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Agglutination
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Agglutination
• The interaction between antibody and a
particulate antigen results in visible clumping
called agglutination.
• Particulate antigen include:
•
•
•
•
bacteria,
white blood cells,
red blood cells,
latex particles
• Antibodies that produce such reactions are
called agglutinins.
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Agglutinin and Agglutinogen
• An agglutinin is an antibody that interacts with
antigen on the surface of particles such as
erythrocytes, bacteria, or latex particles to cause
their agglutination in an aqueous environment
containing electrolyte.
• An agglutinogen is an antigen on the surface of
particles such as red blood cells that react with the
antibody known as agglutinin to produce
agglutination.
The
most
widely
known
agglutinogens are those of the ABO and related
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blood group systems.
• Agglutination is the basis for multiple
serological reactions including:
• blood grouping,
• diagnosis of infectious diseases
• and noninfectious diseases
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Phases of Agglutination
• Agglutination is a two-Phase reaction that results in
the formation of a stable lattice network
• Primary Phase (Sensitization)
• Ab reacts with a single antigenic determinant on the
surface of Ag.
• Secondary Phase (Lattice formation)
• Ab must be able to bridge the gap between particles so that
at least one Fab portion is attached to an antigenic
determinant on each of two adjacent particles, is dependent
on environmental conditions and the relative concentrations
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of antigen and antibody.
This represents what occurs during stage one of agglutination:
Sensitization
Stage 1
Antibody molecules attach to their corresponding Antigenic site
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(epitope) on the red blood cell membrane. There is no visible
clumping.
This represents what occurs during stage 2 of
agglutination: Lattice Formation
Stage 2
Antibody molecules crosslink RBCs forming a lattice that
results in visible clumping or agglutination.
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Direct Agglutination
Agglutination reactions where the antigens are found
naturally on a particle are known as Direct
Agglutination.
a) Slide Agglutination:
• The test is performed in a slide.
• A suspension of bacteria is prepared and is added to
a drop of standarised antiserum.
• Clumping of bacteria is the positive reaction.
• Clumping occurs instantly in a positive result.
Eg: 1) Identification of bacterial strains such as
Salmonella, Vibrio etc.
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2) Blood grouping.
b) Tube Agglutination:
• The test is performed in a tube.
• In these test, the patient’s serum is diluted in
a series of tubes and bacterial antigens
specific for the suspected disease are added
to it.
• Antigen and antibody reactions are
demonstrated by visible clumps of
agglutination.
• It is a standard method used for the
quantitative estimation of antibodies in the
serum.
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Ag-Ab complex
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Passive Agglutination
It employs carrier particles that are coated with soluble antigens.
When antibody instead of antigens is adsorbed on the carrier
particle for detection of antigens, it is called reverse passive
agglutination.
Earlier only RBCs were the major carrier particles used for the
coating of antigens.
Now a variety of particles like polystyrene latex, bentonite and
charcoal are used for this purpose.
The use of synthetic beads or particles provide the advantage of
consistency, uniformity and stability.
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Types of Passive Agglutination
1) Latex Agglutination test
2) Hemagglutination test
3) Coagglutination test
Hemagglutination test:
 RBCs are used as carrier particles.
 RBCs of sheep, human, chick etc are commonly used in this
test.
 When RBCs are coated with antigen to detect antibodies in
the serum, the test is called indirect hemagglutination test
(IHA).
 IHA is used for the dignosis of many parasitic diseases
including amoebiasis, hydatid disease.
 When antibodies are attached to the RBCs to detect microbial
antigen, it is known as reverse passive hemeagglutination12
(RPHA)
Radioimmunoassay (RIA)
 RIA was first described in 1960.
 It was used to measure the amount of endogenous
plasma insulin by Solomon Berson and Rosalyn
Yalow.
 RIA works on the principle of competitive binding.
 In this method, unlabeled antigen competes with
radiolabelled antigen for binding to the antibody.
 When mixtures of radiolabeled and unlabeled
antigen are incubated with the corresponding
antibody, the amount of free radiolabeled antigen is
directly proportional to the quantity of unlabeled
antigen in the mixture.
 The radioactivity can be counted for finding out13the
concentration of unlabeled antigen present.
Uses:
 RIA can be used to detect even nanograms of antigen in the
serum
 Can be used for the quantitation of hormones, drugs and
various viral antigens
Disadvantages:
 Cost of the equipment and reagents are expensive
 Short shelf-life of raidolabeled compounds
 Problems associated with the disposal of radioactive waste.
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