The histolocalization methods of in situ hybridization (ISH)
and immunohistochemistry (MH) are powerful techniques, which have been widely
applied to detect the cellular and tissue sites of specific gene expression.
We have successfully applied these methods to localize the site of expression
of a number of putative antigens of Onchocerca volvulus in particular
as they relate to the development and life cycle of the parasite.
For these procedures, parasite specimens, including adult worms in skin
nodules and larvae (L2, L3, and L4 stages), are fixed with modified Carnoy's
Fixative at -20_C, dehydrated, embedded in Paraplast, and sectioned serially
at 6-8 mm thickness. For ISH, the sections are deproteinized and denatured,
and then hybridized with biotin-labeled DNA probes derived from specific
putative antigen clones isolated by many laboratories. Sites of hybridization
are then visualized by incubating the sections with streptavidin conjugated
with alkaline phosphatase, followed by chromogenic histochemistry. For IMH,
the sections are rehydrated and incubated directly with specific antibodies
prepared against recombinant antigens, followed by enzyme conjugated secondary
antibodies and histochemical reaction. The sections are then observed by
Nomarski differential interference microscopy. At present, we have successfully
applied these procedures to map the gene expression of three candidate antigens,
including O13/O15 (isolated by Dr. Francine Perler), RAL2 (Dr. Thomas Unnasch),
and M3/M4 (Dr. Alan Scott). The resolution is sufficient to localize specific
sites of gene expression such as the hypodermis, microfilarial cuticle,
etc. In addition, the use of serially adjacent sections permits the concomitant
characterization of more than one antigen per specimen.
The success and reproducibility of the ISH procedure are crucially dependent
on the quality of the DNA probe, in the form of either total plasmid or
isolated insert, which directly affects the labeling efficiency and the
resultant specificity and signal intensity. Typically, the DNA probes should
be of CsCl gradient grade or equivalent and a quantity of 10-20 ug is needed
for repetitive experiments. For IMH, the antibodies should be exhaustively
absorbed to remove all non-parasite immunoreactivity and confirmed to specificity
by Western blotting. Parasite specimens are being prepared using stringent,
uniform conditions to ensure preservation of reactivity and morphology.
Generally, once the reagents and the specimens are prepared, ISH and IMH
localization of gene expression are expected to take approximately 3 months
to complete.
The information obtained from ISH and IMH analyses is highly relevant to
and should serve as a useful adjunct in the evaluation of the vaccine candidacy
of a particular antigen, since the expression of the antigen, at both mRNA
transcript and protein levels, may be mapped to a specific stage(s) in the
life cycle of the parasite and to a particular tissue site. For example,
an antigen which is expressed in the body wall structures of L3 larvae would
probably deserve particular attention.
The analyses should also aid in the characterization of monoclonal antibodies
prepared against parasite materials. Finally, such information is invaluable
in understanding the function of specific genes in O. volvulus biology.