structure of epitopes in their native state without the need for crystallization. It can also reveal dynamic properties of the protein interaction and provide information on the conformational flexibility of the antigen, which is often crucial for binding specificity and affinity.
Can NMR analysis map epitopes on large proteins?
NMR can map epitopes on large proteins, though it is more challenging. Advances in isotope labeling and high-field NMR techniques have improved the ability to study larger proteins and complexes. Techniques like TROSY (transverse relaxation-optimized spectroscopy) have been particularly helpful in extending the size limits of proteins amenable to NMR analysis.
How does NMR compare to other epitope mapping techniques?
NMR is complementary to techniques like X-ray crystallography and cryo-electron microscopy. Unlike these methods, NMR does not require the antigen-antibody complex to be crystallized, allowing the study of epitopes in a solution close to physiological conditions. This can provide more realistic insights into the nature of epitope-antibody interactions.
What are the limitations of using NMR for epitope mapping?
NMR spectroscopy requires relatively large amounts of protein, and the complexity of spectra increases with the size of the protein, which can make analysis challenging. Furthermore, the need for isotopic labeling makes this method more expensive and technically demanding than some alternative approaches.
How long does epitope mapping by NMR typically take?
The duration of an epitope mapping project by NMR can vary significantly depending on the complexity of the antigen, the quality of the samples, and the resolution required. Preparing isotopically labeled proteins and optimizing experimental conditions can take weeks to months, with actual NMR data acquisition and analysis taking additional time.
Are there specific types of antigens or antibodies that are better suited for NMR analysis?
NMR is particularly well-suited for mapping epitopes on small to medium-sized proteins or on larger proteins that can be effectively fragmented or truncated. Antibodies or fragments thereof (like Fab or single-domain antibodies) that remain functional and stable under the conditions used in NMR experiments are ideal.