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AGGLUTINATION
By: F.Mirzaalian
Tests Based on Ag/Ab Reactions
• All tests based on Ag/Ab reactions will have to
depend on lattice formation or they will have
to utilize ways to detect small immune
complexes
• All tests based on Ag/Ab reactions can be used
to detect either Ag or Ab
Complex may be directly visible or invisible
Directly visible – agglutination
Invisible
• requires specific probes (enzyme-labelled antiimmunoglobulin, isotope-labelled antiimmunoglobulin, etc.)
• binds Ag-Ab complex and amplifys signals
• signals can be measured by naked eyes or specific
equipment e.g. in ELISA, RIA, IFA
Methods for Ag-Ab detection
• Precipitation
• Agglutination
• Hemagglutination and hemagglutination
inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunoflourescence
• Immunoblotting
• Immunochromatography
• Agglutination
1- Active (direct) agglutination.
2- Passive (Indirect) agglutination.
3- Reverse Passive agglutination.
4- Hemagglutination
5- Hemagglutination inhibition.
6- Viral Hemagglutination.
• Precipitation(Immunodiffusion)
A-Immunodiffusion in gel (Passive e.g RID- Active e.g
electroimmunodiffusion)
B- Fluid phase Immunoprecipitation e.g. nephelometry and
turbidimetry
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Agglutination Tests
Lattice Formation
Agglutination
• The interaction between antibody and a
particulate(Insoluble) antigen results in visible
clumping called agglutination.
• Particulate antigen include:
– bacteria,
– white blood cells,
– red blood cells,
– latex particles
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Agglutination Test
positive.
negative.
antigen
Antibody
.
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 of antigen and antibody.
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This represents what occurs during stage one of agglutination:
Sensitization
Stage 1
Antibody molecules attach to their corresponding Antigenic
site (epitope) on the red blood cell membrane. There is no
visible clumping.
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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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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.
• 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 blood group systems.
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• The following examples of agglutination
reactions :
• 1. Rheumatoid factor latex agglutination
• 2. Bacterial latex agglutination
• 3. Coombs test
• 4. Blood typing
Agglutination
In this test the antigen is particulate (visible,
big and insoluble) (e.g. bacteria and red blood
cells) or an inert particle (latex beads)
coated with antigen.
Antibody is divalent and cross links the
multivalent antigen to form a lattice
network or clumps (agglutination).
This reaction can be performed in a tube
or on a glass slide e.g. ABO blood
grouping.
Slide agglutination is a rapid method to determine the
presence of agglutinating abs
Factors influencing the reaction:
•
•
•
•
Elevation or decrease of temperature.
Motion (shaking,stirring,centrifugation).
PH.
Class of antibody (IgM/IgG).
When Abs & Ags are present in equimolar
ratios They form insoluble complexes that
ppt
(ZONE OF EQUIVALENCE)
• Decreased amounts of ppt are formed in
zones of Ag or Ab excess.
Precipitation Curve
Prozone phenomena
In an agglutination or precipitation reaction, the
zone of relatively high antibody concentrations
within which no reaction occurs. As the antibody
concentration is lowered below the prozone, the
reaction occurs.(solve by dilution)
This phenomenon may be due simply to antibody
excess or it may be due to blocking antibody or to
nonspecific inhibitors in serum.
An example of prozone phenomenon.
Serum dilution
1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 Titr
e
Sample
#1
Sample
#2
4+ 3+ 3+ 2+
--
--
3+ 4+
1+
1+
--
--
--
--
32
4+
3+
3+
2+
1+
--
256
* Sample 2 is an example of the prozone phenomenon
A)Qualitative agglutination tests
Agglutination tests can be used in a qualitative
manner to assay for the presence of an antigen or
an antibody.
The antibody is mixed with the particulate antigen
and a positive test is indicated by the
agglutination of the particulate antigen .
B) Quantitative Agglutination Test
The Ab titre can be determined using serial
dilution of the patient serum.
Serial Dilution
A serial dilution is simply a series of simple dilutions
which amplifies the dilution factor quickly beginning
with a small initial quantity of material (i.e., bacterial
culture, a chemical, orange juice, etc.). The source of
dilution material for each step comes from the diluted
material of the previous. In a serial dilution the total
dilution factor at any point is the product of the
individual dilution factors in each step up to it.
Final dilution factor (DF) = DF1 × DF2 × DF3 etc.
Agglutination/Hemagglutination
Semi-Quantitative agglutination test
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
Titer
Prozone
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
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Direct agglutination
Principle
• combination of an insoluble
particulate antigen with its
soluble antibody
– forms antigen-antibody
complex
– particles clump/agglutinate
• used for antigen detection
Ag-Ab complex
Examples
– bacterial agglutination tests for
sero-typing and sero-grouping
e.g., Vibrio cholerae,
Salmonella spp
Positive
Negative
Passive Agglutination
• Definition - agglutination test done with a soluble
antigen coated onto a particle (Ag is fixed to a
solid surface)
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• Applications
– Measurement of antibodies to soluble antigens
Passive (indirect) agglutination
Principle
- coating antigen onto the surface
of carrier particles like red blood cells,
latex, gelatin, bentonite or charcoal.
-background clears
Examples of types
– latex agglutination
– passive hemagglutination (treated red blood cells
made resistant)
Examples of tests - agglutination for leptospirosis
-Widal test (typhoid fever)
Reverse Passive Agglutination
Antibody attached to carrier particle instead of
antigen. (The Ab is fixed to a solid surface)
Principle:
– antigen binds to soluble antibody coated on
carrier particles and results in agglutination
-detects antigens.
Example
– detecting cholera toxin
Reverse passive agglutination
Positive
Negative
Agglutination:
Performance, applications
Advantages
– sensitive for antibody detection
Limitations
– Prozone phenomenon:
• requires the right combination of quantities of antigen
and antibody
• handled through dilution to improve the match
Time taken
– 10-30 minutes
Advantages:
-Portable.
-Rapid.
-efficient.
-quick and simple.
N.B: they are semiquatitative assays.
Applications:
More than 100 infectious disease.
More than 60 chemical analyte e.g. hCG, CRP,
ASO,fecal occult blood.
Hemagglutination
Principle
- It is a type of agglutination test performed on
RBCs.
- many human viruses have the ability to bind to
the surface structures on red blood cells from
different species thereby causing
agglutination
Example
– influenza virus binds to fowl’s red blood cells
Haemaggultination Tests:
It has two types:
Active: the antigen is the RBC itself.
Viruses can clump red blood cells from
one species or another (active
hemagglutination)
Example is the test used in ABO grouping.
Passive: the antigen here is not the RBC. The
RBC absorbs it and expresses it on the surface.
It will form clumps when mixed with
antibodies.
i.e. red cells are passive carriers .
Haemaggultination Tests
active
passive
Hemagglutination inhibition
Principle
Antibodies to the virus in the
patient serum bind to the
virus; blocks binding sites on
the viral surfaces
– prevents the virus from
agglutinating the red
cells
Example
– detecting antibodies to
influenza and dengue
viruses
• Positive
•
•
Negative
Hemagglutination inhibition for
detection of Dengue antibodies
Hemagglutination:
Performance, applications
Advantages
– highly specific
– can be used as gold standard
Limitations
– technically demanding
– time consuming
– cannot distinguish IgG from IgM
Time taken
– 1 day
– Bacterial infections
–Fourfold rise in titer
• Practical considerations
– Easy
– Semi-quantitative
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
• Applications
– Blood typing
1/2
Agglutination/Hemagglutination
Fluid phase immunoprecipitation
 spectrophotometers and nephelometers can
measure absorbed or scattered light from very
sensitive microsphere agglutination assays.
Turbidity and Nephelometry Light scattering
PRINCIPLES:
Light scattering is the physical phenomenon
resulting from the interaction of light with a
particle(s) in solution.
Dependent on:
•Particle size
•Wavelength
•Distance of observation,
•Concentration of particles
•MW of particles
 In turbidimetry, is the process of measuring
the loss of intensity of transmitted light due
to scattering effect.
 Light is passed through a filter creating a
light of known wavelength which is then
passed through a cuvette containing a
solution.
 A photoelectric cell collects the light
which passes through the cuvette.
 A measurement is then given for the
amount of absorbed light.
 Turbidimetry is measurement of reduction in
the intensity of the transmitted light at 180°.
 Turbidity can be measured on most routine
analysers by a spectrophotometer
(absorbed light).
 In nephelometry, the intensity of the
scattered light is measured at a particular
angle.
The formation of insoluble immune complexes
when a soluble antigen reacts with its specific
antibody produce particles of various sizes that
will reflect light.
The light scattered is proportional to the particle
concentration.
Nephelometry is based on measurement of light
scatter reflectance at a particular angle
nephelometer
.
Examples of Ag and Abs assayed by
nephelometry:
complement components( C 3 and C4)
Immunoglobulin conc (IgA, IgM, IgG)
Albumin and α-1-antitrypsin
acute phase reactants (CRP, transferrin)
Rheumatoid factor