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ANTIGEN- ANTIBODY INTERACTIONS

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Title: ANTIGEN- ANTIBODY INTERACTIONS


1
ANTIGEN- ANTIBODY INTERACTIONS I
2
ANTIGEN- ANTIBODY REACTIONS
  • Antigens and antibodies combine with each other
    specifically and in an observable manner
  • Serological reactions Antigen antibody
    reactions
  • in vitro
  • Uses - In vivo
  • 1. Basis of antibody mediated immunity in
    infectious diseases
  • 2. Tissue injury in hypersensitivity and
    autoimmune diseases
  • In Vitro -
  • 1. Diagnosis of infections in laboratory
  • 2. In epidemiological surveys
  • 3. Detection of non infectious agents - enzymes
  • 4. Detection and quantification of either Ag or
    Ab

3
Stages of Reactions
  • Primary stage
  • - Initial interaction rapid
  • - No visible effect
  • - Reversible
  • - Weaker inter molecular forces
  • - Detected by estimating free and bound Ag or
    Ab
  • Secondary stage
  • - Visible effects
  • - Precipitation
  • - Agglutination
  • - CF
  • - Neutralization
  • - Immobilization
  • - Zinsssers Unitarian hypothesis

4
Comparative efficiency of Immunoglobulin classes
in different serological reactions
IgG IgM IgA
Precipitation Strong Weak Variable
Agglutination Weak Strong Moderate
Complement Fixation Strong Weak Negative
5
General features of Ag Ab reactions
  • Specificity
  • Entire molecules react
  • No denaturation of Ag or Ab
  • Combination occurs on surface
  • Combination is firm but reversible
  • - Affinity Intensity of attraction of Ag or
    Ab molecules
  • - Avidity Strength of bond after formation
    of Ag - Ab complexes, reflect overall
    combining property of various Ab mol in
    antiserum
  • Both Ag and Abs participates
  • Ags and Abs combine in varying proportions, Abs
    are bivalent, Ags are multivalent

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Measurement of Antigen Antibody
  • In terms of Mass, Units or Titre
  • Antibody titre Highest dilution of serum
    showing observable reaction with antigen in a
    test
  • Two parameters of serological tests are
  • 1. Sensitivity Ability of the test to detect
    minute quantities of antigen or antibody
  • Highly sensitive tests False negative result
    is absent
  • 2. Specificity Ability to detect reactions
    between homologous Ags and Abs only , and no
    other
  • Highly Specific test false positive results are
    absent

8
PRECIPITATION
  • Soluble antigen reacts with its antibody in the
    presence of electrolytes at an optimum temp and
    pH, the Ag Ab complex forms an insoluble
    precipitate. Occur in liquid media or in gels
    such as agarose or polyacrylamide
  • Flocculation Instead of sedimenting, if
    precipitate is suspended as floccules eg VDRL
  • Zone phenomenon
  • - Prozone or zone of antibody excess
  • - Zone of equivalence
  • - Postzone or zone of antigen excess
  • Mechanism Lattice hypothesis - Multivalent Ag
    combines with bivalent Abs in varying
    proportions, depending on Ag Ab ration in the
    reacting mixture

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10
Application of Precipitation
  • Qualitative or Quantitative
  • Very sensitive in detecting antigens 1 µg of
    protein
  • Ring test - Eg Ascolis thermo precipitation
    test
  • Slide test - VDRL
  • Tube test - Khan test for syphilis
  • Immuno diffusion (Precipitation in gel)
  • - Single diffusion in one dimension (Oudin)
  • - Double diffusion in one direction (Oakley-
    Fulthorpe)
  • - Single diffusion in two dimensions ( Radial
    ID)
  • - Double diffusion in two dimensions
    (Ouchterlony)
  • Immunoelectrophoresis Grabar Williams
  • - Electrophoretic separation of composite
    antigens
  • - Followed by immunodiffusion against
    antiserum
  • - Enables identification and quantitation of
    various proteins present in serum

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12
IMMUNO ELECTROPHORESIS
IMMUNO ELECTROPHORESIS
13
Electroimmunodiffusion
  • Methods combining electrophoresis with diffusion
  • Counter immuno electrophoresis (CIE)
  • - Electrophoresis of antigen and antibody in gel
    in opposite directions resulting in
    precipitation between them
  • - Visible precipitation in 30 min, 10 times more
    sensitive than double diffusion
  • - Detecting various Ags such as Alphafetoprotein
    in serum
  • - Cryptococcus Ags in CSF
  • Rocket Electroporesis
  • - Quantitative estimation of antigens
  • - Antiserum incorporated in agarose gel
  • - Antigen in increasing concentration placed in
    wells and electrophoresed in gel

14
AGGLUTINATION
  • A particulate Ag combines with its Ab in presence
    of electrolytes at an opt temp and pH resulting
    in visible clumping of particles
  • More sensitive than precipitation for detecting
    Abs
  • 1. Slide Agglutination Blood grouping,
    Bacterial typing
  • 2. Tube Agglutination quantitative method for
    Abs
  • - Serum diluted by doubling dilutions in test
    tubes
  • - Equal volume of particulate Ag is added all
    tubes
  • - Antibody titre Highest dilution of serum at
    which agglutination occurs
  • Eg Widal test, Brucellosis, Weil Felix, Paul
    Bunnel
  • Cold agglutination test

15
Demonstration of hemagglutination
  • Demonstration of hemagglutination using
    antibodies against sheep red blood cells (SRBCs)
  • The control tube (10) contains only SRBCs, which
    settle into a solid button.
  • The experimental tubes 19 contain a constant
    number of SRBCs plus serial two-fold dilutions of
    anti-SRBC serum. The spread pattern in the
    experimental series indicates positive
    hemagglutination through tube 3.

16
ANTIGLOBULIN TEST (COOMBS TEST)
  • For detection of incomplete anti Rh antibodies
  • When mixed with red cells they coat over them but
    do not agglutinate
  • Such coated RBCs treated with antiglobulin or
    Coombs serum (Rabbit antiserum against human
    gamma globulin), cells are agglutinated
  • Two types
  • 1. Direct Coombs test Sensitisation takes
    place in vivo
  • Eg Haemolytic disease of new born
  • 2. Indirect Coombs test Sensitisation of
    RBCs with antibody globulin performed in
    vitro
  • - Used for detecting any type of incomplete or
    non agglutinating antibody, eg Brucelllosis

17
PASSIVE AGGLUTINATION TESTS
  • Precipitation is converted to agglutination
  • Attaching soluble antigen to the surface of a
    carrier particle such as Latex particles,
    Bentonite or RBCs
  • Very sensitive method for detecting antibodies
  • Two types
  • 1. Latex agglutination Polystyrene latex (0.8
    to 1µm diameter , absorb several types of
    antigens
  • - ASO, CRP, RA, HCG
  • 2. Haemagglutination Rose Waaler test in
    Rheumatoid arthritis, an autoantibody (RA
    Factor) appears in serum which acts as
    antibody to gammaglobulin
  • Reverse Passive Agglutination Instead of
    antigen, antibody is adsorbed to carrier
    particles

18
In Agglutination Inhibition, Absence
ofAgglutination Is Diagnostic of Antigen
  • A modification of the agglutination reaction,
    called agglutination inhibition, provides a
    highly sensitive assay for small quantities of
    an antigen
  • One of the early types of home pregnancy test
    kits included latex particles coated with human
    chorionic gonadotropin (HCG) and antibody to
    HCG. The addition of urine from a pregnant
    woman, which contained HCG, inhibited
    agglutination of the latex particles when the
    anti-HCG antibody was added thus the absence
    of agglutination indicated pregnancy

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20
  • Agglutination inhibition assays can also be used
    to determine whether an individual is using
    certain types of illegal drugs, such as cocaine
    or heroin. A urine or blood sample is first
    incubated with antibody specific for the
    suspected drug.
  • Then red blood cells (or other particles) coated
    with the drug are added. If the red blood cells
    are not agglutinated by the antibody, it
    indicates the sample contained an antigen
    recognized by the antibody, suggesting that the
    individual was using the illicit drug.
  • One problem with these tests is that some legal
    drugs have chemical structures similar to those
    of illicit drugs, and these legal drugs may
    cross-react with the antibody, giving a
    false-positive reaction. For this reason a
    positive reaction must be confirmed by a
    nonimmunologic method

21
Agglutination inhibition assays
  • Agglutination inhibition assays are widely used
    in clinical laboratories to determine whether an
    individual has been exposed to certain types of
    viruses that cause agglutination of red blood
    cells
  • If an individuals serum contains specific
    antiviral antibodies, then the antibodies will
    bind to the virus and interfere with
    hemagglutination by the virus. This technique is
    commonly used in premarital testing to determine
    the immune status of women with respect to
    rubella virus
  • The reciprocal of the last serum dilution to show
    inhibition of rubella hemagglutination is the
    titer of the serum. A titer greater than 10 (110
    dilution) indicates that a woman is immune to
    rubella, whereas a titer of less than 10 is
    indicative of a lack of immunity and the need for
    immunization with the rubella vaccine

22
COAGGLUTINATION
  • Protein A on surface of some Staphylococcus
    aureus (Cowan 1 strain)
  • Specific IgG can be coated on these protein A of
    Cowan 1 strains
  • Fc portion binds to Protein A, whereas Ag binding
    Fab remains free
  • The Fab portion binds to specific antigen when
    mixed leads to agglutination
  • Uses Detecting bacterial Ags in blood, Urine,
    CSF for Neisseria gonorrhoea, Strept pyogenes,
    Haemophilus
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