Download Purification of Antibodies

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Purification of Antibodies
Although hyperimmune antisera raised in experimental animals contain very high
concentrations of immunoglobulin directed against the target antigen, such antisera
also always contain antibodies directed against other antigens.
In addition, the immunoglobulins in antisera may bind with low avidity to molecules
that are not true target antigens.
For these and other reasons, antisera can manifest a level of background reactivity
that is unacceptably high.
There are three ways to deal with this problem.
1) An innocuous blocking agent (e.g., bovine serum albumin or normal serum)
can be used to compete with the immunoglobulin for nonspecific binding
sites.
a. Blocking agents are routinely included in solutions used, for example,
in immunological screening of expression libraries constructed in
plasmid or bacteriophage a vectors.
2) Antibodies that are directed against specific contaminating antigens (e.g.
bacterial antigens) can be removed by adsorption.
3) Antibodies directed against the target antigen can be separated from
contaminating antibodies by affinity purification.
For most purposes, antisera need not be fractionated before use.
 If the antisera are to be radiolabeled or conjugated to enzymes such as
horseradish peroxidase (HRP) or alkaline phoshatase, it is necessary to purify
the IgG fraction or, in some cases, to purify the antibody of interest by binding
it to its cognate antigen.
 Many techniques have been developed to purify IgG molecules
 The method of choice is adsorption to, and elution from, beads coated with
protein A, a component of the cell wall of S. aureus (Hjelm et at. 1972).
 For reasons that are not known, this protein (Mr = 42,000) binds strongly to
sites in the second and third constant regions of the Fc portion of the
immunoglobulin heavy chain (Deisenhofer 1981).
 Each IgG molecule therefore contains two binding sites for protein A.
 Because protein A itself has four potential sites for binding sites for IgG
(Sjodahl 1977), it is possible to form multimeric complexes of the two types
of protein.
 Not all immunoglobulins bind to protein A with the same affinity.
 Antibodies from humans, rabbits, and guinea pigs bind most tightly, followed
in decreasing order of affinity by those from pigs, mice, horses, and cows
(Kronvall et al. 1970; Goudswaard et al. 1978).
 Immunoglobulins from goats, rats, chickens, and hamsters, bind in a much
weaker fashion, and a “bridging” antibody is usually required to purify them
by adsorption to protein A.
 Within any one species, different classes of immunoglobulins vary in the
sequences of their Fc regions and consequently bind to protein A with
different affinities.
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o Of the major classes of human IgG, for example, three (IgG1, 2, and 4)
bind with high affinity and one (IgG3) binds very weakly, if at all.
o Similarly, mouse IgG2a binds with high affinity, IgG2b and IgG3 bind
tolerably well, and IgG1 binds poorly (Ey et al. 1978).
These differences are generally unimportant when dealing with polyclonal
sera, where antibodies against the target antigen are distributed throughout all
of the major subclasses of IgG.
Consequently, purification of polyclonal immunoglobulins raised in rabbits,
humans, and mice by binding to protein A may alter the distribution of
subclasses of IgG, but it rarely changes the specificity or avidity of the final
preparation.
Monoclonal antibodies secreted from hybridomas carry only one subclass of
heavy chain.
Before attempting to purify a given monoclonal antibody, it is essential to
determine the subclass of its heavy chain, using commercially available
immunological reagents directed against isotypes of the Fc region.
If the monoclonal antibody falls into a class that binds poorly to protein A
(e.g., human IgG3 or mouse IgG1), it should be purified by another method
(e.g., ammonium sulfate precipitation followed by chromatography of DEAEcellulose).
Alternatively, a bridging antibody can be used to attach the monoclonal
antibody to protein A.