Traditional methods for producing binding molecules for assay development – typically monoclonal or polyclonal antibodies – rely on the immune system of the host animal reacting to the target molecule. This usually results in a significant number of antibodies produced against the target, and the exact binding locations will initially be unknown until a process called epitope mapping is employed. Mapping surface epitopes typically involves laborious techniques such as x-ray crystallography and oligopeptide scanning, which are time-consuming, expensive, and success is not guaranteed.
Our new Epitope Discovery process provides a rapid and cost-effective method for identifying relevant and accessible epitopes in advance of the development of the binding molecules leading to a much more targeted approach. Ideally nanoMIPs™ would be developed, but the information generated could underpin development of other types of binding molecules. We believe that this enables the customer to “start with the end in mind” with their binding molecule needs.
What are nanoMIPs™?
The production of molecularly imprinted polymers by solid-phase synthesis – a process patented by MIP Diagnostics Ltd. – leads to the production of nanoMIPs™. They are synthesized by immobilizing a template (small molecule, peptide etc.) onto a solid phase. Monomers and cross-linker are then added, controlled polymerization is initiated and, ultimately, nanoMIPs™ with high affinity for the template are eluted. The affinity and selectivity of the nanoMIPs™ is typically comparable to that of a monoclonal antibody, but with incredible stability – chemical and thermal – and robustness.
How does the Epitope Discovery process work?
The Epitope Discovery process relies on bringing the target protein into contact with a selection of monomers in solution and subsequently polymerizing to create a protein-polymer complex. The protein is then digested to leave a peptide-polymer complex in which the bound epitope(s) are ‘captured’ within the polymer pockets. The peptide-polymer complexes are then isolated, the peptides removed, and later sequenced using mass spectrometry. The peptide sequence(s) identified correspond to potential binding sites for a binding partner such as an antibody, aptamer, affimer or nanoMIP™. The total process can be completed for each protein target within 1 week.
We have performed the Epitope Discovery process on a number of protein targets, including human galectin-1, gliadin and acetylcholinesterase. In each instance several epitopes corresponding to known antibody epitopes were identified, verifying that the process works correctly. Furthermore, a number of epitopes without any corresponding commercial antibody ligands were also identified, offering opportunity for new binding molecule development. These epitopes were confirmed to be accessible (i.e. on the surface of the protein) and not artefacts due to the digestion process being incomplete.
We have demonstrated that this new method for epitope discovery has identified previously unknown surface epitopes on target biomarkers, which could then be utilized to create an anti-peptide antibody, aptamer, affimer or molecularly imprinted polymer for diagnostic applications. Other potential applications include the preliminary screening of therapeutic antibodies and to support vaccine design.