Protein Protocols Protein Protocols

1014 Fassina et al.
limit the use of this lectin for large-scale purification of monoclonal IgA from cell
culture supernatants. First, jacalin is a biologically active lectin, being a potent T cell
mitogen and a strong B cell polyclonal activator (9), thus requiring a careful control for
ligand leakage into the purified preparation. Second, jacalin binds to the carbohydrate
moiety of IgA, and D-galactose is required to elute IgA from affinity columns, which is
costly and impractical for large-scale operations.
Antibodies of the E class, which represent an extremely important class of immunoglobulins
from a biological and clinical point of view, require complex and timeconsuming
isolation protocols that make their characterization very difficult. The main
purification procedure is represented by immunoaffinity chromatography using anti-
IgE antibodies immobilized on solid supports (10,11). Even if selective enough for
research application, scaling up immunoaffinity chromatography for preparative applications
is very expensive and not easily accomplished. Other approaches for IgE purification
include classical chromatographic protocols based on the combination of
different sequential procedures such as salting out, affinity chromatography on lysine–
Sepharose, ion-exchange, gel filtration, and immuno-affinity chromatography to
remove interfering proteins (12). Studies carried out with immobilized protein A show
that this protein, known to recognize the immunoglubulin Fc region, does not bind to
monoclonal IgE, but binds 12–14% of serum polyclonal IgE. Protein G binds to neither
polyclonal nor monoclonal IgE (13).
IgY is an important class of immunoglobulins obtained from chicken egg yolk that
represents an economical source of polyclonal antibodies (14). Despite the advantages
in production of immunoglobulins from egg yolk in terms of efficiency (15), immunogenicity
against mammal proteins (15), and applications in therapeutics and diagnostics
(16,17), only fewer than 2% of the total number of polyclonal antibodies produced
worldwide and commercially available are raised in chickens. This low diffusion is
related to difficulties in purifying IgY from egg yolk, particularly from the lipidic fraction,
which represents the main contaminant. The complex isolation methods described
in the literature (18–20) and the lack of ligands usable for the affinity purification of
IgY make the isolation of this class of immunoglobulins a laborious process that cannot
be scaled up easily for industrial applications.
A synthetic peptide ligand (PAM, Protein A Mimetic, TG19318) (see Fig. 1),
derived from the synthesis and the screening of a combinatorial peptide library (21),
has been identified in our laboratory for its ability to recognize, as a protein A mimetic, the
constant portion of immunoglobulins. Its applicability in affinity chromatography for
the downstream processing of antibodies has been fully established in studies
examining the specificity and selectivity for polyclonal and monoclonal antibodies
derived from different sources. Ligand specificity is broader than for any existing
ligand, as IgGs derived from human, cow, horse, pig, mouse, rat, rabbit, goat, and
sheep sera (21,22), as well as IgYs derived from egg yolk (23) have been efficiently
purified on PAM affinity columns. The ligand proved useful not only for IgG purification,
but also for IgM (24), IgA (25), and IgE (26) isolation from sera or cell culture
supernatants (see Table 1). PAM ligand, a tetrameric tripeptide, can be produced at
low cost by means of chemical solution phase or solid phase synthesis in large amounts,
does not contain biological contaminants such as viruses, pyrogens, or DNA fragments,