The immune system is an incredible defense system comprised of an integrated network of cells, barriers, and biochemicals, all working collaboratively to protect the host. What I find most remarkable, is the capacity of the immune system to defend against an enormous array of threats (pathogens, toxins, cancer, etc.), with complete ignorance as to what these threats will be prior to exposure. Antibodies are a critical component of the defense arsenal assisting with identifying, capturing, and removing potential threats as well as protecting against future invasions.
Antibodies are gammaglobulin proteins, predominantly referred to as immunoglobulins (Ig). A monomeric antibody is composed of two heavy chains and two light chains covalently linked together through disulfide bonds to generate a Y-shaped structure (Figure 1). This Y-shaped structure provides a bifunctional capacity: 1) antigen-binding through the Fab (antigen binding fragment), and 2) interaction with immune cells and proteins (fragment crystallizable or Fc) to initiate and regulate host defense mechanisms. The Fc region determines the antibody class (or isotype), such as IgM, IgG, IgA, or IgE. The antibody-antigen interaction is driven by specific contacts between the variable region (heavy and light) and the antigen surface. The specific contacts of the antigen are referred to as the epitope, and the contacts on the antibody are called the paratope. An epitope may be a series of amino acids adjacent to one another on a protein, referred to as a linear epitope, or a structural surface defined by the three-dimensional folding of the protein and the contacts are discontinuous in the protein sequence, referred to as a conformational epitope.
II. Antibody Recognition of Antigen:
The Y- shape is due to the fact that there are two Fab regions and one Fc region for each antibody monomer (Figure 2A). The specificity of the antibody is determined by composition of the heavy chain variable region and the light chain variable region. Each of these variable regions are comprised of framework regions(FR) and complementarity determining regions (CDR).(CDR). The greatest variability occurs in the CDRs, which also are the regions that make direct contact with the antigen (Figure 2B). The composition and length of each of the CDRs is unique for each antibody and determines specificity and affinity towards the antigen. So, looking at Figure 2B, you can imagine that relatively minor changes in the CDR sequence and structure could lead to substantial differences in the ability of an antibody to bind a specific structure (or epitope). Through a process known as somatic hypermutation and affinity maturation (discussions for another time), genetic changes are introduced into the fully functional (rearranged)
Ig gene during B cell (the cells that produce antibodies) exposure to the antigen. Those changes that improve the binding or affinity of the Ig survive and expand. This natural selection process then results in the production of the best antibodies to protect the host.
III. The Constant Region Directs Immune Function:
The constant region or Fc region defines how a specific antibody will contribute to an immune response. Specific immune cells have Fc receptors that recognize specific constant regions and regulate (either by enhancing or suppressing) immune functions. Some constant regions also interact with the complement system used to recognize and in some cases kill microbial pathogens. It is the Fc region that defines the class of antibody or isotype (Figure 3).
Immune responses that are mediated by antibodies are referred to as humoral responses; these responses are mediated through the specific recognition of an antigen through the Fab and the regulation of the immune response through interactions in the Fc region. There are five classes of antibodies: IgM, IgD, IgG, IgA, and IgE (Figure 3, IgD not shown). IgD is predominantly found in trace amounts in plasma and on the surface of immature B cells; its function is largely unknown, however, it is generally associated with the mechanisms of B cell development. IgG makes up the majority of Ig in circulation (up to 70%) and is generated during secondary responses. There are four subclasses of IgG (IgG1-4 in humans and IgG1, IgG2a, IgG2b, & IgG3 in mice). These subclasses have different properties relative to interactions with specific Fc receptors and complement which ultimately dictate the type of humoral immune response(s). For example, human IgG1 and IgG3 are much more capable of binding to complement than IgG2, and IgG4 does not bind complement at all, therefore responses that require complement to mediate the host defense require the production of IgG1 or IgG3. The subclasses of IgG differ slightly in structure with different glycosylation patterns (shown as branched lines and circles in Figure 3), disulfide bonding pattern (shown as black lines in Figure 3), and size and structure of the flexible “hinge” region in the CH2 domain. IgM is found as a pentameric structure, mostly in extravascular spaces, although it is found in plasma. IgM is considered the primary antibody response and fixes complement extremely effectively, but does actively regulate cellular immune responses. IgM has an additional CH4 domain in the heavy chain constant region and a J-chain (as does IgA) that is required for multimer formation. IgA has two subclasses—IgA1 and IgA2—and is a secretory Ig, predominantly found in mucous, gastrointestinal, and genitourinary secretions. IgA is a dimer and has an associated J-chain. In addition there is a secretory component that associates with the IgA dimer which plays a role in the secretory process. IgE is only found in trace amounts in circulation, but is located on the cell surfaces of granulocytes (mast cells, basophils, and eosinophils) captured by specific IgE Fc receptors. It contains an additional CH4 domain and has a different glycosylation pattern than that of IgG. IgE plays a major role in fighting parasitic infections by regulating the release of mediators secreted by granulocytes. IgE is also primarily responsible for immediate hypersensitivity responses associated with allergy and asthma.
The structure of the antibody both the Fab and Fc, defines the function it plays in immunological responses. The use of antibodies as research reagents and therapeutics has also taken advantage of these structural features. By developing antibodies with the appropriate specificity (Fab), they can be used to target nearly any structural surface. In addition, specific recognition of the Fc region can be used for capture or detection which is valuable for immunoassays, as well as therapeutics. The Fc structure is also used for the purification of antibodies through the use of protein A and protein G, however, the different isotypes are recognized with varying affinities. Therefore, the class of antibody produced for research reagents, in particular, is important to consider when developing antibody purification strategies. With an understanding of the structural attributes of antibodies and their importance in the immunological response, we can utilize this information when developing monoclonal or polyclonal antibodies for use in research and clinical applications.