Download Agglutination

AGGLUTINATION
Agglutination
• The interaction between antibody and a
particulate antigen results in visible
clumping called agglutination
• Particulate antigen include:
Antigens (Bacteria)
• bacteria,
• white blood cells,
• red blood cells,
• latex particles
• Antibodies that produce such reactions
are called agglutinins
• If an agglutination reaction involves red
blood cells, then it is called
hemagglutination
RBCs Antigens
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Agglutinin and Agglutinogen
• Agglutinin:
• It 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
• Agglutinogen
• It 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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Applications
• Agglutination reactions now have a wide variety
of applications in the detection of both antigens
and antibodies including:
• blood grouping,
• diagnosis of infectious diseases
• measure levels of certain therapeutic drugs, hormones,
and plasma proteins
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Testing Abs & Ags
• The agglutination reaction may be used to
identify the antibody or antigen in a patient
sample
• When testing for antibody, the antigen
concentration is constant for each dilution
being tested
• When testing for antigen, the antibody
concentration is constant for each dilution
being tested
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Agglutination & Precipitation
• Agglutination reactions are similar in principle to
precipitation reactions; they depend on the cross
linking of polyvalent antigens with the exception
that:
• Precipitation reactions involve soluble antigens, while
agglutination involves particulate antigens
• Pecipitation reactions represent a phase change, while
the agglutination reactions manifest as clumping of
antigen/ antibody complexes
• Agglutination is more sensitive than precipitation
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Advantages of Agglutination Techniques
• The agglutination reaction has wide spread use in the clinical
laboratory due to the following reasons:
• they are simple
• inexpensive
• reliable
• The visible manifestation of the agglutination reaction eliminates the need
for complex procedures and expensive instrumentation
• Numerous techniques have been described for agglutination tests,
these techniques may be performed using:
• slides,
• test tubes,
• or micotiter plates, depending on the purpose of the test
• However the principle of the agglutination remain the same
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Qualitative and Quantitative Techniques
• Qualitative agglutination test
• Semi-quantitative agglutination test
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Qualitative Agglutination Test
• 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
• For example, a patient’s red blood cells can be mixed with
antibody to a blood group antigen to determine a person’s
blood type
• In a second example, a patient’s serum is mixed with red blood
cells with virus Ags to assay for the presence of antibodies to
that virus in the patient’s serum
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Semi-Quantitative Agglutination Test
• Agglutination tests can also be used to quantitate the
level of antibodies to particulate antigens
• In this test:
• one makes serial dilutions of a sample to be tested for antibody
• and then adds a fixed number of red blood cells or bacteria or
other such particulate antigen
• then determines the maximum dilution, which gives agglutination
• The maximum dilution that gives visible agglutination is
called the titer
• The results are reported as the reciprocal of the maximal
dilution that gives visible agglutination
• This can be done using a microtiter plate
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Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
Semi-Quantitative Agglutination Test
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
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Steps in Agglutination
• Agglutination is a two-step process that results in the
formation of a stable lattice network
1. Sensitization
• The first reaction involves antigen-antibody combination through
single antigenic determinants on the particle surface and is often
called sensitization
2. Lattice formation
• The second step is the formation of cross-links that form the visible
aggregates
• This represents the stabilization of antigen–antibody complexes
with the binding together of multiple antigenic determinants
• Each stage of the process is affected by different factors,
and it is important to understand these in order to
manipulate and enhance end points for such reactions
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1- Sensitization
• Antibody molecules attach to their corresponding
Antigenic site (epitope) on the particle
• There is no visible clumping
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2- Lattice Formation
Crosslinking Abs
• Antibody molecules crosslink the particles forming a
lattice that results in visible clumping or agglutination
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Factors that Affect Agglutination
• Buffer pH
• The relative concentration of Antibody and Antigen
• Location and concentration of Antigenic Determinants
•
•
•
•
on the Particle
Electrostatic Interactions between Particles
Electrolyte Concentration
Antibody Isotype
Temperature
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Hemagglutination
• The agglutination of red blood cells by either
• direct agglutination
• or indirect agglutination
• Direct agg. Ag is an intrinsic component of RBC
• Indirect agg. Soluble Ags are adsorbed to the RBC
• There are 3 ways in which Ags can be bound to
RBCs:
Spontaneous adsorption of Ags by RBCs
2. Covalent binding using chemical links
1.
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The Latex particles
• Latex particles are usually prepared by emulsion
polymerization
• Styrene (unsaturated liquid hydrocarbon) is mixed
with a surfactant (sodium dodecyl sulfate) solution,
resulting emulsified in billions of micelles extremely
uniform in diameter
• When the polymerization is finished, polystyrene
chains are arranged into the micelles with the
hydrocarbon part in the center and the terminal
sulfate ions on the surface of the sphere, exposed
to the water phase
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The Latex Particles
• Black ball chains represents
polystyrene with sulfate free
radicals (shaded balls)
• Blue balls denote the sulfonic
acid group of the SDS
• Tail represent the hydrocarbon
tail
• The simplest method of
attaching proteins to the
particles is by passive
adsorption
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Latex Agglutination
• In latex agglutination
procedures, an antibody (or
antigen) coats the surface of
latex particles (sensitized latex)
• When a sample containing the
specific antigen (or antibody) is
mixed with the milky-appearing
sensitized latex, it causes visible
agglutination
Positive
Negative
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Viral Hemagglutination
• Many viruses have nonserological hemagglutinating
properties
• They can agglutinate RBCs in the absence of Ab (nonimmune agglutination)
• Mammalian reoviruses agglutinate erythrocytes through
interactions between the viral surface protein sigma 1 and
carbohydrate groups attached to proteins on erythrocyte
membranes
• Hemagglutination (HA) can be used to
• determine titers of certain viruses
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Types of Agglutination
• Direct Agglutination
• Indirect or Passive Agglutination
• Reverse Passive Agglutination
• Agglutination Inhibition
• Coagglutination
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Direct Agglutination
• In this reaction the antigen is an intrinsic
component of the particle
• The agglutination test is used to determine
whether antibody, specific for the antigen
is present in the biological fluids
• serum
• urine
• or CSF
• Direct agglutination tests are used
primarily for diagnosis of infectious
diseases
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Passive or Indirect Agglutination
• Employs particles that are coated with
•
•
•
•
antigens not normally found on their
surfaces
Antigen has been affixed or adsorbed to
the particle surface
A variety of particles, including
erythrocytes, latex and others are used for
this purpose
The use of synthetic beads or particles
provides the advantage of consistency,
uniformity, and stability
Passive agglutination tests have been
used to detect antibodies to viruses such
as:
• cytomegalovirus, rubella, varicella-zoster,
and HIV-1/HIV-2
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Reverse Passive Agglutination
•
•
•
•
•
In reverse passive agglutination,
antibody rather than antigen is
attached to a carrier particle
The antibody must still be reactive
and is joined in such a manner that
the active sites are facing outward
This type of testing is often used to
detect microbial antigens
Latex particle coated with Ab
(known) + serum looking for
particular Ag
If Ag present, then visible
agglutination is observed
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Reverse Passive Agglutination
• Numerous kits are available for rapid identification of
antigens on infectious agents
• Such tests used for organisms that
• are difficult to grow
• or when rapid identification is required
• Testing of specimens for the presence of viral antigens
has still not reached the sensitivity of enzyme
immunoassays
• But for infections in which a large amount of viral
antigen is present, such as rotavirus and enteric
adenovirus in infants, latex agglutination tests are
extremely useful
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Agglutination Inhibition
• Agglutination inhibition reactions are based on
competition between particulate and soluble
antigens for limited antibody-combining sites
• The lack of agglutination is an indicator of a
positive reaction
• The technique is called hemagglutination
inhibition if the particle in the reaction is a RBC
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Agglutination Inhibition - Positive
• Tube containing free known
Ab specific for the Ag to be
detected
• Patient has Ag and will
combine with Ab
• No visible agglutination
• Latex beads coated with same
Ag to be detected is added
• It has nothing to attach to
• No visible reaction
• Therefore agglutination inhibition
is positive
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Agglutination inhibition - Negative
• Tube containing free known Ab
• Patient serum does not contain
Ag
• therefore no combination
• Latex beads coated with same
Ag to be detected is added
• Visible agglutination,
• Therefore agglutination inhibition
is negative
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Hemagglutination Inhibition
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
Positive
Negative
Hemagglutination
inhibition for detection of
influenza antibodies
Coagglutination
• The name given to systems using
bacteria as the inert particles to
which antibody is attached
• Staphylococcus aureus is most
frequently used, because it has a
protein on its outer surface, called
protein A which naturally adsorbs the
FC portion of antibody molecules
• The Fab region is free to interact with
antigens present in the applied
specimens
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HEPATITIS B SURFACE
ANTIBODY DETECTION
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Summary
• Hepatitis B surface antigen (HBsAg) is the first
serologic marker, appearing in the serum 6 to 16
weeks following HBV infection
• In acute cases, HBsAg usually disappears 1 to 2
months after the onset of symptoms with the
appearance of hepatitis B surface antibody (antiHBs)
• Anti-HBs also appears as the immune response
following hepatitis B vaccination
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Principle
• When used by recommended technique, reagent
will agglutinate in presence of Abs to HBV
• No agglutination generally indicates absence of
Abs
• Test cells are preserved avian erythrocytes
coated with Ags of HB
• Any non-specific reaction should be detected
using the control cells
• Avian erythrocytes not coated with Ags of HB
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Procedure for Qualitative Screening
Technique
1.
2.
3.
4.
5.
6.
7.
Each specimen requires 3 wells of a microtiter plate
To well 1 add 190 µl of diluent and 10 µl of serum, -ve or +ve
controls
Using a pipette, mix contents of well 1 and transfer 25 µl to wells 2
&3
Resuspend both test and control cells and then add 75 µl of control
cells to well 2 & 75 µl of test cells to well 3
Tap the plate gently to mix the contents making sure to avoid cross
contamination
Incubate the plate for 45-60 minutes at RT keeping the plate away
from heat, direct sunlight & any source of vibration
Read and record the results, the results are stable for 24 hours if
the plate is covered
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Interpretation of Results
Results
Test cells
Control cells
Strong Positive
Full cell pattern covering the
bottom of the well
No agglutination, tight button
Weak Positive
Cell pattern coves 1/3 of well No agglutination, tight button
bottom
Intermediate
Cell pattern shows distinctly
open center
No agglutination, tight button
Negative
Cells settled to a compact
button
No agglutination, tight button
Non-Specific
Positive reaction
Positive reaction
Reference Values
• Hepatitis B Surface Antibody
• Unvaccinated: negative
• Vaccinated: positive
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