Antigens and Antibody Interactions

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Antigens and Antibody Interactions

• Reading Chapter 6

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• Antigen antibody interaction requires
  noncovalent forces
• Hydrogen bonds
• Ionic bonds
• Hydrophobic interactions
• Van der Waals interactions
• Strong interactions require a close fit and
  operate best over short distances (1 x 10-7 mm)
• CDRs and inducible fit

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• Affinity of an Ab for an epitope
• Combined strength of noncovalent interactions
  between a single antigen-binding site on an Ab
  and a single epitope
• Low Affinity vs. High Affinity
• Ag Ab Ag-Ab
• Ka is equilibrium constant, Ka Ag-Ab
  K1
• 
  AbAg K-1

K-1, and its influence on Ka

• Kd 1 , indicates stability of Ag-Ab complex
• Ka

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• Equilibrium dialysis can determine Ka
• Ka Ag-Ab r_ yields Scratchard
  equasion
• AbAg c(n-r)

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• Scatchard Equasion and Plots
• Repeat dialysis with varying ligand
  concentrations
• Straight slope all antibodies have the same
  affinity
• Curved slopeheterogeneous mixture of Abs
• Intercept reveals the valency of the Ab (IgG2,
  IgM10, IgA4)
• r Kan-Kar
• c
• Importance of Avidity (strength of multiple
  interactions between multivalent Ab and Ag

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• Cross Reactivity
• Antibody elicited by one antigen can cross react
  with an unrelated antigen
• Caused by an identical or similar epitope on the
  antigens
• Ex. ABO blood group antigens
• Antibodies induced by similar epitopes on
  antigens present on common intestinal bacteria
• Ex. M cell antigen and damage of heart and kidney
  tissue following Streptococcus pyogenes infection

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• Precipitation Reactions basis for all
  immunological assays
• Ag-Ab interaction in aqueous solution form
  lattice that can be precipitated
• Precipitins
• Formation of the Ag-Ab lattice depends on the
  valency of the Ab and Ag

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• Precipitation curve for an Ag and its Abs
• Three zones, Equivalence Zone and formation of a
  large multimolecular lattice

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• Immunodiffusion reactions
• Precipitation in region of equivalence
• Used to determine the relative concentrations of
  Ab or Ag, to compare antigens or to determine the
  purity of Ag
• Radial immunodiffusion
• Double Immunodiffusion

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• Radial Immunodiffusion
• Ag sample placed in well within semisolid medium
  with antiserum
• Equivalence zone generates a precipitin ring
• Double Immunodiffusion
• Both Ag and Ab diffuse through semisolid medium
• Equivalence zone generates a precipitin line

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• Immunoelectrophoresis
• Antigen preparation is electrophoresed (charge
  separation)
• Antiserum added to troughs on one or both sides
  of separated Ag
• Equivalence zone forms as lines of precipitation
  (arcs)-qualitative results

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• Individual serum components are idetified with
  antisera specific for a protein or immunoglobulin
  class
• Qualitative assessment of Ab production (detects
  very low or high Ab production)
• Ex. myeloma patient
• Quantitative method rocket electrophoresis (Ag
  must be negatively charged)

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• Agglutination Reactions
• Agglutination- interaction between Ab and Ag
  resulting in visible clumping
• Agglutinins
• Reactions depend on crosslinking of polyvalent
  antigens
• Prozone effect- excess of Ab inhibits
  agglutination
• Incomplete antibodies do not induce and inhibit
  agglutination (often IgG)
• Hemagglutination and blood typing
• Bacterial agglutination to diagnose infection-
  agglutinin titer

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• Agglutination inhibition
• Sensitive assay for small amounts of Ag
• Ex. human chorionic gonadotropin

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• Radioimmunoassay
• Highly sensitive method for detecting antigen or
  antibody
• Competitive binding of radiolabeled Ag to a
  high-affinity Ab
• Detection of hepatitis B in blood

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• Using the linear part of the RIA curve one can
  determine the concentration of Ag in unknown
  serum samples

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• ELISPOT ELISA
• Quantitative determination of the number of cels
  in a population that are producing Ab specific
  for a given Ag or Ag that is specific for a given
  Ab

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• Western Blotting
• Protein separated on an SDS-PAGE gel and detected
  with enzyme-linked antibody
• Allows identification of specific protein in a
  complex mixture of proteins
• Identify specific antibodies in a mixed sample
• Confirmatory testing for HIV

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• Immunoprecipitation
• Sensitive method for detecting Ag in a cell or
  tissue type
• Isolates the Ag of interest for further analysis

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• Use of Ab linked to magnetic beads to detect
  whether Ag is synthesized by a cell or tissue

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• Immunofluorescence
• Function of fluorescent molecules
• Fluorochromes
• Technique emitted light from a fluorochrome is
  viewed with a fluorescent microscope
• Fluorescein (490nm absorption and 517nm emission-
  green)
• Rhodamine (515nm absorption and 546nm
  emission-red)
• Phycoerhthrin (abosorbs and emits much better
  than rhodamine)

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• Ab bound to ? heavy chain are detected with
  rhodamine-conjugated secondary antibody

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• Flow cytometry and fluorescence
• Separating fluorochrome labeled cells to yield
  quantitative data

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• Flow cytometry and leukemia typing
• Determination of the distribution of markers of
  leukemic cells
• Detection of abnormal cells in the blood and bone
  marrow
• Determining what type of leukemia is present for
  viable treatment

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• Immunoelectron Microscopy
• Antibodies and the visualization of specific
  intracellular tissue components
• Ex. B cell lymphoma stained with class II MHC (30
  nm gold particles) molecules and Class I MHC (15
  nm gold particles)