ANTIGEN- ANTIBODY
INTERACTIONS – I
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
Stages of Reactions
Primary stage
Secondary stage
- Initial interaction rapid
- No visible effect
- Reversible
- Weaker inter
molecular forces
- Detected by
estimating free
and bound Ag or Ab
- Visible effects
- Precipitation
- Agglutination
- CF
- Neutralization
- Immobilization
- Zinssser’s Unitarian
hypothesis
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
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
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
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
Application of Precipitation
Qualitative or Quantitative
Very sensitive in detecting antigens – 1 μg of protein
Ring test : - Eg: Ascoli’s 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
IMMUNO
IMMUNO ELECTROPHORESIS
ELECTROPHORESIS
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
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
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 1–9 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.
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 RBC’s 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 RBC’s with
antibody globulin performed in vitro
- Used for detecting any type of incomplete or non
agglutinating antibody, eg: Brucelllosis
PASSIVE AGGLUTINATION TESTS
Precipitation is converted to agglutination
Attaching soluble antigen to the surface of a carrier particle
such as Latex particles, Bentonite or RBC’s
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
In Agglutination Inhibition, Absence of
Agglutination 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
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
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 individual’s 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 (1:10 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
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