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Antigen

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Title: Antigen


1
Antigen Antibody Reactionsor Serological
Reactions
2
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

3
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

6
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
    )

7
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

8
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

9
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)

10
Zone of equivalence
11
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

12
ZONES OF PRECIPITIN FORMATION
Precipitin Curve
13
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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15
Precipitation
  • Precipitation can take place in capillary tubes,
    test tubes, and in gel

16
Precipitation in gel
  • - Double diffusion
  • - Single (radial) diffusion
  • - Combination of diffusion in gel and
  • electrophoresis

17
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

20
RADIAL IMMUNODIFFUSION
Samples
Standards
Precipitin Rings
a b c
A B C
Standard Curve
21
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

29
ELECTROIMMUNOASSAY (ROCKET)
30
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

31
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

32
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

33
33
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

37
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

38
IFE
39
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

40
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

41
WESTERN BLOTTING
42
The Western blot procedure.
42
43
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)

44
FLOCCULATION VS. IMMUNOPRECIPITATION
45
  • Flocculation Tests
  • VDRL (Venereal Disease Research Lab.) test
  • RPR (Rapid Plasma Reagin) test

46
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

47
Agglutination Reactions
48
Agglutination
  • Qualitative slide agglutination
  • - identification of bacteria with antisera
    directed against
  • O, H, K antigens

49
Agglutination
  • Latex agglutination
  • Coagglutination

50
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.

51
1200
1400
1100
Titer
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Agglutination inhibition
54
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

55
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

59
ELISA
60
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
63
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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65
Distribution of selected markers on some leukemia
cell types ? Immunophenotyping using flow
cytometry mAb
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