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.
 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
• Coagglutination and liposome-enhanced incubation of the patient’s specimen with
testing are variations of latex agglutination antihCG, followed by the addition of latex
(Fig. 10-3). Coagglutination uses antibodies particles coated with hCG. If hCG is
bound to a particle to enhance the visibility present, it neutralizes the antibody; thus, no
of agglutination. It is a highly specific agglutination of latex particles is seen. If no
method but may not be as sensitive as latex hCG is present, agglutination occurs
agglutination for detecting small quantities between the anti-hCG and hCG-coated
of antigen. latex particles.
PREGNANCY TESTING PREGNANCY LATEX SLIDE
• The principle of antigen and antibody AGGLUTINATION
interaction has been applied to pregnancy Principle
testing since the first agglutination tests • The rapid, direct, monoclonal latex slide
were developed in the 1960s. These assays agglutination test for detection of hCG is
have replaced animal testing. based on the principle of agglutination
Human Chorionic Gonadotropin between latex particles coated with anti-
• Pregnancy tests are designed to detect hCG antibodies and hCG, if present, in the
minute amounts of human chorionic test specimen.
gonadotropin (hCG), a glycoprotein Results
hormone secreted by the trophoblast of the • Agglutination within 2 minutes represents
developing embryo that rapidly increases in a positive reaction.
the urine or serum during the early stages of • No agglutination represents a negative
pregnancy. This glycoprotein hormone reaction.
consists of two noncovalently linked Technical Sources of Error
subunits, alpha (α) and beta (β). The α unit Reagents should never be expired; latex reagent
is identical to that found in luteinizing must be well shaken and agglutination should be
hormone (LH), follicle-stimulating read within 3 minutes to avoid erroneous results
hormone (FSH), and thyroid-stimulating caused by evaporation.
hormone (TSH). The β subunit has a unique False-Positive Results
carboxy-terminal region. Using antibodies • If a patient has been given an hCG injection
made against the β subunit will cut down on (e.g., Pregnyl) to trigger ovulation or
cross-reactivity with the other three lengthen the luteal phase of the menstrual
hormones. cycle, trace amounts can remain in the
• Accordingly, many pregnancy test kits patient’s system for as long as 10 days after
contain monoclonal antibody (MAb) the last injection. This will produce a false
 positive result. Two consecutive These particles are macroscopically or
quantitative hCG blood assays can microscopically visible only because the
circumvent this problem. If the hCG level precipitated product is forced to remain in a
increases by the second test, the patient is confined space.
probably pregnant. • Flocculation testing can be used in syphilis
• Chorioepithelioma, hydatidiform mole, or serologic testing. These tests are the classic
excessive ingestion of aspirin may give Venereal Disease Research Laboratories
false-positive results. (VDRL) and rapid plasma reagin (RPR)
• In men, a test identical to that used for tests. In the VDRL test, an antibody-like
pregnancy may be performed to detect the protein, reagin, binds to the test antigen,
presence of a testicular tumor. If Mab cardiolipin-lecithin–coated cholesterol
against the β subunit is not used, other particles, and produces the particles that
hormones with the same α unit may cross- flocculate. In the RPR test, the antigen,
react and cause a false-positive reaction. cardiolipin-lecithin–coated cholesterol with
• False-Negative Results choline chloride, also contains charcoal
• Testing before reaching detectable levels particles that allow for macroscopically
of hCG will yield false-negative results. visible flocculation.
DIRECT BACTERIAL AGGLUTINATION
Alternate Procedural Protocols • Direct agglutination of whole pathogens
• Latex agglutination slide tests have been can be used to detect antibodies directed
replaced in many situations (e.g., home against the pathogens. The most basic tests
testing; see Chapter 9) by one-step measure the antibody produced by the host
chromatographic color-labeled to antigen determinants on the surface of a
immunoassays for the qualitative bacterial agent in response to infection with
detection of hCG in urine (e.g., that bacterium. In a thick suspension of the
Clearview hCG II and Clearview hCG bacteria, the binding of specific antibodies
• Easy, Wampole Laboratories, Princeton, to surface antigens of the bacteria causes the
NJ). Another variation is a one-step bacteria to clump together in visible
chromatographic color-labeled aggregates. This type of agglutination is
immunoassay for use with urine or serum called bacterial agglutination.
(e.g., Wampole PreVue hCG Stick or
Cassette, Status hCG).

FLOCCULATION TESTS

• 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
• Flocculation tests for antibody detection
sterile physiologic saline with free positive
are based on the interaction of soluble
ions in the agglutination procedure
antigen with antibody, which results in the
enhances the aggregation of bacteria
formation of a precipitate of fine particles.
because most bacterial surfaces exhibit a
 negative charge that causes them to repel most tests involving erythrocyte antigens.
each other. Because it allows more time for Agglutination is influenced by a number of
the antigen-antibody reaction, tube testing factors and is believed to occur in two
is considered more sensitive than slide stages, sensitization and lattice formation.
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. Sensitization
• By binding different antigens to the RBC • The first phase of agglutination,
surface in indirect hemagglutination or sensitization, represents the physical
passive hemagglutination (PHA), the attachment of antibody molecules to
hemagglutination technique can be antigens on the erythrocyte membrane. The
extended to detect antibodies to antigens combination of antigen and antibody is a
other than those present on the cells (Box reversible chemical reaction. Altering the
10-2). physical conditions can result in the release
• Chemicals such as chromic chloride, tannic of antibody from theantigen-binding site.
acid, and glutaraldehyde can be used to When physical conditions are purposely
cross-link antigens to the cells. Some manipulated to break the antigen-antibody
antibodies (e.g., immunoglobulin G [IgG]) complex, with subsequent release of the
do not directly agglutinate erythrocytes. antibody into the surrounding medium, the
This incomplete or blocking type of procedure is referred to as an elution.
antibody may be detected by using an • The amount of antibody that will react is
enhancement medium such as antihuman affected by the equilibrium constant, or
globulin (AHG) reagent (also known as affinity constant, of the antibody. In most
Coombs reagent). If AHG reagent is added, cases, the higher the equilibrium
this second antibody binds to the antibody constant, the higher is the rate of
present on the erythrocytes 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:
Mechanisms of Agglutination
• Particle charge
• Agglutination is the clumping of particles
• Electrolyte concentration and viscosity
that have antigens on their surface, such as
• Antibody type
erythrocytes, by antibody molecules that
• Antigen-to-antibody ratio
form bridges between the antigenic
• Antigenic determinants
determinants. This is the end point for
 • Physical conditions (e.g., pH, visual observation of an antigenantibody
temperature, duration of incubation) reaction.
Antigen-Antibody Ratio.
• Inert particles such as latex, RBCs, and • Under conditions of antibody excess, there
bacteria have a net negative surface charge is a surplus of molecular antigen-combining
called the zeta potential. The concentration sites not bound to antigenic determinants.
of salt in the reaction medium has an effect Precipitation reactions depend on a zone of
on antibody uptake by the membrane bound equivalence, the zone in which optimum
erythrocyte antigens. Sodium (Na+) and precipitation occurs, because the number of
chloride (Cl−) ions in a solution have a multivalent sites of antigens and antibodies
shielding effect. These ions cluster around are approximately equal. For a precipitation
and partially neutralize the opposite charges reaction to be detectable, the reaction must
on antigen and antibody molecules, which occur in the zone of equivalence. In this
hinders antibody-antigen association. By zone, each antibody or antigen binds to
reducing the ionic strength of a reaction more than one antigen or antibody,
medium (e.g., using low ionic strength respectively, forming a stable lattice or
saline [LISS]), antibody uptake is network (see later). This lattice hypothesis
enhanced. Charges can be overcome by is based on the assumptions that each
centrifugation, addition of charged antibody molecule must have at least two
molecules (e.g., albumin, LISS), or enzyme binding sites and that an antigen must be
pretreatment to permit the cross-linking that multivalent.
results in agglutination
Precipitation Curve

Antibody Type.
• Immunoglobulin M (IgM) antibodies are
more efficient at agglutination because their
large size and multivalency permit more
Zone of Equivalence
effective bridging of the space between
• In the zone of equivalence, the number of
cells caused by zeta potential. IgG
multivalent sites of antigen and antibody
antibodies are too small to overcome
are approximately equal. In this zone,
electrostatic forces between cells. The use
precipitation is the result of random,
of AHG forms cross-links between
reversible reactions whereby each antibody
antibody molecules that have bound to the
binds to more than one antigen and vice
surface of RBCs. This promotes this
versa, forming a stable network or lattice.
formation of agglutination and allows for
 The lattice hypothesis, as formulated by antibody and performing the test again
Marrack, is based on the assumptions that may produce a positive result.
each antibody molecule must have at least • In the postzone, excess antigen may
two binding sites and the antigen must be obscure the presence of a small amount
multivalent. As they combine, this of antibody. Typically, such a test is
arrangement results in a multimolecular repeated with an additional patient
lattice that increases in size until it specimen taken about a week later. The
precipitates out of solution. extra time would allow for the further
• As illustrated by the precipitin curve production of antibody. If the repeated
shown in, when the same amount of test is negative, it is unlikely that the
soluble antigen is added to increasing patient has thatnparticular antibody.
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 To correct the postzone phenomenon, a repeat
occurs, in which antigen combines with blood specimen should be collected 1 or more
only one or two antibody molecules and no weeks later. If an active antibody
cross-linkages are formed. In the prozone, reaction is occurring in vivo, the titer of antibody
usually only one site on an antibody will increase and should be detectable. Repeated
molecule is used and many free antibody negative results generally suggest that the patient
molecules remain in solution. has the specific antibody being tested for by the
• This phenomenon can be overcome by procedure.
serially diluting the antibody-containing Antigenic Determinants.
serum until optimum amounts of antigen The placement and number of antigenic
and antibody are present in the test system. determinants both affect agglutination. For
• At the other side of the zone, where there is example, the A blood group antigen has more
antigen excess, the postzone phenomenon than 1.5 million sites/RBC, whereas the Kell
occurs in which small aggregates are blood group antigen has about 3500 to 6000
surrounded by excess antigen. Again, no sites/RBC. If the number of antigenic sites is
lattice network is formed. In this case, every small or if the antigenic sites are buried deeply in
available antibody site is bound to a single the cell membranes, antibodies will be unable
antigen and no cross-links are formed. physically to contact antigenic sites.
Thus, for precipitation reactions to be • Steric hindrance is an important
detectable, they must be carried out in the physiochemical effect that influences
zone of equivalence. antibody uptake by cell surface antigens. If
• The prozone and postzone phenomena dissimilar antibodies with approximately
must be considered in the clinical setting the same binding constant are directed
because negative reactions occur in both. against antigenic determinants located close
• A false-negative reaction may take place to each other, the antibodies will compete
in the prozone because of high antibody for space in reaching their specific receptor
concentration. If it is suspected that the sites. The effect of this competition can be
reaction is a false negative, diluting out mutual blocking, or steric hindrance, and
 neither antibody type will be bound to its linking is influenced by factors such as the
respective antigenic determinant. zeta potential.
• Steric hindrance can occur whenever there • Methods of Enhancing Agglutination
is a conformational change in the • Techniques used to enhance
relationship of an antigenic receptor site to agglutination include the
the outside surface. In addition to antibody • following:
competition, competition with bound • Centrifugation
complement, other protein molecules, or the • Treatment with proteolytic enzymes
action of agents that interfere with the • Use of colloids
structural integrity of the cell surface can • AHG testing
produce steric hindrance. • Treatment with proteolytic enzymes and
pH. the use of colloids or AHG techniques
• The pH of the medium used for testing could be applied in the immunology
should be near physiologic conditions, or an laboratory. Centrifugation attempts to
optimum pH of 6.5 to 7.5. At a neutral pH, overcome the problem of distance by
high electrolyte concentrations act to subjecting sensitized cells to a high
neutralize the net negative charge of gravitational force that counteracts the
particles. repulsive effect and physically forces the
Temperature and Length of Incubation. cells together
• The optimum temperature needed to reach • Enzyme treatment alters the zeta
equilibrium in an antibody-antigen reaction potential or dielectric constant to enhance
differs for different antibodies. IgM the chances of demonstrable
antibodies are cold-reacting (thermal range, agglutination. Mild proteolytic enzyme
4° C to 22° C [39° F to 72° F]), and IgG treatment can strip off some of the
antibodies are warm-reacting, with an negative charges on the cell membrane
optimum temperature of reaction at 37° C by removing surface sialic acid residues
(98.6° F). The duration of incubation (cleaving sialoglycoproteins from the
required to achieve maximum results cell surface), which reduces the surface
depends on the rate of association and charge of cells, lowers the zeta potential,
dissociation of each specific antibody. In and permits cells to come closer together
laboratory testing, incubation times range for chemical linking by specific antibody
from 15 to 60 minutes. The optimum time molecules.
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
appearance of clumping, may rarely occur
the antibody molecules to bridge the gap
because of rouleaux formation. Rouleaux
and combine with the antigen receptor site
formation can be encountered inpatients
on thesecond cell. As antigens and
with high or abnormal types of globulins in
antibodies combine, a multi molecular
their blood, such as in multiple myeloma or
lattice increases in size until it precipitates
out of solution as a solid particle. Cross-
 after receiving dextran as a plasma
expander.
• On microscopic examination, the
erythrocytes appear as rolls resembling Microplate Agglutination Reactions
stacks of coins. To disperse the • Serologic testing has usually been
pseudoagglutination, a few drops of performed by slide or test tube techniques,
physiologic NaCl (saline) can be added to but the increased emphasis on cost
the reaction tube, remixed, and reexamined. containment has stimulated interest in
This procedure, saline replacement, should microtechniques as an alternative to
be performed carefully after conventional methods. Micromethods for
pseudoagglutination is suspected. It should RBC antigen and antibody testing include
never be done before the initial testing hemagglutination and solid-phase
protocol is followed; a false-negative result adherence assays. These methods are also
may occur from the dilutional effect of the considered to be easier to perform. The use
saline. 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