Antibody structure and function

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