The Immune Response: Antigen-Antibody Reactions

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• The Immune Response Antigen-Antibody Reactions
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• Introduction

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• Antibodies are bifunctional - they bind to the
  target antigen they recognize as foreign, and
  they enable other defense components to react
  with it
• Variable domain (Fab) - binds to target antigen
• Constant domain (Fc) - interacts with cells of
  the immune system and other host defense
  mechanisms

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• Antigen-Antibody Binding

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• occurs within the pocket formed by folding the VH
  and VL regions of the Fab domain

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• Binding is due to weak, noncovalent bonds
• Shapes of epitope and binding site must be
  complementary for efficient binding
• The high complementarity provides for the high
  specificity associated with antigen-antibody
  binding

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• Antigen-Antibody Reactions in the Animal Body (In
  Vivo)

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• The complement system is a series of protein
  components that must be activated in a cascade
  fashion (i.e., the activation of one component
  results in the activation of the next, and so on)
• Results in lysis of antibody-coated bacteria and
  eukaryotic cells
• Mediates inflammation
• Attracts and activates phagocytic cells

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• There are three activation pathways
• Each results in destruction of the target cell,
  but their triggering mechanisms are different
• The classical pathway is dependent on
  antigen-antibody interactions to trigger it it
  is fast and efficient

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• The lectin pathway is activated by
  mannose-binding lectins (MBLs) that have been
  secreted by liver activation leads to
  opsonization
• The alternative pathway does not require
  antigen-antibody binding it is nonspecific and
  inefficient, but contributes to innate resistance

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• ? The final step in the pathway is the formation
  of a membrane attack complex that creates a pore
  in the membrane of the target cell
• ? The pore allows entry of destructive enzymes or
  leads to osmotic rupture of the target cell

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• Ag-Ab Interactions cont.
• Toxin neutralization - antibody (antitoxin)
  binding to toxin renders the toxin incapable of
  attachment or entry into target cells
• Viral neutralization - binding prevents viral
  attachment to target cells
• Adherence inhibiting antibodies - sIgA prevents
  bacterial adherence to mucosal surfaces

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• Antibody-dependent cell-mediated cytotoxicity -
  involves the complement system, NK cells, or
  release of cytotoxic mediators from effector
  cells that attach to the target cell by means of
  Fc receptors
• IgE and parasitic infection - in the presence of
  elevated IgE levels, eosinophils bind parasites
  and release lysosomal enzymes

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• Opsonization
• Prepares the microorganism for phagocytosis
• Phagocytes recognize the Fc portion of IgG or IgM
  antibodies coating the surface of the foreign
  microorganism
• Phagocytosis can also be stimulated by components
  of the complement system, whether initiated by
  the classical or alternative pathways

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• Inflammation can be mediated by IgE attachment to
  mast cells and basophils, or by the binding of
  one of the complement components to mast cells
  and platelets
• This complement component is also a powerful
  chemoattractant for macrophages, neutrophils, and
  basophils

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• Immune complex formation - two or more
  antigen-binding sites per antibody molecule lead
  to cross-linking, forming precipitins (molecular
  aggregates) or agglutinins (cellular aggregates)
• Agglutination that specifically involves red
  blood cells is called hemagglutination
• In vivo testing involves immediate or delayed
  skin testing for the presence of antibodies to
  various antigens

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• Antigen-Antibody Reactions In Vitro (serology)

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• Agglutination - visible clumps or aggregates of
  cells or of coated latex microspheres
• If red blood cells are agglutinated, this is
  called hemagglutination
• Agglutination inhibition can be used to detect
  serum antibodies or to detect the presence of
  specific substances (e.g., illegal drugs) in
  urine samples by a competition assay

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Box 33.2 Rapid urine testing for drugs, e.g
cocaine Abuscreen method
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Figure 33.9 Latex agglutination test for
pregnancy Positive pregnancy test
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Negative pregnancy test
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Figure 33.10 Viral Hemagglutination Some
viruses bind to RBC and cause hemagglutination.
If serum contains anti-viral Abs, then
hemagglutination is inhibition positive test.
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Figure 33.11 Tube agglutination test for
determining antibody titer.
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Q What is the titer in rows A-H?
Figure 33.11 A microtiter plate illustrating
hemagglutination. Ab in wells 1-10. Positive
control row 11, negative control row 12.
RBCs added to each well. If enough Ab is
available to agglutinate the cells, they bind as
a mat to the bottom of the well. If insufficient
Ab is available, they form a pellet at the bottom.
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• Complement fixation
• Irreversible alterations to complement components
  that are initiated by the binding of antibody to
  antigen
• Used to detect the presence of serum antibodies,
  thereby indicating prior exposure to a pathogen

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• If immune complexes are formed, then complement
  is used up and lysis will not occur when
  sensitive indicator cells are added
• If immune complexes are not formed, then
  complement is not used up and lysis will occur
  when sensitive indicator cells are added

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Figure 33.12 Complement fixation
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• Enzyme-linked immunosorbent assay (ELISA)
• Indirect immunosorbent assay - detects serum
  antibody
• Antigen is coated on test wells and serum is
  added
• If antibodies are present, they will bind
  antigen if not, they will wash off

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• Add to the plate an enzyme that is covalently
  coupled to a second antibody against first
  immunoglobulin
• If antigen was present, the second antibody will
  bind if not, it will wash off
• Add colorless substrate (chromogen) for the
  enzyme and measure colored product formation
  spectrophotometrically
• No colored product will form if everything washed
  off

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• Double antibody sandwich assay - detects antigens
  in a sample
• Antibody is coated onto test wells and sample is
  added
• If antigen is present in sample it will bind if
  not it will wash off
• React with antibody against the antigen if
  antigen was present in the sample, this antibody
  will bind if not, it will wash off
• Continue with steps (c), (d), and (e) as in the
  indirect assay

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• Immunodiffusion - involves the precipitation of
  immune complexes in an agar gel after diffusion
  of one or both components
• Single radial immunodiffusion (RID) assay -
  quantitative

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Figure 33.15 Mancini technique Single radial
immunodiffusion assay
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• Double diffusion assay (Ouchterlony technique) -
  lines of precipitation form where antibodies and
  antigens have diffused and met determines
  whether antigens share identical determinants

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Figure 33.15 Ouchterlony technique Double
diffusion agar assay
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• Immunoelectrophoresis - antigens are first
  separated by electrophoresis according to charge,
  and are then visualized by the precipitation
  reaction
• Greater resolution than diffusion assay

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Figure 33.16 Classical Immunoelectrophoresis -
Used to separate major blood proteins in serum
for diagnostic tests Precipitin arcs form where
Ab and Ag precipitate.
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• Immunofluorescence - dyes coupled to antibody
  molecules will fluoresce (emit visible light)
  when irradiated with ultraviolet light
• Direct - used to detect antigen-bearing organisms
  fixed on a microscope slide
• Indirect - used to detect the presence of serum
  antibodies

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Figure 33.17 Direct and Indirect
Immunofluorescence
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• Immunoprecipitation - soluble antigens form
  insoluble immune complexes that can be detected
  by formation of a precipitin

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Figure 33.18 Immunoprecipitation Precipitin
curve and precipitation ring test.
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• Neutralization - an antibody that is mixed with a
  toxin or a virus will neutralize the effects of
  the toxin or the infectivity of the virus this
  is determined by subsequent assay

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• Radioimmunoassay (RIA) - purified antigen labeled
  with a radioisotope competes with unlabeled
  sample for antibody binding
• Serotyping - antigen-antibody specificity is used
  to differentiate among various strains (serovars)
  of an organism