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Obstacles to Antibody Sequencing. Immunoglobulin fine structure ... A type of passive immunotherapy which employs intravenously injecting into ... – PowerPoint PPT presentation

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Title: Antibodies

  • Structure and Function

Chapter 4
  • Basic Structure of Antibodies
  • Obstacles to Antibody Sequencing
  • Immunoglobulin fine structure
  • Antibody-Mediated Effector Functions
  • Antibody Classes and Biological Activities
  • Antigenic Determinants on Immunoglobulins
  • The B-cell Receptor

Enzymatic digestion
  • Rodney Porter and Gerald Edelman derived the
    structure of antibodies by protein enzymatic
    digestion of IgG antibodies.
  • Papain digestion produce two different types of
  • Fab fragments that had antigen binding activity
  • Fc fragments that crystallized in cold storage
  • Pepsin digestion generated a F(ab)2
  • Mercaptoethanol cleave disulfide bonds

Basic Structure of Antibodies
  • Composed of four peptide chains, two identical
    heavy (H) chains and two Identical light (L)
  • Covalent disulfide bonds bind together H and L
    chains to form a (H-L)2. structure.

Basic Structure of Antibodies
  • The first 110 amino acids on the amino terminal
    ends of both the H and L chain are highly
    variable ends.
  • These region are referred to as VH and VL
  • The differences observed between Abs in their Ab
    -Ag binding specificity correspond to differences
    in the V regions.
  • Most differences in the V region fall within the
    complementarity-detemining regions (CDR).

Light chain constant Region
  • The carboxyl half of the light chain is referred
    to as the Constant region or CL
  • There are two different types of CL regions found
    on L chains, kappa (k) and lambda (l).
  • These two different types of constant regions
    give rise to the two different classes of CL
  • Humans have 4 different subgroups of l (l1 l2 l3
  • K light chains predominate.

Heavy Chain Constant (CH) Regions
  • There are five different types of CH regions on H
    chains, m, d, g, a, and e.
  • These five different types of constant regions
    correspond to the five different classes of CH.
  • d, g, and a, H chains are made up of 3 domains
    of 110 amino acids each
  • In addition d, g, and a, contain a proline rich
    hinge region
  • m and e do not contain a hinge region but
    instead have an extra domain (made up of four
    domain of 110 a. acids each).

Immunoglobulin fine structure
  • b pleated sheets contain 3 or 4 antiparallel b
  • Structure is held together by hydrogen bonds in
    neighboring chains
  • The amino acid side groups (R) are arranged
    perpendicular to the sheet.

  • Two beta pleated sheets, each containing 3-4
    anti-parallel strands are folded into a globular
    tertiary structure (domain)
  • Theyre held together by hydrophobic interactions
    and highly conserved disulfide bond.
  • Disulfide bond forms a loop of about 60 amino
  • Residues in the amino terminal loop make up the
    Ag binding site.

Domains, cont.
  • Heavy and light chains are folded into domains,
    each with about 110 amino acid residues and an
    intrachain disulfide bond that forms a loop of 60
    amino acids
  • Amino terminal domains, correspond to the V
    regions, bind to antigen
  • Effector functions are mediated by other domains

CH1 and CL Domains.
  • The function of the CH1 domain and CL domain is
    to extend the Fab arms and increase the maximum
  • The heavy and light chains are held together by
    an intrachain disulfide bond between them

Hinge Region
  • d, g, and a, contain a proline and cysteine rich
    hinge region.
  • The two fab arms are flexible and can work
    independent of each other
  • The large number of proline residues in the hinge
    region gives it an extended polypeptide
    conformation which is vulnerable to enzymatic
    cleavage (pepsin and papain).

CH2/ CH2 domains
  • m and e do not contain a hinge region but
    instead have an extra domain (CH2/ CH2) made up
    of four domain of 110 a. acids each.
  • The function of the extra domain in m and e has
    not been determined
  • The CH2/ CH2 domain protrudes due to a
    carbohydrate interior.
  • This makes this domain much more accessible to
    complement proteins.

The carboxyl terminal end
  • The carboxyl terminal end for d, g, and a is the
    CH3/ CH3 domain.
  • CH4/ CH4 domains are the terminal ends for m and

Secreted vs Membrane Bound Terminal Ends
  • The carboxyl terminal ends in secreted Abs (sIg)
    are different than membrane bound Abs (mIg) .
  • sIg terminal ends have hydrophilic amino acids
    sequences of various lengths.
  • mIg contain three separate regions
  • An extracellular hydrophilic space sequence
    composed of 26 amino acid residues.
  • A hydrophobic transmembrane sequence
  • A short cytoplasmic tail.

Antibody Effector functions
  • Macrophages and neutrophils contain Fc receptors
    (FcR) which bind the constant region of Ig
  • The binding of Ab/Ag complex to the FcR initiates
    a signal-transduction pathway that results in

  • IgM and IgG classes activate the complement
    system by binding to complement proteins.
  • Byproduct of the complement activation pathway is
    C3b, an opsonin
  • Complement C1 binds the CH2 domain

Antibody-dependent cell-mediated cytotoxicity
  • The linking of antibody bound to target cells
    with the FC receptors of a number of effector
    cells focuses the cytotoxic activity of these
  • Eosinophils, NK cells, neutrophils are examples

Antibody classesand
  • Biological Activities

Antibody Classes
  • IgM expressed as membrane bound anitbodies on
  • Pentamer
  • 5 units held together by disulfide bonds (C3 and
    C4 domains)
  • J (Joining) chain functions in the polymerization
    of monomers
  • First immunoglobulin class produced in a primary
    response to an antigen
  • Has 10 anitgen binding sites
  • More effective at stimulating complement
  • Large-size - does not diffuse well
  • The FC receptors on phagocytes bind IgM

  • Found on surface of mature B-cells.
  • Biological function unknown (thought to function
    in activation of B-cells)

  • Most abundant isotype in serum (80)
  • Cross placenta and play important role in
    protecting fetus
  • Placental cells bind the Fc portion of IgG and
    transfer Ab across the placental membrane.
  • Activate complement system
  • Opsoninphagocytosis
  • Four subclass of IgG in humans

  • Mediate the immediate hypersensitivity reactions
    (hayfever, asthma, hives, anaphylactic shock)
  • Mast cells and basophils bind fc portion of IgE
  • Cross-linkage of receptor bound IgE molecules by
    antigen, induces degranulaltion of the Mast and
    basophil cells
  • Parasitic response
  • Eosinophils express receptors for IgE

  • Most abundant Ab in the body
  • Found Predominantly in external secretions i.e.
    Breast Milk, Saliva, tears, mucus.
  • Serum form is a monomer
  • Secretory form is a dimer or tetramer linked
    together via a secretory component and a J
  • J (Joining) chain functions in the polymerization
    of monomers.
  • Secretory component mask sites that are
    susceptible to protease cleavge.
  • Plasma cells that release IgA Abs are
    concentrated along the Mucus Membrane surface.
  • Two subclasses of IgA

Secretory component of IgA
  • The secretory component is a polypeptide produced
    by the epithelial cells of mucus membranes.
  • Secretory IgA is formed during transport through
    mucous membrane epithelial cells.
  • The J chain on a dimeric IgA binds to a poly-Ig
    receptor on the basolateral membrane of an
    epithelial cell and is internalized by receptor
    mediated endocytosis.
  • After transport of the receptor-IgA complex to
    the luminal surface, the poly-Ig receptor is
    enzymatically cleaved releasing the secretory
    component bound to the dimeric IgA.

Mucosal Immunity
  • Question What would be the most effective rout
    to administer an immunization that would enhance
    mucosal immunity?
  • Answer Oral or by a respiritory inhalent.
    Antigen must be one that would stay intact while
    passing through M-cells

  • What is IVIG
  • Answer A type of passive immunotherapy which
    employs intravenously injecting into a recipient
    antibodies with a broad specificity to different
  • intravenous immuneglobulin
  • Patients may receive 200 400 mg / kg of body

Antigenic Determinants on Immunoglobulins
Antigenic determinants on immunoglobulins
  • Antibodies are glycoproteins that like other
    proteins can elicit an immune response.
  • The immune response is carried out by the humoral
    system as it synthesizes Ab against epitopes of
    foreign Abs
  • Epitopes on Abs fall into three main catigories
  • Isotypic
  • Allotypic
  • idiotypic

  • When an antibody from one species is injected
    into a second species the second species will
    make ab specific for the isotypic determinants of
    the first.
  • Isotypic derterminants are found on Hc and Vc
  • The constant regions of the heavy and light chain
    differ from species to species.
  • Anti-isotypic antibodies recognize all Ig of the
    same isotype in all members of the species from
    which the injected antibody came.
  • Anti-isotype Ab is routinely used for research

  • Allotypic determinants differentiate classes of
    constant regions within the same species.
  • In humans, allotypes have been characterized for
    all four IgG subclasses for one IgA subclass and
    for the kappa chain.
  • Anti-allotypic antibodies will recognize Igs of a
    particular isotype only in some members of a
  • How do you produce antibodies to allotypic
  • Example 25 different g-chain (Gm) markers have
    been identified
  • G2m (23)
  • Class subclass (allele )

  • Idiotypic determinants arise from the sequence of
    the heavy and light chain variable regions.
  • Each individual antigenic determinant of the
    variable region is referred to as an idiotope
  • Each Ab will present multiple idiotopes the sum
    of the individual idiotopes is called the
  • Does a B-cell have one or more than one idiotype?

B-Cell Receptor
  • BCR is composed of
  • mIg
  • heterodimers of Ig-a/Ig-b
  • All isotypes of mIg have too short of cytoplasmic
    tails for intracellular signaling molecules
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