Ch08

Clinical Immunology & Serology
A Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis CompanyCopyright © 2010 F.A. Davis Company
Precipitation Reactions
Chapter Eight

Copyright © 2010 F.A. Davis Company
 Precipitation involves combining soluble
antigen with soluble antibody to produce
insoluble complexes that are visible.
 For such reactions to occur, both 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.
 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 epitope and binding site come into close
proximity to each other, several types of
noncovalent bonds hold them together.
 These include ionic bonds, hydrogen bonds,
hydrophobic bonds, and van der Waals forces.
 The more the cross-reacting antigen
resembles the original antigen, the stronger
the bond will be between the antigen and the
binding site.

 However, if the epitope
and the binding site
have a perfect lock-
and-key relationship,
as is the case with the
original antigen, the
affinity will be maximal,
because there is a
very close fit (see Fig .
8-1).

 Avidity represents the sum of all the attractive
forces between an antigen and an antibody.
 This involves the strength with which a
multivalent antibody binds a multivalent
antigen, and it is a measure of the overall
stability of an antigen–antibody complex.
 Avidity is the force that keeps the molecules
together.

 All antigen–antibody binding is reversible and
is governed by the Law of mass action.
 The equilibrium constant (K) represents the
difference in the rates of the forward and
reverse reactions of antigen and antibody
association.
 This constant can be seen as a measure of
the goodness of fit between antigen and
antibody.

 The higher the value of K, the larger the
amount of antigen–antibody complex and the
more visible or easily detectable the reaction.
 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 will
be.

 In addition to the affinity and avidity of the
antibody involved, precipitation depends on
the relative proportions of antigen and
antibody present.
 Optimum precipitation occurs in the zone of
equivalence, in which the number of
multivalent sites of antigen and antibody are
approximately equal.

 As illustrated by the precipitin curve shown in
Figure 8-2, when increasing amounts of
soluble antigen are added to fixed amounts of
specific antibody, the amount of precipitation
increases up to the zone of equivalence.
 Then, when the amount of antigen
overwhelms the number of antibody combining
sites present, precipitation begins to decline.

Figure 8-2

 As can be seen on the precipitation curve,
precipitation declines on either side of the
equivalence zone due to 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.

 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, and again no
lattice network is formed.
 Thus, for precipitation reactions to be
detectable, they must be run 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 due to 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.
 This would give time for the further production
of antibody; if the test is negative on this
occasion, it is unlikely that the patient has that
particular antibody.

 Precipitates in fluids can be measured by
means of turbidimetry or nephelometry.
 Turbidimetry is a measure of the turbidity or
cloudiness of a solution.
 Nephelometry measures the light that is
scattered at a particular angle from the
incident beam as it passes through a
suspension.
 See Figure 8-3 in text.

 Nephelometry can be used to detect either
antigen or antibody, but it is usually run with
antibody as the reagent and the patient
antigen as the unknown.
 Nephelometry provides accurate and precise
quantitation of serum proteins, and due to
automation, the cost per test is typically lower
than other methods.

 The rate of diffusion is affected by the size of
the particles, the temperature, the gel
viscosity, and the amount of hydration.
 Radial immunodiffusion (RID) has been
commonly used in the clinical laboratory.
 In this technique, antibody is uniformly
distributed in the support gel, and antigen is
applied to a well cut into the gel.

 The precipitation of antigen–antibody
complexes can also be determined in a
support medium such as a gel.
 Reactants are added to the gel, and antigen–
antibody combination occurs by means of
diffusion.
 When no electrical current is used to speed up
this process, it is known as passive
immunodiffusion.

 As the antigen diffuses out from the well,
antigen–antibody combination occurs in
changing proportions until the zone of
equivalence is reached and a stable lattice
network is formed in the gel.

 The area of the ring obtained is a measure of
antigen concentration, and this can be
compared to a standard curve obtained by
using antigens of known concentration (see
Fig. 8-4 in text).

 There are two techniques for the
measurement of radial immunodiffusion.
 The first was developed by Mancini and is
known as the end-point method.
 In this technique, antigen is allowed to diffuse
to completion, and when equivalence is
reached, there is no further change in the ring
diameter.

 This endpoint occurs between 24 and 72
hours.
 The square of the diameter is then directly
proportional to the concentration of the
antigen.
 Figure 8-4 depicts some typical results.

 The Fahey and McKelvey method, also called
the kinetic method, uses measurements
taken before the point of equivalence is
reached.
 In this case, the diameter is proportional to the
log of the concentration.

 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.
 Multispecific antibody is placed in the central
well, and different antigens are placed in the
surrounding wells to determine if the antigens
share identical epitopes.

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 epitope.
2. A pattern of crossed lines demonstrates two
separate reactions and indicates that the
compared antigens share no common
epitopes, or nonidentity.

3. fusion of two lines with a spur indicates
partial identity.
 The “spur” in the latter always points to the
simpler antigen.
 See Figure 8-5 for an illustration of these
patterns.
 Ouchterlony double diffusion is still used to
identify fungal antigens such as Aspergillus,
Blastomyces, Coccidioides, and Candida.

Figure 8-5

 Diffusion can be combined with
electrophoresis to speed up or sharpen the
results.
 Electrophoresis separates molecules
according to differences in their electric charge
when they are placed in an electric field.

 One-dimension electroimmunodiffusion, an
adaptation of radial immunodiffusion, was
developed by Laurell.
 Antibody is distributed in the gel, and antigen
is placed in wells cut in the gel, just as in RID.
 Electrophoresis is used to facilitate migration
of the antigen into the agar.

 The end result is a precipitin line that is conical
in shape, resembling a rocket, hence the
name rocket immunoelectrophoresis.
 The height of the rocket, measured from the
well to the apex, is directly in proportion to the
amount of antigen in the sample (see Fig.
8-6).
 This technique has been used to quantitate
immunoglobulins.

Figure 8-6

 Immunoelectrophoresis is a double-diffusion
technique that incorporates electrophoresis
current to enhance results.
 Typically, the source of the antigens is serum,
which is electrophoresed to separate out the
main protein fractions.
 Antiserum is placed in troughs, and the gel is
incubated for 18 to 24 hours.

 Precipitin lines develop where specific
antigen–antibody combination takes place.
 These lines or arcs can be compared in
shape, intensity, and location to that of a
normal serum control to detect abnormalities.
 This procedure has been used as a screening
tool for the differentiation of many serum
proteins, including the major classes of
immunoglobulins (see Fig. 8-7).

Figure 8-7

 Immunofixation electrophoresis 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.
 Immunoprecipitates form only where specific
antigen–antibody combination has taken
place, and the complexes have become
trapped in the gel.

 Patient serum is applied to six lanes of the gel,
and after electrophoresis, five lanes are
overlaid with one each of the following
antibodies: antibody to gamma, alpha, or mu
heavy chains and to kappa or lambda light
chains.
 The sixth lane is overlaid with antibody to all
serum proteins and serves as 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 (see Fig. 8-8).

Figure 8-8

 Immunofixation is especially useful in
demonstrating antigens present in serum,
urine, or spinal fluid in low concentrations.
 Perhaps one of the best-known adaptations of
this technique is the Western blot, used as a
confirmatory test to detect antibodies to HIV-1.

 A mixture of HIV antigens is placed on a gel
and electrophoresed to separate the individual
components.
 The components are then transferred to
nitrocellulose paper by means of blotting or
laying the nitrocellulose over the gel so that
the electrophoresis pattern is preserved.

 Patient serum is applied to the nitrocellulose
and allowed to react by incubation.
 The strip is then washed and stained to detect
precipitin bands.
 Antibodies to several antigens can be detected
in the patient sample .
 Refer to Figure 23-1 for a specific example of
a Western blot used to determine the
presence of antibody to HIV-1.

 Each type of precipitation technique has its
own distinct advantages and disadvantages.
 Table 8-1 presents a comparison of the
techniques discussed in this chapter.

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