Pseudorabies (Aujeszky’s disease) is an economically significant disease
of swine known to cause central nervous disorders, respiratory disease,
reproductive failure and mortality in infected pigs. In attempts to eradicate the
disease from becoming endemic, early detection is important to prevent further
economic losses and to allow for detection and removal of infected pigs in
domestic herds. Thus, a rapid and sensitive technique is necessary for the
detection of the virus. For rapid and simple examination, an immuno –
chromatographic lateral – flow assay system based on immunologic recognition
of specific pseudorabies virus antigen was developed by utilising, as signal
generator, colloidal gold conjugated to secondary antibody to detect primary or
sample antibody in the sera of pseudorabies infected animals. The pseudorabies
virus used as a capture antigen in the test strip was first cultivated in VERO cell
culture and then purified by sucrose gradient separation to produce the viral
protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted
well with the hyperimmune serum (HIS). The antibody detection system is
basically composed of colloidal gold – labelled antibodies fixed on a conjugate
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pad, and the complementary pseudorabies antigen immobilised onto a
nitrocellulose membrane forming capture zone. If the target antibody is present in
a specimen, the colloidal gold-labelled antibody will form a complex with the
antibody sample. Subsequently, the formed complex will migrate to the capture
zone and is then bound to the solid phase via antigen – antibody interaction. As a
result, a signal marker is generated by the accumulation of colloidal gold for
detection confirmation. The results obtained demonstrated that the optimum
combination of pseudorabies antigen needed as the capture reagent and gold
conjugate as secondary antibody recognition marker was at a concentration of
0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid –
based test strip towards pseudorabies antibodies was high with a detection limit
of 1 to 10,000 – dilution factor. The specificity of the assay was 100% with no
cross – reaction being observed with other sera or antibodies. Accurate reading
time needed for confirmation of the assay can be completed in 5 min with a
whole blood sample of 25 ml. The colloidal gold – labelled antibody is stable at
room temperature for 6 months or more (data not shown). Findings from this
study indicated that the solid – based test strip assay system provided high
sensitivity and specificity for the detection of pseudorabies at low levels of
antibody concentration. The assay was rapid, simple, cheap, and does not require
any sophisticated equipment. Thus, the solid based test strip will be a useful
serological screening technique or for rapid diagnosis of an infectious disease in
target populations of animals characterised by heterogeneous antibody responses.