BSMT-3 IMMUNOSEROLOGY Sept.

14, 2020
ANTIGEN-ANTIBODY REACTIONS PART2 Dra. Andrea Villaruel, MD, DPSP

OUTLINE (CHAPTER 8)
play a major role. These are discussed, along
I. Precipitation Reaction with theoretical considerations of binding,
II. Antigen-Antibody Binding including the law of mass action and the
a. Affinity principle of lattice formation. Such
b. Avidity characteristics relate to the sensitivity and
c. Law of Mass Action
specificity of testing in the clinical laboratory.
III. Precipitation Curve
a. Zone of Equivalence
ANTIGEN-ANTIBODY BINDING
b. Prozone and Postzone
IV. Measurement of precipitation by light scattering Affinity
a. Turbidimetry
b. Nephelometry • The primary union of binding sites on antibody
V. Passive immunodiffusion techniques with specific epitopes on an antigen depends
a. Radial Immunodiffusion
on two characteristics of antibody known as
b. Ouchterlony Double Diffusion
VI. Electrophoretic techniques
affinity and avidity.
a. Rocket Immunoelectrophoresis • Affinity is the initial force of attraction that
b. Immunoelectrophoresis exists between a single Fab site on an antibody
c. Immunofixation Electrophoresis molecule and a single epitope or determinant
d. Sources of Error in Electrophoresis site on the corresponding antigen. As epitope
VII. Comparison of precipitation techniques and binding site come into close proximity to
VIII. Summary
each other, several types of noncovalent
IX. References
bonds hold them together. These include ionic
INTRODUCTION bonds, hydrogen bonds, hydrophobic bonds,
and van der Waals forces.
• The combination of antigen with specific • Ionic bonds occur between oppositely charged
antibody plays an important role in the particles.
laboratory in diagnosing many different • Hydrogen bonds involve an attraction between
diseases. Immunoassays have been developed polar molecules that have a slight charge
to detect either antigen or antibody, and they separation and in which the positive charge
vary from easily performed manual tests to resides on a hydrogen atom.
highly complex automated assays. Many such • Hydrophobic bonds occur between nonpolar
assays are based on the principles of molecules that associate with one another and
precipitation or agglutination. Precipitation exclude molecules of water as they do so.
involves combining soluble antigen with • Van der Waals forces occur because of the
soluble antibody to produce insoluble interaction between the electron clouds of
complexes that are visible. Agglutination is the oscillating dipoles.
process by which particulate antigens such as • All of these are rather weak bonds that can
cells aggregate to form larger complexes when occur only over a short distance of
a specific antibody is present. approximately 1 x 10–7 mm, so there must be a
• This chapter focuses on precipitation, and the very close fit between antigen and antibody.
following chapter discusses agglutination. • The strength of attraction depends on the
Precipitation was first noted in 1897 by Kraus, specificity of antibody for a particular antigen.
who found that culture filtrates of enteric One antibody molecule may initially attract
bacteria would precipitate when they were numerous different antigens, but it is the
mixed with specific antibody. For such epitope’s shape and the way it fits together
reactions to occur, both antigen and antibody with the binding sites on an antibody molecule
must have multiple binding sites for one that determines whether the bonding will be
another, and the relative concentration of stable. Antibodies are capable of reacting with
each must be equal. Binding characteristics of
antibodies, called affinity and avidity, also
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ANTIGEN-ANTIBODY REACTIONS PART2 Dra. Andrea Villaruel, MD, DPSP

antigens that are structurally similar to the Law of Mass Action

original antigen that induced antibody
production. This is known as cross-reactivity. • All antigen–antibody binding is reversible and
The more the cross-reacting antigen is governed by the law of mass action. This law
resembles the original antigen, the stronger states that free reactants are in equilibrium
the bond will be between the antigen and the with bound reactants. The equilibrium
binding site. However, if the epitope and the constant represents the difference in the rates
binding site have a perfect lock-and-key of the forward and reverse reactions according
relationship, as is the case with the original to the following equation:
antigen, the affinity will be maximal, because K1
there is a very close fit. Ag + Ab AgAb

K2
Where:
Ag = Antigen
Ab = Antibody
K1 = rate of constant for the forward reaction
K2 = rate of constant for the reverse reaction
• The equilibrium constant is thus;
K = K1/K2 = [AgAb]/[Ab][Ag],
Where [AgAb] = concentration of the antigen-antibody
complex (mol/L)
[Ab] = concentration of antibody (mol/L)
[Ag] = concentration of antigen (mol/L)
• This constant can be seen as a measure of the
goodness of fit. Its value 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, and the value
of K2 decreases. This increases the value of K1.
The higher the value of K, the larger the amount
of antigen–antibody complex and the more
visible or easily detectable the reaction is. 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
Avidity strength of binding. The higher the values are for
both of these and the more antigen–antibody
• Avidity represents the sum of all the attractive
complexes that are formed, the more sensitive
forces between an antigen and an antibody.
the test will be.
This involves the strength with which a
multivalent antibody binds a multivalent PRECIPITATION CURVE
antigen, and it is a measure of the overall
stability of an antigen–antibody complex. In Zone of Equivalence
other words, once binding has occurred, it is
the force that keeps the molecules together. A • In addition to the affinity and avidity of the
high avidity can actually compensate for a low antibody involved, precipitation depends on the
affinity. Stability of the antigen–antibody relative proportions of antigen and antibody
complex is essential to detecting the presence present. Optimum precipitation occurs in the
of an unknown, whether it is antigen or zone of equivalence, in which the number of
antibody. multivalent sites of antigen and antibody are
approximately equal. In this zone, precipitation
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is the result of random, reversible reactions surrounded by excess antigen, and again no
whereby each antibody binds to more than one lattice network is formed. In this case, every
antigen and vice versa, forming a stable network available antibody site is bound to a single
or lattice. The lattice hypothesis, as formulated antigen, and no cross-links are formed. Thus, for
by Marrack, is based on the assumptions that precipitation reactions to be detectable, they
each antibody molecule must have at least two must be run in the zone of equivalence.
binding sites, and antigen must be multivalent. • The prozone and postzone phenomena must be
As they combine, this results in a multimolecular considered in the clinical setting, because
lattice that increases in size until it precipitates negative reactions occur in both. A false-
out of solution. Heidelberger and Kendall negative reaction may take place in the prozone
performed the classic quantitative precipitation due to high antibody concentration. If it is
reactions that established proof for this theory. suspected that the reaction is a false negative,
• As illustrated by the precipitin curve shown in diluting out antibody and performing the test
Figure 8–2, when increasing amounts of soluble again may produce a positive result. In the
antigen are added to fixed amounts of specific postzone, excess antigen may obscure the
antibody, the amount of precipitation increases presence of a small amount of antibody.
up to the zone of equivalence. Then when the Typically, such a test is repeated with an
amount of antigen overwhelms the number of additional patient specimen taken about a week
antibody-combining sites present, precipitation later. This would give time for the further
begins to decline. production of antibody. If the test is negative on
this occasion, it is unlikely that the patient has
that particular antibody.

MEASUREMENT OF PRECIPITATION BY LIGHT

SCATTERING
Turbidimetry

• Precipitation is one of the simplest methods of

detecting antigen–antibody reactions, because
most antigens are multivalent and thus capable
of forming aggregates in the presence of the
corresponding antibody. When antigen and
antibody solutions are mixed, the initial turbidity
is followed by precipitation. Precipitates in fluids
can be measured by means of turbidimetry or
Prozone and Postzone
nephelometry.
• As can be seen on the precipitation curve, • Turbidimetry is a measure of the turbidity or
precipitation declines on either side of the cloudiness of a solution. A detection device is
equivalence zone due to an excess of either placed in direct line with the incident light,
antigen or antibody. In the case of antibody collecting light after it has passed through the
excess, the prozone phenomenon occurs, in solution. It thus measures the reduction in light
which antigen combines with only one or two intensity due to reflection, absorption, or
antibody molecules, and no cross-linkages are scatter. Scattering of light occurs in proportion
formed. This is because usually only one site on to the size, shape, and concentration of
an antibody molecule is used, and many free molecules present in solution. It is recorded in
antibody molecules remain in solution. absorbance units, a measure of the ratio of
• At the other side of the zone, where there is incident light to that of transmitted light.
antigen excess, the postzone phenomenon Measurements are made using a
occurs, in which small aggregates are
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ANTIGEN-ANTIBODY REACTIONS PART2 Dra. Andrea Villaruel, MD, DPSP

spectrophotometer or an automated clinical quantified include complement components, C-

chemistry analyzer. reactive protein, and several clotting factors.
Nephelometry provides accurate and precise
Nephelometry quantitation of serum proteins, and due to
automation, the cost per test is typically lower
• Nephelometry measures the light that is
than other methods.
scattered at a particular angle from the incident
beam as it passes through a suspension5 (Fig.
8–3). The amount of light scattered is an index
of the solution’s concentration. Beginning with
a constant amount of antibody, increasing
amounts of antigen result in an increase in
antigen–antibody complexes. Thus, the
relationship between antigen concentrations,
as indicated by antigen–antibody complex
formation, and light scattering approaches
linearity. Light scatter may be recorded in
arbitrary units of “relative light scatter,” or it
may be directly extrapolated by a computer to
give actual concentrations in milligrams per
deciliter (mg/dL) or international units per
milliliter (IU/mL), based on established values
of standards. Nephelometers measure light
scatter at angles ranging from 10 degrees to
about 90 degrees. If a laser beam is used, light
deflected only a few degrees from the original
path can be measured. Although the sensitivity
of turbidity has increased, nephelometry is
more sensitive, with a lower limit of detection
of 1 to 10 mg/L2
• Nephelometry can be used to detect either PASSIVE IMMUNODIFFUSION TECHNIQUES
antigen or antibody, but it is usually run with
antibody as the reagent and the patient antigen • The precipitation of antigen–antibody
as the unknown. In end point nephelometry, complexes can also be determined in a support
the reaction is allowed to run essentially to medium such as a gel. Agar, a high-molecular-
completion, but large particles tend to fall out weight complex polysaccharide derived from
of solution and decrease the amount of scatter. seaweed, and agarose, a purified agar, are
Thus, another method called kinetic or rate used for this purpose. Agar and agarose help
nephelometry was devised, in which the rate of stabilize the diffusion process and allow
scattering increase is measured immediately visualization of the precipitin bands.
after the reagent is added. This rate change is • Reactants are added to the gel, and antigen–
directly related to antigen concentration if the antibody combination occurs by means of
concentration of antibody is kept constant. diffusion. When no electrical current is used to
Many automated instruments utilize this speed up this process, it is known as passive
principle for the measurement of serum immunodiffusion. The rate of diffusion is
proteins. Quantification of immunoglobulins affected by the size of the particles, the
such as IgG, IgA, IgM, and IgE, and kappa and temperature, the gel viscosity, and the amount
lambda light chains is done almost exclusively of hydration. An agar concentration ranging
by nephelometry, because other methods are from 0.3 percent to 1.5 percent allows for
more labor-intensive.6 Other serum proteins diffusion of most reactants. Agarose is
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preferred to agar, because agar has a strong • The Fahey and McKelvey method, also called
negative charge, while agarose has almost the kinetic method, uses measurements taken
none, so interactions between the gel and the
reagents are minimized. Immunodiffusion
reactions can be classified according to the
number of reactants diffusing and the
direction of diffusion.

Radial Immunodiffusion

• James Oudin was the first to use gels for

precipitation reactions, and he pioneered the
technique known as single diffusion. In single
diffusion, antibody was incorporated into
agarose in a test tube. The antigen was layered
on top, and as the antigen moved down into
the gel, precipitation occurred and moved
down the tube in proportion to the amount of
antigen present. A modification of the single-
diffusion technique, radial immunodiffusion
(RID), has been commonly used in the clinical
laboratory. In this technique, antibody is
uniformly distributed in the support gel, and
before the point of equivalence is reached. In
antigen is applied to a well cut into the gel. As
this case, the diameter is proportional to the
the antigen diffuses out from the well,
log of the concentration. A graph is drawn on
antigen–antibody combination occurs in
semi-log paper by plotting the antigen
changing proportions until the zone of
concentration on the log axis and the diameter
equivalence is reached and a stable lattice
on the arithmetic axis. Readings are taken at
network is formed in the gel. The area of the
about 18 hours.
ring obtained is a measure of antigen
concentration, and this can be compared to a • For either the end-point method or the kinetic
standard curve obtained by using antigens of method, it is essential that monospecific
known concentration. Figure 8–4 depicts some antiserum with a fairly high affinity be used.
typical results. This increases the clarity of the precipitation
reaction. In addition, the precision of the assay
• There are two techniques for the
is directly related to accurate measurement of
measurement of radial immunodiffusion. The
samples and standards. Sources of error
first was developed by Mancini and is known
include overfilling or underfilling the wells,
as the end-point method. In this technique,
nicking the side of the wells when filling,
antigen is allowed to diffuse to completion,
spilling sample outside the wells, improper
and when equivalence is reached, there is no
incubation time and temperature, and
further change in the ring diameter. This
incorrect measurement. Radial
occurs between 24 and 72 hours. The square
immunodiffusion has been used to measure
of the diameter is then directly proportional to
IgG, IgM, IgA, and complement components. It
the concentration of the antigen. A graph is
is simple to perform and requires no
obtained by plotting concentrations of
instrumentation, but it is fairly expensive to
standards on the x-axis versus the diameter
run. Thus, immunodiffusion has largely been
squared on the y-axis, and a smooth curve is fit
replaced by more sensitive and automated
to the points. The major drawback to this
methods such as nephelometry and enzyme-
method is the time it takes to obtain results.

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linked immunosorbent assays except for low detect antibodies to extractable nuclear
volume analytes such as IgD or IgG subclasses. antigens that occur in several autoimmune
diseases. (Refer to Chapter 14 for a discussion
Ouchterlony Double Diffusion of autoimmune diseases.)

• One of the older, classic immunochemical

techniques is Ouchterlony double diffusion. In
this technique, both antigen and antibody
diffuse independently through a semisolid
medium in two dimensions, horizontally and
vertically. Wells are cut in a gel, and reactants
are added to the wells. After an incubation
period of between 12 and 48 hours in a moist
chamber, precipitin lines form where the
moving front of antigen meets that of
antibody. The density of the lines reflects the
amount of immune complex formed.
• Most Ouchterlony plates are set up with a
central well surrounded by four to six
equidistant outer wells. Antibody that is
multispecific is placed in the central well, and
different antigens are placed in the
surrounding wells to determine if the antigens
share identical epitopes. The position of the
precipitin bands between wells allows for the
antigens to be compared with one another.
Several patterns are possible:
1. Fusion of the lines at their junction to
form an arc represents serological
identity or the presence of a common • It is important to perform this technique with
epitope. care, because several problems may arise.
2. A pattern of crossed lines demonstrates Irregular patterns of precipitation may occur
two separate reactions and indicates from overfilling the wells, irregular hole
that the compared antigens share no punching, or nonlevel incubation. Other
common epitopes. factors affecting the accuracy of results include
3. Fusion of two lines with a spur indicates drying out of the gels; inadequate time for
partial identity. diffusion, resulting in weakness of band
• In this last case, the two antigens share a intensity; and fungal or bacterial
common epitope, but some antibody contamination of the gel.
molecules are not captured by antigen and
travel through the initial precipitin line to ELECTROPHORETIC TECHNIQUES
combine with additional epitopes found in the
more complex antigen. Therefore, the spur • Diffusion can be combined with
always points to the simpler antigen1 (Fig. 8– electrophoresis to speed up or sharpen the
5). While of more limited use because it is results. Electrophoresis separates molecules
labor-intensive and requires experience to according to differences in their electric charge
read, Ouchterlony double diffusion is still used when they are placed in an electric field. A
to identify fungal antigens such as Aspergillus, direct current is forced through the gel,
Blastomyces, Coccidioides, and Candida. In causing antigen, antibody, or both to migrate.
addition, this has been a classic technique to As diffusion takes place, distinct precipitin
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bands are formed. This technique can be Immunoelectrophoresis

applied to both single and double diffusion.
• Immunoelectrophoresis is a double-diffusion
Rocket Immunoelectrophoresis
technique that incorporates electrophoresis
• One-dimension electroimmunodiffusion, an current to enhance results. Introduced by
adaptation of radial immunodiffusion, was Grabar and Williams in 1953, this is performed
developed by Laurell in the early 1960s. as a two-step process and can be used for
Antibody is distributed in the gel, and antigen semiquantitation of a wide range of antigens.
is placed in wells cut in the gel, just as in RID. Typically, the source of the antigens is serum,
However, instead of allowing diffusion to take which is electrophoresed to separate out the
place at its own rate, electrophoresis is used to main protein fractions; then a trough is cut in
facilitate migration of the antigen into the the gel parallel to the line of separation.
agar. When the antigen diffuses out of the Antiserum is placed in the trough, and the gel
well, precipitation begins. As the is incubated for 18 to 24 hours. Double
concentration of antigen changes, there is diffusion occurs at right angles to the
dissolution and reformation of the precipitate electrophoretic separation, and precipitin lines
at ever-increasing distances from the well. The develop where specific antigen–antibody
end result is a precipitin line that is conical in combination takes place. These lines or arcs
shape, resembling a rocket, hence the name can be compared in shape, intensity, and
rocket immunoelectrophoresis. The height of location to that of a normal serum control to
the rocket, measured from the well to the detect abnormalities. Changes include bowing
apex, is directly in proportion to the amount of or thickening of bands, or changed mobility.
antigen in the sample. If standards are run, a Interpretation may take considerable
curve can be constructed to determine experience.
concentrations of unknown specimens (Fig. 8- • This procedure has been used as a screening
6). tool for the differentiation of many serum
proteins, including the major classes of
immunoglobulins. It is both a qualitative and a
semiquantitative technique and has been used
in clinical laboratories for the detection of
myelomas, Waldenstrom’s
macroglobulinemia, malignant lymphomas,
and other lymphoproliferative disorders. (See
Chapter 15 for more details on
lymphoproliferative diseases.) In addition,
immunodeficiencies can be detected in this
• Rocket immunoelectrophoresis is much more manner, if no precipitin band is formed for a
rapid than RID, with results available in just a particular immunoglobulin. Deficiencies of
few hours. It is essential, however, to complement components can also be
determine the net charge of the molecules at identified. Figure 8–7 shows examples of a
the pH used for the test, because this normal pattern and an IgM monoclonal
determines the direction of migration within gammopathy, an immunoproliferative
the gel. Typically, a pH is selected so that disorder. However, this technique is relatively
antibodies in the gel do not move, but the insensitive, with a detection limit of
antigens become negatively charged. Antigens approximately 3 to 20 uL concentration.
then migrate toward the positive anode.4 This Interpretation can also be difficult and may
technique has been used to quantitate take considerable experience. Therefore, it has
immunoglobulins, using a buffer of pH 8.6. largely been replaced by immunofixation

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electrophoresis, which gives quicker results and to kappa or lambda light chains. The sixth
and is easier to interpret. lane is overlaid with antibody to all serum
proteins and serves as the reference lane.
Reactions in each of the five lanes are
compared to the reference lane.
Hypogammaglobulinemias will exhibit faintly
staining bands, while polyclonal
hypergammaglobulinemias show darkly
staining bands in the gamma region.
Monoclonal bands, such as found in
Waldenström’s macroglobulinemia or multiple
myeloma, have dark and narrow bands in
specific lanes (Fig. 8–8).

Immunofixation Electrophoresis

• Immunofixation electrophoresis, as first

described by Alper and Johnson, is similar to
immunoelectrophoresis except that after
electrophoresis takes place, antiserum is
applied directly to the gel’s surface rather than
placed in a trough. Agarose or cellulose
• This method is especially useful in
acetate can be used for this purpose.
demonstrating those antigens present in
Immunodiffusion takes place in a shorter time
serum, urine, or spinal fluid in low
and results in a higher resolution than when
concentrations. Cerebrospinal fluid is the
antibody diffuses from a trough. Because
specimen of choice for diagnosing multiple
diffusion is only across the thickness of the gel,
sclerosis, and urine is used to detect the
approximately 1 mm, the reaction usually
presence of Bence-Jones proteins that are
takes place in less than 1 hour.
found in multiple myeloma. Although
• Most often, an antibody of known specificity is
immunofixation is more sensitive than
used to determine whether patient antigen is
immunoelectrophoresis, dilutions may be
present. The unknown antigen is placed on the
necessary to avoid the zones of antigen excess,
gel, electrophoretic separation takes place,
which may occur if concentrations of
and then the reagent antibody is applied.
monoclonal antibody are very high. Perhaps
Immunoprecipitates form only where specific
one of the best-known adaptations of this
antigen–antibody combination has taken
technique is the Western blot, used as a
place, and the complexes have become
confirmatory test to detect antibodies to
trapped in the gel. The gel is washed to remove
human immunodeficiency virus 1 (HIV-1). A
any nonprecipitating proteins and can then be
mixture of HIV antigens is placed on a gel and
stained for easier visibility. Typically, patient
electrophoresed to separate the individual
serum is applied to six lanes of the gel, and
components. The components are then
after electrophoresis, five lanes are overlaid
transferred to nitrocellulose paper by means
with one each of the following antibodies:
of blotting or laying the nitrocellulose over the
antibody to gamma, alpha, or mu heavy chains
gel so that the electrophoresis pattern is
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preserved. Patient serum is applied to the SUMMARY

nitrocellulose and allowed to react. The strip is
then washed and stained to detect precipitin • Immunoassays in which antigen–antibody
bands. It is simpler to visualize the reaction on combination occurs have been developed to
the nitrocellulose, and in this manner, detect either antigen or antibody, and they
antibodies to several antigens can be play an important role in diagnosing diseases.
detected. Refer to Figure 23-1 for a specific Many such assays are based on the principle of
example of a Western blot used to determine precipitation, which involves combination of
the presence of antibody to HIV-1. This soluble antigen with soluble antibody to
technique is characteristically used to produce insoluble complexes that are visible.
determine the presence of antibodies to • Union of antigen and antibody depends on
organisms of complex antigenic composition. affinity, or the force of attraction that exists
If antibodies to more than one disease- between one antibody-binding site and a
associated antigen are identified in patient single epitope on an antigen. Avidity is the sum
serum, this usually confirms presence of the of all attractive forces occurring between
suspected disease. multiple binding sites on antigen and antibody.
For testing purposes, it is important to have an
SOURCES OF ERROR IN ELECTROPHORESIS antibody with high affinity and high avidity for
the antigen in question.
• Many of the sources of error are similar for all
• Maximum binding of antigen and antibody
the electrophoretic techniques, so they are
occurs when the aggregate number of
discussed together here. One problem that can
multivalent sites of antigen and antibody are
arise is applying the current in the wrong
approximately equal. The concentrations of
direction. If this occurs, samples may either
antigen and antibody that yield maximum
run off the gel or not be separated. Incorrect
binding represent the zone of equivalence.
pH of the buffer and incorrect electrophoresis
When antibody is in excess (the prozone) or
time also hinder proper separation.
antigen is in excess (the postzone),
Concentrations of antigen and antibody must
manifestations of antigen–antibody
be carefully chosen so that lattice formation
combination such as precipitation and
and precipitation is possible. If either is too
agglutination are present to a much lesser
concentrated, no visible reaction will result.
degree. All testing should take place in the
The amount of current applied also influences
zone of equivalence, where a reaction is most
the efficiency of the separation. If the current
visible.
is not strong enough, separation may be
incomplete. On the other hand, if the current • Precipitation can be detected in the laboratory
is too strong, heat will be generated and may by several different means. Light scatter
denature proteins. As in diffusion techniques, produced by immune complexes in solution
wells must be carefully filled. can be measured as a reduction in light
intensity (turbidimetry) or as the amount of
COMPARISON OF PRECIPITATION light scattered at a particular angle
TECHNIQUES (nephelometry). Several automated
instruments are based on these principles.
• Each type of precipitation technique has its • Other precipitation techniques utilize a
own distinct advantages and disadvantages. support medium such as a gel, and antigen–
Some techniques are technically more antibody combination takes place by means of
demanding, and others are more automated. passive diffusion. In single diffusion, only one
Each type of precipitation testing has of the reactants travels, while the other is
particular applications for which it is best incorporated in the gel. An example is radial
suited. Table 8–1 presents a comparison of the immunodiffusion, in which antibody is
techniques discussed in this chapter. incorporated in a gel in a plate or a petri dish.
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• The amount of precipitate formed is directly

TABLE 8-1 Comparison of Precipitation Techniques
related to the amount of antigen present.
• Ouchterlony diffusion is a double-diffusion TECHNIQUE APPLICATION SENSITIVITY COMMENT
technique in which both antigen and antibody (ug ab/ml)
diffuse from wells and travel toward each Immunoglobulins 1-10 Automated,
Nephelometry
other. Precipitin lines may indicate identity, , complement, C- sensitive,
nonidentity, or partial identity, depending on reactive protein, expensive
the pattern formed. other serum equipment
proteins. needed
• Electrophoresis can be combined with
Radial Immunoglobulins 10–50 Quantitative,
diffusion to speed up the process and enhance
immunodiffusio , complement slow, not as
the reaction patterns. Rocket n sensitive as
immunoelectrophoresis applies an electric nephelometry
charge to single diffusion, with resultant Ouchterlony Complex antigens 20–200 Semiquantitati
precipitation patterns that look like rockets double such as fungal ve, slow, may
diffusion antigens. be difficult to
that project upward from the sample wells. In interpret.
immunoelectrophoresis, the antigen is first Rocket Immunoglobulins 2 Fast,
electrophoresed by itself to separate out electrophoresis , complement, quantitative,
different components, and then antibody is alpha-fetoprotein technically
placed in a trough to allow diffusion to take demanding
Immunoelectro Differentiation of 20–200 Slow,
place. Specific reaction patterns indicate phoresis serum proteins semiquantitati
presence or absence of certain antigens. A ve, difficult to
related technique, immunofixation interpret
electrophoresis, differs in that antibody is Immunofixation HIV, Lyme Variable Fairly rapid,
applied directly to the gel after electrophoresis electrophoresis disease, syphilis semiquantitati
ve, sensitive
has taken place. Compared to HIV = Human immunodeficiency virus; Ab = antibody
immunoelectrophoresis, precipitation occurs Adapted from Kindt, TJ, Goldsby, RA, and Osborne, BA. Kuby Immunology, ed. 6. WH Freeman
and Co., New York, 2007, p. 152.
in a shorter time, and bands with higher
resolution are obtained.
• Precipitation is adaptable to both automated
technologies and individually processed
testing. Thus, it is a versatile and practical
method for use in the clinical laboratory.

REFERENCE

• Stevens, C. D. (2010). Clinical immunology &

serology: A laboratory perspective.

Happy Medtech Week! :)

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