Bio-Rad Affi-Gel 15 Gel - Manual (Page 18)

1. Specific Elution with excess antigen or antibody
should be considered first, because, in theory, it will
always work. It is often impractical due to the cost
and availability of the specific eluant. Another disad-
vantage is that an antigen-antibody complex will be
eluted and the dissociation of this complex may be
necessary and difficult to achieve.
2. Acid Elution is the most commonly employed des-
orption method and is frequently very effective.
Eluants such as glycine-HCl, pH 2.5, 20 mM HCl,
and sodium citrate, pH 2.5, can be used to disrupt the
antigen-antibody interactions. Acid elution can give
low recoveries due to hydrophobic interactions
between the antigen and the antibody. An eluant such
as 1 M propionic acid, or the addition of 10% diox-
ane
3 or ethylene glycol to the acid eluant, is more
effective in dissociating such complexes.
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9.3 Elution Strategies
Elution is usually the most difficult step in
immunoaffinity chromatography. The objective is to
obtain high purity and high recovery of a stable and active
product. Attempting to maximize yields, elution condi-
tions which denature the proteins are often chosen.
Antigens and antibodies are bound to each other by a
combination of ionic bonding, hydrogen bonding, and
hydrophobic interactions.
2 The strength of different anti-
gen-antibody complexes varies widely. Other parameters
such as ligand density, steric orientation, and nonspecific
interactions can also be important. Many solvents have
been used as eluants in immunoaffinity chromatography,
and the choice of an effective eluant often appears to be
empirical. There is, however, a logical strategy, or
sequence of eluants to consider when approaching a new
immunoaffinity application.
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