PRECIPITATION AND AGGLUTINATION antigen resembles the original antigen, the stronger the bond of antigen and antibody
er the bond of antigen and antibody present. Optimum precipitation
REACTIONS
The combination of an antigen with a specific antibody
plays an important role in the laboratory in diagnosing
many different diseases. Immunoassays have been
developed to detect either antigen or antibody and vary
from easily performed manual tests to highly complex
automated assays. The first such assays were based on the
principles of precipitation or agglutination. Precipitation
involves combining soluble antigen with soluble antibody to
produce insoluble complexes that are visible. Agglutination
is the process by which particulate antigens such as cells
aggregate to form larger complexes when a specific
antibody is present. Precipitation and agglutination are
considered unlabeled assays because a marker label is not
needed to detect the reaction. Labeled assays, which were
developed much later, will be considered in Chapter 11.
Precipitation was first noted in 1897 by Kraus,
who found that culture filtrates of enteric bacteria would
precipitate when they were mixed with specific antibodies.
For such reactions to occur, both the antigen and antibody
must have multiple binding sites for one another and the
relative concentration of each must be equal. Binding
characteristics of antibodies, called affinity and avidity, also
play a major role.
I. Antigen-Antibody Binding
The primary union of binding sites on an antibody with
specific epitopes on an antigen depends on two
characteristics of antibody known as affinity and avidity.
Such characteristics are important because they relate to the
sensitivity and specificity of testing in the clinical
laboratory.
Affinity
Affinity is the initial force of attraction that exists between
a single Fab site on an antibody molecule and a single
epitope or determinant site on the corresponding antigen. As
the epitope and binding site come into close proximity to
each other, they are held together by rather weak bonds
occurring only over a short distance of approximately 1 x
10–7 mm. The strength of attraction depends on the
specificity of antibody for a particular antigen. One
antibody molecule may initially attract numerous different
antigens, but it is the epitope’s shape and the way it fits
together with the binding sites on an antibody molecule that
determines whether the bonding will be stable. Antibodies
are capable of reacting with antigens resembling the
original antigen that induced antibody production, which is
known as cross-reactivity. The more the cross-reacting
will be between the antigen and the binding site. However,
if the epitope and the binding site have a perfect lock-and-
key fit, as is the case with the original antigen, the affinity
will be maximal. When the affinity is higher, the assay
reaction is more sensitive because more antigen–antibody
complexes will be formed and visualized more easily.
Avidity
Avidity represents the overall strength of antigen–antibody
binding and is the sum of the affinities of all the individual
antibody–antigen combining sites. Avidity refers to the
strength with which a multivalent antibody binds a
multivalent antigen and is a measure of the overall stability
of an antigen–antibody complex. In other words, once
binding has occurred, it is the force that keeps the molecules
together. A high avidity can actually compensate for a low
affinity. Different classes of antibodies actually differ in
avidities. The more bonds that form between antigen and
antibody, the higher the avidity is. IgM, for instance, has a
higher avidity than IgG because IgM has the potential to
bind 10 different antigens. Both affinity and avidity
contribute to the stability of the antigen–antibody
complexes, which is essential to detecting the presence of
an unknown, whether it is antigen or antibody.
Law of Mass Action
All antigen–antibody binding is reversible and is governed
by the law of mass action. This law states that free
reactants are in equilibrium with bound reactants. The
equilibrium constant K represents the difference in the rates
of the forward and reverse reactions according to the
following equation: K = [AgAb] / [Ab] [Ag]
The value of K depends on the strength of binding
between antibody and antigen. As the strength of binding,
or avidity, increases, the tendency of the antigen–antibody
complexes to dissociate decreases, which increases the
value of K. When the value of K is higher, the amount of
antigen–antibody complex is larger and the assay reaction is
more visible or easily detectable.
The ideal conditions in the clinical laboratory would be to
have an antibody with a high affinity, or initial force of
attraction, and a high avidity, or strength of binding. The
higher the values are for both of these and the more
antigen–antibody complexes that are formed, the more
sensitive the test.
II. Precipitation Curve
In addition to the affinity and avidity of the antibody
involved, precipitation depends on the relative proportions
occurs in the zone of equivalence.
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, 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
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.
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 that particular
antibody.
III. Principles of Agglutination Reaction
Whereas precipitation reactions involve soluble antigens,
agglutination is the visible aggregation of particles caused
by combination with specific antibody. Antibodies that
produce such reactions are often called agglutinins.
Because this reaction takes place on the surface of the
particle, antigen must be exposed and able to bind with
antibody. Types of particles participating in such reactions
include erythrocytes, bacterial cells, and inert carriers such
as latex particles. Each particle must have multiple
antigenic or determinant sites, which are cross-linked to
sites on other particles through the formation of antibody
bridges.
In 1896, Gruber and Durham published the first
report about the ability of antibody to clump cells, based on
observations of agglutination of bacterial cells by serum.
This finding gave rise to the use of serology as a tool in the
diagnosis of disease and also led to the discovery of the
ABO blood groups. Widal and Sicard developed one of the
earliest diagnostic tests in 1896 for the detection of
antibodies occurring in typhoid fever, brucellosis, and
tularemia. Agglutination reactions now have a wide variety
of applications in the detection of both antigens and
antibodies. Such testing is simple to perform and the end
points can easily be read visually.
Agglutination, like precipitation, is a two-step
process that results in the formation of a stable lattice
network. The first reaction, called sensitization, involves
antigen–antibody combination through single antigenic
determinants on the particle and is rapid and reversible. The
second step, or lattice formation, 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.
Sensitization is affected by the nature of the
antigens on the agglutinating particles. If epitopes are sparse
or if they are obscured by other surface molecules, they are
less likely to interact with antibody. Additionally, red blood
cells (RBCs) and bacterial cells have a slight negative
surface charge; because like charges tend to repel one
another, it is sometimes difficult to bring such cells together
into a lattice formation.
The class of immunoglobulin is also important; IgM with a
potential valence of 10 is over 700 times more efficient in
agglutination than is IgG with a valence of 2. Antibodies of
the IgG class often cannot bridge the distance between
particles because their small size and restricted flexibility at
the hinge region may prohibit multivalent binding. IgM
antibodies, on the other hand, are strong agglutinins because
of their larger size.
Achieving visible reactions with IgG often requires
the use of enhancement techniques that vary
physicochemical conditions such as the ionic strength of the
solution, the pH, and the temperature. Antibodies belonging
to the IgG class agglutinate best at 30°C to 37°C, whereas
IgM antibodies react best at temperatures between 4°C and
27°C. Because naturally occurring antibodies against the
ABO blood groups belong to the IgM class, these reactions
are best run at room temperature. Antibodies to other
human blood groups usually belong to the IgG class;
reactions involving these must be run at 37°C. These latter
reactions are the most important to consider in selecting
compatible blood for a transfusion because these are the
ones that will actually occur in the body.
In addition to temperature considerations, detection
of IgG antibodies often requires the use of a second
antibody, antihuman immunoglobulin, to visualize a
reaction. Anti-human immunoglobulin is also known as
Coombs reagent and is used frequently in blood bank
testing. Coombs reagent will attach to the Fc portion of IgG
and help to bridge the gap between RBCs so a visible
agglutination reaction will occur. Figure 10–9 demonstrates
how Coombs reagent works.
IV. Types of Agglutination Reactions
Agglutination reactions are easy to carry out, require no
complicated equipment, and can be performed as needed in
the laboratory without having to batch specimens. Batching
specimens is done if a test is expensive or complicated; in
this case, a large number are run at one time, which may
result in a time delay. Many kits are available for standard
testing, so reagent preparation is minimal. Agglutination
reactions are a frequently employed serological test; they
can be used to identify either antigen or antibody. Typically,
most agglutination tests are qualitative, simply indicating
the absence or presence of antigen or antibody, but dilutions
can be made to obtain semi quantitative results. Many
variations exist that can be categorized according to the type
of particle used in the reaction and whether antigen or
antibody is attached to it.
Direct Agglutination
Direct agglutination occurs when antigens are found
naturally on a particle. One of the best examples of direct
agglutination testing involves known bacterial antigens used
to test for the presence of unknown antibodies in the patient.
Typically, patient serum is diluted into a series of tubes or
wells on a slide and reacted with bacterial antigens specific
for the suspected disease. Detection of antibodies is
primarily used in diagnosis of diseases for which the
bacterial agents are extremely difficult to cultivate. One
such example is the Widal test, a rapid screening test used
to help determine the possibility of typhoid fever. A
significant finding is a fourfold increase in antibody titer
over time when paired dilutions of serum samples are tested
with any of these antigens. Although more specific tests are
now available, the Widal test is still considered useful in
diagnosing typhoid fever in developing countries and
remains in use in many areas throughout the world.
If an agglutination reaction involves RBCs, then it
is called hemagglutination. The best example of this
occurs in ABO blood group typing of human RBCs, one of
the world’s most frequently used immunoassays. Patient
RBCs mixed with antisera of the IgM type can be used to
determine the presence or absence of the A and B antigens;
this reaction is usually performed at room temperature
without the need for any enhancement techniques. Group A
RBCs will agglutinate with anti-A antibody and Group B
RBCs will agglutinate with anti-B antibody. This type of
agglutination reaction is simple to perform, is relatively
sensitive, and is easy to read.
A titer that yields semiquantitative results can be
performed in test tubes or microtiter plates by making serial
dilutions of the antibody. The reciprocal of the last dilution
still exhibiting a visible reaction is the titer, indicating the
antibody’s strength. Interpretation of the test is done on the
basis of the cell sedimentation pattern. If there is a dark red,
smooth button at the bottom of the microtiter well, the result
is negative. A positive result will have cells that are spread
across the well’s bottom, usually in a jagged pattern with an
irregular edge. Test tubes also can be centrifuged and then
shaken to see if the cell button can be evenly resuspended.
If it is resuspended with no visible clumping, then the result
is negative. Positive reactions can be graded to indicate the
strength of the reaction.
Passive Agglutination
Passive, or indirect, agglutination employs particles that
are coated with antigens not normally found on their
surfaces. A variety of particles, including erythrocytes,
latex, and gelatin, are used for passive agglutination. The
use of synthetic beads or particles provides the advantages
of consistency and uniformity Reactions are easy to read
visually and give quick results. Many antigens, especially
polysaccharides, adsorb to RBCs spontaneously, so they are
also relatively easy to manipulate. Particle sizes vary from 7
μm for RBCs down to 0.8 μm for fine latex particles.
 In 1955, Singer and Plotz found by happenstance
that IgG was naturally adsorbed to the surface of
polystyrene latex particles. Latex particles are inexpensive,
are relatively stable, and are not subject to cross-reactivity
with other antibodies. A large number of antibody
molecules can be bound to the surface of latex particles, so
the number of antigen-binding sites is large. Additionally,
the large particle size facilitates reading of the test.
Latex agglutination tests have been used to detect
rheumatoid actor, antibodies to Group A Streptococcus
antigens, and antibodies to viruses such as rotavirus,
cytomegalovirus, rubella, and varicella zoster.
Hemagglutination kits are available for detection of
antibodies to hepatitis B virus (HBV), hepatitis C virus
(HCV), and human immunodeficiency virus (HIV) I and II,
to cite just a few examples.
Because many of these kits are designed to detect
IgM antibody and there is always the risk of nonspecific
agglutination caused by the presence of other IgM
antibodies, reactions must be carefully controlled and
interpreted. Commercial tests are usually performed on
disposable plastic, cardboard cards, or glass slides. Kits
contain positive and negative controls; if the controls do not
give the expected results, the test is not valid. Such tests are
typically used as screening tools, which are followed by
more extensive testing if the results are positive.
Reverse Passive Agglutination
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. Adsorption may be
spontaneous, or it may require some of the same
manipulation as is used for antigen attachment. This type of
testing is often used to detect microbial antigens. Figure
10–12 shows the differences between passive and reverse
passive agglutination.
Numerous kits are available for the rapid
identification of antigens from such infectious agents as
Group B Streptococcus, Staphylococcus aureus,
streptococcal groups A and B, rotavirus, and Cryptococcus
neoformans. Rapid agglutination tests have found the
widest application in detecting soluble antigens in urine,
spinal fluid, and serum. The principle is the same for all
these tests: Latex particles coated with antibody are reacted
with a patient sample containing the suspected antigen. In
some cases, an extraction step is necessary to isolate antigen
before the reagent latex particles are added. Organisms can
be identified in a few minutes with fairly high sensitivity
and specificity, although this varies for different organisms.
The use of monoclonal antibodies has greatly cut down on agglutinate are added to the mixture. If antibody is present,
cross-reactivity, but there is still the possibility of it will attach to the viral particles and prevent agglutination,
interference or nonspecific agglutination. so a lack of or reduction in agglutination indicates the
Such tests are most often used for organisms that presence of patient antibody. Controls are necessary
are difficult to grow in the laboratory or for instances when because there may be a factor in the serum that causes
rapid identification will allow treatment to be initiated more agglutination or the virus may have lost its ability to
promptly. Direct testing of specimens for the presence of agglutinate.
viral antigens has still not reached the sensitivity of enzyme
immunoassays; however, for infections in which a large
amount of viral antigen is present, such as rotavirus and
enteric adenovirus in infants, latex agglutination tests are
very useful. Reverse passive agglutination testing has also
been used to measure levels of certain therapeutic drugs,
hormones, and plasma proteins such as haptoglobin and C-
reactive protein.
Agglutination Inhibition
Agglutination inhibition reactions are based on
competition between particulate and soluble antigens for
limited antibodycombining sites. A lack of agglutination is
an indicator of a positive reaction. Typically, this type of
reaction involves haptens that are complexed to proteins;
the hapten–protein conjugate is then attached to a carrier
particle. The patient sample is first reacted with a limited
amount of reagent antibody that is specific for the hapten
being tested. Indicator particles that contain the same hapten
one wishes to measure in the patient are then added. If the
patient sample has no free hapten, the reagent antibody is
able to combine with the carrier particles and produce a
visible agglutination. In this case, however, agglutination is
a negative reaction, indicating that the patient did not have
sufficient hapten to inhibit the secondary reaction. Either
antigen or antibody can be attached to the particles. The
sensitivity of the reaction is governed by the avidity of the
antibody itself. It can be a sensitive assay capable of
detecting small quantities of antigen. Tests used to detect
illicit drugs such as cocaine or heroin are examples of
agglutination inhibition tests.
Hemagglutination inhibition reactions use the
same principle, except RBCs are the indicator particles.
This type of testing has been used to detect antibodies to
certain viruses, such as rubella, influenza, and respiratory
syncytial virus (RSV). RBCs have naturally occurring viral
receptors. When virus is present, spontaneous agglutination
occurs because the virus particles link the RBCs together.
Presence of patient antibody inhibits the agglutination
reaction.
To perform a hemagglutination inhibition test,
dilutions of patient serum are incubated with a viral
preparation. Then RBCs that the virus is known to