Bio 151 lec 2 2012 2013

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  • 1. ANTIGENS & ANTIBODIES Biology 151 lecture 2 AY 2012-2013_1st Main Reference for this Topic: Immunology by KUBY et al.Monday, June 25, 2012
  • 2. ANTIGENSMonday, June 25, 2012
  • 3. What are antigens? • any molecule that can bind specifically to an antibody • substances that can be recognized by the immunoglobulin receptor of B cells or by the T-cell receptor when complexed with MHC • name arises from their ability to generate antibodiesMonday, June 25, 2012
  • 4. What are antigens? • includes sugars, lipids, intermediary metabolites, hormones, complex carbohydrates, phospholipids, nucleic acids and proteins • found in surface or parts of a microbe or from the environment (e.g. food, pollen, etc)Monday, June 25, 2012
  • 5. Immunologic Properties of Antigens • Allergenicity: • having the capacity to induce allergy (hypersensitivity) • Tolerogenicity: • capable of inducing immunological tolerance (immune system does not attack the antigen) • Immunogenicity* • Antigenicity*Monday, June 25, 2012
  • 6. Biology 151 Introduction to Immunology ARE YOU ALLERGIC TO SOME DRUGS?Monday, June 25, 2012
  • 7. Monday, June 25, 2012
  • 8. Monday, June 25, 2012
  • 9. Antigens and Immunogens • ANTIGEN • A molecule which can be specifically recognized and bound by an antibody • “Antigenic” • IMMUNOGEN • A molecule which can elicit the production of specific antibody upon injection into a suitable host • “Immunogenic” • ALL molecules that are immunogenic are also antigenic..BUT...not all antigenic molecules are immunogenic! (Example: HAPTENS!)Monday, June 25, 2012
  • 10. Antigens: Haptens & Immunogens • HAPTENS (incomplete antigen) • antigens that by themselves do not elicit antibody production • unable to induce an immune response alone but able to react with products (Abs) • have the property of antigenicity but not immunogenicity (elicit immune response) • NOTE: could be rendered immunogenic by covalently linking them to a carrier moleculeMonday, June 25, 2012
  • 11. Monday, June 25, 2012
  • 12. Monday, June 25, 2012
  • 13. Antigens: Haptens & Immunogens • IMMUNOGENS (complete antigen) • antigens that can elicit antibody production • stimulate B and/or T cell arms of the immune response and react with products (Abs) • both induces an immune response and reacts with the products of it (Abs)Monday, June 25, 2012
  • 14. Factors that Influence Immunogenicity • Nature of the Immunogen • foreignness • molecular size • chemical composition and heterogeneity • lipids as antigens • susceptibility to antigen processing and presentationMonday, June 25, 2012
  • 15. Foreigness • Recall: “in order to elicit an immune response, a molecule must be recognized as NON-SELF by the biological system” • tolerance for SELF-antigens • The greater the phylogenetic distance between two species, the greater the structural disparity between them • EXAMPLE: bovine serum albumin not immunogenic to cow bt is on chicken (cow > goat > chicken)Monday, June 25, 2012
  • 16. Molecular Size • The most ACTIVE immunogens: • 100,000 Da • Substances with a molecular mass of 5,000-10,000 Da are poor immunogens • EXEMPTIONS: few substances with a molecular mass less than 1,000 Da have proven to be immunogenicMonday, June 25, 2012
  • 17. Chemical Composition & Heterogeneity • chemical complexity contributes to immunogenicityMonday, June 25, 2012
  • 18. Lipids as Antigens • appropriately presented lipoidal antigens can induce both B-cell and T-cell responses • Example: lipid-protein conjugates (lipids are used as haptens)Monday, June 25, 2012
  • 19. Lipids as AntigensMonday, June 25, 2012
  • 20. Susceptibility to Antigen Presentation & Processing • the development of both humoral and cell-mediated immune responses requires interaction of T-cells with antigen that has been processed and presented together with MHC molecules • LARGE, INSOLUBLE macromolecules are generally more immunogenic than SMALL, SOLUBLE ones • larger molecules are more readily phagocytosed and processed • degradative enzymes within antigen-presenting cells can degrade only proteins containing L-amino acids, polymers of D-amino acids cannot be processedMonday, June 25, 2012
  • 21. Factors that Influence Immunogenicity •Biological System • genotype of the recipient animal • immunogen dosage and and route of administration • adjuvantsMonday, June 25, 2012
  • 22. Genotype of Recipient Animal • MHC gene products = determines the degree to which an animal responds to an immunogen (immune responsiveness) • response influenced by genes that encode B-cell and T-cell receptors • response influenced by genes that encode various proteins involved in immune regulatory mechanisms • THUS: genetic variability affects immunogenicity in different animalsMonday, June 25, 2012
  • 23. Dosage and Route • experimental immunogen exhibits unique dose- response curve • DOSE • insufficient dose will not stimulate an immune response (fails to activate lymphocytes or tolerance) • excessively high dose = tolerance • THUS....repeated adminsitrations or BOOSTERS are done if a single dose will not induce a strong response = increase clonal proliferation of antigen- specific T cells or B-cells = increase the lymphocyte populations SPECIFIC for the immunogenMonday, June 25, 2012
  • 24. Dosage and Route • experimental immunogen exhibits unique dose-response curve • ROUTE • generally administered parenterally = other than the GIT • Subcutaneous route > Intramuscular > Intraperitoneal > Intraveous > Oral route • Can you recall the route of your vaccine shots? • strongly influence which immune organs and cell populations will be involved in the response • intravenous = carried first to the spleen • subcutaneous = moves first to local lymph nodesMonday, June 25, 2012
  • 25. Adjuvants• substances that, when mixed with an antigen and injected with it, ENHANCE the immunogenicity of that antigen• used to boost the immune response when an antigen has LOW IMMUNOGENICITY or when only SMALL AMOUNTS of an antigen is availableMonday, June 25, 2012
  • 26. AdjuvantsMonday, June 25, 2012
  • 27. IN SUMMARY... Parameter Increased Decreased Immunogenicity Immunogenicity Size Large >10,000; Small MW<2500 best >100,000 Dose Intermediate High or Low Route Subcutaneous/IM > Intravenous > Oral or Intraperitoneal > intragastric Composition Complex Simple Form Particulate Soluble Denatured Native Similarity to self proteins Multiple differences Few differences Adjuvants Slow release Rapid release Bacteria No bacteria Interaction with host MHC Effective IneffectiveMonday, June 25, 2012
  • 28. EPITOPES: Antigenic DeterminantsMonday, June 25, 2012
  • 29. Antigenic Determinants or Epitopes • DEFINITION: • immunologically active regions of an immunogen that interacts with the specific antigen binding site in the variable region of the antibody molecule (PARATOPE) or to secreted antibodies • EXCELLENT FIT between epitope and paratope: based upon their 3-D interaction and non covalent union • are discrete site on the macromolecule recognized by the lymphocytes (B- lymphocytes/ T-lymphocytes) • NOTE: B and T cells recognize DIFFERENT epitopes on the SAME antigenic molecule • THUS: the ability to function as a B-cell epitope is determined by the nature of the ANTIGEN-BINDING site of the antibody molecules DISPLAYED by B-cellsMonday, June 25, 2012
  • 30. Antigenic Determinants or Epitopes • An antigen molecule has 2 or more epitopes or antigenic determinants per molecule • Epitopes consist of approximately 6 amino acids or 6 monosaccharides • Immunodominant Epitope: stimulate a greater antibody responseMonday, June 25, 2012
  • 31. Monday, June 25, 2012
  • 32. APPLICATIONS: Epitope Mapping • Assignment: Diagram an experiment on how to carry out epitope mapping and determination of epitopes • QUESTION: How will you know which fraction/ epitope will be the most highly immunogenic/ antigenic? • Handwritten: Yellow Paper to be submiited on TuesdayMonday, June 25, 2012
  • 33. Antigen Classification • T cell dependent Ags or TD • much more complex than TI Ags • usually proteins • stimulate a full complement of immunoglobulins with all five classes represented • elicit an anamnestic or memory response & are present in most pathogenic organismsMonday, June 25, 2012
  • 34. Viral proteins = HA and NA bacterial protein = flagellin Fungal proteins = keratinasesMonday, June 25, 2012
  • 35. Antigen Classification •T cell independent Ags or TI • often polysaccharides or lipopolysaccharides • elicit an IgM response only and fail to stimulate an amnestic responseMonday, June 25, 2012
  • 36. Bacterial membrane/OM components capsular polysaccharide Fungal Mannans & GlucansMonday, June 25, 2012
  • 37. Example: Fungi Group of fungi Fungi Polysaccharide Ag (TI) Protein Ag (TD) Molds Aspergillus galactomannan glycoproteins Yeasts Candida albicans Alpha mannan proteinase Yeasts Cryptococcus neoformans galactoxylomannan Dermatophytes Trichophyton Galactomannan peptides Keratinases I, II. III Zygomycetes Rhizopus peptidofucomannan Proteinase Dimorphic systemic fungi Histoplasma capsulatum galactomannan “h” & ‘m” factors “ Paracoccodioides galactomannan E2 factor “ Coccidioides imitis Methyl-mannose polymer Coccidiodin factor Subcutaneous Sporothrix schenckii Peptido-L-rhamno-D-mannanMonday, June 25, 2012
  • 38. Summary of Antigen Classification Thymus dependent Thymus independent Structural Complex Simple properties Chemistry Proteins; protein- Polysaccharide of nucleoprotein conjugates; pneumococcus; dextran glycoproteins; polyvinyl pyrolidone; lipoproteins bacterial Lipolysaccharide Antibody class IgG, IgM, IgA, IgM induced (+ IgD and IgE) Immunological YES NO memory response Present in most YES NO pathogenic microbesMonday, June 25, 2012
  • 39. Superantigens • a substance such as a bacterial toxin capable of stimulating MANY CD4+ T lymphocytes leading to the release of relatively large quantities of cytokines that provoke pathophysiologic manifestations • NOTE: Superantigens are TD antigens = THUS, Do not require phagocyte processing • stimulate multiple T cells that augment a protective T & B cell responseMonday, June 25, 2012
  • 40. SuperantigensMonday, June 25, 2012
  • 41. Superantigens • Examples: • Staphylococcal enterotoxins (food poisoning) • Staphylococcal toxic shock toxin (toxic shock syndrome) • Staphylococcal exfoliating toxins (scalded skin syndrome) • Streptococcal pyrogenic exotoxins (shock) • The diseases associated with exposure to superantigens are, in part, due to hyper activation of the immune system and subsequent release of biologically active cytokines by activated T cellsMonday, June 25, 2012
  • 42. Mitogens • substance, often derived from plants, that causes DNA synthesis and induces blast transformation and division by mitosis • Lectins, representing plant-derived mitogens or phytomitogens are used in experimental and clinical immunology to evaluate T and B lymphocyte function in vitroMonday, June 25, 2012
  • 43. Mitogens Characteristic Concanavalin A Phytohemagglu Pokeweed (Con A) tinin (PHA) mitogen (PWM) Source Jack beans Kidney beans Pokeweed Molecular Tetramer Tetramer Polymeric Structure Ligand A-D-mannose & a- N- Di-N- D-glucose acetylgalactosamine acetylchitobiose Target cell/s T cells T cells T cells and B cellsMonday, June 25, 2012
  • 44. SPECIAL ISSUE: Antigenic VariationMonday, June 25, 2012
  • 45. Monday, June 25, 2012
  • 46. How are antigens recognized? • Pattern Recognition ReceptorsMonday, June 25, 2012
  • 47. DIFFER IN INNATE AND IN ADAPTIVE RESPONSESMonday, June 25, 2012
  • 48. Monday, June 25, 2012
  • 49. ANTIBODIES (Immunoglobulins or Igs)Monday, June 25, 2012
  • 50. What are antibodies? • Antibodies : antigen-binding proteins present on the B-cell membrane and secreted by plasma cells • when bound confers antigenic specificity on B-cells • Common to all antibodies: • structural features • binds to antigen • participate in effector functionMonday, June 25, 2012
  • 51. Antibody: Name Game Antitoxin Neutralize toxins Agglutinins Clumps cells Precipitins Ppt soluble antigens Lysins Lyses cells Opsonins ! phagocytosis Neutralizing antibodies Neutralize viruses Complement fixing Activate complement antibodiesMonday, June 25, 2012
  • 52. Ab-mediated Effector Functions • Opsonization: promotion of phagocytosis of antigens by macrophages and nuetrophils • Fc receptors (in surfaces of macrophages and nuetrophils) bind to Ig molecules which induces a signal transduction pathway resulting in phagocytosis of the Ag-Ab complex • Actions: enzymatic digestion, oxidative damage, membrane-disrupting effects of bacterial peptidesMonday, June 25, 2012
  • 53. Monday, June 25, 2012
  • 54. Ab-mediated Effector Functions • Complement Activation (IgM and IgG): induces a collection of proteins that can perforate cell membranes • C3b: important by-product; binds non- specifically to cell and Ag-Ab complexes • many cell types have receptors for C3b (e.g. macrophages leading to phagocytosis) • significance: removal and killing of pathogensMonday, June 25, 2012
  • 55. Ab-mediated Effector Functions • Antibody-dependent cell-mediated Cytotoxicity (ADCC): kills cells • Ab acts as a newly acquired receptor enabling the attacking cell to recognize and kill the target cell • Pre-requisite: linking of Ab bound to target cells (e.g. virus in host cells) with the Fc receptor of many cell types can direct the cytotoxic activities of the effector cell against the target cellMonday, June 25, 2012
  • 56. Monday, June 25, 2012
  • 57. Basic Structure of Antibodies • Antibodies found in serum protein fractions • electrophoretic mobility revealed four peaks: albumin, alpha, beta and gamma • Gamma globulin factors = immunoglobulins (IgG) which contains serum antibodiesMonday, June 25, 2012
  • 58. Antibodies are Heterodimers • consist of two identical side light chains & two identical heavy chains linked by disulfide bonds • heavy chain: has an amino-terminal variable region followed by a constant regionMonday, June 25, 2012
  • 59. Antibodies are HeterodimersMonday, June 25, 2012
  • 60. Immunoglobulin Classes • in any given antibody molecule, the constant region contains one of five basic heavy chain sequences called isotypes • the heavy chain isotype determines the class of an antibodyMonday, June 25, 2012
  • 61. Immunoglobulin Classes • IgG • IgD • IgE • IgA • IgMMonday, June 25, 2012
  • 62. Antibody ClassesMonday, June 25, 2012
  • 63. Antibody Classes : IgGMonday, June 25, 2012
  • 64. Antibody Classes : IgG • PROPERTIES: • major serum immunoglobulins (systemic immunity) • major immunoglobulin in extravascular spaces • does not require antigen binding during placental transfer (IgG2) • fixes complement (IgG4) • binds to Fc receptors (iGg2 and IgG4) • phagocytes - opsonization • Killer cells - ADCCMonday, June 25, 2012
  • 65. Antibody Classes : IgM • PROPERTIES • pentamer • 3rd highest serum immunoglobulin • first immunoglobulin made by fetus and B cells C1r C1 s • fixes complement C1q C1r C1 s • Figure: fixation of C1 by IgG C1q and IgM • No activation Activation agglutinating immunoglobulin • binds to Fc receptors • B-cell surface immunoglobulinsMonday, June 25, 2012
  • 66. Antibody Classes : IgM & IgGMonday, June 25, 2012
  • 67. Antibody Classes : IgA PROPERTIES OF IgA • serum monomer • secretions (sIgA) • 2nd highest serum Secretory Piece J Chain immunoglobulins • major secretory Ig (tears, saliva, gastric and pulmonary secretions) = mucous and local immunity • DO NOT fix complement (unless aggregated • binds to Fc receptors on some cellsMonday, June 25, 2012
  • 68. Antibody Classes : IgA FORMATION OF SECRETORY IgAMonday, June 25, 2012
  • 69. Antibody Classes : IgAMonday, June 25, 2012
  • 70. Antibody Classes : IgD PROPERTIES OF IgD • monomer • tail piece • 4th highest serum Ig • B-cell surface Ig Tail Piece • DOES NOT BIND complementMonday, June 25, 2012
  • 71. Antibody Classes : IgE PROPERTIES OF IgE • monomer • with extra domain • least common serum Ig • binds to basophils and mast cells (DO NOT require antigen binding) • allergic reaction C!4 • parasitic infections (helminths) • binds to Fc receptors on eosinophils • DOES NOT fix complementMonday, June 25, 2012
  • 72. Allergies IMMUNOGLOBULINS & ALLERGIESMonday, June 25, 2012
  • 73. Biology 151 Introduction to Immunology Georges Kohler and MONOCLONAL Cesar Milstein in 1975 ANTIBODIES Polyclonal antibodies : arise from MANY B- cell clones and have a HETEROGENOUS collection of binding sites Monoclonal antibodies : derived from a SINGLE B-cell clone and is a HOMOGENOUS collection of binding sitesMonday, June 25, 2012
  • 74. Biology 151 Introduction to Immunology CLINICAL UTILITY OF MONOCLONAL ANTIBODIESMonday, June 25, 2012
  • 75. Biology 151 Introduction to Immunology CLINICAL UTILITY OF MONOCLONAL ANTIBODIES • DIAGNOSTICS: detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits (Biotech, 1989); diagnosis of AIDS by the ELISA test. • THERAPEUTICS: radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells (Chaudhari et al, 1994); cancer drug based on monoclonal antibody technology = Ritoxin, approved by the FDA in November 1997 (Orrs, 1997) radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells (Chaudhari et al, 1994); cancer drug based on monoclonal antibody technology = Ritoxin, approved by the FDA in November 1997 (Orrs, 1997) viral diseases, traditionally considered "untreatable" = AIDS (P/S/L, 1997). • TRANSPLANTATION OKT3, an antibody to the T3 antigen of T cells, is used to alleviate the problem of organ rejection in patients who have had organ transplants (Transweb, 1996). Parungao-Balolong 2010Monday, June 25, 2012
  • 76. Biology 151 Introduction to Immunology NEXT MEETING Innate and Adaptive ResponseMonday, June 25, 2012