Title: ANTIGEN- ANTIBODY INTERACTIONS
1ANTIGEN- ANTIBODY INTERACTIONS I
2ANTIGEN- 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
3Stages 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
4Comparative efficiency of Immunoglobulin classes
in different serological reactions
IgG IgM IgA
Precipitation Strong Weak Variable
Agglutination Weak Strong Moderate
Complement Fixation Strong Weak Negative
5General 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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7Measurement 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
8PRECIPITATION
- 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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10Application 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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12IMMUNO ELECTROPHORESIS
IMMUNO ELECTROPHORESIS
13Electroimmunodiffusion
- 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
14AGGLUTINATION
- 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
15Demonstration 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.
16ANTIGLOBULIN 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
17PASSIVE 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 -
18In 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
21Agglutination 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
22COAGGLUTINATION
- 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