Shotgun Mutagenesis Epitope Mapping Services
High-resolution epitope mapping with no purified protein needed and >95% success, even for complex proteins and conformational epitopes
Epitope Mapping for Challenging Proteins
Integral Molecular’s proprietary Shotgun Mutagenesis technology is the only high-throughput technique that can reliably map conformational epitopes at single amino acid resolution. And unlike other technologies, like H/D exchange and cryo-EM, our platform does not require purified proteins—only sequences. Because your protein is produced and mapped directly within human cells, it is always folded correctly.
To date, we have mapped over 1,500 epitopes with >95% success. We specialize in:
- Epitopes on GPCRs, transporters, and viral Envelope proteins
- Conformational epitopes
- Epitopes on multi-subunit proteins
- Receptor state-dependent epitopes
Our Epitope Mapping platform can provide important insights into mechanism of action (left, adapted from Fong et al.), as well as key data for patent applications (right, adapted from Deng et al. (PDF)). To learn more about our epitope mapping technology, see Davidson et al.
For those seeking enhanced patent protection for their antibody intellectual property, we also offer Paratope-PLUS comprehensive CDR-Scanning services.
"The epitope mapping that Integral Molecular has done has greatly supported both our mechanistic understanding and the intellectual property claims of our proprietary antibodies."
Epitope Mapping Technology & Project Deliverables
Our high-throughput Shotgun Mutagenesis platform allows us to identify target residues that are critical for binding of antibodies, small molecules, and other biotherapeutics. We introduce hundreds of specified mutations into the target protein and individually express each mutant within live human cells. Then we use high-throughput flow cytometry to precisely measure binding.
Project Deliverables:
Before preparing a mutation library, we will express and validate your wild type target protein and determine the optimal screening conditions. You’ll receive an Assay Setup Report, summarizing the results.
Upon project completion, you’ll receive a Final Report with:
- Fully analyzed data package
- Amino-acid resolution epitope
- Publication-ready figures
Available Epitope Mapping Services
Custom Epitope Mapping Services
Expedited ReadyMap Epitope Mapping Services
Mapping Small Molecule Binding Sites
Featured Case Studies
“Integral Molecular was able to identify critical binding residues for an antibody for which no mapping had been successfully done after years in development. Excellent team, excellent communication, results when promised, results with impact to drive decision making."
Frequently Asked Questions
How does Integral Molecular perform its Epitope Mapping service?
Our epitope mapping approach utilizes Shotgun Mutagenesis, a proprietary platform for high-throughput mutagenesis and cellular expression. We create a comprehensive mutagenesis library by mutating each residue of the full-length target to alanine. Next, each variant is expressed in live human cells within individual wells of a 384-well plate. Using high-throughput flow cytometry, we determine the binding of your antibody to each variant. Mutated positions that no longer support antibody binding are identified as putative epitope residues. Importantly, our platform includes critical controls to ensure that identified residues are part of the epitope rather than artifacts. To date, our Shotgun Mutagenesis Epitope Mapping platform has been used to identify 1,500+ epitopes with a >95% success rate, even for conformational antibodies and highly complex proteins.
What are the benefits of Shotgun Mutagenesis Epitope Mapping compared to other methods, such as peptide mapping, H/D exchange, and cryo-EM?
Shotgun Mutagenesis Epitope Mapping can be used to identify both linear and conformational epitopes at amino-acid resolution, even for complex, multipass membrane proteins. By contrast, peptide mapping determines antibody binding to short peptides, independent of protein tertiary structure, precluding its use for mapping conformational epitopes. Compared to H/D exchange, Shotgun Mutagenesis Epitope Mapping does not require a soluble or purified form of the target protein and identifies amino acid resolution epitopes rather than antibody footprint regions. Cryo-EM requires large quantities of correctly folded and natively processed target protein and does not identify the energetically critical binding residues of epitopes, as Shotgun Mutagenesis does. Shotgun Mutagenesis Epitope Mapping is suited to mapping antibody epitopes on any protein target, including GPCRs, ion channels, and proteins with complex folds, such as viral Envelopes and immuno-oncology targets, without the need for protein purification or cell surface expression.
How do you control for mutations that cause defects in protein folding, expression, and/or trafficking?
Control antibodies are used to validate that the mutated protein exhibits proper trafficking, expression, and folding. Typical control antibodies include commercially available reagent antibodies or the customer’s noncompeting antibody against the same target. If a control is not available, then an epitope-tag (FLAG, V5, HIS6, etc.) can be added to the target protein and used to control for expression and trafficking. In some cases, we use the function of the target protein as a control for proper folding.
What are the deliverables of a Shotgun Mutagenesis Epitope Mapping project?
The deliverables for a project are an Assay Setup Report and a Final Report containing publication-ready figures. The Assay Setup Report is delivered before initiation of mutagenesis and determines the optimal high-throughput antibody screening conditions, which are established using a wild-type protein construct. Upon completion of the mapping project, a Final Report is delivered. This report includes a graphical representation of the data, identities of the critical interacting residues, and a structural representation of the epitope mapped on the target protein. As part of the project, Integral Molecular assists customers in preparing high-quality figures or tables for publications, presentations, and patent/regulatory filings.
What kinds of proteins can be tested?
A Shotgun Mutagenesis Epitope Mapping project can be initiated on any protein that can be expressed in human cells, including soluble proteins and conformationally complex proteins, such as GPCRs, ion channels, transporters, and viral Envelope proteins. Both extracellular and intracellular proteins can be epitope mapped just as easily.
What is the typical turnaround time for a Shotgun Mutagenesis Epitope Mapping project?
Custom epitope mapping projects typically take 3 months from project initiation to delivery of the Final Report. ReadyMap projects from our catalog of available libraries typically take 4–6 weeks. Please contact us to discuss your project. A more accurate timeline will be established after initial discussions.
What is needed to start a Shotgun Mutagenesis Epitope Mapping project?
To begin an epitope mapping project, we require the sequence of the target protein and 500 µg of each antibody that will be mapped. Purified target antigen protein is never required.