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It is not normally prudent to employ biospecifi c affi nity chromatography as an initial purifi ca-
tion step, as various enzymatic activities present in the crude fractions may modify or degrade
the expensive affi nity gels. However, it should be utilized as early as possible in the purifi cation
procedure in order to accrue the full benefi t afforded by its high specifi city.
6.6.5 Immunoaffi nity purifi cations
Immobilized antibodies may be used as affi nity adsorbents for the antigens that stimulated their
production ( Figure 6.15). Antibodies, like many other biomolecules, may be immobilized on a
sui table support matrix by a variety of chemical coupling procedures.
Immunoaffi nity chromatography is amongst the most highly specifi c of all forms of biospe-
cifi c chromatography. The high affi nity with which an antibody normally binds its ligand can
often make subsequent ligand desorption from the column diffi cult. Desorption can require
conditions that result in partial denaturation of the bound protein. This is often achieved by
alteration of buffer pH or by employing chemical disrupting agents, such as urea or guanidine.
One of the most popular elution methods employed involves irrigation with a glycine-HCl
buffer at pH 2.2-2.8. In some cases, elution is more readily attainable at alkaline pH val-
ues. Specifi c examples have been documented in which protein elution was performed under
relatively mild conditions, such as a change of buffer system or an increase in ionic strength;
however, such examples are exceptional. The inclusion of an immunoaffi nity step in the purifi -
cation of recombinant blood factor VIII used to treat haemophilia (Chapter 12) is one example
of the industrial usage of this technique
6.6.6 ProteinAchromatography
Most species of Staphylococcus aureus produce a protein known simply as protein A. This protein
consists of a single polypeptide chain of molecular weight 42 kDa. Protein A binds the F c region
(the constant region) of immunoglobulin G (IgG; Chapter 13) obtained from human and many
other mammalian species with high specifi city and affi nity. Immobilization of protein A on chro-
matography beads provides a powerful affi nity system that may be used to purify IgG. There is,
however, a considerable variation in the binding affi nity of protein A for various IgG subclasses
obtained from different mammalian sources. In some cases another protein, protein G, may be
used instead of protein A. Most immunoglobulin molecules that bind to immobilized protein A do
so under alkaline conditions, and may subsequently be eluted at acidic pH values.
6.6.7 Lectinaffi nity chromatography
Lectin affi nity chromatography may be used to purify a range of glycoproteins. Lectins are a
group of proteins synthesized by plants, vertebrates and a number of invertebrate species. Espe-
cially high levels of lectins are produced by a variety of plant seeds. Plant lectins are often termed