may be required for living and fixed tissues for the same protein, as fixation may
destroy the structure of the epitopes in some cases. Fixed tissues are prepared by
standard histological methods. The tissue is fixed with a preservative which kills the
cells but maintains structure and makes the cell membranes permeable. The sample is
embedded in wax or epoxy resin and fine slices are taken using a microtome and they
are then mounted onto microscope slides. The antibodies that are used for immuno-
pathology may carry enzyme, fluorescent markers or labels such as gold particles.
They may also be unconjugated and in this case would require a secondary antibody
conjugate and solid substrate to visualise them. It is important to remember that
enzymes such as alkaline phosphatase may beendogenous(found naturally) in
mammalian tissue samples and their activity is not easily blocked. Often horseradish
peroxidase is used as an alternative. Any endogenous peroxidase activity in the
sample can be blocked by treating the sample with hydrogen peroxide. Antibodies
may recognise structural proteins within the cells and can access them in fixed tissues
through the permeabilised membranes. More than one antibody can be used to
produce a composite stain with more than one colour of marker being used. Combi-
nations of fluorescent and enzyme staining may also be used but this has to be carried
out sequentially. Fluorescent stains can also be used in conjunction with standard
histological stains viewed with a microscope equipped with both white and ultraviolet
light. Fluorescence will decay in time and although anti-quench products can be used
the specimens should not be considered to be permanent. Photographs can be taken of
slides through the microscope and kept as a permanent record.
7.10 Immunocapture polymerase chain reaction (PCR)
Immunocapture PCRis a hybrid method which uses the specificity of antibodies to
capture antigen from the sample and the diagnostic power ofPCRto provide a result.
The method is particularly of use in diagnostic virology where the technique allows
the capture of virus from test samples and subsequent diagnosis by PCR. It is useful
where levels of virus are low such as in water samples and other non-biological
sources. The technique can be carried out in standard PCR microtitre plates or in
PCR tubes. The antibody is bound passively to the plastic of the plate or tube and the
sample incubated afterwards (see Fig. 7.18). After washing to remove excess sample
material the PCR reagents can be added andthermocyclingcarried out on the bound
viralnucleic acid. RNA viruses will require an additionalreverse transcriptionstep
prior to PCR (see also Section 6.8.1).
7.11 Immunoaffinity chromatography (IAC)
Immunoaffinity chromatographycan be used for a number of applications. The
principle is based on the immobilisation of antibody onto amatrix, normally beads,
which are then placed into a chromatography column. Antibody may be permanently
295 7.11 Immunoaffinity chromatography (IAC)
