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Antibody structure and function

Antibody structure and function. Parham – Chapter 4. Outline. Antigens Antibody structure Antigen-antibody interactions Applications - immunoassays Generation of antibody diversity Isotype switching. Immunoglobulins – membrane-bound and soluble receptors. Epitopes.

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Antibody structure and function

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  1. Antibody structure and function Parham – Chapter 4 H. HogenEsch, 2009

  2. Outline • Antigens • Antibody structure • Antigen-antibody interactions • Applications - immunoassays • Generation of antibody diversity • Isotype switching H. HogenEsch, 2009

  3. Immunoglobulins – membrane-bound and soluble receptors H. HogenEsch, 2009

  4. Epitopes • Epitope (antigenic determinant) is the part of an antigen to which an antibody binds. • Most antigens have multiple epitopes (multivalent) • Usually carbohydrate or peptide. Fig. 2.9 http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html H. HogenEsch, 2009

  5. Epitopes recognized by antibodies are usually located at the antigen’s surface. Fig. 2.8 H. HogenEsch, 2009

  6. Conformational vs. linear epitopes H. HogenEsch, 2009

  7. Epitopes heat, acid Conformational epitopes - destroyed by denaturation Linear epitopes - unaffected by denaturation H. HogenEsch, 2009

  8. Epitope recognition H. HogenEsch, 2009

  9. Haptens Small molecules that are not immunogenic by themselves, but can bind immunoglobulins or TCRs. Haptens can induce an immune response when linked to a larger protein (carrier). H. HogenEsch, 2009

  10. Hapten Parham Fig. 10.25 H. HogenEsch, 2009 Fig. 12.26

  11. Hapten Parham Fig. 10.26 Fig. 12.27 H. HogenEsch, 2009

  12. Basic structure of immunoglobulins • 2 light chains • lambda () • kappa (κ) 5 heavy chains - mu (μ) - gamma (γ) - alpha (α) - epsilon (ε) - delta (δ) Fig. 4.2 H. HogenEsch, 2009

  13. Basic structure of immunoglobulins Fig. 4.2 H. HogenEsch, 2009

  14. Antigen-binding Fragment Crystallizable Fragment H. HogenEsch, 2009

  15. H. HogenEsch, 2009

  16. Hinge region provides flexibility to antigen-binding sites H. HogenEsch, 2009

  17. Structure of immunoglobulins H. HogenEsch, 2009

  18. Structure of immunoglobulins H. HogenEsch, 2009

  19. Hypervariable and framework regions HV = CDR = complementarity -determining region Fig. 2.7 H. HogenEsch, 2009

  20. Immunoglobulin classes (isotypes) L-chain: k or l H. HogenEsch, 2009

  21. Differences between immunoglobulins H. HogenEsch, 2009

  22. Allotypes • Small differences (few base pairs/amino acids) • May affect half life • May affect subclass distribution • Mendelian inheritance – autosomal dominant • Different distributions among ethnic groups • Associated with susceptibility to infectious diseases and autoimmune diseases H. HogenEsch, 2009

  23. Antibody-antigen interaction H. HogenEsch, 2009 Fig. 2.10

  24. Antibody-antigen interaction • Non-covalent binding: • Electrostatic • Hydrogen bonds • Van der Waals forces • Hydrophobic forces • Affinity: Strength of interaction between epitope and one antigen-binding site • Avidity: Strength of the sum of interactions between antibody and antigen Short range H. HogenEsch, 2009

  25. Crossreactivity Antiserum raised against antigen A reacts also with antigen B Antigen A and B share epitopes Antigen A and B have similar (but not identical) epitopes H. HogenEsch, 2009

  26. Crossreactivity H2N2 H2N3 Influenza virus H. HogenEsch, 2009

  27. Monoclonal antibodies • Immortalization of a single clone of antibody-secreting cells • Fusion of B cells with neoplastic plasma (myeloma) cells H. HogenEsch, 2009

  28. Monoclonal antibodies H. HogenEsch, 2009

  29. Polyclonal vs. monoclonal antibodies • Polyclonal antibodies • purified from serum of immunized animals, often goats or rabbits. • Multiple specificities and affinities • Variation from batch to batch • Monoclonal antibodies • Produced by immortalized plasma cells, usually mouse origin. • Single specificity and affinity • Unlimited supply of identical antibody molecules H. HogenEsch, 2009

  30. Examples of monoclonal antibodies as therapeutics H. HogenEsch, 2009

  31. Types of therapeutic monoclonal antibodies H. HogenEsch, 2009

  32. Rituximab in autoimmune disease(pemphigus vulgaris) Rituximab + IVGG Ahmed et al. NEJM, 355, 1772, 2006 Approximate cost: $3,976 per infusion or $15,904 for a four-dose course H. HogenEsch, 2009

  33. Immunoassays • Precipitation assay • Agglutination assay • Enzyme-linked immunosorbent assay (ELISA) • Radioimmunoassay (RIA) • Western blotting • Immunofluorescence • Flow cytometry H. HogenEsch, 2009

  34. Sensitivity of immunoassays precipitation - 30 mg/ml agglutination - 1 mg/ml radioimmunoassays, ELISA - 1 pg/ml H. HogenEsch, 2009

  35. Precipitation reaction Aggregates formed by interaction of multivalent antibodies and multivalent macromolecular antigens. H. HogenEsch, 2009

  36. Antigens have multiple epitopes H. HogenEsch, 2009

  37. Hemagglutination H. HogenEsch, 2009

  38. Coombs test • Direct: Add anti-human immunoglobulin antibodies (Coombs’ reagent) to red blood cells. Agglutination occurs if the red blood cells are coated with antibodies. • Indirect: Incubate test serum with red blood cells. Wash red blood cells. Add anti-human immunoglobulin antibodies. H. HogenEsch, 2009

  39. Rhesus factor H. HogenEsch, 2009

  40. 1 2 3 4 Enzyme-linked immunosorbent assay (ELISA) Principle of ELISA/RIA • Coat wells with antigen • Add serum sample • Add enzyme-labeled • anti-human IgG • Add substrate H. HogenEsch, 2009

  41. Western blotting Western blot H. HogenEsch, 2009

  42. Immunofluorescence H. HogenEsch, 2009

  43. Flow cytometry H. HogenEsch, 2009

  44. Flow cytometry for CD4 T cells H. HogenEsch, 2009

  45. Monitoring CD4 T cells in HIV infection H. HogenEsch, 2009

  46. Immunoglobulin genes H. HogenEsch, 2009

  47. V k J k C k germline DNA // 5’ 3’ 1 2 3 4 5 n 1 2 3 4 5 rearrangement 5’ 3’ B cell DNA V2J3 transcription 5’ 3’ primary RNA transcript splicing mRNA V2J3C translation k chain polypeptide V k C k V-region domains are constructed from gene segments H. HogenEsch, 2009

  48. VL and VH-region domains are constructed from gene segments H. HogenEsch, 2009

  49. H. HogenEsch, 2009

  50. Recombination Signal Sequences H. HogenEsch, 2009

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