Faculty of Allied Medical Sciences
Clinical Immunology & Serology Practice
(MLIS 201)
IMMUNOGENS & ANTIGENS
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: elgreatlyem@hotmail.com
OBJECTIVES:
BY THE END OF THE LECTURE THE STUDENTS WILL BE ABLE TO
1. To Define the immunogen, antigen, and
hapten
 2. To describe the factors influencing
immunogenicity
 3. To characterize antigenic determinants
 4. To introduce the concept of hapten-carrier
conjugates
 5. To know the types of antigens

COMPONENTS OF ANTIGEN-ANTIBODY
REACTIONS
1. Immunogens & Antigens
2. Immunoglobulins (Antibodies)
3. Complement
Definitions

IMMUNOGEN:
A substance, usually foreign, which when introduced into an animal
can generate a specific immune response and binding specifically
to immune components.

ANTIGEN:
A substance, usually foreign, which may not always induce an
immune response in all animals, however is capable of binding
specifically to immune components when present.
6
Definitions (cont.)
Depending on the nature of the immune response the
substance may be classified as:



an Immunogen/ antigen – a protective effect
a Tolerogen – depending on the nature of the tolerogen,
its effect on the individual may be harmful, beneficial or
neutral.
an Allergen – generally the effect is harmful.
7
[1] Immunogenicity
“Ability of a substance to stimulate the production of
antibodies and/or cell-mediated immune response”
Immunogens can be classified into:
Complete immunogen
Incomplete immunogen, also known as hapten
Haptens = molecules that can bind to antibodies or
surface receptors (antigenic). However, they cannot
induce specific immune response alone (non
immunogenic)
8
HAPTENS
Low mw molecules.
e.g. Antibiotics, drugs
Not immunogenic, unless...
HAPTENS
HAPTENS
Low mw molecules.
E.g. Antibiotics, drugs
Not immunogenic, unless...
conjugated to high mw compounds
(carriers) to gain immunogenicity
CARRIER
HAPTENS
[2] Antigenicity
“Ability of a substance to combine
specifically with the final product of the
immune responses” (i.e. antibodies and/or
cell surface receptors)
All immunogenic molecules are antigenic
but
Reverse is not true
11
What affects
Immunogenicity/Antigenicity ?
1) Foreignness
Ability of lymphocyte to recognise self antigen
occurs during MATURATION
Any molecule not exposed to immature lymphocytes during this
critical period = nonself or foreign
The greater the phylogenetic distance between two species, the
greater the genetic and antigenic disparity between them
Degree of immunogenicity depends on degree of foreignness
13
FOREIGNNESS (CONT)
Response to
Immunization
Cow
NO
Bovine serum
Albumin (BSA)
Sheep
Yes
++
Chicken
Yes
+++++
Bovine serum albumin (BSA) = more immunogenic
in chicken than sheep
FOREIGNESS & ANTIBODY PRODUCTION:
Rabbit
Antibody response
to human serum
Chimpanzee
Time
15
2)
Size (i.e.Molecular Weight)
molecular weight
<1000D-Not Immunogenic (penicillin)
1000-6000D May or May not be immunogenic (insulin)
>6000D-Generally immunogenic (albumin, Tetanus toxin)
Rabbit
Antibody response
to Antigen
0
1.0 2.0 5.0 10 25
Molecular Weight x103
100
500
16
3)CHEMICAL COMPEXITY & HETEROGENEITY
Proteins
Carbohydrates
glycoproteins
Lipids
Nucleic Acids
almost always immunogenic
potentially immunogenic
usually immunogenic
poorly immunogenic
poorly immunogenic
Chemically complexity (cont.)
The more chemically complex the substance the
stronger the immunogenicity
homopolymers not generally immunogenic Ex:
Polylysine-30,000 D, Poly-D-Glutamic acid-50,000D
 Primary, secondary, tertiary and quaternary
structures of proteins affect immunogenicity
CHEMICAL COMPLEXITY (CONT.)
Levels of Protein Structure
CHEMICAL COMPLEXITY (CONT.)





ANTIGENIC DETERMINANT OR EPITOPE :
A specific small structural shape on the surface of an
immunogen or antigen, and usually limited to those portions
of the antigen that are accessible to antibodies
It physically interact with paratopes (combining sites) of Abs
Therefore actually "determine" antigen specificity
Epitopes may be Linear or Discontineous
Ag
Epitope
A model of a substance
with four epitopes
NUMBER OF ANTIGENIC DETERMINANTS
(i.e. epitopes)
Small mass = fewer
antigen determinants
Large mass = greater number of
antigen determinants
21
4) Degradability
Macromolecules that cannot be degraded and presented by
APC are poor immunogens
22
5) Genotype Of The Recipient Animal
Mouse 1
Mouse 1
Ag X
Ag X
 Level of Ab
 Level of Ab
Cross
F1 Generation
Ag X
Intermediate Level of Ab
23
6) Immunogen Dosage
Low dose  failure to activate enough lymphocytes or induces
Nonresponsiveness i.e. Low dose tolerance
High dose  lymphocytes enter nonresposnsivess state
i.e. High dose tolerance
Single dose usually not enough to induce reaction
Repeated doses over a period of weeks to induce a
strong immune response
24
Repeated administration of antigen is required to stimulate
a strong immune response
7) Route Of Administration
Route of administration determines which immune organ and
cell population will be deployed
Intravenous administration  carried first to spleen
Subcutaneous = Move first to lymph nodes; strongest response
Gastrointestinal route – GALT; may induce tolerance
Intranasal route – MALT; may elicit allergic responses
This generated differences due to differences in residing
populations of cells
Route of Administration: SC>IP>IV>Intragastric
26
8) ENHANCING IMMUNOGENICITY
BY ADJUVANTS

Adjuvants are “Substances that enhance
immunogenicity of antigen when mixed and
injected with it”

Adjuvants used to boost immune response when:
immunogen has low immunogenicity
Small amounts of immunogen are available






Examples:
Incomplete Freund’s adjuvant : oil-in- water emulsion
Complete Freund’s adjuvant: oil-in-water emulsion plus
dead Mycobacteria
Aluminum hydroxide gel
TYPES OF ANTIGENS ACCORDING TO
THEIR ORIGINS :

1-Exogenous antigens
Exogenous antigens are antigens that have
entered the body from the outside by inhalation,
ingestion, or injection.
e.g. Viruses, bacteria, food allergens,
 2-Endogenous
antigens
Endogenous antigens are antigens that
have been generated within the cell, as a
result of normal cell metabolism, or
because of viral or intracellular bacterial
infection.
 3-Autoantigens
An autoantigen (self antigen)is usually a normal self
protein or complex of proteins (and sometimes DNA
or RNA) that becomes immunogenic due to break
down of normal immunological tolerance for such an
antigen.
When recognized by the immune system of patients
it results in an autoimmune disease.
 4-Tumor
antigens
Tumor antigens are those antigens that
are presented on the surface of tumor
cells.
e.g. Alpha fetoprotein, Carcinoembryonic antigen, prostate specific
antigen
FACTORS INFLUENCING IMMUNOGENICITY
(SUMMARY)
1-Foreigness :
Foreign substances are immunogenic
2- Molecular size:
High molecular weight increase immunogenicity
3- Chemical structure complexity:
High complexity increase immunogenicity
4- Route of administration:
Parenteral routes are more immunogenic to oral route
SC>IP>IV>Intragastric
FACTORS INFLUENCING IMMUNOGENICITY
(SUMMARY) (CONT.)
5- Degradability of the immunogen
6-Genotype of the recipient
7- immunogen dose:
Appropriate dose
Low dose
High dose
optimum antigenicity
low- zone tolerance
high-zone tolerance
8- Adjuvant:
Substance when injected with an immunogen
enhance immunogenicity
IMMUNOGLOBULINS:
STRUCTURE & FUNCTIONS
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: elgreatlyem@hotmail.com
TEACHING OBJECTIVES:
1. To discuss the general properties of all
immunoglobulins
 2. To describe the basic structure of
immunoglobulins
 3. To relate immunoglobulin structure with
function
 4. To define immunoglobulin classes and
subclasses, types and subtypes
 6. To describe the structures and properties of
immunoglobulin classes

IMMUNOGLOBULINS
Definition:
Glycoprotein molecules in serum and tissue fluids
that are produced by plasma cells in response to an
immunogen and which function as antibodies.
They react specifically with antigen
Five classes of Antibodies:
IgG
IgM
IgA
IgD
IgE
Serum Protein Electrophoresis
-
+
globulins
Immune serum
γ
β
α1
Ag adsorbed serum
Mobility
α2
Amount of protein
albumin
Serum Protein Electrophoresis
Amount of protein
albumin
globulins
-
Mobility
+
TWO FORMS OF IMMUNOGLOBULIN
Membrane-bound receptor
Soluble antibody
IMMUNOGLOBULINS
MEMBRANE-BOUND AND SOLUBLE RECEPTORS
Structure of Immunoglobulin
The Four-Chain Basic Unit

Immunoglobulins are composed of two identical heavy (H) and
two identical light (L) polypeptide chains. Each H and L chain
has an amino-terminal variable (V) region and a carboxylterminal constant (C) region.
IMMUNOGLOBULINS
FOUR-CHAIN BASIC UNIT
VL
CL
CL
VL
IMMUNOGLOBULINS: STRUCTURAL REGIONS
IMMUNOGLOBULINS: STRUCTURAL DOMAINS
IMPORTANT TERMS
Antibody – immunoglobulin secreted by B cells
 Antigen (antibody generator) – any substance capable of

Binding with specific antibody
Epitope – region of the antigen recognized by an antibody
 Paratope – region of the antibody that binds the epitope

Paratope
Antibody is a flexible molecule
(Hinge Region)
IMMUNOGLOBULIN CLASSES AND
SUBCLASSES
Immunglobulin molecules are divided into distinct classes
and subclasses in terms of the differences in amino
acid sequence of constant region of heavy chains into:
1- 5 distinct classes i.e. γ,α, μ,δ, and ε chains.
2- Subclasses:

IgG has a family of subclass, IgG1, IgG2, IgG3, IgG4.

IgA is divided into two subclasses, IgA1 and IgA2.
Immunoglobulin classes
IgG subclasses
Light Chains

All light chains have protein molecular
weights of approximately 23,000.

Divided into two distinct types, namely
κ (Kabba) chain and λ (Lumbda) chain
with a ration of 2:1
Functions of the domains on Ig：
VH, VL
antigen binding sites
CH1～3, CL
genetic markers of Ig
CH2(IgG), CH3(IgM)
C1q binding sites
CH2～CH3(IgG)
binding to placenta
CH3(IgG)
Fcγ Receptor binding site
CH4(IgE)
Fcε Receptor binding site
FUNCTIONS OF IMMUNOGLOBULINS
(FAB)

Recognition of antigen

Activation of complement

Opsonization

Neutralization

Antibody-dependent cell-mediated
cytotoxicity,ADCC

Mediate hypersensitivity type I
SPECIFIC ANTIGEN BINDING WITH ANTIGEN BINDING SITE
Paratope
Complement activation
Opsonization
ADCC
PROPERTIES OF IMMUNOGLOBULINS
Property
IgG
Heavy chain
symbol
γ
α
µ
ε
δ
Molecular
weight
150
KDa
170-400
KDa
900
KDa
190
KDa
180
KDa
Percentage
in serum
75 %
15 %
10 %
0.004 %
% 0.2
Complement
fixation
Yes
No
Yes
No
No
Transplacental
passage
Yes
No
No
No
No
Opsonization
Yes
No
No
No
No
IgA
IgM
IgE
IgD
EFFECTOR FUNCTIONS OF IGG







Major serum Ig
Major Ig in extravascular spaces
Opsonization of bacteria for phagocytosis by
macrophages and neutrophils
Neutralization of toxins and microbes
Activation of the classical pathway of complement
Antibody dependent cell mediated cytoxicity (ADCC) by
NK cells.
Transfer of maternal antibody across the placenta (i.e.
The only Placental transfer Ig)
IgA
EFFECTOR FUNCTIONS OF IGA
-Found in serum and body secretions (Tears, saliva,
gastric and pulmonary secretions)
 Major secretory Ig on Mucous surfaces gives Local
Immunity by coating bacteria or viruses preventing
their adherence to mucosal cells and is chiefly
derived from local synthesis and is mainly dimeric
 Does not fix complement (unless aggregated)
 Present in colostrum and mother milk protect newly
born.
 Neutralization of bacteria, viruses and toxins
 In humans, the IgA in serum is chiefly monomeric,
comprising ~90% IgA1 and 10% IgA2
EFFECTOR FUNCTIONS OF IGM
Elevated level of IgM usually indicated either
recent infection or recent exposure to the
antigen.
 Isohemagglutinins (Blood group antibodies).
 Antigen receptor of B lymphocyte
 Activation of classical pathway of complement
 IgM is the first antibody produced in a primary
response to an antigen.

EFFECTOR FUNCTIONS OF IGD
IgD,
together with IgM, is expressed
by mature B cells as a B cell surface Ig.
Present in very small amount in serum
Does not bind complement
IgE
EFFECTOR FUNCTIONS OF IGE
 Least
common serum Ig (Binds to basophils and
mast cells)
 Mediate immediate hypersensitivity reaction.
 Mediate antibody dependent cell mediated
cytotoxicity (ADCC) to parasites involving
eosinophils Binds to Fc receptor on eosinophils
 Does not fix complement
ENZYMATIC DIGESTION PRODUCTS OF
IMMUNOGLOBULINS
ANTIBODY PRODUCTION

Polyclonal antibodies: antibodies produced
against antigen by multiple B cells, have
different paratopes

Monoclonal antibodies: antibodies secreted
from a single B cell, have identical paratopes
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: elgreatlyem@hotmail.com
TEACHING OBJECTIVES:
1. To know the general properties of complement
system
2. To know different components of complement
3. Understand different pathways of complement
activation.
4. Know the biological Functions of C activation
products.
73
Complement: history
Discovered in 1894 by Bordet
 Sheep antiserum could lyse Vibrio
cholera
 Its lysing activity was destroyed when
heated at 56C° for 30 min
 Added fresh serum (with no cholera
antibodies) to heated serum restored
the lysing ability!!!!
 Complement activity in serum that
completes the action of antibodies.

The complement system
Important effector in both innate and acquired immunity
Heat-labile serum proteins that ‘complemented’ antibody
activity
is present in serum and all tissue fluids except urine and
CSF
Over 30 circulating and membrane-bound proteins
synthesized maily in liver and other cells : immune and
epithelial
There are three activation pathways: classical, alternative
and MBL
Acts as a cascade (one event must occur before another
takes place) i.e. Activation of complement is essentially a
proteolytic chain reaction
Cascade:
Many of the components are enzymes that become
activated when cleaved into two peptides :
One peptide binds to the immune complex and becomes a
functional part of it
The other peptide diffuses away and can become an
inflammatory mediator (binds to a receptor)
Letter “b” is usually added to the larger, membranebinding, peptide
and “a” to the smaller peptide (e.g., C3b/C3a, C4b/C4a,
C5b/C5a),
EXCEPT C2 (the larger, membrane-binding moiety is C2a;
the smaller on is C2b)
Activated component are usually over-lined: e.g. C1qrs
Proteins of the complement
system (nomenclature)
C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9
factors B, D, H and I, properdin (P)
mannose binding lectin (MBL), MBL associated
serine proteases (MASP-1 MASP-2)
C1 inhibitor (C1-INH, serpin), C4-binding
protein (C4-BP), decay accelerating factor
(DAF),
C1 receptor (CR1), protein-S (vitronectin)
Complement activation
1-classical pathway which is activated
by Ab bound to Ag
 2-the lectin pathway activated by
carbohydrates (Mannose & Fucose)
 3-Alternative pathway activated in the
presence of various microbial pathogen

Pathways of complement
activation
CLASSICAL
PATHWAY
antibody
dependent
LECTIN
PATHWAY
ALTERNATIVE
PATHWAY
antibody
independent
Activation of C3 and
generation of C5 convertase
activation
of C5
LYTIC ATTACK
PATHWAY
Components of the Classical
Pathway
C3
C1 complex
C4
Classical Pathway
Generation of C3-convertase
Classical Pathway
Generation of C3-convertase
C4b2a is C3 convertase
C4b
Classical Pathway
Generation of C5-convertase
C4b2a3b is C5 convertase;
it leads into the Membrane
Attack Pathway
C4b
C3b
Components of mannose-binding
lectin pathway
M
M
Bacteria
MBL
MASP1
Mannose-binding lectin pathway
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1
MBL
M M
Bacteria
Components of the
alternative pathway
C3
C3b stabilization and
C5 activation
C3b finds an activator
(protector) membrane
This is stable C5 convertase
of the alternative pathway
C3b
b
C3 b
C5-convertase of the two
pathways
C5-convertase of the
Classical and lectin
Pathways
C3b
C4b
C5-convertase of the
Alternative Pathway
C3b
C3b
Lytic pathway
Generation of C5 convertase
leads to the activation of the
Lytic pathway
Components of the lytic pathway
C7
C6
C
9
Lytic pathway
C5-activation
b
C4b
C3b
Lytic pathway
assembly of the lytic complex
C6
C7
b
Lytic pathway:
insertion of lytic complex into cell membrane
C6
C7
CC C C
C9 9 9 9C
9C C C9
9 9 9
b
MAC PORES
C9 complexes on RBC
Complement Activation
Classical Pathway
MBL Pathway
C1
C4
C2
C3
Alternative pathway
C5
C6
C7
C8
C9
Membrane damage
Classic And Alterenative
pathways
Classic Pathway
Alternative pathway
* Specific acquired immunity
* Non-specific innate immunity
* Initiated by antibody
* Bacterial endotoxin, capsule
* Interaction of all components
* Properdin system not involved
* C1, C4, C2 are by-passed
* Properdin system is involved
Biological functions of
Complement
Lysis of bacteria and infected cells
Opsonization to enhance phagocytosis
Phagocyte attraction and activation
Clearance of immune complexes
Regulation of antibody responses
Inflammation and anaphylaxis
The complement system
Figure 2-18
Study Questions:
Write a short note about Factors
Influencing Immunogenicity .
 Define: Epitope – Paratope.
 Compare between: Classic and
Alterenative pathways of complement
activation.

99
‫‪Assignment:‬‬
‫‪Immunoglobulins Classes and‬‬
‫‪subclasses‬‬
‫‪‬‬
‫‪‬‬
‫‪‬‬
‫‪‬‬
‫‪‬‬
‫‪100‬‬
‫امال عرابى الكردى‬
‫امل رأفت محمد‬
‫امنية بدوى احمد‬
‫امنية صبرى محمد‬
‫امنية عبد الحى عبد الوهاب‬
‫‪‬‬
Thanks