Antibody structure and function

1. Antibody structure and function Parham – Chapter 2 H. HogenEsch, 2005

2. Outline • Antibody structure • Antigens • Antigen-antibody interactions • Generation of antibody diversity • Isotype switching • Applications - immunoassays H. HogenEsch, 2005

3. Immunoglobulins – membrane-bound and soluble receptors H. HogenEsch, 2005

4. Basic structure of immunoglobulins Fig. 2.2 H. HogenEsch, 2005

5. Basic structure of immunoglobulins Fig. 2.2 Fig. 2.2 H. HogenEsch, 2005

6. Antigen-binding Fragment Crystallizable Fragment H. HogenEsch, 2005

7. H. HogenEsch, 2005

8. g a m d e H: Immunoglobulin classes (isotypes) L-chain: k or l H. HogenEsch, 2005

9. Structure of immunoglobulins H. HogenEsch, 2005

10. Structure of immunoglobulins H. HogenEsch, 2005

11. Hypervariable and framework regions CDR = complementarity -determining region Fig. 2.7 H. HogenEsch, 2005 Fig. 2.7

12. Differences between immunoglobulins H. HogenEsch, 2005

13. 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 H. HogenEsch, 2005

14. Immunoglobulin epitopes are usually located at the antigen’s surface. Fig. 2.8 H. HogenEsch, 2005

15. Conformational vs. linear epitopes Fig. 2.11 H. HogenEsch, 2005

16. Epitopes heat, acid Conformational epitopes - destroyed by denaturation Linear epitopes - unaffected by denaturation H. HogenEsch, 2005

17. Epitope recognition H. HogenEsch, 2005

18. 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. H. HogenEsch, 2005

19. Hapten Parham Fig. 10.25 H. HogenEsch, 2005

20. Hapten Parham Fig. 10.26 H. HogenEsch, 2005

21. Antibody-antigen interaction H. HogenEsch, 2005 Fig. 2.10

22. 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, 2005

23. 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, 2005

24. A B Crossreactivity H. HogenEsch, 2005

25. Immunoglobulin genes Fig. 2.13 H. HogenEsch, 2005

26. Somatic recombination – light chain Fig. 2.14 H. HogenEsch, 2005

27. Somatic recombination – Heavy chain Fig. 2.14 H. HogenEsch, 2005

28. Number of gene segments Fig. 2.15 H. HogenEsch, 2005

29. Recombination Signal Sequences Fig. 2.16 H. HogenEsch, 2005

30. Recombination V(D)J – recombinase Fig. 2.17 H. HogenEsch, 2005

31. V k J k C k k chain polypeptide V 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 H. HogenEsch, 2005

32. Generation of diversity • k chain: 40 V x 5 J = 200 Vk • l chain: 30 V x 4 J = 120 Vl • H chain: 65 V x 27D x 6 J = 10,530 VH • (200 + 120) x 10,530 = 3.4 x 106 combinations H. HogenEsch, 2005

33. Mechanisms for additional diversity in immunoglobulins • Imprecise joining of gene segments • Random nucleotide addition at joining regions • terminal deoxynucleotidyl transferase (TdT) Fig. 2.17 H. HogenEsch, 2005

34. Generation of diversity • Multiple gene segments: • - k chain: 40 V x 5 J = 200 Vk • - l chain: 30 V x 4 J = 120 Vl - H chain: 65 V x 27D x 6 J = 10,530 VH • Combination of H and L chain: (200 + 120) x 10,530 = 3.4 x 106 combinations • Imprecise joining and nucleotide addition > 108 different specificities H. HogenEsch, 2005

35. Organization of CH genes Fig. 2.19 H. HogenEsch, 2005

36. Naïve mature B cells express IgM and IgD Fig. 2.20 H. HogenEsch, 2005

37. Allelic exclusion Allelic exclusion ensures that the B lymphocyte expresses immunoglobulin molecules with only one specificity. Mechanism: Successful rearrangement of immunoglobulin gene segmentsone allele shuts down the rearrangement process of the other allele. l k H 16 6 12 H. HogenEsch, 2005

38. B cell receptor complex Fig. 2.21 H. HogenEsch, 2005

39. Changes in B cells after activation by antigen • Somatic mutation – additional diversity • Isotype switching H. HogenEsch, 2005

40. Somatic hypermutation Fig. 2.24 H. HogenEsch, 2005

41. Hypervariable and framework regions CDR = complementarity -determining region Fig. 2.7 H. HogenEsch, 2005

42. Isotype switching IgG1 IgG2 IgG3 IgG4 IgM+/IgD+ IgA1 IgA2 IgE H. HogenEsch, 2005

43. Organization of CH genes Fig. 2.19 H. HogenEsch, 2005

44. Isotype switching H. HogenEsch, 2005

45. Physical properties of immunoglobulins H. HogenEsch, 2005

46. IgM • Membrane-bound monomer and secreted pentamer. • First immunoglobulin to be synthesized during ontogeny and in the immune response. • Activates complement pathway; agglutination. • Can be transported into mucosal secretions. H. HogenEsch, 2005

47. IgG • Highest concentration in serum. • Four subclasses: IgG1 - 4 • Activates complement • Binds to Fcg -receptors on neutrophils, macrophages and NK cells H. HogenEsch, 2005

48. IgA • Usually dimer • Secretory IgA is a dimer with a secretory component. • Two subclasses: IgA1 and IgA2 • Major immunoglobulin in mucosal secretions • Neutralization; Prevents binding of micro-organisms to receptors • Not effective activator of complement H. HogenEsch, 2005

49. IgE • Very low serum concentration in healthy individuals. • Concentration is higher in patients with helminth infections and often in patients with allergies. • Lacks hinge region; extra CH domain • Binds to Fce receptor on mast cells and basophils. Cross-linking results in degranulation and release of pro-inflammatory mediators. H. HogenEsch, 2005

50. IgD • Very low concentration in serum • Primarily found with IgM on naïve mature B cells • Function is unknown H. HogenEsch, 2005