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

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

3. Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com Immunogens & ANTIGENS

4. 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 OBJECTIVES:By the end of the lecture the students will be able to

5. 1. Immunogens & Antigens 2. Immunoglobulins (Antibodies) 3. Complement COMPONENTS OF ANTIGEN-ANTIBODY REACTIONS

6. Definitions • IMMUNOGEN: • A substance, usually foreign, which when introduced into an animal can generatea 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 specificallyto immune components when present.

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.

8. [1] Immunogenicity “Ability of a substance to stimulate the production of antibodiesand/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)

9. Low mw molecules. • e.g. Antibiotics, drugs • Not immunogenic, unless... Haptens HAPTENS

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

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

12. What affects Immunogenicity/Antigenicity?

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 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

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

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

16. 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 25 100 500 Molecular Weight x103

17. Proteins almost always immunogenic • Carbohydratespotentially immunogenic • glycoproteinsusually immunogenic • Lipidspoorly immunogenic • Nucleic Acidspoorly immunogenic 3)Chemical Compexity & Heterogeneity

18. 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

19. Chemical Complexity (cont.) Levels of Protein Structure

20. Epitope Ag • 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 Chemical Complexity (cont.) A model of a substance with four epitopes

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

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

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

24. 6) Immunogen Dosage Low dose  failure to activate enough lymphocytes or induces Nonresponsiveness i.e. Low dose tolerance High dose  lymphocytes enter nonresposnsivessstate 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

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

26. 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

27. Adjuvants are “Substances that enhance immunogenicity of antigen when mixed and injected with it” • Adjuvantsused 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 8) Enhancing Immunogenicity by Adjuvants

28. 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, Types of Antigens according to their origins :

29. 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.

30. 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.

31. 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

32. 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)

33. 5- Degradability of the immunogen 6-Genotype of the recipient 7- immunogendose: Appropriate dose optimum antigenicity Low dose low- zone tolerance High dose high-zone tolerance 8- Adjuvant: Substance when injected with an immunogen enhance immunogenicity Factors influencing Immunogenicity (Summary) (cont.)

34. Take some rest

35. Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com Immunoglobulins:Structure & Functions

36. 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 TEACHING OBJECTIVES:

37. Definition: Glycoproteinmolecules in serum and tissue fluids that are produced by plasma cellsin response to an immunogen and which function as antibodies. They react specifically with antigen Five classes of Antibodies: IgG IgM IgA IgD IgE Immunoglobulins

38. - + albumin Amount of protein globulins Immune serum Ag adsorbed serum Mobility Serum Protein Electrophoresis γβα2 α1

39. Serum Protein Electrophoresis albumin Amount of protein globulins - + Mobility

40. Two Forms of Immunoglobulin Membrane-bound receptor Soluble antibody

41. Immunoglobulinsmembrane-bound and soluble receptors

42. 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 carboxyl-terminal constant (C) region.

43. ImmunoglobulinsFour-Chain Basic Unit VL CL VL CL

44. Immunoglobulins: Structural Regions

45. Immunoglobulins: Structural Domains

46. Antibody – immunoglobulin secreted by B cells Antigen (antibodygenerator) – 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 Important Terms Paratope

47. Antibody is a flexible molecule (Hinge Region)

48. 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 and Subclasses

49. Immunoglobulinclasses

50. IgG subclasses