Antibodies

Introduction to Antibodies

Antibodies are proteins made by B cells of the immune system in response to an immune challenge. They can be defined as an immunoglobulin (Ig) capable of interacting specifically with the antigen that caused its formation. This antigen is usually a high molecular weight polypeptide or polysaccharide, but other molecules such as lipids or nucleic acids can also function as an antigen. Smaller molecules (termed haptens) can also serve as antigens if they are coupled to a larger carrier protein such as BSA.

The basic structural unit of an antibody molecule consists of 4 polypeptides: 2 identical light chains (L) and 2 identical heavy chains (H). The light chains fall into either the "kappa" (κ) or "lambda" (λ) class, while the heavy chains are classified as "alpha" (α), "gamma" (γ), "delta" (δ), "epsilon" (ε), and "mu" (μ), corresponding to the IgA, IgG, IgD, IgE, and IgM classes of antibodies, respectively.

Each class of antibodies possess distinctive biological properties and are expressed at different places and times. In addition, several classes can be further divided into subclasses. IgM molecules are the main antibody produced in the primary immune response. These molecules form pentameric structures which efficiently activate the complement system of the immune system. IgG antibodies constitute the major class of immunoglobulin in the blood and are produced mainly during the secondary immune response. After binding to their antigen, these molecules bind to specific receptors of phagocytic cells, increasing the efficiency with which these cells ingest and destroy the microorganisms that produced the antigen in question. IgA molecules are found primarily in secreted fluids, and the IgE is thought to be involved in allergic reactions. The role of IgD antibodies is largely unknown.

The 4 antibody chains are held together by a combination of noncovalent interactions and covalent bonds (disulfide linkages) in such a way that they form a Y-shaped molecule. The 2 antigen binding sites are at the a-terminus of the light and heavy chains at the ends of the "arms" of the "Y". Mild digestion of the IgG antibody with the proteolytic enzyme papain will cleave molecule into the 2 antigen binding regions (termed F(ab)) and the base of the "Y" (termed Fc). It is the Fc region of the antibody that is recognized by specific macrophage receptors during the immune response.

In the laboratory, antibodies are often classified as polyclonal or monoclonal. Polyclonal antibodies are a heterogeneous population of antibodies that recognize multiple regions (termed epitopes) of the same antigen. These antibodies often give a more robust response but are more likely to cross-react with similar proteins. Monoclonal refers to the fact that the antibodies are produced from a single clone and recognize the exact same epitope of the antigen. These molecules are usually very specific, but are more sensitive to loss of an epitope through modification of the antigen.

The ability of antibodies to recognize specific proteins is useful in many different laboratory procedures such as immunoblotting (Western Blots), FACS, immunohistochemistry (IHC), and ELISAs. Generally, these procedures use 2 different antibodies: the primary, which recognizes the protein in question, and the secondary antibody, which is specific for antibodies from the organism that generated the primary antibody. The secondary antibody is usually linked to either a chemical or enzyme to amplify the resulting signal.