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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
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
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
Basic structure of immunoglobulin • Fab ; fragment antigen binding • Fc ; fragment crystallizable • H chain ; 50 kDa • L chain ; 25 kDa
Immunoglobulin fold ; multiple domain Hinge : flexible antigen binding
Immunoglobulin isotypes (5 classes) • 5 heavy chain types : Isotype – determine the class of antibody • IgM (m), IgG (g), IgA (a), IgD (d), IgE (e)
IgG 80% of the total serum Igs Different biological activity
IgM 5-10% of total Igs J chain ; polymerization Pentameric structure ; 10 Ag binding sites more efficient than IgG
IgA 10-15% of total Igs Predominant in external secretion ; mucosal transport Monomer, polymer (2-4) ; J-chain Poly-Ig receptor – secretary component
IgE Immediate hypersensitivity reaction ; Allergy P-K reaction Allergen - IgE/Fc ; degranulation
IgD Membrane bound Ig in mature B cell
Hypervariable regions • CDR : complementarity determining region
Production of hybridoma Select in HAT(hypoxanthine, aminopterin, thymidine) medium
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)
chimeric: mouse V region + human C region humanized: mouse CDR region + all other human region
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
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)
Organization of imunoglobulin genes leader (L) peptide ; signal for transport to ER V (variable), J (joining), D (diversity), C (constant)
Gene rearrangement Somatic recombination of gene segments
Multigene organization of Ig genes Gene segment (coding sequence)
Joining the gene segments by recombination RAG : recombination activating gene
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
Junctional flexibility P- & N- addition ; different reading frame Productive rearrangement ; increase antibody diversity Non productive rearrangement
Membrane-bound vs. Secreted form of Ig ; alternative polyadenylation
Heavy chain gene expression by RNA processing ; alternative splicing
Immature B cell ; gene rearrangement, RNA processing, mIgM, mIgD
Mature B cell ; Antigen stimulation Activation & differentiation, Class switching
Neutralization: • for masking • opsonization: • for efficient phagocytosis