Ch08

Clinical Immunology & SerologyA 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 Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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).



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation ReactionsFigure 8-2



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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).



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation ReactionsSeveral 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.



Precipitation Reactions3. 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.






Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.






Precipitation Reactions 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.



Precipitation Reactions 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).






Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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).






Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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.



Precipitation Reactions 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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