linked to the beads by covalent linkage to reactive sites on a resin or bound using
protein A or G. Usually, antibody is permanently bound to column beads for most
applications as this is a more stable linkage and allows repeated use of the columns
following regeneration. IAC may be used as a clean-up method in analytical chemistry
to extract small quantities of chemical residues such aspesticidesfrom wastewater
and other sources. The method also works well for the extraction of biological
compounds such as hormones from patient samples. The columns are made by
reacting highly purified antibody (monoclonal or polyclonal) with the chromato-
graphy beads to form the affinity matrix. Harsh conditions have to be avoided as
denaturation of the antibody molecules could occur. A number of proprietary resins
are available which have reactive sites suitable for antibody immobilisation. The
affinity matrix is loaded into chromatography columns prior to use. Antibody binding
of antigen generally occurs best at around pH 7.4 but individual monoclonal
antibodies may vary considerably from this pH. Once the sample has been loaded
onto the column it should be washed to remove contaminating material from sample
fluid. Conditions for elution vary according to individual antibodies and antigens
but pH 2.0 buffer, methanol and 10% acetonitrile have all been used successfully.
The column can be regenerated after elution by incubating with pH 7.4 buffer.
The technique works extremely well for clean-up and concentration of sample
from dilute sources prior to additional analysis. Samples eluted from IAC columns
may be tested further by high-performance liquid chromatography, ELISA or other
analytical techniques.
7.12 Antibody-based biosensors
Abiosensoris a device that is composed of a biological element and aphysicochemical
transductionpart which converts signal reception by the biological entity into an
electrical impulse. A number of biosensor devices are available that use enzymes as
the biological part of the device. The enzyme is used to catalyse a chemical reaction
which generates an electrical charge at an electrode. Antibodies have the potential to
Plate coated
with antibody
Sample lysed to
release nucleic acid
followed by PCR with
specific primers
Sample added and
antigen captured
Fig. 7.18Immunocapture PCR.
296 Immunochemical techniques
