Antibody Structure

There are 5 classes of human antibodies: IgG, IgM, IgA, IgD, and IgE. The simplest antibodies, such as IgG, IgD, and IgE, are "Y"-shaped macromolecules called monomers (def). A monomer is composed of four glycoprotein chains: two identical heavy chains (def) and two identical light chains (def). The two heavy chains have a high molecular weight that varies with the class of antibody. The light chains come in two varieties: kappa or lamda and have a lower molecular weight. The four glycoprotein chains are connected to one another by disulfide (S-S) bonds (def) and noncovalent bonds (see Fig. 1).

Additional S-S bonds fold the individual glycoprotein chains into a number of distinct globular domains (see Fig. 2). The area where the top of the "Y" joins the bottom is called the hinge. This area is flexible to enable the antibody to bind to pairs of epitopes various distances apart on an antigen.

The two tips of the "Y" monomer are referred to as the Fab portions (def) of the antibody (see Fig. 1 and Fig. 2). The first 110 amino acids or first domain of both the heavy and light chain of the Fab region of the antibody provide specificity for binding an epitope (def) on an antigen.

The amino acid sequence of the first domain of both the hight chain and the heavy chain shows tremendous variation from antibody to antibody and constitutes the variable domains of the antibody. This is because each B-lymphocyte, early in its development, becomes genetically programmed through a series of gene-splicing reactions to produce a Fab with a unique 3-dimensional shape capable of fitting some epitope with a corresponding shape. The various genes the cell splices together determine the order of amino acids of the Fab portion of both the light and heavy chain; the amino acid sequence determines the final 3-dimensional shape (see Fig. 3). Therefore, different antibody molecules produced by different B-lymphocytes will have different orders of amino acids at the tips of the Fab to give them unique shapes for binding epitope. (More on this below.) The antigen-binding site is large enough to hold an epitope of about 5-7 amino acids or 3-4 sugar residues.

The bottom part of the "Y", the C terminal region of each glycoprotein chain, is called the Fc portion (def). The Fc portion, as well as one domain of both the heavy and light chain of the Fab region has a constant amino acid sequence that defines the class and subclass of each antibody. The Fc portion is responsible for the biological activity (def) of the antibody (see Fig. 1 and Fig. 2). Depending on the class and subclass of antibody, biological activities of the Fc portion of antibodies include the ability to:

• activate the complement pathway (def) (IgG & IgM);
• bind to phagocytes (IgG); or
• bind to mast cells, basophils, and eosinophils (IgE);
• bind to NK cells (def) (IgG).

Individual "Y"-shaped antibody molecules are called monomers (def) and can bind to two identical epitopes. Antibodies of the classes IgG, IgD, and IgE are monomers.

Two classes of antibodies are more complex. IgM (see Fig. 4) is a pentamer (def), consisting of 5 "Y"-like molecules connected at their Fc portions by a "J" or joining chain. Secretory IgA (see Fig. 5) is a dimer (def) consisting of 2 "Y"-like molecules connected at their Fc portions by a "J" chain and stabilized to resist enzymatic digestion in body secretions by means of a secretory component.

The 5 Classes or Isotypes of Human Antibodies (Immunoglobulins)

a. IgG (Immunoglobulin G; 4 subclasses)

• IgG makes up approximately 80% of the serum antibodies.
  
• IgG has a half-life of 7-23 days depending on the subclass.
  
• IgG is a monomer and has 2 epitope-binding sites (see Fig. 1).
  
• The Fc portion of IgG can activate the classical complement pathway.
  
• The Fc portion of IgG can bind to macrophage and neutrophils for enhanced phagocytosis.
  
• The Fc portion of IgG can bind to NK cells for antibody-dependent cytotoxicity (ADCC).
  
• The Fc portion of IgG enables it to cross the placenta. (IgG is the only class of antibody that can cross the placenta and enter the fetal circulation.)

b. IgM (Immunoglobulin M)

• IgM makes up approximately 13% of the serum antibodies and is the first antibody produced during an immune response.
  
• IgM has a half-life of about 5 days.
  
• IgM is a pentamer and has 10 epitope-binding sites (see Fig. 4).
  
• The Fc portions of IgM are able to activate the classical complement pathway. (IgM is the most efficient class of antibody for activating the classical complement pathway.)
  
• Monomeric forms of IgM are found on the surface of B-lymphocytes as B-cell receptors or sIg.

c. IgA (Immunoglobulin A; 2 subclasses)

• IgA makes up approximately 6% of the serum antibodies where it has a half-life of approximately 5 days.
  
• IgA is found mainly in body secretions (saliva, mucous, tears, colostrum and milk) as secretory IgA (sIgA) where it protects internal body surfaces exposed to the environment by blocking the attachment of bacteria and viruses to mucous membranes. While only 6% of the antibodies in the serum are IgA, secretory IgA is the most immunoglobulin produced.
  
• IgA is made primarily in the mucosal-associated lymphoid tissues (MALT).
  
• IgA appears as a dimer of 2 "Y"-shaped molecules and has 4 epitope-binding sites and a secretory component to protect it from digestive enzymes in the secretions (see Fig. 5).
  
• The Fc portion of secretory IgA binds to components of mucous and contributes to the ability of mucous to trap microbes.
  
• IgA can activate the alternative complement pathway.
  

d. IgD: (Immunoglobulin D; 2 subclasses)

• IgD makes up approximately 0.2% of the serum antibodies.
  
• IgD is a monomer and has 2 epitope-binding sites.
  
• IgD is found on the surface of B-lymphocytes (along with monomeric IgM) as a B-cell receptor or sIg where it may control of B-lymphocyte activation and suppression.
  
• IgD may play a role in eliminating B-lymphocytes generating self-reactive autoantibodies.

e. IgE (Immunoglobulin E)

• IgE makes up about 0.002% of the serum antibodies with a half-life of 2 days.
  
• Most IgE is tightly bound to basophils and mast cells via its Fc region.
  
• IgE is a monomer and has 2 epitope-binding sites.
  
• IgE is made in response to parasitic worms (helminths) and arthropods (def). It is also often made in response to allergens(def). (Allergens are antigens causing allergic reactions.)
  
• IgE may protect external mucosal surfaces by promoting inflammation, enabling IgG, complement proteins, and leucocytes to enter the tissues.
  
• The Fc portion of IgE can bind to mast cells and basophils (def) where it mediates many allergic reactions. Cross linking of cell-bound IgE by antigen triggers the release of vasodilators for an inflammatory response.
  
• The Fc portion of IgE made against parasitic worms and arthropods can bind to eosinophils enabling opsonization. This is a major defense against parasitic worms and arthropods.

Each day an average adult produces approximately three grams of antibodies, about two-thirds of this IgA.

 

Ways in which Antibodies Protect the Body

The antibodies produced during humoral immunity ultimately defend the body through a variety of different means. These include:

1. Opsonization
2. MAC Cytolysis
3. Antibody-dependent Cellular Cytotoxicity (ADCC) by NK Cells
4. Neutralization of Exotoxins
5. Neutralization of Viruses
6. Preventing Bacterial Adherence to Host Cells
7. Agglutination of Microorganisms
8. Immobilization of Bacteria and Protozoans.

A. Opsonization

1. Opsonization with IgG, C3b, and C4b

The process starts with IgG or IgM being made against a surface antigen of the organism or cell to be phagocytosed. The Fab portion (def) of the antibody reacts with epitopes of the antigen. The Fc portion (def) of IgG can then bind to neutrophils and macrophages thus sticking the antigen to the phagocyte (see Fig. 6).

Alternately, IgG and IgM can activate the classical complement pathway and C3b or C4b (def) can stick the antigen to phagocytes (see Fig. 6). Like IgG, C3b, and to a lesser extent C4b, can function as opsonins, that is, they can attach antigens to phagocytes. One portion of the C3b binds to proteins and polysaccharides on microbial surfaces; another portion attaches to CR1 receptors on phagocytes, B-lymphocytes, and dendritic cells for enhanced phagocytosis. (see Fig. 7). (Remember that C3b and C4b are also produced during the alternative complement pathway and the lectin pathway as was discussed in Unit 2.)

Actually, C3b molecule can bind to pretty much any protein or polysaccharide. Human cells, however, produce Factor H that binds to C3b and allows Factor I to inactivate the C3b. On the other hand, substances such as LPS on bacterial cells facilitate the binding of Factor B to C3b and this protects the C3b from inactivation by Factor I. In this way, C3b does not interact with our own cells but is able to interact with microbial cells.

Attachment then promotes destruction of the antigen. Microorganisms are placed in phagosomes (see Fig. 8) where they are ultimately digested by lysosomes (see Fig. 9). If the antigen is a cell too large to be ingested - such as virus-infected host cells, transplant cells, and cancer cells - the phagocyte empties the contents of its lysosomes directly on the cell for extracellular killing (see Fig. 10 and Fig. 11).

Animation of opsonization and intracellular destruction.

Animation of opsonization and extracellular destruction.

Opsonization is especially important against microorganisms with antiphagocytic structures such as capsules since opsonizing antibodies made against the capsule are able to stick capsules to phagocytes (see Fig. 12).

Animation showing opsonization of an encapsulated bacterium.

2. Opsonization with IgE

IgE is made against parasitic worms (helminths) and arthropods. The Fab portion of IgE bind to epitopes on the helminth or arthropod and the Fc portion binds to receptors on eosinophils enabling opsonization. In otherwords, IgE sticks phagocytic eosinophils to helminths and arthropods for the extracellular killing of that organism (see Fig. 13). IgE also promotes inflammation for the recruitment of phagocytes.

B. MAC Cytolysis (def)

The process starts with IgG or IgM being made against epitopes on membranes. The Fab portion (def) of IgG or IgM reacts with the epitopes on the membrane and the Fc portion (def) of the antibody then activates the classical complement pathway. C5b6789_n (the membrane attack complex or MAC) (def) then puts holes in the membrane. (Remember that MAC is also produced during the alternative complement pathway and the lectin pathway as was discussed in Unit 2.)

In the case of bacteria, MAC can put holes in the outer membrane and possibly the cytoplasmic membrane of the gram-negative cell wall (see Fig. 14) causing lysis (see Fig. 15). With enveloped viruses, the MAC can damage the viral envelope (see Fig. 16 and Fig. 17). In the case of "foreign" human cells - virus-infected cells, transplanted cells, transfused cells, cancer cells - the MAC causes direct cell lysis (see Fig. 18 and Fig. 19).

Animation showing cytolysis of a gram-negative bacterium by MAC.

Animation showing cytolysis of an infected human cell by MAC.

C. Antibody-Dependent Cellular Cytotoxicity (ADCC) by NK cells

NK cells are capable of antibody-dependent cellular cytotoxicity (def) or ADCC. NK cells have receptors on their surface for the Fc portion (def) of IgG. When IgG is made against epitopes on "foreign" membrane-bound cells, e.g., virus-infected cells and cancer cells, the Fab (def) portions of the antibodies react with the "foreign" cell. The NK cells then bind to the Fc portion of the antibody.

The NK cell then releases pore-forming proteins called perforins, proteolytic enzymes called granzymes, and chemokines. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation (see Fig. 20 and Fig. 21). As a result, the cell breaks into fragments that are subsequently removed by phagocytes. Perforins can also sometimes result in cell lysis. (When NK cells are carrying out ADCC, they are sometimes also referred to as killer cells.)

Animation of ADCC lysis by NK cells

Animation of ADCC apoptosis by NK cells

D. Neutralization of Exotoxins

For an exotoxin to cause harm it must first bind to receptors on a susceptible host cell. Antitoxin antibodies, mainly IgG, are made against protein exotoxins (def). They combine with the exotoxin molecules before they can interact with host target cells and thus neutralize the toxin (see Fig. 22).

Animation showing neutralization of an exotoxin.

E. Neutralization of Viruses

In order for viruses to infect a cell and replicate, they must first adsorb to receptors on the host cell's plasma membrane. Antibodies made against the epitopes on viral capsids or on viral envelope glycoproteins that function in the attachment of the virus to the host cell receptor prevent viral adsorption (see Fig. 23). Neutralizing antibodies are especially important in preventing viral reinfection.

Animation showing neutralization of a virus.

F. Preventing Bacterial Adherence

One of the body's innate defenses is the ability to physically remove bacteria from the body through such means as the constant shedding of surface epithelial cells from the skin and mucous membranes, the removal of bacteria by such means as coughing, sneezing, vomiting, and diarrhea, and bacterial removal by bodily fluids such as saliva, blood, mucous, and urine. Bacteria may resist this physical removal producing pili, cell wall adhesin proteins, and/or biofilm-producing capsules.

Antibodies are made against pili (def), capsules (def), and adhesins (def) to prevent bacteria from adhering to and colonizing host cells (see Fig. 24).

G. Agglutination of Microorganisms

Agglutination is mainly a function of antibodies with multiple reactive Fab sites such as IgM and IgA. The antibodies link microorganisms together (cause them to agglutinate) so they can be filtered out of the lymph and blood and be phagocytosed more effectively by the fixed macrophages of the mononuclear phagocytic (reticuloendothelial) system (see Fig. 25).

H. Immobilization of Bacteria and Protozoans

Flagella and cilia are organelles of locomotion and enable motile microorganisms to move towards or away from environmental molecules through a process called taxis (def). Antibodies made against the flagella of motile bacteria or the flagella or cilia of motile protozoans bind to these locomotor organelles, arrest the organism's movement, and may block its ability to spread.