MICR 304 Immunology &

Serology

MICR 304 Immunology &

Serology

Lecture 7B Antibodies Part IChapter 3.1 – 3.9

Lecture 7B Antibodies Part IChapter 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

Innate Adaptive

Cells PhagocytesEpithelial Cells

NK Cells

Lymphocytes(B-Ly, T-Ly)

Effector Molecules

ComplementAntimicrobial (Poly)PeptidesAntimicrobial

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

SpikesDisappear

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

Pro

tru

din

g S

urf

ace

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 inAntigen-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

LightChain

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