Immunoprecipitation
Introduction
The combination of antigen with specific antibody can be thought of as in
three distinct phases: primary, secondary and tertiary. The primary
phenomenon involves the combination of an individual binding site on an
antibody molecule with a single epitope or determinant site on an antigen.
These reactions are reversible and not easily detectable, although they can be
measured indirectly by techniques such as immunofluoresence,
radioimmunoassay and enzyme immunoassay. Secondary phenomena, can
be measured more readily and these include precipitation, agglutination and
complement fixation. Inflammation, phagocytosis, deposition of immune
complexes, immune adherence and chemotaxis are all in vivo reactions that
are classified as tertiary phenomena.
Precipitation was first noted in 1897 by Kraus, who found that culture filtrates
of enteric bacteria would precipitate when they were mixed with specific
antibody. 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 that is affinity and avidity
also play a major role.
Immunoprecipitation (IP) is the technique of precipitating an antigen out of
solution using an antibody that specifically binds to that particular antigen.
This process can be used to isolate and concentrate a particular antigen from
a sample containing many thousands of different antigens.
Antigen-Antibody Binding
Antigen-Antibody binding is mediated by the sum of many weak interactions
between the antigen and antibody. These weak interactions include hydrogen
bonds, van der Waals forces, and ionic and/or hydrophobic interactions.
These interactions can only take place if the antigen and antibody molecules
are close enough for some of the individual atoms to fit into complementary
cavity. The complementary regions of an antibody are its 2 antigen binding
sites (thus the antibody is said to be bivalent). The corresponding region(s) of
the antigen is referred to as an antigenic determinant. Most antigens have
multiple determinants; if 2 or more are identical, the antigen is said to be
multivalent. As the binding of an antibody (ab) to its antigen (ag) is reversible,
the binding reaction can be expressed as:
ab + ag ↔ ab:ag
The strength of the interaction is expressed as the affinity constant Ka, where:
Ka = [ab:ag]/[ab][ag]
Immunoprecipitation
In this equation, [ab:ag] is the molar concentration of the antibody-antigen
complex, and [ab] and [ag] are the molar concentrations of the antibody and
antigen, respectively. Affinity constants can vary widely between different
antibodies and antigens, and are affected by pH, temperature, and solvent.
Affinity
Antibody affinity is the strength of the
reaction between a single antigenic
determinant and a single combining site
on the antibody. It is the sum of the
attractive and repulsive forces operating
between the antigenic determinant and
the combining site of the antibody as
illustrated in Figure 1.1.
Avidity - Avidity is a measure of the
overall strength of binding of an antigen
with many antigenic determinants and
multivalent
antibodies.
Avidity
is
influenced by both the valence of the
antibody and the valence of the antigen.
Avidity is more than the sum of the
individual affinities. This is illustrated in
Figure 1.1.
Figure 1.1. Affinity and avidity of ag-ab interactions.
Factors affecting measurement of Ag/Ab
reactions
The only way that one knows that an antigenantibody reaction has occurred is to have
some means of directly or indirectly detecting
the complexes formed between the antigen
and antibody. The ease with which on can
detect antigen-antibody reactions will depend
on a number of factors.
1. Affinity - The higher the affinity of the
antibody for the antigen, the more stable
will be the interaction. Thus, the ease with
which one can detect the interaction is
enhanced.
2. Avidity - Reactions between multivalent
antigens and multivalent antibodies are
more stable and thus easier to detect.
Figure 1.2. The sizes of the complexes formed
3. Ag:Ab ratio - The ratio between the is related to the concentration of the antigen and
antigen and antibody influences the antibody.
detection of Ag/Ab complexes because the sizes of the complexes formed
is related to the concentration of the antigen and antibody. Figure 1.2.
4. Physical form of the antigen - The physical form of the antigen influences
how one detects its reaction with an antibody. If the antigen is part of a
cell, one generally looks for agglutination of the antigen by the antibody. If
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Immunoprecipitation
the antigen is soluble one generally looks for the precipitation of the
antigen after the production of large insoluble Ag/Ab complexes.
The precipitation reaction occurs when a soluble antigen reacts with a
homologous antibody to form immunocomplexes. The structures and
solubilities of immune complexes are dependent on the natures and relative
amounts of the reacting antigens and antibodies, and also on the numbers of
combining sites on each. Antigens may have one to numerous antibodybinding sites, on the other hand, IgG antibodies are bivalent having 2 antigenbinding sites. Repeated antigen-antibody linkages can result in large insoluble
complexes at antibody optimal ratio (AOR). The AOR is the ideal
antibody/antigen ratio for formation of insoluble immune complexes
(equivalence). When the number of antigen particles is much higher than the
number of antibody molecules, many antibody binding sites on the antigen will
remain empty. The complexes that are formed are small and soluble, not
visible with the naked eye. When number of antibody molecules is much
higher than the number of antigen molecules, there is not enough antigen to
form cross-linkages. Again the complexes that are formed are small and
soluble (Figure 1.3).
A visible antigen-antibody precipitation
reaction occurs in agarose gel at AOR
(optimal antigen/ antibody ratio) or
equivalence. A single diffusing antibody
meeting its cognate antigen will
produce a single line of precipitation. A
number of currently used techniques
make use of this principle: double
immunodiffusion, single radial diffusion,
and immunoelectrophoresis.
Precipitation Curve
Prozone and Postzone
On either side of the equivalence zone,
precipitation is actually prevented
because of an excess of either antigen
or antibody. In the case of antibody Figure 1.3. Schematic complexes of antigen and bivalent
excess, the prozone phenomenon antibody
occurs, in which antigen combines with
only one or two antibody molecules, and so no cross-linkages are formed
(Figure 1.3). 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.
Zone of Equivalence
In addition to the affinity and avidity of the antibody involved, precipitation
depends on the relative proportions of antigen and antibody present. The
zone in which optimum precipitation occurs is called the zone of equivalence,
in which the number of multivalent sites of antigen and antibody are
approximately equal. Precipitation is the result of antibody binding to more
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Immunoprecipitation
than one antigen and vice versa, forming a stable network or lattice. In this
case each antibody must have at least two binding sites and antigen must be
multivalent. As they combine this results in a multimolecular lattice that
increases in size until precipitate out of solution.
Double Immunodiffusion (The Ouchterlony Technique)
This procedure was developed by Ouchterlony (1966). Wells are cut in a gel
(agar or agarose); one well contains the antibody and the other well contains
the antigens. Antigens and antibodies diffuse towards each other at rates that
increase in proportion to their concentrations in the well but decrease in
proportion to their sizes. They form a line of precipitation (precipitin line)
where they meet at equivalence. Generally the agar does not interfere with
the diffusion of the two species. The initial formation of the precipitin line
moves the equilibrium between the antibody and antigen towards formation of
more precipitation increasing the flow of reactants into the zone of
precipitation, (Figure 1.4a). It also acts as
a
barrier
across
which
neither
components can pass. The double
diffusion
technique
has
many
applications:
1. Determine the homogeneity of
antigen-antibody complexes.
2. Immunodiffusion can also be used to
follow the purification of an antigenic
mixture.
3. Determine whether a given antigen
a
shares
structural
characteristics
(cross-reacts) with other molecules of
interest.
Double diffusion in two dimensions is a
useful technique for comparing antigens
for the number of identical or crossreacting determinants. If a solution of
antigen is placed in two adjacent wells
and the homologous antibody is placed in
the center well (Figure 1.4b), the two
precipitin bands that form will join at their
closest ends and fuse. This is known as a
reaction of identity (Figure 1.5 a).
When unrelated antigens are placed in
b
adjacent wells and the center well is filled
Figure 1.4. Mechanism of precipitation formation
with antibodies for each antigen, the
in. the Ouchterlony Technique "a". Double
diffusion plate "b".
precipitin bands will form independently
of each other and will cross. This is
known as a reaction of non-identity (Figure 1.5 b).
If two purified antigens cross-react, then placing them in adjacent peripheral
wells with antibody to one in the central well will give a single band with the
homologous and cross-reacting antigen. Since the crossreacting antigen lacks
some of the antigenic determinants present in the homologous antigen, it is
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Immunoprecipitation
not able to precipitate all of the antibody. The remaining antibody will diffuse
beyond the line of cross-reacting precipitate to react with the homologous
antigen to produce a spur. The spur that forms projects toward the antigen
with the fewer determinants, i.e., the cross-reacting antigen. This is called a
reaction of partial identity. Since these non-cross-reacting antibodies often are
only a fraction of the total antibody involved in the homologous precipitin
reaction, the spur is usually less dense (often difficult to visualize) than the
precipitin band from which it projects (Figure 1.5 c).
a
b
c
Figure 1.5. Three basic patterns of precipitation for double immunodiffusion, (a) identilty, (b) nonidentity, and (c) partial identity.
Single Radial Immunodiffusion
(SRID)
The single radial immunodiffusion
technique is used to measure the
concentration of a particular
substance in solution that is mixed
with other substances when
appropriate antiserum is available.
Figure 1.6. A sample containing the antigen is placed in
In this technique, which was
a well cut in the gel containing the antibody and allowed
developed by Mancini, a gel
to diffuse out. As the antigen meets its cognate antibody
(usually agarose) is prepared that
a visible precipitate is formed as a halo around the well.
contains antiserum. A sample
containing the antigen is placed in
a well cut in the gel and allowed to diffuse out. As the antigen meets its
cognate antibody a visible precipitate is formed as a halo around the well
(Figure1.6). As more antigen diffuses into the equivalence zone, there is now
excess antigen and some of the precipitin in this area dissolves. The antigen
diffuses further outwards until concentration drops to that necessary for
equivalence, and is reprecipitated by the antibody. This precipitation, partial
redissolving, reprecipitation continues until antigen is too low to redissolve the
rim of the precipitin halo. The precipitin halo stops increasing in diameter.
There is a linear relationship between the amount of antigen and diameter of
the halo. Because the actual diameter of the halo is also a function of
temperature and other factors, SRID experiments always include a set of
known varying concentration of the antigen. A standard curve is plotted to
determine the concentration of unknown sample.
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