ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

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ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL
DIVERSITY
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WHAT ARE ANTIBODIES?

• Antigen specific proteins produced by plasma
  cells
• Belong to immunoglobulin superfamily
• Located in blood and extravascular tissues,
  secretions and excretions
• Bind pathogenic microorganism and their toxins in
  extracellular compartments
• Secreted form of immunoglobulins

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WHAT ARE IMMUNOGLOBULINS?

• Antigen specific proteins produced by B
  lymphocytes
• Belong to immunoglobulin superfamily
• Bound to surface of B lymphocytes
• Function as binding (receptor) sites for specific
  antigens
• Antigen receptor sites on mature B lymphocytes
• IgM
• IgD
• Membrane-bound form of immunoglobulins

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WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY

• Proteins with structural feature first defined in
  immunoglobulins
• Characteristic structural feature
• Sequence of Domains providing stable conformation
• Domain
• Polypeptide (100 to 110 amino acids) chain folded
  into sandwich (2 slices of bread) held together
  by disulfide bond
• IG superfamily members
• Antibodies, B cell receptors, T cell receptors,
  MHC molecules and others

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STRUCTURE OF ANTIBODIES

• Antibodies are glycoproteins composed of
• Polypeptide chains and carbohydrate
• Monomeric structure
• Polypeptide chains
• 2 identical heavy chains
• 2 identical light chains
• Polypeptide chains joined by disulfide bonds
• Carbohydrate

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STRUCTURE OF ANTIBODIES

• Polypeptide chains have variable and constant
  regions
• Variable
• N (amino)-terminal of polypeptide chain
• Antigen binding site
• Constant
• C (carboxyl)-terminal of polypeptide chain
• Binding sites for cell surface receptors and
  complement
• Structure represented by the letter Y
• Y shaped molecule cleaved by protease papain
• Fragment antigen binding (Fab)
• Fragment crystallizable (Fc)

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CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

• Classes based on constant region of heavy chains
• Immunoglobulin A (IgA)
• Immunoglobulin D (IgD)
• Immunoglobulin E (IgE)
• Immunoglobulin G (IgG)
• Immunoglobulin M (IgM)
• Differentiation of heavy chains
• Length of C region, location of disulfide bonds,
  hinge region, distribution of carbohydrate
• Classes have different effector functions

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CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

• Additional classification based on light chains
• Kappa
• Lambda
• Each IG has either kappa or lambda, not both
• IgG kappa
• IgG lambda
• No functional differences between light chains

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ITS GREEK TO ME

• Heavy chains, light chains and other molecules of
  the immune system identified using letters of the
  Greek alphabet

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THREE DIMENSIONAL STRUCTURE OF ANTIBODIES

• Antibodies function in setting of infectious
  process
• Proteolytic enzymes, salt and pH differences
• Antibodies remain stable based on
• Sequence of domains
• Single domain consists of
• 100 110 amino acids folded into compact and
  stable conformation
• Domains
• Variable (V)
• Single V domain in H and L chains
• Constant (C)
• Single C domain in L chains
• Three to four (C) domains in H chains

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ANTIGEN BINDING SITES OF IMMUNOGLOBULINS

• Antigen binding sites formed from hypervariable
  regions
• Heavy chain V domain
• Light chain V domain
• Hypervariable regions of V domains
• Amino acid sequence differences concentrated
• Flanked by less variable framework regions
• Three hypervariable regions in each V domain
• Hypervariable regions also called
• Complementarity-determining regions (CDR)

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ANTIGEN BINDING SITES OF IMMUNOGLOBULINS

• Antigen binding sites vary with size and shape of
  antigen
• Part of antigen to which antibody binds
• Antigenic determinant (Epitope)
• Antigen-Antibody binding based on non-covalent
  forces
• Hydrogen bonds
• Affinity
• Strength of binding of one molecule to another by
  a single binding site
• Avidity
• Overall strength of binding between two molecules

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ANTIBODIES AS DIAGNOSTIC AND THERAPEUTICS AGENTS

• Based on specificity and affinity of antibodies
• Both applications require large quantities of
  identical antibodies
• Monoclonal antibodies
• Monoclonal antibodies are produced using
  hybridoma cell line
• Hybridoma cell line
• Derived from single antibody producing cell fused
  with myeloma cell (neoplastic plasma cell)

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IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE
ENCOUNTER WITH ANTIGEN

• Immune system capable of producing a limitless
  number of different immunoglobulins/antibodies
• Mechanism
• Genes for IG organized differently
• Genes exist as nonfunctional segments
• Variable (V), Joining (J), Diversity (D),
  Constant (C)
• Genes are inherited in this form
• Germline form (germline configuration)

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IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE
ENCOUNTER WITH ANTIGEN

• Expression
• Gene segments must be rearranged into functional
  gene
• Gene Rearrangement
• Takes place during development of B-cells
• Mechanism of somatic recombination
• Genes for IG located at 3 chromosomal locations
• Heavy chain locus on chromosome 14
• Kappa light chain locus on chromosome 2
• Lambda light chain locus on chromosome 22

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GERMLINE ORGANIZATION OF HUMAN IG HEAVY CHAIN AND
LIGHT CHAIN LOCI

• Lambda light chain locus
• Gene segments
• 30 (V), 4 (J) and 4 (C)
• Kappa light chain locus
• Gene segments
• 40 (V), 5 (J) and 1 (C)
• Heavy chain locus
• Gene segments
• 65 (V), 27 (D), 6 (J) and 9 (C)

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CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN
VARIABLE REGIONS

• Light chain
• Constructed from 2 segments
• 1 (V) segment
• 1 (J) segment
• Heavy chain
• Constructed from 3 segments
• 1 (V) segment
• 1 (D) segment
• 1 (J) segment

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SOMATIC RECOMBINATION

• Performed by enzymes with cut and rejoin DNA
• Directed by
• Recombination signal sequences (RSS)
• Recombination signal sequences
• Recognition sites for enzymes
• Recombination occurs between different types
• 9 / 12 / 7
• 9 / 23 / 7
• Mechanism follows the 12/23 rule
• Ensures segments joined in correct order

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MECHANISMS OF GENETIC DIVERSITY IN V-REGION OF
IMMUNOGLOBULINS

• Random combination of
• V and J segments in light chain genes
• V, D and J segments in heavy chain genes
• Addition of P (palindromic) and N (non-templated)
  nucleotides at junctions of gene segments during
  recombination
• Junctional diversity
• Association of H and L chains in different
  combinations

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CONSTRUCTION OF B-CELL SURFACE IMMUNOGLOBULINS

• Following rearrangement of VH gene segments, two
  CH loci are transcribed
• IgM
• IgD
• M and D constant segments
• Located nearest variable segments
• M and D transcript processed by
• Cleavage, polyadenylation and splicing
• IgM and IgD enter endoplasmic reticulum

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SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS
TO COMPLETE ANTIGEN RECEPTOR

• In ER, IgM and IgD associated with transmembrane
  proteins
• Ig-alpha
• Ig-beta
• Transmembrane proteins
• Transport M and D to B cell surface
• Communication of antigen binding to inside of B
  cell
• Tails interact with intracellular signaling
  molecules
• Complex of IgM and IgD with Ig-alpha and Ig-beta
  forms
• B-cell receptor

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DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS
ENCOUNTER ANTIGEN

• Mature, naïve B cell has membrane bound IgM and
  IgD antigen receptors
• Binding of antigen initiates proliferation and
  differentiation of B-cells into plasma cells
• During differentiation, B cells switch from
  making immunoglobulin to antibody M and D
  isotypes
• IgM
• Produced in large amounts
• Provides protective immunity
• IgD
• Produced in small amounts
• No known function

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MECHANISM OF SWITCHING FROM IMMUNOGLOBULIN TO
ANTIBODY

• Surface and secreted forms derived from same
  heavy chain gene by alternative RNA processing
• Each heavy chain C gene has
• Membrane coding (MC) region
• Secretion coding (SC) region
• Mechanism involves a switch in cleavage,
  polyadenylation and splicing
• From pAm region to pAs region

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DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS
ENCOUNTER ANTIGEN

• Following antigen activation of B-cells,
  additional diversification occurs in V domain by
• Somatic hypermutation
• Somatic hypermutation
• Introduction of random single nucleotide
  substitutions (point mutations) throughout V
  regions of H and L chains
• Mechanism poorly understood
• More common in hypervariable regions (CDRs)

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OUTCOME OF SOMATIC HYPERMUTATION

• Gives rise to some antibodies with higher
• Affinity for antigen
• Affinity
• Strength of binding of one molecule to another by
  a single binding site
• Higher affinity antibodies are produced as immune
  response proceeds
• Affinity maturation

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THE PRIMARY HUMORAL IMMUNE RESPONSE

• Immune response initially produces IgM antibodies
  then switches to IgG antibodies
• Question
• Why switch from IgM to IgG?
• Answer
• Limited effector mechanisms for IgM
• Range of effector mechanisms for IgG
• Mechanism
• Isotype or class switching

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ISOTYPE OR CLASS SWITCHING

• Process by which B cell changes class of IG
  produced while preserving antigenic specificity
• Involves somatic recombination which attaches
  different heavy chain constant region to variable
  region
• Occurs only during active immune response
• Mechanisms involves recombination between
• Switch sequences (regions)

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CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF
IMMUNOGLOBULINS

• Subclasses are numbered according to plasma
  concentration

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FUNCTIONS AND PROPERTIES OF ANTIBODY

• Neutralization
• Direct inactivation of pathogen or toxin thereby
  preventing its interaction with human cells
• Opsonization
• Coating of pathogens for more efficient
  phagocytosis
• Activation of complement
• More efficient phagocytosis
• Direct killing

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IgM ANTIBODY OF THE IMMUNE RESPONSE

• First isotype produced in primary response
• May or may not be produced in secondary response
• Produced before B cells undergo somatic
  hypermutation
• Occurs as pentamer with J chain
• Found primarily in blood and lymph
• Multiple binding sites confers high avidity and
  compensates for low affinity of monomers
• Highly effective in complement activation
• Functions as rheumatoid factor

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IgG ANTIBODY OF THE IMMUNE RESPONSE

• Second isotype produced in primary response
• Primary isotype of
• Secondary immune response
• Memory immune response
• Represents approximately 75 of total serum IG
• Four subclassses (1-4)
• Different effector functions
• Transported across placenta
• Functions as rheumatoid factor

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IgA ANTIBODY OF THE IMMUNE RESPONSE

• Two subclasses (IgA1 and IgA2) and two forms
  (monomeric and dimeric)
• Monomeric
• Located in blood and extracellular spaces
• Predominately IgA1
• Ratio of IgA1 to IgA2 is 101
• Functions as rheumatoid factor
• Dimeric
• Located in mucous membranes and secretions
• Predominately IgA2
• Ratio of IgA2 to IgA1 is 32
• J chain like IgM

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IgE AND IgD ANTIBODIES OF THE IMMUNE RESPONSE

• IgE
• Binds with high affinity to receptors on mast
  cells, basophils and activated eosinophils
• Longer half-life when cell bound
• Initiates a strong inflammatory reaction to
  parasites
• Involved in allergic reactions
• IgD
• Antigen receptor on mature B-cells
• No other known function

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