Structural Biochemistry/Protein function/Epitope

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Epitope, or antigenic determinant, is a small, specific portion of an antigen recognized by the immune system such as antibodies. A single antigen usually has several different epitopes. The region on an antibody which recognizes the epitope is called a paratope. Antibodies fit precisely and bind to specific epitopes.

Chem114A epitopes.jpg

Epitopes exist as tertiary structures of amino acids and are not recognized by antibodies with the same specificty or affinity when denatured by pH or temperature. Immunoglobins which recognize the epitopes will also denature under similar conditions. There is a distinction in the naming of native epitopes and denatured linear epitopes. Epitopes on natural tertiary structures are named cryptotopes and that of linear sequences is called unfoldons. There are two different yet effective ways to study epitope and map their locations. These methods are x-ray crystallography and monoclonal antibodies. The immune response of an animal produces many different types of antibodies that recognize different epitopes with a range of affinity. These various antibodies are called polyclonal antibodies and are found in the serum taken from the blood. A monoclonal antibody is just one of these antibodies out of the many polyclonal antibodies, and it is this monoclonal antibody that is used to define specific epitopes.

Epitope Mapping

The challenges in mapping epitopes and identifying its location becomes challenging because different monoclonal antibodies will recognize different or similar epitopes. An epitope cannot exist without there being a corresponding antibody that recognizes it specifically. This definition makes it harder to specifically identify the range of any given epitope since monoclonal antibodies begin first and polyclonal antibodies all with different affinities and specificity. Some of these antibodies will have different leniency for amino acids and others will overlap in the epitope they recognize. It then becomes troublesome in deciding which polyclonal antibody should correspond to the specific epitope as many recognize the same sequence.

Often the means of the monoclonal antibody, or Mab, influences the definition of the epitope. Because Western Blotting’s SDS-PAGE partially denatures the protein this will affect the recognition of MAb to epitope. Other procedures that utilize native conformations such as liquid phase immunoassays and or frozen tissue samples will simulate in vivo affinities. This creates three artificial categories of Mab to eptiope recognition. Monoclonal antibodies that recognize only partially denatured epitopes, those that recognize nativetertiary structure of epitopes, and those that recognize both.

Disulfide bridges that are distinctly visible can be isolated through the precise mapping of Epitope. However, epitopes can be recognized through various different monoclonal antibodies. The specificity and recognition of these features become more pronounced in the tissues. Monoclonal antibodies are rather selective in recognizing Epitope despite the structural subtle and differences that may arise in the conformational change of this protein. Despite the domain not having a concrete protein residue or subunit cavity, the structure can still be detected under x-ray crystallography.

X-ray crystallography is the most precise method in determining native tertiary contact between the epitope and monoclonal antibody. Challenges exist in defining how close the antibody must be to be in “contact” and even if the two are in contact it does not necessarily mean there is binding. Also x-ray crystallogoraphy is an expensive method and requires the antibody to be in crystal form. Methods that circumvent these challenges include NMR which can be used commonly but it sacrifices the precision of x-ray crystallography. NMR is hindered by the size of the antigen. If the antigen is too large then electron-microscopy can be used because it requires that the antigen be sizable.

References

Glen, Morris E. "Choosing a Method for Epitope Mapping." Methods in Molecular Biology. 1996. Epitope Mapping Protocols. 03 Dec. 2008 <http://springerlink.com/content/t5u6u667k23g1725/fulltext.html>.