Epitopes

What is an epitope?

Antibodies interact with antigens at distinct sites referred to as antigenic determinants or epitopes, thus, an epitope is defined as the antigenic site where the paratope of an antibody interacts with its respective antigen.

Epitopes are classified accordingly as:

  • linear or continuous epitopes, consisting of sequential amino acids in the primary structure of the protein and constitute typically 20 % of epitopes identified
  • conformational or discontinuous epitopes, formed by two to six regions separated in the primary structure, which are brought into proximity in the native protein structure. These epitopes constitute approximately 80 % of epitopes identified 
 
 

Continuous epitopes typically comprise 5-15 amino acids. Although primarily surface accessible, some continuous epitopes are occasionally hidden within the protein structure, thereby forming hidden or cryptic epitopes. These epitopes only become accessible to antibody binding upon denaturation of the native protein structure. Continuous epitopes are typically of flexible nature, although a number of different conformations, such as α-helices and β-sheets may comprise these epitopes as well. Continuous epitopes depending on their secondary structure for reactivity are typically referred to as continuous conformational epitopes or semi-conformational epitopes.

Discontinuous epitopes constitute the majority of epitopes identified and are composed of several peptide fragments. As a consequence, are they often slightly longer than continuous epitopes and typically comprise to 10-25 amino acids. The amino acid residues that constitute these epitopes are not held together by internal chemical bonds, rather they possess a collective identity recognized by antibodies using the entire protein as a scaffold, thus discontinuous epitopes are not intrinsic structures of the antigen, they are only defined through the interaction with their antibodies and typically not possible to isolate as an entity independent from the rest of the antigen. As a consequence, these epitopes are usually defined in structural terms, for example by analyzing antibody-antigen complexes by X-ray crystallography.

The distinction between continuous and discontinuous epitopes is not always clear-cut, as some discontinuous epitopes contain segments of contiguous residues segments that occasionally may be able to bind to antibodies raised against the protein and given the status of a continuous epitope. 

Typical epitope characteristics

Epitopes typically reside on the surface of an antigen, making them highly accessible for antibody interactions, as a consequence, epitopes often protrude from surfaces as turns, loops and hairpins structures. A limited number of epitopes is located within the component core, only becoming accessible upon defolding or fragmentation.   

Epitopes are often enriched in hydrophilic and charged residues, especially Arg, Asp and Glu, and depleted of aliphatic hydrophobic amino acids. Moreover, specific amino acids such as Tyr and Trp have been found to be overrepresented in epitopes due to their capability to form a multitude of interactions. Amino acids such as Pro and Gly are often found in epitopes as well, as these amino acids often are represented in turns and flexible regions. Besides individual amino acid preferences, specific amino acid pairs have been observed in epitopes, suggesting that some amino acid pairs work cooperatively in mediating antibody binding.

Typical characteristics: 

  • Epitope Length: 5-25 amino acids
  • Estimated size: 600-1000 Å2
  • Surface area shape: Flat rugged area, flat oblong (ellipse) shaped area
  • Segmentation: > 60 % of epitope residues exists in linear stretches of three or more residues. 85 % of epitopes has a linear stretch of five or more residues
  • Structure: loops, turns, pins, flexible regions, occasionally strands and helices
  • Localization: Surface exposed, protrude from the antigen surface (unless hidden in the primary structure and only exposed upon defolding)
  • Amino acid composition: Enriched in charged and polar residues, often depleted of hydrophobic amino acids
  • Amino acid cooperativeness: pairs of Tyr:Tyr, Cys:Pro, Asn:Tyr, Asp:Pro, Thr:Tyr, Arg:Tyr, Asn:Tyr, His:Tyr and His:Met are more frequently observed

How are epitopes identified?

Epitope mapping determines the antigenic binding site. Several approaches for epitope mapping exist such as:

  • peptide scanning and peptide libraries
  • phage display libraries “bio-panning”
  • X-ray crystallography
  • Mass spectrometry
 
For several decades synthetic peptides have been employed for epitope mapping. The advantage of using synthetic peptides is that peptides are easily synthesized and that a variety of post-translational and chemically modified amino acids can be introduced. Peptide scanning is often used, where antibody reactivity is analysed to overlapping peptides of varying length, which may cover a complete protein or region of interest. Even though this method primarily is limited to determine continuous epitopes, pepscans can also identify components of discontinuous epitopes in the event that two distant peptides each contain sufficient structural elements that allow them to bind the antibody separately, indicating that these peptides contribute to the intact epitope. 

More sophisticated methods, such as combinatorial peptide libraries, have successfully been used for epitope mapping as well. Here, heterogeneous combinatorial peptide libraries are synthesized and then screened for antibody reactivity followed by isolation and sequencing of reactive peptides. Peptide libraries in combination with phage display have been applied for epitope mapping as well. This approach, referred to as “bio-panning”, employs antibodies immobilized on a solid surface, which react with a comprehensive library of random peptides displayed on the phage surface. Peptides that resemble the specific epitope are recognized by the antibody, whereas the remaining phage-peptide complexes are washed away.

The golden approach for epitope identification is X-ray crystallography of antibody-antigen complexes, which constitute a more sophisticated technique of epitope mapping. In this approach highly purified antigen-antibody crystals are interrogated with x-rays and due to the highly structured repeated nature of the crystal unit, diffraction patterns are generated, which are applied to determine the electron densities of the amino acids composing the antigens and its bound antibody. This method provides atomic resolution of the epitope compared to the remaining methods, which only determine the specific amino acid sequence. Although very efficient, this method requires a large amount of highly purified antibody and antigen to obtain an appropriate crystal for this level of detailed analysis.

Mass spectrometry approaches such as hydrogen-deuterium-exchange mass spectrometry may be used for epitope mapping of conformational epitopes. This approach measures the exposure levels of amide hydrogen atoms in an antibody-antigen complex. Initially, the exposed hydrogen atoms are exchanged with deuterium, except from hydrogen atoms in the antibody-antigen complex. By examining the protein or peptide using bottom-up mass spectrometry and comparing them with the amino acid sequence, it is possible to determine which amino acids constitute the antibody-antigen interface.