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