Chapter 4 Antigens and Antibodies

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Chapter 4 Antigens and Antibodies
Ab
Ag
Complementarity of interacting surfaces of Ab and
Ag
Oct 17, 19 24, 2006
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• ????????
• 1.??????? (immunogen) ???????
• 2. ????????? (epitope)?
• B-cell epitope ?????
• 
  
• 4. ?????????????????
• 5. ????????????
• 6. ??????????

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Outline 1.
Immunogenicity versus antigenicity 2. Epitopes

3. Basic structure of antibodies (Abs)
4. Ab-binding site 5. Ab-mediated effector
functions 6. Ab classes and biological
activities 7. Antigenic determinants on
immunoglobulins (Ig) 8. The B-cell receptor 9.
The Ig superfamily 10. Monoclonal Abs
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Immunogenicity vs. Antigenicity
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Immunogenicity ??? the ability to induce
an Ab and/or cell-mediated immune
response Antigenicity ??? the ability to
combine specifically with Ab and/or
cell-surface receptors (Ig/TCR)
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• - Although a substance that induces a specific
• immune response is usually called an antigen,
• it is more appropriately called an immunogen.
• - Although all molecules that have the property
• of immunogenicity also have the property of
• antigenicity, the reverse is not true.
• Some small molecules, called haptens, are
• antigenic but incapable, by themselves, of
• inducing a specific immune response. In other
• words, they lack immunogenicity.

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DNP dinitrophenol
(bovine serum albumin)

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Landsteiners work demonstrated both the
specificity of the immune system for small
structural variations on haptens and the enormous
diversity of epitopes that the immune system is
capable of recognizing.
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Factors That Influence Immunogenicity
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Intrinsic properties of an immunogen -
Foreignness - Molecular size - Chemical
composition and heterogeneity - Susceptibility to
antigen processing and presentation The
biological system - Genotype of the recipient
animal - Immunogen dosage and route of
administration - Adjuvants
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• Foreignness
• Generally, the greater the phylogenetic
• distance between two species, the greater the
• structural (and therefore the antigenic)
• disparity between them.
• Some macromolecules (e.g., collagen and
• cytochrome c) were highly conserved
• throughout evolution and therefore display
• very little immunogenicity across diverse
• species lines.

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• Conversely, some self-components (e.g.,
• corneal tissue and sperm) are effectively
• sequestered from the immune system, so
• that if these tissues are injected even into
• the animal from which they originated,
• they will function as immunogens.

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Molecular size - There is a correlation between
the size of a macromolecule and its
immunogenicity. - The best immunogens tend to
have a molecular mass gt100,000 daltons (Da). -
Generally, substances with a molecular mass
less than 5,000 10,000 Da are poor
immunogens.
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• Chemical composition
• and complexity
• - Synthetic homopolymers tend to lack
• immunogenicity regardless of their size.
• All 4 levels of protein organization
• primary, secondary, tertiary and quaternary
• contribute to the structural complexity of a
• protein and hence affect its immunogenicity.

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Four levels of protein organizational structure
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For Ab (B cell) responses Proteins are the
most potent immunogens, with polysaccharides
ranking second. Lipids and nucleic acids of an
infectious agent generally do not serve as
immunogens unless they are complexed with
proteins or polysaccharides. For T cell
responses Only proteins and some lipids
(glycolipids and phospholipids) serve as
immunogens.
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• Susceptibility to antigen
• processing and presentation
• The development of both Ab-mediated and
• T-cell-mediated immune responses requires
• interaction of T cells with Ag that has been
• processed and presented together with MHC
• molecules.

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• Large, insoluble macromolecules generally
• are more immunogenic than small, soluble
• ones because the larger molecules are more
• readily phagocytosed and processed.
• Molecules that cannot be degraded (e.g.,
• polymers of D-amino acids) and/or cannot
• be presented with MHC molecules are
• poor immunogens.

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• The biological system contributes
• to immunogenicity
• Genotype of the recipient animal
• - Immunogen dosage and route of administration
• - Adjuvants

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Immunogen dosage and route of
administration Doses too low ? no
response, too high ? tolerance Exposure
repeated administration (booster) over a period
of time is usually more
effective Routes orally (????)
parenterally (?????)
- intravenous (iv) into a vein
- intradermal (id) into the
skin - subcutaneous (sc)
beneath the skin -
intramuscular (im) into a muscle
- intraperitoneal (ip) into the
peritoneal cavity
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??? Ag ??????? ? ?? ???????????
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• Adjuvants ??
• Latin adjuvare, to help
• Substances that, when mixed with an
• antigen and injected with it, enhance
• the immunogenicity of that antigen.

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Effects of adjuvants 1. antigen
persistence is prolonged - slower release
of antigen at the injection site e.g.,
alum ?? AlK(SO4)2, 2. costimulatory signals (p.
159) are enhanced - increased expression of
B7 molecules on APC ? maximal
activation of TH cells 3. local inflammation is
increased 4. nonspecific proliferation of
lymphocytes is stimulated - formation of a
dense, macrophage-rich mass of cells
called a granuloma ??? e.g., incomplete
Freunds adjuvant (IFA),
complete Freunds adjuvant (CFA)
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1. ???????????,???? CFA (complete Freunds
adjuvant),????? ??? 2. ??????????? Ab ???
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Epitopes
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• Lymphocytes do not interact with, or recognize,
• entire immunogen molecules instead, they
• recognize discrete sites on the macromolecule
• called epitopes, or antigenic determinants.
• epitopes immunologically active regions of an
• immunogen, that bind to
  Ag-specific
• membrane receptors on
  lymphocytes
• or to secreted Abs.

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T-cell and B-cell epitopes - The recognition of
antigens by T cells and B cells is
fundamentally different. - Because B cells bind
antigen that is free in solution, the
epitopes they recognize tend to be highly
accessible sites on the exposed surface of
the immunogen. - T-cell epitopes are peptides
combined with MHC molecules. Thus, there is
no requirement for solution
accessibility such as B-cell epitope.
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• Properties of B-cell epitopes
• B-cell epitopes on native proteins generally
• are composed of hydrophilic a.a. on the
• protein surface that are topographically
• accessible to membrane-bound or free Ab.
• 2. B-cell epitopes may be composed of
• sequential or nonsequential amino acids.

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Sperm whale myoglobulin contains 5 sequential
B-cell epitopes
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Hen egg-white lysozyme (HEL) composes one
nonsequential (conformational) epitope
Contact with Ab light chain
Contact with Ab heavy and light chains
Contact with Ab heavy chain
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Ab to native HEL does not bind to reduced HEL
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• B-cell epitopes tend to be located in flexible
  regions
• of an immunogen and display site mobility.
• - site mobility of epitopes maximizes
  complementarity
• with the Abs binding site
• Complex proteins contain multiple overlapping
• B-cell epitopes, some of which are
  immunodominant.
• - Most of the surface of a globular
  protein is potentially
• immunogenic.
• - Some epitopes, called immunodominant,
  induce a more
• pronounced immune response in a
  particular animal than
• other epitopes.

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Basic structure of Abs
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Electrophoresis of immune serum (Tiselius
Kabat, 1939)
Immune sera
after reaction with Ag
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• g- globulin (gG)
• Immunoglobulin (Ig)
• IgG, IgM, IgA, IgE, IgD
• Antibody (Ab)

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Ab molecules contain 4 peptide chains - A
dimer of heterodimers
V variable C constant
(H)
m IgM g IgG a IgA d IgD e IgE
(L)
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100 kDa
150 kDa
? numerous small peptides
Fab fragment antigen binding 45 kDa
22 kDa
Fc fragment crystallizable 50 kDa
50-55 kDa
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• Antibody to the Fab fragment could react with
  both the H and L chains, whereas antibody to the
  Fc fragment reacted only with the H chain.
• ? Fab consists of portions of an H and a L chain.
  
• ? Fc contains only H chain components.

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- A heterogeneous spectrum of antibodies in
the serum g-globulin fraction - Multiple myeloma
a cancer of Ab- producing plasma cells -
Myeloma protein 95 of the serum Ig -
Bence-Jones proteins the excess light chains
in the urine. - MOPC mineral-oil induced
plasmacytoma in mice
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Heavy and light chains are folded into
domains
(IgG, IgD, IgA)
(IgM, IgE)
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Associations between domains of an Ab molecule
2 Ag-binding sites
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Immunoglobulin Domains - each contains about
110 a.a. residues and an intrachain disulfide
bond that forms a loop of 60 a.a. Variable-Region
Domains - hypervariable regions (15 -
20 of the variable domain)
complementarity-determining regions (CDR)
CDR1, CDR2, CDR3 - framework regions (FR) -
diversity in the VH domain is concentrated in CDRs
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Variability of a.a. residues in the VH and VL
domains
CDRs bind Ags
number of
different amino acids at a given
position Variability ________________________
_____________________________
frequency of the most common amino acid
at a given position
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Conformational changes may be induced by antigen
binding
CDRs of L chain L1, L2, L3 CDRs of H chain
H1, H2, H3
- after binding to the Ag - before binding
to the Ag
Fab
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Ab-mediated effector functions
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• - Antibodies generally do not kill or remove
• pathogens solely by binding to them.
• While V regions bind to Ag, the CH region
• is responsible for a variety of collaborative
• interactions with other proteins, cells, and
• tissues that result in the effector functions
• of the Ab responses.

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• Ab-Mediated Effector Functions
• Opsonization (????) is promoted by Ab
• Abs activate complement (?? ) (chapter 7)
• Antibody-dependent cell-mediated cytotoxicity
• (ADCC) kills cells (p. 366 chapter 14)
• - Some Abs can cross epithelial layers by
  transcytosis

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Ab activates complement-mediated cytolysis or
promotes opsonization through a binding to C
receptors on phagocytes
Ab promotes opsonization through a binding to Fc
receptors (FcR) on phagocytes
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Antibody-dependent cell-mediated cytotoxicity
(ADCC)
(Figure 14-15)
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• Transcytosis
• - movement of Ab across epithelial layer
• - delivery of IgA to the mucosal surfaces of
  the
• respiratory, gastrointestinal, and
  urogenital tracts,
• as well as its export to breast milk
• - transplacental transport of IgG from mother
  to fetus

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Secretory IgA in Breast Milk
Bind to microbes in babys digestive tract and
thereby prevent their attachment to the walls
of the gut and their subsequent passage
into the bodys tissues.
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Ab classes and biological activities
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5 major classes of secreted antibody
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IgG

• most abundant in serum
• 80 of total serum Ig
• 4 IgG subclasses

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4 subclasses of human IgG

• size of the hinge region
• no. position of the interchain -S-S- bond
• - IgG1gtIgG2gtIgG3gtIgG4 in serum conc.
• 90 - 95 homologous in DNA sequences
• varied effectiveness in placenta transfer and C
  activation

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IgM Pentamer
- monomer on the membrane pentamer in
secretion - 5 - 10 serum Ig
- 1st Ab in neonates - 1st Ab
in primary response - more
efficient in agglutination C fixation -
J (joining) chain is required for
polymerization of the monomers to form
pentameric IgM - also present in mucosal
surfaces
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IgA Dimer

• 10 - 15 of total serum Ig
• monomers, dimers, trimers and
• tetramers in serum
• - predominant in external secretions,
• e.g., breast milk, saliva, tears, and
• mucus of the bronchial, genitourinary,
• and digestive tracts

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Secretory IgA
Dimers and tetramers in secretion with a
secretory component
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Formation of Secretory IgA
Transcytosis
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IgE

• potent biological activity
• extremely low conc. in serum
• - mediates the immediate hyper-
• sensitivity reactions
• responsible for the symptoms
• of hay fever, asthma, hives, and
• anaphylactic shock

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Allergen cross-linkage of receptor- bound IgE on
mast cells
- induces degranulation, causing release of
substances that mediate allergic
manifestations
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IgD

• 0.2 of total serum Ig
• - together with IgM, is the
• major membrane-bound Ig
• on mature B cells
• thought to function in the
• activation of B cells
• no biological effector
• function has been identified

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Antigenic determinants on Igs
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3 Antigenic Determinants of Immunoglobulins Isot
ypes Allotypes Idiotypes
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• Isotypic Determinants
• constant-region determinants that collectively
  define
• each H-chain class and subclass, and each
  L-chain
• type and subtype within a species

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Allotypic Determinants -
differences in amino acids in C regions, which
occur in some, but not all, members of a species
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• Idiotypic Determinants
• The unique amino acid sequence of the V regions
  of
• a given Ab. In some cases, an idiotype is the
  actual
• antigen-binding site

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B-cell Receptor
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Fc receptors bound to Fc regions of Abs
neonatal Fc receptor
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• Fc receptors - essential for many of the
• biological functions of
  Abs
• movement of Abs across cell membranes, e.g.,
• the transfer of IgG from mother to fetus across
• the plancenta
• passive acquisition of Ab by many cell types,
• including B and T lymphocytes, neutrophils,
• mast cells, eosinophils, macrophages, and
  natural
• killer cells (a linker between Ab molecules and
• various types of cells)

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The Ig superfamily
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The Ig superfamily -1
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The Ig superfamily -2
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Monoclonal Abs
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Clonal Selection of B Lymphocytes
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Production of monoclonal antibodies (mAb)
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• Questions
• How to predict whether a molecule is a good
  immunogen?
• What are the differences between B-cell epitopes
  and T-cell epitopes?
• Draw a schematic diagram of a typical IgG
  molecule and label each of the chains, bonds,
  regions, sites, fragments and domains.
• How does Ab kill or remove pathogens?
• What is the principal of making monoclonal
  antibody?