Antibody Characteristics

When an animal is immunized, typically a mouse, rabbit, or goat, an immune response results; the antibodies are predominantly found in the serum fraction of the blood. Serum containing antigen-specific antibodies is called antiserum. Five main types of antibodies have been identified, and these types are separated into five classes based upon their unique properties. These five classes of immunoglobulins are: IgM, IgG, IgA, IgD, and IgE.

All antibodies share the same basic structure which consists of four polypeptide chains held together by disulfide bonds. These four polypeptide chains form a symmetrical molecular structure (see figure below) comprised of two identical halves with the antigen binding sites formed between the ends of the heavy and light chains on both sides. The antibody has a hinged region in its center between the heavy chains that allows flexibility to articulate antigen binding. The two light (L) chains are identical to each other and are about 220 amino acids long while the two heavy (H) chains are about 440 amino acids in length and are also identical to each other. These chains are bound together by covalent and non-covalent disulfide bonds, and the number and type of disulfide bonds vary between classes of immunoglobulin.

There are two types of light chain among all classes of immunoglobulin, a lambda chain and a kappa chain; and there are no functional differences known between the two. However, there are five main heavy chain classes, or isotypes, which are unique to each class of immunoglobulin, and some isotypes have several different subtypes. These isotypes determine the biological properties of the antibody in an immune response.

An antibody’s function in the immune system is to specifically bind foreign particles and to signal other cells to eliminate the foreign matter. An antibody’s ability to bind specifically is due to how strongly it can interact with the antigen. An antibody is able to bind with its antigen binding sites that are at the amino-terminal end of each branch of the molecule. The strength of the interaction between antibody and antigen is determined by the strength of interaction between the antibody and a single binding site and by the number of binding sites on the antigen. The strength of binding between the antibody and a single binding site is known as the antibody’s affinity for the antigen, and the binding is reversible. The affinity between the antibody and the antigen binding site is determined by the type of bond formed. Because an antigen can have multiple different epitopes, a number of antibodies can bind to the protein. When two or more antigen binding sites are identical, an antibody can form a stronger bond with the antigen than if only one of the antibody’s sites is bound. Antigens with multiple identical binding sites are called multivalent, and antibodies are able to bind it more strongly. This total binding strength is known as avidity, and is a measure of the strength of the interaction between the antibody and antigen.

The antigen binding portion of an antibody varies extensively among secreted antibodies, and this length of sequence is known as the variable region. The variable region construction during antibody production in the B cell is what enables antibodies to be generated against an infinite variety of antigens. The other biological properties of the antibody and its role in signaling to other immune cells are determined by the constant regions of the heavy chains.

Antibody molecules break into fragments when digested with proteolytic enzymes. The fragments are two Fab fragments (fragment of antigen binding), and one Fc fragment (fragment crystallizable). The Fab fragment contains the entire light chain and the amino-terminal portion of the heavy chain with the antigen binding site between the chains. The Fc fragment contains the carboxy-terminal portion of both heavy chains, and it has no antigenic binding sites. In technical applications, it is the Fc portion that allows the antibody to be bound to matrices for plate based assays such as sandwich ELISAs, or allows the antibody to be used in immunoprecipitations and be recognized by secondary antibodies, or to be affinity purified with protein A or G.