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Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

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3 Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

4 Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail:

5  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

6 1. Immunogens & Antigens 2. Immunoglobulins (Antibodies) 3. Complement

7 6 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.

8 7 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.

9 8 [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)

10  Low mw molecules.  e.g. Antibiotics, drugs  Not immunogenic, unless... HAPTENS

11  Low mw molecules.  E.g. Antibiotics, drugs  Not immunogenic, unless... conjugated to high mw compounds (carriers) to gain immunogenicity HAPTENS CARRIER

12 11 [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

13 What affects Immunogenicity/Antigenicity ?

14 13 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

15 Bovine serum Albumin (BSA) Response to Immunization NO Yes ++ Cow Sheep Chicken Yes +++++ Bovine serum albumin (BSA) = more immunogenic in chicken than sheep

16 15 FOREIGNESS & ANTIBODY PRODUCTION: Time Antibody response to human serum Rabbit Chimpanzee

17 16 Molecular Weight x10 3 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)

18  Proteins almost always immunogenic  Carbohydrates potentially immunogenic  glycoproteins usually immunogenic  Lipids poorly immunogenic  Nucleic Acids poorly immunogenic

19 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

20 Levels of Protein Structure

21  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

22 21 NUMBER OF ANTIGENIC DETERMINANTS (i.e. epitopes) Small mass = fewer antigen determinants Large mass = greater number of antigen determinants

23 22 4)Degradability Macromolecules that cannot be degraded and presented by APC are poor immunogens

24 23 5) Genotype Of The Recipient Animal Mouse 1  Level of Ab  Level of Ab Cross F1 Generation Ag X Intermediate Level of Ab Ag X

25 24 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

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

27 26 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 residing populations 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

28  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

29  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,

30  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.

31  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.

32  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

33 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

34 5- Degradability of the immunogen 6-Genotype of the recipient 7- immunogen dose: A ppropriate dose optimum antigenicity Low dose low- zone tolerance High dose high-zone tolerance 8- Adjuvant: Substance when injected with an immunogen enhance immunogenicity


36 Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail:

37  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

38 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

39 Immune serum Ag adsorbed serum + - albumin globulins Mobility Amount of protein γ β α2 α1 Serum Protein Electrophoresis

40 Amount of protein Mobility albumin globulins +- Serum Protein Electrophoresis

41 Membrane-bound receptor Soluble antibody


43  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




47  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

48 Antibody is a flexible molecule (Hinge Region)

49 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.

50 Immunoglobulin classes

51 IgG subclasses

52  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

53 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 the domains on Ig :

54  Recognition of antigen (FAB)  Activation of complement  Opsonization  Neutralization  Antibody-dependent cell-mediated cytotoxicity,ADCC  Mediate hypersensitivity type I

55 Paratope

56 Complement activation

57 Opsonization



60 Property Property IgG IgG IgA IgA IgM IgM IgE IgE IgD IgD Heavy chain Heavy chain symbol symbol γ α µ ε δ Molecular Molecular weight weight 150 150 KDa KDa170-400 900 900 KDa KDa 190 190 KDa KDa 180 180 KDa KDa Percentage Percentage in serum in serum 75 % 75 % 15 % 15 % 10 % 10 % 0.004 % 0.004 % % 0.2 % 0.2 Complement Complement fixation fixation Yes Yes No No Yes Yes No No Transplacental passage passage Yes Yes No No Opsonization Opsonization Yes Yes No No

61  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)

62 IgA


64 - 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


66  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.

67  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

68 IgE

69  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


71  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


73 Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail:

74 TEACHING OBJECTIVES: 73 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.

75 n Discovered in 1894 by Bordet n Sheep antiserum could lyse Vibrio cholera n Its lysing activity was destroyed when heated at 56C° for 30 min n Added fresh serum (with no cholera antibodies) to heated serum restored the lysing ability!!!! n Complement activity in serum that completes the action of antibodies. Complement: history

76  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

77 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

78 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)

79 Complement activation n 1-classical pathway which is activated by Ab bound to Ag n 2-the lectin pathway activated by carbohydrates (Mannose & Fucose) n 3-Alternative pathway activated in the presence of various microbial pathogen

80 Pathways of complement activation CLASSICAL PATHWAY ALTERNATIVE PATHWAY activation of C5 LYTIC ATTACK PATHWAY antibody dependent LECTIN PATHWAY antibody independent Activation of C3 and generation of C5 convertase

81 Components of the Classical Pathway C4 C2 C3 C1 complex Ca ++ C1r C1s C1q

82 Ca ++ C1r C1s C1q C4 C4a b Classical Pathway Generation of C3-convertase

83 C4b Mg ++ C4a Ca ++ C1r C1s C1q C2 C2b a C2 a C4b2a is C3 convertase

84 Classical Pathway Generation of C5-convertase C4b Mg ++ C4a Ca ++ C1r C1s C1q C2b C2 a C3 C3a b C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway

85 Components of mannose-binding lectin pathway Bacteria M C4 MBL C2 MASP1 MASP2 M

86 Mannose-binding lectin pathway Bacteria C4 MBL C4b C4a C4b C2 C2b C2a C4b2a is C3 convertase; it will lead to the generation of C5 convertase MASP1 MASP2 MM

87 Components of the alternative pathway C3 B D P

88 C3b stabilization and C5 activation C3b C3b finds an activator (protector) membrane C3 C3a b B D b P This is stable C5 convertase of the alternative pathway

89 C5-convertase of the two pathways C3b BbBb C5-convertase of the Alternative Pathway C4b C2a C3b C5-convertase of the Classical and lectin Pathways

90 Generation of C5 convertase leads to the activation of the Lytic pathway

91 Components of the lytic pathway C6 C9C9 C8 C7 C5

92 Lytic pathway C5-activation C3b C2 a C4b C5 b C5a

93 Lytic pathway assembly of the lytic complex C5 b C6 C7

94 Lytic pathway: insertion of lytic complex into cell membrane C5 b C6 C7 C8 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9 C9C9

95 MAC PORES C9 complexes on RBC

96 Complement Activation Classical Pathway C 1 MBL Pathway C 4 C 2 C 3 Alternative pathway C 5 C 6 C 7 C 8 C 9 Membrane damage

97 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

98 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

99 Figure 2-18 The complement system

100 Study Questions: n Write a short note about Factors Influencing Immunogenicity. n Define: Epitope – Paratope. n Compare between: Classic and Alterenative pathways of complement activation. 99

101 Assignment: n Immunoglobulins Classes and subclasses n امال عرابى الكردى n امل رأفت محمد n امنية بدوى احمد n امنية صبرى محمد n امنية عبد الحى عبد الوهاب 100

102 Thanks

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