popular. Linkage is achieved by simple chemistry to provide stable antibody–enzyme
conjugates. Glutaraldehyde is a cross-linking compound and conjugation to HRP is
carried out in two stages. Firstly the glutaraldehyde is coupled to reactive amino
groups on the enzyme. The HRP–glutaraldehyde is then purified bygel permeation
chromatographyand added to the antibody solution. The glutaraldehyde reacts with
amino groups on the antibody forming a strong link between the antibody and HRP.
Alkaline phosphatase can be linked to antibody by glutaraldehyde using a one-step
conjugation. The linkage is achieved through amino groups on the antibody and on
the enzyme coupled with the glutaraldehyde.
A number of proprietary labelling reagents are also available for making antibody
enzyme conjugates.
Fluorescent labels can also be added for use in immunofluorescent assays; usually
fluoresceinis the molecule of choice.Fluorescein isothiocyanate(FITC) is often the
derivative used to label antibodies. FITC is a fluorescein molecule with an isothiocya-
nate reactive group (–N¼C¼S) replacing one of the hydrogens. This derivative is
reactive towards primary amines on proteins and will readily react with antibodies to
produce fluorescent conjugates.
It is also possible to link antibodies to gold particles for use inimmunosorbent
electron microscopy(ISEM) andlateral flow devices. Gold particles are prepared by
citrate reduction of auric acid. The size of particle is predictable and can be controlled
by pH manipulation. The gold particles are reactive and will bind antibodies to their
surface formingimmunogold. The immunogold particles are stable and can be stored
at 4C until required.
Rare earth lanthanidescan also be used as labels and have the advantage that a
single assay can be used to detect two or three different antibody bindings. The
lanthanides are attached to the antibodies as a chelate. The commonest of the chelat-
ing compounds used is diethylenetriamine pentaacetate (DTPA). Each lanthanide
fluoresces at a different light frequency and so multiple assays can be carried out
and the individual reactions visualised by the use of a variable wavelength spectro-
photometer. Antibodies can also be attached to latex particles either by passive
absorption or to reactive groups or attachment molecules on the surface of the latex.
These can be used either as the solid phase for an immunoassay or as markers
in lateral flow devices. Magnetised latex particles are available allowing the easy
separation of latex particle/antibody/antigen complex from a liquid phase. Latex and
magnetic particles may be purchased which have protein A covalently attached to
their surface. Protein A binds the Fc portion of the antibody which orientates the
molecules with the binding sites facing outwards.
7.3 Immunoassay formats
The first immunoassay formats described were methods based on theagglutination
reaction (Fig. 7.10). The reaction between antibody and antigen can be observed when
agglutination occurs and is characterised either by a gel formation in a liquid phase or
283 7.3 Immunoassay formats
