Indirect, or two stage,
immunochemistry procedures are often easier, are more widely used and have
the inbuilt advantage of allowing easy multi-probe comparative controls. Indirect immunochemistry
procedures involve the first stage incubation of a primary probe (antibody
or CBM) and, after washing away unbound probe, bound probe is detected by a
secondary detection probe that is usually a further antibody. For a rat
monoclonal antibody such as anti-xylan LM11 this can be
anti-rat-immunoglobulin-linked to-FITC or anti-his-tag for CBM detection.
This system allows the facile assessment of several probes which can often
act as controls for each other as well as easy assessment of the signal from
a no antibody control (that is needed to see the extent of autofluorescence
of cells walls that can be a problem in some systems). The system also
avoids the possible varied efficiencies of direct coupling procedures with a
wide set of probes. Shown below are two primary antibodies binding to
equivalent sections and detected with
identical secondary antibody incubation procedures. A vast array of fluorophores and secondary detection reagents are available commercially
allowing varied detection strategies. The same pot of antibody can be
readily used for fluorophore or gold or enzyme detection strategies without
the need to prepare specific coupled probes.
anti-arabinan LM6 / anti-rat-IgG:FITC
anti-xylan LM11 / anti-rat-IgG:FITC
immunohistochemical analyses of sections of plant
materials are often done with fluorescence and gold detection in conjunction
with light and
electron microscopies respectively. A section through a plant organ can
reveal all cell wall layers from the plasma membrane to intercellular matrices
of all cells. It is becoming clear however, that if a probe
does not bind to a cell wall the interpretation is not as straightforward as
perhaps previously thought. The lack of probe binding may indicate the
presence of no antigen or low levels of an antigen. Alternatively, it may
indicate that an antigen is soluble and is lost during the extensive washing steps inherent in immunolabelling procedures. This is the case with some pectins and these may
be detected by tissue/organ printing techniques that pick up soluble antigens.
Another possibility for the lack of a probe binding may be that the epitope is masked
by proximity to or possibly covalent association with another polymer. This has recently
been shown to be the case for xyloglucan in some cell walls where xyloglucan
epitopes are revealed by pectic enzyme deconstruction of cell walls (Marcus et al. 2008).
Work with xylan-directed CBMs indicate that polymer context within cell
walls, varying between cell types and species, can also influence probe recognition (McCartney
et al. 2006).