1. Ch4. Antibody Structure and the Generation of B-cell diversity

2. B-Cell Receptor (BCR) Immunoglobulin(Ig), antibody specificity: one antibody for one antigen or a few different antigens diversity: as high as 10 16 of antibody repertoire; in practice about 10 9 B cell is stimulated to differentiate into plasma cells

3. B cell activation

4. Basic structure of Immunoglobulin Heterodimers ; 2 light + 2 heavy chains by disulfide and non-covalent bond V (variable) region ; Ag binding site (CDR) C (constant) region ; effector function(host cell receptor binding), glycosylation

5. Basic structure of immunoglobulin Fab ; fragment antigen binding Fc ; fragment crystallizable H chain ; 50 kDa L chain ; 25 kDa

6. Immunoglobulin fold ; multiple domain Hinge : flexible antigen binding

7. Immunoglobulin isotypes (5 classes) 5 heavy chain types : Isotype – determine the class of antibody IgM (  ), IgG (  ), IgA (  ), IgD (  ), IgE (  )

9. IgG 80% of the total serum Igs Different biological activity

10. IgM 5-10% of total Igs J chain ; polymerization Pentameric structure ; 10 Ag binding sites more efficient than IgG

11. IgA 10-15% of total Igs Predominant in external secretion ; mucosal transport Monomer, polymer (2-4) ; J-chain Poly-Ig receptor – secretary component

12. Resist to protease

13. IgE Immediate hypersensitivity reaction ; Allergy P-K reaction Allergen - IgE/Fc ; degranulation

14. IgD Membrane bound Ig in mature B cell

16. Structure of heavy and light chain

17. Hypervariable regions (HV)

18. Hypervariable regions CDR : complementarity determining region

19. Epitope : antigenic determinant

20. Epitopes vs. Ab

21. Monoclonal antibody

22. Production of hybridoma Select in HAT(hypoxanthine, aminopterin, thymidine) medium

23. Clinical use of mAbs Diagnostic reagent ; pregnancy, pathogen, blood level of drugs, tumor Ag Imaging ; detect tumor metastasis Therapeutic reagents ; immunotoxin Abzyme ; catalytic antibody (Ab + enzyme)

25. FACS

26. chimeric: mouse V region + human C region humanized: mouse CDR region + all other human region

27. Genetic regulation Immunoglobulin gene expression Recombination : genetic material is broken and then joined to different region heavy and light chain gene rearrangement isotype switching (class switching) Alternative splicing: different combinatorial use of exons IgM and IgD switching Alternative polyadenylation: different use of polyadenylation sites; transmembrane region

28. Genetic model for Ig structure Vast diversity of antibody specificities Maintaining the constancy in constant (C) region Isotypes with the same antigenic specificity Germ-line vs. somatic variation Germ-line theory ; genome of germ cells contains a large repertoire of Ig genes Somatic-variation theory ; relatively small number of Ig gene on genome mutation and gene rearrangement Two gene model ; two genes encode a single polypeptide many V region + 1 copies of C region class gene Ig gene rearrangement during th B cell differentiation (by Tonegawa)

29. Organization of imunoglobulin genes leader (L) peptide ; signal for transport to ER V (variable), J (joining), D (diversity), C (constant)

30. Gene rearrangement Somatic recombination of gene segments

31. Gene rearrangement

32. Multigene organization of Ig genes Gene segment (coding sequence)

33. Recombination signal sequences (RSS)

35. Joining the gene segments by recombination RAG : recombination activating gene

36. Generation of antibody diversity Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P (palindromic) nucleotide addition (P-addition) N (nontemplated) nucleotide addition (N-addition) Somatic hypermutation Combinatorial association of light and heavy chains

37. Junctional flexibility

38. P- & N- addition ; different reading frame Productive rearrangement ; increase antibody diversity Non productive rearrangement

39. Generation of junctional diversity : 3 X 10 7

41. Somatic hypermutation increase diversity

42. Membrane-bound vs. Secreted form of Ig ; alternative polyadenylation

43. Heavy chain gene expression by RNA processing ; alternative splicing

44. Class switching (Isotype switching)

45. Function and properties of the human Ig isotypes

46. Immature B cell ; gene rearrangement, RNA processing, mIgM, mIgD

47. Mature B cell ; Antigen stimulation Activation & differentiation, Class switching

49. Fine tuning of antibody function

50. Neutralization: –for masking opsonization: –for efficient phagocytosis

51. Membrane-bound immunoglobulin (BCR) Intracellular signaling

52. Ig superfamily Common evolutionary ancestor ; gene duplication & divergence Immunoglobulin fold (~110 a.a) domain

53. Fc receptors (FcR)

54. Allelic exclusion Product of Ig gene inhibit further gene rearrangement of counter allele Antigenic specificity