Antibodies (Ab's) are a class of proteins secreted by B-Lymphocytes (B-Cells) in response to the presence of a foreign substance or Angtigen. A very wide variety of antigens will stimulate the production of antibodies; virtually any foreign protein, many types of carbohydrates and even some nucleic acids and nucleotides. Fig. 7-32,33,34,35
- typically a large molecule or supramolecular assembly containing multiple
- epitopes&endash;structures to which antibodies bind; may be a group of amino acids (for a protein antigen) or a small organic group called a hapten. Haptens by themselves are not large enough to generate production of Abs, but if covalently bound to a large carrier protein and injected into an animal,
Abs which bind the hapten will be produced.
- When an antigen (Ag) is injected into an animal (e.g. rabbit, mouse, goat etc.), the B-Cells of the animal produce a large repertoire of Ab's against the various epitopes of the Ab. Furthermore, different B-cells will produce different Ab's that bind to a particular epitope, these are called polyclonal Ab's.
- monoclonal Ab's (see below and Box 7-4) are produced by special fused hybridoma cells grown in culture
- Hybridoma Cells-mouse lymphocytes that produce Ab's fused with mouse myeloma cells to produce cells that can grow in cell culture
- Each line of hybridoma cells produces one species of Ab against a particular epitope
B. Basic Structure - H2L2 Fig. 7-33-35
1. 2 identical heavy chains (H-chains) 53-75 kD
2. 2 identical light chains (L-chains) ~23kD
3. connected by noncovalent interactions and by disulfide bonds
IgG Antibody Structure: light chains are in green and dark blue, heavy chains in light blue and orange, disulfide bonds in yellow spacefill, carbohydrate in red wireframe. Click on the image to download the three-dimensional structure file for display with RasMol.
C. Five classes of Ab determined b y type (class) of H-chain - Table 7-2
1. IgA (a- H-chain): H2L2; (H2L2)2; Fig. 34-19
2. IgD (d-H-chain): H2L2
3. IgE (e-H-chain): H2L2
4. IgG (g-H-chain): H2L2
5. IgM (m-H-chain): (H2L2)5
D. Different Classes of Ab have different locations and functions
1. IgA - intestinal tract, tears, sweat, saliva, milk
2. IgD - blood
3. IgE - blood; protects agianst parasite --> role in allergic reactions
4. IgG - blood and interstitial fluid; most common type of Ab
5. IgM - blood; 1st class of Ab produced
E. H-chains and L-chains have Variable and Constant Sequences Fig 7-34
1 Variable Regionsa. at N-termini -- approx. 110 amino acid residues
b. hypervariable regions -- 3 each on H-chain and L-chain; called CDR for Complementary Determining Regions
2. Constant Regions Fig. 7-34a. H-chain has 3: CH1, CH2, CH3
b. L-chains has 1
F. Fragments produced by proteolysis with Papain
1. Fab: two fragments which bind Ag are produced from each (H-L)2
2. Fc: one produced for each Ab, does not bind Ag but can be crystallized
3. cleavage at Hinge region connecting the three types of fragments; actually more like a tether
G. Precipitin Rreaction - Fig. 7-37
1. Requires at least a divalent Ab (Fab doesnt work)
2. Maximal when [Fab] ~ [Ag]
3. basis for early tests of Ab binding
Antibody Fab fragments complexed with peptide Epitopes: Left - two Fab fragments, the one in front has VH-chain in green and VL-chain in yellow with peptide Epitope in Orange (Fab in back has VH and VL chains in dark and light blue and Epitope in aqua).
Right - one Fab fragment with VH and VL chains in darke blue and aqua and peptide epitope (part of hemagglutinin) in yellow; all displayed in spacefill. Click on the images to download the three-dimensional struture files for display with RasMol.
H. Ag-Binding sites are complementary to Ag Epitope structure - Fig. 7-36
1. binding site is in a cleft at the end of Fab formed by VL and VH
2. hypervariable regions determine size, shape, and binding of Ab-epitope
3. binding interactions are: van der Waals forces, hydrophobic interactions, and H-bonds.
4. Epitopea. for a protein is typically 14-16 surface residues; can come from distant parts of 1° structure
b. entire Ag surface may be antigenic containing many different epitopes