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

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

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

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  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 1016 of antibody repertoire; in practice about 109 • 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 (m), IgG (g), IgA (a), IgD (d), IgE (e)

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

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

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

  11. Resist to protease

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

  13. IgD Membrane bound Ig in mature B cell

  14. Structure of heavy and light chain

  15. Hypervariable regions (HV)

  16. Hypervariable regions • CDR : complementarity determining region

  17. Epitope : antigenic determinant

  18. Epitopes vs. Ab

  19. Monoclonal antibody

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

  21. 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)

  22. FACS

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

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

  25. 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)

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

  27. Gene rearrangement Somatic recombination of gene segments

  28. Gene rearrangement

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

  30. Recombination signal sequences (RSS)

  31. Recombination signal sequences (RSS)

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

  33. 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

  34. Junctional flexibility

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

  36. Generation of junctional diversity : 3 X 107

  37. Somatic hypermutation increase diversity

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

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

  40. Class switching (Isotype switching)

  41. Function and properties of the human Ig isotypes

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

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

  44. Fine tuning of antibody function

  45. Neutralization: • for masking • opsonization: • for efficient phagocytosis

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