Overview of Epitope Mapping Technologies



Epitope mapping has become one of the key elements of both vaccine and drug development.  Epitope mapping is the process of identifying the binding site of a protein to its corresponding antigen. FDA and EMEA guidelines requires molecular analysis of the interaction site between a drug and its target for the regulatory filing. In addition, a better understanding of the interaction site is useful to develop more potent drugs and to protect products with patents.

More detailed information on the CovalX techniques available can be seen here.

Linear vs Conformational

Epitopes of can be defined by two main categories, linear or conformational. A linear (or sequential) epitope is composed of a linear stretch of amino acids in the sequence which have no 3-dimensional structure. A conformational epitope is one that requires tertiary folding to create the proper binding region.  Conformational epitopes are most often found in regions that have a break in the sequence of amino acids binding (discontinuous) requiring the folded structure be present to bring the regions into proximity. Conformational mapping techniques can be used for identifying either linear or conformational epitopes, however linear techniques can not map conformational epitopes.  Generally conformational epitope mapping is more complex, time consuming and require more material then linear epitope mapping.  There are various techniques for mapping linear epitopes available, some of which provide "conformational-like" data through mimicking structure.  These linear techniques will not be discussed here in detail as the vast majority of epitopes are conformational.

 
Linear vs Conformational Epitopes

Comparison of Conformational Techniques

Depending on the "resolution" of the technique utilized the region identified can be individual amino acids, small groups of amino acids or regions of the protein.  Generally, higher resolution techniques are more complex, time consuming and require more material/time.

X-ray co-crystallography

The "gold-standard" for epitope mapping is X-ray co-crystallography.  If successful, this technique can provide single amino acid resolution with a high degree of confidence, although flexible regions can be missed.  However, this technique is not always possible as crystal generation from a protein complex tends to be difficult and in many cases impossible.  In cases where crystal formation is difficult then smaller protein segments or modified proteins may be mapped.  Additionally, x-ray analysis requires large costly equipment and large volumes of high purity material.

Peptide Scanning

In this technique an array of overlapping peptides is generated from the antigen.  These peptides are then analyzed to see if they disrupt the formation of a complex between the antibody and antigen.  This is one of the most common techniques due to its relatively low cost and ability to analyze large numbers of antibodies quickly.  However, as this technique relies upon peptides it is not possible to reliably map conformational epitopes.

Mutagenesis

With this technique specific amino acid residues of the antigen are mutated or changed (often to alanine) and the presence of the complex is detected (often with fluorescence).  This technique provides high amino acid resolution of the epitope; however, generating the mutations is labor-intensive and slow.  In order to optimize the process many plasmid clones can be generated in a library format and using computers to perform statistical calculations of the database.  This automated mutagenesis process is termed "shotgun mutagenesis".

Crosslinking coupled Mass Spectrometry

This technique begins by binding the antibody and the antigen with a mass labeled chemical crosslinker. Next the presence of the complex is confirmed using high mass MALDI detection. Because after crosslinking chemistry the Ab/Ag complex is extremely stable, many various enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high resolution mass spectrometry and MS/MS techniques.  Identification of the crosslinked peptides is determined using mass tag linked to the cross-linking reagents.  After MS/MS fragmentation and data analysis using specific interaction softwares, both epitope and paratope are determined in the same experiment.  Because of the highly sensitivity and accuracy of mass spectrometry detection, very little quantity of material is required.  CovalX is offering this technique through our contract research services facility, more information can be seen here.

Hydrogen Deuterium Exchange (HDX)

This technique measures the availability of hydrogen molecules in the backbone of a protein structure.  During analysis both the unbound antigen and the bound antibody-antigen complex are incubated in deuterated water in order to exchange any hydrogens from exposed amino acids of the proteins backbone.  By comparing the unbound antigen with the bound antibody-antigen complex the residues of the epitope can be determined.  This technique requires careful control of temperature, pH and time of the reactions which can be highly optimized using modern robotics and softwares.  CovalX is offering this technique through our contract research services facility, more information can be seen here.

 

 

Linear

Conformational

Cost

Sample Consumption

Analysis Time

Amino Acid Resolution

Peptide Scanning YES NO $ LOW Weeks Several AA
Mutagenesis YES NO $ LOW Weeks Single AA
X-ray co-crystallography YES YES $$$ VERY HIGH Months-Years Single AA
Crosslinking coupled Mass Spec YES YES $$ LOW Several Weeks Few AA
Hydrogen Deuterium Exchange YES YES $$ MEDIUM Few Months Several AA

 

CovalX provides various techniques for mapping of conformational epitopes using the most advanced mass spectrometry equipment.  More detailed information on the CovalX techniques available can be seen here.

determined using mass tag linked to the cross-linking reagents.  After MS/MS fragmentation and data analysis using specific interaction softwares,