Faculty of Allied Medical Sciences

Clinical Immunology & Serology Practice

(MLIS 201)

IMMUNOGENS & ANTIGENS

Prof. Dr. Ezzat M HassanProf. of Immunology

Med Res Inst, Alex UnivE-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

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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.

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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.

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[1] Immunogenicity

“Ability of a substance to stimulate the production of antibodies and/or cell-mediated immune response”

Immunogens can be classified into:Complete immunogenIncomplete 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)

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

HAPTENS

CARRIER

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[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 antigenicbut

Reverse is not true

What affects Immunogenicity/Antigenicity ?

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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

Degree of immunogenicity depends on degree of foreignness

The greater the phylogenetic distance between two species, the greater the genetic and antigenic disparity between them

FOREIGNNESS (CONT)

Bovine serum Albumin (BSA)

Response toImmunization

NO

Yes++

Cow

Sheep

Chicken Yes+++++

Bovine serum albumin (BSA) = more immunogenic in chicken than sheep

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FOREIGNESS & ANTIBODY PRODUCTION:

Time

Antibody response to human serum

Rabbit

Chimpanzee

16Molecular Weight x103

Antibody response to Antigen

Rabbit

0 1.0 2.0 5.0 10 25 100 500

molecular weight<1000D-Not Immunogenic (penicillin) 1000-6000D May or May not be immunogenic

(insulin)>6000D-Generally immunogenic (albumin,

Tetanus toxin)

2) Size (i.e.Molecular Weight)

3)CHEMICAL COMPEXITY & HETEROGENEITY

Proteins almost always immunogenic

Carbohydrates potentially immunogenic glycoproteins usually immunogenic

Lipids poorly immunogenic Nucleic Acids 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

AgEpitope

A model of a substance with four epitopes

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NUMBER OF ANTIGENIC DETERMINANTS(i.e. epitopes)

Small mass = fewer antigen determinants

Large mass = greater number of antigen determinants

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4) Degradability

Macromolecules that cannot be degraded and presented by APC are poor immunogens

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5) Genotype Of The Recipient Animal

Mouse 1 Mouse 1

Level of Ab Level of Ab

Cross

F1 Generation

Ag X Ag X

Intermediate Level of Ab

Ag X

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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

Repeated administration of antigen is required to stimulate a strong immune response

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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

This generated differences due to differences in residingpopulations of cells

7) Route Of Administration

Route of Administration: SC>IP>IV>Intragastric

Gastrointestinal route – GALT; may induce tolerance

Intranasal route – MALT; may elicit allergic responses

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, Carcino-

embryonic 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 optimum

antigenicity Low dose low- zone tolerance High dose high-zone tolerance

8- Adjuvant: Substance when injected with an immunogen enhance immunogenicity

Take so

me rest

IMMUNOGLOBULINS:STRUCTURE & FUNCTIONS

Prof. Dr. Ezzat M HassanProf. of ImmunologyMed Res Inst, Alex UnivE-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

Immune serum

Ag adsorbed serum

+-

albumin

globulins

Mobility

Am

ount of protein γ β α2 α1

Serum Protein Electrophoresis

Am

ou

nt

of

pro

tein

Mobility

albumin

globulins

+-

Serum Protein Electrophoresis

TWO FORMS OF IMMUNOGLOBULIN

Membrane-bound receptor

Soluble antibody

IMMUNOGLOBULINS MEMBRANE-BOUND AND SOLUBLE RECEPTORS

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 carboxyl-terminal constant (C) region.

Structure of Immunoglobulin The Four-Chain Basic Unit

IMMUNOGLOBULINSFOUR-CHAIN BASIC UNIT

VLCL

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

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

Light Chains

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 siteCH4(IgE) Fcε Receptor

binding site

Functions of the domains on Ig：

FUNCTIONS OF IMMUNOGLOBULINS

Recognition of antigen (FAB) 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

IgA IgM IgE IgD

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

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 HassanProf. of ImmunologyMed Res Inst, Alex UnivE-mail: elgreatlyem@hotmail.com

TEACHING OBJECTIVES:

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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.

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.

Complement: history

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

The complement system

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, membrane-binding, 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 complementsystem (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

CLASSICALPATHWAY

ALTERNATIVEPATHWAY

activationof C5

LYTIC ATTACKPATHWAY

antibodydependent

LECTINPATHWAY

antibodyindependent

Activation of C3 andgeneration of C5 convertase

Components of the Classical Pathway

C4C2 C3

C1 complex

Ca++

C1r C1s

C1q

Ca++

C1r C1s

C1q

C4

C4a

b

Classical Pathway Generation of C3-convertase

Classical Pathway Generation of C3-convertase

C4b

Mg++

C4a

Ca++

C1r C1s

C1q

C2

C2ba

C2a

C4b2a is C3 convertase

Classical Pathway Generation of C5-convertase

C4b

Mg++

C4a

Ca++

C1r C1s

C1q

C2b

C2a

C3

C3a

b

C4b2a3b is C5 convertase; it leads into the Membrane

Attack Pathway

Components of mannose-binding lectin pathway

Bacteria

M

C4

MBL C2 MASP1

MASP2

M

Mannose-binding lectin pathway

Bacteria

C4

MBL

C4b

C4a

C4b

C2

C2b

C2a

C2a

C4b2a is C3 convertase; it will lead to the generation of

C5 convertaseMASP1

MASP2

M M

Components of thealternative pathway

C3 B

D

P

C3b stabilization andC5 activation

C3b

C3b finds an activator (protector) membrane

C3

C3a

bB

D

b

PThis is stable C5 convertase

of the alternative pathway

C5-convertase of the two pathways

C3b Bb C3b

C5-convertase of the Alternative Pathway

C4b C2a C3b

C5-convertase of the Classical and lectin

Pathways

Generation of C5 convertase leads to the activation of the

Lytic pathway

Lytic pathway

Components of the lytic pathway

C6

C9

C8

C7

C5

Lytic pathwayC5-activation

C3b C2 aC4b

C5 b

C5a

Lytic pathwayassembly of the lytic complex

C5 b

C6

C7

Lytic pathway:insertion of lytic complex into cell membrane

C5 b

C6

C7C8

C9C

9C9 C

9C9

C9 C

9 C9

C9

MAC PORES

C9 complexes on RBC

Complement Activation Classical Pathway C1

MBL Pathway 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 * C1, C4, C2 are by-passed

* Properdin system not involved * Properdin system is involved

Biological functions of Complement

Lysis of bacteria and infected cellsOpsonization to enhance phagocytosisPhagocyte attraction and activationClearance of immune complexesRegulation of antibody responsesInflammation and anaphylaxis

Figure 2-18The complement system

Study Questions:

Write a short note about Factors Influencing Immunogenicity .

Define: Epitope – Paratope. Compare between: Classic and

Alterenative pathways of complement activation.

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Assignment:

Immunoglobulins Classes and subclasses

الكردى عرابى امال محمد رأفت امل احمد بدوى امنية محمد صبرى امنية الوهاب عبد الحى عبد امنية

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Thanks