It is typical to have about 6 residues in an epitope, although a smaller number can be sufficient. They do not have to be contiguous. It is possible to have a single-residue epitope be recognized by an antibody if the residue has a non-natural or post-translationally-modified side chain. A good example is phosphotyrosine. Another is N-acetyl-lysine.

http://www.ncbi.nlm.nih.gov/pubmed/2481233

Here is an example of a 4-residue epitope.

http://www.ncbi.nlm.nih.gov/pubmed/2481233

One way to test for specificity of your antibody is to perform a Western blot with it on an extract of the source material. If you only see 1 band, the antibody is probably specific, at least at that particular dilution. Unfortunately, if you see multiple bands, you can't be sure if the antibody is non-specific or if the antigen is degraded or post-translationally modified (e.g. glycosylated). Ideally, you would be able to produce a matching sample from which the target antigen was lacking because of a genetic deletion and show that there was no detection of anything by Western blot.

Since the apparent degree of specificity depends upon how much antibody and antigen were present, you may be able adjust these parameters (downwards) to improve the specificity of detection. This assumes that the antibody's affinity is highest for the target protein.

It is typical to have about 6 residues in an epitope, although a smaller number can be sufficient. They do not have to be contiguous. It is possible to have a single-residue epitope be recognized by an antibody if the residue has a non-natural or post-translationally-modified side chain. A good example is phosphotyrosine. Another is N-acetyl-lysine.

http://www.ncbi.nlm.nih.gov/pubmed/2481233

Here is an example of a 4-residue epitope.

http://www.ncbi.nlm.nih.gov/pubmed/2481233

One way to test for specificity of your antibody is to perform a Western blot with it on an extract of the source material. If you only see 1 band, the antibody is probably specific, at least at that particular dilution. Unfortunately, if you see multiple bands, you can't be sure if the antibody is non-specific or if the antigen is degraded or post-translationally modified (e.g. glycosylated). Ideally, you would be able to produce a matching sample from which the target antigen was lacking because of a genetic deletion and show that there was no detection of anything by Western blot.

Since the apparent degree of specificity depends upon how much antibody and antigen were present, you may be able adjust these parameters (downwards) to improve the specificity of detection. This assumes that the antibody's affinity is highest for the target protein.

Dear Paulo,

The following publication covers the answer to your question. I have copied the important paragraphs for quick view.

Immunology and Evolution of Infectious Disease.

http://www.ncbi.nlm.nih.gov/books/NBK2396/

4.1. Antigens and Antibody Epitopes

An antigenic molecule stimulates an immune response. Each specific subset of an antigenic molecule recognized by an antibody or a T cell receptor defines an epitope. Each antigen typically has many epitopes. For example, insulin, a dimeric protein with 51 amino acids, has on its surface at least 115 antibody epitopes (Schroer et al. 1983). Nearly the entire surface of an antigen presents many overlapping domains that antibodies can discriminate as distinct epitopes (Benjamin et al. 1984).

Epitopes have approximately 15 amino acids when defined by spatial contact of antibody and epitope during binding (Benjamin and Perdue 1996). Almost all naturally occurring antibody epitopes studied so far are composed of amino acids that are discontinuous in the primary sequence but brought together in space by the folding of the protein.

The relative binding of a native and a mutant antigen to a purified (monoclonal) antibody defines one common measure of cross-reactivity. The native antigen is first used to raise the monoclonal antibody. C50mut is the concentration of the mutant antigen required to cause 50% inhibition of the reaction between the native antigen and the antibody. Similarly, C50nat is the concentration of the native antigen required to cause 50% inhibition of the reaction between the native antigen and the antibody (self-inhibition). Then the relative equilibrium binding constant for the variant antigen, C50nat/C50mut, measures cross-reactivity (Benjamin and Perdue 1996).

Site-directed mutagenesis has been used to create epitopes that vary by only a single amino acid. This allows measurement of relative binding caused by an amino acid substitution. Studies differ considerably in the methods used to identify the amino acid sites defining an epitope, the choice of sites to mutate, the amino acids used for substitution, and the calculation of changes in equilibrium binding constants or the free-energy of binding. Benjamin and Perdue (1996) discuss these general issues and summarize analyses of epitopes on four proteins.

Five tentative conclusions about amino acid substitutions suffice for this review. First, approximately 5 of the 15 amino acids in each epitope strongly influence binding. Certain substitutions at each of these strong sites can reduce the relative binding constant by two or three orders of magnitude. These strong sites may contribute about one-half of the total free-energy of the reaction (Dougan et al. 1998).

Second, the other 10 or so amino acids in contact with the antibody may each influence the binding constant by up to one order of magnitude. Some sites may have no detectable effect.

Third, the consequences of mutation at a particular site depend, not surprisingly, on the original amino acid and the amino acid used for substitution.

Fourth, theoretical predictions about the free-energy consequences of substitutions based on physical structure and charge can sometimes be highly misleading. This problem often occurs when the binding location between the antibody and a particular amino acid is highly accessible to solvent, a factor that theoretical calculations have had difficulty incorporating accurately.

Fifth, antibodies raised against a particular epitope might not bind optimally to that epitope—the antibodies sometimes bind more strongly to mutated epitopes. In addition, antibodies with low affinity for an antigen can have higher affinity for related antigens (van Regenmortel 1998).

Hoping this will be helpful,

Rafik

Many thanks to Rafik for his helpful reply. Could he help us further with the literature demonstrating that "Almost all naturally occurring antibody epitopes studied so far are composed of amino acids that are discontinuous in the primary sequence but brought together in space by the folding of the protein."? And the immunological mechanism on how such antibodies with structural epitopes come about (not in anything I have seen)? Obviously the paratope (antibody's binding site) has only that much space for an antigen's epitope.