A description of what an antigen is and how to choose one to make an antibody. 

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​The basic principle of any immunohistochemical technique is that a specific antibody will combine/bind with its specific antigen to give an exclusive antibody-antigen complex.

The classic definition of "antigen" is any foreign substance that elicits an immune response (e.g. the production of specific antibody molecules) when introduced into the tissues of a susceptible animal, and is capable of combining with the specific antibodies formed.

Antigens are generally of high molecular weight and are commonly proteins or polysaccharides. Polypeptides, lipids, nuclear acids and many other materials can also function as antigens.

Immune responses may also be generated against smaller substances, called haptens, if these are chemically coupled to a larger carrier protein. Common carrier proteins are bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or other synthetic matrices.

A variety of molecules such as drugs, simple sugars, amino acids, small peptides, phospholipids or triglycerides may function as haptens. Thus, given enough time, just about any foreign substance will be identified by the immune system and evoke specific antibody production.

However, this specific immune response is highly variable and depends much in part on the size, structure and composition of antigens. Antigens that elicit strong immune responses are said to be strongly immunogenic.

For efficient interaction to occur between the antigen and the antibody, the epitope (a part of an antigen that the specific antibody recognizes and binds to) 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 blot but very good in immunohistochemistry because in the latter procedure, a complex antigenic site might be maintained in the tissue, whereas in the former procedure the process of sample preparation alters the protein conformation sufficiently to destroy the antigenic site and hence eliminate antibody binding. 

Thus, the epitope may be present in the antigen's native, cellular environment, or only exposed when denatured. In their natural form they may be cytoplasmic (soluble), membrane associated or secreted. The number, location and size of the epitopes depends on how much of the antigen is presented during the antibody making process.

If a gene product of interest is present in extremely low concentrations, one may choose to use known nucleotide sequence information to derive a corresponding peptide for generating sequence-specific antibodies (e.g. peptide specific antibodies). In some instances, peptide antigens have advantages over whole protein antigens in that the antibodies generated may be targeted to unique sequence regions. This is especially useful when investigating proteins that belong to families of high sequence homology.

Characteristics of a good antigen include:

  • Areas of structural stability and chemical complexity within the molecule.
  • Significant stretches lacking extensive repeating units.
  • A minimal molecular weight of 8,000–10,000 Da, although haptens with molecular weights as low as 200 Da have been used in the presence of a carrier protein.
  • The ability to be processed by the immune system.
  • Immunogenic regions that are accessible to the antibody-forming mechanism.
  • Structural elements that are sufficiently different from the host.
  • For peptide antigens, regions containing at least 30% of immunogenic amino acids: lysine, arginine, glutamic acid, aspartic acid, glutamine, aspargine.
  • For peptide antigens, significant hydrophilic or changed residues.

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