Agglutination Method

PRINCIPLES OF AGGLUTINATION
• Precipitation and agglutination are the
visible expression of the aggregation of
antigens and antibodies through the
formation of a framework in which antigen
particles or molecules alternate with
antibody molecules
• Precipitation and agglutination are
considered unlabeled assays because a
marker label is not needed to detect the
reaction.
• Precipitation is the term for the
aggregation of soluble test antigens. Artificial carrier particles may be needed to
Precipitation is the combination of indicate visibly that an antigen-antibody
soluble antigen with soluble antibody to reaction has taken place; examples include latex
produce a visible insoluble complex. particles and colloidal charcoal. Cells unrelated
• It involves combining soluble antigen to the antigen, such as erythrocytes coated with
with soluble antibody to produce antigen in a constant amount, can be used as
insoluble complexes that are visible. biological carriers. Whole bacterial cells can
contain an antigen that will bind with antibodies
produced in response to that antigen when it is
introduced into the host

• Agglutination is the process whereby

specific antigens (e.g., red blood cells)
aggregate to form larger visible clumps
when the corresponding specific antibody is
present in the serum.
• It is the process by which particulate
antigens such as cells aggregate to form
larger complexes when a specific antibody The quality of test results depends on the
is present. following technical factors:
• Time of incubation with the antibody
source (e.g patient
• Amount and avidity of an antigen
conjugated to the carrier
• Conditions of the test environment (e.g.,
pH, protein concentration) serum
 LATEX AGGLUTINATION
• In latex agglutination procedures antibody
molecules can be bound to the surface of
latex beads. Many antibody molecules can
be bound to each latex particle, increasing
the potential number of exposed antigen-
binding sites.
• If an antigen is present in a test specimen
such as C-reactive protein, the antigen will
bind to the combining sites of the antibody
exposed on the surface of the latex beads,
forming visible cross-linked aggregates of
latex beads and antigen.
• In some procedures (e.g., pregnancy testing,
rubella antibody testing), latex particles can
be coated with antigen. In the presence of
serum antibodies, these particles
agglutinate into large visible clumps.

• Coagglutination and liposome-enhanced
testing are variations of latex agglutination
(Fig. 10-3). Coagglutination uses antibodies
bound to a particle to enhance the visibility
of agglutination. It is a highly specific
method but may not be as sensitive as latex
agglutination for detecting small quantities
of antigen.
PREGNANCY TESTING
• The principle of antigen and antibody
interaction has been applied to pregnancy
testing since the first agglutination tests
were developed in the 1960s. These assays
have replaced animal testing.
Human Chorionic Gonadotropin
• Pregnancy tests are designed to detect
minute amounts of human chorionic
gonadotropin (hCG), a glycoprotein
hormone secreted by the trophoblast of the
developing embryo that rapidly increases in
the urine or serum during the early stages of
pregnancy. This glycoprotein hormone
consists of two noncovalently linked
subunits, alpha (α) and beta (β). The α unit
is identical to that found in luteinizing
hormone (LH), follicle-stimulating
hormone (FSH), and thyroid-stimulating
hormone (TSH). The β subunit has a unique
carboxy-terminal region. Using antibodies
made against the β subunit will cut down on
cross-reactivity with the other three
hormones.
• Accordingly, many pregnancy test kits
contain monoclonal antibody (MAb)
directed against the β subunit to increase the
specificity of the reaction.
• For the first 6 to 8 weeks after conception,
hCG helps maintain the corpus luteum and
stimulate the production of progesterone.
As a general rule, the level of hCG should
double every 2 to 3 days. Pregnant women
usually attain serum concentrations of 10 to
50 mIU/mL of hCG in the week after
conception. If a test is negative at this stage,
the test should be repeated within a week.
Peak levels are reached approximately 2 to
3 months after the last menstrual period
(LMP).
Agglutination Inhibition
• The determination of in vitro
agglutination inhibition depends on the
incubation of the patient’s specimen with
antihCG, followed by the addition of latex
particles coated with hCG. If hCG is
present, it neutralizes the antibody; thus, no
agglutination of latex particles is seen. If no
hCG is present, agglutination occurs
between the anti-hCG and hCG-coated
latex particles.
PREGNANCY LATEX SLIDE
AGGLUTINATION
Principle
• The rapid, direct, monoclonal latex slide
agglutination test for detection of hCG is
based on the principle of agglutination
between latex particles coated with anti-
hCG antibodies and hCG, if present, in the
test specimen.
Results
• Agglutination within 2 minutes represents
a positive reaction.
• No agglutination represents a negative
reaction.
Technical Sources of Error
Reagents should never be expired; latex reagent
must be well shaken and agglutination should be
read within 3 minutes to avoid erroneous results
caused by evaporation.
False-Positive Results
• If a patient has been given an hCG injection
(e.g., Pregnyl) to trigger ovulation or
lengthen the luteal phase of the menstrual
cycle, trace amounts can remain in the
patient’s system for as long as 10 days after
the last injection. This will produce a false
 positive result. Two consecutive
quantitative hCG blood assays can
circumvent this problem. If the hCG level
increases by the second test, the patient is
probably pregnant.
• Chorioepithelioma, hydatidiform mole, or
excessive ingestion of aspirin may give
false-positive results.
• In men, a test identical to that used for
pregnancy may be performed to detect the
presence of a testicular tumor. If Mab
against the β subunit is not used, other
hormones with the same α unit may cross-
react and cause a false-positive reaction.
• False-Negative Results
• Testing before reaching detectable levels
of hCG will yield false-negative results.
Alternate Procedural Protocols
• Latex agglutination slide tests have been
replaced in many situations (e.g., home
testing; see Chapter 9) by one-step
chromatographic color-labeled
immunoassays for the qualitative
detection of hCG in urine (e.g.,
Clearview hCG II and Clearview hCG
• Easy, Wampole Laboratories, Princeton,
NJ). Another variation is a one-step
chromatographic color-labeled
immunoassay for use with urine or serum
(e.g., Wampole PreVue hCG Stick or
Cassette, Status hCG).
FLOCCULATION TESTS
• Flocculation tests for antibody detection
are based on the interaction of soluble
antigen with antibody, which results in the
formation of a precipitate of fine particles.
These particles are macroscopically or
microscopically visible only because the
precipitated product is forced to remain in a
confined space.
• Flocculation testing can be used in syphilis
serologic testing. These tests are the classic
Venereal Disease Research Laboratories
(VDRL) and rapid plasma reagin (RPR)
tests. In the VDRL test, an antibody-like
protein, reagin, binds to the test antigen,
cardiolipin-lecithin–coated cholesterol
particles, and produces the particles that
flocculate. In the RPR test, the antigen,
cardiolipin-lecithin–coated cholesterol with
choline chloride, also contains charcoal
particles that allow for macroscopically
visible flocculation.
DIRECT BACTERIAL AGGLUTINATION
• Direct agglutination of whole pathogens
can be used to detect antibodies directed
against the pathogens. The most basic tests
measure the antibody produced by the host
to antigen determinants on the surface of a
bacterial agent in response to infection with
that bacterium. In a thick suspension of the
bacteria, the binding of specific antibodies
to surface antigens of the bacteria causes the
bacteria to clump together in visible
aggregates. This type of agglutination is
called bacterial agglutination.
• The formation of aggregates in solution is
influenced by electrostatic and other forces;
therefore, certain conditions are usually
necessary for satisfactory results. The use of
sterile physiologic saline with free positive
ions in the agglutination procedure
enhances the aggregation of bacteria
because most bacterial surfaces exhibit a
 negative charge that causes them to repel
each other. Because it allows more time for
the antigen-antibody reaction, tube testing
is considered more sensitive than slide
testing. The small volume of liquid used in
slide testing requires rapid reading before
the liquid evaporates.
HEMAGGLUTINATION
• The hemagglutination method of testing
detects antibodies to erythrocyte antigens.
The antibody-containing specimen can be
serially diluted and a suspension of red
blood cells (RBCs) added to the dilutions. If
a sufficient concentration of antibody is
present, the erythrocytes are cross-linked
and agglutinated. If non reacting antibody
or an insufficient quantity of antibody is
present, the erythrocytes will fail to
agglutinate.
• By binding different antigens to the RBC
surface in indirect hemagglutination or
passive hemagglutination (PHA), the
hemagglutination technique can be
extended to detect antibodies to antigens
other than those present on the cells (Box
10-2).
• Chemicals such as chromic chloride, tannic
acid, and glutaraldehyde can be used to
cross-link antigens to the cells. Some
antibodies (e.g., immunoglobulin G [IgG])
do not directly agglutinate erythrocytes.
This incomplete or blocking type of
antibody may be detected by using an
enhancement medium such as antihuman
globulin (AHG) reagent (also known as
Coombs reagent). If AHG reagent is added,
this second antibody binds to the antibody
present on the erythrocytes
Mechanisms of Agglutination
• Agglutination is the clumping of particles
that have antigens on their surface, such as
erythrocytes, by antibody molecules that
form bridges between the antigenic
determinants. This is the end point for
most tests involving erythrocyte antigens.
Agglutination is influenced by a number of
factors and is believed to occur in two
stages, sensitization and lattice formation.
Sensitization
• The first phase of agglutination,
sensitization, represents the physical
attachment of antibody molecules to
antigens on the erythrocyte membrane. The
combination of antigen and antibody is a
reversible chemical reaction. Altering the
physical conditions can result in the release
of antibody from theantigen-binding site.
When physical conditions are purposely
manipulated to break the antigen-antibody
complex, with subsequent release of the
antibody into the surrounding medium, the
procedure is referred to as an elution.
• The amount of antibody that will react is
affected by the equilibrium constant, or
affinity constant, of the antibody. In most
cases, the higher the equilibrium
constant, the higher is the rate of
association and the slower the rate of
dissociation of antibody molecules. The
degree of association between antigen
and antibody is affected by a variety of
factors and can be altered in some cases
in vitro by altering some of the factors
that influence antigen-antibody
association, including the following:
• Particle charge
• Electrolyte concentration and viscosity
• Antibody type
• Antigen-to-antibody ratio
• Antigenic determinants
 • Physical conditions (e.g., pH,
temperature, duration of incubation)
• Inert particles such as latex, RBCs, and
bacteria have a net negative surface charge
called the zeta potential. The concentration
of salt in the reaction medium has an effect
on antibody uptake by the membrane bound
erythrocyte antigens. Sodium (Na+) and
chloride (Cl−) ions in a solution have a
shielding effect. These ions cluster around
and partially neutralize the opposite charges
on antigen and antibody molecules, which
hinders antibody-antigen association. By
reducing the ionic strength of a reaction
medium (e.g., using low ionic strength
saline [LISS]), antibody uptake is
enhanced. Charges can be overcome by
centrifugation, addition of charged
molecules (e.g., albumin, LISS), or enzyme
pretreatment to permit the cross-linking that
results in agglutination
Precipitation Curve
Antibody Type.
• Immunoglobulin M (IgM) antibodies are
more efficient at agglutination because their
large size and multivalency permit more
effective bridging of the space between
cells caused by zeta potential. IgG
antibodies are too small to overcome
electrostatic forces between cells. The use
of AHG forms cross-links between
antibody molecules that have bound to the
surface of RBCs. This promotes this
formation of agglutination and allows for
visual observation of an antigenantibody
reaction.
Antigen-Antibody Ratio.
• Under conditions of antibody excess, there
is a surplus of molecular antigen-combining
sites not bound to antigenic determinants.
Precipitation reactions depend on a zone of
equivalence, the zone in which optimum
precipitation occurs, because the number of
multivalent sites of antigens and antibodies
are approximately equal. For a precipitation
reaction to be detectable, the reaction must
occur in the zone of equivalence. In this
zone, each antibody or antigen binds to
more than one antigen or antibody,
respectively, forming a stable lattice or
network (see later). This lattice hypothesis
is based on the assumptions that each
antibody molecule must have at least two
binding sites and that an antigen must be
multivalent.
Zone of Equivalence
• In the zone of equivalence, the number of
multivalent sites of antigen and antibody
are approximately equal. In this zone,
precipitation is the result of random,
reversible reactions whereby each antibody
binds to more than one antigen and vice
versa, forming a stable network or lattice.
 The lattice hypothesis, as formulated by
Marrack, is based on the assumptions that
each antibody molecule must have at least
two binding sites and the antigen must be
multivalent. As they combine, this
arrangement results in a multimolecular
lattice that increases in size until it
precipitates out of solution.
• As illustrated by the precipitin curve
shown in, when the same amount of
soluble antigen is added to increasing
dilutions of antibody, the amount of
precipitation increases up to the zone of
equivalence. When the amount of antigen
overwhelms the number of antibody-
combining sites present, precipitation
begins to decline because fewer lattice
networks are formed.
Prozone and Postzone
• As can be seen on the precipitin curve,
precipitation declines on either side of the
equivalence zone because of an excess of
either antigen or antibody. In the case of
antibody excess, the prozone phenomenon
occurs, in which antigen combines with
only one or two antibody molecules and no
cross-linkages are formed. In the prozone,
usually only one site on an antibody
molecule is used and many free antibody
molecules remain in solution.
• This phenomenon can be overcome by
serially diluting the antibody-containing
serum until optimum amounts of antigen
and antibody are present in the test system.
• At the other side of the zone, where there is
antigen excess, the postzone phenomenon
occurs in which small aggregates are
surrounded by excess antigen. Again, no
lattice network is formed. In this case, every
available antibody site is bound to a single
antigen and no cross-links are formed.
Thus, for precipitation reactions to be
detectable, they must be carried out in the
zone of equivalence.
• The prozone and postzone phenomena
must be considered in the clinical setting
because negative reactions occur in both.
• A false-negative reaction may take place
in the prozone because of high antibody
concentration. If it is suspected that the
reaction is a false negative, diluting out
antibody and performing the test again
may produce a positive result.
• In the postzone, excess antigen may
obscure the presence of a small amount
of antibody. Typically, such a test is
repeated with an additional patient
specimen taken about a week later. The
extra time would allow for the further
production of antibody. If the repeated
test is negative, it is unlikely that the
patient has thatnparticular antibody.
To correct the postzone phenomenon, a repeat
blood specimen should be collected 1 or more
weeks later. If an active antibody
reaction is occurring in vivo, the titer of antibody
will increase and should be detectable. Repeated
negative results generally suggest that the patient
has the specific antibody being tested for by the
procedure.
Antigenic Determinants.
The placement and number of antigenic
determinants both affect agglutination. For
example, the A blood group antigen has more
than 1.5 million sites/RBC, whereas the Kell
blood group antigen has about 3500 to 6000
sites/RBC. If the number of antigenic sites is
small or if the antigenic sites are buried deeply in
the cell membranes, antibodies will be unable
physically to contact antigenic sites.
• Steric hindrance is an important
physiochemical effect that influences
antibody uptake by cell surface antigens. If
dissimilar antibodies with approximately
the same binding constant are directed
against antigenic determinants located close
to each other, the antibodies will compete
for space in reaching their specific receptor
sites. The effect of this competition can be
mutual blocking, or steric hindrance, and
 neither antibody type will be bound to its
respective antigenic determinant.
• Steric hindrance can occur whenever there
is a conformational change in the
relationship of an antigenic receptor site to
the outside surface. In addition to antibody
competition, competition with bound
complement, other protein molecules, or the
action of agents that interfere with the
structural integrity of the cell surface can
produce steric hindrance.
pH.
• The pH of the medium used for testing
should be near physiologic conditions, or an
optimum pH of 6.5 to 7.5. At a neutral pH,
high electrolyte concentrations act to
neutralize the net negative charge of
particles.
Temperature and Length of Incubation.
• The optimum temperature needed to reach
equilibrium in an antibody-antigen reaction
differs for different antibodies. IgM
antibodies are cold-reacting (thermal range,
4° C to 22° C [39° F to 72° F]), and IgG
antibodies are warm-reacting, with an
optimum temperature of reaction at 37° C
(98.6° F). The duration of incubation
required to achieve maximum results
depends on the rate of association and
dissociation of each specific antibody. In
laboratory testing, incubation times range
from 15 to 60 minutes. The optimum time
of incubation varies, depending on the class
of immunoglobulin and how tightly an
antibody attaches to its specific antigen.
LATTICE FORMATION
• Lattice formation, or the establishment of
cross-links between sensitized particles
(e.g., erythrocytes) and antibodies, resulting
in aggregation, is a much slower process
than the sensitization phase. The formation
of chemical bonds and resultant lattice
formation depend on the ability of a cell
with attached antibody on its surface to
• Pseudoagglutination, or the false
come close enough to another cell to permit
the antibody molecules to bridge the gap
and combine with the antigen receptor site
on thesecond cell. As antigens and
antibodies combine, a multi molecular
lattice increases in size until it precipitates
out of solution as a solid particle. Cross-
linking is influenced by factors such as the
zeta potential.
• Methods of Enhancing Agglutination
• Techniques used to enhance
agglutination include the
• following:
• Centrifugation
• Treatment with proteolytic enzymes
• Use of colloids
• AHG testing
• Treatment with proteolytic enzymes and
the use of colloids or AHG techniques
could be applied in the immunology
laboratory. Centrifugation attempts to
overcome the problem of distance by
subjecting sensitized cells to a high
gravitational force that counteracts the
repulsive effect and physically forces the
cells together
• Enzyme treatment alters the zeta
potential or dielectric constant to enhance
the chances of demonstrable
agglutination. Mild proteolytic enzyme
treatment can strip off some of the
negative charges on the cell membrane
by removing surface sialic acid residues
(cleaving sialoglycoproteins from the
cell surface), which reduces the surface
charge of cells, lowers the zeta potential,
and permits cells to come closer together
for chemical linking by specific antibody
molecules.
appearance of clumping, may rarely occur
because of rouleaux formation. Rouleaux
formation can be encountered inpatients
with high or abnormal types of globulins in
their blood, such as in multiple myeloma or
 after receiving dextran as a plasma
expander.
• On microscopic examination, the
erythrocytes appear as rolls resembling
stacks of coins. To disperse the
pseudoagglutination, a few drops of
physiologic NaCl (saline) can be added to
the reaction tube, remixed, and reexamined.
This procedure, saline replacement, should
be performed carefully after
pseudoagglutination is suspected. It should
never be done before the initial testing
protocol is followed; a false-negative result
may occur from the dilutional effect of the
saline.
Microplate Agglutination Reactions
• Serologic testing has usually been
performed by slide or test tube techniques,
but the increased emphasis on cost
containment has stimulated interest in
microtechniques as an alternative to
conventional methods. Micromethods for
RBC antigen and antibody testing include
hemagglutination and solid-phase
adherence assays. These methods are also
considered to be easier to perform. The use
of microplates allows for the performance
of a large number of tests on a single plate,
which eliminates time-consuming steps
such as labeling test tubes

A microplate is a compact plate of rigid or

flexible plastic with multiple wells. The wells
may be U-shaped or have a flat bottom
configuration. The U-shaped well has been used
most often in immunohematology. The volume
capacity of each well is approximately 0.2 mL,
which prevents spilling during mixing. Samples
and reagents are dispensed with small-bore
Pasteur pipettes. These pipettes are recommended
because they deliver 0.025 mL, which prevents
splashing. After the specimens and reagents are
added to the wells, they are mixed by gentle
agitation of the plates. The microplate is then
centrifuged for an immediate reading