Antibodies

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Antibodies

• Structure and Function

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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

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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
  fragments.
• 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

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Basic Structure of Antibodies

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

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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).

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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
  l4)
• K light chains predominate.

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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).

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Immunoglobulin fine structure

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

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Domains

• 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
  acids
• Residues in the amino terminal loop make up the
  Ag binding site.

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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

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CH1 and CL Domains.

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

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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).

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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.

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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
  e.

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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.

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Antibody Effector functions
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Opsonization

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

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Complement

• 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

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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
  cells
• Eosinophils, NK cells, neutrophils are examples

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Antibody classesand

• Biological Activities

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Antibody Classes
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IgM

• IgM expressed as membrane bound anitbodies on
  B-cells
• 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
  (opsinization)

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IgD

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

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IgG

• 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

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IgE

• 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

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IgA

• 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
  chain.
• 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

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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.

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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

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Question

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

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Antigenic Determinants on Immunoglobulins
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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

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Isotype

• 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
  domains
• 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
  purposes.

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Allotype

• 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
  species.
• How do you produce antibodies to allotypic
  determinants?

• Example 25 different g-chain (Gm) markers have
  been identified
• G2m (23)
• Class subclass (allele )

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Idiotype

• 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
  idiotype

• Does a B-cell have one or more than one idiotype?

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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