Antibody Structure, Function, and Categorizations
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 or KLH.
The basic structural unit of an antibody molecule is compromised of 4 polypeptides:
- 2 identical light chains (L)
- 2 identical heavy chains (H).
The light chains fall into either the "kappa" (κ) or "lambda" (λ) class. 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 possesses distinctive biological properties and is expressed at different places and times. In addition, several of the classes can be further divided into subclasses, as seen below.
The basic structure, however, remains the same: 4 antibody chains are held together by a combination of non-covalent 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.
Polyclonal Versus Monoclonal
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.
An antigen is defined as a substance that causes an immune response when introduced into an organism. It is also capable of binding with the specific antibodies. This binding is mediated by the sum of many weak interactions between the antigen and antibody. These weak interactions include hydrogen bonds, van der Waals forces, and ionic and/or hydrophobic interactions.
Interactions can only take place if the antigen and antibody molecules are close enough for some of the individual atoms to fit into complementary recesses. The complementary regions of an antibody are its 2 antigen binding sites (thus the antibody is said to be bivalent). The corresponding region(s) of the antigen is referred to as an antigenic determinant. Most antigens have multiple determinants; if 2 or more are identical, the antigen is said to be multivalent.
As the binding of an antibody (ab) to its antigen (ag) is reversible, the binding reaction can be expressed as:
ab + ag ↔ ab:ag
strength of the interaction is expressed as the affinity constant Ka, where:
Ka = [ab:ag]/[ab][ag]
In this equation, [ab:ag] is the molar concentration of the antibody-antigen complex. [ab] and [ag] are the molar concentrations of the antibody and antigen, respectively. Affinity constants can vary widely between different antibodies and antigens, and are affected by pH, temperature, and solvent. The affinity of an antibody reflects how well the antigenic determinant fits in the antigen binding site of antibody. It is also independent of the number of binding sites.
Another way to measure the antibody-antigen interaction is the avidity of the binding. This reflects the overall stability of the antibody-antigen complex. Avidity is defined as the total binding strength of all of its binding sites together. Thus, it is influenced by the affinity of the antibody for its antigen and the valencies of both the antibody and the antigen. A typical IgG molecule will bind up to 10,000 times more strongly to a multivalent antigen if both antigen-binding sites are engaged than if only one site binds the antigen.
The multivalent nature of most antibodies and antigens often causes secondary reactions such as precipitation, cell clumping, and complement fixation in an organism. In the laboratory, we can make use of these sorts of reactions in techniques such as western blotting, ELISAs, and immunoprecipitation.
A more detailed description of how antibodies interact with certain antigens can be found in: Davies DR and Cohen GH. (2004). Interactions of protein antigens with antibodies. Proc. Natl. Acad. Sci. USA 93:7-12.