Antigen

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Antigen Antibody Reactionsor Serological
Reactions
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Antigen-Antibody interactions

• Characterized as
• Non-covalent interaction (similar to lock and
  key fit of enzyme-substrate)
• Do not lead to irreversible alteration of Ag or
  Ab
• This exact and specific interaction has led to
  many immunological assays that are used to
• detect Ag or Ab
• diagnose disease
• measure magnitude of humoral IR
• identify molecules of biological and medical
  interest

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Introduction

• Ag Ab reactions are one of the most specific
  noncovalent biochemical reactions known
• The forces that hold the reactants together are
• - van der Waals forces
• - Electrostatic forces
• - Hydrophobic forces
• They can be represented by the simple formula
• Ag Ab ? AgAb
• The reaction is driven to the right but it is
  reversible

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Strength of Reaction

• The strength of the reaction (how far it is
  driven to the right) is referred to as affinity
• Antibody affinity
• - A quantitative measure of binding strength
• - Combined strength of the noncovalent
• interactions between a binding site on an Ab
  
• monovalent Ag
• - Affinity varies broadly among immunoglobulins

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Strength of Reaction

• Antibody avidity
• - Avidity is often used to describe the
  collective affinity of multiple binding sites on
  an antibody molecule
• - True strength of the Ab -Ag interaction
  within
• biological systems
• - The interaction at one site will increase
  the possibility
• of reaction at a second site
• - High avidity can compensate for low affinity
  (secreted
• pentameric IgM has a higher avidity than IgG
  )

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CROSS REACTIVITY

• Antibody elicited by one Ag can cross-react with
  a related Ag.
• Occurs if two different Ags share identical or
  very similar epitope
• 1- Vaccinia virus and smallpox virus
• 2- Rabies JE vaccine
• 3- Streptococcus pyogenes infection heart
  
• Kidney damage following infection
• 4- Original antigenic sin.
• 5- Bacterial Ag and sugars on RBC

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STAGES OF Ag - Ab REACTIONS

• Primary reactions Vs secondary reactions Small
  Ag - Ab complexes Vs large complexes (The Lattice
  hypothesis)
• Development of macroscopic manifestations
  reactions (e.g. immunoprecipitation)
• Ag Ab reactions involving IgM are confined to
  the blood stream, while those of lower molecular
  weight (IgG and IgE) can leave the vasculature
  and enter tissues
• Time required is hours to days for precipitin
  formation leading to irreversible
  immunoprecipitates

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LATTICE THEORY

• Lattice formation (visible Ag - Ab aggregates)
  occurs when
• Ag is multivalent (contains more than 2 identical
  epitopes)
• Cross-linking of Ags by specific Abs (2 or more
  antigen-binding sites)
• Molar ratios of epitopes and antigen-binding
  sites are optimal (zone of equivalence)

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Zone of equivalence
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LATTICE THEORY

• Zones of lattice formation
• Far Ag excess (no ppt. formed free Ag in
  supernatant) -- postzone
• Ag excess (sub-optimal ppt. free Ag in spnt.)
• Zone of equivalence (maximum ppt. no Ag or Ab in
  spnt.)
• Ab excess (sub-optimal ppt Ab in spnt.)
• Far Ab excess (no ppt Ab in spnt.) -- prozone

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ZONES OF PRECIPITIN FORMATION
Precipitin Curve
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METHODS THAT DETECT Ag- Ab REACTIONS

• Primary Reactions
• - Immunofluorescence (IF)
• - Radioimmunoassay (RIA)
• - Enzyme immunoassay (EIA)
• - Immunonephelometry (measures picogram to
• nanogram quantities of analyte)
• Secondary Reactions
• - Agglutination Techniques
• - Precipitation Techniques Electrophoresis

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Precipitation

• Precipitation can take place in capillary tubes,
  test tubes, and in gel

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Precipitation in gel

• - Double diffusion
• - Single (radial) diffusion
• - Combination of diffusion in gel and
• electrophoresis

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SINGLE VS. DOUBLE DIFFUSION

• Single diffusion
• Supporting medium (gel) contains one reactant at
  a uniform concentration
• Only the unknowns move through the medium
• Double diffusion
• Gel is inert (contains no reactants)
• Both Ag and Ab travel through the medium

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RADIAL IMMUNODIFFUSION

• Ab uniformly distributed in gel Ag diffuses
  outward from a well (single diffusion)
• Ag- Ab complexes form as concentric rings around
  the well at zone of equivalence
• At a set time, ring diameters are measured
• Ag is directly proportional to the ring d2
• Unknown value is determined by comparing to a
  3-standard curve

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RADIAL IMMUNODIFFUSION
Samples
Standards
Precipitin Rings
a b c
A B C
Standard Curve
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RADIAL IMMUNODIFFUSION

• Fahey method (kinetic)
• Read at 18 hours
• Plot std vs. ring diameter on semi-log paper
• Mancini method (endpoint)
• Read at 48 or 72 hours
• Plot std vs. ring diameter squared on graph
  paper
• Results reliable only if the ring size is within
  the range of the standards if greater than
  highest std, dilute and repeat test
• Used to measure IgM, IgG, C4,C3,transferrin, CRP,
  others

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OUCHTERLONY DOUBLE DIFFUSION

• Ag Ab placed in wells cut into an agarose gel
  (both reactants diffuse)
• Precipitin line (or arc) indicates Ab has
  specificity for Ag
• Position of precipitin between wells depends on
  MW and concentration of reactants
• 3 possible patterns of reaction identity,
  non-identity, partial identity

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OUCHTERLONY DOUBLE DIFFUSION
Ouchterlony Plates Precipitin
Patterns
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ELECTROIMMUNOASSAY (ROCKET)

• Electrophoresis hastens movement of Ag (placed in
  wells) through Ab -imbedded gel (single
  diffusion)
• Selected pH (8.6) keeps Abs at their isoelectric
  point they will not move
• Rocket-shaped precipitin bands will form at zone
  of equivalence (changes as reactants move)
• Ag proportional to length of rocket
• Unknowns compared to standards

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ELECTROIMMUNOASSAY (ROCKET)
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ELECTROIMMUNOASSAY (ROCKET)

• May be used to quantitate plasma proteins such as
  coagulation factors, alpha-fetoprotein, C3, C4,
  CRP, haptoglobin
• Compared with RID
• faster
• similar sensitivity
• requires electrophoretic equipment and more
  technological finesse
• Largely replaced by immunonephelometry

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IMMUNONEPHELOMETRY

• Ag Ab ? AgAb ? microscopic Ag - Ab complexes
• Microcomplexes cause light moving through the
  suspending solution to scatter
• Nephelometer detects light scattered at a 90o
  angle
• Amount of light scattered at 90o is proportional
  to Ag - Ab complexes formed
• Sensitive and quantitative technique used for
  measurement of many serum proteins

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IMMUNOELECTROPHORESIS (IEP)

• Electrophoresis and double diffusion
• 2 stages
• Proteins separated by electrophoresis
• Antiserum placed in trough parallel to separated
  proteins all reactants diffuse in all directions
• Precipitin forms at zones of equivalence
• Trough may be filled with simple or complex
  antisera yielding simple to complex patterns

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Immunoelectrophoresis of normal human serum.
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IMMUNOELECTROPHORESIS (IEP)

• Qualitative to semi-quantitative
• Serum, urine, or CSF may be analyzed
• Complex patterns may be difficult to interpret
• Useful to detect
• missing proteins
• abnormal proteins
• normal proteins in abnormal concentrations
• Used to evaluate conditions such as multiple
  myeloma
• Largely replaced by immunofixation

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IMMUNOFIXATION ELECTROPHORESIS (IFE)

• Proteins that were separated by electrophoresis
  are exposed to Ab directly, instead of through
  diffusion
• Steps
• Electrophoresis of protein mixture in gel (use
  serum or urine samples)
• Paper strips imbedded with specific Ab are
  blotted onto gel Ags transfer to paper and
  bind to Abs
• Strips washed (unbound material washes away)
• Strips stained to reveal precipitin bands

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IFE
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IMMUNOFIXATION ELECTROPHORESIS (IFE)

• Used to detect the presence of Igs in conditions
  like multiple myeloma
• Fairly sensitive - Ab is highly specific,
  electrophoresis leaves Ag isolated and accessible
• Faster and easier to interpret than IEP
• Only 1 Ab may be used per strip

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WESTERN BLOTTING

• Similar to IFE but the unknown is Ab rather than
  Ag
• Steps
• Separation of complex antigenic material (eg.,
  viral proteins) by electrophoresis
• Separated components transferred from gel to
  nitrocellulose paper by blotting
• Unknown (or control) sera (which may have Abs)
  incubated with paper strips Ag - Ab complexes
  ppt. at site of transfer
• Strips washed staining reveals complexes

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WESTERN BLOTTING
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The Western blot procedure.
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FLOCCULATION

• Immunoprecipitation (or agglutination) of
  insoluble particles
• Characterized by very sharp pro- and postzones
• No precipitin formed in zones of Ab or Ag excess,
  only in zone of equivalence
• Clinically important examples, VDRL and RPR tests
  (screening tests for syphilis)

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FLOCCULATION VS. IMMUNOPRECIPITATION
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• Flocculation Tests
• VDRL (Venereal Disease Research Lab.) test
• RPR (Rapid Plasma Reagin) test

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Agglutination

• Titer
• Zeta potential
• Types of Agglutination
• - Direct agglutination or hemagglutination
• - Indirect (passive) agglutination or
  hemagglutination
• - Agglutination or hemagglutination inhibition
• The Coombs test
• - Direct
• - Indirect

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Agglutination Reactions
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Agglutination

• Qualitative slide agglutination
• - identification of bacteria with antisera
  directed against
• O, H, K antigens

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Agglutination

• Latex agglutination
• Coagglutination

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Agglutination

• Tube agglutination tests
• - Gruber-Widal typhoid fever (S. typhi)
• - Weil-Felix typhus (Rickettsia)
• - Wright brucellosis
• Identify and titrate antibodies in the patients
• serum.
• Titre is defined as the reciprocal of the
• highest dilution of serum showing agglutination.

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1200
1400
1100
Titer
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Agglutination inhibition
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Hemagglutination Inhibition Test

• To Detect Antibodies (Rubella)
• - Serum (Ab) HA RBCs No Hemagglutination
• Positive Test
• - Serum (No Ab) HA RBCs Hemagglutination
  Negative Test
• To Detect Antigen (HBsAg)
• - Serum (HBsAg) Anti HBsAG HBsAg coated RBCs
  No Hemagglutination Positive Test
• - Serum (No HBsAg) Anti HBsAG HBsAg coated
  RBCs Hemagglutination Negative Test

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Use of Labels in Ag Ab Reactions

• Immunoassays
• - Radioimmunoassay (RIA)
• - Enzyme Immunoassys (EIA)
• Immunofluorescence (IF)
• - Direct IF
• - Indirect IF
• Flow cytometry and Cell Sorting (FACS)

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Immunologic Tests

• 4) Radioimmunoassay (RIA) a very sensitive test
  used for measuring hormones, serum proteins,
  drugs, etc. at low concentrations ( 0.001ug/ml)
• measures competitive binding of
  radiolabelled Ag unlabelled (test) Ag to high
  affinity Ab

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ELISA
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ELISA tests

• Depend on enzyme conjugated to 2 Ab reacting with
  a specific substrate to produce a color reaction.
  
• Variations of ELISAs Allows for qualitative or
  quantitative testing. Each one can be used for
  qualitative detection of Ag or Ab
• Also, a standard curve based on known
  concentrations of Ag/Ab can be prepared and an
  unknown concentration can be determined
• Indirect ELISA
• Sandwich ELISA
• Competitive ELISA

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Direct and indirect Immunofluorescence
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Immunoprecipitation

• Provides a quick and sensitive test for finding
  proteins/Ags especially in low concentrations
• Binds Ab to synthetic bead support ? centrifuged
• Or 2 Ab with bead or magnetic bead and collect
  by magnetism

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Distribution of selected markers on some leukemia
cell types ? Immunophenotyping using flow
cytometry mAb
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