College of Technology Medical

Department of Medical Laboratory

STAGE(3)

IMMUNOLOGY
(Epitopes)

Preparation:
‫مهند حمد حمادي‬

Supervised by:
DR.NAWFAL AL DABBAGH

Epitopes
The small site on an antigen to which a complementary antibody may
specifically bind is called an epitope or antigenic determinant. This is
usually one to six monosaccharides or five to eight amino acid residues on the
surface of the antigen. Because antigen molecules exist in space, the epitope
recognized by an antibody may be dependent upon the presence of a specific
three dimensional antigenic conformation (e.g., a unique site formed by the
interaction of two native protein loops or subunits). This is known as a
conformational epitope. The epitope may also correspond to a simple linear
sequence of amino acids and such epitopes are known as linear epitopes.
The range of possible binding sites on a target molecule (antigen) is enormous,
with each potential binding site having its own structural properties derived from
covalent bonds, ionic bonds, hydrophilic, and hydrophobic interactions. Indeed,
this has important ramifications for antibody choice and performance. For
efficient interaction to occur between the target antigen and the antibody, the
epitope must be readily available for binding.
If the target molecule is denatured, e.g., through fixation, reduction, pH changes,
or during preparation for gel electrophoresis, the epitope may be altered and this
may affect its ability to interact with an antibody. For example, some antibodies
are ineffective in Western blotting (WB) but are suitable for
immunohistochemistry (IHC) applications, because, in the IHC procedure, a
complex antigenic site might be maintained in the tissue, whereas in the WB
procedure, the process of sample preparation alters the protein conformation
sufficiently to destroy the antigenic site, and hence eliminates antibody binding.
In a denatured protein, only the linear epitope may be recognized. Hence, in
protocols where a denatured protein is used, such as in Western blotting, an
antibody that recognizes a linear epitope is preferred. Sometimes an epitope is
on the interior of a folded protein. The epitope is then inaccessible to the
antibody in a nondenaturing protocol, such as immunoprecipitation. A
conformational epitope, by definition, is on the outside of the folded protein. An
antibody that recognizes the conformational epitope is suitable for mild,
nondenaturing  procedures, such as immunoprecipitation or flow cytometry.
Optimally, an antibody that recognizes a linear epitope on the surface of a
normally folded protein will work well in both nondenaturing and denaturing
protocols. Thus, the epitope may be present in the antigen’s native, cellular
environment, or it may be exposed only when denatured. In their natural form,
antigens may be cytoplasmic (soluble), membrane-associated, or secreted. The
number, location and size of the epitopes depend on how much of the antigen is
presented during the antibody-making process.
Knowledge about the target protein, the epitope recognized by the antibody,
sequence conservation, and the technique principles are valuable in making
good antibody and protocol choices. Actual epitope mapping or sequence data,
though useful, are not needed, however, to be confident in antibody specificity.