Download Lab.4: agglutination

AGGLUTINATION
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Agglutination
• The interaction between an antibody and a particulate
antigen results in visible clumping called agglutination
Antigens (Bacteria)
• Particulate antigen include:
• 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
• 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
• Antibody molecules attach to their corresponding Antigenic
site (epitope) on the particle
• There is no visible clumping
Crosslinking Abs
• Antibody molecules crosslink the particles forming a lattice that
results in visible clumping or 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
• 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
• Prozone – antibody excess, many antibodies coat all antigen sitesresults in false negative
• Postzone – antigen excess, antibody coats antigen but cannot get
lattice formation, results in false negative
• Zone of Equivalence – antigen and antibody present in optimal
proportions to bind and give visible reaction
Conc. Of Ag-ab Complexes
Increasing Antigen Concentration (Antibody Concentration Is Constant)
• Qualitative agglutination test
• Semi-quantitative agglutination test
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
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
• Then add 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
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
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6
7
8
128
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• Direct Agglutination
• Indirect or Passive Agglutination
• Reverse Passive Agglutination
• Agglutination Inhibition
• Coagglutination
• 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
• 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
•
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
• 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
• Agglutination inhibition reactions are based on competition
between particulate and soluble antigens for limited antibodycombining 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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• 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
No Agglutination Occur
• 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
Agglutination Occur
Hemagglutination inhibition
Antibodies to the virus in the patient
serum bind to the virus; blocks
binding sites on the viral surfaces
Positive
Negative
• prevents the virus from agglutinating
the red cells
Example
• detecting antibodies to influenza
Hemagglutination inhibition
for detection of influenza
antibodies
• 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
• 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 2 ways in which Ags can be bound to RBCs:
1.
Spontaneous adsorption of Ags by RBCs
2. Covalent binding using chemical links
• 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
• 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
• 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
• 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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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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Slides, Test tubes
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MICOTITER PLATES
• Agglutination reactions now have a wide variety of applications in the
detection of both antigens and antibodies including:
• Blood grouping,
• Diagnosis of infectious & non-infectious diseases
• Measure levels of certain therapeutic drugs, hormones, and plasma
proteins
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Detection of Abs or 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
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 (anti-HBs)
• Anti-HBs also appears as the immune response following hepatitis
B vaccination
• 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
1. Each specimen requires 12 wells of a microtiter plate
2. Add 50 µl of diluent to wells A1 – A10 for each sample
3. Add 50 µl of sample to well A1, mix well and transfer 50 µl to well A2, mix well
and transfer 50 µl to well A3, till well A10
4. Transfer 25 µl of A1 to B1, 25 µl of A2 to B2, till A10 to B10
5. Add 25 µl of +ve control to well A11 and 25 µl of –ve control to well A12
6. Resuspend test cells and then add 75 µl of test cells to wells A1 – A12
7. Tap the plate gently to mix the contents making sure to avoid cross
contamination
8. Incubate the plate for 30-45 minutes at RT keeping the plate away from heat,
direct sunlight & any source of vibration
9. Read and record the results, the results are stable for 24 hours if the plate is
covered
Sample 1 Dil.
Sample 1
Sample 2 Dil.
Sample 2
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Sample
1/2
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Results
Test cells
Strong Positive
Full cell pattern covering the bottom of the well
Weak Positive
Cell pattern coves 1/3 of well bottom
Intermediate
Negative
Cell pattern shows distinctly open center
Cells settled to a compact button
Reference Values
• Hepatitis B Surface Antibody
• Unvaccinated: negative
• Vaccinated: positive
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