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Antibodies PowerPoint Presentation

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On : Mar 14, 2014

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  • Slide 1 - Antibodies Secreted by B lymphocytes Great diversity and specificity: >109 different antibodies; can distinguish between very similar molecules Tag particles for clearance/destruction Protect against re-infection (vaccines)
  • Slide 2 - Antibody Structure Ig domain: 110 amino acids; globular domain used in many proteins. Variable domains, Constant domains, Hinge. Fab: fragment antigen binding Fc: fragment crystallizable (effector functions) © New Science Press Ltd. 2003
  • Slide 3 - ppt slide no 3 content not found
  • Slide 4 - The Immunoglobulin Superfamilya few examples
  • Slide 5 - Variability in antibodies is clustered in the loops in the variable domains of the heavy and light chains (green); these regions are responsible for binding to antigen.
  • Slide 6 - Antibody Classes: Structure © New Science Press Ltd. 2003
  • Slide 7 - Affinity and Avidity Affinity: the strength of binding between a single binding site and a single ligand. [A][B] [AB] Avidity: the strength of binding between a molecule and a complex ligand, e.g. if there are multiple binding sites then the avidity may be increased by increasing the number of binding sites or by increasing the affinity of those binding sites. KD =
  • Slide 8 - Affinity and Avidity, continued IgM is produced early in an immune response when the affinity for antigen often is low; as an immune response continues, antibody affinity is improved, this is combined by “class switching” to the use of smaller molecules (IgG, IgE and IgA). The increased affinity compensates for the decrease in number of binding sites in maintaining the overall avidity for antigen.
  • Slide 9 - © New Science Press Ltd. 2003
  • Slide 10 - Monoclonal Antibodies Single antibody (all same H and L chains) Made by fusion of B cells to a transformed cell line of the plasma cell type and selection for “hybridomas” that produce antibody with the desired properties Standardized, unlimited reagent for diagnosis or therapy (human antibodies or “humanized” antibodies can be made)
  • Slide 11 - Generation of Monoclonal Antibodies © New Science Press Ltd. 2003
  • Slide 12 - Polyclonal vs. Monoclonal Antibodies Immunize Individual Serum Polyclonal antibodies 7+ days Purify antibodies B lymphocytes 3 days Immortalize “hybridoma” cDNA cloning Monoclonal Antibodies
  • Slide 13 - Monoclonal antibodies used in medicine Standardized, unlimited amounts of reagents for diagnosis or therapy (human antibodies or “humanized” antibodies can be made).
  • Slide 14 - Immunoglobulin genes Generation of Ig diversity in B cells before encounter with antigen (Primary Repertoire) the body can produce billions of different antibodies (although a single B cell produces one specificity only) part of this diversity is produced by the various combinations of H and L chain polypeptides no ready made genes in the germ line Ig heavy and light chain loci consist of families of gene segments. Some of these segments are rearranged to somatically generate the immunoglobulin genes in B lymphocytes (only) the rearrangement process generates the enormous diversity
  • Slide 15 - Recombination of gene segments produces a single gene encoding H chain, and a single gene encoding L chain in a given B lymphocyte This unique process of DNA rearrangement happens (daily) when a hemopoetic stem cell differentiates into a B lymphocyte Note: Unfortunately, the one gene segment that needs to be joined to the D or J segments is called V, similar to the V region of the H or L chain polypeptide. However, it encodes only part of the V polypeptide sequence.
  • Slide 16 - Summary — Making IgM from DNA to RNA to Protein
  • Slide 17 - There are several copies of the V, D, and J segments, respectively. Different B cells choose different copies. This creates diversity in the antigen binding sites of antibodies generated by all B cells, even though an individual B cell produces only one kind of antibody.
  • Slide 18 - Generation of Antibody Diversity  light chains: 40 V x 5 J = 200  light chains: 30 V x 4 J = 120 H chains: 40 VH x 27 DH x 6JH = 6,480 320 L chains x 6,480 H chains = 2.1 x 106 Junctional diversity (addition or deletion of nucleotides at recombination sites, especially of H chain), estimated to add 3x107 fold to overall diversity.
  • Slide 19 - Mechanism of V(D)J recombination CACCGTG Recombination signals Rag-1/Rag-2/Artemis Non-homologous end joining proteins defects: Severe Combined Immunodeficiency (SCID) © New Science Press Ltd. 2003
  • Slide 20 - Recombination enzymes produce additional diversity in the antigen-binding sites of Igs, termed Junctional Diversity
  • Slide 21 - Creation of Junctional Diversityby P-regions and TdT
  • Slide 22 - Discovery of Rag1, 2 genes “Recombination Activating Gene”
  • Slide 23 - Defects in Lymphocyte development leading to severe combined immunodeficiency (SCID) Note: SCID can also result from defects that interfere with lymphocyte activation (adenosine deaminase deficiency, purine nucleotide phosphorylase deficiency, MHC defects, etc.)
  • Slide 24 - Allelic and isotypic exclusion At each of the loci encoding Igs, only one (at most) of the two alleles is functional in any one lymphocyte; this is called allelic exclusion, and it ensures that all of the antibody molecules produced by a cell have the same specificity. Furthermore, in a given lymphocyte, either  or  light chain, but not both, can combine with heavy chain to form a complete Ig molecule; this is called L chain isotypic exclusion.
  • Slide 25 - Allelic and isotypic exclusion, continued The precise mechanism for H chain allelic exclusion is not known, although a feedback mechanism generally is assumed. The H genes are formed before the L genes. After a functional antigen receptor is formed, the RAG genes are turned off; this ends Ig gene rearrangement and mediates L chain allelic and isotypic exclusion.
  • Slide 26 - Lymphoid malignancies resulting from errors in V(D)J recombination © New Science Press Ltd. 2003 VDJ Recombination (and switch) reactions contribute to translocation leading to over-expression of a cellular growth or survival promoting gene CH CH
  • Slide 27 - Generation of Ig diversity in B cells after encounter with antigen (Secondary Repertoire)
  • Slide 28 - Red boxes - somatic mutations; CDR, complementarity determining regions Rearranged V-region gene segments are further diversified by somatic hypermutation This leads to antibodies with increased affinity for the inducing antigen
  • Slide 29 - from Longacre and Storb Cell 102: 541, 2000.
  • Slide 30 - The rearrangement of V,D, and J segments produces a functional exon that encodes the variable region of an Ig chain; together with the nearby exons encoding the constant region it constitutes the complete Ig gene. At the H chain locus, the constant region gene segments (defining the Ig classes) are arranged next to each other.
  • Slide 31 - When a B cell expands into a clone, it may switch its Ig class. When this happens, the variable region of the antibody stays the same, but the constant region changes.
  • Slide 32 - Isotype switching involves recombination between specific switch regions Switch regions are repetitive DNA sequences at the 5’ side of each C region Switching occurs by recombination between switch regions, with deletion of the intervening DNA
  • Slide 33 - ppt slide no 33 content not found
  • Slide 34 - Comparison of VDJ recombination, class switch recombination and somatic hypermutation Deamination of Cytidine
  • Slide 35 - Activation-induced cytidine deaminase (AID) Discovered as an induced gene in a cell line with inducible class-switch recombination (subtractive hybridization) Transfection into B cell lines induces class switch recombination AID KO mice have no class switch recombination AND no somatic hypermutation Hyper-IgM syndrome type 2 (autosomal) is due to mutation in AID; very similar phenotype to mice (no IgG, IgA, IgE; very much reduced somatic mutation)
  • Slide 36 - AID: How does it work? AID is closely related to ABOBEC-1, a cytidine deaminase that edits mRNA for Apolipoprotein B indirect action or direct action in class switch and hypermutation? AID could edit mRNAs for factors that act in class switch and factors that act in class switch OR it could act directly in both processes
  • Slide 37 - AID as a mutator of DNA AID is mutagenic in bacteria; and mutations are increased by deficiency in Uracil-DNA glycosylase (enzyme that removes U from DNA and triggers DNA repair) Class switch is inhibited and hypermutation perturbed in UNG-deficient mice These results favor the hypothesis that AID directly acts on C residues in DNA to promote class switch and hypermutation
  • Slide 38 - In hypermutation: dU in DNA causes mutations through pairing with A, rather than with G, as C does; and more mutations are introduced via mismatch repair and/or error-prone DNA polymerases. In class switch recombination: dU in DNA could lead to nick formation by repair enzymes: nicks on both strands-->ds breaks-->recombination
  • Slide 39 - Evolution of adaptive immunity Lymph nodes LRR humoral immunity
  • Slide 40 - Shark Ig gene cassettes Repeating cassettes of unrearranged and/or pre-rearranged VDJC heavy chain or VJC light chain genes. How is expression controlled?
  • Slide 41 - Chickens create variability by gene conversion (AID-dependent)
  • Slide 42 - To construct an immune system
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