This article explains what an antigen is and the best method to choose an antigen against which to raise an antibody.
All immunohistochemical techniques are based upon the same principle: a specific antibody binds with its specific antigen to produce a unique antigen-antibody complex.
What is an Antigen?
An antigen is simply any substance foreign to the immune system that causes an immune response, such as specific antibody production, when it has been brought into close contact with the tissues of any animal that is sensitive to it, and can combine with the specific antibodies that is formed.
Most antigens have a large molecular weight and are chemically composed of proteins or polysaccharides, but may also be lipids, polypeptides, or nuclear acids, among others.
There are low-molecular weight substances which are capable of producing an immune response, called haptens. These can be conjugated by chemical reaction to a larger protein which acts as a carrier, some examples being bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), as well as some synthetic substrate molecules.
Many molecules have hapten capability, such as drugs, small peptides, triglycerides, simple sugars, or amino acids, which means that if the period of exposure is sufficient, almost any substance foreign to the body will be recognized as such by the immune system and stimulate the production of specific antibodies.
Factors that Affect Antigen-Antibody Interaction
The nature of the immune response varies with the size, composition and structure of the antigen. A strong immunogen is an antigen that provokes a powerful immune response.
Antigen-antibody interaction is optimal when the epitope, or antibody recognition/binding site on the antigen, is open to the surroundings and therefore available for the antibody to bind it. This may change if the target is denatured by any process such as pH changes, reduction or fixation, or sample preparation for gel electrophoresis. In this case the epitope may no longer be able to bind the antibody.
Some antibodies are thus excellent for antibody detection in immunohistochemistry where the antigenic site is well maintained, but perform poorly in Western blotting, which requires sample preparation that changes the conformation of the protein and in so doing, alters the antigen site at which antibody binding occurs.
Some epitopes are found to occur only in the native or unprocessed state of the antigen, while others require the antigen to be denatured before they are exposed. They may be soluble, and therefore found in the cytoplasm, or they may be secreted, or associated with the membrane. The number and location of the epitopes, along with the size, varies with the extent of antigen presentation during the process of antibody production.
If a protein is present but at minute concentrations, the knowledge of the nucleotide sequence can be used to generate the same type of peptide so that antibodies specific for that sequence may be produced, called peptide-specific antibodies. These peptide antigens are sometimes superior to the whole proteins because they can produce antibodies that are directed against unique regions of the sequence, particularly when the proteins under study are part of a family of extremely homologous proteins.
Characteristics of a Good Antigen
A good antigen shows the following features:
- Intramolecular areas of stable structure and complex chemical bonding
- Large stretches which are not composed of long repeating units
- A molecular weight of at least 8 000 to 10 000 Da (however, it must be noted that haptens of only 200 Da molecular weight have been conjugated with a carrier protein)
- Can undergo processing by the immune system
- Has regions that can be presented to the antibody-forming process to stimulate the immune system
- Has structural dissimilarity with the host
- Peptide antigens should contain immunogenic regions with at least 30% of amino acids such as lysine, glutamine, arginine, glutamic acid, aspargine and aspartic acid, called immunogenic amino acids, as well as sufficiently high numbers of hydrophilic or charged functional groups
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