Technical Guide: Epitope Mapping Techniques

Whether you’re developing a novel vaccine or designing antibody therapeutics, chances are you have used several epitope mapping techniques to learn more about how your antibodies are behaving. However, this crucial step in many research applications often comes with complications when working with challenging proteins, such as those with non-linear binding regions. An ELISA has historically been the go-to method for epitope mapping but frequently falls short due to its limitations, such as a lack of direct binding affinity measurements. Fortunately, as the field of structural biology advances, solutions to these limitations become increasingly available to researchers.

Nicoya’s mission is to improve human life by helping scientists succeed. To help you to get the best data from your epitope mapping studies, we examine the advantages and limitations of traditional epitope mapping techniques. Furthermore, we discuss how surface plasmon resonance (SPR) can get you the data needed to expedite your antibody therapeutics development.

Epitope Mapping in a Nutshell

Before diving into epitope mapping techniques, let’s briefly address what epitope mapping is and why it’s so important. An epitope is the region of an antigen that participates in binding with an antibody. Therefore, epitope mapping is the experimental process of locating the epitope of a particular antibody on an antigen surface (Rockberg and Nilvebrant, 2018). A linear (or continuous) epitope is a peptide chain in an antigen that can usually be mimicked by peptide sequences, while a non-linear (discontinuous) epitope consists of distant amino acids that are brought together when the protein is in its folded state (Rockberg and Nilvebrant, 2018). In both cases, a knowledge of each binding partner’s structure, function and their binding kinetics are all essential in sufficiently mapping the interaction between an antigen epitope and its antibody. Epitope mapping is crucial when developing antibody therapeutics, conducting vaccine candidate identification and even when performing disease diagnosis (Rockberg and Nilvebrant, 2018; Karlsson, 2013)

Different Epitope Mapping Techniques

Here we highlight several common techniques used for epitope mapping. While this is by no means an exhaustive list of epitope mapping techniques, we cover several different types of data typically collected when epitope mapping.

In-silico (or computational) techniques are commonly used for preliminary epitope binding investigation. Most efforts in this area take advantage of existing protein structural databases to predict the binding event between two proteins. Computational techniques are a fast and cost-effective way to predict a large number of binding events but should be backed by experimental data for credibility.

is a peptide-based approach for epitope mapping. On a high level, a series of overlapping peptides are screened for antibody binding. This can be as simple as a single isolated segment on an antigen and can also be a large-scale microarray of synthesized peptides to be used for high-throughput screening (Rockberg and Nilvebrant, 2018). An ELISA can investigate large scale of binding events at a relatively low cost but is limited in the information that is provided. A colour change can indicate yes/no binding, analyte concentration can be quantified, and EC50 can be calculated, but kinetics (such as binding affinity) cannot be directly measured with this technique. ELISAs can also be less reliable when attempting to identify complicated epitope regions (Rockberg and Nilvebrant, 2018). This being said, there are many published examples of ELISAs and surface plasmon resonance being used in tandem to provide highly credible epitope mapping data (Thomsen and Gurevich, 2018; Bhandari, Chen, Hamal, and Bridgman, 2019).

X-ray crystallography is an approach that can provide highly detailed structural information about the studied epitope. When applicable, X-ray crystallography is an excellent technique to use in tandem with surface plasmon resonance in order to obtain both structural and binding kinetics data. However, this strategy requires pure, crystallized proteins which can be incredibly challenging and time consuming to produce (Rockberg and Nilvebrant, 2018). Other structural techniques exist as well, such as Nuclear magnetic resonance (NMR) and mass-spectroscopy. However, these are often limited to small, simple protein structures (Rockberg and Nilvebrant, 2018).

Epitope Mapping using Surface Plasmon Resonance

Surface Plasmon Resonance (SPR) is a quantitative technique used to measure the binding kinetics between two interacting macromolecules. SPR has become the preferred epitope mapping technique as it provides quantitative binding kinetics data about an epitope and its antibody in real-time and without the use of labels (Karlsson, 2013; Thomsen and Gurevich, 2018; Bhandari et al., 2019).

The real-time measurements provided by SPR allow each step in the mapping experiment to be followed (Karlsson, 2013), providing quantitative data for the on-rate, off-rate, and overall affinity of the interaction between an antibody and its epitope. SPR is also advantageous for its high sensitivity compared to a technique such as ELISA (Bhandari et al., 2019), making it a versatile technique. However, SPR works best with purified protein samples and does not provide structural techniques. For these reasons, SPR is often used to characterize the best candidates from a higher-throughput method (such as ELISA) and can be used in tandem with a structural technique to provide a highly detailed mapping of an antibody interaction (Thomsen and Gurevich, 2018; Bhandari et al., 2019).

Measure Epitope Binding Quantitatively with OpenSPR™

We’ve spoken with many researchers who shy away from incorporating SPR into their epitope mapping studies due to its high cost and difficulty to use. Nicoya helps scientists obtain crucial binding data by providing them with accessible surface plasmon resonance instrumentation. The OpenSPR was designed to be user-friendly and is priced so that researchers can study epitope binding on their own lab bench. Recently, Dr. Stasevich and Dr. Simonds both used the power of OpenSPR™ to publish quantitative binding kinetics data in Nature Communications and Communications biology!

Accelerate your antibody therapeutics development with OpenSPR™



  1. Rockberg, J., Nilvebrant, J. (2018). Epitope Mapping Protocols (3rd ed., Vol. 1785). Doi: 10.1007/978-1-4939-7841-0_1
  2. Karlsson, R. (2013). Surface Plasmon Resonance in Binding Site, Kinetic, and Concentration Analyses. In Wild, D. (Ed.) The Immunoassay Handbook (Fourth Edition) (pp. 209-221). Doi: 10.1016/B978-0-08-097037-0.00015-4
  3. Thomsen, L., Gurevich, L. (2018). A surface plasmon resonance assay for characterisation and epitope mapping of anti‐GLP‐1 antibodies. J Mol Recognit. 31(8). Doi: 10.1002/jmr.27114.       Bhandari, D., Chen, F., Hamal, S., Bridgman, R. (2019). Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor. Antibodies, 8(1). Doi: 10.3390/antib8010022
  4. Bhandari, D., Chen, F., Hamal, S., Bridgman, R. (2019). Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor. Antibodies, 8(1). Doi: 10.3390/antib8010022