references
 
 
 

ANTIBODIES

History

  • Serum components with activity known for centuries.
  • 1939: Rabbit immunized with egg white albumin and extract reactive globulins.
  • Further separation by electronic field (electrophoresis) indicates the gamma-globulins increased with immunization.
  • Further purification of the gamma-globulin protein aided characterization of their structure. Myeloma patients, who have B-cell tumors secreting large amounts of one type of antibody, excrete this protein in their urine. Bence and Jones purified these proteins from patient urine (the so-called Bence-Jones proteins) and identified them as individual chains from immunoglobulins, abbreviated Ig. Experiments by Edelman and Porter in the 1950's-60's characterized basic protein structure (Nobel Prize 1972), showing a papain cleavage site and two different chains linked by disulfide bonds.
  • Different research groups began to report structural differences among Bence-Jones proteins isolated from different individuals. This indicated that there were different classes of Ig or isotypes of Ig. Reports indicated that subtle differences between individuals occur within the same isotypes (allotypes).

IgG structure

  • Heavy and Light chains : Denaturing agents known to break disulfide bonds converted pure Ig into two fractions of molecular weights 53kD (heavy) and 22kD (light) respectively. Calculations indicated that each Ig had two heavy chains and two light each, a total of four chains.
  • Antigen binding Site : Located in the Fab (fragment antigen binding), an Ig has two antigen binding sites and is termed divalent.
  • The Fc region (fragment-crystalizable) is monovalent, made up of only heavy chains, determines the isotype, and confers the functional aspects of the antibody. It has two constant domains, the complement receptor and a site for the addition of carbohydrate/sugars.

Other structural aspects:

  • Ig Domain: This common structural feature of many molecules in the immune system is 100-115 amino acids long, forms a stable globular loop with intrachain disulfide bonds. If analysis indicates that the structure is similar in many types of receptors the domain may be termed constant . If variation is observed between different types, the domain is termed variable.
  • The Hinge Region: a short flexible region with cysteine and proline residues allows for steric torque within this large molecule, allowing binding across distances that vary.
  • Complimentarity-determining Regions (CDRs ) is the precise area where antibody binds antigen in the Fab's V-LC and V-HC's domains. Analysis indicates that there is incredible genetic diversity in certain sites that determine which amino acid are available for contact with antigen. These hypervariable regions are most interesting and we will touch on them later. Structurally, loops of the V-regions of both heavy and light chains form this binding site. Antibodies with different specificity have different CDRs, but some antibodies that bind the same epitope of an antigen also have different CDRs--there's power in diversity.
  • Within the Fc region, different combinations of disulfide bonds, Ig domains and carbohydrates delineate different classes or isotypes of antibody. These different isotypes have unique specialized functions, allowing different isotypes to carry out certain tasks in the immune response to differing pathogens (more later, see Benjamini Chapter 5).
    • IgG (G-class, with gamma heavy chains) is the predominant class in blood, as four subclasses , crosses the placenta, and fixes complement well.
    • IgM (M-class with mu heavy chains) is the first produced by a B cell as it matures. It is often found in a Pentameric form, joined by J-chain protein and this pentameric form is the best complement fixer. It does NOT cross the placenta, but binds large, repetitive antigens.
    • IgA (A-class with alpha heavy chains) is found in secretions including sweat and saliva. It is the major Ig in the colostrum, passed from mother to child in milk, conferring passive immunity. In secretions, it is often found as a dimer, two IgA molecules linked by the same J-chain as used by IgM, with another protein know as secretory component. In blood, IgA is usually monomeric.
    • IgD (D-class using delta heavy chains) is also found on immature B cells. The function is unclear at the moment and the molecule is degraded quickly.
    • IgE (E-class using epsilon heavy chains) Fc portion binds high affinity receptors on mast cells and Basophils, causing mast cell degranulation and release of histamines. IgE is useful for combating multicellular organisms like helminths, but harmful in allergies.

 

MONOCLONAL ANTIBODIES (MABs)

Monoclonal antibodies are produced by cells that are all derived from a single antibody -producing cell. This means that they are exceptionally pure and highly specific. This specificity allows them to be used in humans and in animals for disease diagnosis and treatment.

Producing MABs

  • Producing MABs requires first immunizing an animal, usually a mouse. The mouse is injected with a specific antigen and the animal's immune system recognizes and respond to it by producing antibodies.
  • After the mouse’s immune system develops antibodies against the antigen, antibody producing B-cells are removed from the mouse’s spleen.
  • These cells are then fused with myeloma cells to make them immortal. The fused cells is called a hybridoma. These cells secrete MABs.