Epitope mapping services

Our epitope mapping approach utilizes Shotgun Mutagenesis, a proprietary platform for high-throughput mutagenesis. Essentially, we take the sequence of your protein and synthesize variants that have each amino acid individually mutated to alanine (or serine if it is already alanine). Each of these variants, which encode the full-length protein with only a single alanine mutation, is then arrayed into individual wells of a 384-well plate and expressed in human cells. Using high-throughput flow cytometry we can then determine the binding of your antibody to each individual protein variant and control for defects in expression, trafficking and folding. Accordingly, this method identifies the amino acids critical for the binding of your antibody to the target protein. The ability to map antibody epitopes in the context of full-length protein using an alanine-scanning approach allows us to rapidly identify linear and conformational epitopes at an amino acid resolution with a 95% success rate.

Shotgun Mutagenesis Epitope Mapping works by individually mutating each amino acid of the target protein in the context of the full-length protein, expressing these variants in human cells and measuring binding of your antibody to each variant. Using this method, both linear and conformational epitopes can be identified at amino acid resolution. This is in contrast to peptide mapping, which determines binding of an antibody 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 form of the target protein and relies on easily interpretable flow cytometry data, simplifying analysis and interpretation. Ultimately, Shotgun Mutagenesis Epitope Mapping is ideally suited to mapping antibody epitopes on any protein target including multipass membrane proteins such as GPCRs and ion channels, and proteins with complex folds such as immuno-oncology targets.

Control antibodies are used to validate that the mutated protein exhibits proper trafficking, expression, and folding. Typically, a commercially available reagent antibody, or a non-competing antibody generated by the customer against the same target will be used as a control. If no control antibodies exist for a given protein, an epitope-tag (FLAG, V5, HIS6, etc) can be added to the target protein and used to as a control for expression and trafficking.

The first deliverable is an Assay Setup report, showing optimal high-throughput screening conditions for each antibody using a wild-type protein construct prior to initiation of mutagenesis. Upon completion of the project, a Final Report is compiled that includes a graphical representation of the data, identification of critical interacting residues, and the epitope mapped onto the target protein structure (if available). If required, Integral Molecular is happy to assist customers in preparing epitope mapping data for publication, presentation or patent/regulatory filings.

Epitope mapping can be done on any protein that can be expressed in mammalian cells, including soluble proteins and conformationally complex proteins such as GPCRs, ion channels, transporters and viral envelope proteins.

Custom epitope mapping projects typically take 3-4 months. Please contact us if you have tight filing/regulatory deadlines.

We require the protein sequence and 500 µg of each antibody that is being mapped.

Any cell-based assay that is adapted to microplate format can be used for Shotgun Mutagenesis. Prior projects have commonly included calcium-flux, viral infection, and flow-cytometry- based immunofluorescence assays.