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Agglutination
Serology and Immunology
Agglutination
• is the aggregation of particulate matter caused
by the combination with specific antibody
• 1896: First observed by Gruber and Durham
when serum antibody was found to react with
bacterial cells
Agglutination
• Agglutinins
▫ Antibodies that produce such reactions
• Involves two-step process:
▫ Sensitization or initial binding
▫ Lattice formation or formation of large
aggregates
Agglutination
• Types of particles that participate
in such reactions:
▫ Erythrocytes
▫ Bacterial cells
▫ Inert carriers such as latex particles
Steps in Agglutination
• Primary phenomenon
(SENSITIZATION)
 First reaction involving Ag-Ab combination
 Single antigenic determinant on the surface
particle
1) Initial reaction: rapid and reversible
2) Cross link formation  visible aggregates
(stabilization)
SENSITIZATION
Secondary phenomenon:
LATTICE FORMATION
▫ Ab + multivalent Ag  stable network
(visible reaction)
▫ conc. of Ag and Ab
▫ Governed by physiochemical factors:
 Ionic strength of milieu
 pH
 temperature
Secondary Phenomenon
• Lattice Formation
• The Fab portion of the Ig molecule attaches to
antigens on 2 adjacent cells-visible results in
agglutination
• If both antigen and antibody are SOLUBLE reaction
will become visible over time, ie, precipitation
• Antibody:
▫ Complete
 primary and secondary interactions  visible agg
▫ Incomplete
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Non-agglutinating  no visible aggregation
Thought to have one active site
Now known: two active sites  no lattice formed
IgG class (small size = ↓flexibility of hinge region)
HEMAGGLUTINATION
• Detects antibody to erythrocyte antigens
▫ sufficient concentration of antibody present->
antibody cross-link= agglutination
▫ non-reactive/insufficient antibody present= no
agglutination
• Binding different antigens on the RBC surface =
detect antibodies to antigen other than those
present in the cells
HEMAGGLUTINATION
• chromic chloride, tannic acid, and
glutaraldehyde= cross- link antigens to the cell
• IgG (does not agglutinate directly)-> need
enhancement medium-> AHG
• AHG binds to the second antibody present on
the erythrocyte.
DIRECT AGGLUTINATION
-test patient serum against large, cellular antigens
to screen for the presence of antibodies.
• Antigen is naturally present on the surface of the
cells.
• In this case, the Ag-Ab reaction forms an
agglutination, which is directly visible.
DIRECT AGGLUTINATION
• The particle antigen may be a bacterium.
e.g.: Serotyping of E. coli, Salmonella using a
specific antiserum
• The particle antigen may be a parasite.
e.g.: Serodiagnosis of Toxoplasmosis
• The particle antigen may be a red blood cell.
e.g.: Determination of blood groups
DIRECT AGGLUTINATION
• These reactions can be performed on slides
(rapid tests) or on microliter plates or tubes for
Antibody titration if required.
Passive Agglutination
• An agglutination reaction that employs particles
that are coated with antigens not normally found
in the cell surfaces
• Particle carriers include:
▫
▫
▫
▫
Red blood cells
Polystyrene latex
Bentonite
charcoal
Passive Agglutination
• Passive agglutination has been used in the
detection of :
▫
▫
▫
▫
Rheumatoid factor
Antinuclear antibody in LE
Ab to group A streptococcus antigens
Ab to Trichinella spiralis
The latex particles are coated with IgG and mixed
with the patient's serum
REVERSE PASSIVE
• Antibody rather than antigen is attached to a
carrier particle
• For the detection of microbial antigens such
as:
▫
▫
▫
▫
▫
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Group A and B streptococcus
Staphylococcus aureus
Neisseria meningitidis
Haemophilus influenzae
Rotavirus
Cryptococcus neoformans
Mycoplasma pneumoniae
Candida albicans
REVERSE PASSIVE
• PRINCIPLE: latex particles coated with antibody
are reacted with a patient sample containing
suspected antigen
Agglutination Inhibition
• Based on the competition between particulate and
soluble antigens for limited antibody combining site
• Lack of agglutination is indicator of a positive reaction
• Usually involves haptens complexed with proteins
Agglutination Inhibition
• Pregnancy Testing
-classic example of agglutination inhibition
▫ Human chorionic gonadotropin (hCG)
 Appears in serum and urine early in pregnancy
Agglutination Inhibition
Urine
No hCG in urine:
Anti-hCG free
Carriers coated with hCG added
AGGLUTINATION of carriers:
Negative test for hCG
NOT PREGNANT
Antiserum
hCG in urine:
Anti-hCG neutralized
Carriers coated with hCG added
NO AGGLUTINATION of carriers:
Positive test for hCG
PREGNANT
Coagglutination
• Name given to systems using inert bacteria as
the inert particles to which the antibody is
attached
• S.aureus: most frequently used because it has
protein A in its outer surface that naturally
adsorbs the Fc portion of the antibody
• Highly specific but not very sensitive in
detecting small quantities of antigen
Reading/Grading Agglutination
Reactions
• done by gently shaking the tubes containing the
serum and cells, and observing the cell button as
it is dispersed
• Hard shaking must be avoided because this may
yield to false result
• Attention should also be given to whether
discoloration of the supernatant is present
(Hemolysis).
Latex Agglutination
• Antibody molecules can be bound to each latex beads
• It will increase the potential number of exposed antigenbinding sites.
• When an antigen is present in test specimen, it may bind
to the latex bead thus forming visible cross-linked
aggregates.
• Latex particles can be coated with antigen (pregnancy
testing, rubella antibody testing)
Coagulation and Liposome-enhanced
testing
• Are variations of latex agglutination
• uses antibodies bound to a particle to enhance the
visibility of agglutination
• is a highly specific method but may not be sensitive.
Pregnancy Testing
• Designed to detect minute amounts of hCG.
• Based on the principle of agglutination between
latex particles coated with anti-hCG antibodies
and hCG, if present.
False-Positive Result
• If injected with hCG to trigger ovulation or to
lengthen luteal phase of menstrual cycle.
• Chorioepithelioma, hydatidiform mole or
ingestion of aspirin
• To detect the presence of a testicular tumor in
men
False Negative
• Testing before reaching detectable levels of hCG.
Reading/Grading Agglutination
Reactions
• Rupture or hemolysis of red blood cells is as
important as finding agglutination
• The strength of agglutination is graded from 0
(negative or no agglutination) to 4+
(agglutination or all erythrocytes clumped).
Reading/Grading Agglutination
Reactions
• Pseudoagglutination or the Rouleaux Formation
also occurs
▫ Red blood cells appear as stacks of coins.
• Addition of physiologic NaCl will disperse
pseudoagglutination
• Saline Replacement is done after
pseudoagglutination is observed so that it may
not give false negative result due to the dilution
effect of the saline
GRADING AGGLUTINATION REACTIONS
GRADE
DESCRIPTION
Negative (-)
No aggregates
Weak (+/-)
Tiny aggregates that are
barely visible
macroscopically; turbid
and reddish supernatant
1+
A few small aggregates
just visible
macroscopically; turbid
and reddish supernatant
Appearance
2+
Medium-sized aggregates;
clear supernatant
3+
Several large aggregates; clear
supernatant
4+
One solid aggregate; clear
supernatant