Comparison of Conformational Epitope Mapping Technologies

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.




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