Agglutination

Suryarini Trisa
• Agglutination is clumping and sedimentation
of particulate antigen/antibody complexes.
• Particulate antigen ex: bacteria, white blood
cells, red blood cells, latex particles.
• Agglutinins: antibodies that agglutinate
antigen
• Agglutinogen: antigens that become
agglutinate.
 Principles of agglutination
• Agglutination occurs only when the properties
and relative concentrations of antigen and
antibody allow sufficient lattice formation
• Lattice: large immune complexes that form as
a result of antibody interaction with
multivalent antigens.
 Report strength of agglutination
reaction

Strength of
agglutination Explanation
reaction
+1 Small aggregates

+2 Medium size aggregates

+3 Several large aggregates

+4 One solid aggregates

• Those aggregates are visible with naked eye.
• Very tiny aggregates are visualized with
microscope and reported as weak aggregates.
Factors affecting agglutination

1. Buffer pH
2. The relative concentrations of antibody and
antigen
3. Location and concentration of antigenic
determinants on the particles
4. Electrostatic interactions between particles
5. Electrolyte concentrations
6. Antibody isotype
7. Temperature
 Agglutination techniques
• Agglutination may be performed using:
1. slides
2. test tube
3. microtiter plates
 Agglutination tests
Particles Detect Positive
reaction
Direct (active) Intrinsic antigen on Antibodies Agglutination
agglutination microbe, cell, or other
particulate antigen
Indirect (passive) Soluble antigen is Antibodies Agglutination
agglutination affixed or absorbed to
the particle
Reverse Antibody is affixed or Antigens Agglutination
agglutination absorbed to the
particles
Latex particle Initially the soluble Antigen NO agglutination
agglutination antigen and antibody
inhibition interact.
Incubation with
antigen coated latex
particle is a second
 Particles Detect Positive
reactions
Direct (active) Antigen is an IgM antibodies Agglutination
hemagglutination intrinsic part of
the red blood cell

Antiglobulin test Red blood cell to Testing for IgG Agglutination

(direct Coombs’ which IgG has bound to antigen
test) bound to an on red blood cell
antigen

Variation of Red blood cell, Testing for serum Agglutination

antiglobulin test intrinsic antigen IgG that binds
(indirect Coombs’ antigen on red
test) blood cell
 Particles Detect Positive reaction
Non Immune Red blood cell is the Antigen (virus) Agglutination
(viral) particle but it doesn’t
agglutination posses either antigen or
antibody
Viral Stage 1: viral particles Antibodies NO agglutination
hemagglutination incubated with sample. If it
inhibition contains antibodies, they
bind viral particles.
Stage 2: red blood cells are
added to the mixture.
Agglutination occurs if viral
particles havn’t bound to
antiviral antibodies assay in
the first stage.
 Direct agglutination
• Antigen is an intrinsic component of particle
(e.g., bacteria)
• Agglutination test is used to determine
whether antibody specific for the antigen, is
present in biological fluids.
• In case in which the particle is a red blood cell,
the agglutination reaction is reffered to as
hemagglutination
 Indirect agglutination
• Antigen has been affixed or absorbed to the
particle surface.
• Soluble antigen can serve as agglutinogen
when complexed to a particle.
• Particles used in indirect agglutination: red
blood cells, latex, charcoal, colloidal gold,
bentonite)
• To test patient serum for the presence of
antibodies, serum is mixed with latex spheres
with the soluble antigens attached.
• Antibodies will then cause visible
agglutination of the latex spheres with the
soluble antigens attached.
 Reverse agglutination
• Antibody is attached to the particle and
sample is tested for the presence of antigen.
 Latex particle agglutination inhibition
reaction
• Competitive binding assay
• Differs from assays based on direct/indirect
agglutination, agglutination indicates a
negative results
• It is two stage technique designed to detect
the presence of antigen.
• Stage 1: patient’s sample and antibodies
(provided in test kit) are incubated together
allowing formation of soluble antigen-
antibody complexes. When these complexes
form, the antibody Fab sites are no longer
available for binding antigen coated latex
particles. If no antigen present in sample, then
all Fab sites remain free of antigen and
available for binding antigen coated latex
particles.
• Stage 2: antigen coated latex particles are
added to microtiter wells and agglutination
occurs if the Fab sites are unoccupied.
therefore, agglutination denotes the absence
of antigen in sample.
 Viral agglutination/nonimmune
agglutination
• Many viruses have nonserological
hemagglutinating properties. They can
agglutinate red blood cells in the absence of
antibody. Agglutination by viruses is reffered
to viral hemagglutination. Because no
antibodies are required, it is classed as
nonimmune agglutination.
• Viral hemagglutination assay is performed in
V-bottom microtiter plated using dilutions of
attenuated viral particles and a constant
concentration of red blood cells.
• Viral agglutination manifests as adherence of
red blood cells to the bottom of V-shaped
well, which doesn’t flow when the plate is
tilted.
• For most part, titer of viral particles in body
fluids is not sufficient to detect in viral
hemagglutination assay.
• Antiviral antibodies however, may be present
in sufficient quantities and these can be
detected using viral hemagglutination
inhibition test.
Viral hemagglutination inhibition assay
• It is a competitive binding assay
• Used to detect anti-viral antibodies in serum
Viral antigen concentration determination
•Perform viral hemagglutination test in which viral
concentration is titrated
•The last well in which agglutination is observed is
given a value of one hemagglutinin unit
•The immediately previous wells are then reported
as having in sequence 2, 3, 4 units, respectively
•Four units of agglutination is the concentration
selected for use in viral hemagglutination inhibition
assay
Remove nonspecific inhibitory
 Stage 1
• Allow patient’s antibody bind to viral particles
and form immune complexes.
• Serially diluted serum samples using two fold
dilution in wells 2 through 12 of microtiter plate.
Add 4 units of attenuated virus in each well
• Cover and incubate on rotator for 1-2 hours to
allow antiviral antibodies bind to viral particles
• If antiviral antibodies are present in patents
serum, viral particles and antibodies will form
soluble complexes
 Stage 2
• Add red blood cells to each sample well and incubate
for 1-2 hours on rotator
• Viral particles that did not bind to antibodies are
available to bind red blood cells andcause
agglutination.
• In the absence of free viral particles, agglutination will
not occur so the amount of free particles available
depends on amount or absence of antiviral antibodies
present in patient’s serum
• Therefore, maximal agglutination is observed in a
negative sample
 Detection
• Agglutination is determined by viewing
microtiter plates from beneath by reflection in
the mirror
• Agglutination of red blood cells is visualized a
crosslinked layer of cells that settles
differently from unagglutinated cells
 Expected results
• Absence of agglutination is a positive result
indicating that all viral particles were bound
by antibody present in serum
• Endpoint hemagglutination titer is the
reciprocal of highest dilution of patient’s
serum that completely inhibits
hemagglutination
TERIMAKASIH