Antigens and Antibodies

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• Chapter 3
• Antigens and Antibodies

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• Hallmark molecules of adaptive immunity
• Antibody and T-cell receptor
• Antibody is part of the B cell receptor
• Innate immunity recognizes patterns, whereas
  antibodies and T cell receptors have high degree
  of specificity

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• Antibodies and T cell receptors
• Recognize epitopes
• Immunologically active regions of immunogen that
  bind to antigen-specific antibodies or T-cell
  receptors

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Antibodies (Abs)

• Epitope binding proteins
• Membrane bound on B cells OR
• Secreted in blood
• Humoral immunity
• Share structural features, bind to antigen, and
  participate in number of effector functions
• Known collectively as Immunoglobulins (Igs)
• Abs dont kill anything, their job is to plant
  the kiss of death on an invader

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T cell Receptor

• T Cell Receptor
• Expressed on surface of T cells
• Recognize processed antigen complexed with MHC
  molecules

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• Immunogenicity
• Ability to induce humoral and/or cell-mediated
  immune response
• Immunogen is substance that induces response
• Antigenicity
• Ability to combine specifically with Abs or
  T-cell receptor/MHC
• Not all antigens are immunogenic
• Haptens

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Haptens

• Hapten too small, lack immunogenicity
• If hapten is coupled to carrier protein, immune
  response can be induced
• Hapten-carrier conjugate
• Produces 3 types of antigenic determinants
• Antibodies to hapten
• Antibodies to carrier
• Antibodies to hapten-carrier conjugate

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Properties of Immunogen contribute to
Immunogenicity

• 4 Properties
• Foreignness
• Molecular size
• Chemical composition and complexity
• Ability to be processed and presented on MHC

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• Foreignness
• Lymphocytes that do not bind to self antigens are
  allowed to further develop
• Therefore they will later only recognized nonself
  antigens
• For example
• Bovine serum albumin (BSA) is not immunogenic
  when injected into cow but is when injected into
  chicken
• Some macromolecules are highly conserved
  throughout evolution and display little
  immunogenicity
• Cytochrome c, collagen

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• Molecular Size
• Active (good) immunogens
• gt 100,000 Daltons
• Poor immunogens
• lt 5,000-10,000 Daltons

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• Chemical Composition
• Polymers composed of multiple copies of same
  amino acid or sugar tend to be poor immunogens
• Lipids are haptens and need to be congugated with
  carrier to produce antibodies
• Important for assays for detection of some
  steroids, vitamins

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• Susceptibility to antigen processing
• Large, insoluble macromolecules are more likely
  to be phagocytized for processing

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The biological system contributes to
immunogenicity

• Host Genetic make-up
• Manner in which material is presented
• Use of agents (adjuvants) to enhance
  immunogenicity

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• Genotype of recipient animal
• Genes of MHC
• Genes in coding for specific antibodies

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• Material presentation immunogen dosage and
  route of administration
• Too low or high of dosage can induce tolerance
• Single dose is often not enough booster is
  needed
• Route
• Intravenous (iv)
• Intradermal (id)
• Subcutaneous (sc)
• Intramuscular (im)
• Intraperitoneal (ip)
• Antigen administered iv would travel to spleen
  administered sc would travel to lymph nodes

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• Adjuvants
• Enhance immunogenicity
• Not exactly sure how they work but are recognized
  by Toll-like receptors
• Water-in-oil adjuvants
• Freunds incomplete adjuvant antigen in aqueous
  solution, mineral oil, and emulsifying agent
• Antigen is then released very slowly from
  injection site
• Based on Freunds complete adjuvant - also
  contained heat killed Mycobacteria

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Epitopes

• Antigenic determinants recognized by B cells and
  T cells
• B cell epitopes tend to be on the outside of the
  antigen
• For example, the hydrophilic amino acids on a
  proteins surface
• T cell epitopes from proteins derived from
  enzymatic digestion of peptide and then
  association with MHC

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Receptor-Ligand Interactions

• Antigen receptors of the adaptive immune system
  are transmembrane proteins
• B cells the B cell receptor
• T cells the T cell receptor
• Multiple noncovalent bonds
• Hydrogen bonds
• Ionic bonds
• Van der Waals
• Hydrophobic interactions

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• Receptor-ligand interactions induce signal
  transduction pathways
• Translated to biochemical change within affected
  cell
• Ligand binding can
• Induce conformational changes in receptor
• Alter receptor location within membrane
• Phosphorylation is an early step in signaling
  pathways
• Phosphorylation of certain amino acids on
  enzymes can activate or deactivate them
• Phosphorylation of tyrosine on some molecules is
  seen early, serine and threonine later
• PIP2 in cell membrane phosphorylated to PIP3,
  serves as binding site for other proteins in
  membrane
• PIP2 also hydrolyzed by other enzyme to IP3 and
  DAG
• IP3 interacts with endoplasmic reticulum
  vesicles, release of stored calcium, altering
  activity of other proteins
• For example in lymphocytes, calcium ions bind
  calmodulin altering its conformation allowing
  dephosphorylation of NFAT (nuclear factor of
  Activated T cells)

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Immunoglobulin Superfamily

• All have similar structures
• Examples
• Antibodies
• T-cell receptors
• Class I and II MHC molecules
• Part of B cell receptor
• Most members of immunoglobulin superfamily cannot
  bind antigen

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Antibodies and B cell Receptor

• B cell epitopes have characteristic properties
• Located on surface of immunogen accessible to
  antibody
• When talking about proteins, the epitopes can be
  sequential or nonsequential (referring to amino
  acid sequence) depending on protein folding

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Basic Structure of Antibodies

• Known since late 19th century that antibodies are
  in serum
• Serum is fluid phase that remains after plasma is
  allowed to clot
• Antibodies are also found in other secretions

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• Antibodies are heterodimers
• 2 light chains
• 22, 000 daltons each
• 2 heavy chains
• 55,000 daltons each
• First 110 aa of amino-terminal end of heavy and
  light chain vary depending on antibody specificity

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• Different digestion procedures reveal different
  fragments
• F(ab)2 still shows antigen binding capability

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Light Chains

• When aa sequences of light chains from several
  individuals were sequenced, pattern emerged
• Amino-terminal end (110 aa) varied
• Other part remained constant
• Were found to be either kappa (?) OR
• Lambda (?)
• In mice and humans, different lambda subtypes
  have been found

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Heavy Chains

• Amino-terminal end also shows variability
• 5 different heavy chain constant regions
  (isotypes)
• IgM µ
• IgG ?
• IgA a
• IgD d
• IgE e
• Some subisotypes have been discovered in some
  species
• Each antibody has 2 identical heavy chains, 2
  identical light chains

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Overall structure of immunoglobulin

• Primary sequence of amino acids
• Secondary folding into series of ß pleated
  sheets
• Tertiary compact globular domains
• Quarternary adjacent light and heavy chains
  interact

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Secondary
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Quartenary Structure
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• Hypervariable regions complimentarity-determinin
  g regions (CDRs)
• Complimentary to epitopes that they will bind

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• Ab-antigen interaction
• Smaller antigens will fit in pockets in the
  variable regions of Abs
• Larger antigens will interact with flatter
  regions of the variable region
• 15-22 amino acid residues on antibody will
  interact with residues on antigen

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• Hinge Region
• ? (gamma), d (delta), and a (alpha) heavy chains
  have extended peptide sequence
• Rich in proline and cysteine
• Gives flexibility
• Immunoglobulins can be secreted or membrane-bound
• Membrane-bound differ in the carboxyl-terminal
  end
• Extracellular spacer of 26 aa
• Hydrophobic transmembrane sequence
• Cytoplasmic tail

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B Cell Receptor (BCR)

• Heavy chain portion of membrane-bound antibody
  does not extend far enough through the cell
  membrane for signaling
• Membrane bound antibody is accompanied by Iga and
  Igß

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Antibody-mediated Effector Functions

• Remember, they plant kiss of death on an
  invader
• In addition to binding antigen, Abs can
• Promote phagocytosis (opsonization)
• Activate complement
• Antibody dependent cell mediated cytotoxicity
  (ADCC)
• Natural killer cells have receptor for Fc portion
  of antibody
• Some can cross epithelial layers to be excreted
  through mucous or across placenta

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• Monomeric IgM expressed on B cells
• Secreted is pentameric
• 1st class produced in primary response
• Activates complement
• Very good at agglutination
• 5 times the binding sites

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• Membrane bound on B cells

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• Most abundant
• 4 human subclasses
• Crosses placenta
• Involved in complement
• Gives us our immunity

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• Involved in allergic reactions
• Involvement in parasitic infections

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• Predominant class in secretions
• J chain and secretory component helps with
  transport across intestinal wall
• J chain makes IgA more resistant to acids and
  enzymes found in digestive tract
• IgA and macrophages restrict commensal bacteria
  that occasionally enter the tissues from the
  intestines
• Better for IgA to interact than IgG this is
  because the Fc portion of IgG has high affinity
  for receptors of immune cells and would
  constantly trigger inflammatory responses
• Can cross-link large antigens
• Exists as dimer

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• Immunoglobulins when injected into another
  species can be immunogenic
• Isotypic differences in constant region from
  one species to another
• Allotypic differences (alleles) that occur in
  some individuals
• Idiotypic differences in variable regions will
  differ even on Abs of same isotype

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Monoclonal Antibodies

• Most antigens offer multiple epitopes
• However, a single B cell will only produce
  antibody specific to single epitope
• Antibodies found in serum are from many different
  B cells
• Polyclonal antibodies
• However, for diagnostic uses, monoclonal
  antibodies are needed

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T cell receptor vs B cell receptor

• T cell receptor is only membrane bound
• Doesnt appear in soluble form like antibodies so
  more difficult to assess its structure
• Antigen binding of T cell receptor is weaker than
  that of antibodies
• Antigen recognized by T cells is not antigen
  alone but antigen associated with MHC molecules

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• (a) T cell receptor (TCR) is specific for peptide
  A
• (b) Right MHC haplotype but wrong antigen
  (peptide B)
• (c) Right antigen (peptide A) but wrong haplotype

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• T cell receptor (TCRs)
• TCR heterodimers are similar to immunoglobulins
• Therefore they are classified in immunoglobulin
  superfamily
• Resembles Fab fragment

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• TCRs
• Associate with MHC aß TCR
• Do not associate with MHC ?d TCR
• Much remains to be learned of function of ?d TCR

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TCR-CD3 Complex

• Accessory molecules help in signal transduction
  after interaction of T cell with antigen
• 2 Zeta ?? chains
• Heterodimer of delta epsilon ?e chains
• Heterodimer of delta epsilon ed chains

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T cell accessory molecules

• T cells can be divided into 2 populations
• CD4
• Recognize antigen associated with Class II MHC OR
• CD8
• Recognize antigen associated with Class I MHC
• CD4 and CD8 function as coreceptors and assist
  with signal transduction

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• Affinity of TCR for peptide-MHC complexes is
  enhanced by coreceptors

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• Allogenic genetically different individuals of
  same species
• Alloreactivity of T cells is puzzling
• Evidence supports that T cells can only respond
  to antigenMHC
• However, T cells can recognize a foreign MHC
  molecule alone
• As with transplants