ANTIGEN-ANTIBODY
REACTIONS
Babitha Elias
• Antigens and antibodies combine with
each other specifically and in an
observable manner.
• In the body, they form the basis of antibody
mediated immunity in infectious diseases,
or hypersensitivity and autoimmune
diseases.
• In laboratory, they help in diagnosis of
infections, in epidemiological surveys, in
the identification of infectious agents,
enzymes.
• Antigen – antibody reactions in vitro are
known as serological reactions.
Stages of Ag – Ab reactions
Primary stage:
• Initial interaction between Ag & Ab – invisible
• Rapid, occurs at low temperatures & obeys the
general laws of physical chemistry &
thermodynamics.
• Reaction is reversible.
• Ag & Ab is bound to each other by weak Van der
Waal’s forces, Ionic bonds & Hydrogen bonding.
Secondary stage:
• Demonstrable events – Precipitation,
agglutination, lysis of cells, killing of live antigens,
neutralization of toxins, complement fixation,
immobilization of motile organisms &
enhancement of phagocytosis.
Precipitin – Ab participate in precipitation
Agglutinin - Ab participate in agglutination
Precipitinogen – Ag participate in precipitation
Agglutinogen - Ag participate in agglutination
Tertiary stage:
• Includes neutralization or destruction of
injurious agents or tissue damage.
• Also includes humoral immunity against
infectious diseases as well as clinical
allergy & other immunological diseases.
GENERAL FEATURES OF Ag – Ab
REACTIONS
1. The reaction is specific.
2. Entire molecules react and not the fragments
3. There is no denaturation of the antigen or
antibody during the reaction.
4. The combination occurs at the surface. So
surface antigens are immunologically relevant.
5. The combination is firm but reversible. The
firmness is influenced by the affinity & avidity of
the reaction.
Affinity – refers to the intensity of attraction between
the antigen & antibody molecules. It is the function
of closeness of fit between the epitope & antigen
binding region of its Ab.
Avidity – strength of the bond after the formation
Ag-Ab complexes.
6. Both Ags & Abs participate in the formation of
agglutinates or precipitates.
7. Antigens & antibodies can combine in varying
proportions. Both Ags & Abs are multivalent.
MEASUREMENT OF ANTIGEN & ANTIBODY
• Measurement may be in terms of mass or
more commonly as units or titre.
• The Antibody titre of a serum is the highest
dilution of the serum which shows an
observable reactions with the antigen in a
particular test.
Two important parameters in serological tests –
Sensitivity & Specificity
Sensitivity: The ability of the test to detect
even very minute quantities of antigen or
antibody.
• When the test is highly sensitive, false negative
results may be absent or minimal.
Specificity: The ability of the test to detect
reactions between homologous Ags & Abs
only, and with no other.
• In highly specific test, false positive reactions are
absent or minimal.
Types of Antigen – Antibody Reactions
1.
2.
3.
4.
5.
6.
7.
8.
9.
Precipitation reaction
Agglutination reaction
Neutralization reaction
Complement fixation test
Immobilization test
Opsonisation
Immunofluorescence
Radioimmuno assay
Enzyme immunoassay
PRECIPITATION REACTION
PRINCIPLE: When a soluble Ag combines with its Ab
in the presence of electrolytes (NaCl) at a suitable
temperature & pH, the Ag-Ab complex forms an
insoluble precipitate.
• When instead of sedimenting, the precipitate
remains suspended as floccules – Flocculation
reaction
• Precipitation can take place in liquid media or in
gels such as agar, agarose or polyacrylamide.
ZONE PHENOMENON
• The amount of precipitate formed is
greatly influenced by the relative
proportions of Ags & Abs.
• If increasing quantities of Ags are added to
the same amount of antiserum in different
tubes, precipitation is found to occur most
rapidly & abundantly in the middle tubes.
– Preceding tubes – Ab excess (Prozone)
– Middle tubes – Ag & Ab in equivalent
proportions (Zone of equivalence)
– Later tubes – Ag excess (Post zone)
Mechanism of precipitation
• Marrack (1934) proposed the lattice
hypothesis – mechanism of precipitation
• The multivalent antigens combine with
bivalent Abs in varying proportions,
depending on the Ag – Ab ratio on the
reacting mixture.
• Precipitation results when a large lattice is
formed consisting of alternating Ag & Ab.
Marrack’s hypothesis
Applications of Precipitation reaction
• It can be carried out as either a
quantitative or qualitative test.
• Sensitive for the detection of Ags.
Types of precipitation reactions:
– Ring test
– Slide test
– Tube test
– Immunodiffusion
– Electroimmunodiffusion
RING TEST:
• Consists of layering Ag solution over a column of
antisera in a narrow tube.
Eg: Ascolis thermoprecipitin test, Grouping of
Streptococci by Lancefield technique
SLIDE TEST:
• When a drop of Ag & antiserum is placed
on a slide & mixed by shaking, floccules will
appear.
• Eg: VDRL test & RPR test for syphilis
TUBE TEST:
• This is employed for the standardization of
toxins & toxoids.
• Serial dilutions of toxin/toxoid are added to
the tubes containing a fixed quantity of
antitoxin.
• The amount of toxin that flocculates
optimally with one unit of the antitoxin – Lf
dose.
• Eg: Kahn test for syphilis.
IMMUNODIFFUSION (precipitation in gel)
Advantages of immunodiffusion:
• Reaction is visible as a distinct band of
precipitation.
• Stable, can be stained for preservation.
• Indicates identity, cross reactions, non
identity between different Ags.
1. Single diffusion in one dimension (Oudin
procedure)
• Ab is incorporated in agar gel in a test
tube & Ag solution is layered over it.
• Ag diffuses downward through the agar
gel – forming a line of precipitation.
2. Double diffusion in one dimension
(Oakley-Fulthorpe procedure)
• Ab is incorporated in agar gel
• Above which is placed a column of plain
agar.
• The Ag is layered over it.
• The Ag & Ab move towards each other
through the intervening column of plain
agar & form the precipitate.
3. Single diffusion in two dimensions (Radial
immunodiffusion)
• Here the antisera is incorporated in a gel &
poured on a flat surface.
• Wells are cut on the surface to which Ag is
added.
• It diffuses radially from the well & forms
ring shaped bands of precipitation
concentrically around the well.
4. Double diffusion in two dimensions
(Ouchterlony procedure)
• Helps to compare different antisera &
antigens directly.
• Agar gel is poured on a slide & wells are
cut .
• Antiserum – central well
• Different Ags in the surrounding wells.
Reaction of identity
Lack of relatedness
Partial identity
Elek’s gel precipitation
5. Immunoelectrophoresis
• Graber & Williams devised this technique.
• This involves the electrophoretic separation of
composite Ag into its constituent proteins,
followed by immunodiffusion against its
antiserum – separate precipitin lines.
• It is performed on an agarose gel with an Ag well
& Ab trough cut on it.
• The test serum is placed in the antigen well &
electrophoresed for about 1 hour.
• Ab against human serum is placed in the trough
& diffusion allowed for 18 – 24 hrs.
Immunoelectrophoresis
ELECTROIMMUNODIFFUSION
•
•
The development of precipitin lines can
be speeded up by electrically driving the
Ag & Ab.
Two types
1. Counterimmunoelectrophoresis (One
dimensional double electroimmunodiffusion)
2. Rocket electrophoresis (One dimensional
single electroimmunodiffusion)
1. Counterimmunoelectrophoresis (CIE)
•
•
•
This involves simultaneous electrophoresis of Ag
& Ab in gel in opposite directions resulting in
precipitation at a point between them.
Produce precipitation lines within 30 mins.
Clinical application: detecting Ags like
alphafetoprotein in serum, Ags of Cryptococcus &
Meningococcus in the CSF.
2. Rocket electrophoresis
• Used for quantitative estimation of Ags.
• The antiserum to the Ag to be quantitated is
incorporated in agarose gel on a slide.
• Ag in increasing concentrations, is placed in wells
punched in the set gel.
• The Ag is electrophoresed into the Ab containing
agarose.
• The pattern of immunoprecipitation resembles a
ROCKET.
Rocket electrophoresis
Laurell’s two dimensional electrophoresis
• Variant of rocket electrophoresis.
• The Ag mixture is electrophoretically
separated in a direction perpendicular to
that of the final rocket stage.
• Used to quantitate each of the several Ags
in a mixture.
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