Epitope mapping is the process of identifying the binding sites, or ‘epitopes’, of antibodies on their target antigen

Antigen

An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

s (which are proteins).

Identification and characterization of the binding sites of antibodies can aid in the discovery and development of new therapeutics, vaccines, and diagnostics.

Epitopes (the binding sites on the protein) can be divided into linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

, while conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding

Protein folding

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....

. The vast majority of antigen-antibody interactions have conformational epitopes.

Methods for epitope mapping

Epitope mapping of complex target antigens, such as integral membrane protein

Membrane protein

A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes.-Function:...

s or multi-subunit

Protein subunit

In structural biology, a protein subunit or subunit protein is a single protein molecule that assembles with other protein molecules to form a protein complex: a multimeric or oligomeric protein. Many naturally occurring proteins and enzymes are multimeric...

 proteins, is often challenging because of the difficulty in expressing and purifying these types of antigens.

There are several methods available for mapping antibody epitopes on target antigens :

• The gold standard approach is X-ray co-crystallography
  X-ray crystallography
  X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
  
  , which allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, requires large amounts of purified protein, and can be time-consuming and expensive.

• Array
  Protein microarray
  A protein microarray, sometimes referred to as a protein binding microarray,provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, or to identify the targets of biologically active...
  
  -based oligo-peptide
  Peptide
  Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
  
   scanning (sometimes called pepscan analysis
  Pepscan
  Pepscan is a procedure for mapping and characterizing epitopes involving the synthesis of overlapping peptides and analysis of the peptides in enzyme-linked immunosorbent assays . The method is based on combinatorial chemistry and was pioneered by Mario Geysen and coworkers ....
  
  ): This technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein and tests for their ability to bind the antibody of interest. This method is fast and relatively inexpensive, and specifically suited to profile epitopes for large number of candidate antibodies against a defined target. By combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (such as by using CLIPS scaffolds), discontinuous epitopes can be mapping with very high reliability and precision.

• Site-directed mutagenesis
  Mutagenesis
  Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...
  
   : Using this approach, systematic mutations of amino acids are introduced into a protein sequence followed by measurement of antibody binding in order to identify amino acids that comprise an epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and slow, typically limiting analysis to a small number of amino acid residues.

• Mutagenesis Mapping. This approach utilizes a comprehensive mutation library, with each clone
  Cloning
  Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
  
   containing a unique amino acid mutation and the entire library covering every amino acid in the target protein. Amino acids that are required for antibody binding can be identified by a loss of reactivity and mapped onto protein structures to visualize epitopes. This approach has recently been used to epitope map a panel of antibodies against human CCR5
  CCR5
  C-C chemokine receptor type 5, also known as CCR5, is a protein that in humans is encoded by the CCR5 gene. CCR5 is a member of the beta chemokine receptors family of integral membrane proteins...
  
  , a GPCR coreceptor for HIV
  HIV
  Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
  
   entry.

• Other methods, such as phage display
  Phage display
  Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...
  
  , and limited proteolysis
  Proteolysis
  Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
  
  , provide high throughput but lack reliability, especially for conformational epitopes.