Republic of the Philippines

ISABELA STATE UNIVERSITY

City of Ilagan Campus

IMMUNOLOGY (BS PSYCH 2)

ANTIBODIES
Prepared by: Charles Z. Ariola Jr., MSN, LPT

OBJECTIVES:

1. Describe with the aid of a simple diagram the immunoglobulin molecule, identifying the antigen-binding site (Fab)
and Fc portions of the molecule.

2. Briefly describe the properties of the antigen-binding site.

3. Distinguish between antibody affinity and avidity.

4. List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their
individual structure.

Overview

What is an antibody?
• A protein that is produced in response to an antigen
• Binds specifically to the antigen
• Form the class known as IMMUNOGLOBULINS
• Large family of soluble GLYCOPROTEINS
• Produced by B lymphocytes
• Found in serum
• Deficiency is life threatening
• After binding antigen, initiate secondary effector functions
- Complement activation
- Opsonisation
- Cell activation via specific antibody-binding receptors (Fc receptors)

Structure
• symmetrical
• Each chain has amino and carboxyl terminal
• Chains held together by disulphide bridges
• Different antibodies therefore have different charges

The discovery of antibody structure

• Rodney Porter
• Limited the digestion of gamma-globulin with purified
papain, which produced 3 fragments in equal amounts
1
 • 2 fragments had antigen binding activity (Fab)
• The third did not, but formed protein crystals (Fc)

Flexibility
• There is a hinge in the antibody which allows flexibility
between the two Fab

• This allows the angle between the two antigen binding sites to change
angle depending on the proximity of cell surface determinants, i.e.
how close together antigens are

Note:
• Both light and heavy chains can be divided into variable (where the
sequences are different) and constant (same sequence) regions
• Each IG (immunoglobulin/antibody) domain, e.g. variable light, has
INTRAMOLECULAR DISULPHIDE BONDS to maintain their specific 3D
structure required for antigen binding
• Many cell surface proteins also have IG-like domains, and are said to belong to the IG super family
• The constant region binds to Fc receptors, which can lead to cell activation, e.g. NK cells (secondary effector
functions in immune response)

Antigen-binding site

• Antigen binding occurs at 3 HYPERVARIABLE regions, known as COMPLEMENTARITY DETERMINING REGIONS

(CDR’s)
• These have specific residue positron numbers
• The region of binding is a large undulating 3D structure (~750A = 10 -10m), so is highly specific and there are a
significant number of interactions between the antibody and antigen surface

Forces involved
• Hydrogen bonds
• Ionic bonds
• Hydrophobic interactions
• Van der Waals interactions

Are non-covalent, therefore are relatively weak. This means that in order to have a HIGH AFFINITY, there can only be
a short distance between the antigen and antibody, highly complementary nature, and a significant number of
interactions.

Antibody Affinity
The strength of the total non-covalent interactions between a single antigen binding site and a single epitope on
the antigen.

The affinity association constant K can be calculated:

K varies from 104 to 1011 L/mol

Antibody Avidity
The overall strength of multiple interactions between an antibody with multiple binding sites and a complex antigen
with multiple epitopes

• This is a better measure of binding capacity in biological systems

• Monovalent interactions have a low affinity
• Bivalent interactions have a high affinity
2
 • Polyvalent interactions have a very high affinity

Cross-Reactivity
Antibodies elicited in response to one antigen can also recognise a different antigen, for example:

1. Vaccination with cowpox induces antibodies which are able to recognise smallpox

2. ABO blood group antigens are glycoproteins on red blood cells. Antibodies made against microbial agents on
common intestinal bacteria may cross-react with the glycoproteins, which poses a problem for blood
transfusions.

Isotypes and Allotypes

• Isotypes are antibodies who are present in everybody, with a constant region.
• Allotypes are antibodies that contain single amino acid mutations, giving allelic polymorphisms which vary in
the population

Immunoglobulin Classes

Different classes of antibodies differ in the constant regions of their heavy chains
Class IgG IgA IgM IgD IgE
Heavy chain γ α µ Δ ε
CH Domains 3 3 4 3 4
Light Chain κ/λ κ/λ κ/λ κ/λ κ/λ

IgG and IgA have subclasses

Class IgG IgA
Subclass IgG1, IgG2, IgG3, IgG4 IgA1, IgA2
H chain γ1, γ2, γ3, γ4 Α1, α2

IgG IgA IgM

• γ heavy chain
• most abundant
• monomer
• 4 subclasses- variability mainly
located in hinge region and
effector function domains
• Actively transported across the
placenta- protection from
mother to newborn
• Found in Blood and
extracellular fluids
• Major activator of classical
complement pathway (mainly
IgG1 and IgG3)
• Subclasses decrease in
proportion from 1-4
•  heavy chain
• Second most abundant
• monomer (blood)
• dimer (secretions)
• Major secretory
immunoglobulin
• Protects mucosal surfaces from
bacteria, viruses and protozoa
• Secretory IgA: joined by J chain
and secretory component.
Plasma cell secretes dimeric
form without secretory. This
bonds to poly-Ig receptor and is
endocytosed and secreted into
lumen. The poly-Ig receptor is
cleaved and becomes the
secretory component
• µ heavy chain
• pentameric
• 5 monomers joined by J chain
(10 x Fab)
• mainly confined to blood
(80%)
• first Ig synthesised after
exposure to antigen (primary
antibody response)
• multiple binding sites
compensate for low affinity
• efficient at agglutination of
bacteria
• activates complement

• The secretory component

protects IgA from being
degraded in the lumen, by
proteases etc

IgD IgE
3
 • δ heavy chain
• extremely low serum concentrations
• least well characterised
• surface IgD expressed early in B cell
development
• involved in B cell development and activation
•  heavy chain
• present at extremely low levels
• produced in response to parasitic infections and
in allergic diseases
• binds to high affinity Fc receptors of mast cells
and basophils

cross-linking by antigen triggers mast cell activation and

histamine release

Selective Immunoglobulin Distribution

• IgG and IgM in blood

• IgG in extracellular fluid
• Dimeric IgA in secretions across epithelia, including breast milk
• Maternal IgG in foetus via placental transfer
• IgE with mast cells below epithelium
• Brain devoid of antibodies

Antibody effector functions

Effector Function Activity Example Antibody
Class
Neutralization of toxins Inhibits toxicity Tetanus toxin Mainly IgG
Neutralization of viruses Inhibits infectivity Measles Mainly IgG
Neutralization at body Inhibits infectivity of Polio Secretory
surfaces bacteria & viruses Salmonella IgA
Agglutination Ag-Ab complexes/ Bacteria & RBC IgM, IgG
Lattice formation
Opsonization Promotes Bacteria, fungi IgG
phagocytosis
Complement activation Classical Pathway Ag-Ab complex IgM, IgG

Mast Cell sensitisation & Expulsion Parasites IgE

triggering Hypersensitivity Pollen
NK cell Cytotoxicity Virus infected Mainly IgG
ADCC cells

Summary

Antibodies:
 In defence
- targeting of infective organisms
- recruitment of effector mechanisms
- neutralisation of toxins
- removal of antigens
- passive immunity in the new born
 In medicine
- levels used in diagnosis and monitoring
- pooled antibodies for passive therapy/protection
 In laboratory science
- vast range of diagnostic and research applications