MICR 304 Immunology & Serology Lecture 7B Antibodies Part I Chapter 3.1 – 3.9 Lecture 7B Antibodies Part I Chapter 3.1 – 3.9

Overview of Today’s Lecture Basic structure of antibodies Generation of antibodies Structural variations in the constant regions

Key Players in Immunology InnateAdaptive Cells Phagocytes Epithelial Cells NK Cells Lymphocytes (B-Ly, T-Ly) Effector Molecules Complement Antimicrobial (Poly)Peptides Antimicrobial lipids? Antibodies

What is an Antibody? Glycoprotein Binds to antigen Consists of 2 heavy chains (  ) and 2 light chains (  ) connected by disulfide bonds Variable domains (antigen binding) Constant regions (effector function)

Antibody Classes Determined by the type of constant chain   : IgG   : IgM   : IgA   : IgE   : IgD Used to explain principle make up of an ab

Basic Structure of an Antibody Molecule 2 light and 2 heavy chains Disulfide bonds Hinge region N-terminus: variable, antigen binding C-terminus: constant region, effector function

Proteolytic Cleavage of the Antibody Molecule Variable regions intact Antigen binding without effector function Constant regions intact Crystallizable Variable regions intact and additional amino acids Antigen binding without effector function Single site Multiple sites pepsin

Antibody Molecules are Flexible Spikes Disappear after treatment with pepsin Antibody arms are joined by a flexible hinge.

The Structure of Immunoglobulin Constant and Variable Domains Immunoglobulin superfamily domain (4 strand + 3 strand) of (4 strand + 5 strand) Found in many other proteins Anti-parallel  strands forming 2  sheets that assume a  barrel structure V domain is larger!

Localized Regions of Hypervariable Amino Acid Sequences Form the Antigen Binding Site Hypervariable regions (HV1- 3) of light chain and heavy chain create the antigen binding site –Surface complementary to the antigen –Complementarity- determining regions (CDRs) –Combinatorial diversity Framework regions (FR1-4) provide structural frame work Variability Plot Compares aa sequences of many different V regions

Hypervariable Regions Lie in the Discrete Loops Different aa in different CDRs form different surfaces and bind different antigens.

Antigen Binding Sites Assume Varying Shapes Pocket Groove Extended Surface Protruding Surface

Hapten Antigen that is too small to elicit antibody response by itself When coupled to carrier protein antibodies can be generated Once antibodies are generated they can recognize and bind to uncoupled hapten

Epitop Domain on antigen which actually binds to antibody binding site Antigenic determinant Two types of epitops –Conformational discontinuous aa sequence –Linear continuous aa sequence Y Y Y Y

Non-Covalent Forces in Antigen-Antibody Interactions (Salt bridges) (Electric dipoles) Short distance Overall interaction

Antibody-Antigen Interactions Never covalent! Reversible Depends on the actual antigen and antibody Single amino acid changes can cause loss of recognition

Lysozyme Heavy Chain Light Chain Glutamine residue (Ly) making hydrogen bonds

Active Learning Exercise How can the interaction between antibody and antigen be disrupted and antigen released from the antibody?

Today’s Take Home Message The IgG antibody molecule consists of 2 identical light chains and 2 identical heavy chains connected by disulfide bridges. Each chain contains at the N-terminus a variable region for antigen binding and at the C-terminus a constant region for effector function. The variable regions of the light and heavy chain form antigen binding sites. Each antibody molecule has two identical antigen binding sites allowing for cross linking of antigen. Hypervariable loops comprise the complementarity determining regions that form the surface interacting with the antigen