Chapter 4 Antigens and Antibodies

1. Chapter 4 Antigens and Antibodies Ag Ab Complementarity of interacting surfaces of Ab and Ag Oct 17, 19 & 24, 2006

2. 你需要學習的課題: • 1.「好」的免疫原 (immunogen)有些什麼特質？ • 2. 什麼叫做抗原決定區 (epitope)？ • B-cell epitope 有何特性？ • ********************************************************* • 4. 抗體分子的基本構造及各部位的名稱。 • 5. 抗體的種類、特性及功能。 • 6. 單株抗體與多株抗體。

3. Outline 1. Immunogenicity versus antigenicity 2. Epitopes ********************************************************** 3. Basic structure of antibodies (Abs) 4. Ab-binding site 5. Ab-mediated effector functions 6. Ab classes and biological activities 7. Antigenic determinants on immunoglobulins (Ig) 8. The B-cell receptor 9. The Ig superfamily 10. Monoclonal Abs

4. Immunogenicity vs. Antigenicity

5. Immunogenicity免疫性: the ability to induce an Aband/or cell-mediated immune response Antigenicity抗原性: the ability to combine specifically with Ab and/or cell-surface receptors (Ig/TCR)

6. - Although a substance that induces a specific • immune response is usually called anantigen, • it is more appropriately called animmunogen. • - Although all molecules that have the property • of immunogenicity also have the property of • antigenicity,the reverse is not true. • Some small molecules, calledhaptens,are • antigenic but incapable, by themselves, of • inducing a specific immune response. In other • words, they lack immunogenicity.

7. DNP: dinitrophenol (bovine serum albumin) {

8. Landsteiner’s work demonstrated both the specificity of the immune system for small structural variations on haptens and the enormous diversity of epitopes that the immune system is capable of recognizing.

9. Factors That Influence Immunogenicity

10. Intrinsic properties of an immunogen: - Foreignness - Molecular size - Chemical composition and heterogeneity - Susceptibility to antigen processing and presentation The biological system: - Genotype of the recipient animal - Immunogen dosage and route of administration - Adjuvants

11. Foreignness • Generally, the greater the phylogenetic • distance between two species, the greater the • structural (and therefore the antigenic) • disparity between them. • Some macromolecules (e.g., collagen and • cytochrome c) were highly conserved • throughout evolution and therefore display • very little immunogenicity across diverse • species lines.

12. Conversely, some self-components (e.g., • corneal tissue and sperm) are effectively • sequestered from the immune system, so • that if these tissues are injected even into • the animal from which they originated, • they will function as immunogens.

13. Molecular size - There is a correlation between the size of a macromolecule and its immunogenicity. - The best immunogens tend to have a molecular mass >100,000 daltons (Da). - Generally, substances with a molecular mass less than 5,000 – 10,000 Da are poor immunogens.

14. Chemical composition • and complexity • - Synthetichomopolymerstend to lack • immunogenicity regardless of their size. • All 4 levels of protein organization – • primary, secondary, tertiary and quaternary – • contribute to the structural complexity of a • protein and hence affect its immunogenicity.

15. Four levels of protein organizational structure

16. For Ab (B cell) responses: Proteinsare the most potent immunogens, withpolysaccharidesranking second. Lipids and nucleic acids of an infectious agent generally do not serve as immunogens unless they are complexed with proteins or polysaccharides. For T cell responses: Onlyproteins andsome lipids(glycolipids and phospholipids) serve as immunogens.

17. Susceptibility to antigen • processing and presentation • The development of both Ab-mediated and • T-cell-mediated immune responses requires • interaction of T cells with Ag that has been • processed and presented together with MHC • molecules.

18. Large, insoluble macromolecules generally • are more immunogenic than small, soluble • ones because the larger molecules are more • readily phagocytosed and processed. • Molecules that cannot be degraded (e.g., • polymers of D-amino acids) and/or cannot • be presented with MHC molecules are • poor immunogens.

19. The biological system contributes • to immunogenicity • Genotype of the recipient animal • - Immunogen dosage and route of administration • - Adjuvants

20. Immunogen dosage and route of administration Doses:too low  no response, too high  tolerance Exposure:repeated administration (booster) over a period of time is usually more effective Routes:orally (從口入的) parenterally (非從口入的) - intravenous (iv) : into a vein - intradermal (id) : into the skin - subcutaneous (sc) : beneath the skin - intramuscular (im) : into a muscle - intraperitoneal (ip) : into the peritoneal cavity

21. 為什麼 Ag 的量、接觸次數 及 路徑 與免疫反應的強度有關？

22. Adjuvants佐劑 • Latin adjuvare, tohelp • Substances that, when mixed with an • antigen and injected with it, enhance • the immunogenicity of that antigen.

23. Effects of adjuvants 1. antigen persistence is prolonged - slower release of antigen at the injection site e.g., alum 明礬[AlK(SO4)2], 2. costimulatory signals (p. 159) are enhanced - increased expression of B7 molecules on APC  maximal activation of TH cells 3. local inflammation is increased 4. nonspecific proliferation of lymphocytes is stimulated - formation of a dense, macrophage-rich mass of cells called agranuloma 肉芽腫 e.g., incomplete Freund’s adjuvant (IFA), complete Freund’s adjuvant (CFA)

24. 1. 在實驗動物中製備抗體時，經常使用 CFA (complete Freund’s adjuvant)，其作用機轉 為何？ 2. 為何局部發炎反應能增強 Ab 反應？

25. Epitopes

26. Lymphocytes do not interact with, or recognize, • entire immunogen molecules; instead, they • recognize discrete sites on the macromolecule • calledepitopes, orantigenic determinants. • epitopes: immunologically active regions of an • immunogen, that bind to Ag-specific • membrane receptors on lymphocytes • or to secreted Abs.

27. T-cell and B-cell epitopes - The recognition of antigens by T cells and B cells is fundamentally different. - Because B cells bind antigen that is free in solution, the epitopes they recognize tend to be highly accessible sites on theexposed surface of the immunogen. - T-cell epitopes are peptides combined withMHC molecules. Thus, there is no requirement for solution accessibility such as B-cell epitope.

28. Properties of B-cell epitopes • B-cell epitopes on native proteins generally • are composed ofhydrophilic a.a. on the • protein surface that are topographically • accessibleto membrane-bound or free Ab. • 2. B-cell epitopes may be composed of • sequential ornonsequentialamino acids.

29. Sperm whale myoglobulin contains 5 sequential B-cell epitopes

30. Hen egg-white lysozyme (HEL) composes one nonsequential(conformational) epitope Contact with Ab light chain Contact with Ab heavy and light chains Contact with Ab heavy chain

31. Ab to native HEL does not bind to reduced HEL

32. B-cell epitopes tend to be located inflexible regions • of an immunogen and display site mobility. • - site mobility of epitopes maximizes complementarity • with the Ab’s binding site • Complex proteins contain multipleoverlapping • B-cell epitopes, some of whichare immunodominant. • - Most of the surface of a globular protein is potentially • immunogenic. • - Some epitopes, called immunodominant, induce a more • pronounced immune response in a particular animal than • other epitopes.

34. Basic structure of Abs

36. Electrophoresis of immune serum (Tiselius & Kabat, 1939) Immune sera after reaction with Ag

37. g- globulin (gG) • Immunoglobulin (Ig): • IgG, IgM, IgA, IgE, IgD • Antibody (Ab)

38. Ab molecules contain 4 peptide chains - A dimer of heterodimers V: variable C: constant (H) m : IgM g :IgG a :IgA d : IgD e : IgE (L)

39. 100 kDa 150 kDa → numerous small peptides Fabfragment: antigenbinding 45 kDa 22 kDa Fcfragment: crystallizable 50 kDa 50-55kDa

40. Antibody to the Fab fragment could react with both the H and L chains, whereas antibody to the Fc fragment reacted only with the H chain.  Fab consists of portions of an H and a L chain.  Fc contains only H chain components.

41. - Aheterogeneousspectrum of antibodies in the serum g-globulin fraction - Multiple myeloma: a cancer of Ab- producing plasma cells - Myeloma protein: 95% of the serum Ig - Bence-Jones proteins: the excess light chains in the urine. - MOPC:mineral-oil inducedplasmacytoma in mice

43. Heavy and light chains are folded into “domains” (IgG, IgD, IgA) (IgM, IgE)

44. Associations between domains of an Ab molecule 2 Ag-binding sites:

45. Immunoglobulin Domains - each contains about 110 a.a. residues and an intrachain disulfide bond that forms a loop of 60 a.a. Variable-Region Domains -hypervariable regions: (15% - 20% of the variable domain) =complementarity-determiningregions(CDR) CDR1, CDR2, CDR3 - framework regions (FR) - diversity in the VH domain is concentrated in CDRs

46. Variability of a.a. residues in the VH and VL domains CDRs bind Ags number of different amino acids at a given position Variability = _____________________________________________________ frequency of the most common amino acid at a given position

47. Conformational changes may be induced by antigen binding CDRs of L chain : L1, L2, L3 CDRs of H chain : H1, H2, H3 ─: after binding to the Ag ─: before binding to the Ag Fab

48. Ab-mediated effector functions

49. - Antibodies generally do not kill or remove • pathogens solely by binding to them. • While V regions bind to Ag, theCH region • is responsible for a variety of collaborative • interactions with otherproteins,cells, and • tissuesthat result in the effector functions • of the Abresponses.

50. Ab-Mediated Effector Functions • Opsonization (調理作用)is promoted by Ab • Abs activate complement (補體 ) (chapter 7) • Antibody-dependent cell-mediated cytotoxicity • (ADCC) kills cells (p. 366 chapter 14) • - Some Abs can cross epithelial layers by transcytosis